This book examines the rapidly growing field of functional foods in the prevention and management of chronic and infectious diseases. Chapters explore the varied sources, biochemical properties, metabolics, health benefits, and safety of bioactive ingredients of nutraceutical and functional food products. Special emphasis is given to linking the molecular and chemical structures of biologically active components in foods to their nutritional and pharmacological effects on human health and wellness. In addition to discussing scientific and clinical rationales for different sources of functional foods, the book also explains in detail scientific methodologies used to investigate the functionality, effectiveness, and safety of bioactive ingredients in food.
The chapter authors discuss advanced nanocarriers for nutraceuticals based on structured lipids and nonlipids, nanoparticulate approaches for improved nutrient bioavailability, adulteration and safety issues, nanodelivery systems, microencapsulation, and more. The book discusses some particular health benefits from nutrition nutraceuticals, including probiotic dairy and non-dairy products and bioactive proteins and peptides as functional foods. The volume also gives an overview of emerging trends, growth patterns, and new opportunities in the field of nutraceuticals and functional foods.
ADVANCES IN
NUTRACEUTICALS AND
FUNCTIONAL FOODS
Concepts and Applications
ADVANCES IN
NUTRACEUTICALS AND
FUNCTIONAL FOODS
Concepts and Applications
Edited by
Sreerag Gopi, PhD
Preetha Balakrishnan, PhD
First edition published 2022
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Library and Archives Canada Cataloguing in Publication
Title: Advances in nutraceuticals and functional foods : concepts and applications / edited by Sreerag Gopi, PhD, Preetha
Balakrishnan, PhD.
Names: Gopi, Sreerag, editor. | Balakrishnan, Preetha, editor. Description: First edition. | Includes bibliographical references and index.
Identifiers: Canadiana (print) 20210367172 | Canadiana (ebook) 20210367296 | ISBN 9781774637524 (hardcover) |
ISBN 9781774637531 (softcover) | ISBN 9781003277088 (ebook)
Subjects: LCSH: Functional foods. | LCSH: Functional foods—Health aspects. | LCSH: Functional foods—Nutritional
aspects.
Classification: LCC QP144.F85 A38 2022 | DDC 613.2—dc23
Library of Congress Cataloging-in-Publication Data
CIP data on file with US Library of Congress
ISBN: 978-1-77463-752-4 (hbk)
ISBN: 978-1-77463-753-1 (pbk) ISBN: 978-1-00327-708-8 (ebk)
About the Editors
Sreerag Gopi, PhD
Chief Scientific Officer, ADSO Naturals, India; Vice President, CureSupport, The Netherlands, Mobile: +91-8594023331,
E-mail: Sreeraggopi@gmail.com
Sreerag Gopi, PhD, is a Chief Scientific Officer at ADSO Naturals, India, and Vice President at CureSupport, The Netherlands. He graduated with a degree in Chemistry from Calicut Univer sity, Kerala, India, and an advanced degree from Madras Christian College, Chennai, India. He is a recipient of a prestigious Erasmus Mundus Fellowship from the European Union during his PhD period. He is a materials chemist and nanomaterials scientist and has expertise in nanomaterial synthesis, characterization, biocomposites for natural products, and biomedical and water purification experiments. He has published over 20 peer-reviewed international papers and several book chapters and also has book projects in the works with several publishers, including the Royal Society of Chemistry, Springer, Wiley. He was selected as an Associate Member of the Royal Society of Chemistry in 2018, and he is a chartered member of Royal Austra lian Chemical Institute.
Preetha Balakrishnan, PhD
Principal Scientist, QA, QC, ADSO Naturals India, and Curesupport, The Netherlands,
Mobile: +91-7025921175
E-mails: preetha@adsonaturals.com
Preetha Balakrishnan, PhD, is the principal scientist, QA, QC, at ADSO Naturals, India, and at CureSupport,
Netherlands. She graduated in Chemistry from Calicut University, Kerala, India, and earned her postgraduate degree at Mahatma Gandhi University, Kerala, with a gold medal and first rank. She is a recipient of prestigious INSPIRE Fellowship from the Government of India. She was a postdoctoral
vi About the Editors
researcher in the research group of Professor Sabu Thomas, Vice Chancellor, a renowned scientist in this area who has sustained international acclaims for his work in polymer science and engineering, polymer nanocomposites, elastomers, polymer blends, interpenetrating polymer networks, polymer membranes, green composites and nanocomposites, nanomedicine, and green nanotechnology. She completed her PhD in Chemistry at Mahatma Gandhi University under Dr. Thomas’s guidance. She has visited many foreign universities as a part of her research activities and has published over 15 research articles and over 20 book chapters. She has edited ten books with leading publishers, including Elsevier, Springer, Wiley, and the Royal Society of Chemistry. Dr. Balakrishnan has received a number of national and international presentation awards. She also worked as a guest lecturer in chemistry at the Department of Chemistry, Morning Star Home Science College, Angamaly, Kerala, India.
Contents
Contributors ix
Abbreviations xiii
Preface xvii
. Introduction to Functional Foods and Nutraceuticals
Luana Pulvirenti and Angela Paterna
. Advanced Nanocarriers for Nutraceuticals Based on Structured
Lipid and Nonlipid
Shafiullah, Syed Wadood Ali Shah, Ismail Shah, Shujat Ali, Aziz Ullah,
Samiullah Burki, and Mohammad Shoaib
. Nanoparticulate Approaches for Improved Nutrient Bioavailability..
Abdul Qadir, Mohd. Aqil, and Dipak Kumar Gupta
. Adulteration and Safety Issues in Nutraceuticals and Functional
Foods
Shujat Ali, Syed Wadood Ali Shah, Muhammad Ajmal Shah, Muhammad Zareef,
Muhammad Arslan, Md. Mehedi Hassan, Shujaat Ahmad, Imdad Ali, Mumtaz Ali,
and Shafi Ullah
. Nutraceuticals-Loaded Nano-Sized Delivery Systems: Potential
Use in the Prevention and Treatment of Cancer
Mohammed Jafar, Syed Sarim Imam, Sultan Alshehri, Chandra Kala, and Ameeduzzafar Zafar
. Nutrition Nutraceuticals: A Proactive Approach for Healthcare
Conor P. Akintola, Dearbhla Finnegan, Niamh Hunt, Richard Lalor,
Sandra O’Neill, and Christine Loscher
. Bioactive Proteins and Peptides as Functional Foods
Deepa Thomas and M. S. Latha
. News and Trends in the Development of Functional Foods:
Probiotic Dairy and Non-Dairy Products
Eliane Maurيcio Furtado Martins, Wellingta Cristina Almeida do Nascimento Benevenuto, Aurélia Dornelas de Oliveira Martins, Augusto Aloيsio Benevenuto Junior, Isabela Campelo de Queiroz, Thainل de Melo Carlos Dias, Daniela Aparecida Ferreira Souza, Daniele De Almeida Paula, and Maurيlio Lopes Martins
viii Contents
. Microencapsulation: An Alternative for the Application of
Probiotic Cells in the Food and Nutraceuticals Industries
Daniele De Almeida Paula, Carini Aparecida Lelis, and Nataly De Almeida Costa
. Nutraceuticals-Based Nano-Formulations: An Overview
Through Clinical Validations
Shelly Singh and Shilpa Sharma
. Growth Patterns, Emerging Opportunities, and Future Trends in
Nutraceuticals and Functional Foods
Asad Ur Rehman, Salman Akram, and Thierry Vandamme
Index 347
Contributors
Shujaat Ahmad
Department of Pharmacy, Shaheed Benazir Bhutto University Sheringal, Dir (Upper), Khyber Pakhtunkhwa, Pakistan
Conor P. Akintola
Immune Modulation Group, School of Biotechnology, Dublin City University, Dublin, Ireland
Salman Akram
University of Strasbourg, CNRS 7199, Faculty of Pharmacy, 74 Route du Rhin, CS - 60024, 67401 ILLKIRCH CEDEX, France
Imdad Ali
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi - 75270, Pakistan
Mumtaz Ali
Department of Chemistry, University of Malakand, Khyber Pakhtunkhwa - 18800, Pakistan
Shujat Ali
School of Food and Biological Engineering, Jiangsu University, Zhenjiang - 212013, P. R. China; College of Electrical and Electronic Engineering, Wenzhou University, Wenzhou 325035, PR China, E-mail: shujat86@yahoo.com
Sultan Alshehri
Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
Mohd. Aqil
Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Deemed University), M. B. Road, New Delhi - 110062, India, E-mail: maqil@jamiahamdard.ac.in
Muhammad Arslan
School of Food and Biological Engineering, Jiangsu University, Zhenjiang - 212013, P. R. China
Wellingta Cristina Almeida do Nascimento Benevenuto
Federal Institute of Southeast of Minas Gerais, Food Science and Technology Department (DCTA/IF Sudeste MG), Rio Pomba, MG, CEP - 36180-000, Brazil
Samiullah Burki
Department of Pharmacology, Faculty of Pharmacy, Federal Urdu University of Arts, Science, and Technology, Karachi, Pakistan
Nataly De Almeida Costa
Department of Food Technology, Federal University of Viçosa (UFV), P.H. Rolfs Avenue, Campus, Viçosa - 36570-900, MG, Brazil
Thainل de Melo Carlos Dias
Federal Institute of Southeast of Minas Gerais, Food Science and Technology Department (DCTA/IF Sudeste MG), Rio Pomba, MG, CEP - 36180-000, Brazil
Dearbhla Finnegan
Immune Modulation Group, School of Biotechnology, Dublin City University, Dublin, Ireland
x Contributors
Dipak Kumar Gupta
Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Deemed University), M. B. Road, New Delhi - 110062, India
Md. Mehedi Hassan
School of Food and Biological Engineering, Jiangsu University, Zhenjiang - 212013, P. R. China
Niamh Hunt
Immune Modulation Group, School of Biotechnology, Dublin City University, Dublin, Ireland
Syed Sarim Imam
Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
Mohammed Jafar
Assistant Professor, Department of Pharmaceutics, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University, P.O. Box - 1982, Dammam - 31441, Saudi Arabia, Mobile: +966502467326, E-mail: jafar31957@gmail.com mjomar@iau.edu.sa
Augusto Aloيsio Benevenuto Junior
Federal Institute of Southeast of Minas Gerais, Food Science and Technology Department (DCTA/IF Sudeste MG), Rio Pomba, MG, CEP - 36180-000, Brazil
Chandra Kala
Faculty of Pharmacy, Maulana Azad University, Jodhpur - 342802, Rajasthan, India
Richard Lalor
Fundamental and Translational Immunology Group, School of Biotechnology, Dublin City University, Dublin, Ireland
M. S. Latha
Department of Chemistry, Sree Narayana College, Chathannur, Kollam, Kerala, India; Department of Chemistry, Sree Narayana College, Kollam, Kerala, India, E-mail: lathams2014@gmail.com
Carini Aparecida Lelis
Center for Food Analysis (NAL), Technological Development Support Laboratory (LADETEC), Federal University of Rio de Janeiro (UFRJ), Cidade Universitلria, Rio de Janeiro, RJ, 21941-598, Brazil
Christine Loscher
Immune Modulation Group, School of Biotechnology, Dublin City University, Dublin, Ireland
Aurélia Dornelas de Oliveira Martins
Federal Institute of Southeast of Minas Gerais, Food Science and Technology Department (DCTA/IF Sudeste MG), Rio Pomba, MG, CEP - 36180-000, Brazil
Eliane Maurيcio Furtado Martins
Federal Institute of Southeast of Minas Gerais, Food Science and Technology Department (DCTA/IF Sudeste MG), Rio Pomba, MG, CEP - 36180-000, Brazil, E-mail: eliane.martins@ifsudestemg.edu.br
Maurيlio Lopes Martins
Federal Institute of Southeast of Minas Gerais, Food Science and Technology Department (DCTA/IF Sudeste MG), Rio Pomba, MG, CEP - 36180-000, Brazil
Sandra O’Neill
Fundamental and Translational Immunology Group, School of Biotechnology, Dublin City University, Dublin, Ireland, E-mail: Sandra.oneill@dcu.ie
Angela Paterna
Institute of Biophysics, National Research Council, Via Ugo La Malfa - 153,90146, Palermo, Italy
Contributors xi
Daniele De Almeida Paula
Federal Institute of Sمo Paulo (IFSP), Campus Avaré - Av. Professor Celso Ferreira da Silva, 1333, Jardim Europa, CEP 18707-150, SP, Brazil, E-mail: daniele.paulaufv@gmail.com
Luana Pulvirenti
Department of Chemical Sciences, University of Catania, Viale Andrea Doria-6, 95125, Catania, Italy [Dipartimento di Scienze Chimiche, Università Degli Studi di Catania, Viale A. Doria - 6,95125, Catania, Italy], E-mail: luanapulvirenti@unict.it
Abdul Qadir
Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Deemed University), M. B. Road, New Delhi - 110062, India
Isabela Campelo de Queiroz
Federal Institute of Southeast of Minas Gerais, Food Science and Technology Department (DCTA/IF Sudeste MG), Rio Pomba, MG, CEP - 36180-000, Brazil
Asad Ur Rehman
University of Strasbourg, CNRS 7199, Faculty of Pharmacy, 74 Route du Rhin, CS - 60024, 67401 ILLKIRCH CEDEX, France; University of Paris Descartes, UTCBS CNRS UMR 8258-INSERM U1267, Faculty of Pharmacy, 4 Avenue de l’Observatoire, Paris - 75006, France
Shafiullah
Department of Pharmacy, University of Malakand, Chakdara, Dir Lower - 18300, Khyber Pakhtunkhwa, Pakistan, E-mail: shafi_ullah34@yahoo.com
Ismail Shah
Department of Pharmacy, Abdulwali Khan University, Mardan - 23200, Khyber Pakhtunkhwa, Pakistan
Muhammad Ajmal Shah
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Government College University, Faisalabad, Pakistan
Syed Wadood Ali Shah
Department of Pharmacy, University of Malakand, Chakdara, Dir Lower - 18300, Khyber Pakhtunkhwa - 18800, Pakistan
Shilpa Sharma
Department of Biological Sciences and Engineering, Netaji Subhas University of Technology, Dwarka, New Delhi, India
Mohammad Shoaib
Department of Pharmacy, University of Malakand, Chakdara, Dir Lower - 18300, Khyber Pakhtunkhwa, Pakistan
Shelly Singh
Department of Biological Sciences and Engineering, Netaji Subhas University of Technology, Dwarka, New Delhi, India
Daniela Aparecida Ferreira Souza
Federal Institute of Southeast of Minas Gerais, Food Science and Technology Department (DCTA/IF Sudeste MG), Rio Pomba, MG, CEP - 36180-000, Brazil
Deepa Thomas
Research and Post Graduate Department of Chemistry, Bishop Moore College, Mavelikara, Alappuzha, Kerala, India
Aziz Ullah
Department of Pharmaceutics, Faculty of Pharmacy, Gomal University, D.I. Khan, Khyber Pakhtunkhwa, Pakistan
Shafi Ullah
Department of Pharmacy, University of Malakand, Khyber Pakhtunkhwa - 18800, Pakistan; H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi - 75270, Pakistan, E-mail: Shafi_ullah34@yahoo.com
Thierry Vandamme
University of Strasbourg, CNRS 7199, Faculty of Pharmacy, 74 Route du Rhin, CS - 60024, 67401 ILLKIRCH CEDEX, France
Ameeduzzafar Zafar
Department of Pharmaceutics, College of Pharmacy, Jouf University, Sakaka, Aljouf, Saudi Arabia
Muhammad Zareef
School of Food and Biological Engineering, Jiangsu University, Zhenjiang - 212013, P. R. China
Abbreviations
ABC ATP-binding cassette
ACE angiotensin-converting enzyme
ACE angiotensin I-enzyme
ADA American Dietetic Association
Ag silver
AI artificial intelligence
ALA alpha-linoleic acid
ARA arachidonic acid
Au gold
AuNPs gold nanoparticles
BDPP bioactive dietary polyphenol preparations
CAG compound annual growth
CAGR compound annual growth rate
CAPE caffeic acid phenethyl ester
CCPs caseinophosphopeptides
CD Crohn’s disease
CdS cadmium sulfide
CLA conjugated linoleic acid
CLNA conjugated α-linolenic acid
CPSC consumer product safety commission
CST critical solution temperature
CVD cardiovascular diseases
DALYs disability-adjusted life years
DE dextrose equivalent
DHA docosahexaenoic acid
DIM diindolylmethane
DPP4 dipeptidyl peptidase-IV
DSC differential scanning calorimetry
DTA differential thermal analysis
EC European Commission
EE encapsulation efficiency
EFSA European Food Safety Authority
EGCG epigallocatechin gallate
EGFR epidermal growth factor receptor
xiv Abbreviations
EMS eosinophilia-myalgia-syndrome
Eos essential oils
EPA eicosapentaenoic acid
EPA Environmental Protection Agency
EPS exopolysaccharides
FAO Food and Agriculture Organization
FAS fatty acid synthase
FDA Food and Drug Administration
FIM foundation for innovation in medicine
FOSHU foods for specified health use
FPHs fish protein hydrolysates
FSA Food Standards Agency
Gas glycoalkaloids
GI gastrointestinal
GIP glucose-dependent insulinotropic polypeptide
GIT gastrointestinal tract
GLP-1 glucagon-like peptide-1
GRAS generally recognized as safe
HCl hydrochloric acid
HSE health service executive
IBD inflammatory bowel disease
IBS inflammatory bowel syndrome
IGFR insulin-like growth factor receptor
IL interleukin
ILSI International Life Sciences Institutes
IPP Ile-Pro-Pro
IPP isopentenyl diphosphate
JECFA Joint FAO/WHO Expert Committee on Food Additives
LAB lactic acid bacteria
LCST lower critical solution temperature
LMP low methoxyl pectin
LUVs large unilamellar vesicles
MEP methylerythritol phosphate
Met-S metabolic syndromes
MLV multilamellar vesicles
MMPs matrix metalloproteinases
MPS mononuclear phagocyte system
MRP multidrug resistance protein
MTSG1 mitochondrial tumor suppressor 1
Abbreviations xv
MVA mevalonic acid
NA nicotinic acid
NCDs non-communicable diseases
NEs nanoemulsions
NIOSH National Institute for Occupational Safety and Health
NLCs nanostructured lipid carriers
NPs nanoparticles
NREA Nutraceutical Research and Education Act
NSAIDs non-steroidal anti-inflammatory drugs
O/W oil-in-water
O/W/O oil-in-water-in-oil
OCP office of combination products
OSHA Occupational Safety and Health Administration
OsLu lactulose-derived oligosaccharide
PA palmitic acid
PAA poly(acrylic acid)
PAAM poly(acrylamide-co-butyl methacrylate)
PBS phosphate buffer solution
PC phosphatidylcholine
PCADK poly(cyclohexane-1,4-diyl acetone dimethylene ketal)
PCL polycaprolactone
Pd palladium
PDEAEM poly(N,N9-diethylaminoethyl methacrylate)
PDEAM poly(N,N-diethylacrylamide)
PDMAEMA poly[2-(dimethylamino)ethyl methacrylate]
PE phosphotidyl ethanolamine
PECs polyelectrolyte complexes
PEG poly(ethylene glycol)
PGA poly(glycolic acid)
PK polyketals
PLA polylactic acid
PLGA poly(lactic-co-glycolic acid)
PNIPAM poly(N-isopropylacrylamide)
PNPs polymeric nanoparticle systems
PPARγ peroxisome proliferator-activated receptors
Pt platinum
PUFAs polyunsaturated fatty acids
PVCL poly(N-vinylcaprolactam)
xvi Abbreviations
PVCL-PVA-PEG polyvinyl caprolactam-polyvinyl acetate-polyethylene
glycol
RA rheumatoid arthritis
RES reticuloendothelial system
RESS rapid expansion of supercritical solution
RNA ribonucleic acid
ROS reactive oxygen species
SERS surface-enhanced Raman spectroscopy
siRNA small interfering RNA
SLN solid lipid nanoparticles
SMEDDS self-micro emulsifying drug delivery system
SOD superoxide dismutase
SUVs small unilamellar vesicles
T2DM type 2 diabetes mellitus
TEM transmission electron microscopy
TJs tight junctions
TMC N-trimethyl chitosan
TNF tumor necrosis factor
TNF-α tumor necrosis factor-alpha
UC ulcerative colitis
UCST upper critical solution temperature
US United States
USA United States of America
USDA US Department of Agriculture
USPTO US Patent and Trademark Office
VEGF vascular endothelial growth factor
VPP val-pro-pro
W/O/W water-in-oil-in-water
WHO World Health Organization
Preface
In recent years there is a growing interest in nutraceuticals, which provide health benefits and are alternative to modern medicine. Nutrients, herbals, and dietary supplements are significant constituents of nutraceuticals which make them instrumental in maintaining health, act against various disease conditions, and thus promote the quality of life. The explosive growth, research developments, lack of standards, marketing zeal, quality assurance, and regulation will play a vital role in its success or failure.
The demand for foods with a positive impact on human health and well ness has exploded globally over the past two decades. This growth is driven by socioeconomic and scientific factors, including increases in population, disposable income, life expectancy, and healthcare costs. Advancements also enhance the market for healthier foods in our understanding of dietary bioactive ingredients and their effects on various aspects of human health at a systems and molecular level. This book examines the rapidly growing field of functional foods to prevent and manage chronic and infectious diseases. It attempts to provide a unified and systematic account of functional foods by illustrating the connections among the different disciplines needed to under stand foods and nutrients, mainly: food science, nutrition, pharmacology, toxicology, and manufacturing technology. Advances within and among all these fields are critical for the successful development and application of functional foods. Chapters in the present volume explore the varied sources, biochemical properties, metabolism, health benefits, and safety of bioactive ingredients. Special emphasis is given to linking the molecular and chemical structures of biologically active components in foods to their nutritional and pharmacological effects on human health and wellness. In addition to discussing scientific and clinical rationales for different sources of functional foods, the book also explains in detail the scientific methodologies used to investigate the functionality, effectiveness, and safety of bioactive ingredi ents in food.
CHAPTER 1
Introduction to Functional Foods and Nutraceuticals
LUANA PULVIRENTIand ANGELA PATERNA
1Department of Chemical Sciences, University of Catania, Viale Andrea Doria-6, 95125, Catania, Italy
2Institute of Biophysics, National Research Council,
Via Ugo La Malfa - 153,90146, Palermo, Italy
ABSTRACT
Nowadays, the term functional food gained more attention, especially by the younger generations, since are certainly more informed about the increasingly close correlation between food and health. This term was first used in Japan in 1980 and since that time it has been possible to record a growing interest from the scientific community around the world, in order to clarify their potential role in the prevention of chronic diseases and in the maintenance of good health of a population with a longer life expectancy than in the past. In this context, this chapter aims to offer a simple and comprehensive overview about definitions and classifications of functional food. Furthermore, atten tion was focused on the close relationship that exists between the chemical composition of a food in terms of ‘functional’ chemical compounds known today as nutraceuticals, and the ability of the food to play a functional role.
.1 INTRODUCTION
Today, the common thought is that foods together with a good lifestyle may be able to prevent diseases or physiological disorders. This belief is actu ally much older than might think, and even Hippocrates about 2500 years ago claimed, “Let food be thy medicine and medicine be thy food.” The
Advances in Nutraceuticals and Functional Foods
aforementioned concept is particularly felt mostly by the younger generations, who represent a new class of consumers, of course, more health-conscious than before. More in general people take more into account the strict rela tionship between diet and health. The reason is that they are more informed about it, thanks also to many scientific and popular magazines, tv programs, social media posts, and blogs which often deal with topics concerning the content of bioactive chemical substances in foods and their potential activity as chemopreventive agents of degenerative diseases. Therefore, these foods defined ‘functional foods’ are considered desirable in a good diet. The term ‘functional foods,’ used for the first time in Japan in 1980 [1, 2], includes every food or food ingredients exerting a nutritional function but at the same time express promising healthy effect when eaten regularly in a varied diet [3]. The above consideration allows to enclose in this group not processed foods such as fruit and vegetable, but also foods formulated with a specific health purpose.
In recent years a renew attention has been registered on functional foods from researchers in the world working in different fields of science due to a growing global interest for these foods. Indeed, the fields of investiga tion involving functional foods are manifold and often linked together; for example, using on Scopus.com the index term ‘functional food’ about 61.000 documents (articles, chapters, and books) were found, published between 1980 and 2019, with an increasing number of publication year by year, confirming the growing scientific interest. Furthermore, it is noteworthy that analyzing quickly the results of search by subject area, it is possible to observe that this topic involves many scientific fields (Figure 1.1).
Therefore, it is not surprising that the functional foods development has required interconnection with related field like food chemistry, biology, nutrition, pharmacology, and statistics [4].
In this context, important contributions have been made by many epide miological studies reported in literature with the purpose to evaluate the relationship between diet habits and the risk of contracting a large share of the global diseases, through conditions such as high blood pressure and elevated blood glucose and cholesterol levels. Two famous examples are the Mediterranean-style diet and high-fat diets, also well known as the French paradox, incorporating moderate red wine intake are reported to benefit to human health [5]. In particular French paradox refers to the lower risk of the French people towards cardiovascular diseases (CVD), despite their high fat diet, attributed to their habitual but moderate consumption of red wine. Also, the Mediterranean-style diet, expressed by a reduced drinking of alcohol, a
Introduction to Functional Foods and Nutraceuticals
balanced eating of meat and its subproducts, an increased ingestion of fruits, vegetables, and extra-virgin olive oil, is widely recognized to have beneficial effects on CVD.
FIGURE 1.1 Total number of articles (a) by year; and (b) subject area. Source: Published from: 1980 to 2019 at Scopus.com using the index term ‘functional food.’
Nowadays, is commonly approved that the helpful outcomes of functional foods can be attributed to the chemical substances’ characteristic of their composition, well known with the term ‘nutraceuticals,’ for which it was registered an increased interest corroborated by the growth of the nutraceu tical trend aiding the growth of the global market. The global nutraceutical market size was valued to grow from about $209 billion in 2017 to $373 billion in 2025, predicting a spread at a CAG (compound annual growth) rate of 8.3% over the estimated period [6].
The growing scientific interest in the development of functional foods and nutraceuticals also goes hand in hand with the increased life expectancy average at the global level (around 70-80 years), and with the necessity in maintaining a good overall health status in the years. Therefore, both are considered a valid and safe help that together with a healthy lifestyle they can prevent chronic diseases, very frequent in the elderly.
Advances in Nutraceuticals and Functional Foods
The purpose of this chapter is to offer a comprehensive overview of functional foods and nutraceuticals, briefly mentioning about definitions, classifications, and their potential role in the chemoprevention of chronic diseases such as diabetes, obesity, CVD, and cancer.
.2 FUNCTIONAL FOODS
1.2.1 CONCEPTUAL DEFINITION AND CLASSIFICATION
Since 1980, when the term “functional food” was used for the first time [1, 2], inaugurating a new sector of food sciences around the world, researchers, government agencies, and national and international organizations have tried to formulate their own definition. This circumstance certainly created little clarity and many opportunities to generate confusion due to the large number of definitions and their large variations of meaning that make it difficult to provide industry partners with solid information on market trends and potential or adequately protect consumers through legislation.
In this context, the lack of an official or commonly accepted formal meaning for functional foods has promoted the opportunity of an inter national debate involving many researchers. Scientists have accepted the challenge of trying to make a concise analysis of all the definitions in the literature, trying to clarify at least a conceptual level of which type of food should be considered “functional” and which scientific requirements should support them.
The monograph of International Life Sciences Institutes [7], and the work done by Doyon and Labrecqueri [8], respectively, were really important in the formulation of the conceptual meaning of functional foods, and have allowed to summarize the most important phases of their development. Table 1.1 offers only a quick overview of the most important definitions found in literature for “functional food” formulated over the past 30 years from different countries and health institutions. Therefore, it is not surprising that there is ambiguity among the highest government offices, public health professionals, and of course the population.
Furthermore, it is noteworthy to highlight that currently, Japan is the unique country that has defined a precise regulatory recommendation for functional foods practices well known with the acronym FOSHU (foods for specified health use) [9], while in the rest of the world the boundary between conventional and functional foods remains undeciphered and make trouble also experts such as nutritionists and health experts. It is obvious to think that
Introduction to Functional Foods and Nutraceuticals
the lack of specific legislative regulation was, on the one hand, a limitation for the definition of coherent guidelines to be followed in their development and, on the other hand, did not allow the release of health claims that were regulated.
TABLE 1.1 Various Definitions of Functional Foods
Year Definition of Functional Food Source/Reference(s)
‘Foods which are, based on the knowledge between FOSHU, Japanese
foods or food components and health, expected to have Ministry of Health
certain health benefits, and have been licensed to bear and Welfare
a label claiming that a person using them for specified
health use may expect to obtain the health use through
the consumption thereof.’
‘Foods that encompass potentially healthful products, National Academy of
including any modified food or food ingredient that may Sciences Food and
provide a health benefit beyond that of the traditional Nutrition Board
nutrients it contains.’
‘Food which could be regarded as ‘functional’ as The European
being one that has been satisfactorily demonstrated to Commission
beneficially affect one or more functions in the body, Concerted Action
beyond adequate nutritional effects, in a way which is Group on Functional
relevant to either an improved state of health and well Food Science in
being and/or a reduction of risk. It is consumed as part Europe (FUSOSE),
of a normal food pattern. It is not a pill, a capsule or any International Life
form of dietary supplement.’ Sciences Institute
(ILSI) [10]
‘Modified foods or food ingredients that provide health Adelaja and Schilling
benefits beyond their traditional nutrients.’
‘Foods or food components that may have health benefits National Institute of
that reduce the risk of specific diseases or other health Nutrition
concerns.’
‘A food component (being a nutrient or not) which Roberfroid
affects one or a limited number of function(s) in the body
in a targeted way so as to have positive effects that may
justify health claims.’
‘Functional foods serve naturally primarily the supply of European food
nutrients, but additionally they offer a special advantage information council
for the health.’
‘Substances that provide essential nutrients often beyond Institute of Food
quantities necessary for normal maintenance, growth, Technologists (IFT)
and development, and/or other biologically active components that impart health benefits or desirable physiological effects.’
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TABLE 1.1 (Continued)
Year Definition of Functional Food Source/Reference(s)
‘Foods that may provide health benefit beyond basic International Food
nutrition.’ Information Council
(IFIC) (IFIC
Foundation (2006)
‘A functional food is a conventional food or a food Health Canada
similar in appearance to a conventional food, it is part of a regular diet and has proven health-related benefits and (or) reduces the risk of specific chronic diseases above its basic nutritional functions.’
‘Natural or processed foods that contains known or The Functional Food
unknown biologically-active compounds; which, in Center (FFC)
defined, effective non-toxic amounts, provide a clinically proven and documented health benefit for the prevention, management, or treatment of chronic disease.’
The analysis of these definitions is not the purpose of this chapter, but briefly, it is clear that the main characteristics that can be extrapolated in order to define a food as “functional” include the type of the food and the relationship with their potential health benefits and consumption pattern.
It is widely recognized that the general perception is that a functional food is any healthy food consumed regularly during the daily life, and is declared to possess a physiological advantage such as health-promoting or diseasepreventing properties beyond the basic function of supplying nutrients. On this basis, functional foods can be classified in different classes depending on the origin, modification, and their potential biological activities.
In the following are reported practical examples of foods that should be considered “functional foods” classified based on their possible modifica tion [11]:
• Not processed natural food (or conventional food), such as fruit,
vegetable, or fish, well known for their promising content of bioactive natural products;
• A natural food which may be modified during plant breeding or other
technological procedures in order to improve desired characteristics; • A modified food by adding a bioactive component; • A modified food by removing or reducing component.
As regards the composition of functional foods often substances were eliminated or incorporated into foods targeted for specific group of consumers.
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In the development and production of this kind of food products, food tech nology plays a key role, considering always palatability and convenience as essential requirement for the success of the product on the reference market. Among the technologies that allow the modification of the food composition, fortification, and extraction are very important. In food technology, the term fortification is used to indicate the enhancement of a product with a specific nutrient before to be processed. Whereas, the simply use of the term “enrich ment” indicates a product in which a component, not normally present in the food in the original composition, is added. Extraction and purification are techniques used in the field of food technology with two main purposes: obtaining bioactive substances from plant, food or waste materials possessing special activity related to health and well-being, in order to add them to food products; eliminating a component that interferes with the optimal nutritional value of food product. Therefore, an assortment of functional foods can be developed and classified [11] as reported in Table 1.2.
TABLE 1.2 Different Types of Functional Foods
Types of
Description Practical Example
Functional Foods
Product with an increased content of a Vitamins in juice
Fortified products
component.
Product with a new component in its Spread with added
Enriched products
content. phytosterols
Product in which a component is removed,
Altered products Fiber in meat products
and replaced with a beneficial one.
Product in which the nutrient composition Vitamins in fruit and Enhanced products
is altered by raw commodities. vegetables
Thus, the term functional foods can include traditional foods like fruit, vegetable, fish, meat, and derivatives and modified foods (fortified, enriched, altered, and enhanced) which have proven nutritional and preven tive qualities. The consumption could therefore improve well-being, prolong existence, and prevent the development of chronic diseases.
Many of conventional foods considered functional for their beneficial qualities are known since the tradition in which they were used together with spices, medicinal herbs, and roots for the preparation of recipes deemed capable of treating and preventing health-related ailments. Nowadays, thanks to the scientific progress, it is possible to demonstrate the role of food on human health and not just hypothesize it.
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A growing attention has been registered in particular on plant based functional food such as fruit, vegetables, and spices, also supported by the health claims issued by the most authoritative health organizations such as World Health Organization (WHO). In fact, among the WHO guide lines, to guarantee a healthy diet it is necessary to introduce minimum five portions of fruit and vegetables daily [12]. It was also highlighted an increased demand for plant-based foods from consumers more attracted to the opportunity to have health benefits in a natural way. A wide range of examples can be cited in this regard (Table 1.3). Among foods possessing a “functional” aspect in the body, pomegranate (Punica granatum), a fruit widely consumed as fresh fruit and juice, is known since the traditional medicine for its therapeutic qualities in the treatment of diarrhea, diabetes, hemorrhage, and inflammation [13]. In recent years, studies both in vitro and in vivodemonstrated its antioxidant, antidiabetic, hypolipidemic, and shows antibacterial, anti-inflammatory, antiviral, and anticarcinogenic activities [14]. Many studies have suggested a beneficial potential on health in dietary grape consumption; in particular cardiovascular benefits and cancer chemopreventive are only some of the potential disease prevention activities of grape [15]. Grapes are also the raw material used to obtain red wine, whose cardioprotective potential has been widely studied in people who consume it in a habitual but moderate way to accompany main meals [5]. Special mention must be made for Citrus fruit, for which the annual total global production is estimated to be about 120 million tons [16]. The interest in consumption of these fruit is certainly favored by the delicious flavors, but more importantly, their nutritional value and the health promotion effects are of considerable impact. The human health-promoting by Citrus fruit has been the object of many scientific investigations that highlighted the efficacy against various diseases including cardiovascular, cancer, and inflammatory diseases [17]. There are several works in literature focused on characterizing the intrinsic health-promoting potential of blueberry, today recognized by media as “superfruit” for its several health benefits that include maintenance of blood sugar levels, reduction of oxidative stress, anti-inflammatory effect, prevention of CVD, antimicrobial, and antitumor activity [18]. Another example of functional food much cited in the litera ture is tomato (Lycopersicon esculentum) that represents an important and significant part of the human diet, and its regular consumption has been related with a risk reduction to various types of cancers and CVD [19]. In particular, there is scientifically supported epidemiological evidence, which suggests a reduction in the risk of susceptibility to certain types of cancer,
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in more exposed subjects such as smokers [20]. Furthermore, is noteworthy a recent study published on “Public Health Nutrition” associates a lower risk of cancer mortality increasing the consumption of tomato [21]. Since the 2000s was denoted a growing interest in ginger (9.247 document results using scopus.com as source), known for its beneficial properties for health already in traditional oriental medicine to treat different illnesses. Today is widely used as spice in foods and beverages and strongly recommended as a functional ingredient in our daily diet for its nutraceutical attributes that include digestive stimulant action, anti-inflammatory influence, and anticancer effect [22]. It is interesting to note that although ginger performs a digestive action capable of promoting the absorption of nutrients, in conditions of high-fat diet this spice is able to suppress the accumulation of cholesterol and fats in the body suggesting a potential role in weight management and hence in preventing risk of obesity [23] and CVD. Among the foods that have promising properties in the prevention of the risk of chronic diseases such as diabetes, obesity, and CVD we can also mention broccoli (Brassica oleracea) [24], recently studied by Aranaz et al. [25]
that provided new knowledge about their potential role in the prevention of metabolic syndrome. Most of the foods plant-based discussed until now are low in calories, with the exception of Hass Avocado (1.7 kcal/g) very popular as the main ingredient of the avocado-based guacamole. Hass Avocado is a tropical nutrient-dense fruit that has a high oil content composed of highly digestible unsaturated fatty acids; due to these nutritional characteristics, it could be a valid substitute for not very healthy high-calorie snacks carrying out a protective and preventive action against CVD. In fact, an avocadorich diet has been shown to reduce blood cholesterol, preserving the level of high-density lipoproteins, significantly reducing low density lipoproteins [26] and diminishing the risk of metabolic syndrome [27]. Taking into account beverages, tea infusion is one of the most popular widely consumed worldwide, known also to be linked to the promising activity of prevention of many types of cancer and to reduce the risk of chronic diseases [28]; for all the health-promoting qualities, reviewed in the last 30 years, tea is recognized as functional food by many authors in literature. Moreover, a recent epidemiologic study on the Japanese population showed that a higher consumption of green tea is associated with lower risk of mortality for heart and cerebrovascular diseases, and a moderate consumption decreased the risk of total cancer and respiratory disease mortality [29]. Another spice for which there has been greater interest in recent years is cinnamon; its introduction into weight-reducing diets has proved positive, improving the
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weight reduction effect, moreover, many studies have supported its activity as an antiobesity [30]. These are just a few examples of foods that could have a functional role if inserted in a balanced diet.
TABLE 1.3 List of Conventional Foods and Their Beneficial Qualities
Food Plant-Based Beneficial Qualities References
Antioxidant, antidiabetic, hypolipidemic, Pomegranate (Punica
antibacterial, anti-inflammatory, antiviral, [14]
granatum L.)
anticarcinogenic
Cardioprotective, decreased platelet
Grapes (Vitis vinifera) aggregation, antihypertensive, anticancer, [15]
antioxidant
Citrusfruits (orange,
Cardioprotective, anti-inflammatory,
tangerine, lime, lemon, [17]
anticancer grapefruit)
Anti-hyperglycemic, antioxidant, anti
Blueberry inflammatory, cardioprotective, antimicrobic, [18]
anti-mutagenic, antitumoral Tomato (Lycopersicon
Antioxidant, cardioprotective, anticancer [19]
esculentum)
Antibacterial, antiviral, analgesic, antipyretic, carminative, anti-inflammatory,
Ginger (Zingiber
immunomodulator, antitumorigenic, [22]
officinale)
antihyperglycemic, anti-lipidemic, antidiabetic
Broccoli (Brassica
Antioxidant, cardioprotective, anticancer [24]
oleracea)
avocado (Persea Antioxidant, cardioprotective, LDL-oxidation,
[26]
Americana) immune system, diabetes, cancer
Antioxidant, cardioprotective, anticancer,
Green tea (Camellia anti-inflammatory, antiarthritic, antibacterial,
[28]
sinensis) antiangiogenic, antioxidative, antiviral,
neuroprotective
Stringent, carminative, antiseptic, antifungal, Cinnamon (Cinnamomum
antiviral, digestive, antihyperlipidemic, [30]
zeylanicum)
antihyperglycemic, antiobesity
In this chapter, we wanted to highlight the close correlation between healthy food and the prevention of chronic degenerative diseases. In this context, however, it should not be forgotten that the opposite is also true, namely that bad eating habits and unregulated lifestyles are among the main
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causes of twentieth-century diseases. Especially in economically developed western countries, the diseases with a higher incidence are obesity, meta bolic syndrome, and diabetes, in which weight management represents a key strategy in prevention. In particular, obesity is today considered one of the main public health problems worldwide [31] due to its high incidence and represents an important risk factor for diseases such as type 2 diabetes, CVD, and tumors [32]. Therefore, functional foods that have a preventive action in this sense, such as the above-cited foods, are considered desirable in the diet also in view of the pandemic impact of chronic diseases that are becoming the leading causes of global morbidity and mortality. Finally, the most pragmatic and widely scientifically-supported recommendation for populations in general is a balanced diet, with an emphasis on fruit and vegetables and increased physical activity.
.3 NUTRACEUTICALS
1.3.1 DEFINITION AND REGULATORY ASPECTS
Today it is known that functional foods owe their beneficial qualities to the biologically active chemicals that make them up; in particular, edible plants and fruit together with their agro-industrial waste are considered promising sources of potential chemopreventive agents for degenerative diseases. Among the food sciences, food chemistry, thanks also to the development of analytical techniques, has invested many efforts on the evaluation and char acterization of the molecular composition of foods. Despite, nowadays, the chemical composition of most of these foods is known, the study of potential biological activities has become the object of interest. In this regard, a lot of efforts has been made to isolate and characterize the chemical compounds believed to be beneficial to health in order to perform in vitroand in vivo studies aiming to demonstrate their potential role on human health.
Natural products, both isolated compounds from foods and comestible plants, able to achieve a “functional” role in the body are called “nutraceuti cals.” Currently, there is a growing global interest in nutraceuticals due to the recognition that they may play a major role in health enhancement and they are considered to be a promising source of potential chemotherapeutic agents. Many studies support the hypothesis that they are capable to counteract pathologies such as inflammation, obesity, diabetes, carcinogenesis, or neurodegenera tive disorders; moreover, many of those perform a very effective antioxidant
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activity (radical scavengers). For this reason, these natural compounds have also been exploited in drug discovery, developing new synthetic analogs with the purpose to improve their biological activity, bioavailability, the route of administration, etc. A variety of examples could be cited in this regard. In 1989 Stephen De Felice, Chairman of the Foundation for Innovation in Medicine (FIM), coined the term nutraceutical combining the terms “Nutrition” and “Pharmaceutical,” to highlight the strict connection between some groups of food molecular components and their capability to act with effects attributable to drugs. Therefore, it can be considered nutraceutical any substances, part of a food, able to contribute to well-being as well as prevention and treatment of diseases. In summary, the concept of nutraceutics integrates, in its definition, the union between food (or rather nutrition), understood as a generic intake of substances that allow the body to function, and the pharmaceutical that has to do with substances or components synthesized or isolated for therapeutic purposes, going for the first time to sanction a fundamental concept that sees food as a container of potentially biologically active substances. According to this principle, food can be medicine but at the same time poison depending on how they are composed.
As a consequence of misinformation and maybe the lack of specific regulations, there is a lot of confusion regarding the boundary between nutra ceuticals and pharmaceuticals. From a practical point of view, recognized by many scientists, the difference between these two product groups is patent coverage that supports the pharmaceutical function of drugs [33]. Indeed, both pharmaceuticals and nutraceuticals have the ability to cure or prevent diseases; only pharmaceuticals have the authoritative approval. The use of nutraceuticals with a potential therapeutic effect has also met the interest of pharmaceutical companies that are more incentivized in the development of this type of product, which requires a lower basic investment if compared to pharmaceutical ones. This data is also confirmed by the size of the global nutraceutical market, which has been estimated to be $230.9 billion in 2018 and should reach $336.1 billion by 2023 with a compound annual growth rate (CAGR) of 7.8% [34]. According to the Food and Drug Administration (FDA) regulations, nutraceuticals in the Unites States would be recognized as “dietary supplements” comprising vitamins, minerals, herbs, and extracts which are able to provide nutrients [35]. Instead, the safety assessment and regulation of nutraceuticals in the European Community are special product category regulated by the European Food and Safety Authority (EFSA) called “food supplements” concentrated sources of nutrients or other substances with beneficial nutritional effects (Directive 2002/46/EC) [36]. Thanks to constant progress in scientific research, today we are able to accurately assess
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the influence of different nutraceuticals on the normal physiological functions of the body. However, it was highlighted that most part of nutraceuticals can have a multiple therapeutic effect, and more in general, they can have a role in the protection against obesity, diabetes, metabolic syndrome, etc. Therefore, it is not so surprising that since prehistoric times, humans have been able to draw most part of their medicines from foods and plants in order to treat multiple pathologies. At this regard, the Ebers Papyrus (1550 BC) is rich of examples. Nutraceuticals can be classified differently, according to the needs of the discussion; thus, we could classify them on the basis of the potential biological activity and the potential sector of use, but this would make the discussion more complicated since, as already said, most of these substances can have a multiple biological effect. For that reason, the classification that from our point of view is more exemplary is that which takes into account the biogenetic origin and therefore the chemical class to which they belong.
1.3.2 NUTRACEUTICALS AND CHEMICAL CLASSIFICATION
All the living organisms such as plants, animals, and microorganisms require a wide number of organic compounds to live, grow, and reproduce. The metabolic pathways are responsible, through a complex system of enzyme-mediated chemical reactions, to provide the essentially molecules carbohydrates, proteins, fats, and nucleic acids. The synthesis, transforma tion, and degradation of these compounds is called primary metabolism and the molecules originated are primary metabolites. Moreover, in organisms occur the secondary metabolism contributing to the production of the most pharmacologically active natural compounds described as secondary metab olites. These bioactive compounds are distributed and sometimes confined in specific organisms as a consequence of the environmental context, defense, or resistance against insects and are classified according to their biosyn thetic pathways. The classification of these compounds comprises phenolic compounds, terpenoids, alkaloids, and fatty acids [37].
1.3.2.1 POLYPHENOLS
Polyphenols, belonging to an important family of natural products, are compounds that should be found in fruits, vegetables, herbs; moreover, in foods and beverages derived from them, have been the subject of studies indicating their role in the chemoprevention of degenerative diseases, such as
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CVD, cancer or Alzheimer’s disease. Some of these polyphenols are consid ered nutraceuticals because they are constituents of foods and drinks acting as and beverages able to play a ‘functional’ role in the body. Several studies support the hypothesis that polyphenols, derived from natural sources, are potent antioxidants (radical scavengers) and are able to counteract patholo gies such as inflammation, diabetes, carcinogenesis, or neurodegenerative disorders. Phenolic compounds are widespread mainly in the Plant Kingdom and include more than 8000 known compounds. Their role in the plant is presumably defensive, but they may also have other biological activities in interspecies relationships. This group of compounds is one of the most studied worldwide, and many publications report beneficial effects of polyphenols on various aspects of human health and well-being [38].
The growing interest in (poly)phenolic compounds and their exploitation in the fields of agro-food, cosmetic, and drug industry has led to a broader (and sometimes inappropriate) use of the term ‘polyphenols’ with respect to the original definition of ‘plant polyphenols,’ later expanded by E. Haslam [39], and recently by S. Quideau [40]. Originally, the ‘plant polyphenols’ were substantially equivalent to ‘vegetable tannins,’ with reference to the tanning action of some plant extracts that had been employed for centuries in the leather-making process. However, this definition has subsequently been broadened in the common use to include low-molecular-weight phenolic molecules as well, not necessarily water-soluble or exerting a ‘tanning’ action. Consequently, the common feature of polyphenols has been reconfigured with regard to their biosynthetic origin, thus including phenolic metabolites biosynthetically derived through the shikimate and/or the acetate/malonate pathways. Scheme 1.1 briefly reviews the biosynthesis of phenolic compounds, mainly through the shikimate pathway (Scheme 1.1) [41, 42]. Some examples of bioactive polyphenols are reported below.
Resveratrol is the widely recognized polyphenol, should be found in grapes and red wine, considered cardioprotective and anticarcinogenic, which has become very popular due to the so-called French paradox, already discussed above. J. Pezzuto, in a recent review, cites about 512 references on its ability to prevent cancer [15]. A further well-known phenolic compound is Genistein, an isoflavone present in soybean (Glycine soja), with estrogenlike activity able to relieve menopause symptoms and prevent some estrogendependent cancers, such as breast cancer [43]. Tannins are another class of natural polyphenols known for their several biological activities related to their antioxidant [44], antiviral [45], host-mediated antitumor activities [46, 47], moreover, recently they are reported for their promising antidiabetic
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properties [48]. Many polyphenols are esters or amides of phenolic acids such as CAPE (caffeic acid phenethyl ester), found in substance produced by bees, known as propolis; this compound is capable of acting as a potent antioxidant, reported also for its promising antitumor properties [49].
Scheme 1.1 Biosynthesis of phenolic compounds; shikimate pathway.
1.3.2.2 TERPENOIDS
Terpenoids represent a wide and diversified class of secondary metabolites derived from C5 isoprene units joined together. Classification of terpenoids is based on the number of carbon skeleton (C5)nlinked through a linear
head-to-tail organization leading to monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesterterpenes (C25), triterpenes (C30) and
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tetraterpenes (C40). Two different metabolic pathways should be involved in the synthesis of the isoprenoids units-dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP)-the mevalonic acid (MVA) pathway and the 2-C-methyl-d-erythritol 4-phosphate (methylerythritol phosphate: MEP)
pathway (Figure 1.2).
FIGURE .2 Terpenoids biosynthesis, mevalonic acid pathway, and methylerythritol phosphate pathway.
MAV and MEP pathways are responsible for providing the isoprene units for the biosynthesis of particular classes of terpenes; in particular animals and fungi utilize the mevalonate pathway exclusively, instead MEP is presents in plants, algae, and bacteria. In plants, both pathways are present and compartmentalized, MAV in the cytosol and MEP in the plastids. Thus, triterpenes, and sesquiterpenes are formed by the mevalonate pathway, mono-di-, and tetraterpenes are MEP derived [50].
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Among the natural products scaffolds, terpenoids play a crucial role in a wide variety of therapeutic indications. Essential oils (EOs) are secondary metabolites produced by plants, consist of a mixture of volatiles terpenoids, phenylpropanoids, and short-chain aliphatic hydro carbon derivatives containing a major constituent up to 85%, while other constituents are present in traces. They are usually extracted by steam distillation from natural sources (flowers, seeds, leaves, bark, herbs, wood, fruits, and roots) and have been used since ancient times because of their perfumes, flavors, and preservatives features [51]. EOs are char acterized by high chemical diversity and biochemical specificity being responsible for their biological activities. EOs extracted from Origanum vulgare, Thymus vulgarisand Rosmarinus officinalishave been shown to
possess antibacterial activity against Staphylococcus aureus and Listeria monocytogenes[52]. EOs of the fresh leaves, unripe, and ripe fruit peels of Citrus reshnihave been displayed potential antiviral activity against avian influenza virus A (H5N1 subtype) [53]. Moreover, EOs extracted from plants exhibited antifungal [54], insecticidal [55, 56] and anticancer activities [57]. In drug discovery programs, diterpenes represent another important class of terpenoids. Macrocyclic diterpenes presenting lathy rane and jatropha scaffolds, extracted from Euphorbiaspecies, displayed a potential anticancer activity as MDR inhibitors in multidrug resistance, acting through P-gp modulation [58-61]. Moreover, Rosmarinus offici nalisextracts exhibited high antioxidant properties due to the presence of phenolic diterpenes [62].
1.3.2.3 ALKALOIDS
Alkaloids are compounds characterized for their basicity and the presence of nitrogen atoms in the molecule. Morphine were the first alkaloid discovered obtained from plants, consequently the early definition of alkaloids included these three characteristics nitrogen-containing, basicity, and plant origin. Successively, the theory of being derived from amino acids was added, together with the idea that the nitrogen should be in a heterocyclic ring [63]. Alkaloids are frequently classified based on the structure, such as the presence of the nitrogen in the ring. Some different amino acid precursors are involved in alkaloid biosynthesis (Figure 1.3).
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FIGURE 1.3 Alkaloids biosynthesis and basic structures.
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Nevertheless, a large group of alkaloids is found to acquire their nitrogen atoms via transamination reactions, incorporating only the nitrogen from an amino acid, whilst the rest of the molecule may be derived from acetate or shikimate; others may be terpenoid or steroid in origin [64].
Indole alkaloids are plant-derived compounds, comprising over 3000 members, characterized by a wide range of biological activities (Figure 1.4), including cytotoxic and anti-inflammatory [63, 65, 66]. Vincristine and vinblastine, clinically important anticancer agents, are examples of useful bioactive indole alkaloids from Apocynaceae [67]. Plant species belonging to the Apocynaceae family are important source of these secondary metabo lites. Rubiaceae, Loganiaceae, and Nyssaceae families are also known for synthesize bioactive indole alkaloids (Figure 1.4) [68-71].
FIGURE .4 Representative terpene indole alkaloids, with the corresponding biological function.
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Moreover, Capsicumgenus fruits (chili peppers) are food ingredients and additives used widespread. The principal secondary metabolite present is capsaicin, a phenylalanine derived alkaloid well-known for the mucosal irri tant peculiarity and beyond food flavoring possess multiple health benefits like obesity, cardiovascular, and gastrointestinal (GI) disorders cancer [72] and in vivoantioxidant activity [73].
Glycoalkaloids (GAs), are secondary metabolites synthesized by plant belonging to the Solanaceaefamily (i.e., tomato, potato, and eggplant). The two major GAs present in potato (Solanum tuberosum) are α-solanine and α-chaconine, tomato plants (S. lycopersicum) present α-tomatine and dehydro tomatine and in eggplant fruits (S. melongena) are found solanine and solamar gine. These bioactive compounds, besides have antifungal, antimicrobial, and insecticidal properties as a protective activity against several insects, pests, and herbivores; several studies reported their potential anticancer activity [74].
1.3.2.4 FATTY ACIDS
The acetate pathway is involved in the biosynthesis of fatty acids, another crucial class of nutraceuticals. Fatty acids are classified in saturated and unsaturated; their formation is catalyzed by the enzyme fatty acid synthase (FAS) and natural saturated fatty acids may contain from 4 (butyric acid) to 30 (melissic acid), or even more, carbon atoms. The unsaturated fatty acids, usually containing one or more double bonds in a non-conjugated pattern, occurs in animals and plants. Omega 3 and omega 6 are polyunsaturated fatty acids (PUFAs), also called essential fatty acids because of our organism is not able to synthesize them ex Novoand have beneficial effects in human health. Is important to assume them through the diet. PUFAS are well known to take part in cellular physiology, are involved in energy storage and are structural components of cell membrane conferring fluidity, thickness, stability, and permeability [75]. Moreover, PUFAs like arachidonic acid (ARA, C20:4n-6), eicosapentaenoic acid (EPA, C-20:5n-3) and docosahexaenoic acid (DHA, C-22:6n-3) are precursors of specific lipid mediators with a potent pro- and anti-inflammatory activity. The inflammation resolution pathways need numerous biochemical signals fundamental to achieve the inflammatory response and the lipid mediators’ synthesis, including prostaglandins, leukot rienes, resolvins, lipoxins, maresins, and protectins. All these compounds are mono-, di-, and tri-hydroxylated and epoxidized derivatives of PUFAs. PUFAs and the corresponding derived lipid mediators possess a strategic
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function as potential pharmaceutical and nutraceutical targets in the preven tion and treatment of several chronical immune diseases [76].
FIGURE 1.5 Illustrative description of phospholipid and liposomes.
1.3.3 NUTRACEUTICALS AND NANOTECHNOLOGY
As mentioned above, nutraceuticals possess a wide array of chemical and physical properties, features which often do not allow to use these products directly in their pure form. Nowadays, nanotechnology techniques are tools used by food and nutraceuticals industries to develop new products with improved characteristics. Encapsulation methods are useful to separate and entrap the biologically active compound from the outer part conferring many
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benefits. These benefits include mix incompatible ingredients, reduce toxicity, enhance solubility and stability, improve effectiveness, prevent chemical degradation in food matrix, increase bioavailability and lead to targeted delivery; moreover, in food can change color, smell, and flavor [77]. Among all the encapsulation techniques, the one which fascinate researchers and still capture attention in the literature is the use of liposomes. Liposomes are spher ical-shaped structures consisting of a central aqueous compartment enclosed by one or more concentric phospholipid layers. The phospholipid vesicles are characterized by hydrophilic (aqueous cavity) and hydrophobic (within lipidic bilayer) elements consenting to amphiphilic bioactive substances to be incorporated within these structures. Liposomes were usually classified on the basis of their size and number of bilayers, the unilamellar vesicles are identified in small, large, and giant (SUV: 20-100 nm; LUV: > 100 nm; GUV: > 1000 nm) and multilamellar vesicles (MLV): > 500 nm (Figure 1.5) [78].
Food industries exploit nutraceuticals entrapped in liposomes to produce functional foods, reinforcing the nutritional efficacy, enhancing the health of buyers, and reducing the risk of some diseases. Encapsulation of phenolic compounds from Pistachio green hulls of Ahmad aghaeivariety, possessing antioxidant and antimicrobial properties, could enhance the bioavailability of the extract [79]. Vitamins are another vast class of nutrients involved in several biochemical functions such as preventing cancer and cardiovascular disorders and improving the immune system. These bioactive compounds are produced in few amounts by our body leading to a necessary implementation of vitamins through food supplements. Vitamins are classified in two groups water-soluble and fat-soluble, encapsulation of vitamins in liposomes is a suit able technique to increase their stability and solubility [80]. PUFAs are another class of nutraceuticals widely used and are susceptible to autoxidation reac tions which is considered the key disadvantage to overcome. Encapsulation of PUFAs get better foods in terms of sensory parameters, like smell and flavor, and in terms of stability [81]. Furthermore, betalains are natural pigments extensively used as colorants for food products, and despite this use, some studies disclosed about the antioxidant, anti-inflammatory, anticancer, and antidiabetic properties. The weakness of betalains is the limited oral bioavail ability, encapsulation in liposomes increased its stability and enhanced the antioxidant activity [82]. Turmeric plant (Curcuma longa) is the source of curcumin, a yellow phenolic compound, known worldwide for its antioxidant and anti-inflammatory properties and used in Asian traditional medicine for the multiple health benefits. Although curcumin exhibit numerous thera peutic effects possess some disadvantages limiting the real effectiveness, has
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not a good solubility in water, low bioavailability and is quickly metabolized and eliminated. Several studies proved that encapsulation of curcumin in liposomes increase anticancer activity by improving pharmacokinetics and pharmacodynamics and reducing the dose required [83].
.4 CONCLUSION
Although the use of plants in traditional medicine to prevent and treat several diseases have been known and employed by indigenous people since ancient times; just a while ago, the effectiveness of nutraceuticals has been supported by scientific reports. The enlarged scientific interest in nutraceuticals and functional foods led food industries in a growing attention to develop healthpromoting ingredients. Despite regulation of nutraceuticals and functional foods is still ambiguous and varies from country to country, an expanded global market of nutraceuticals and functional foods has been documented together with a recent customer conscience and careful to choose healthy food and a constant search for high dietary intake of nutraceuticals. An important challenge and still a topic of discussion concerns improving the characteristics of this bioactive compound and promote their biological efficacies. Nanotechnology techniques are employed to enhance stability, solubility, bioavailability, targeted delivery, smell, color, and flavor.
KEYWORDS
• cardiovascular diseases • compound annual growth rate • European Food and Safety Authority • isopentenyl diphosphate
• mevalonic acid
• World Health Organization
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. Kwak, N. S., & Jukes, D. J., (2001). Functional foods. Part 1: The development of a
regulatory concept. Food Control, 12(2), 99-107.
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CHAPTER 2
Advanced Nanocarriers for
Nutraceuticals Based on Structured Lipid and Nonlipid
SHAFIULLAH,SYED WADOOD ALI SHAH,ISMAIL SHAH,SHUJAT ALI,,6AZIZ ULLAH,SAMIULLAH BURKI,and
MOHAMMAD SHOAIB
1Department of Pharmacy, University of Malakand, Chakdara, Dir Lower - 18300, Khyber Pakhtunkhwa, Pakistan
2Department of Pharmacy, Abdulwali Khan University, Mardan - 23200,
Khyber Pakhtunkhwa, Pakistan
3School of Food and Biological Engineering, Jiangsu University,
Zhenjiang - 212013, P. R. China
4Department of Pharmaceutics, Faculty of Pharmacy, Gomal University,
D.I. Khan, Khyber Pakhtunkhwa, Pakistan
5Department of Pharmacology, Faculty of Pharmacy, Federal Urdu
University of Arts, Science, and Technology, Karachi, Pakistan 6College of Electrical and Electronic Engineering, Wenzhou University,
Wenzhou 325035, PR China
ABSTRACT
Nanocarriers-based therapeutics are gaining greater attention and research trends in the biomedical field because they have excellent commercialization potential. In this context, an upsurge in nanocarriers based on commercially available products has been observed. Nanotechnology basically deals with the engineering of particles at molecular levels, and this term was originally the subject of building nanoscale devices and machines. In recent years,
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nanotechnology has broached in various unfamiliar fields of Pharmaceuti cals and nutrition. Nutraceuticals have immense importance among both consumers and researchers because of the growing interest in alternative medicinal sources. Nutraceuticals are foods, or foods’ parts, that provide health or medical benefits, including the treatment and prevention of diseases. Nutraceuticals is a broad term where foods, i.e., dietary supplements, antioxi dants, dairy products (fortified), and minerals, vitamins, herbals, citrus fruits, cereals, and milk comes under this umbrella. Due to the proven healthcare and fitness benefits of nutraceuticals, researchers as well as ordinary people are engrossed towards these natural dietary agents. The increasing interest in such products is also due to the innate biological activities (i.e., antioxidant activities), biocompatibility, and non-toxic nature of these phytochemicals. The World Health Organization (WHO) has started a worldwide strategy to cope with traditional medicines-related issues due to this growing public interest in nutraceuticals. Nutraceuticals from various sources have shown to suppress pro-inflammatory pathways for treating cancer and similarly in other ailments; however, their low in vivobioavailability limits their use. Research on nano-encapsulation of nutraceuticals has been the recent trend to resolve the limitations associated with them and improve their health benefits. This chapter gives an overview of nutraceuticals, different types of nanocar riers, recent developments in the field of nanocarriers based nutraceuticals delivery, their absorption mechanisms, and major challenges in the way of commercialization of nanocarriers based nutraceuticals.
.1 INTRODUCTION
Natural products are the main sources of bioactives and are regarded as the key discovery of modern medicine [1, 2]. Search for novel bioactive compounds and pharmacophores has always been in regular practice because still organic and synthetic medicinal chemists yet have to find replacement for many natural compounds [3]. The field of drug delivery is revolution ized by nanotechnology-based drugs where bioactive compounds are encapsulated in nanocarriers for addressing various bioactive related issues. Several phytochemicals face the issue of poor bioavailability due to their low intrinsic solubility. Encapsulating such phytochemicals and bioactive compounds in appropriate nanocarriers can enhance their bioavailability by virtue of altered pharmacokinetics and biodistribution profiles in nanosized delivery carriers [4]. Similarly, it has also been shown that using nanocar riers based delivery systems, the drug is localized to specific tissues, and the therapeutic index of drugs is increased [5, 6]. The availability of nanocarriers
Advanced Nanocarriers for Nutraceuticals Based on Structured Lipid and Nonlipid
based products in the market strongly suggests that they have commercializa tion potentials. At present, researchers as well as the ordinary population is attracted towards agents from natural dietary sources because of their proven effectiveness in fitness and healthcare [7]. As an example, due to the proven health benefits of the cactus plant (having taurine as the key constituent) such as anti-diabetic, anti-viral, and anticancer potentials, it has become an active constituent of nutraceuticals. WHO has started a worldwide strategy to resolve issues related to traditional medicines keeping in view the growing researchers and scientists’ interest in nutraceuticals. Similarly, the European Commission (EC) has also decided to put the disease risk reduction at priority in future plans. Research on encapsulation and delivery of nutraceu ticals in nanocarriers-based systems has been the subject of interest for many researchers to address the issues associated with nutraceuticals and enhance their healthcare outcomes. This chapter describes nutraceuticals; different types of nanocarriers, recent developments in the field of nanocarriers based nutraceuticals delivery, their absorption mechanisms, and major challenges commercialization of nanocarriers based nutraceuticals.
.2 NUTRACEUTICALS
The word nutraceutical is the combination of two terms, i.e., nutrition, and pharmaceuticals. Nutraceuticals are food or food products offering nutritional as well as pharmaceutical benefits, i.e., give nutrients to the body, provide resistance against various diseases, and also help in treating certain ailments [8]. Long ago, people having knowledge and working in the field of medicine thought to develop and search such foods that may be served as medicine for treatment and prevention of diseases. Ultimately, such sparkling ideas led to the development of nutraceuticals, whereas the term nutraceuticals were first coined by Dr. Stephen DeFelice in 1989 by combining nutrition and pharmaceuticals [9]. There are three main classes of nutraceuticals, i.e., functional foods, dietary supplements, and functional beverages. The dietary supplements can be further sub-divided into mineral supplements, protein supplements, vitamins, herbal supplements, and plant extracts.
Probiotics and omega fatty acid foods fall in the category of functional foods, while functional beverages are sub-classified into fortified juices, sports drinks, and energy drinks. Dietary supplements, functional food, multi-func tional food, etc., are some of the common words used as synonyms or related to nutraceuticals. Functional foods are just the basic foods; however, some special or specific ingredients are incorporated in them for providing health benefits to the body along with nutrients [9]. Functional foods exclusively
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designed for promoting good health in human beings is made possible due to recent technological advancements in the field of food technology. Isolation, identification, characterization, and purification are some basic requirements to be considered while incorporating food components in functional foods. Moreover, characterization of incorporated food components in terms of medicinal values, nutritional values, etc., are also important. Our body need primary food components composed of proteins, carbohydrates, and lipids for normal energy and proper body functioning. Some secondary food elements, i.e., vitamins, are commonly not produced in the human body, and are neces sary for proper body function, thus these components must be taken in food diet. Nutraceuticals are also minor food elements that improve the body func tion by virtue of fighting against some chronic disease conditions [10].
The therapeutic efficacy of any drug, food product or nutraceuticals is dependent on its bioavailability. In pharmacological perspectives, bioavail ability is the rate and extent to which the drug reaches systemic circulation or its site of action, while in terms of nutritional concept-bioavailability means some nutrients in food are partially available. When orally admin istered, certain parameters, e.g., low solubility and/or permeability within the gastrointestinal tract (GIT), less gastric residence time and instability in GIT or under food processing conditions limit their activity. The increasing popularity and ever-growing public interests in nutraceuticals as preventive medicine demands and put pressure on manufacturers and regulators of health-related products to address their bioavailability-related issues [11].
.3 NANOCARRIERS FOR NUTRACEUTICALS DELIVERY
Nanotechnology-based delivery systems, e.g., nanoparticles (NPs), vesicles, hydrogels or microparticles are attaining promising place in pharmaceutical industry and food technology because they offer tools for enhancing therapeutic efficacy drugs and nutraceuticals. Nanotechnological tools are mainly applied to those drugs or nutraceuticals having poor aqueous solubility, low bioavail ability, and GI stability problems. As an example, the stability of flavonoids and anthocyanidins depends upon GI pH whereas nearly 60% of probiotic bacteria cannot survive in GI environment. Thus, to protect nutraceuticals or drugs from harsh GI environment and to improve their bioavailability, their delivery in suitable carrier-based system is highly demanding [12].
Nanotechnology-based products possess great commercialization potential and a multifold increase of such products in the market is expected in coming years because such products overcome many of the limitations associated
Advanced Nanocarriers for Nutraceuticals Based on Structured Lipid and Nonlipid
with them [13]. Nevertheless, the safety of newly developed nanocarriers based systems must be ensured prior to their incorporation in commercial food products. Various desirable features of nanocarriers-based systems must be considered prior to the construction of such systems. Firstly, as the size of the drug/nutraceuticals is reduced to nano-size range, the behavior of this system will be different from the conventional particles in GIT [14, 15].
Toxicity issues may occur if the degradation products of the nanocarriers based delivery system is different from conventional particulate matter. Thus, to ensure safety, it is imperative to evaluate toxicity profiles to ensure the safety of such food-grade nanoscale devices. For the fabrication and construction of nanocarriers-based delivery systems, the application of food-grade materials is highly preferable. In addition, these nano-formulations must be strong enough to withstand storage conditions, economically feasible as well as have strong potentials for practical applications. Moreover, the quality of products should not be adversely affected by the incorporation of such materials into final food products. Figure 2.1 shows the advantages of using nanocarriers’based delivery systems over other conventional dosage forms. However, the nanotechnology-based nutraceuticals delivery systems should be:
FIGURE .1 Advantages of using nanocarriers’-based delivery systems over other conventional dosage forms.
Advances in Nutraceuticals and Functional Foods
• Physicochemically stable to environmental conditions and should
preserve its functional properties [16];
• Capable of improving GI stability of labile bioactive components; • Capable of maintaining constant dose levels in systemic circulation; • Capable of facilitating lymphatic transport in case of highly lipophilic
compounds;
• Capable of extending gastric retention times [17].
Several researchers have reported the application of nanotechnology for nutraceuticals over the last few years. The increase in oral bioavailability and absorption and thus the nutraceutical effects of certain phenolic compounds have been reported [18, 19]. The advantages of nanocarriers-based delivery systems for nutraceuticals is summarized in Figure 2.2. The nanocarriers based nutraceuticals have been mainly prepared using polymers (natural or synthetic polymers, polysaccharides, proteins) or lipids-based systems (liposomes, solid lipid nanoparticles (SLN), nanoemulsions (NEs)). These polymers and lipids-based delivery systems for nutraceuticals are discussed in the following subsections.
FIGURE 2.2 The advantages of nanocarriers-based delivery systems for nutraceuticals.
Advanced Nanocarriers for Nutraceuticals Based on Structured Lipid and Nonlipid
2.3.1 POLYMERS
In targeted drug delivery, polymers are extensively employed as delivery vehicles due to their specialized flexible structures. In addition, polymers prevent nutraceuticals from severe conditions of the GI tract due to their entrapment of nutraceuticals. Polymers ideally deliver nutraceuticals to the targeted site either in natural or synthetic form, usually in nano or microsized particles [20].
2.3.1.1 BIODEGRADABLE POLYMERS
Biodegradable synthetic or natural polymers have been extensively used in the field of tissue engineering and drug delivery as nanocarriers for nutraceuticals/drug delivery because of their unique characteristics such as biodegradability, biocompatibility, and flexible nature [21]. Polymers with biodegradability characteristics certainly degrade via normal biological and chemical processes in the body. Specifically, in most cases, the synthetic polymers are engineered in such a way that they biodegrade by hydrolysis phenomena. It should also be kept in mind that the degradation product of a synthetic biodegradable polymer should also be biocompatible and biode gradable. These biodegradable polymers encapsulate the nutraceuticals and are tailored in such a way that the encapsulated product is released upon degradation of the polymer in the desired site.
2.3.1.2 NATURALLY OCCURRING BIODEGRADABLE POLYMERS
Though, the naturally occurring biodegradable polymers have certain limi tation such as structural complexities, poor biomechanical characteristics, etc., still they are considered as nanocarriers for numerous attractive reasons like their commercial availability, capability of structural modification and outstanding biocompatible and biodegradation potentials. Moreover, the natural polymers based nanocarriers could easily be metabolized by the host successfully and cleared from the body. The important naturally occurring biodegradable polymers include proteins such as collagen, gelatin, albumin, elastin, globulin, zein, gliadin, and casein. Chitosan, carrageenan, chitin, alginate, dextran, and hyaluronic acid are classified as naturally occurring polysaccharides-based polymers [22].
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2.3.1.2.1 PROTEINS BASED NATURAL POLYMERS
Proteins are long-chain macro-molecules comprised of one or more amino acids chains and are considered vital for basic biological and normal physi ological functions of living systems. Proteins are macromolecules not only important for nutritional point of view, but also extensively used in drug/ nutraceuticals delivery systems, especially as an encapsulating and coating materials for nutraceuticals. Among animal proteins; gelatin [23, 24], casein [25], collagen [26-28], albumin [29, 30] and whey proteins [31, 32] have been extensively used as delivery vehicles, while glycinin [33], zein [34] and wheat gliadin [35] are proteins obtained from plant sources that have been investigated for drug and nutraceuticals delivery to the target sites.
Owing to non-immunogenic and biodegradable nature of collagen and gelatin, they have been extensively used in drugs/nutraceuticals delivery and tissue engineering. Several delivery systems of collagen in the form of nano-films, nano-gels, and nano-sponges have been investigated for the delivery of nutraceuticals and drugs (antibiotics anti-inflammatory drugs) [36]. The applicability of collagen as a nanocarrier has not been too much attractive, and this might be due to difficulty in entrapping the bioactive molecules in collagen. However, more widespread applications of gelatin (a derivative of collagen) have been reported for encapsulation of bioactives as nanotechnology-based systems [37]. Another animal protein; albumin is also widely used as a natural polymer in drugs/nutraceuticals delivery systems due to its outstanding non-toxic nature, and acceptable non-immunogenicity, biocompatibility, and biodegradability features. Examples of albumin-based nanoparticulate systems that have been investigated for the delivery of key anticancer drugs such as doxorubicin for chemotherapy of breast cancer and noscapine have been reported. Similarly, anti-inflammatory drugs like SB202190 delivery in albumin-based NPs have also been reported for inhi bition of p38 mitogen-activated protein kinase and multiple inflammatory cytokines’ secretion [38, 39].
Oral-based nano-drug delivery systems have also been constructed from Gliadin and Zein proteins. These are prolamin proteins used by plants for storage purposes. Key examples of prolamin proteins extensively employed and investigated as ideal nanocarriers are Zein from corn, hordein from barley and gliadin from wheat. Beside these proteins; the antioxidant proteins-inhibiting catalase, superoxide dismutase (SOD), and scavenging free radicals have also been regarded as applicable in delivery systems. On
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the other hand, these antioxidant proteins are prone to degradation in the harsh GI environment because of its low GI pH and the presence of protein degrading enzymes, i.e., trypsin and pepsin. The antioxidant proteins coated with gliadin, zein or other prolamin proteins successfully protect them from harsh GIT environment [40].
2.3.1.2.2 POLYSACCHARIDES BASED NATURAL POLYMERS
These are long-chain molecules composed of monosaccharide units. Primarily their function in plants is related to storage and provides firm struc ture to the plants. Polysaccharide polymers are usually obtained from both plants’ sources such as inulin fiber, pectin, and starch and animals’ sources like chondroitin sulfate, glycogen, and chitosan. The biotransformation or breakdown of polysaccharide polymers can be accomplished in different parts of GI by normal bacterial flora. Nutraceuticals are protected from harsh GI conditions when encapsulated and delivered in polysaccharides-based drug delivery systems. When the polysaccharides-based nanocarriers reach the colon portion of GIT, the polysaccharides are hydrolyzed, and the encap sulated nutraceuticals are released in the colon. Probiotics like bifidobacteria and lactobacilli delivery is mainly accomplished with the application of polysaccharides-based nanocarriers [41].
2.3.1.3 SYNTHETIC BIODEGRADABLE POLYMERS
For the delivery of nutrients/nutraceuticals, the synthetic biodegradable polymers can be used to enhance the encapsulation efficiency (EE), and they can also release the nutraceuticals/bioactives for a couple of days to weeks. Comparatively, polymers from natural sources have a relatively short period of time for drug release and commonly confined by the use of organic solvents and need comparatively harder formulation conditions. Synthetic polymers are able enough to control or sustainly release the bioactives for a couple of days to weeks as compared to natural polymers. Nevertheless, synthetic polymers could potentially lead to chronic inflammation and toxicity. Thus, if synthetic polymers have to be used for nano-encapsulation of nutraceuticals, their immunogenicity and toxicity should be taken into consideration and should be properly evaluated [42].
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2.3.2 ESTER, ANHYDRIDE, AND AMIDE FUNCTIONAL GROUPS’
BASED POLYMERS
Synthetic polymers which are biodegradable include polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic) acid (PLGA), polyanhy drides, polyorthoesters, and polyamide. Ester-based polymers like PLGA, PGA, and PLA have been extensively used in the field of nanotechnology [43]. These polymers have a number of useful applications in the field of biomedical science as drug delivery carriers, artificial-tissue materials, and resorbable sutures. PLGA is a copolymer composed of lactic acid and glycolic acid in which a different make-up of two monomers represents the polymer properties. PGA is a hard, tough, and crystalline polymer due to glycolic acid composition only. PGA has excellent fiber-forming properties but is not soluble in almost all those common polymer solvents that affect its application for drug carriers, as it cannot be made into films, rods, or capsules. PLA is a thermoplastic biodegradable polymer and is degraded by hydrolysis because of lactic acid composition only. PLGA has been used as a nanocapsule to increase the hydrophilicity, bioavailability, and anticancer property of lipophilic molecules such as lutein [44].
For controlled delivery of nutraceuticals/drugs, different nano-systems like microspheres, coatings, tubes, and disks can be constructed from biocompatible poly-anhydrides. The antibiotics like ampicillin or nonsteroidal anti-inflammatory drugs (NSAIDs; e.g., salicylic acid) has been encapsulated in polyanhydrides and the payload was released as the polymers degraded [45]. At room temperature, the polyorthoesters based polymers are stable in dry conditions. NSAIDs were encapsulated in Polyorthoesters and they released the active drug by surface erosion. These polyorthoester polymers have the advantage of prolonged drug release rates from nano-/ microspheres formulation from a couple of days up to months [46]. Even polyamide is biodegradable; however, its application has been confined due to its immunogenicity and poor mechanical characteristics.
2.3.3 SMART/STIMULI-RESPONSIVE POLYMERS
2.3.3.1 TEMPERATURE-SENSITIVE POLYMERS
Temperature-sensitive polymers are those that show a drastic change in their physical properties (mostly solubility) with temperature. The phase change
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behavior of such polymers can be due to the disruption of intra- and inter molecular interactions at their critical solution temperature (CST) causing expansion or collapse in the polymer within the aqueous solution. Tempera ture sensitive polymers with a lower critical solution temperature (LCST) will show phase separation (e.g., precipitation) above a specific temperature, while those with an upper critical solution temperature (UCST) will display phase separation (e.g., precipitation) below a specific temperature. These polymers are mostly used for the construction of hydrogels, and thus, hydrogels formulated from such polymers are called smart gels that show sole-gel transition at a specific temperature [47-49]. Polymeric hydrogels form a loosely cross-linked three-dimensional polymeric network, which absorbs a sufficient quantity of water by hydration as they have a large number of hydrophilic groups. Sol is regarded as a stable colloidal suspension (0.1-1 mm) of solid particles or polymers in a liquid.
Poly(N-isopropylacrylamide) (PNIPAM) is one of the temperaturesensitive polymers which is currently used mostly in hydrogels. Below its LCST, PNIPAM is soluble in water and this solubility is due to the change in phase of PNIPAM from a hydrated swollen state to a dehydrated shrunken state upon temperature change [50]. In this case when the hydrogel is in solu tion form, the volume of hydrogel changes up to 90% at approximately 32°C (LCST for PNIPAM) [51]. PNIPAM can be used for such drugs or nutraceu ticals where the release of the payload is dependent on the temperature of the body or specific tissue. Hence, it can be used for drugs or nutraceuticals for inflammation and cancer where specific body part or tissue temperature will be comparatively higher, and the drug/nutraceuticals will be released at the target site. As the nutraceuticals/drugs that are encapsulated in PNIPAAM will be stable above LCST; however, the release of nutraceuticals/drugs will be started at a temperature above LCST due to swelling of the polymer/ hydrogel [52]. Examples of thermo-sensitive polymers with LCST consist of poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA), poly(N vinylcaprolactam) (PVCL), and poly(N,N-diethylacrylamide) (PDEAM). Thermo-sensitive polymers with UCST have opposite phase transition compared to LCST hydrogels. Hydrogels with UCST swell and are soluble in water above their UCST. Below UCST, the solution of such hydrogels will give a cloudy appearance. Examples of UCST hydrogels are polyacrylamide (PAAM), poly(acrylamide-co-butyl methacrylate) or poly(acrylic acid) (PAA). Smart hydrogels not merely show response to a change in temperature but will also show response to other conditions like change in pH, enzyme, or electrical potential depending on polymer and hydrogel nature [53, 54].
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2.3.3.2 PH-SENSITIVE POLYMERS
Almost all polymers with pH-sensitivity characteristics have either basic (ammonium salts) or acidic (sulfonic or carboxylic acid) functional groups which are responsible for changes in environmental pH [53, 54]. Poly(sulfonic acid) and PAA polymers are the examples of polyanions polymers employed in drug delivery. At alkaline, neutral, or high pH, the acidic functional groups containing polymers are dissolved due to ionization polymers swelling or complete dissolution. Whereas; polycationic polymers, e.g., poly(N,N9 diethylaminoethyl methacrylate) (PDEAEM) swell or dissolve at low pH [55]. Polycationic base polymer hydrogels have been mainly used for drug delivery in the stomach because of their swelling in acidic environments. Such type of hydrogels can best be used for antibiotics (e.g., metronidazole, amoxicillin, etc.), delivery to the stomach for the treatment of stomach infections such as Helicobacter pyloriinfection. Many nutraceuticals, e.g., Phytosterols [56], carotenoids, vitamins, and lutein efficient delivery through such polymers have also been reported [57].
Polyketals (PK), is a new class of synthetic acid-responsive polymers having ketal linkages in their backbone. For drug/nutraceuticals delivery, they are designed to hydrolyze by macrophages in the acidic environment of phagosome after phagocytosis. Thus, this class of polymers can successfully be used for enhancing intracellular delivery of therapeutic drugs/nutraceu ticals. As an example, the poly(cyclohexane-1,4-diyl acetone dimethylene ketal) (PCADK) based microparticles significantly enhanced the activity of SOD; superoxide scavenging enzyme) to scavenge reactive oxygen species (ROS) produced by macrophages [58]. Polyketal PK3 is another polymer that has been efficiently used for the delivery of tumor necrosis factoralpha (TNF-α)-small interfering RNA (siRNA) to Kupffer cells in vivoand successfully inhibited gene expression in the liver [59].
2.3.4 LIPOSOMES
Liposomes are lipid bilayer vesicles consisting of phosphatidylcholine (PC) based phospholipids. A liposome may also comprise of lipid or lipid chains like phosphatidylethanolamine (PE), cholesterol, sphingolipids, and longchain fatty acids. Liposome exist in spherical shape with an inner aqueous core and hydrophobic membrane layer in the middle of the bilayer [60, 61]. In vivo, liposomes are cleared by the reticuloendothelial system (RES) of
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the liver and mononuclear phagocyte system (MPS) very rapidly, and due to this limitation, the application of liposomes is slightly hampered. However, this selective clearance of liposomes by MPS can certainly be exploited to target cells of the MPS, particularly macrophages and carry drugs to MPS with high proficiency. Liposomes can be pegylated to reduced their clear ance by the RES and extend their systemic circulating times. Consequently, pegylation enhances the efficiency of liposomes for drugs’ delivery. Simi larly, liposomes can also be fabricated with a targeting species including a ligand or an antibody to target specific kinds of tissue or cells [62].
Liposomes have been applied for the delivery of a number of therapeutic drugs for central nervous system illnesses such as brain infection, ischemia, and brain tumors [63, 64]. Drugs encapsulated within liposomal vesicles can be administrated through different routes, i.e., intracerebrally, intraven tricularly, or intravenously. Liposomes are among the most widely explored delivery systems owing to their biocompatibility and low immunogenicity characteristics; however, they face few limitations such as high cost of production, low stability, short shelf life and rapid removal by RES after intravenous injection [65].
2.3.5 SOLID LIPID NANOPARTICLES (SLNS)
SLNs are aqueous surfactant solutions or colloidal dispersions of lipids in water [66]. They have several advantages such as excellent stability of the encapsulated material, controlled release, easy to scale-up, protection of incorporated molecules from the external environment, and ability to carry both hydrophilic as well as lipophilic drugs. These lipid-based systems can also serve to improve functional and organoleptic properties. Furthermore, these systems contain the species with generally recognized as safe (GRAS) status [67]. Shortcomings such as aggregation, flocculation, increased particle size, comparatively high-water content and compound release may happen during storage [68].
2.3.6 NANOEMULSIONS (NES)
Emulsions are fundamentally bi-phasic structures which are comprised of an outer phase, i.e., continuous phase and an inner phase, i.e., dispersed phase, whereas, surfactant molecules make an interphase. NEs are emulsions which have very small sizes and appear translucent or transparent. They occur in
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much smaller size as compared to the conventional emulsions, i.e., size range from 50 to 200 nm [69]. Generally, a micelle is 5 nm or more in diameter and a surfactant molecule have a length of 2 nm. However, incorporation of oil phase into micellar core may result in increase in its size occasion ally to a large range [4]. NE is a good choice to incorporate nutraceuticals with poor solubility into food matrix, and it is understood that maximum biologically active phytochemicals are either lipophilic in nature or having low solubility. Systemic bioavailability of these bioactive phytochemicals is significantly influenced by their low solubility, because their characteristics including lipophilicity, partition coefficient, solubility, etc., dictate their way of transport, administration, and target sites. Modification of these bioactives into NEs can offer the advantages such as increase surface area (the small particle size of NEs), thereby resulting in improved epithelium cell perme ability, rapid diffusion across mucus membrane and enhanced digestion rates [17, 70, 71].
Furthermore, as may protect chemically reactive compounds from oxida tion, and hence resulting in minimum degradation in the GIT and increased shelf life [72, 73]. Several reports have been published on the entrapment of bioactives into NEs, and current drifts have revealed the application of food-grade NEs [74]. Carrier oil is a significant constituent in the synthesis of food-grade NEs, as it regulates the bioavailability of encapsulated compo nents [75, 76]. However, the carrier oil must have the ability to form mixed micelles, should be fully digestible and have a high solubilization capacity for active components [77]. Different types of nanocarriers commonly used as nutraceuticals/drug delivery vehicles are given in Figure 2.3.
FIGURE .3 Different types of nanocarriers-based delivery systems for nutraceuticals/ drugs.
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.4 EXAMPLE OF NANOCARRIERS BASED NUTRACEUTICALS
DELIVERY
Probiotics keep the digestive system healthy by controlling microbial balance. Though, most of the probiotic bacteria (about 60%) cannot endure in the gastric environment. Only a limited number of bacterial species (0-10
CFU/mL) can exist in the stomach (due to the low intragastric pH). The main bacterial species in the stomach are Enterobacteriaceae, lactobacilli, staphylococci, streptococci, and yeasts. Most of bacterial species, about 500 strains live in the intestinal microbiota. More probiotic bacteria can live in the small intestine and their number further increase in the duodenum (0-10CFU/g) to ileum (10CFU/g) and colon (10-10CFU/g) [78].
Hence, a delivery system for nutraceuticals is crucial to protect nutraceu ticals or probiotic bacteria from the severe gastric environment. Micro- or nano-encapsulation of probiotics can keep safe these biological cells in an unpleasant environment.
By using different gel-forming approaches, probiotic bacteria have been entrapped in the gel matrix. Probiotic cells can be encapsulated by emulsion, extrusion, and spray drying approaches. Extrusion is a conventional method for probiotic formulation. Using an extrusion method for alginate capsule, cell suspension is obtained by the addition of probiotic cells into hydrocolloid solution. Then, to produce droplets, the cell suspension is passed through the syringe needle and are directly dropped into the hardening solution comprising cations such as calcium. In the hardening solution, the alginate polymers in the cell suspension are crosslinked by the cations which result in the alginate capsule. Finely, the as-prepared alginate capsule is obtained and dried by applying a suitable approach.
Emulsion is a suitable means for the encapsulation of lactic acid bacteria (LAB). In this approach, polymer slurry and a dispersed phase that contain a small volume of cells are emulsified into a continuous phase that having a large volume of vegetable oil such as sunflower, soy oil, light paraffin, and corn oil. The gel formation of emulsion is done by various cross-linking approaches including interfacial, enzymatic, and ionic polymerization.
During the drying process, starches, and gums tend to produce sphereshaped microparticles. In spray drying approach, dissolved polymer matrix and probiotic cells of starches or gum Arabic is obtained. The spray drying approach can successfully produce microparticles; but, during the drying process, probiotic cells may be damaged because of physical injury to microparticles and heat generation. In order to minimize the damage of
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probiotic cells, the outlet and inlet heating for spray drying must be adjusted and appropriate cryoprotectant must be applied during freeze-drying.
Probiotic bacteria are thought to influence the immune system; hence their delivery is important. To efficiently deliver probiotic bacteria, suitable encapsulating materials and encapsulation methods should be used. The probiotic bacteria should be released to changes of osmotic force, time, temperature, environmental pH, enzymatic activity, and mechanical stress. During the formation, several parameters such as heat generation should also be regulated to improve the viability of probiotic bacteria.
According to the Food Standards Agency (FSA), titanium nitride, nano clay on silver and fumed silica are the nanomaterials that are allowed to be applied in food if they follow the pertinent legislation. Center for Food Safety has generated a database which registers about 300 foodstuff contact products that use nanotechnology [79]. In addition, Chinese nano tea, nano gold, and nanosilver have been applied as mineral supplements. Similarly, carotenoid NPs have been used in fruit drinks and patented “Nanodrop” delivery structures have been applied for encapsulation of vitamins, etc., and Nanoclusters or Nano cages have been applied in nanoceutical foodstuffs such as chocolate drink, thus, giving sweetness without addition of any sweeteners or sugar [80].
.5 ABSORPTION MECHANISMS OF NANOCARRIERS BASED
NUTRACEUTICALS
The small intestine portion of GIT is regarded as the major site for absorp tion of nanocarriers/nutraceuticals. The wall of the intestine is an active and complex structure that regulates nutrients absorption, immunes system and interactions between intestinal microflora, thus guarantees intestinal equilibrium [81]. A nanocarrier/nutraceuticals must first diffuse through the thick intestinal mucus layer prior to reaching the endothelial cells. The goblet cells synthesize this mucus layer and is composed of lipids and glycoproteins combination [82]. Various parameters, e.g., charge, size, and viscosity influence the passage of nutrients/drug to pass through this layer. The interaction of this layer with mucoadhesive hydrophobic materials or large molecules may reduce their permeability. Permeation of nutraceuticals through gut endothelia may be restricted; however, lipophilic components (e.g., and kaempferol and resveratrol) can pass through the mucus layer when mixed with bile salts, free fatty acids, and phospholipids [83]. Alternatively,
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nutraceuticals can adhere or pass-through mucus layer when encapsulated in polymeric nanocarriers, e.g., chitosan, lectin, polyethylene glycol (PEG) and gelatin thus can allow the uptake of nutraceuticals [84]. Nanocarriers with negative surface charges are repelled by the mucus layer, and their cellular uptake is decreased because of less residence time in the epithelial cells. Estradiol intracellular uptake was increased when it was encapsulated in positively charged poly(lactic-co-glycolic acid) (PLGA) NPs as compared to neutral or negatively NPs [85]. As the mucus layer barrier is overcome by nanocarrier/nutraceuticals, they may then cross the epithelium barrier via either through paracellular (through tight junctions) or transcellular (including M-cell-mediated transport) transport routes.
2.5.1 PARACELLULAR ROUTE
Passive transport of materials (drugs/nutraceuticals) via passive diffu sion occurs through inter-cellular spaces of epithelial cells of the intestine [86]. Epithelial tight junctions (TJs) are present at the intercellular spaces, and these TJs are composed of proteins (e.g., claudin, and occludin and claudin), making it a complex structure [87]. These TJs regulates intestinal permeability of substances, intercellular adhesion, paracellular transport, mediate passage of molecules from lumen to lamina propria and impede in the access of microbes to host cells and tissues [88]. Polar molecules and small water-soluble molecules such as sugars, water, amino acids, ions, and peptides having molecular weight less than 500 Da can pass through these TJs [89]. The function of TJs is increased by certain agents (e.g., polyphe nols) while others, e.g., caprylic acid can decrease the function of the TJ barrier thus enhances the small molecules’ uptake [90]. Paracellular trans port can potentially decrease intracellular metabolism, which is important for nutraceuticals. However, it has also been reported that polyphenols (i.e., quercetin, chrysin, caffeic acid, rutin, gallic acid, and resveratrol) are poorly transported through passive diffusion both in Caco-2 cells monolayer and similar artificial membrane permeability assays [91]. Moreover, TJs cannot open more than 20 nm; thus, the transport of nanocarriers across intestinal epithelium through paracellular route is very low, and this space impedes most of the nanocarriers based delivery systems [92].
Nano-systems with less than 20 nm size can adversely affect TJs and release the payload to systemic circulation. Later, the TJs restore their func tion to their original regular position. In addition, positively charged particles
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can easily be transported via paracellular transport because the negatively charged membrane surface will attract them. Nanosystems with cationic chitosan have the ability to open TJs and thus induce paracellular transport. As an example, the tea catechins delivery and transport is enhanced when encapsulated in poly(glycolic acid) (PGA) and chitosan-based NPs and cross the intestinal barrier through paracellular transport [93].
2.5.2 TRANSCELLULAR ROUTE
The transcellular absorption mechanism is dependent on the active or passive transport of molecules through cells via endocytosis. Most of the nutraceuticals are believed to be absorbed simply by passive transport without the involvement of carrier or receptor via transcellular route [94]. This mechanism has been proposed for non-polar polyphenol aglycones and carotenoids [90]. For instance, Guri reported the curcumin transport through passive diffusion in Caco-2 cells when loaded in SLNs [95]. In contrast, some charged and polar biomolecules bind to a specific receptor (receptor mediated transport) or naturally-occurring membrane protein transporter (carrier-mediated transport) located in the apical cell membrane. These molecules are then transported against concentration gradient within the intestinal cells with expenditure of energy; a phenomenon known as active transport [96], rather, they might not cross the cell membrane [97]. Such receptors and membrane carriers are vital for the uptake of numerous nutra ceuticals. For example, fatty acids, vitamin C, and some peptides are carried via fatty acid-binding proteins, sodium vitamin C co-transporter and protoncoupled peptide transporters, respectively of [83]. Moreover, the capacity of stimulation or inhibition of membrane transporters may be affected by many polyphenols and the stimulation or inhibition potential depend on the polyphenol concentration, form, exposure time, etc., [90].
Molecules that attach to specific carriers at the apical cell membrane or cell membrane receptors are internalized to the cell by endocytosis mechanisms (including pinocytosis or phagocytosis) [98]. Entry to M-cells of the Peyer’s patch (specialized in antigen sampling) is mainly based on phagocytosis (M-cells mediated transport), thus offering a supposed route for nanocarriers-based delivery systems [96, 99]. When the expression of M-cells comes under less than 1% of total intestine area, then transport through these cells becomes very difficult [100]. Nanocarriers can also be internalized by pinocytosis mechanism where they bind to complementary
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cell surface receptors (like lectins, lactoferrin, and α5β1 integrin) [101]. Some nanocarriers designed for nutraceuticals have been reported using specific ligands on their surface for specific receptors to achieve enhanced intracellular delivery both in M-cells and enterocytes [101]. For example, the gambogic acid transport in lactoferrin-based NPs resulted in enhanced trans port through cell membrane because of the presence of lactoferrin receptor [102]. It is worthy to mention that some nutraceuticals are excreted back into the lumen of GIT after their absorption by efflux pumps (efflux transporter) present lipid bilayers of the cell membrane, thus, limit the bioavailability of such nutraceuticals [103]. Therefore, the knowledge and understanding of various transport mechanisms across GIT is vital for the successful develop ment of nanocarriers-based nutraceuticals delivery system.
.6 RELEASE MECHANISMS OF NUTRACEUTICALS FROM
NANOCARRIER
The knowledge and understanding of release mechanisms are very important for the development of controlled and tailored nano-based delivery systems. On the basis of a good understanding of release mechanisms, one can predict ways for better protection of the payload in the nanocarrier, their absorp tion as well as optimize their release from the system [104]. Release of the encapsulated material from the carrier can occur through various processes depending on the nature of the encapsulated molecule, composition of the carrier, loaded amount, the release media, and the particle’s geometry. The release of encapsulated drug/nutraceutical from the carrier may certainly fall in one of the following four main mechanisms:
. Diffusion: The drug/nutraceutical molecules simply diffuse out
from the intact non-biodegradable biopolymers to the surrounding medium. This diffusion can take place via homogeneous matrix, water-filled pores, or via an external shell from an internal reservoir. The overall rate of mass transfer is dependent on the solubility of drug/nutraceuticals in the matrix, geometry, and size of the carrier as well as on its diffusion coefficient through the matrix. The diffusion coefficient in turn is affected by various environmental and particle parameters like porosity (porosity ∝diffusion coefficient), tortuosity (tortuosity 1/∝diffusion coefficient) and temperature (temperature ∝ diffusion coefficient).
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. Erosion: Another mechanism for payload release from nanocarriers
is the erosion. The encapsulated drug/nutraceuticals are released to the medium either via homogenous (occurring in the bulk volume of the nanocarrier; or heterogeneous erosion (occurring at the nanocarrier surface. Enzymatic and/or chemical processes can induce the erosion process. Bulk erosion is the process in which the nanocarriers’ size remains almost the same where the external fluid goes inside the nanocarrier via breaking of the physical or chemical bonds. In contrast, the surface erosion is the process where the nanocarrier (usually a biopolymer) size is gradually reduced through erosion at the external surface [105]. The rate of erosion is depen dent on various parameters, i.e., physicochemical stability, polymer molecular weight (erosion 1/∝ molecular weight), size (erosion 1/∝ size) and the release medium [106].
. Swelling-Shrinkage Mechanism: In this phenomenon, when the drug
or other payload dimensions (e.g., size) is higher than the nanocar riers’ pore size, they are entrapped within the nanosystem. Then the nano-system conditions are changed through different triggers (e.g., temperature, water activity, ionic strength, or pH) that cause the swelling of the nanosystem leading to increase in the nanocarriers’ pore size and ultimately release of the entrapped payload takes place. On the other hand, in shrinkage-induced release mechanism, the payload is entrapped in the nanosystem initially upon its swelling and then released upon shrinkage via altering the solution conditions [107].
. Fragmentation: In this case, the entrapped drug/nutraceuticals are
released through physical disruption of the nanocarrier to medium. The physical disruption of the nanocarriers may be either fragmen tation or fracturing through shear or compression mechanisms in the mouth and gastrointestinal (GI) environments or during processing [108].
It is noteworthy that diffusion is always involved in each of the above mechanisms. For the design of the efficient delivery system with desired EE and release profiles, mathematical modeling is also of prime impor tance [109]. A preliminary understanding of the drug release mechanism is required for the selection of an appropriate release model. For a full understanding of the release mechanisms, various parameters are consid ered such as nanocarrier size, concentration, solubility of the entrapped substance in the release medium and nanocarrier matrix, the porosity,
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pore size distribution along with the effective diffusion coefficients. Such parameters can often be found for drug delivery systems; however, only limited data have been published for nutraceuticals delivery systems in recent years [110, 111].
.7 REGULATORY ASPECTS
Regulatory issues for medicinal products should have to be resolved regarding quality, efficacy, safety, testing, and marketing authorization processes for nutraceutical products claiming medicinal benefits [112]. The application and designing of nano-delivery systems not only for fresh foods but also for healthier foods has been the recent trend, however several of such products can pose serious threats to peoples’ safety [113]. Several governing bodies like the European Food and Safety Authority (EFSA), United States Food and Drug Administration (FDA), Environmental Protection Agency (EPA), Occupational Safety and Health Administration (OSHA), National Institute for Occupational Safety and Health (NIOSH), US Department of Agriculture (USDA), US Patent and Trademark Office (USPTO) and Consumer Product Safety Commission (CPSC) regulates the application of nanosystems in food [114].
FDA published guidance documents about nanotechnology in 2012. According to FDA guidelines, the dietary supplements are considered a a category of food, so these documents have no specific mentions for dietary supplements. Rather, they talk about food and cosmetics. Moreover, FDA guidance papers have mentioned that if chemical or physical properties of food substance is changed its bioavailability will also be changed. Addition ally, such physical or chemical changes in food products can potentially lead to toxicity. In a later FDA draft guidance for new dietary ingredient, the agency pointed nanotechnology as a process that construct new dietary ingredients thus should notify FDA properly. However, nanoceuticals can be brought to market with little or no safety verification because they are not properly regulated. The FDA anticipated that nanocarriers-based products should come under the jurisdiction of the Office of Combination Products (OCP) [115]. When nanotechnology-based products are aimed for food applications, such products should be designed from non-toxic, mycotoxins, and heavy metals free materials as per EC Food Law Regulation [116]. The directive 89/107/EEC further states that nanomaterials for food packaging application should first be as a direct food additive [117].
Advances in Nutraceuticals and Functional Foods
.8 CONCLUSION
Nutraceuticals are foods or food parts that provide health or medical benefits, including basic nutrition as well as the treatment and prevention of chronic disease conditions. Medicinally important nutraceuticals include natural antioxidant foods or essential minerals, vitamins, functional foods, pre/probi otics, and phytochemicals. For efficient delivery to target tissue or systemic circulation, nutraceuticals should be encapsulated in a biocompatible, safe, and targeted delivery system. Polymers offer unique characteristics as delivery vehicles that cannot be attained by using any other material. Particularly, stimuli-responsive, and biodegradable polymers have been the subject of interest for controlled drug delivery systems. On the other hand, nanocarriers made of amphiphilic materials such as liposomes and micelles possess lower serum stability. Such systems have also been extensively employed for the delivery of small molecules, siRNA, antisense nucleotides, and small proteins. Production of nanotechnology-based products via eco-friendly processes is quite a promising research area for the development of various food products. Though, to a large extent, important goals have been reached in achieving food products with controlled release characteristics, the cost of such products production is still the overriding factor that hinders the introduction of more sophisticated controlled release technologies in food technology. The potential health benefits of probiotics and nutraceuticals are very well known. Thus, the addition of nutraceutical ingredients and nanocarriers for maintaining the stability of these materials will justify the additional cost of nanoencapsulation technology. Nanocarriers-based delivery systems will be more commercially available in the markets than in the past, as indicated from the published literature. It looks like these new technologies are feasible and promising tools for the food product industries and convince and persuade manufacturers to introduce nanocarriers-based ingredients into their food products as a part of their marketing strategy. Nanocarriers-based technologies can minimize various unique problems such food products via safeguarding their stability and preserving safety, appeal (texture, color, odor, and taste), stability, low cost, and nutritional value. Thus, it is concluded from published literature that nanocarriers based delivery systems will have more commercial status in the market in the near future.
Advanced Nanocarriers for Nutraceuticals Based on Structured Lipid and Nonlipid
KEYWORDS
• critical solution temperature •European Commission •gastrointestinal tract
•lower critical solution temperature • non-steroidal anti-inflammatory drugs •polyglycolic acid
•solid lipid nanoparticles
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CHAPTER 3
Nanoparticulate Approaches for Improved Nutrient Bioavailability
ABDUL QADIR, MOHD. AQIL, and DIPAK KUMAR GUPTA
Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Deemed University), M. B. Road, New Delhi - 110062, India
ABSTRACT
Nanoparticles (NPs) are described as minute dispersions particles or solid particles with a size varying 10-1000 nm. The NPs are made either by dissolving, entrapping, encapsulating, or attaching to a nanoparticle matrix. Currently, biodegradable polymeric NPs, especially those coated with a hydrophilic polymer like poly ethylene glycol are quite a hit as potential drug delivery devices due to their advantages. Oral delivery of NPs is a desirable route to dispense therapeutics or bioactive compounds in long-term treatments. In order to reduce the carrier-induced undesirable cytotoxicity, food polymers are best to employ in designing such delivery systems. Different methods of preparation of NPs include: Nanoprecipitation, nano emulsion technique and reverse-phase evaporation etc. The parameters used to evaluate the NPs are: particle Size, surface charge, surface morphology, encapsulation efficiency, differential thermal analysis (DTA) and differential scanning calorimetry (DSC) etc. Methods to enhance oral bioavailability of nutraceuticals include: safety of labile compounds, delay of gastric retention time, lymphatic uptake etc., In the food industry, the application of nano technology is at the infant stage due to insufficient knowledge regarding the safety of NPs. Food proteins have exhibited potential for development and incorporation in nutraceuticals and provide controlled release via the oral route. To make NPs widely amenable, it is important to explore and develop methods for assuring their safety and characterization. Combinato rial techniques can be applied for characterization of NPs in food matrices.
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Future research should focus on the development and validation of methods for analysis of NPs in food and other samples.
.1 INTRODUCTION TO NANOPARTICLES (NPS)
Nanoparticles (NPs) are described as minute dispersions particles or solid particles with a size varying 10-1000 nm. The NPs are made either by dissolving, entrapping, encapsulating, or attaching to a nanoparticle matrix. NPs are defined as per the method of preparation used, such as NPs, nanospheres, or nanocapsules. In nanocapsules systems, API is entrapped in a cavity enclosed by a unique polymer membrane, while in nanospheres system API is uniformly suspended physically in a matrix system. Currently, biodegradable polymeric NPs, especially those coated with a hydrophilic polymer like poly (ethylene glycol) (PEG) identified as long-circulating particles are quite hit as potential drug delivery devices due to their numerous advantages such as the capability to disseminate for a prolonged period of time, targeted drug delivery, as transporters of DNA in gene therapy, ability to deliver proteins, peptides, and genes [1-5].
Numerous kinds of nanoscale materials are produced by nanotechnology [6]. Researchers discovered the versatility of NPs when they found the nanosize alter the physiochemical characterizes of a substance, e.g., the optical properties. The physical properties of 20 nm gold (Au), platinum (Pt), palladium (Pd), and silver (Ag) NPs are wine red color, yellowish gray, black, and dark black colors, respectively. These unique properties of NPs such as colors, size, and shape, are utilized in bio-imaging applications [7]. Any changes in these properties alter the absorption properties of the NPs, and therefore different absorption colors are detected. NPs are not simple molecules but complex one and contain three layers:
. Upper Layers: i.e., the surface layer, which are activated by type of
small molecules, metal ions, surfactants, and polymers. . The Shell Layer: Completely different from the core layer in chemi
cally material.
. The Core: Fundamentally central portion of the NP and typically
refers to the NP itself [8, 9].
NPs possess numerous benefits such as: reduce the toxicity, improve bioactivity, advance targeting, and offer multipurpose means to control the release profile of the encapsulated moiety [10].
Nanoparticulate Approaches for Improved Nutrient Bioavailability
Oral delivery of NPs is a desirable route to dispense therapeutics or bioactive compounds in long-term treatments due to various advantages, which include: patient compliance and ease of administration. Drugs that are made on polymer-based delivery systems have been explored extensively for the biomedical and pharmaceutical sectors in order to enhance the targeted delivery of bioactive compounds and to protect them as well. The key mode of actions entailed for modification of bioactive molecule absorption by polymeric NPs are:
• Safeguarding the API or bioactive molecule from the abrasive envi
ronment of the GI tract;
• Extending residence time in the gut via mucoadhesion; • Endocytosis of the particles and Permeabilizing effect of the polymer.
In order to reduce the carrier-induced undesirable cytotoxicity, food polymers are best to employ in evolving such delivery systems in oral consumption. Food origin polymers are best due to their property similar to soft condensed matter with which we interact daily. Another added advantage is: natural, soft materials, biodegradable, biocompatible, and bio functional. These food-based polymers are optimum choices to deliver API in therapeutics and functional foods. These include nanostructured vehicles like association colloids, lipid-based nano-encapsulator, bio-polymeric NPs, nanotubes, nanoemulsions (NEs), and nano-fibers made from food-grade ingredients such as food biopolymers (proteins, carbohydrates), fats, low molecular weight surfactants and co-polymers (protein-carbohydrate conju gates) [11-13].
.2 PREPARATION OF NANOPARTICLES (NPS)
Pharmaceutical industries have vested their interest in the effective delivery of bioactive agents, peptides, and APIs to the systemic circulation and even tually to the targeted organ or cells due to current progress and development in biotechnology.
3.2.1 NANOPRECIPITATION
This method is applicable to lipophilic drugs because of the miscibility of the solvent with the aqueous phase [14]. In short, organic solvents such
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as acetone are used to dissolve the lipids and the drug. Then this organic mixture is mixed with water containing surfactant. Promptly after this step, the organic solvent is separated from the colloidal suspension under reduced pressure by Rota evaporation. The ensuing particle suspension is filtered via a 1.0-mm cellulose nitrate membrane filter; tailored in size by mechanical extrusion to obtain a nanoparticle formulation [15].
3.2.2 NANOEMULSION TECHNIQUES
Among many methods for preparing NPs, nanoemulsion is one. It is defined as the heterogeneous mixture of different oils with minute-diameter oil droplets in water (20-500 nm). They have potential application in numerous chemicals, pharmaceutical, and cosmetic industries due to their safe trans dermal applications worldwide. There are numerous benefits of NEs like the possibility to solubilize hydrophobic compounds in the oil phase, the ability to customize the surface of the oil droplets with polymers to prolong circula tion times, and passive targeting of tumors and/or actively targeting ligands [14]. NEs composed of oils are formulated by coarse homogenization trailed by high-energy ultrasonication method [17, 18]. Briefly, the aqueous phase is prepared by adding soya lecithin into the deionized water, and stirred at high speed. Organic solvents are used to dissolve the candidate drug and then dispersed in oil. Subsequently, evaporation of the aqueous phase is done by heating at 70-75°C. The remainder of the oil phase which comprises the entrapped drug is slowly added to the aqueous phase to make a uniform solu tion which eventually makes the coarse oil-in-water (O/W) emulsion [19]. The obtained coarse emulsion is ultrasonicated to get the desired nano-sized oil droplets.
3.2.3 REVERSE-PHASE EVAPORATION
This is a widely used technique to make NPs of various types of drugs. Lipids such as selective phospholipids, in pure form or mixed with other lipids like cholesterol or long-chain alcohols are used. This lipid combination is further mixed with organic solvent, afterwards the solvent is isolated under reduced pressure via Rota evaporator. The resultant system is then purged with nitrogen to get reverse-phase vesicles which are formed after re-dissolving the lipids in the organic phase. To enhance the solubility of lipids in ether, chloroform or methanol can be added. The system is preserved under nitrogen
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and the water phase and the resulting two-phase system is sonicated for 2-5 min, until the mixture becomes either clear or a homogeneous opalescent dispersion that does not separate for at least 30 min after sonication. The organic solvent is then separated by Rota vapor under reduced pressure. After removal of bulk of the solvent, viscous gel appears; later an aqueous suspension is formed after 5-10 minutes. Finally, the obtained product is either dialyzed or centrifuged to eliminate non-encapsulated material and residual organic solvent [15].
.3 EVALUATION OF NANOPARTICLES (NPS)
Evaluation of NPs can be performed for some parameters, which are discussed in subsections.
3.3.1 PARTICLE SIZE AND SURFACE CHARGE
Determination of particle size and zeta potential of the NPs was done by photon correlation spectroscopy and laser Doppler Anemometry, using a Mastersizer 2000 and Zetasizer 2000, respectively (Malvern Instruments, South borough, MA). Before analysis, samples were diluted with suitable media and filtered (0.22 mm pore size) to obtain an appropriate range. The size analysis was performed at 25°C. It was recorded for 180s for each measurement. The polydispersity values of nanoparticle dispersions after homogenization varied between 0.2 and 0.5. The mean hydrodynamic diam eter was generated by cumulative analysis. The zeta potential measurement was analyzed using an aqueous dip cell in the automatic mode. Particles with zeta potentials > +30 mV or < −30 mV generally marks the stability of the formulation [20, 21].
3.3.2 SURFACE AND INTERIOR MORPHOLOGY
Nanoparticle morphology was analyzed under a transmission electron microscopy (TEM): The freshly-prepared NPs suspension diluted with suit able media and put on a copper grid sealed with nitrocellulose and allowed to get dry then stained with phosphotungstic acid (1% w/v). It is further analyzed by a transmission electron microscope [20].
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3.3.3 ENCAPSULATION EFFICIENCY (EE)
The preparation of nanoparticles is a blend of coated and uncoated (free drug) medicament portions [22]. The separation between the coated and uncoated medicament is the initial step of the technique that can be determined by using a dialysis membrane in which the nanoparticle sample is immersed in a phosphate buffer solution (PBS) for 120 minutes [23].
3.3.4 DIFFERENTIAL THERMAL ANALYSIS (DTA) AND
DIFFERENTIAL SCANNING CALORIMETRY (DSC)
Measure the temperature and heat flow difference between a sample and a reference material. They can be used to measure phase changes, melting point, purity, evaporation, sublimation, crystallization, pyrolysis, heat capacity, polymerization, aggregation, compatibility, etc. The methods can be used to track the degradation process of NPs by identifying the formed by-product, and simultaneously to inspect the food quality change along with the addition of NPs [24].
3.3.5 STORAGE STABILITY
Nanoparticle dispersions were stored at 4°C for 20 days. Particle size and turbidity were analyzed immediately after preparation and after storage for 1, 3, 10, and 20 days [20].
3.3.6 IN VITRO RELEASE STUDIES
The in-vitrodrug release study was performed by the dialysis tube diffusion method. Some milliliters of the formulation should be placed in the dialysis bag that should be tied in such a way that air could not pass through it. The dialysis bag placed in the cell containing the suitable aqueous medium and should be maintained at 37°C with continuous agitation. The cell should be closed to avoid vaporization of the aqueous medium. Samples of the dialysate are then taken out at different time intervals, and at the same time, the same amount of same fresh sample should be added to keep the volume of the cell constant. Withdrawing of samples should be performed in triplicate and then analyzed for the estimation of drugs by using any chromatographic technique [25, 26].
Nanoparticulate Approaches for Improved Nutrient Bioavailability
.4 BIOAVAILABILITY OF NUTRACEUTICALS
According to the FDA, the definition of bioavailability is the rate and amount of API absorbed from the dosage form and becomes available at the site of action. Bioavailability in its definition explains two key points: (i) the absorption rate-how quickly the bioactive agent which goes into the systemic circulation and (ii) the absorption extent-the amount of bioactive available in the systematic circulation. Poor bioavailability indicates the inability of the API or bioactive agents to reach the targeted site hence reducing the therapeutic efficacy, which leads to failed biological results. Typically, some different steps determine the biological fate of bioactive agents after ingestion:
• release of the bioactive agent from the dietary matrix; • Digestion by enzymes within the intestine;
• Adherence and uptake by the mucosal layer of the intestine; • Transfer across the gut wall (passing through and/or between the
epithelium cells) to the lymphatic system or portal vein; • Systemic distribution and deposition (storage); • Metabolic and functional use;
• Excretion (via urine or feces).
The efficacy of nutraceutical products in preventing diseases depends on protective the bioavailability of the active ingredients. This represents a difficult challenge, given that only a small proportion of molecules remain available following oral administration, due to insufficient gastric residence time, low permeability and/or solubility within the gut, as well as instability under conditions encountered in food processing or in the gastrointestinal (GI) tract, all of which limit the activity and potential health benefits of nutraceutical molecules [18].
A number of external and internal factors affect the overall bioavail ability rate of consumed drug. The external factors consist of: nature of the bioactive agent, composition, and structure of the food matrix; whereas internal factors comprise gender, age, health, nutrient status, and life phase. Several definitions of bioavailability are only confined it to nutrients which is defined as the amount of the nutrient used, stored, absorbed, or excreted. Macronutrient’s nutrients such as: carbohydrates, proteins, and fats typically have very high bioavailability, which is more than 90% of the consumed amount in the gut. While the bioavailability of micronutrients (vitamins and
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minerals) and nutraceuticals (flavonoids and carotenoids) differ depending on their molecular and physicochemical properties. For instance, lipophilic bio-actives have limited or poor bioavailability due to their poor solubility, high melting point, chemical instability, and ingredient interactions. On the whole, numerous components regulate the bioavailability of nutrients and nutraceuticals as mentioned in Figure 3.1.
Physicochemical/nutraceutical Physiological Factors
factors
First-pass
metabolism Low apparent
solubility High gastric emptying rate
High molecular weight of
nutraceuticals
Effect of food
Inappropriate
partition
Restriction by the coefficient
intestinal barrier
Enzymatic degradation
of bioactives in GIT
FIGURE 3.1 Factors determining the bioavailability of nutraceuticals and other bioactive components.
In this chapter, we focus on the effective nanoparticle delivery systems for nutraceuticals and related active ingredients; it is necessary to understand the biological processes that regulate uptake and bioavailability [27].
.5 EXTERNAL FACTORS AFFECTING THE NUTRACEUTICAL
BIOAVAILABILITY
Bioaccessibility is an important phase in the bioavailability of nutraceuticals in foods. There are several factors that influence the bioaccessibility of nutraceuticals, such as those factors that act before food ingestion (external
Nanoparticulate Approaches for Improved Nutrient Bioavailability
GIT factors) and factors that manifest during the food digestion (internal GIT factors). The previous studies provide more focus about the effect of GIT mechanisms and its environment, which affects the bioavailability of bioactive agents and their transport to tissues and organs. Additionally, external GIT factors should also be considered as they have a major influ ence on nutraceuticals bioavailability that act on the food matrix prior to oral consumption. Study and evaluation of such factors play an important role in the development of medical and functional foods designing via reverse engineering methods. With the advancement in technology, the food prod ucts during the process can be customized to get the best nutritional values, attractive sensory attributes along and health benefits. Major external GIT factors altering the bioavailability of nutraceuticals are physicochemical properties of nutraceuticals, characteristics of food matrices, properties of nutraceutical delivery systems, level of processing, and conditions of food storage. For instance, nutraceuticals solubility is a key factor that influences food processing and preparation of food nano-carriers as well as the behavior of nutraceuticals within the GIT [28].
.6 METHODS TO ENHANCE ORAL BIOAVAILABILITY OF
NUTRACEUTICALS
Nutraceuticals go through various physiological and physicochemical barriers after consumption that which decrease the dose reaching the systemic circulation. To increase the bioavailability of bioactive; many researchers have designed numerous products with diverse delivery systems to minimize or overcome these limiting factors. In this method, we will explore different techniques that investigators applied while designing an optimum delivery system for nutraceuticals.
3.6.1 SAFETY OF LABILE COMPOUNDS
Nutraceuticals on oral intake pass through complicated digestion processes which include physiological or physiochemical environmental changes. From the mouth to the colon, the vast GIT tract environment may cause instability to the chemical structures of active ingredients. Enumerate factors including pH variations, ionic strength, enzyme degradations, mechanistic motilities, etc., are possibly responsible for the degradation of nutraceuticals.
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Hence, dosage forms which can avoid or protect these gastric instabilities and promote effective oral dosing are encouraged [29].
3.6.2 DELAY OF GASTRIC RETENTION TIME
The oral digestion process involves various complex steps that make the mate rial pass through various sites in the GIT. Gastric retention time is not always sufficient for proper absorption to allow desired results of nutraceuticals which cause incomplete absorption of nutrients, excessive compound excretion, and a decrease in the dose-responsive efficiency of therapeutic purposes. Developing and designing such delivery systems which offer the following advantages is highly acceptable: reduce the gastric movement with higher viscosity or ability to slow down the gastric movement of bioactive compounds, enhances the residence time in the GI tract and make a greater percentage of bioactives available at the targeted site for absorption prior to gastric emptying [29].
3.6.3 IMPROVEMENT OF AQUEOUS SOLUBILITY
The bioactive compounds need to be solubilized, suspended, or dispersed in the aqueous environment of the GIT. Hydrophilic nutrients are easy to solubilize in aqueous environment compared to lipophilic compounds, which show poor solubility and often get precipitate as clusters after adding to the aqueous environment. Formation of these big clusters, which lack the desired particle size requirement, prevents intestinal absorption of lipophilic nutrients, and the latter get eliminated quickly via excretion mechanisms. Hence, low aqueous solubility is a key aspect which precincts the absorption of lipophilic compounds. Dosage forms which can overcome this hindrance and increase solubility or dispersion of such ingredients will invariably increase the concentration of the bioactive at the required site in the body; thus, will yield the desired biological results [29].
3.6.4 CONTROLLED/DELAYED RELEASE
A controlled release dosage form is required to maintain the desired concentra tion of the bioactive in the systemic circulation in order to obtain optimum therapeutic results. The novel controlled/delayed drug delivery systems are capable of providing this uniform and continuous release of bioactive substances
Nanoparticulate Approaches for Improved Nutrient Bioavailability
in the system for prolonged time, simultaneously preventing the GIT complex environment and ensuring proper absorption. In this kind of delivery system, the vehicle carrying the active moiety gets disintegrated by the enzymes present in the GI milieu. The rate and time of release of bioactive compounds can be regulated by choosing the correct material with more tolerance to the digestive system and applying more protective layers on vehicle surfaces [29].
3.6.5 LYMPHATIC UPTAKE
Typically, the majority of the compounds with good aqueous solubility get absorbed in the blood via portal vein in the small intestine; then they are metabolized in the liver. For lipophilic substances; the lymphatic uptake system is a better option as it skips the first-pass metabolism and increases the bioavailability of parenteral drugs. The extent of lymphatic uptake depends on the capability of bioactive compounds to couple with lipoprotein within enterocyte. The lipid-based delivery systems with nanoscale particle size have been reported as an effective way to enhance direct intestinal lymphatic uptake of lipophilic compounds [29].
3.6.6 IMPROVEMENT OF INTESTINAL PERMEABILITY
Diverse form of materials has exhibited the ability to alter the physical barrier function of the intestinal wall. Consumption of dietary lipids can influence the intestinal membrane fluidity also by interaction with mucoadhesive polymers. While designing delivery vehicles, the focus should be kept on all components making up vehicle which can provide maximum support to intestinal membrane fluidity. For example, chitosan is a positively-charged mucoadhesive polymer which mitigates intestinal membrane integrity and tight junction widening that permits the paracellular absorption of lipophilic compounds [29].
3.6.7 MODULATION OF METABOLIC ACTIVITIES
The first barrier which diminishes the bioavailability is the limited absorption; the second one being the first-pass metabolism that decreases the systemic dosage level of nutraceuticals. Including materials which can prevent physical or chemical activity of metabolic enzymes on the delivery vehicle may reasonably improve the bioavailability of bioactive in the systemic
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circulation. While working on it, the safety should be kept on priority as these enzyme inhibitors may sometimes produce toxicity due to impaired detoxification activity [29].
.7 RELEASE MECHANISMS OF NANOPARTICLES (NPS) FOR
NUTRACEUTICALS
One of the main objectives of controlling drug release is to retain the drug concentration within the therapeutic range in the blood. Therefore, it is ideal to make drug carriers that have low dosing rate and provide controlled drug release. Zero-order drug release profile is aimed to get the controlled release in which the drug is uniformly released. Drug release from a nanocarrier is affected by various factors drug, polymer, and excipient, the ratio of ingredi ents, physical or chemical interaction among components, and manufacturing methods, etc. Drug release can be categorized into four segments: diffusion, solvent, chemical interaction, and stimulated release determined by the mechanism of drug discharge from the vehicle as shown in Figure 3.2 [30].
FIGURE 3.2 Various mechanisms of drug release from nano-carriers.
Nanoparticulate Approaches for Improved Nutrient Bioavailability
3.7.1 DIFFUSION-CONTROLLED RELEASE
In this mechanism, in a capsule-like systems, the drug is released where the drug is either melted or dispersed in a core. The movement of the drug happened due to difference in concentration gradient across the membrane. Initially, the drug gets dissolved in the central part and afterwards diffuses via membrane. The matrix type nanospheres do not have membrane barriers but have a diffusioncontrolled release profile, in which the drug molecules are continuously released in the polymer matrix. Consequently, these systems usually have high initial release, but then over time, the release rate decreases due to an increase in the drug molecule diffusion distance inside the carrier [30].
3.7.2 SOLVENT-CONTROLLED RELEASE
The solvent-controlled release depends on osmosis-controlled release and swelling controlled release. The former happens in a vehicle covered with a semi-permeable polymeric membrane, in which water moves from outside of the carrier to the inside core loaded with drug, i.e., from low drug concen tration to high drug concentration. This mechanism causes a zero-order release profile until the constant concentration gradient is maintained across the membrane. The hydrophilic polymeric systems get easily diffused into the system in an aqueous solution, including body fluids. The water diffused across the system causes swelling of the polymeric particles, which leads to drug release, and this kind of release is known as a swelling-controlled release system. Diffusion rate of water and the chain relaxation rate of polymers determine the drug release rate. The swelling-controlled systems consist of polymeric materials with three-dimensionally crosslinked network like hydrogels. In this, mesh size plays a key role in regulating the drug release. Semi-empirical Peppas model can be employed to calculate drug release from hydrogels. This model helps in defining the release mechanism (e.g., Fickian or non-Fickian diffusion) [31, 32]. Zero-order drug release can be achieved by swelling-controlled systems subjected to the initial drug distribution or polymer composition in the system [33].
3.7.3 DEGRADATION-CONTROLLED RELEASE
Biodegradable polymers are used for drug carrier constitution, for example, polyesters, polyamides, and polysaccharides which release the drug via
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enzymatic decomposition, which in turn cause ester or amide bond degradation or hydrolysis. A matrix composed of polymers such as poly-lacticcoglycolic acid (PLGA), poly-lactic acid (PLA), or polycaprolactone (PCL)conse quently goes through the process of degradation, and altogether the matrix is deteriorated simultaneously. Contrarily, the matrix consisting of polymeric anhydrides or ortho-esters ordinarily erodes from the surface towards the center and results in faster degradation of the polymer than water diffuses into the matrix. However, in the NPs, there is a small-sized matrix and hence shows a low diffusion length for water and a restricted zone of crystallization. Overall polymer degradation continues to stimulate the release process rather than only surface erosion. Polymer systems that are biodegradable are usually preferred because they can degrade in the body [30].
3.7.4 STIMULI-CONTROLLED RELEASE
Internal or external stimuli controlled the release of drug from nano-carriers that are stimuli-responsive, like ionic strength, temperature, pH, sound, and electric or magnetic fields.
As it is feasible to confine the stimuli, these carriers for target-specific delivery of drugs have been explored. For example, for tumors site specific delivery of drugs, nanocarriers having pH sensitive linkers have been developed as well as to take advantage of weakly acidic pH of various solid tumors. To increase the difference between drug release, i.e., extracellular drug release and intracellular drug release pH-sensitive carriers are estab lished. Temperature induced phase transition of the polymer results in the drug release from carriers that are thermo-sensitive [33].
.8 ROLE OF NUTRACEUTICALS LOADED NANOPARTICLES (NPS)
IN VARIOUS DISEASE CONDITIONS
General principles of nanotechnology are usually followed for nanoformula tion of nutraceuticals. Consequently, the nanotechnology platforms are being exceedingly used to develop the delivery systems for nutraceuticals and natural bioactive products having poor solubility in water. The market predic tion for these technologies proposes a multiplex increase in their marketing potential over the next 5 years. A list of nutraceuticals, materials used in the development of NPs, NPs size and their targets like specific tissue/tumor, are summarized in Table 3.1.
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TABLE 3.1 The Phytochemicals and the Materials Used for Preparing Nanoparticles for
Various Diseases
Phytochemicals Targets Site Materials Used
Curcumin Leukemia, colon, PLGA
breast, prostate cancer
cells
Cervical cancer cells Alginate-chitosan
Breast cancer cells Silk
Cervical cancer cells Casein
Ellagic Acid Kidney PLGA-polycaprolactone
Dibenzoylmethane Cervical cancer cells Polylactic acid
Eugenol Bacteria Chitosan
Ferulic acid Liver Bovine serum albumin
Naringenin Liver Polyvinyl acetate
Quercetin Brain Polylactide
Brain, liver PLGA
Stomach, intestine Glyceryl monoste
Resveratrol Neuronal cell line Poly-caprolactone-PEG
Simvastatin Plasma Glyceryl monooleate/poloxamer 407
Thymoquinone Leukemic cells PLGA
Ursolic acid Liver Soybean phospholipid-poloxamer 188
For example, low systemic bioavailability is shown by curcumin; by developing several nanoparticle formulations; its biologic activity and bioavailability can be greatly increased. Various studies have proposed curcumin for chemoprevention and as a safe alternative for cancer therapy with its anticancer properties and anti-inflammatory properties, yet the compound has not been accepted unequivocally by the cancer community. A clinical trial of colorectal cancer patients showed that the postoperatively administered curcumin’s systemic bioavailability is less in humans [34].
Nanoparticulate curcumin has been developed by Bisht et al. by utilizing the cross-linked polymeric NPs that are composed of N-vinyl-2-pyrrolidone, PEG acrylate, and N-isopropyl acrylamide which has been subjected to pancreatic cancer cell lines test [35].
A purified compound viz. triptolide found in Chinese traditional origin medicine has immunosuppressive, antineoplastic, anti-inflammatory, and antifertility properties [36]. A study by Mei et al. reported penetration of
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triptolide into the skin and its anti-inflammatory efficacy got increased by the preparation of SLN (solid lipid nanoparticles) for transdermal delivery [37, 38]. It is predicted that the above strategy enhances the bioavailability of drug at the site of action, as well as reduces the dose required, and also reduces side effects that are dose-dependent such as stinging and irritation.
Another group of nutraceuticals, i.e., polyphenols have established antiinflammatory properties and thus have high capability for cancer therapy. The low bioavailability and short half-life of polyphenols is a challenge for the treatment of cancer. Polyphenol-loaded NPs is one of the substitutes to free compounds [16].
.9 CONCLUSION
In the food industry, the application of nanotechnology is at the infant stage due to insufficient knowledge regarding the safety of NPs in food and food-related products like: (a) which hinders its further development; (b) consumers are also cautious to consume NPs food products due to its uncertain safety profile and potential health risk; (c) this constrains authori ties in developing proper legislation. Food proteins have exhibit potential to get developed and incorporate in nutraceuticals and provide controlled release via the oral route. Food proteins are also suggested to be safe. The well-defined advantages of food protein matrices are high nutritional value, abundant renewable sources, consumer acceptability due to their natural and easy digestion by the digestive enzyme.
.10 FUTURE PERSPECTIVE
To make NPs widely available, it is important to explore and develop methods for assuring their safety and characterization of NPs. As highlighted in this chapter, techniques are not available to singly detect and characterize all the vital attributes of NPs used in food, nutraceuticals, and food additives. Combinatorial techniques can be applied for characterization of NPs in food matrices. Future research should focus on the development and validation of methods for analysis of NPs in food and other samples. In most cases, pre treatment of solid food samples is inevitable, but caution is recommended to select appropriate methods and develop applicable protocols that result in minimal disturbance of the NPs within a sample.
Nanoparticulate Approaches for Improved Nutrient Bioavailability
KEYWORDS
• characterization of nanoparticles • method of preparation
• nanoparticles
• nutrient bioavailability
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Abbruzzese, J. L., et al., (2008). Phase II trial of curcumin in patients with advanced pancreatic cancer. Clin. Cancer Res., 14, 4491-4499.
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agent purified from a Chinese herb Tripterygium wilfordiihook F. Leuk Lymphoma, 42, 253-265.
. Mei, Z., Chen, H., Weng, T., Yang, Y., & Yang, X., (2003). Solid lipid nanoparticle and
microemulsion for topical delivery of triptolide. Eur. J. Pharm. Biopharm., 56, 189-196. 38. Barras, A., Mezzetti, A., Richard, A., Lazzaroni, S., Roux, S., & Melnyk, P. (2009).
Formulation and characterization of polyphenol-loaded lipid nanocapsules. Int J Pharm, 379, 270-277.
CHAPTER 4
Adulteration and Safety Issues in Nutraceuticals and Functional Foods
SHUJAT ALI,,7SYED WADOOD ALI SHAH,MUHAMMAD AJMAL SHAH,MUHAMMAD ZAREEF,MUHAMMAD ARSLAN,MD. MEHEDI HASSAN,
SHUJAAT AHMAD,IMDAD ALI,MUMTAZ ALI,and SHAFI ULLAH,5
1School of Food and Biological Engineering, Jiangsu University, Zhenjiang - 212013, P. R. China
2Department of Pharmacy, University of Malakand,
Khyber Pakhtunkhwa - 18800, Pakistan
3Department of Pharmacognosy, Faculty of Pharmaceutical Sciences,
Government College University, Faisalabad, Pakistan
4Department of Pharmacy, Shaheed Benazir Bhutto University Sheringal,
Dir (Upper), Khyber Pakhtunkhwa, Pakistan
5H.E.J. Research Institute of Chemistry, International Center for Chemical
and Biological Sciences, University of Karachi, Karachi - 75270, Pakistan 6Department of Chemistry, University of Malakand,
Khyber Pakhtunkhwa - 18800, Pakistan
7College of Electrical and Electronic Engineering, Wenzhou University,
Wenzhou 325035, PR China
ABSTRACT
The consumption of nutraceuticals and functional foods, especially those that originated from plants, has been increasing owing to the communal concept that they are natural substances and are free from hazards. However, adul teration and safety issues in the production and selling of these substances are a universal concern for consumers, health professionals, regulators, and
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stakeholders. Particularly, adulteration by the unlawful addition of other ingredients is of main concern since dishonest manufacturers can misrep resent these substances to offer speedy effects and to promote sales. This illegal practice extremely endangers human health with several chronic and acute diseases and disregards the public rights for safer food. The intent of this chapter is to offer a base reference document for understanding adulteration and safety issues in nutraceuticals and functional foods, their evaluation, impacts on human health, and ways to prevent these issues. This will offer a background for future quantitative and innovative research. The adulteration and safety issues are described in terms of economically and criminally motivated adulteration, unintentional adulteration, undeclared labeling, and regulatory issues. The study provides major causes and evalu ation of adulteration and safety issues. In the later part of the chapter, their impacts on public health and ways to prevent these issues are discussed. This study provides a foundation for future research regarding food safety, food adulteration, and food defense.
.1 INTRODUCTION
Besides, food is something having general nutrition, aroma, and taste; the additional categories of food have been recognized, such as “nutraceuticals” and “functional foods.” These are the substances that have more advantages than simple foods and are possibly equivalence with formally recognized “vitamins” [1].
A worldwide debate concerning nutraceuticals, functional food, and dietary supplements is whether they should be regarded as medicine or food. Likewise, it is difficult to distinguish between functional foods, nutraceuticals, herbal medicines, food additives, or nutrients owing to their drug-like health-related properties [2]. Other terms such as “medicinal foods and dietary supplements” are also used to refer to these substances. Health Canada defines a nutraceutical as a substance that is obtained from food and supposed to have advantages to health and/or prevent chronic diseases [3]. In other words, any safe food extract additive that has logically established health advantages for the prevention and treatment of illnesses [4]. Zeisel defined the term nutraceuticals as the diet supplements that bring a concen trated form or isolated form of a reputed bioactive ingredient from a food, obtainable in a nonfood matrix and could be applied to encourage health [5]. American Dietetic Association (ADA) described the nutraceuticals as any
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substance having a food-constituent and give health or medical benefits, such as treatment and prevention of diseases, for example, minerals (selenium), vitamins, and animal (carnitine, carnosine, chitosan), and plants (ginger, garlic, Ginkgo biloba) extracts [6].
Similarly, the term functional food is defined as food that should have an appropriate effect on health or well-being and minimize the risk for disease [7]. In another study, the term is defined as foods like conventional substances that are used up as a constituent of a usual diet and beyond basic nutritional functions have established biological advantages and/or mini mize the hazard of long-lasting disease [8]. Food for special dietary use and foods for specified health use (FOSHU) have highlighted those functional foods are regarded as foods, determine their properties in amounts that can usually be projected to be used in the diet, and are eaten as a constituent of a conventional food form [9]. Natural and traditional foods can be sold or advertised as functional foods, provided they are attended by the somewhat new representation of their health advantages [10]. Functional food constituents offer health-promoting properties other than usual nutri tion and when compared to dietary supplement constituents, have unique regulatory requirements, and need diverse safety measures. Functional food constituents are not the same as a dietary supplement, while they may have the same chemical functional groups and may have same health benefits [11]. In a simple way, functional food ranges from any improved food or food products that may offer health advantages, while in another aspect, foods that have possibly disease-preventing and health-promoting properties [12]. Furthermore, the term functional food sometimes is confusing, as nearly all foods, irrespective of whether they have additional constituents, somehow affect health by offering nutrients and calories and can be regarded as “func tional.” Nevertheless, effective, or not, the terminology of functional food has emerged as the main one and must be elucidated in order to educate its scope and its spot, as well as to ease the improvement of a generally recognized regulatory outline [13].
Nutraceuticals and functional foods are natural constituents that may be consumed in combination, individually, or added to beverage for health benefits or technologic purposes and essentially have a suitable safety outline that determines safe for eating by humans [14]. Medical drugs have the risk of adverse effects or toxicity, hence, the search for harmless functional food and nutraceutical-based tactics are of great interest for maintaining the good health of human beings. This led to a worldwide revolution in functional food and nutraceuticals. The option of disease management and health regulation
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by natural methods has been assumed by a substantial percentage of the global population [15]. Previously, researchers have tried to standardize the definitions of functional foods and nutraceuticals. The Nutraceutical Research and Education Act (NREA) was proposed by Stephen De Felice in 1999 [1]. However, the proposal was for the time being laid to rest, and no considerable contribution was established regarding this. Since then, the terms functional foods and nutraceuticals have been inflated. Generally, the dietary supplements are presented to be safer and natural, hence most of the worldwide population favor these substances for health care advancement over pharmacological medicines [16]. Quick growth in study on nutraceuti cals and functional foods is a vital and integral component of the revolution. For the successful use of functional foods and nutraceuticals in the manage ment of human health the safety and efficacy are two essential key sets for the purpose [17]. The safety and efficacy are rapidly improving due to the sophisticated and modern technologies.
The problem of adulteration and safety in the nutraceuticals and func tional food occurs from manufacturing level to consumption. Some food processors, restaurant owners, manufacturers, and transporters are respon sible for this wrong act of adulteration. Nutraceuticals and functional foods may be adulterated by means of different inexpensive substances, toxic artificial colors, and harmful chemicals [18]. Uses of harmful chemicals to get quick effects and to attract consumers is among the commonly used practices [19]. The consumption of such unsafe substances negatively affects public health with frequent chronic and acute infections. Adulteration of nutraceutical and functional food has been observed several years ago, and this unethical act is growing day by day, especially in developing countries [20]. Major causes of nutraceutical and functional food adulteration include dishonest importers, traders, cultivators, manufacturers, and processing agencies [21]. Particularly, in developing countries, these unethical practices are involved in the adulteration of functional foods and nutraceuticals, and there is no proper and strict laws and principles to control the adulteration and safety issues [22]. The rules may comprehend the offenses like lack of hygiene, fake licenses, poor quality of food, substandard infrastructure food impurity, food adulteration, selling products with expired dates and incor rect information on food packages. However, the issue is the appropriate and sustained implementation of the rules and regulations by the dependable establishments. Also, shortage of test instruments, reagents, and skillpersons is much noticeable.
Adulteration and Safety Issues in Nutraceuticals and Functional Foods
This chapter provides a base reference document to understand adultera tion and safety issues in nutraceuticals and functional foods, their evaluation, impacts on human health, and ways to prevent these issues. The adulteration and safety issues are described in terms of economically and criminally motivated adulteration, unintentional adulteration, undeclared labeling, and regulatory issues. Various approaches to the evaluation of adulteration and safety issues in nutraceuticals and functional foods are critically discussed. In the last part of the chapter, the impacts of adulteration and safety issues on public health and ways to prevent these issues are discussed (Figure 4.1). We believe that the study provides a background for future innovative and quantitative research. Furthermore, it may assist researchers to understand the foundation for future research regarding food safety, food adulteration, and food defense.
FIGURE 4.1 Understanding the terms nutraceuticals and functional food, adulteration, and safety issues, evaluation of adulterating substances, impact on human health, and ways to prevent adulteration and safety issues.
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.2 WHAT ARE NUTRACEUTICALS AND FUNCTIONAL FOODS?
Nutraceutical is a pragmatic term, and its meaning is not uniform globally, the term has different descriptions worldwide [1]. This terminology was presented by Stephen DeFelice in 1989, Chairman and Founder of the Foun dation for Innovation in Medicine (FIM) [23]. Basically, the word nutraceu tical originated from “nutrition” and “pharmaceutical” and followed the way of the cosmeceuticals term, which was introduced by Albert Kligman in 1980 and Raymond Reed in 1961 [24]. According to DeFelice, “nutraceuticals are food or food constituent that offers health advantages and medicinal values, counting the prevention and/or treatment of a disease” [1]. Regardless of the substantial use of the term nutraceutical in common practice and selling, it has no clear regulatory definition or absolute legal standing [25]. Nutraceu ticals are health-indorsing substances, sold with the health-promoting claims of improving different mental and physical actions of the body, generally without pharmaceutical constituents [26]. A nutraceutical is almost molding drug and food into one formulation, which acts neither as a pharmaceutical product nor a simple food [23]. Nevertheless, it is a wide-ranging word that includes minerals, amino acids, vitamins, botanicals, and herbs [27]. Thus, both fortified food products and dietary supplements can be considered as nutraceuticals [28]. Nutraceuticals can be divided into two classes, estab lished nutraceuticals and potential nutraceuticals. An established nutraceu tical is one that holds enough clinical data to demonstrate health and medical benefits, while a potential nutraceutical is one that has a potential for health benefits. Generally, majority of nutraceuticals exist in the ‘potential’ class and waiting to declare established [29]. In broader sense, nutraceuticals can be classified into these three groups [30], i.e., (1) Nutrients, substances with recognized nutritional roles, such as vitamins, minerals, amino acids, and fatty acids; (2) Herbals, concentrated botanical extracts and products; and (3) Dietary supplements, substances obtained from other sources, for example, chondroitin sulfate, pyruvate, and steroid hormone. They offer functions and advantages such as weight-loss supplements, meal replacements and sports nutrition.
The word functional food was familiarized for the first time in the mid1980s in Japan, referred to as treated food comprising ingredients that can affect body functions [31, 32]. The definition of functional food is not the same all over the world, and the term is occasionally used miscellaneous with the other terms related to food [1]. Certainly, a wide variety of food products are categorized as functional foods, with various constituents,
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both classified and not classified as nutrients, regulate body activities relevant to the reduction of the risk of disease. Hence, functional food is to be understood as a concept and no universally accepted and simple defini tion of the term exists until now. Some elaborate definitions of the term are: (a) daily diet foodstuffs, resulted from naturally available substances and when ingested possess certain biological advantages [33]; (b) daily diet foodstuffs, obtained from naturally occurring substances and when ingested can help to regulate body process [34]; (c) daily diet foodstuffs, and when ingested establish physiological advantages and minimize the risk of chronic illness [35]; and (d) daily diet foodstuffs, that may offer health advantages beyond that of the traditional nutrients it contains [35]. Based on the definitions, functional food appears as a sole concept that justifies a category of its own, different from designer food, vita-food, pharma food, nutraceutical, and medi-food. Functional food is also a concept that is related to nutrition and not pharmacology. It is not drugs and must be food. Furthermore, their role concerning disease is reducing the risk rather than treatment. However, it should be underlined that a functional food will not essentially be equally efficient for all members of a population, and that corresponding individual biological needs with selected food constituent consumptions may develop a key task on their body response [36]. Functional food can be classified into different categories; based on the bioactive ingredients, it can be divided into fibers, probiotics, phyto chemicals, minerals, vitamins, herbs, and proteins, etc. Functional foods offer physiological advantages that distinguish them from normal foods. Its effectiveness is resulting from bioactive constituents and depends on numerous technical features. The bioactive constituents in functional foods assist in the stoppage of infections and improve body performance of the individual beyond their recognized nutritional role. They directly involved in adjusting body systems, such as the endocrine, circulatory, nervous, digestive, and immune systems [37].
All the aforementioned definitions for nutraceuticals and functional food pointed out that there are no consent and a certain definition of nutra ceuticals and functional food. Consistently, there is a need to emphasize the terms nutraceuticals and functional foods as drug or food; in fact, it blurs the demarcation between food and drug (Figure 4.2). For example, cholesterin, obtained from red yeast rice is a cholesterol-lowering ingre dient and is essentially a supplement identical to lovastatin [38]. Similarly, tryptophan, an amino acid derivative, is necessary for metabolism in small amounts, while it, at higher doses, in the form of 5-hydroxy-L-tryptophan
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behave like a drug for the treatment of insomnia via enhancing brain serotonin production [39]. But it was legally banned from the market since tryptophan administration resulted in the eosinophilia-myalgia syndrome (EMS). Nutraceuticals are considered to have at least one constituent of essential macro or micro-nutrients that are the active part of foods. Consequently, many nutraceuticals having food phytochemicals such as sulfur compounds (from garlic), carotenoids (lycopene from tomato), curcumin, glycosinolates, isoflavonoids, phytosterols, essential fatty acids, proanthocyanins, proteins, vitamins, amino acids (e.g., arginine), peptides (e.g., carnosine) antioxidants and polysaccharides, etc., are now existing in the market [40, 41]. The exact value that any consumer would place on nutraceuticals and functional foods directly depends on the consumer’s self-image. For example, a hypothetical consumer might realize himself as normally existing within a range of 75-85% efficiency [42]. Below 75% efficiency, there is no equilibrium and no longer feeling good himself and even in low range one may feel sick, motivating a need for some sort of medication and treatment. However, within this range (75-85%) one feels in equilibrium with his surroundings. The high level of this range (85%) is the best one and give healthy lifestyle habits, and good feeling. The goal is to uphold oneself in the 75-85% range, but environment consideration of limited food choice or limits on physical action disturbs to stay within this optimal range. Hence, nutraceuticals, and functional foods may put that goal within easier reach to be accomplished.
FIGURE 4.2 Demarcation between nutraceuticals and functional food and drug.
Adulteration and Safety Issues in Nutraceuticals and Functional Foods
.3 ADULTERATION AND SAFETY ISSUES
Nutraceuticals and functional foods are marketed as natural products deprived of side effects and are sold with therapeutic claims. Unfortunately, any unac knowledged ingredients may cause dangerous hazards to consumer’s health. Furthermore, according to some reports, they can be substandard, adulter ated, falsified, counterfeit, and unregistered [43]. Regarding the definitions, there is an enduring debate concerning the exact descriptions for adulterated nutraceuticals and functional food. According to the World Health Organi zation (WHO), adulterated products are impure formulations, debased, or corrupted [44]. Adulteration may occur due to the addition of an external or inferior element or substance. Adulteration in nutraceuticals and functional food can also be determined as the existence of an undeclared material, or a component is changed from its standard limits, and that a profile is improbable to happen [45]. Adulteration may be unintentionally or purpose fully. The motivation behind purposeful adulteration in nutraceuticals and functional foods is ultimately for economic income [46]. Natural products have conventionally used in drug research, especially in the treatment of metabolic syndrome disorders, immunosuppression, and malignant diseases [47]. Worldwide request for plant phytochemicals for use in functional foods and nutraceuticals has been rapidly increasing. The sources of many phyto chemicals are restricted, and the preparation of such substances with desired molecules are cost-consuming and involves a lengthy process, so this can lead to intentional or purposefully adulteration [18]. Plants are among the most common source of nutraceuticals and functional food, these sources may be contaminated during plantation and manufacturing, and by heavy metals, fertilizers, microbial agents, pesticides, etc. All these incidences may result in food-borne illnesses such as liver injury, gastric complaints, and other life-threatening infections [48]. Hence, the safety and security of fresh and processed nutraceuticals and functional food are essential by defining specifications in appropriate detail. Furthermore, issues related to the stability of active ingredients and pathogenic control have also been observed. It should also be highlighted that safety issues do not occur only with pesticides, synthetic drugs, and additional species; but also, pollens, insects, dust, parasites, microbes, rodents, molds, fungi, heavy metals, and toxins pose a serious problem with herbal formulations related nutraceuticals and functional foods [49]. Other serious hazards may result from synthetic chemicals, which are unsafe and hence not permissible in order to intensify a claimed biological effect or to change an immediate physiological action
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[50]. The consumption of nutraceuticals and functional foods, particularly those having plants phytochemicals as constituents, has been rising owing to the public concept that they are natural substances and pretense no hazards to human health. Adulteration by the unlawful addition of medicinal ingredients or their equivalents is of main concern since dishonest manufacturers can misrepresent these substances to provide speedy effects and to upsurge sales. The adulteration of functional foods and nutraceuticals is an emerging issue and that an operative check by food regulatory establishments is required to protect consumers. Some important issues regarding adulteration and safety of nutraceuticals and functional foods are given below.
4.3.1 ECONOMICALLY MOTIVATED ADULTERATION
This kind of adulteration is the origin of public health hazards, and the term was demarcated in the Open Meeting on Economically Motivated Adul teration in May 2009. According to this definition, economically motivated adulteration is “the fake, intended addition or substitution of material in a product for reducing the cost of its production or to enhance the seeming value of the product” [51]. Economically motivated adulteration may include dilution of foodstuffs with amplified amounts of cheap substances as well as the substitution or addition of an ingredient to hide dilution, and as a result, this may pose a possible or known health hazard to consumers [52]. The result or impact of economically motivated adulteration is an actual public health hazard and the misbranding or adulteration creates the potential for harm. Similarly, it may threaten more risk than conventional adulteration and safety because the contaminants are not traditional. Fraudsters may apply additives which are not registered among those predictable food safety adulterants. For instance, fraudsters applied melamine since it inventively mimicked high-quality protein in common protein content quality control tests, it was an unpredicted food contaminant; meanwhile it is a plasticizer and used in making plastic products [53]. The concept of safety covers all threats associated with consumers’ health, irrespective of the source, produc tion, processing, and traditional efforts. This is the commonsense develop ment for an impression as well as for well-known, the adulteration response now starts at the interference stage (that is, to know about the risks) then passes to the reply stage (that is, public-private mutual coordination). When the response stage develops well, fraud attention will logically change to the preclusion stage [54].
Adulteration and Safety Issues in Nutraceuticals and Functional Foods
4.3.2 CRIMINALLY MOTIVATED ADULTERATION
This kind of adulteration is carried by professional people for their own benefits. Criminals form a system to perform a food-adulteration crime, and when some action take place against them, they disperse, however they return to their usual and re-organize into a new criminal system for perpetrating a new fraud [55]. Since the chance is for a minor fraud to be circulated across consumers, less erudite criminals who are planned should not be neglected. As compare to old-style organized crimes, these are frequently networks or groups, interrupting any single connection in the network will not certainly break the chain or the capability for a new fraud [54, 56]. It is significant to highlight that there could be proficient hurdles and protectors in place, but the nature of a developing and evolving danger is that new slits always happen. People associated with adulteration are not all a civil law violators or criminals and may not be measured wrong in many cultures [57]. An infinite number of producers may relate to fraud, increased brand recogni tion and brand growth of a product, and hence essentially rises the fraud opportunity [58]. Then, the guardian led to a big fraud occasion. These are objects that protect or monitor the foodstuffs and may include individual companies, customs, local or federal law enforcement, non-governmental organizations, and trade associations, and so on to minimize such incidences. Furthermore, the risk of detection should be increase, the necessary tech nology that commit the fraud should be make expensive and strict laws and regulations should be implemented. Steps have been proposed to decrease the chances of fraud, but narrowing of focus in detection and modification to a procedure could unintentionally create new gaps that may be used by food criminals. Food adulteration by criminals is resourceful in nature and denotes an important challenge to both government and industry [59]. The detection and exploring process is further complicated because the fraud network generally is intelligent, clandestine, resilient, and sophisticated at stealthily sidestepping detection [60].
4.3.3 UNINTENTIONAL ADULTERATION
Unintentional adulteration of nutraceuticals and functional food may occur from naturally occurring substandard, poor storage conditions, drought, lack of rainfall, etc. Furthermore, unintentional adulteration may be owing to lack of knowledge about the authentic source, the similarity of sources in aroma
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or morphology, non-availability of the reliable source, careless processing, confusion in language names between indigenous systems and local dialects and other unidentified reasons [61]. Not all adulterations are intentional, it is noted that the nutraceuticals and functional foods are also adulterated unintentionally, sometimes, suppliers are uneducated and not aware of their counterfeit supply [62].
In other words, unintentional adulteration is the addition of unsolicited substances due to carelessness, ignorance, lack of proper hygiene, and lack of facilities during the processing of nutraceuticals and functional foods. This can be developed from contamination by the entry of harmful residues from packing material, dust, and stones, spoilage of food by rodents, fungi, and bacteria [63]. Similarly, inherent adulteration such as the presence of organic compounds, certain chemicals, or radicals naturally happening in foods like poisonous varieties of mushrooms, pulses, fish, and seafoods may also occur [64]. Other possible sources for unintentional adulteration are operations carried out for veterinary medicine and animal husbandry, crop husbandry, treatment, manufacture, processing, preparation, packaging, packing, and transport, etc., [64].
Nowadays, in developed countries, modern instruments and techniques are using to maintain high-quality standards. WHO rejects any raw material regarding medicinal plants which has more than 5% of any other part of the same authentic plant [65]. According to these standards, adulteration, whether unintentional or intentional, should be excluded. Also, traders and suppliers must be educated about the control and management of uninten tional adulterations.
4.3.4 UNDECLARED LABELING
Most of the problems associated with the use of functional foods and nutraceuticals arise mainly from the organization of many of these products as dietary supplements or foods in some countries. Furthermore, the pres ence of unidentified allergens is ubiquitous, and several such substances have been found in packaged foodstuffs without preventive labeling [66]. Consequently, food products without precautionary or mandatory labeling are not safe for allergic consumers. Hence, safety quality, and efficacy of these ingredients should be considered before marketing [67]. Similarly, production standards, quality tests, and appropriate labeling are less precise and, in some cases, manufacturers, and producers may not be licensed and
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certified. Therefore, the undeclared labeling has become a foremost concern to both the general public and national health authorities [68]. It is necessary that nutraceuticals and functional foods be accompanied by comprehensive information for safe use, such as how to store the product, how to use the product, regulatory information, and side effects. The information needs to be labeled on the packaging or leaflet put into the product package [67].
Although producers may use health benefits to sponsor their foodstuffs, the ultimate determination of health claims is to help consumers by offering detail on healthy eating outlines that may assist in minimizing the risk of cancer, heart sickness, high blood pressure, osteoporosis, dental cavities, and birth defects. Various kinds of health-related claims are permissible in food labeling. This information represents about ingredients or other nutrients in food and its health-related effects [69]. Indirect health claims may also be specified, which indirectly declares a disease-diet relationship. Indirect claims may seem in symbols, vignettes, and brand names, when used with detailed nutrient information. Though, all labels having indirect claims must also stand for the full health claim [12]. Foods nominated as “functional food” and credited definite health claims should obtain logical scrutiny prior to specific health benefits are allowed. In countries, where rich cultural tradition belief occurs, the health-promoting features of various food components are related to specific health claims, but these claims may not be scientifically proved to establish experience with these practices [70]. Some health claims have been legally documented, and future research and studies will fully document and approve or disprove the supposed health benefits [71]. Nutraceuticals and functional foods-related professionals should continue to recommend validified and hazard-free items and follow the rules of food components and specific foods as both preventative and treatment therapy for health problems. With the new substitute methods for defining the technical basis for health benefits, attention in health claims is expected to remain high, and innovative claims seem to influence food labels within the conceivable future [12].
.4 REGULATORY ISSUES
Several studies have been dedicated to nutraceuticals and functional foods, research publications in reputed journals regarding nutraceuticals and functional foods show auspicious projections for the applications of these constituents in foodstuffs, hence create worth for producers and advantages
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for consumers health [72]. This scenario results in a crucial demand for regulation, which would make safe this new collection of foods [67]. There were no specific guidelines or was registered by any health establishments in Europe to monitor nutraceuticals and functional foods until 1997 when “Green Paper on Food Law” started a new provocation to the foundation of European Food Law [18]. Afterward, in 2000 this law was favored by the “White Paper on Food Safety.” Since then, most of the “White Paper” proposals have been applied. Though there is no uniform regulatory frame work for nutraceuticals and functional foods, the guidelines to be applied are abundant and related to the kind and origin of the foodstuff. Furthermore, regulations on food supplements, novel foods, and nutritional foods may also be appropriate to nutraceuticals and functional foods depending on the kind and origin of the product as well as on their use. The foundation of European Union regulation on food products, such as nutraceuticals and functional foods is ‘safety.’ Conclusions on the safety basis of regulations are based on risk investigation, in which logical risk analysis is achieved by the European Food Safety Authority (EFSA) and risk management was made by the Euro pean Commission (EC). In the risk management stage, both the safety value and other authentic factors were measured in selecting a suitable way to deal with adulteration and safety issues [67]. Now, the EU implemented an instruction to harmonize the regulation of food products across the EU and initiated a basic licensing system to assist the public make knowledgeable choices about the consumption of nutraceuticals and functional foods [73]. However, in the developing countries where poorly regulated food products and many unregistered foodstuffs are sold freely on the market with no or slight limitation. Furthermore, the public misconception that natural prod ucts are not contaminated and are free of adverse effects often result in the unrestrained intake and inappropriate use, and this has caused hazards and health difficulties. This misconception is not only in developing countries, but it also occurs in developed countries, where the people frequently resort to “natural” products without any suitable information or awareness on the related risks [74].
.5 EVALUATION OF ADULTERATING SUBSTANCES
The analytical technique chosen for the evaluation of adulterating substances and detection of the adulterants depends on several factors such as required sensitivity, number of targeted substances, nature of the substances, the
Adulteration and Safety Issues in Nutraceuticals and Functional Foods
complexity of the formulation and physical behaviors (i.e., liquids, solids, gas, etc.), [75]. Generally, sample preparation approaches include the extraction of adulterating substance by organic solvent. The suitable solvent system is to be chosen, and the suspension or solution is to be sonicated, agitated, centrifuged, filtered, and further diluted. Despite the fast and simple procedure for sample preparation, complexity in the co-extraction of several different compounds should be considered, as this can influ ence the determination of the targeted analyte [56]. Hence, in addition to simple extraction methods, pre-concentration steps and clean-up measures are required based on the detection procedure applied for evaluation. For example, if mass spectrometry is applied, co-extracted matrix-compounds can possibly affect the evaluation of target substances and result in matrix effects through the ionization process and cause enhancement or suppres sion of signal, and hence make inaccurate analytical results [76]. This effect can be minimized by applying sophisticated techniques such as solid-phase extraction [77], liquid-liquid extraction [78], and surface-enhanced Raman spectroscopy (SERS) procedure [79]. SERS technology is based on definite vibrational spectroscopy with very high sensitivity at the molecular level constructed on Raman peaks for the detection of targeted substances. This technique offers a rapid, accurate, simple way for real samples examination [80, 81].
Chemometrics, a multivariate data scrutiny tool generally used in combi nation with data-rich instrumental approaches such as nuclear magnetic resonance, infrared spectroscopy, and mass spectrometry [82]. This is a prevailing data minimizing tool regarding food fraud, used qualitatively for classifying or grouping unknown samples with analogous features and quantitatively for analyzing adulterants in food samples [83]. Reports have demonstrated the application of partial least squares multivariate models of the infrared spectra and main component analysis to detect contaminants in various samples [84-86].
In addition to the technologies such as nuclear magnetic resonance spec troscopy, mass spectrometry, near-infrared spectroscopy, Raman spectros copy, Fourier transform infrared spectroscopy, etc., many others approach also exist including electronic noses and tongues [87, 88], electrochemical detection [89], nanosensors, and nanoparticle-based detection systems [90], apt sensor-based detection [89] and quantification via ELISA [91]. In the current age of systems-level thinking and the resultant interdisciplinary collaborations between the physical sciences, biology, and engineering (Figure 4.3), it is clear that novel developments and research will establish
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an easy and cost-effective technology for determination of adulteration and safety issues in nutraceuticals and functional food [92, 93].
FIGURE 4.3 Various methods used for the detection of adulterants.
.6 IMPACTS ON HUMAN HEALTH
Adulteration and safety issues in nutraceuticals and functional foods are food hazards that gaining concern and recognition. Irrespective of the reason of the risk, adulteration, and safety issues, evaluation is the responsibility of both the government and industries. Food fraud and adulteration is an inten tional practice for financial gain, while safety issues may be unintentional acts with unintentional hazards. Food-associated public health hazards are more dangerous than conventional food safety issues due to unconventional contaminants. Up-to-date intervention systems are not well-planned to mark a huge number of potential contaminants. Consumption of adulterated nutra ceuticals and functional foods may cause serious complications [18, 94].
Adulteration and Safety Issues in Nutraceuticals and Functional Foods
The majority of the nations have considered this problem as a priority and initiated strict efforts to control food-related issues, specifically in the devel oping countries. Safety issues in developing countries are of interest to many international organizations and are broadly recognized [95]. Efforts to cope with the demand of nutraceuticals and functional foods in the developing regions will apparently not work in the absence of a deliberate approach to monitor safety issues. Significant causes of illnesses such as diarrhea, kidney failure, digestive problems, have been raised in different regions of the world. These adulterants are reported to be responsible for the increasing incidents of diseases.
.7 WAYS TO PREVENT ADULTERATION AND SAFETY ISSUES
There are challenges to proactively notice and completely eradicate adul terants and safety issues from nutraceuticals and functional foods. Ethical manufacturers ensure and precisely determine the chemicals and ingredients present in the products, but they do not always account for the hazard of unexpected or unknown substances. Consumers, scientists, and regulators are at a disadvantage when they are experienced by un-wanted substances that can lead to shocking health issues and subsequently undermine public trust. Reports have shown that many such foodstuffs claiming to be safe and beneficial have been exposed with a compelling indication of adulteration and safety issues. In other words, the phenomena of adulteration and safety issues becoming dangerous in different parts of the world, especially in developing nations. Hence, all countries are suggested to impose more harsh rules and regulations and licensing measures to advance regulatory ethics to create suitable pre- and post-marketing checkups for analysis and to protect their public. The whole supply chain from the manufacturers and importers via wholesalers to sellers will have to be monitored properly because inspec tion at the selling level only will not cause enough positive influences. Regular inspection by authoritative agencies should do it in a designed way for monitoring adulteration and safety issues. Similarly, a consumer aware ness campaign is to be initiated to alert people about the risks associated with adulterated nutraceuticals and functional foods. Adequate actions by the related civil societies, agencies, social organizations, electronic, and print media, and consumers can make changes to ensure the safety and security of such food items. In this way, a combined effort is to be set to obtain safe food for a healthy life. For example, the Food and Drug Administration (FDA) of
Advances in Nutraceuticals and Functional Foods
the United States (US), contained trained personnel with the competence of implementation of laws to screen the quality of drugs and foods existing in the US markets [96].
Testing nutraceuticals and functional food against publicly available standards are effective measures in responding to contaminants and adulter ants arising from new processing methods or new sources of raw materials [97]. Sensitive evaluation procedures can be developed or modified in a community way and rigorous standards to notice new impurities. To date, no legal requirements mandate food ingredients analysis before product use [97]. Both public health and public confidence are threatened by the lack of standards. Hence, nutraceuticals and functional food manufacturers should provide appropriate specifications by using quality monographs. Stakeholders can contribute to set standards, and this will ensure that manu facturers, distributors, importers, exporters, and consumers know that the nutraceuticals and functional food possess appropriate quality features.
Adulteration and safety issues that result in public health risks are often unknown till it is too late and may only be known by chance rather than from a proper risk-based study; hence it is required to develop analytical models for the future. Several approaches are in use to detect the presence of adulterants and safety issues in nutraceuticals and functional foods; however, these tactics rely on the adulterant or their sources, and on this basis, the substances could not be declared totally free of adulteration. Currently, technologies available for the detection of adulteration and safety issues include the vibrational spectroscopies: mid-infrared, near-infrared, Raman; mass spectrometry, as well as NMR spectroscopy. More sophisticated techniques are now developing to verify the provenance claims made about nutraceuticals and functional foods [43]. Previously, analytical screening approaches have been applied to recognize adulteration and safety issues, these techniques worked well when the nature of the adulterant was known. The evolving forensics methods such as spectroscopy or isotope analysis do not require the adulterants to be known and are frequently applied for adul teration analysis [98, 99]. However, these assessments are costly and could not be used as a tool for simple verification and not as a form of screening for routine batch release. Hence, for common practice, these approaches are not used as online, real-time monitoring, either a preventative control within an established quality plan [56]. Concurrent risk analysis studies on social and economic factors such as animal disease outbreaks, pressure on food prices and nature actions causing crop harm, etc., together with related predictive modeling can be used to forecast the potential for adulteration and safety
Adulteration and Safety Issues in Nutraceuticals and Functional Foods
issues. Policy measures have announced the need for the implementation of both predictive procedures, detection, and reaction approaches. Prediction of adulteration depends on the suitable investigation of intelligence through the application of expert knowledge and predictive tools [100].
.8 CONCLUDING REMARKS AND OUTLOOK
Nutraceutical and functional foods are natural substances and may be consumed in combination, individually, and may be added to beverage or food for specific health benefits or technologic purposes, must have an acceptable safety profile signifying the safety for consumption by the public. The risk of adverse effects and toxicity of general medicines leads us to the attention of harmless functional food and nutraceutical-based tactics for health management. Nutraceuticals and functional foods are in high demand by consumers throughout the world. These products are usually active to maintain a healthy life by preventing diseases. However, adulteration, and safety issues are growing problems globally, and reports have exposed numerous ambiguities with respect to their definition, registration, claim, sales tactics, safety, and efficacy. Consumption of adulterated nutraceuticals and functional foods badly affects human health by causing many acute illnesses. The benefits and risks of these substances are not as recognized as for conventional drugs, and there is a lack of described possible adverse effects regarded the misuse of such items.
Although functional foods and nutraceuticals have significant potential in the health management and prevention of diseases. However, nutrition ists, regulatory toxicologists and health professionals should strategically work together to design proper regulation to provide the desired health and therapeutic benefits to the public. The effects of various processing approaches on the effectiveness and biological availability of nutraceuticals and functional foods remain to be determined. Governmental authorities and lawmakers are more must reinforce the current regulation for healthpromoting beverages and other food items, specifically with food labeling. Laws and regulations regarding functional foods and nutraceuticals may be modified in the better interest of public health. For safety, proper manufac turing methods, patenting focus, formula, and formulations and specified applications should be evaluated and modified. An adequate and enabling documented explanation is essential for getting a license. Inventors should periodically revise their findings and adopt whether to pursue a patent for
Advances in Nutraceuticals and Functional Foods
maintaining as trade secrets or to use for new discoveries. Comprehensive analysis and searches on related technology would support inventors to pinpoint the market position and assess the patentability of their products. Furthermore, frequent evaluations and revisits of developments in patenting policy and regulatory should be endorsed for setting up reasonable research and trading strategy for the encouragement of safer business. The future is fertile with opportunities to adopt and measure established frameworks and systems from the more sophisticated regulatory environments of the developed markets such as in Europe and North America. The time is right for businesses and entrepreneurs from around the globe to take advantage of the situation and launch services and protocols with the potential to become existent standards in the developing countries’ regulatory networks.
KEYWORDS
• adulteration
• Eosinophilia-Myalgia-syndrome • European Commission
• functional foods • nutraceuticals • safety issues
• surface-enhanced Raman spectroscopy
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CHAPTER 5
Nutraceuticals-Loaded Nano-Sized Delivery Systems: Potential Use in the Prevention and Treatment of Cancer
MOHAMMED JAFAR,SYED SARIM IMAM,SULTAN ALSHEHRI,CHANDRA KALA,and AMEEDUZZAFAR ZAFAR
1Department of Pharmaceutics, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia 2Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
3Faculty of Pharmacy, Maulana Azad University, Jodhpur - 342802,
Rajasthan, India
4Department of Pharmaceutics, College of Pharmacy, Jouf University,
Sakaka, Aljouf, Saudi Arabia
ABSTRACT
Cancer is considered as one of the most life-threatening diseases, wherein uncontrolled growth of abnormal cells takes place. Nutraceutical is any compound which is a nutritious food or a fraction of nutritious food which gives health or clinical boons, together with the prevention and treatment of disease. This chapter aims to provide to its readers a key scientific knowledge about how important are these nutraceuticals in terms of effectively preventing and treating various types of cancer via a novel nanocarrier strategy. Various types of lipid type, polymeric type, and inorganic nanocarriers have been investigated to improve the bioaccessibility and therapeutic success of nutraceuticals. It is also briefly explained in this chapter in its subsections, the importance of the combination approach of nutraceuticals and chemo therapeutic agents utilizing nanocarriers, over simple nutraceutical loaded
Advances in Nutraceuticals and Functional Foods
nanocarriers. Regardless of efficient manufacturing procedures of nano nutraceutical delivery systems, the caliber, strength, potency, and untoward effects should work out and discourse on top preference. To make use of the full prospective of nanocarriers, additional preclinical and clinical investiga tions are required for nanoformulation nutraceuticals. It is apprehended that the continued attempts in the field of nutraceutical delivery using the variety of novel nanocarriers would yield many rewarding outcomes.
.1 INTRODUCTION
Cancer is considered as one of the most life-threatening diseases, wherein unrestrained growth of abnormal cells takes place [1]. According to an International cancer research institution, GLOBOCAN, it is estimated that about 18.1 million new cancer cases were identified throughout the world in 2018 and approximately 9.6 million cancer deaths took place. As per one of the important reports of the American Cancer Society, the second most general cause of mortality in the US is cancer, which is surpassed only by heart disease [2]. It is also reported that behavioral factors, mainly substandard nutrition, high alcohol intake, physical inertia, and high weight gain, are responsible for 25% of incident cancers that takes place in the US, and therefore, these can be prevented [3, 4]. It is not only sufficient to modify the lifestyle to prevent cancer, but also it is required to minimize the spread of cancer in individuals. Therefore, the search for newer strategies for the effective treatment of cancer is in progress. However, nutraceuticals are emerging as a new approach in decreasing the progression of cancer.
Nutraceutical, a composite expression from ‘nutrition’ and ‘pharmaceu tical,’ was coined by Stephen L. DeFelice, chairman of the Foundation for Innovation in Medicine (FIM), Cranford, in 1989. According to Stephen nutraceutical is any compound which is a nutritious food or a fraction of nutritious food which gives health or clinical boons, together with the prevention and treatment of disease [5-7]. In the recent past, nutraceuticals have acquired ample recognition in the field of cancer investigation due to their pleiotropic sequel and reasonably innocuous nature [8]. These compounds include vitamins, carotenoids, prebiotics, probiotics, dietary fiber, phenolics, and fatty acids [9]. Nutraceuticals prevent cancer through many different mechanisms such as inhibiting efflux transporters such as Breast Cancer Resistance Protein (BCRP), P-glycoprotein (P-gp), multidrug
Nutraceuticals-Loaded Nano-Sized Delivery Systems
resistance protein (MRP), inhibiting cell proliferation and differentiation, or by reducing the harmful effects of anticancer drugs [10, 11].
The vast majority of nutraceuticals have been explored for the prevention of cancer throughout the world, but most of them suffer from poor bioavailability in humans due to their poor aqueous solubility and poor permeability [12, 13]. Some of the other factors responsible for poor rates and extents of absorp tion of nutraceuticals are: (i) abrupt delivery of active constituents from the nutritious foodstuff [14]; (ii) Insoluble complex formation with the different constituents of GIT; and/or (iii) Intestinal first-pass metabolism [15-17].
To conquer these obstacles nanoformulations have transpired as compe tent vehicles because of their nano size and other promising attributes. Nano formulations improve aqueous solubility and stability, provide moisture protection to foodstuff, prolongs drug release, manipulate texture and flavor. Moreover, nanocarriers can influence the pharmacodynamic and pharma cokinetic profile of nutraceuticals [18]. Different types of nanocarriers viz liposomes, micelles, polymeric NPs, etc., have been used to improve the biological performance of nutraceuticals.
.2 NUTRACEUTICALS IN CANCER PREVENTION AND
TREATMENT
It is mainly the non-toxic nature of nutritional substances, which makes them gain plenty of engrossment for their capability in cancer prevention and treat ment. It is reported that nutraceuticals by the modulation of miRNAs, cellular signaling, and epigenome, could prevent cancer progression in individuals [19]. Moreover, another interesting property of nutraceuticals is that they are pleiotropic, i.e., they can down-regulate several signaling pathways. All of these good qualities make nutraceuticals excellent suitor for accomplishing greater treatment outcomes in cancer patients, as solely targeted moiety usually malfunction in clinical trials [20, 21]. The major signaling pathways influenced by nutraceuticals are Pi3K/Akt/mTOR pathway, insulin-like growth factor receptor (IGFR), MAPK/ERK pathway, Ras/Raf signaling pathway, epidermal growth factor receptor (EGFR) family receptors, B-catenin signaling pathway sonic hedgehog signaling pathway, etc., [7, 22]. By acting against these molecular targets, nutraceuticals inhibit the rapid increase of cancer cells, elicit cell cycle arrest, and conquer angiogenesis/invasion/metas tasis. Thus, the cytotoxic outcomes of nutraceuticals are arbitrated through the action against different factors, viz survivin, vascular endothelial growth
Advances in Nutraceuticals and Functional Foods
factor (VEGF), matrix metalloproteinases (MMPs), etc. Many different nutra ceuticals such as curcumin, 3,30-diindolylmethane (DIM), resveratrol, indole 3-carbinol, epigallocatechin-3-gallate, lycopene, and curcumin are known to down-regulate the signal transductions like Pi3K, NF-kB, Akt and other signal transduction pathways which are required for the spread of cancer [23]. Besides restraining these traditional targets, nutraceuticals are also been showed to harmonize the pharmacokinetics of anticancer drugs by regulating ATP-binding cassette (ABC) such as MRP, P-gp, BCRP, etc. [83]. The simul taneous oral administration of curcumin increases the AUC and C maxof etopo
side by 1.50 and 1.36-fold, respectively [24]. It is not only that the nutraceuticals only enhance the bioavailability of simultaneously administered anticancer drugs, but they also extenuate the toxic effects of administered drugs. It is reported that a simultaneous intake of co-enzyme Q10 prevents anthracyclineinduced cardiotoxicity [25]. Nevertheless, the majority of nutraceuticals, because of their poor aqueous solubility and poor permeability, exhibit poor bioavailability and thus low therapeutic response. To overcome these hurdles, nanocarriers strategies have appeared to be more effective approaches and showed their capability to improve the clinical performance of nutraceuticals, and the same is explained in the following section.
.3 NANOCARRIERS-BASED NUTRACEUTICALS FOR CANCER
PREVENTION AND TREATMENT
Among the novel anticancer drugs, 40% are lipophilic, and this nature is a big hindrance for a new medicine invention project [26]. The traditional formulation methods to handle these drugs involve pH adjustment, use of cosolvents, particle size reduction, and use of surfactants which are generally harmful. In one of the commercial paclitaxel i.v. formulation (Taxol®), they used Cremophor EL, a surfactant which is linked with acute hypersensitivity reactions, and neurotoxicity [27]. Furthermore, the major hurdle with these conventional formulations is that they showed a huge amount of “collateral damage” to some normal cells, and this is due to their abnormal distribution in few compartments of the body [28].
The abnormal pharmacokinetic pattern showed by the nutraceuticals after their oral administration could be greatly minimized by encapsulation them into an emerging novel nano-drug carrier systems-based approach [29]. In recent years different types of nano-drug carrier systems, for instance, liposomes, solid lipid nanoparticles (SLNs), micelles, polymeric
Nutraceuticals-Loaded Nano-Sized Delivery Systems
nanoparticles (NPs), polymeric conjugates, carbon nanotubes, quantum dots, etc., have been investigated to regulate both pharmacokinetics and pharmacodynamics of the drug.
Many different mechanisms have been suggested for improving the oral bioavailability of these nanocarriers. One important example is chitosan based nanocarriers can cross the tight-junctions via paracellular route and ability to modulate P-gp present on epithelial cells. Another interesting example is polymeric NPs prepared using PLGA are absorbed through a distinctive Payer’s patches. Liposomes have the potential to regulate P-gp and/or CYP450 to enhance intracellular concentration and stimulate lipo protein/chylomicron production. Moreover, entrapment of nutraceuticals into novel nanocarriers could safeguard them from the harsh gastrointes tinal (GI) environment. In the latest report, it is explained that incorpora tion of epigallocatechin gallate (EGCG) in liposomes greatly decreases its degradation in an artificial intestinal fluid by approximately 10-folds [30]. Few nutraceuticals like fatty acids, Vitamin E, etc., are also used as additives in the formulation of nanocarriers, and in these systems besides their additive role, the nutraceuticals play a vital role in improving the oral bioavailability of several chemotherapeutic agents. For example, the use of d-a-Tocopheryl polyethylene glycol 1000 succinate (Vitamin E TPGS), a non-ionic surfactant, inhibits P-glycoprotein via ATPase inhibition and thus regulates the pharmacokinetics of the administered P-gp substrate [31]. These novel nanocarrier systems also exhibit improved permeation and retention effect through active and also passive targeting [32]. Because of the leaky vasculature of cancer endothelial cells, these nanocarriers could efficiently gain entry into cancer cells using passive targeting. Nevertheless, these novel nano-drug delivery carriers have the potential for the site-specific delivery of chemotherapeutic agents by binding a suitable directing moiety (ligands) on the exterior of nanocarriers utilizing active targeting strategy [10, 33]. Hyaluronic acid, RGD peptides, and folic acids are few among the various ligands widely used for the targeted delivery of chemotherapeutic agents because of their overexpression on cancer cells [34-36].
5.3.1 LIPID BASED NANOCARRIERS
The different types of lipid-based nanocarriers like liposomes, SLNs, nano structured lipid carriers (NLCs), self-emulsifying systems have been used
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for improving the bioavailability and therapeutic effects of nutraceuticals (Table 5.1).
The lipid-based nanocarriers usually contain a solid lipid core, which has potential in accumulating medications with hydrophilic and lipophilic nature into its lipid fabric. SLN is accurately embraced with more than a single solid lipid, which shows a melting point of 40°C and even higher. Subsequently, at the beginning of the 1990s, the benefits of control release property of SLNs have been emerged [48], including cellular toxicity, augmented compat ibility, and high in vivo tolerance [49, 50]. Compared to SLN, NLCs, which contain suitable blends of both liquid and solid lipids seems to possess the benefits of elevated medication carrying potential, improved storage steadi ness, and efficient drug discharging characteristics [51, 52]. These newer lipid-based NPs have the potential to circumvent P-gp through paracellular penetration and capability to uptake by microfold cell [53]. One of the recent studies conducted on SLN reports that the in-vitrocytotoxicity of a nutraceutical aloe-emodin against human breast cancer MCF-7 cells and human hepatoma HepG2 cells was drastically increased compared to its pure form, after encapsulation of it into SLNs [41], on the other hand, there was a great reduction in its toxicity on normal human mammary epithelial MCF-10A cells was recorded. This could be due to high cellular ingestion of SLNs as compared to the pure aloe-emodin formulation. In another similar study conducted by Ramalingam and Ko, it is reported about the enhanced bioavailability of resveratrol from SLNs formulated using N-trimethyl chitosan (TMC)-g-palmitic acid (PA) [54]. This investigation showed that the relative bioavailability of resveratrol from TMC-g-PA SLNs was 3.8-fold higher than that from its conventional suspension dosage form.
Liposomes are colloidal vesicular transporters, which are produced by the hydration of phospholipids. The nanosized liposomes are made up of phospholipids composed of the polar head as well as non-polar fatty acid chains, which aids them accommodated in individual minor structural phospholipid units both the hydrophilic and hydrophobic drug molecules accessing their delivery to the targeted sites [55]. Liposomes are amenable to surface modification with various targeting ligands such as sialic acid, aptamers, folic acid, etc., [56]. However, to make liposomes long circulatory in-vivo their surface has been modified by incorporating in its formulation polyethylene glycol. In an investigation, it is showed that PEG-modified liposome of ursolic acid has improved in vitrocytotoxicity in EC-304 cells as compare to pure ursolic acid [57].
TABLE 5.1 Outline of Few Current Investigations on Various Types of Nanocarriers Utilized in the Delivery of Nutraceuticals in Cancer Management
Nutraceuticals Chief Excipients In-Vitro/In-VivoModel Developments References
Apigenin -Distearoyl Human colon cancer cell Enhanced in-vitrocytotoxic activity of apigenin [37]
sn-glycero-3 lines HCT-15, and HT-29 and 5-fluorouracil combination was attributed to
phosphocholine maximal reversal of Warburg effect.
In-vivonude mice
(DSPC)
xenograft model The increased in-vivochemotherapeutic potential
of apigenin was due to the passive targeting
achieved by the liposomal drug-loaded nanocarrier.
Synergistic effect of apigenin with 5-fluorouracil.
Quercetin Compritol Human MCF-7 and Quercetin-Solid lipid nanoparticles (QT-SLNs) [38]
MCF-10 A cell lines inhibited MCF-7 cells growth with a low IC(50%
inhibitory concentration) value, compared to the free QT. QT-SLNs induced a significant decrease in the viability and proliferation of MCF-7 cells, compared to the free QT.
Curcumin Krill lipids A549 lung cancer cells Sustained in-vitrodrug release Powerful [39]
antioxidant activity.
Human umbilical vein
endothelial cells Improved in-vitrocytotoxic activity against
specific cancer cells
Resveratrol ,2-dipalmitoyl Human colon cancer cells Improved in-vitrocytotoxicity against human colon [40]
sn-glycero-3 HT-29 cancer cells
phosphocholine
(DPPC)
Aloe-emodin Lecithin, Ploxomer MCF-7, MCF-10A, and Improved in-vitrocytotoxicity of Aloe-emodin [41]
, Poloxomer 407 HepG2 cell lines solid lipid nanoparticles as compare to free
Aloe-emodin
TABLE 5.1 (Continued)
Nutraceuticals Chief Excipients In-Vitro/In-VivoModel Developments References
Fisetin Polylactic acid (PLA) Xenograft mouse model of Improved bioavailability [42]
breast cancer cells
Reduced toxicity
Inhibited tumor volume and weight without altering body weight
Quercetin Chitosan Tumor xenograft mice with Decreased tumor weight and volume [85]
A549 and MDA MB 468
cells.
Epigallocatechin Polyethylene glycol Rv1 cells implanted Decreased tumor size and volume [43]
-gallate (PEG), Polylactic tumor xenograft in athymic
Reduced prostate-specific antigen levels in serum
acid (PLA) nude mice
Genistein Mannitol, PLGA, HepG2 cells Surface modified nanoparticles showed superior [44]
TPGS in-vitrocytotoxicity, and in-vivoantitumor activity
than plain or linear nanoparticles of genistein
Thymoquinone Polyethylene glycol MCF-7, and HBL-100 Improved in-vitrocytotoxicity of Thymoquinone [45]
(PEG), Polyvinyl nanoparticles than pure thymoquinone
pyrrolidone (PVP)
Curcumin AUNPs Human breast epithelial Folate-coated cancer cell targeting using [46]
and mouse fibroblast cell CurAu-PVP NCs is a promising approach for
Folic acid
lines. tumor-specific therapy of breast cancer without
Polyvinyl pyrrolidone
harming normal cells. Improved in-vivoactivity Breast cancer orthotopic
(PVP)
mouse model
Resveratrol AUNPs Human breast (MDAMB Improved anticancer effect [47]
231), pancreatic (PANC-1), and prostate (PC-3) cancers cell lines
Nutraceuticals-Loaded Nano-Sized Delivery Systems
The isotropic mixture of surfactant, co-surfactant, and oil constitutes a novel self-emulsifying drug delivery system SNEDDS [58]. These novel nanocarriers were proved to be highly effective in the delivery of nutraceu ticals of various potential including chemoneutraceuticals. In some recently reported investigations, it was found that well-known nutraceuticals like curcumin, piperine, and naringenin, when given in the form of SNEDDS their bioavailability was increased many folds as compared to their tradi tional formulations. Moreover, the bioavailability of these nutraceuticals was doubled when they are administered in combination in SNEDDS [59, 60]. This could be attributed to the synergistic effect of neutraceuticals. One more investigation showed that self-micro emulsifying drug delivery system (SMEDDS) of Brucea javanica oil significantly inhibited the growth of tumor cells and drastically decreased S180 cancer [61].
Polymers show different characteristics in their composition. For attaining favorable drug delivery to the targeted area, the best suitable option is the polymeric NPs of colloidal nanosized systems (1 nm<d<1000 nm) [62]. Based on the structural differences, the polymeric NPs are clas sified as nanospheres and nanocapsules. Nanospheres generally consist of a polymeric matrix with three drug-loading patterns: (1) to encapsulate drugs into the spheres; (2) to absorb drugs onto the surface; (3) to disperse drugs within the polymeric network.
In contrast to nanospheres, nanocapsule score-shell possesses the ability to dissolve drugs in the core or to absorb drugs on the shell when present in the drug-loading form [63, 64].
A variety of polymers such as biodegradable polymers, polysaccharides polymers have been extensively used to deliver nutraceuticals to their specific site. Some of the reported biodegradable polymers exploited for the delivery of nutraceuticals are polylactic acid co-glycolic acid (PLGA), polycaprolactone (PCL), polylactic acid (PLA) and their copolymers are poly(ethylene glycol) (PEG), d-a-tocopheryl polyethylene glycol 1000 succinate (TPGS), etc., [65]. Similarly, polysaccharides-based polymers viz alginate, chitosan, pectin, etc., have also been used in the encapsulation and site-specific delivery of nutraceuticals [66, 67]. Polymeric NPs possess many benefits over conventional nanocarriers such as pH-dependent prolonged drug release, amenable to surface modification because of the existence of functional groups for site-specific drug delivery systems. Moreover, few polymers such as chondroitin sulfate, and hyaluronic acid have the potential to target CD44 overexpressing cancer cells [68, 69]. Jiang et al. [86] developed bovine serum albumin PCL NPs of curcumin and showed
Advances in Nutraceuticals and Functional Foods
enhancement in inhibition of the growth of three-dimensional LNCaP multicellular tumors as compared to native curcumin. This improvement in the cytotoxicity could be attributed to the efficient cellular uptake of the NPs via caveolin endocytosis. Some of the recently developed polymeric NPs for delivery of nutraceuticals are summarized in Table 5.1.
Nano-micelles consisting of polymeric and surfactant nano-micelles are known to be as rising novel carrier systems for nutraceuticals delivery. Apart from their smaller size, improved drug solubility and stability [70], and lower adverse effects and high biocompatibility [71] aids them to become potential candidates for poorly aqueous soluble drug delivery. Fewer amphiphilic molecules, when added to special solvents, adapt to self-assemble and results in core-shell monomers called nano-micelles [72]. Liu et al. [52] described novel curcumin containing a nano micelle formula tion using a polyvinyl caprolactam-polyvinyl acetate-polyethene glycol (PVCL-PVA-PEG) graft copolymer. Nano micelle curcumin was formulated and optimized and then further evaluated for in vitrocytotoxicity, in vitro cellular uptake, in vitroantioxidant activity, and also various in-vivoactivi ties. The solubility, chemical stability, and antioxidant activity of curcumin were greatly improved after the encapsulation of it into the PVCL-PVA-PEG nanomicelles. Moreover, the formulated curcumin nanomicelles are stable at storage conditions, they had good cellular tolerance, and also their in-vitro and in-vivo activities were significantly improved when compared with a free curcumin solution.
Polymeric hydrogels are cross-linked water-loving polymer meshes, and they possess the potential to pledge localized, prolonged release of nutra ceuticals. These novel polymeric hydrogels possess a great attraction to water, but they are protected from solubilizing because of either by physical entanglements, covalent cross-linking, or by non-covalent attractions [73, 74]. These polymeric hydrogels could also undergo phase transition while exposed to atmospheric temperature [67]. A most useful characteristic of these polymeric hydrogels is that they are amenable for surface modifica tion by active targeting ligands. It is reported that folate functionalized PEG cross-linked acrylic polymer (FA-CLAP) hydrogel were successfully utilized for the targeted delivery of curcumin [75]. Moreover, it is proved in the study that folate-functionalized hydrogel demonstrated higher uptake in HeLa cell lines than non-functionalized hydrogels. One more investiga tion carried out on the same nutraceutical curcumin, curcumin hydrogels were formulated using gelatin, hyaluronan, and showed improved in vitro cytotoxicity against A549 lung adenocarcinoma cells than pure curcumin
Nutraceuticals-Loaded Nano-Sized Delivery Systems
[76]. Moreover, these hydrogels showed comparatively greater apoptosis rates than pure curcumin demonstrated via Annexin V-FITC/PI analysis.
5.3.2 INORGANIC NANOCARRIERS
Inorganic type of nanocarriers which could deliver nutraceuticals include quantum dots, nanosilica, carbon nanotubes, magnetic nanomaterials, silver, and gold nanoparticles (AuNPs) [77, 78]. These nanocarriers possess some unique properties like smaller size, different shapes, varying content, high surface volume ratio, and most importantly, their potential for surface modi fication makes them excellent candidates to be extensively used in nutra ceutical delivery. Carbon nanotubes, are hydrophobic tubular meshworks of carbon atoms showing length and diameter of around 1 to 100 nm and 1 to 4 nm respectively, turned out to be utilized for the delivery of nutraceuticals [79, 80]. The main problems with these carbon nanotubes are, they are highly insoluble in almost all solvents and they are held by several toxicity issues, but these problems could be overcome by modifying them chemically and thus ameliorating their biocompatibility, alleviating their toxicity and make them water-soluble carriers [1].
Among the various inorganic nanocarriers AuNPs have been volumi nously investigated for nutraceuticals delivery because of their well-defined surface chemistry, simplicity in synthesis, and magnificent biocompatibility [57]. In one of the reported investigations, apigenin AuNPs were formulated and biocompatible property of prepared NPs was shown by non-toxicity on normal epidermal cells (HaCat). Moreover, these NPs showed in vitro compatibility with squamous epidermal cancer (A431) and also with human cervical squamous cell cancer (SiHa) cells. Epigallocatechin-3-gallate and green tea consolidated AuNPs have also been designed and adjudged particularly harmful towards MCF-7 cells, and Ehrlich’s Ascites Carcinoma, while showing no toxicity in normal primary mouse hepatocytes and the reason attributed to it is that they possess greatest antioxidant characteristics [81]. Magnetic NPs, besides their small size, possess excellent magnetic properties, because of which they were successfully utilized in the delivery of nutraceuticals. In one of the reported research study curcumin magnetic NPs were designed and expanded with the aim to enhance its bioavailability and thus efficacy [82]. The investigation showed a 2.5-fold increase in oral bioavailability of curcumin as compare to pure curcumin. Moreover, this nanoparticle formulation significantly reduced pancreatic tumor growth in
Advances in Nutraceuticals and Functional Foods
an HPAF-II xenograft mouse model and increases the life span of mice by slowing down the cancerous cell growth.
.4 CONCLUSION AND FUTURE PROSPECTS
Scientific investigations done during the last few decapods have given substantial affirmation of the appreciable flexibility of nutraceuticals and the various targets that make them highly potential candidates for cancer prevention and treatment. While, preclinical and cell culture studies showed that nutraceuticals have potential antitumor activity and other fitness boons, therapeutic application of the aforesaid compounds is lean. An application of novel nanocarrier built delivery systems has facilitated scientists to conquer the physicochemical and biological hindrances of nutraceuticals. Nearly all of polymers and other additives used in the design and develop ment of nanocarriers have been approved as safe by FDA. Moreover, these novel carrier systems result in site-specific delivery of nutraceuticals in cancer tissue because of improved tissue permeation and retention effect. The most interesting and important characteristics of nanocarriers are that their surfaces could be modified using specific ligands in order to attain cancerous cell-specific delivery of nutraceuticals. During the past few years, a combined approach of nutraceuticals and anticancer drugs utilizing nanocarriers has gained much attention. It is apprehended that the continued attempts in the field of nutraceutical delivery using the variety of novel nanocarriers and also combination-based strategies would yield many rewarding outcomes.
KEYWORDS
• cancer
• chemotherapy
• insulin-like growth factor receptor • nanoparticles
• nutraceuticals
• targeted delivery systems • vascular endothelial growth factor
Nutraceuticals-Loaded Nano-Sized Delivery Systems
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CHAPTER 6
Nutrition Nutraceuticals: A Proactive Approach for Healthcare
CONOR P. AKINTOLA,DEARBHLA FINNEGAN,NIAMH HUNT,
RICHARD LALOR,SANDRA O’NEILL,and CHRISTINE LOSCHER1Immune Modulation Group, School of Biotechnology, Dublin City University, Dublin, Ireland
2Fundamental and Translational Immunology Group,
School of Biotechnology, Dublin City University, Dublin, Ireland
ABSTRACT
The important link between nutrition and health has led to the discovery of nutraceuticals which are food derived products that exhibit health boosting properties beyond their nutritional value. This chapter analyses the current body of data describing the benefits of the most commonly studied nutra ceuticals including the new generation of protein-derived nutraceuticals that are currently under development. Many of the nutraceuticals are found in abundance in superfoods such as milk, fish oils, tomatoes, berries and dark chocolate, which are promoted as health boosting foods. This chapter provides evidence of their health protecting aspects including anti-inflam matory, anti-oxidant, pro-anabolic, liporegulatory, and glucoregulatory that have the potential to promote metabolic, cardiovascular, and tissue health when consumed in the right amounts. Furthermore, nutraceuticals such as curcumin, have shown synergistic effects with established chemotherapeutic strategies to treat cancer. Despite the strong evidence of their beneficial properties, this chapter identifies several important questions which remain unanswered such as whether there is a need for regulation of nutraceuticals to the same extent as drug products and the role of these nutraceuticals in the management of chronic diseases. There is no clinical data to examine whether or not, supplementing a diet, with a given nutraceutical, has any
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long-term adverse effects while many over the counter food supplements have health claims with no robust evidence, The European Commission is working to address this issue and in the context of health promotion or disease management strong clinical evidence with medical oversight or clear communication and education from the manufacturer with regard to its use will be required. Age is a major factor when it comes to the prevalence of chronic disease where the use of nutraceuticals to promote healthy aging is an area where it can have the greatest impact, particularly since the World Health Organization estimates that by 2050, there will be over two billion people worldwide aged over 60. Future efforts at mining food sources for health boosting bioactives should focus on the hunt for anti-inflammatory and antioxidant nutraceuticals to support healthy aging in this ever-growing population.
.1 INTRODUCTION
Two commonly quoted phrases, whose meanings are still highly relevant today are, “you are, what you eat,” which has its roots in the 1826 publica tion “The Physiology of Taste” by Anthelme Brillat-Savarin and “Let food be thy medicine, and medicine be thy food” a phrase allegedly spoken by Hippocrates in 400 BC [1]. An individual’s health status and the develop ment of human diseases are influenced by genetics, in addition to environ mental factors, where diet is a major contributing factor [2]. Nutritional status, notably, malnutrition plays a critical role in the development of human disease [3]. The lack of essential micronutrients such as vitamins has long been associated with the development of disease. For example, vitamin C deficiency causes a breakdown of epithelial junctions in the body resulting in the onset of scurvy [4], while deficits in vitamin D causes weak bone structure and increased incidences of rickets and osteopathologies [5]. There are also detrimental effects noted with protein malnutrition, including dysfunction in skeletal muscle physical and metabolic functions, and compli cations for critical organ health [6]. Often overlooked, is the phenomenon of “overnutrition,” which is a problem associated with middle-to high-income populations. Overnutrition is a consequence of direct, easy access to more food than is nutritionally required and similar to malnutrition, it plays an integral role in the incidence of human disease. In developed countries, diets high in refined sugar, salt, and “bad fats” such as saturated fats or trans-fatty acids correlate directly to increased rates of dietary-related morbidity such as
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hypertension, diabetes, cancer, and cardiovascular vascular disease (CVD) [7]. Excess intake of dietary protein has also been linked with the occurrence of renal disease [8].
In 2020, the World Health Organization (WHO) estimates that the global population will approach 7.8 billion, with an estimated 1.9 billion adults’ overweight, of which 650 million are classed as obese. In the United States of America (USA), 27.6% of adult men and 33.2% of adult women in the USA are obese [9, 10]. An increased incidence of obesity amongst a popu lation will place additional pressure on global resources, most notably the health care sector. Perhaps more worryingly, is that obesity rates in children and adolescents are on the rise. In the USA, approximately 11.6% of children and adolescents are overweight [9, 10], and similar percentages are observed in studies on children and adolescents in other developed countries [11]. However, recent studies have shown that the rates of childhood obesity have plateaued in developed countries [12]. Lack of exercise and unhealthy eating habits both directly influence national and global rates of obesity. In 2018, a survey by Sport Ireland and the health service executive (HSE), found that only 17% of primary school and 10% of post-primary school children engage in at least 60 minutes of exercise per day. Eating habit surveys conducted by the HSE also note an increased prevalence of unhealthy eating habits [13].
Higher rates of obesity are correlated with increased incidences of chronic, non-communicable diseases (NCDs) such as CVD, metabolic syndromes (Met-S), cancers, and immune-mediated disorders [14]. According to the WHO, 71% of all global deaths are attributed to NCDs, accounting for 41 million deaths per year. NCDs can manifest in several forms, such as type 1 diabetes mellitus and asthma, which are conditions that arise from birth [15]. However, there is a continuing and concerning rise in dietary-related NCDs globally [16, 17] that have a massive economic burden to global healthcare systems and have significant impacts on disability-adjusted life years (DALYs) [18]. Moreover, pharmaceutical, therapeutic strategies for treating dietary-related NCDs present several challenges in terms of effi cacy and side effects. For example, Infliximab, a tumor necrosis factor-alpha (TNF-α) agonist commonly employed to treat rheumatoid arthritis (RA) and Crohn’s disease (CD), displays immunogenic properties in humans, which results in resistance to the treatment over time. While combination therapies with other immunosuppressive drugs improve Infliximab efficacy, these therapies are associated with increased susceptibility to viral and bacterial infections [19]. Another example is the use of nicotinic acid (NA), a drug to treat hyperlipidemia that exhibits common side effects including skin itch
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and flushing [20]. Therefore, for many NCDs, newer therapies with fewer side effects are required.
Nutraceutical is a term derived from “nutrition” and “pharmaceutical” that applies to products that are isolated from herbal, dietary supplements, and functional foods such as dairy, cereals, and beverages. These prod ucts exhibit properties beyond nutritional value such as health-boosting properties [21]. The supplementation of diets with nutraceuticals derived from functional foods or other sources have been shown to be useful in the treatment of chronic human NCDs such as CVD, Met-S, and chronic inflammatory diseases [22]. In today’s society, there is a trend in consumers becoming increasingly health-conscious with a desire to acquire products that are derived from natural sources. The popularity of functional foods in the nutraceutical industries are therefore increasing as consumers seek viable alternatives to conventional therapies that are often more expensive, high-tech treatments that have unforeseen or undesirable side effects. Nutraceuticals have received considerable interest due to their safety and therapeutic effects as unlike their pharmaceutical counterparts, no studies have observed the accumulation of nutraceuticals in the body’s tissues during prolonged usage. As a consequence, it is unlikely that there will be no long-term negative side effects, however this remains to be investigated [23-25]. Consequently, these industries are rapidly expanding with a net value of $230.9 billion globally in 2018, which is projected to reach $336.1 billion by 2023 [26].
There has been major investment in the research and development of nutraceuticals for the prevention and treatment of NCDs as a proactive approach to healthcare. In this chapter, we will discuss nutraceutical strate gies that may be used to prevent or ameliorate the symptoms and treat the major NCDs that are highly prevalent in today’s society, such as CVD, Met-S, cancers, and immune disorders. Specifically, the current nutraceu ticals that will be discussed are, curcumin, omega 3 polyunsaturated fatty acids (PUFAs), carotenoids, flavonoids, and specific amino acids, due to the fact that these are functional molecules that are naturally occurring in foods deemed “superfoods” and also available as individual purified supplements in health food stores and pharmacies. Technological strategies used to mine food for bioactive nutraceuticals will be discussed, specifically highlighting the work ongoing into uncovering and screening of bioactive peptides from both plant-based and animal sources. We will also highlight clinical trials that have investigated the potential benefits of nutraceuticals in the preven tion and treatment of NCDs.
Nutrition Nutraceuticals: A Proactive Approach for Healthcare
.2 NATURAL NUTRACEUTICALS
6.2.1 CURCUMIN
Curcumin is a natural bioactive that has received a lot of attention as a nutra ceutical strategy for several human diseases. It has been described to possess several health boosting properties including immunomodulatory, anticancer, anti-cardiovascular disease, anti-diabetic, antioxidant, and anti-ageing [26, 27]. Curcumin is a polyphenolic compound found in the commonly avail able spice, turmeric [26] that was shown to target multiple cell signaling pathways as well as intracellular and extracellular molecules. In particular, it is thought to target nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), a pathway critical to driving a pro-inflammatory immune response [28]. Chronic activation of NFκB and consequent chronic inflam mation is linked with several human diseases including cancer, inflammatory disease, CVD, and metabolic disease [28-30].
Curcumin modulates several immune cell types, including dendritic cells, cells critical to the activation of the adaptive immune response [31]. The activation of dendritic cells with bacterial lipopolysaccharide induces a pro-inflammatory state where the activation of NFκB is a critical part of this process [32]. These activated cells secrete a panel of inflammatory media tors, including cytokines such as interleukin (IL)-12 and TNF, and costimu latory cell surface markers required for the activation of adaptive immune responses [33]. Curcumin inhibits the secretion of these cytokines and the expression of the co-stimulatory cell surface markers MHCI/II, CD80, and CD86 on LPS activated dendritic cells. Curcumin impairs the translocation of the NFκB p65 subunit which is an important process in the activation of inflammatory responses [31]. Curcumin similarly inhibits the activity of macrophages, cells important in the induction and maintenance of adaptive immune responses. Macrophages can be differentiated into different cell phenotypes and studies demonstrate that curcumin can skew macrophages from a pro-inflammatory M1 phenotype towards an anti-inflammatory M2 phenotype [34].
Immune-mediated inflammatory disorders, such as RA and inflamma tory bowel disease (IBD), share common underlying mechanism that involve pro-inflammatory cytokines such as TNF and the activation of NFκB. Given the anti-inflammatory potential of curcumin, several studies were designed to investigate the efficacy of curcumin in treating inflammatory disorders. In a rat model of RA, curcumin supplementation of 200 mg/kg of body
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weight for three weeks resulted in reduced infiltration of inflammatory cells into the synovium of the knee joint. Furthermore, curcumin reduced mTOR signaling within the synovium, which is often overexpressed in RA lesions, thought to propagate the disease [35]. In a pilot study in humans, curcumin reduced RA severity compared to diclofenac sodium, a NSAID commonly used to treat RA [36].
Curcumin has also been documented as improving the clinical symptoms of IBD, through ant-inflammatory activity and by directly and beneficially influencing the gut microbiota, which alleviates the severity of the disease in patients suffering from IBD [27, 37]. The gut microbiome comprises of several trillion bacterial cells of varying genus’ that line the walls of the human digestive tract. The microbiome plays several roles in maintaining human health including; digesting dietary molecules we are unable to digest normally into useable metabolites, and in preventing the colonization of the gut by potential pathogenic bacteria [38, 39]. In the context of IBD, imbal ances in specific bacterial populations associated with the incidence of IBD. Patients presenting with IBD have frequently shown that increased popu lations of Enterobacteriaceae and decreases in Clostridium, Firmicutes,
Bifidobacterium, and Lactobacilluspopulations [40]. Curcumin has been documented to promote the growth of Lactobacillus and Bifidobacterium [41]. In a mouse model of IBD, researchers demonstrated that nanoparticle delivery of curcumin increased butyrate concentrations in the gut, believed to be sourced from commensal Clostridiumclusters, which in turn promoted the activity of gut T-regulatory lymphocytes and reductions in gut inflam matory markers [42].
The NFκB pathway has a critical role in cell survival and apoptosis, programmed cell death [28] and given the inhibitory effect of curcumin on this pathway, it has become a molecule of interest in the context of cancer treatment and prevention. Cancer in its simplest terms can be described as a division of abnormal cells associated with prolonged cell survival and an evasion of apoptosis, a natural process in an organism’s growth and develop ment [43]. Curcumin was shown to have a notable effect on a variety of cancer types [27]. It has been demonstrated in vitrothat curcumin, in the presence of paclitaxel (a chemotherapy medication used to treat a number of types of cancer) has the ability to suppress antiapoptotic genes, prolif erative genes and metastatic genes in a paclitaxel-resistant breast cell line via the NFκB signaling pathway. In the same study, using a human breast cancer xenograph model, researchers discovered that oral administration of curcumin decreased the occurrence of lung metastases in mice [44]. Further
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synergy with the chemotherapeutic docetaxel was also demonstrated in vitro using a metastatic prostate cancer cell line. Curcumin-docetaxel treatment increased the efficacy of docetaxel compared to curcumin and docetaxel treatment alone [45].
Curcumin can suppress angiogenesis, a key hallmark in cancer patho genesis and therefore could preventing the formation of new blood vessels inhibiting the delivery of oxygen and nutrients to tumor tissue. VEGF-A is the principal mediator of angiogenesis and curcumin is thought to prevent angiogenesis through the modulation of the VGEF signaling pathway [30, 46]. In a model of murine Dalton’s lymphoma, it was discovered that curcumin can promote the activity of the tumor suppressor gene p53, suggesting a role in cancer prevention [47]. A direct role in the killing of cancer cells has also been described in vitro, by causing cell cycle arrest, apoptosis, and inducing autophagy in pancreatic cancer cells [48]. This finding is significant given the poor prognosis associated with pancreatic cancer due to the lack of effec tive treatments [49].
Curcumin displays potential beneficial effects in protecting against the development of atherosclerotic plaques in human arteries. Atherosclerosis is a degenerative condition in which excess circulating lipids are deposited in the artery walls, causing the development of atherosclerotic plaques. These plaques cause narrowing of the artery, resulting in hypertension. Injury to the arterial wall and the presence of ectopic lipids in the blood leads to platelet aggregation and clotting, where the resulting clot blocks the flow of blood in the artery resulting in stroke or myocardial infarction [50]. Inflam matory M1 macrophages are the predominant immune cell during plaque formation. The chronic activation of M1 macrophages impairs the healing of the damaged cell wall, promoting the development of atherosclerotic lesions. The protective effects of curcumin in this context may be multifaceted. One mechanism by which curcumin may protect against atherosclerosis is by modulating the immune microenvironment, which can influence plaque formation. As previously mentioned, curcumin is believed to skew macro phage from an inflammatory M1 phenotype towards an anti-inflammatory M2 phenotype, which promotes wound healing [34, 51]. Furthermore, the serum lipid-lowering effects of curcumin have also been extensively docu mented. This effect may be two-fold as it may lead to a reduced presence of lipid activated M1 inflammatory macrophage in atherosclerotic lesions and the reduction of excess serum lipid concentrations reduces the risk of a major cardiac event [51, 52]. In vivomurine models of atherosclerosis appear
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to support these mechanisms, however further investigation in both human and animal models is required [53].
Curcumins therapeutic potential also extends as a possible intervention to improve the clinical symptoms of type 2 diabetes mellitus (T2DM). Inflammation is hypothesized to play an integral role in the development of T2DM, through the induction of adipose tissue and skeletal muscle insulin resistance, a symptom often seen in pre-diabetic individuals [54, 55]. By reducing chronic systemic inflammation, curcumin treatment may act as a strategy to prevent the onset of T2DM. This is exemplified in a human study, of pre-diabetic patients as supplementation with 250 mg/day of curcumi noids, prevented the onset of T2DM in the treatment group, whereas 16.4% of patients in the placebo group were diagnosed with T2DM. Those who received the Curcumin treatment exhibited improved sensitivity to insulin, improved pancreatic β-cell function, and improved inflammatory profiles [56]. In summary, the direct effects of curcumin in both in vitroand in vivo human and animal trials included, decreased levels of circulating LDL, and triglycerides, anti-hyperglycemic activity, improved fasting blood glucose concentrations, antioxidant potential, and decreased circulating inflamma tory markers [57, 58].
To date, the recommended for daily intake for curcumin ranges from of 0 to 3 mg/kg of body weight, as approved by the European Food Safety Authority (EFSA) and the Joint FAO/WHO Expert Committee on Food Additives (JECFA) [59]. However, at high dose intakes can have several adverse effects of excessive intakes of the nutraceutical. For example, at daily doses ranging between 1000 mg and 12,000 mg can result in yellowing of the stool, diarrhea, and a rash [60].
6.2.2 OMEGA-3 POLYUNSATURATED FATTY ACIDS (PUFAS)
Perhaps one of the longest standing health boosting bioactives are the omega-3 PUFAs. Long believed to be of great benefit for cardiovascular health, omega-3 PUFAs are found predominantly in the flesh of oily fish such as salmon, trout, and mackerel. Fish oils are also abundant with high levels of omega-3 PUFAs, particularly cod liver oil, a popular over the counter supplement [61]. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are two of the main omega-3 PUFAs shown to have protective effects against a variety of chronic diseases, which are found in varying amounts depending on the source [62]. EPA and DHA are 20 and 22 carbon fatty
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acids, respectively, and are isolated primarily from marine sources, but can also be found in animal sources such as eggs [63]. However, small quantities of EPA and DHA can be synthesized endogenously through consumption of foods rich in another essential omega-3 PUFAs known as alpha-linoleic acid (ALA) [64], which in its own right has had noticeable benefits for several aspects of human health, most notably, brain health.
The most common omega-3 fatty acid is ALA, which can be sourced from vegetable oils, nuts (especially walnuts), flax seeds and flaxseed oil, leafy vegetables, and some animal fat, especially in grass-fed animals [65, 66]. Data suggests that ALA plasma status is inversely associated with the incidences of stroke in adult men [67]. Furthermore, animal models have demonstrated that ALA plays an active role in inducing recovery from stroke [68]. Interestingly, ALA supplementation has also been linked with potential applications for improving mental health [69]. In a mouse model of depres sion, diets high in ALA were capable of inducing anti-depressive behaviors. Phenotypic changes in the hippocampus and changes in gene expression in the brain were also observed, suggesting a potential therapeutic avenue for mental health disorders [70]. However, comprehensive human trials will need to be conducted to uncover the potential of ALA for neuroprotection and mental health applications.
Marine sourced omega-3 PUFAs have a long-documented history in the treatment of several risk factors linked to CVD. Documented modalities of omega-3 PUFAs include modulation of vascular endothelial cell function, anti-hypertensive activity, anti-hypercholesterolemia activity, reduction in platelet aggregation, and modulation of heart rate to reduce tachycardia [62]. A study conducted in an American cohort demonstrated an associa tion between plasma concentrations of omega-3 PUFAs, including EPA and DHA, and a decreased likelihood of death as a result of a major cardiac event [71]. A meta-analysis study of randomized placebo-control clinical trials noted that considerable improvements in circulating triglyceride levels, improvements in blood pressure and heart rate were among the big indica tors for improved cardiovascular health. A decrease in mortality as a result of major cardiac events was also observed following consumption of marine sourced omega-3 PUFAs [72]. Given the direct effects of CVD risk factors such as hypertension and hyperlipidemia, omega-3 PUFAs also display anti-inflammatory properties, which may indirectly improve cardiovascular health by modifying the release of inflammatory eicosanoids, which are lipid derived modulators that regulate several aspects of cellular signaling and immune function [62, 73].
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Many studies have examined the effect of omega-3 PUFAs on immune function, and in summary, they conclude that that omega-3 PUFAs exert potent anti-inflammatory effects in vitroand in vivo[65, 74, 75]. The mecha nisms by which omega-3 PUFAs influence inflammatory cell function has yet to be clarified, however it is believed to exert its effect through several mechanisms including; direct signaling through fatty acid receptors, incor poration into membrane phospholipids that consequently effects membrane fluidity and extracellular signaling, and influence hormonal pathways linked to inflammatory processes [74]. An in vitrostudy using THP-1 macrophage demonstrated that EPA exhibits potent anti-inflammatory properties, suppressing the expression of genes associated with inflammatory cyto kines and chemokines, and genes involved in the NFκB signaling cascade. Furthermore, enhanced expression of mitochondrial tumor suppressor 1 (MTSG1), a candidate tumor suppressor protein, and an inverse suppression of NOS2, indicate that EPA is capable of relieving oxidative stress in inflam matory states [76]. A separate study, also using THP-1 macrophages showed that along with cytokine suppression, NF-κB p65 transcription factor was also suppressed in EPA and DHA treated cells [77]. DHA has also displayed similar activity in an ex vivo human study that compared macrophages from healthy control patients, and from patients suffering with small abdominal aortic aneurism. Isolated macrophage was treated with DHA and suppres sion of inflammatory signaling was observed with upregulation of free radical scavenging activity in both healthy and diseased states [78].
The in vitro trend of consistent anti-inflammatory activity by omega-3 PUFAs has been examined in numerous human studies [79, 80]. For example, while there may be several beneficial uses of omega-3 PUFAs, one that is being explored is its use in promoting healthy ageing. As we age, our bodies become increasingly inflammatory, with noted increases in circulating inflammatory biomarkers. This phenomenon known as “inflam maging,” is a chronic, but low-grade upregulation of inflammation that can have a detrimental effect on the body leading to the onset of several chronic diseases [81, 82]. Inflammaging has been linked with the onset of a disease known as sarcopenia that causes a degenerative loss of muscle mass over time [83]. Sarcopenia is a deleterious condition that impacts DALYs [84-86]. It is believed that inflammation may directly impact the anabolic sensitivity of skeletal muscle, which is crucial for muscle health as it promotes anabolic pathways that allow muscle tissue to maintain its mass and function. Since, animal and human studies have observed a negative correlation between the anabolic capacity of muscle and the concentration of circulating inflammatory
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markers [84, 87], omega-3 PUFA supplementation is one of several strategies being considered to treat sarcopenia in older adults. Emerging data suggest that omega-3 PUFA ingestion is correlated with a decrease in circulating inflammatory markers in older adults [88, 89]. A direct mechanism for promoting the anabolic capacity of muscle by omega-3 PUFAs has also been described. In C2C12 myotubes, treatment with EPA and DHA was found to enhance phosphorylated activation of mTOR signaling [90]. mTOR is considered as the master regulator of protein synthesis in skeletal muscle [91]. Decreased activity of this pathway leads to reduced protein synthesis and subsequent reduced muscle mass and strength, which can significantly impact on an individual’s quality of life [86]. In vivo, after eight weeks of consumption of an EPA and DHA rich supplement, researchers found that in older adults, mTOR signaling, the primary anabolic pathway in muscle, was upregulated suggesting that omega-3 PUFA ingestion could be an avenue to promote muscle protein synthesis in sarcopenic patients [92].
The anti-inflammatory nature of omega-3 PUFAs can be utilized to treat a variety of chronic immune disorders. IBD is an overarching term that describes several diseases, including CD and ulcerative colitis (UC), where chronic inflammation in the gut leads to adverse health conditions. Numerous studies have demonstrated that ingestion of omega-3 PUFAs reduce intestinal inflammation [93]. Furthermore, a study with a European cohort demonstrated an inverse relationship between the consumption of DHA and the occurrence of CD [94]. A case report concerning 22 females with IBD presented an imbalance of omega-6 PUFA and omega-3 PUFA with co-occurring vitamin D deficiency. Rebalancing the ratio of omega-6 PUFA and omega-3 PUFA with an EPA and DHA supplement with co-ingestion of vitamin D reduced disease severity and symptoms [95]. The imbalance of the Omega-6/3 PUFAs is related to several chronic disease states such as CVD, auto-immune conditions, and metabolic disorders [96]. Westernized diets are often deficient in omega-3 PUFAs, which are also associated with the increased prevalence of chronic diseases such as CVD and CID. Omega-3 PUFA supplements may be used to restore this balance to ameliorate and prevent the onset of chronic disease [97].
Omega-3 PUFA containing fish oil supplements are widely available in pharmacies and health food stores, commonly seen at 1000 mg. The WHO advises 250 mg/day of EPA and DHA for men and women, and it is recom mended to increase intake to 300 mg/day for pregnant and lactating women [98]. Few adverse effects are associated with regular intake of marinederived omega-3 PUFA. Although one study reported that over a four-week
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period, in patients diagnosed with CVD, intake of 1.7 g/day of an EPA and DHA containing capsule resulted in 12% of the participant groups suffering from adverse gastrointestinal (GI) symptoms such as abdominal pain, diar rhea, and GI bleeding [99]. Omega-3 PUFA supplements derived from fish oils are not suitable for certain groups such as vegans or some vegetarians. However, algae are potent sources of omega-3 PUFAs and could be a poten tial source for these cohorts [100, 101]. However, there appears to be a gap in the literature, comparing the efficacy of fish-derived omega-3 PUFAs and algae-derived omega-3 PUFAs, thus work is required in this regard.
6.2.3 CAROTENOIDS
Carotenoids are a class of pigmented phytochemical found almost exclu sively in plants [102]. Carotenoids have a forty-carbon skeleton of isoprene units and maybe cyclized at one or both ends, with various hydrogenation levels or oxygen-containing functional groups, primarily in the transform occurring naturally [103]. Carotenoid-rich foods include carrots, tomatoes, spinach, and apricots [102]. The most commonly studied carotenoids are lycopene and β-carotene, and they have been shown to display beneficial properties inducing antioxidant and immunomodulatory properties.
6.2.3.1 LYCOPENE
Lycopene is an acylated carotenoid that is found in abundance in tomatoes and tomato-based processed foods [104]. As well as exhibiting cholesterollowering bioactivity, it displays potent antioxidant and anti-inflammatory properties and an ability to protect cells from oxidative damage [46]. Lycopene inhibited the inflammatory cytokines, IL-1β, IL-6, and TNFα, the activity of iNOS and disrupted NFκB signaling in LPS stimulated RAW264.7 macrophages [105, 106]. In RAW264.7 macrophage, treatment with lyco pene and β-carotene suppressed reactive oxygen species (ROS) production in vitro [107]. Furthermore, a study in THP-1 macrophage demonstrated the negative regulation of oxidative pathways by lycopene as it reduced basal ROS production and ROS production in 7-ketocholesterol stimulated THP-1 macrophages [108]. The antioxidant and immunomodulatory properties of lycopene may protect against the development of cancer, CVD, and Met-S.
Lycopene has also been investigated as a nutraceutical intervention for the treatment of CVD. In rabbit models, lycopene supplementation has
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been found to decrease total circulating cholesterol levels, LDL levels, and triglyceride levels [109]. Furthermore, some research has associated lycopene intake with a reduction in the size of atherosclerotic lesions [110]. However, there are conflicting reports in the literature on the beneficial properties of lycopene, with one study associating increased lycopene intake with a decreased incidence of CVD [111] while conversely, a second study concerning 39,876 women over the age of 45, found no correlation between lycopene intake and CVD incidence. Researchers did note, however, that there was an inverse correlation between tomato product intake and CVD incidence, suggesting that a synergistic contribution with other phytochemi cals found in tomato-based foodstuffs may be beneficial [112].
Lycopene is perhaps most recognized for its alleged role in the prevention of cancer with several studies linking lycopene intake to a decreased risk of prostate cancer [113]. In a randomized control trial, ingestion of a lycopene rich supplement twice daily was associated with a decreased incidence of prostate cancer in patients diagnosed with high-grade prostate intraepithelial neoplasia, a precursor condition to prostate cancer, compared to the placebo group [114]. Another study found that increased lycopene intake was associ ated with a reduced risk of severe prostate cancer development [115]. Despite its obvious bioactivity as a potent antioxidant and anti-inflammatory, there are questions as to whether lycopene alone, is capable of reducing the risk of cancer development. Studies investigating the anticancer potential of lycopene have either used purified lycopene or a tomato-based foodstuff as a vector to deliver the lycopene [116]. For example, in male rats, consumption of a tomatobased powder and not purified lycopene contributed to a reduced risk of death from prostate cancer [117]. There is no significant contribution of diets rich in lycopene in lowering the risk of ovarian cancer, whereas other carotenoids such as alpha-carotene, beta-carotene, lutein, and beta-cryptoxanthin were associated with a decreased risk [118]. It is possible that the effects of lycopene on cancer could be mechanistically specific to prostate cancer. However, there is some preliminary evidence that lycopene can inhibit the growth of endometrial cancer cell line [119] and therefore more longitudinal studies in the context of other cancer types is required to determine this.
6.2.3.2 Β-CAROTENE
Like its counterpart lycopene, β-carotene, historically was discussed extensively in the context of cancer prevention [120]. Recently, β-carotene
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containing creams have been documented to decrease the absorption and penetration of UV light into the epidermis of pig’s ears, suggesting a poten tial protective effect against sunlight-induced melanomas [121]. Epidemio logical studies that garnered support for β-carotene in cancer prevention were based primarily on retrospective and prospective questionnaires concerning dietary intake. However, a meta-analysis of randomized control studies has indicated that dietary intake of β-carotene had no effect on the incidence of pancreatic, colorectal, prostate, breast, melanoma, and non-melanoma cancers [122].
β-carotene is a pro-vitamin A molecule that possesses many of the immu noregulatory and antioxidant properties of vitamin A [123]. In RAW264.7 macrophage, treatment with β-carotene suppressed ROS production in vitro [107].The antioxidant potential of β-carotene was demonstrated in vivo in humans suffering from chronic lead poisoning. Twelve weeks of supple mentation with a β-carotene supplement called Beta Karoten® reduced oxidative profiles in a cohort of men frequently exposed to lead in their place of work [124].
However, there is evidence to suggest that direct β-carotene supple mentation may be detrimental to human health under certain conditions. In the context of cardiovascular health, supplementation with β-carotene has no effect and even worsening effects on the likelihood of a major cardiac event in certain groups. In smokers, for example, β-carotene supplementa tion increased the likelihood of death or a cardiovascular event [125]. Some studies also suggest that β-carotene may increase the likelihood of lung cancer development in smokers [126]. However, the anticancer effects of β-carotene are well documented, believed to exert its effects through epigen etic modification [127, 128]. Thus, supplementation with β-carotene may not be suitable for specific cohorts, therefore ensuring that adequate dietary carotenoid levels are obtained from whole food sources such as tomatoes.
6.2.3.3 FLAVONOIDS
Flavonoids are a group of polyphenolic metabolites found primarily in edible fruits, vegetables, and plants [129]. Bioactive properties of flavo noids include antioxidant, anti-inflammatory, and anti-thrombotic effects [130]. Flavonoids can be divided into several groups such as flavanones, flavone, flavonols, isoflavones, and chalcones. These flavonoid subgroups differ based on their molecular structure, yet all exhibit similar bioactivities
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in the range of immunomodulatory, antioxidant, chemoprotective, and cardioprotective properties [131]. Dietary flavonoids come from a variety of plant-based sources, including red wine, tea, peppers, blueberries, and citrus fruits [132].
Several studies have identified the increased dietary intakes on flavo noids was correlated with decreased risk of mortality as a result of a cardiovascular event [133]. It has been suggested that the protective effects of flavonoids may be mediated through blocking the oxidation of LDL and improving vascular smooth muscle cell tolerance to oxidized-LDL [134]. Anti-hypertensive effects are also theorized to play a role in reducing CVD mortality [135, 136]. The ability of flavonoids to regulate oxidative metabo lisms are also postulated to play an integral role in the prevention of cancer [137]. Furthermore, several flavonoids play a direct role in the destruction of cancer cells, through promoting apoptosis [138].
Flavonoids have a long-documented role in the attenuation of chronic inflammation, which enables flavonoids to be potentially employed to treat the symptoms of inflammatory disorders [139, 140]. Apigenin is a flavone derived molecule found in abundance in fruit skins [132]. Murine derived bone marrow dendritic cells activated with LPS, exhibited a reduction in inflammatory cytokines and cell surface expression of the key co-stimu latory molecules CD80, CD86, and CD40 when treated with apigenin. In the same study, researchers using the mouse model of collagen-induced arthritis demonstrated that apigenin supplementation improved the clinical symptoms of CIA employing a similar mechanism of action that targeted inflammatory cytokines and co-stimulatory marker expression [141]. There is a lack of clinical studies examining the beneficial effects of apigenin in human disease, therefore there is no evidence that these in vitrostudies and in vivo findings translate to humans. However, there is promising data in a randomized control study examining the topical administration of apigenin rich chamomile oil in osteoarthritic subjects for three weeks who displayed reduced pain levels as measured by a reduction in the need for pain-relieving medication [142].
The anti-inflammatory properties of flavonoids also mean that they may have an application in promoting healthy ageing, by modulating the phenom enon of “inflammaging” which has been previously discussed. Flavonoids are known to modulate the expression and activity of NFκB, COX-2 and iNOS, therefore reducing inflammatory activity [132, 143]. Retrospective studies have demonstrated that increased dietary intake of flavonoids is associated with an increased quality of life as we age [144]. This is also
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supported mechanistically, as diets high in flavonoids are associated with a decreased circulating levels of c-reactive proteins, IL-6, IL-8, IL-18, and TNF-R2 [145]. Through modulating inflammation, there is a decreased like lihood of developing chronic diseases such as cancers, CVD, and metabolic disease as we age.
There has also been much discussion around using flavonoids to treat the clinical symptoms of diabetes. Several flavonoids have been found to drive improvements in glycemic control, in vitro and in vivo through several mechanisms of action, such as; promoting GLUT4 activity, boosting insulin secretion, downregulating inflammation, and oxidation pathways, and modulating circulating lipid profiles [146]. In a diabetic rat model, researchers demonstrated that ingestion of an apigenin analog was able to enhance glucose uptake and reduce circulating glucose concentrations [147]. However, as outlined in Al-Ishaq et al. [146], there is a wealth of in vivo data concerning the effects of flavonoids supplementation in rodent models of diabetes and metabolic disease, however, there is a gap in the literature concerning the effects in humans. Comprehensive studies are required to determine the efficacy of flavonoid supplementation in the case of T2DM.
.3 AMINO ACIDS
Amino acids are the fundamental building blocks of life, forming peptide bonds to create structural units that make up proteins. There are 20 amino acids that are incorporated into protein molecular structures during synthesis, despite many more been described, however only 9 amino acids are consid ered essential as they cannot be synthesized by the body [148]. Amino acids have an array of functions, both enabling cellular function and supporting cellular function through nutrition. With regards to important nutraceutical amino acids, studies have shown that L-glutamine and leucine amino acids have beneficial effects in diseases such as inflammatory bowel syndrome (IBS) and metabolic health.
6.3.1 L-GLUTAMINE
L-glutamine is one such example of a nutraceutical amino acid that plays a biological role beyond being a protein building block. Glutamine is the most abundant amino acid found in the human body. It is an L-α-amino acid, which is not one of the essential amino acids [149]. L-glutamine has many
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biological properties, which make it a potential health-boosting supplement. One such activity is the ability to modulate cellular and tissue integrity, which in the context of gut health has been widely explored. Tight junction proteins are essential in maintaining tissue integrity, and in the gut, sustained expression and activity of these proteins ensure that the gut lining remains selectively permeable, protecting the host from immunogenic microbiota and chronic bowel conditions [150]. In a study that encompassed several clinical symptoms of human IBS, researchers found that the expression of tight junc tion proteins, ZO-1, occludin, and claudin-1, in IBS presenting individuals were negatively regulated, increasing gut permeability [151]. IBS is very different to IBD in that both are chronic conditions that cause abdominal pains, cramping, and urgent bowel movements; however, IBS does not cause inflammation or destruction of the bowel wall, which leads to diseases such as CD and colitis [152]. Several in vitrostudies using the colonic cell line CaCO-2, have remarked that L-glutamine supplementation improves tight junction protein expression and function [149]. Similar effects on claudin-1 expression were observed in colonic explants derived from patients suffering from diarrhea dominant IBS [153]. L-glutamine supplementation improved human gut permeability and reduced disease severity [154]. This informa tion, combined with knowledge that L-glutamine supplementation was not associated with any side effects, suggests that L-glutamine supplementation may be a therapeutic strategy to improve gut layer integrity in disease states. L-glutamine displays potent immunomodulatory potential. During an immune response, active immune cells require a high output energy source. Studies have shown that glutamine and glucose are required in equal measure to promote the appropriate activity of both innate and adap tive immune cell function [155]. Appropriate endogenous glutamine stores are therefore essential to promote effective immune function. In healthy individuals, glutamine synthase inhibition or poor plasma concentrations may result in impaired immune function and resolution of inflammation, which can have detrimental effects over time [156, 157]. Similar to other nutraceuticals, L-glutamine was shown to skew macrophage phenotypes; however, in this context, it switched cells from an M2 to M1 phenotype through the inhibition of glutamine synthase. In the context of this study, this inhibition was beneficial as the M1 macrophage induced T-cell activa tion and reduced cancer metastasis [158]. There is evidence to suggest that L-glutamine supplementation during chemotherapy treatment may reduce the severe side effects associated with chemotherapeutic infusions [159]. Studies have documented that oral supplementation of an L-glutamine rich
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source improves chemotherapy side effects such as poor gut function, muco sitis, and weight loss [155].
The anti-inflammatory effect in the human gut was demonstrated in vitro using colonic explants. Researchers showed that pro-inflammatory cytokines were down regulated, following 2 weeks of supplementation [153]. Furthermore, dual supplementation with L-glutamine and L-alanine, reduced inflammation in rat skeletal muscle in response to injury, potentially protecting surrounding tissue from further damage [160]. Similar effects were also seen in an elderly cohort who undertook 30 days of glutamine supplementation combined with exercise. Compared to the control exercise group (no glutamine), those that exercised and ingested glutamine showed improved oxidative and inflammatory balance, suggesting an application to promote healthy aging [161].
6.3.2 LEUCINE
Leucine is a branched-chain amino acid and is one of the essential amino acids required by humans [148] that is of interest with regards to metabolic health in humans. Leucine is particularly important with regard to muscle metabolic, and subsequent locomotive functions. Leucine was identified to play an important role in modulating muscle protein synthesis in skeletal muscle. Leucine was identified to promote muscle protein synthesis through the activation and activity of mTOR and 4E-BP1 [162]. This was confirmed in a human exercise model, where subjects fed a leucine rich and carbohy drate mix supplement exhibited enhanced post-exercise activation of mTOR signaling [163]. In the context of human health, leucine supplementation was found to improve muscle strength and reduce baseline inflammation in patients diagnosed with cerebral palsy, improving quality of life [164]. In contrast, a study involving post-exercise supplementation with a leucine enriched whey protein source versus a standard whey protein source found that there was no difference in the ability of the enriched source to boost muscle protein post-exercise, despite an increase in plasma leucine concen trations. However, the leucine enriched whey protein source was associated with a more sustained activation of muscle protein synthesis pathways up to five hours post-exercise [165].
Leucine also plays a role in regulating glucose metabolism as it can modulate the activity of glucose transport proteins GLUT1 and GLUT4 that regulates insulin secretion and glucose intake pathways [166]. In a mouse
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model of obesity, ingestion of leucine improved the glycemic profile by improving insulin sensitivity and glucose tolerance [167]. The anabolic and glucoregulatory activity has made leucine a promising candidate for the treatment of Met-S such as sarcopenia. However, a long-term 3-month study conducted in older men, found that consumption of 7.5 g of leucine daily, had no significant impact on the regulation of muscle mass or glucoregulatory activity [168]. There is a lack of long-term studies concerning the effects of leucine on metabolic health and its suitability as a nutraceutical intervention, therefore more data and in-depth studies are required (Table 6.1) [169].
.4 PROTEIN AND BIOACTIVE PEPTIDES
Proteins are three-dimensional macromolecular structures that are one of the most fundamental building blocks of life. They are multifunctional mole cules, involved in every aspect of cellular function, including influencing gene expression, functioning as structural proteins, intercellular communica tion signals, and intracellular signal transducers. Proteins can be synthesized de novo, or essential amino acids are obtained from dietary sources. When consumed, whole-food proteins are digested, and peptide bonds hydrolyzed to form small peptide sequences or amino acids that are subsequently absorbed and used as the building blocks for endogenous protein synthesis. Deviation in protein structures because of protein gene mutations, errors in ribonucleic acid (RNA) translation, or protein misfolding in the endoplasmic reticulum can drastically affect how a protein functions, and is at the root of many acquired and inherited diseases.
Deficiency in dietary protein plays a considerable role in the development of human disease having major implications for human health. Consequently, the lack of protein and essential amino acid dietary intake can lead to a lowering of health quality including decreased strength, decreased muscle function, and immunodeficiency [177]. For example, in C57/Bl6 mice, a protein-deficient diet increased the susceptibility of infection by influenza, which could be the result of reduced immune function [178]. This observa tion was supported in a study in older women who consumed a proteindeficient diet exhibited decreased immune function compared to those who consumed a protein sufficient diet [179]. Conversely, excess intake of dietary protein can also have negative implications for human health as it is linked with the increased incidence of hepatic and renal disease [8].
TABLE 6.1 Summary of Key Beneficial Properties of Fatty Acid, Plant Derived Nutraceuticals and Amino Acids
Bioactive Food Source RDA (WHO) Health Boosting Properties In Vitro In Vivo Application to NCD
Molecule Treatment
Curcumin Turmeric -3 mg/kg of Anti-inflammatory [31] [36] CID
body weight Antioxidant [170] [172] Caner
Glucoregulatory [171] [56] T2DM
Chemoprotective [47] [173] CVD
Adjunct chemotherapeutic [48] [45]
Omega 3 PUFAs Salmon mg/day Cardioprotective n/a [72] CID
(EPA, DHA, Trout Combinations Anti-inflammatory [77] [89] Sarcopenia
ALA) Mackerel of EPA and Antioxidant [76] [92] CVD
Fish oils DHA, of which Pro-anabolic [90] [70] Cancer
should be Anti-depressive behavior n/a
predominately
DHA
Flavonoids Edible To be Antioxidant [134] [175] Cancer
fruits and determined Anti-inflammatory [141] [145] CID
veg Cardioprotective [134] [133] CVD
Glycemic control [174] [147] T2DM
Carotenoids: Tomatoes To be Antioxidant [108] [124] Cancer
Lycopene Tomato- determined Anti-inflammatory [105] [95] CID
B-Carotene based Cardioprotective n/a [109] CVD
products Chemopreventive n/a [113]
L-glutamine Dietary To be Immunomodulatory [151] [161] CID
Protein determined Tissue integrity [149] [154]
Leucine Dietary To be Pro-anabolism [91] [164] Sarcopenia
Protein determined Glucoregulatory [176] [167] T2DM
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Balanced protein intake, based on physical activity is critical to human health in the long-term. Currently, the recommended daily intake of dietary protein for healthy adults is 0.8 g/kg of body weight [6]. This increases to 1.2 g/kg to 2.0 g/kg for those undertaking frequent intense aerobic and/or resis tance exercise [180]. Bovine milk is one of the most protein dense nutrient sources available. There are two primary protein families in bovine milk, whey protein and casein protein, which account for 80% and 20% of the total milk protein content, respectively. When consumed, whey and casein proteins are hydrolyzed and broken down into peptides. It is believed that the subsequent release of these peptides, which are known to possess specific bioactivities, are the drivers of health boosting properties [181]. Although these bioactive peptide sequences can be released through the natural digestive process, there are questions as to whether they are sufficiently and frequently bioavailable to exert health boosting effects. Consequently, there is a worldwide push to mine protein-rich sources for bioactive healthboosting peptides using bioinformatic and laboratory-based approaches [182]. Mining protein-rich sources can produce two potential nutraceuticals; specific bioactive peptide sequences or hydrolysates, which are a mixture of several different bioactive peptides that work together to produce health benefits. Although bovine milk is highly protein-dense, only a small fraction of the milk protein is initially digestible. Whey protein is acid-soluble and is digested and absorbed rapidly by the body. Conversely, casein protein, which comprises a majority of the protein content of milk, is not acid-soluble and tend to coagulate in the stomach, reducing surface area digestibility. Due to this fact, it is believed that undigestible whey and casein components may harbor a wealth of bioactive peptide or hydrolysates that can beneficially modulate several aspects of human health [183, 184].
6.4.1 WHEY PROTEIN
Whey protein comprises 20% of the total protein content of milk and is a major by-product generated by the cheese making industry. Whey protein is comprised of a mixture of several proteins, peptides, and enzymes including; β-lactoglobulin, α-lactalbumin, bovine serum albumin, lactoferrin, immuno globulins, and lysozyme with other growth factors such as TGFβ, IGF-I, and IGF-II [185-187].
A number of formulations exist on the international market that supply bioactive peptides derived from whey proteins that positively
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affect cardiovascular and metabolic health in humans [188]. For example, NOP-47™ is a bioactive peptide derived from whey protein that is produced by Glanbia Nutritionals. Two in vivohuman studies, in both men and women concerning the use of NOP-47™, found that noticeable improvements in cardiovascular function in the groups that were fed NOP-47™ compared to the placebo groups [189, 190]. Whey protein hydrolysates also display angiotensin-converting enzyme (ACE) inhibitory properties, an important regulator of blood pressure. ACE converts Angiotensin I to Angiotensin II, which in turn exerts contractile effects on the vascular system, causing an increase in blood pressure which contributes to hypertension that subse quently has long-term implications on the cardiovascular system [191]. Several ACE inhibitory bioactive peptides have been isolated from the whey proteins α- and β-Lactoglobulin [192, 193]. BioZate®is a second whey
protein-based product that in humans has shown to positively regulate blood pressure through ACE inhibition [188, 194].
Consumption of whole whey protein has been shown to increase the whole insulin sensitivity in obese, healthy, and insulin-resistant subjects [195]. Furthermore, whey protein hydrolysates have exhibited the capacity to drive the translocation of the primary insulin-stimulated glucose transporter GLUT-4 to a greater degree, in a rat exercise model [196]. It is understood that whey protein possesses the ability to suppress appetite, which, by default, may aid in weight loss due to reduced calorie intake, subsequently improving obesity, which is a significant risk factor for cardiovascular and metabolic health [195, 197].
6.4.2 CASEIN PROTEIN
Casein is composed of four protein subunits (αs1-, αs2-, β-, and к-casein) constituting about 80% of the total protein content in bovine milk [181] and like whey, casein has been shown to display nutraceutical properties. Hydrolysates derived from these subunits have been extensively explored for their immunomodulatory and anti-inflammatory potential [181]. Several studies have cited the anti-proliferative effects of casein hydrolysates in lymphocytes [198]. A к-casein fragment has also been shown to induce an M2-like phenotype in macrophages and suppress LPS induced cytokine signaling by abrogating the NFκB signaling pathway. Researchers also demonstrated that к-casein treated macrophages and dendritic cells also displayed a reduced capacity to induce robust T lymphocyte responses [21].
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Furthermore, unspecified casein hydrolysates of varying sizes, have been shown to suppress the expression of inflammatory cytokines on the colonic CaCOcell line and ex vivocolonic explants [199, 200]. These findings
suggest that an immunomodulatory casein hydrolysates or peptides could modulate the gut microenvironment through direct interaction with immune cells and the gut epithelial cells to suppress inflammation and restore gut homeostasis, potentially improving IBD/IBS symptoms [201].
Casein hydrolysates may also be beneficial in boosting skeletal muscle anabolic signaling via amplification of mTOR signaling. In a human exercise trial in trained cyclists, researchers showed that ingestion of a casein hydro lysate in combination with a carbohydrate source was able to significantly enhance the activity of an mTOR signal transducer, called 4E Binding protein 1 (4E-BP1), compared to the whole protein [202]. In an inactive state, 4E-BP1 is bound to the translation inhibitor eukaryotic translation initiation factor 4E (eIF4e), once phosphorylated 4E-BP1 and eIF4e dissociate and 4E-BP1 moves to initiate translation [203]. This upregulation of 4E-BP1 is therefore corre lated with improved protein synthesis in skeletal muscle, helping to maintain and improve muscle mass and function. In the elderly, 4E-BP1 signaling is blunted and the anabolic capacity of muscle is down regulated, leading to syndromes such as sarcopenia [84]. As has been demonstrated with whey protein ingestion previously, combinatory interventions with resistance exer cise and hydrolysate consumption may improve skeletal muscle and overall health in the elderly, drastically improving the quality of life [204].
Whole Casein protein, however, has not had any notable effects on glucoregulatory pathways; however, several studies concerning casein isolated peptides have demonstrated that they are capable of inducing insulin sensitivity. In vitro, a casein-derived macro peptide, was capable of inducing insulin sensitivity in the human hepatic cancer cell line, HepG2 via AMPK amplification [205]. Similarly, in an in vivoanimal model, rats fed a high-fat diet exhibited superior glucose tolerance when administered a casein hydrolysate over the whole protein prior to exercise [206]. Given this preliminary evidence, further research into the glucoregulatory potential of casein hydrolysates is required.
6.4.3 PLANT-BASED PROTEIN
Despite animal products being the richest source of high-quality protein, plant-based protein is also a good source of quality protein. Although the
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pro-anabolic effects of plant protein are measurably less than animal protein, in terms of the whole food consumption, protein-rich plant foods, such a green leafy vegetable, often contain phytochemicals, and are rich in minerals and trace nutrients that have added health-boosting properties [207]. Never theless, several sources of plant-based proteins have exhibited promising health-boosting bioactivities. Vegetarians and vegans alike, despite social misconceptions, are capable of gathering enough dietary protein to suffi ciently support their bodies. Legumes such as chickpeas, beans, lupins, and lentils are a rich source of high-quality protein [208]. Peas, soya beans, and lupins also have comparable levels of amino acids, such as leucine, lysine, and isoleucine, histidine, and phenylalanine, to animal-based sources, such as eggs [209]. Furthermore, lupin protein hydrolysates have been shown to be ACE inhibitory, suggesting a potential application to treat hypertension in humans [210].
In particular, pea is a commonly occurring food on dinner tables across the world that is a good source of plant protein. Similar to cow’s milk proteins, after ingestion, the pea undergoes hydrolysis into amino acids and peptides, and this process is identical to all plant proteins. Peptides isolated from the yellow pea following enzymatic hydrolysis of whole protein yielded a mixture of peptides that we’re able to suppress M1 macrophage function. These peptides suppressed IL-6 and TNFα and reduced iNOS activity in vitro. In the same study, female BALB/c mice were administered oral supplements of these peptides enhancing the phagocytic activity of gut peri toneal macrophage, whilst also increasing the number of IgA, IL-4, IL-10, and IFNγ producing immune cells in the gut. This study suggests that pea hydrolysates may also act as anti-inflammatory, antioxidant chemoprotec tive ingredients, and an immune-boosting ingredient to protect the host from gut pathogens [211].
As previously mentioned, the anabolic capacity of plant protein is less efficient than animal-based counterparts. However, pro-anabolic protein sources can be found in plant-based foods, and may offer an avenue to boost the anabolic potential of plant-based foods. In a recent study, involving young healthy female subjects, individuals were fed an isolated potato protein and its ability to induce muscle protein synthesis was examined. The sample group who consumed the protein isolate twice daily for two weeks exhibited elevated levels of muscle protein synthesis both at rest and after exercise [212]. These findings are very important, as it may offer a way to improve muscle health and function over time, even while resting.
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Nuritas is an Irish-based company involved in mining different sources of food for functional bioactive peptides with health-boosting and pharma cological applications. Nuritas is unique as the company employs the use of a patented artificial intelligence (AI) model to identify peptides of interest. Touted as the world’s first bioactive hydrolysates identified by AI, PeptAIde contains peptides isolated from brown rice. The effects of PeptAIdewere examined in a kinetic study using healthy adults. This study demonstrated that PeptAIdewas indeed immunomodulatory, having the ability to decrease inflammatory cytokine and chemokine secretion. Although, levels of circu lating inflammatory markers returned to baseline after 24 hrs., it demon strated that a single 20 g dose was able to suppress whole body inflammations [213]. These studies demonstrate that several NCDs can be prevented using an anti-inflammatory peptide and that these new technologies can fast track the identification of novel peptides (Table 6.2).
TABLE 6.2 Summary of Key Beneficial Properties of Proteins and Bioactive Peptides
Protein Bioactive Health Boosting Mechanisms Application Refs.
Source Content Properties for NCD
Treatment
Bovine Whey protein Insulin sensitizing Enhancing T2DM [196]
whey hydrolysate GLUT4
function
NOP-47 Cardioprotective Modulating CVD [189]
hydrolysate vascular
endothelial cell
function
BioZate Anti-hypertensive ACE inhibitory CVD [194]
hydrolysate
Bovine к-casein Immunomodulatory Suppression of CID [21]
casein Subunit NFкB
Heterogenous Anti-inflammatory Suppression of IBD [199]
hydrolysates inflammatory
gene expression in colonic tissue
Glucoregulatory Improvements T2DM [206]
whole
Pre-diabetes body glucose tolerance
Pro-anabolic Enhanced Sarcopenia [202]
mTOR signaling
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TABLE 6.2 (Continued)
Protein Bioactive Health Boosting Mechanisms Application Refs.
Source Content Properties for NCD
Treatment
Yellow Hydrolyzed Immunomodulatory, Inhibition of CID [211]
pea yellow pea antioxidant M1 Macrophage
Chemo
protein function,
protective
improvements of pathogen/ monitoring immune
function in the gut
Potato Potato protein Pro-anabolic Enhanced Sarcopenia [212]
isolate muscle protein
synthesis post
exercise and at
rest
Brown PeptAIde Anti-inflammatory Acute CID [213]
rice (Nuritias) suppression of
inflammatory cytokines and chemokines in healthy subjects
.5 COMPOUNDS CURRENTLY IN CLINICAL TRIALS
The bioactivity associated with the nutraceuticals discussed in this chapter, provides a wealth of information concerning the potential application of nutraceutical interventions to chronic human disease. However, as also mentioned, the efficacy of some of these nutraceuticals remains to be fully elucidated, and thus, their translation into a clinical setting cannot be fully determined until they have been examined robustly under clinical trial condi tions. While there are many clinical trials currently underway to examine the benefits of nutraceuticals, this chapter will discuss a range of clinical trials to provide some insight into the activity that is ongoing in the clinical setting and the range of molecules that are currently being examined.
6.5.1 RESVERATROL
Plant-derived polyphenols and phytochemicals continue to be examined extensively for their clinical applications. There are several ongoing clinical
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trials investigating the potential application of resveratrol, a compound found in abundance in grapes. In vitroand in vivostudies have demonstrated the antioxidant, anti-inflammatory, and anti-tumorigenic properties of this compound [214]. The in vitroand in vivoanticancer properties of resve
ratrol are reviewed extensively in the following review chapter [215]. In brief, resveratrol is believed to be both a chemopreventative and a potential chemotherapeutic strategy in cancer as its potent antioxidant and free radical scavenging activity is believed to protect cells from oxidative damage and subsequent DNA damage that may result in carcinogenesis [216]. Furthermore, resveratrol was found to suppress the growth of neoblastoma xenographs in mice at serum concentrations of 2-10 µmol/L. Resveratrol treatment exhibited reduced tumor cell viability in vitro,[217] while in humans, resveratrol taken over a 10-year period reduced the likelihood of breast cancer development [218].
The antioxidant and anti-inflammatory properties of resveratrol are believed to exert protective effects in a variety of NCDs, including CVD, neurodegenerative disease, and metabolic disease [214, 219]. Several recruiting and ongoing clinical trials concerning resveratrol aim to uncover the potential application of the compound in chronic human disease states (Table 6.3). Resveratrol administration reduced disease severity in humans with mild to moderate Alzheimer’s disease [220], while a clinical trial (NCT03762096), examined the benefits of resveratrol supplementation in individuals that suffer from diabetes that carry an increased risk of devel oping CVD. Individuals who received resveratrol supplementation of 2 g/ day over six weeks, seemed to have improved cardiac function, cardiac metabolism, and immune function. A second study (NCT03525379) is examining whether a resveratrol supplement (500 mg per dose) taken twice daily for eight weeks has any improvement in blood flow, vascular function, and oxygen uptake in the skeletal muscle of patients with heart failure. The findings from this trial are yet to be published.
6.5.2 BIOACTIVE DIETARY POLYPHENOL PREPARATIONS (BDPP)
Bioactive dietary polyphenol preparations (BDPP) are also receiving much
attention as another nutraceutical therapeutic. It consists of a combination of grape-derived bioactive polyphenolic compounds, one of which is resvera trol, touted to have an array of bioactivities consistent with other polyphe nols. BDPP therapeutic strategy is to exploit this compound in order to treat
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metabolic and neurodegenerative disease. BDPP relieves pain in a rat model of intervertebral disc degeneration, suggesting a potential application as a strategy to treat chronic pain in humans [221]. Furthermore, it is believed that BDPP supplementation may delay the transition from mild cognitive impair ment to fully active Alzheimer’s disease [222, 223]. BDPP was found to improve brain synaptic brain function in mice, while also improving several aspects of metabolic disease, suggesting that Met-S may put individuals at risk of developing neurodegenerative disease [224].
Clinical translation to human disease models is lacking in the context of BDPP, however there are clinical trials currently recruiting to model the efficacy of BDPP in metabolic and neurodegenerative disease. In a phase 1 clinical trial (NCT02502253), researchers aim to answer several outstanding questions of BDPP supplementation in humans who display mild cogni tive impairment. Over a four-month period, participants will receive low, medium, and high doses of BDPP. Side effects of BDPP ingestion at the varying doses will be monitored, which will be a critical point to determine BDPPs suitability for human consumption. Furthermore, due to the effect of BDPP in mouse models of Alzheimer’s disease, several outputs of cognitive function will be monitored for any changes due to BDPP ingestion. Related to the first study, a second phase 1 study (NCT04421079) will examine the pharmacokinetics of BDPP ingestion. At low, medium, and high doses, researchers aim to assess the bioavailability of active BDPP metabolites over five weeks, specifically, dihydrocaffeic acid (DHCA). Researchers will subsequently assess whether there is any correlation between BDPP intake, DHCA bioavailability and blood serum concentrations of IL-6.
6.5.3 NUTRAFOL
Nutrafol is a commercially available nutraceutical that has been documented to improve hair growth in women [225]. One of the primary bioactive compounds in Nutrafol is a curcumin extract, of which bioactivity has been discussed in detail previously. The cosmetic benefits of hair growth extend beyond the surface and hair loss can often have negative psychological impacts in both men and women [226]. Clinical trials are currently ongoing with the Nutrafol product line and its application to promoting hair growth in menopausal and pre-menopausal women (NCT04048031). Participants in this study will receive either 4 x Nutrafol capsules daily or a placebo for a six-month period. During this time, researchers will monitor the effects of Nutrafol supplementation on hair growth and volume.
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6.5.4 KB220
Addiction is a growing pandemic in the 21st century, and given the lack of
effective treatments, any benefit that a nutraceutical could provide would be very novel. KB220 is a glutaminergic-dopaminergic compound that contains an array of amino acids, such as L-glutamine, L-phenylalanine, and tryptophan, in combination with several other compounds and trace minerals that act as dopamine and neurotransmitter precursors. It is believed that this nutraceutical cocktail can boost dopamine synthesis in the brain and balance the brain reward circuitry system, which is often imbalanced in cases of addiction [227, 228]. Potential applications of KB220 are believed to be helping to resolve alcohol, nicotine, and opioid addictions, of which widespread usages are linked with chronic diseases, such as CVD and cancer. Clinical trials are ongoing in an African American population with opioid addiction (NCT03861832) in the USA. Researchers hypothesize that there are genetic variants associated with low dopamine status, which causes imbalances within the brain reward circuitry system, resulting in increased incidences of addiction. This may be present in a higher frequency in African American population compared to European Americans. Researchers aim to both assess the genetic variations addressed above and whether administration with KB220 can improve the brain reward circuitry balance in opioid addicts to reduce dependencies and relapses in opioid use.
6.5.5 OMEGA 3-PUFAS
As discussed in detail previously, omega-3 PUFAs have a long history of health boosting properties in the context of several disease states, notably CVD. Despite the number of studies to date, many questions remain unan swered. One of which is whether or not genotype plays a role in how a person responds to omega-3 PUFAs. An active phase 1 clinical trial is attempting to answer whether or not the response of measurable risk factors for CVD and metabolic disease, such as blood pressure and glycemic status, are affected by the genetic variations in fatty acid sensor genes (NCT01343342). Over a six-week period, participants will ingest 1.9 g of EPA and 1.1 g of DHA in combination with 5 g of fish oils per day. Researchers will measure changes, if any, in blood pressure, serum lipid profiles and monitor changes in fatty acid sensor gene expression.
TABLE 6.3 Current Clinical Trials Examining the Benefits of Nutraceuticals
Compound Sources Bioactivity Clinical Trial Status Clinical Trials
Government ID
Resveratrol Grape skin Anticancer, Short interval resveratrol trial in cardiovascular Recruiting, active NCT03762096
Seeds Antioxidant, surgery
Neuroprotective
Anti-inflammatory
Cardioprotective
Evaluating the clinical efficacy of resveratrol Recruiting, active NCT03525379
improving metabolic and skeletal muscle
function in patients with heart failure
BDPP Grapeseed Anti-inflammatory BDPP treatment for mild cognitive impairment Recruiting, active NCT02502253
Antioxidant, (MCI) and prediabetes or Type 2 diabetes
Neuroprotective mellitus (T2DM)
Metabolism of bioactive dietary polyphenol Not yet recruiting, NCT04421079
preparation (BDPP) active
Nutrafol Phytochemical Anti-inflammatory, Efficacy and safety of a nutraceutical Recruiting, active NCT04048031
supplement antioxidant supplement with standardized botanicals in
(including peri-menopausal and menopausal women with
curcumin) thinning hair
KB220 Glutaminergic Neuroprotective SMART brain health in African-Americans Recruiting, active NCT03861832
dopaminergic Mood altering (SMART)
supplement Anti-addiction
Omega-3 EPA/DHA Cardioprotective Genes, omega-3 fatty acids and CVD risk Not recruiting, NCT01343342
fatty acids Cod liver oil factors (FAS) active
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.6 CONCLUSION AND FUTURE DIRECTIONS
This chapter highlighted the significant amount of data on commonly avail able nutraceuticals and the new generation of protein-derived nutraceuticals that are currently under development. It also provided evidence on the health protecting aspects of these nutraceuticals with regard to the prevention and treatment of NCDs that are prevalent in the 21stcentury [22]. The beneficial
properties of these nutraceuticals include anti-inflammatory [31], anti-oxidant [124], pro-anabolic [164, 212], liporegulatory, [72] and glucoregulatory [171] that have the potential to promote metabolic [91, 147], cardiovascular [72], and tissue health [154] when consumed in the right amounts. Further more, nutraceuticals such as curcumin, have shown synergistic effects with established chemotherapeutic strategies to treat cancer [44, 45]. Many of the nutraceuticals are found in abundance in superfoods such as milk, fish oils, tomatoes, berries, and dark chocolate, which are promoted as health boosting foods [229]. Despite the strong evidence of their beneficial proper ties, several important questions remain unanswered, such as whether there is a need for regulation of nutraceuticals to the same extent as drug products and the role of these nutraceuticals in the management of NCDs.
One concern is that the consumption of nutraceuticals is not regulated in the same regard as pharmaceutical products, thus potentially anyone can freely consume these concentrated products without medical oversight. There is strong evidence that over consumption of vitamins leads to hyper vitaminosis such as hypervitaminosis A that can lead to vitamin A toxicity, which caused symptoms such as changes to vision, bone pain, and skin changes. In extreme cases it can lead to liver damage and increased cranial pressure [230]. Although the adverse effects due to over consumption of nutraceuticals may not be as severe as drug therapy, they are still relevant, and the side effects observed can be considered mild to moderate. No clinical information is available that answers whether or not, supplementing a diet, with a given nutraceutical, has any long-term adverse effects if taken over a considerable length of time.
There are many over the counter food supplements that have health claims with no robust evidence to support these claims. However, the European Commission (EC) is working to address the issue of health claims associated with nutraceuticals. The EFSA is responsible for evaluating the scientific evidence supporting health claims ensuring that health claims provided are based on sound scientific evidence that can be easily understood by consumers. The suitability of certain nutraceuticals is also of concern, such
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as the increased risk of developing lung cancer with increased β-carotene consumption in smokers. Consequently, it may be necessary to regulate the sale and availability of concentrated nutraceuticals. There are no studies that have evaluated the prescribing of nutraceuticals by medical professionals to treat NCDs and if they are observing any beneficial effects. A recent clinical trial is currently recruiting volunteers that aims to address that question (NCT04161859). Researchers will aim to evaluate the clinical usage of several common nutraceutical compounds such as; Omega 3 PUFAs, resve ratrol, curcumin, and alpha-linolenic acid, to name but a few. Specifically, researchers will be monitoring the clinical usage of these nutraceuticals with regard to the treatment of CVD.
The use of nutraceuticals in the context of disease management is a very important issue that needs to be addressed fully. This will require strong clinical evidence with medical oversight or clear communication and educa tion from the manufacturer with regard to its use. In the same regard that athletes will periodize their training and nutrition over a calendar year based on competition schedule, a similar tactic could be employed with regard to prescribed usages of nutraceuticals. For example, rather than take Vitamin C supplements all year, periodizing consumption during periods of increased likelihood of contracting cold and influenza and decreasing supplemented consumption outside of this period. This is a strategy that several international athletes have employed when traveling abroad for competition concerning the use of probiotics, to reduce the likelihood of contracting GI infection abroad [231-233]. However, NCDs are very complex, and many can remain asymptomatic or exhibit vague symptoms until the disease enters the more chronic stages of disease. Thus, without constant health monitoring, this strategy or periodization may not be useful. Rather, ensuring that adequate dietary requirements and limits are adhered to through consumption of whole foods naturally containing these nutraceuticals may be a strategy for preventing the onset of chronic NCDs later in life.
Age is a major factor when it comes to the prevalence of NCD, and the use of nutraceuticals to prevent age-related diseases is the area where it can have the greatest impact. As we age, our metabolism slows, and our dietary calorie intake reduces [234], which makes it difficult to consume a diet that has optional nutrition. Supplementing food with nutraceuticals means that we can enhance the health benefits of food. As mentioned previ ously, “inflammaging” a phenomenon in which the basal immune state of an individual leans towards a more inflammatory state as the body ages [82]. This phenomenon over chronic low-grade inflammation appears to underly
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the development of several NCDs, including cancer, diabetes, and CVD [81, 235, 236]. The WHO estimates that by 2050, there will be over 2 billion people worldwide aged over 60, so it stands to reason that we may see an increase in NCD prevalence in the not-so-distant future. With this in mind, the beneficial effects of anti-inflammatory nutraceuticals are clear, in the potential to delay the onset and development of disease as we age. Future efforts at mining food sources for health-boosting bioactives should focus on the hunt for anti-inflammatory and antioxidant bioactives so we can support an ever-growing aging population.
KEYWORDS
• cardiovascular vascular disease • Crohn’s disease
• metabolic syndromes • nicotinic acid
• non-communicable diseases • rheumatoid arthritis • tumor necrosis factor
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neuroblastoma and mediates apoptosis by directly targeting mitochondria. Clin. Cancer Res., 13, 5162-5169. https://doi.org/10.1158/1078-0432.CCR-07-0347.
218. Levi, F., Pasche, C., Lucchini, F., Ghidoni, R., Ferraroni, M., & La Vecchia, C., (2005).
Resveratrol and breast cancer risk. Eur. J. Cancer Prev., 14, 139-142. https://doi. org/10.1097/00008469-200504000-00009.
219. Pasinetti, G. M., Wang, J., Ho, L., Zhao, W., & Dubner, L., (2015). Roles of resveratrol
and other grape-derived polyphenols in Alzheimer’s disease prevention and treatment. Biochim. Biophys. Acta-Mol. Basis Dis., 1852, 1202-1208. https://doi.org/10.1016/j. bbadis.2014.10.006.
220. Zhu, C. W., Grossman, H., Neugroschl, J., Parker, S., Burden, A., Luo, X., & Sano, M.,
(2018). A randomized, double-blind, placebo-controlled trial of resveratrol with glucose and malate (RGM) to slow the progression of Alzheimer’s disease: A pilot study. Alzheimer’s dement. Transl. Res. Clin. Interv., 4, 609-616. https://doi.org/10.1016/j. trci.2018.09.009.
221. Lai, A., Ho, L., Evashwick-Rogler, T. W., Watanabe, H., Salandra, J., Winkelstein, B. A.,
Laudier, D., et al., (2019). Erratum: Dietary polyphenols as a safe and novel intervention for modulating pain associated with intervertebral disc degeneration in an in-vivorat model (Plos One, 14, 10 (E0223435)). doi: 10.1371/journal.pone.0223435). PLoS One 14, 1-24. https://doi.org/10.1371/journal.pone.0225674.
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(Vitis viniferaL.) derived polyphenols against Alzheimer’s dementia: Treatment and mechanisms. Bull. Natl. Res. Cent., 43,108. https://doi.org/10.1186/s42269-019-0149-z.
223. Wang, J., Bi, W., Cheng, A., Freire, D., Vempati, P., Zhao, W., Gong, B., Janle, E. M.,
et al., (2014). Targeting multiple pathogenic mechanisms with polyphenols for the
treatment of Alzheimer’s disease-experimental approach and therapeutic implications. Front. Aging Neurosci., 6, 1-10. https://doi.org/10.3389/fnagi.2014.00042.
224. Wang, J., Tang, C., Ferruzzi, M. G., Gong, B., Song, B. J., Janle, E. M., Chen, T. Y.,
et al., (2013). Role of standardized grape polyphenol preparation as a novel treatment to improve synaptic plasticity through attenuation of features of metabolic syndrome in a mouse model. Mol. Nutr. Food Res., 57, 2091-2102. https://doi.org/10.1002/ mnfr.201300230.
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controlled study evaluating the safety and efficacy of a nutraceutical supplement for promoting hair growth in women with self-perceived thinning hair. J. Drugs Dermatology, 17, 558-565.
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P., & Jones, G., (2018). Vitamin D toxicity-a clinical perspective. Front. Endocrinol. (Lausanne). 9, 1-7. https://doi.org/10.3389/fendo.2018.00550.
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237. Lalor, R., (2019). Immunomodulatory Properties of Bovine Caseins on Innate Immune
Cells. Dublin City University.
CHAPTER 7
Bioactive Proteins and Peptides as Functional Foods
DEEPA THOMASand M. S. LATHA,3
1Research and Post Graduate Department of Chemistry,
Bishop Moore College, Mavelikara, Alappuzha, Kerala, India 2Department of Chemistry, Sree Narayana College, Chathannur, Kollam, Kerala, India
3Department of Chemistry, Sree Narayana College, Kollam, Kerala, India
ABSTRACT
The quality of food plays an important role in the prevention of disease. Functional food describes “food that can provide health benefits beyond basic nutrition.” Bioactive proteins and peptides from an important fortifying ingredient for functional food. Apart from providing nutri tional benefits, they resist invasion of disease, inhibit pathophysiological pathways, and suppress pathogenic molecular activity. They also exhibit antioxidant, antihypertensive, antimicrobial, antimutagenic, anti-inflam matory, immune, and cytomodulatory and mineral-binding property. Dairy products, eggs, fish, wheat, maize, soy, rice, and mushrooms are the major sources of these functional ingredients. Fortification with bioactive proteins and peptides enhances the desirable physiological and immu nological effects of the food system. Development of fully integrated bioprocesses for the large-scale manufacture and refinement of these significant biomolecules would accelerate their introduction to the main consumer markets. Therefore, effective measures are required to imple ment economically viable methods for the production of these functional foods on an industrial scale.
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.1 INTRODUCTION
Functional food is a concept defined as “foods that may provide health benefits beyond basic nutrition.” A functional food may be derived from nature, a food from which an element has been eliminated or added, or a food in which the formulation of one or more constituents has been altered, or a food wherein the bioavailability of one or more elements or some combina tion thereof has been altered. ‘Functional food science’ helps to foster the development of functional food. The essential strategies for the production of functional foods are fortification, enrichment, modification, and enhance ment through new feed composition, unique growing conditions, or genetic manipulation [1, 2]. Bioactive proteins and peptides form an essential ingre dient of functional foodstuffs. They not only provide nutritional benefits but also help to resist the development of disease, inhibit pathophysiological pathways, or suppress pathogenic molecular activity. They exhibit specific bioactivities such as improving nutrient intake, growth enhancement, enzyme inhibition, protection against pathogenic agents and immune system modu lation. They are capable of exhibiting local effects in the gastrointestinal (GI) system or having systemic effects following intestinal absorption and circulation. They display numerous functions, including antioxidant, antihy pertensive, antimicrobial, antimutagenic, antioxidative, anti-inflammatory, immune, and cytomodulatory and mineral binding activity. They are able to produce beneficial effects on major body systems such as digestive, immune, cardiovascular, nervous, and endocrine systems. Biologically active peptides may be generated from precursor proteins in the following ways: (a) diges tive enzymatic hydrolysis, (b) fermentation with proteolytic starter cultures, (c) proteolysis by microorganism or plant-derived enzymes. An arrangement of the above-mentioned methods has proven successful in many studies in generating short functional peptides [3].
.2 INGREDIENTS IN FUNCTIONAL FOOD
There are many methods for producing functional foods, such as food processing modification, genetic engineering, etc., which enables the food industry to produce new products with added market value. Probiotics, prebiotics, biogenic, and nutrients are the most important components which can be added to food.
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7.2.1 PROBIOTICS
They are living microorganisms such as lactobacilli and bifidobacteria that have several immune-enhancing effects on host health and these microbial supplements assistant naturally with the intestinal mucosa, build up the intes tinal microbial balance. Probiotic bacteria have been gradually introduced into a variety of items, including milk powders, yogurts, cheeses, ice cream, frozen dairy desserts, fermented vegetables, and meats because of their perceived health benefits. The survival and multiplication of probiotic microorganisms in the host significantly influence their advantages. In addition to the immu nological benefits and the prevention, defense, and elimination of pathogenic bacteria, probiotic bacteria are also associated with cancer treatment and are found to be beneficial in patients with elevated cholesterol rates [4].
7.2.2 PREBIOTICS
They are nondigestible food elements, such as dietary fibers and oligosaccha rides that are capable of stimulating the growth of beneficial intestinal bacteria in the colon, by offering growth enhancers and nutrients to probiotic bacteria. They are short-chain carbohydrates that may be fermented in the broad bowel and promote the production of significantly valuable probiotics [5].
7.2.3 NUTRIENTS
They include fatty acids, minerals, and vitamins that are specific and targeted action. A lot of these nutrients are used in foodstuffs such as cereals, beverages, dairy products, bakery items, etc., due to their significant role in preventing disease and wellness promotion [5].
7.2.4 BIOGENICS
These are biologically active molecules includes peptides, proteins, enzymes, carotenoids, phenolic acids, and flavonoids that benefit the host through direct immunostimulation, suppression of mutagenesis, peroxidation, tumorigen esis, or intestinal putrefaction. Such functional components operate biologi cally within the GI tract and are capable of modifying the gut microbiota, influencing endotoxin translocation and eventual immune activation, and
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promoting host nutrition [6, 7]. They are also related to the neutralization of reactive species that target cell molecules and in the prevention of several oxidative and nitrosative stress-related diseases such as cardiovascular diseases (CVD), cancer, hypertension, atherosclerosis, diabetes mellitus, and neurological disorders. To improve functional activities, they are included in various foods such as pasta, fish products, ice cream, yogurt, and cheese. Among biogenic, proteins, and peptides are important class of ingredients for functional foods. They activate physiological intrinsic behaviors which make them useful as therapeutic agents. Such bio functionalities can be exploited in therapeutic products and for immune-nutrition as well. They are obtainable from the most varied natural sources.
.3 BIOLOGICALLY ACTIVE PEPTIDES AND PROTEINS: FUNCTIONS
Bioactive proteins and peptides have the potential to arrest certain diseases. It is found that certain proteins and peptides extracted from foods such as milk, egg, meat, pulses, algae, and fungi help to delay the development of disease and inhibit the process of pathophysiology. They possess antidisease char acteristics such as: antihypertensive activity; antimicrobial activity; antican cerous and antitumorigenic activity; antiobesity activity; anti-inflammatory activity; mineral binding activity; immune- and cytomodulatory activity; and antioxidant activity which are depicted in Figure 7.1.
. Antihypertensive Activity: Hypertension is a medical disorder that
refers to chronic high blood pressure in the arteries and initiates substantial risk aspects for heart disease and stroke. Angiotensin I-enzyme (ACE), a dipeptidyl carboxypeptidase plays a pivotal role in the control of blood pressure. The switching of angiotensin-I, a decapeptide to angiotensin-II, an octapeptide is catalyzed by ACE. Inactivating ACE is known to be the first step of therapy to treat hypertension. Therefore, ACE-inhibitory components are benefited to reduce the blood pressure in hypertensive patients [7, 8]. In addi tion to promote improvements in the general lifestyle, attempts have been engendered to develop functional foods that possess elements that help to reduce blood pressure and maybe a supplement or alter native to the prescribed hypertension treatment. Bioactive proteins and peptides with ACE inhibitory and antihypertensive activity have been the subject of special attention. They are isolated from plants, animals, aquatic, or microbial sources [9].
Bioactive Proteins and Peptides as Functional Foods
FIGURE 7.1 Functions of bioactive proteins and peptides.
. Antimicrobial Activity: Antimicrobial peptides and proteins are
an integral part of living organisms in providing natural protec tion against foreign pathogenic substances. The great advantage of antimicrobial peptides produced from food proteins is that they are derived from harmless substances, so their safety can be expected for use in medicines and the food industry. They show a wide range of activity against a vast array of microorganisms, including Grampositive and Gram-negative bacteria, yeast, protozoa, and fungi. They protect the GI tract from invasive viruses and bacteria. They function meanderingly by promoting the development of advanta geous microorganisms in the gut or explicitly by performing an antimicrobial operation or neutralizing the attachment or penetration pathways of pathogenic substances. The antimicrobial peptides work by disintegrating the microorganism’s cell membrane. Many anti microbial peptides are cationic and have an alpha-helical structure. The cationic properties of peptides allow attachment with the anionic cell membrane, which is in the lipid-rich nature and the initiation of cell membrane lysis through three possible mechanisms includes the formation of toroidal pores, barrel stave formation and carpet formation. Numerous species of antimicrobial proteins and peptides have been reported from insects to plants and animals [10].
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. Anticancerous and Antitumorigenic Activity: Cancer is a chronic
disease which appears to be one of the world’s leading causes of human death. Bioactive peptides and proteins extracted from food have been clinically proven to be effective substitutes for cancer control. Research results showed that some of their attributes such as smaller sizes, better synthesis, specificity, modification facility, and better penetration into cell membranes make them suitable candidates for cancer management. Pharmaceutical research and development related to peptide anticancer is likely to attract considerable attention. A number of anticancer protein and peptides from natural sources such as milk, rice, corn, mushrooms, soybean, chickpea, and egg have been reported [11, 12]. The phosphate and calcium content of casein is responsible for the anticarcinogenic capacity of milk [11, 13-15]. The anticancer action of peptides derived from food is rooted in their structural features such as composition, length, sequence, overall charge, and hydrophobicity of amino acids. The positive charge and hydrophobicity are responsible for the amphipathic attachment of target cells to the membrane. Phosphatidylserine is possessed by the outer layer of cancer cells. It is a phospholipid possesses a negative charge and is responsible for electrostatic attraction between cancer cells and peptides. Improving the hydrophobicity of peptides has been demonstrated to be good in making them extra stable in the serum and boosting their anticancer efficacy. Hydrophobic amino acids such as proline, glycine, alanine, leucine, and one or more residues of arginine, serine, lysine, glutamic acid, threonine, and tyrosine are the prevailing amino acids of food protein-derived anticancer peptides [13, 16, 17].
. Antiobesity Activity: Research has shown that eating foods with
functional benefits as part of a balanced diet on a daily basis can help to minimize the risk or control a variety of health problems. Over the years, it has been observed that diet and physical activity remain the best and most efficient options for maintain body weight in overweight and obese individuals. Dietary maintenance can be accomplished by recognizing bioactive functional food ingredients that could be useful in modulating molecular pathways and functions of genes and proteins along with calorie restriction and exercise [18]. Moderate protein consumption displays a key role in reducing and maintaining the body weight. Studies have shown that functional peptides derived from food proteins play a significant role in the loss of body weight and regulation of lipid metabolism.
Bioactive Proteins and Peptides as Functional Foods
. Anti-Inflammatory Activity: The therapeutic uses of natural
compounds and their derivatives are becoming increasingly important as healthier alternatives to the currently available anti-inflammatory drugs. Because of their food sources and the perceived lack of severe side effects, bioactive peptides and proteins can potentially provide a safer alternative to conventional pharmaceuticals for inhibition and treatment of inflammation. Different studies have revealed that inflammatory markers include IL -1, IL -6, IL -8, TNF -α and CRP and various transcription elements such as STAT and NF -π B are critical components regulating inflammatory diseases. Studies show that bioactive peptides derived from food proteins show significant anti-inflammatory activity by suppressing or decreasing the expres sion of the inflammatory biomarkers and/or by reducing the function of those transcription factors [19].
. Mineral Binding Activity: Some minerals, like Zn, Fe, and Ca are
vital to life, and their deficiencies may result in a variety of health problems. Since bioactive peptides have mineral binding potential, they may be used as a functional ingredient to improve mineral availability and open up new food supplementation opportunities. Metals exist in a soluble form during complexation between metals and peptides that is readily accessible to the organism. The mineralbinding activity is correlated with the peptide molecular weight and amino acids in the peptide sequence. The peptide-metal bond is formed by the interaction between the electron donator group of the ligand surface (peptide) and electron receptor (metallic ion) with one or more available coordination sites. The complex making specificity is regulated by the spatial arrangement of the ligands and hence the sequence of peptide. The interactions can be altered by varying the amino acid residues sequences. Peptide surface charge also plays a crucial role for determining the metal complex stability [20].
. Immuno- and Cytomodulatory Activity: The immune system
performs an essential role in protecting the body from invading pathogens [21-24]. Immunomodulator is any material that can
control or adjust the functions of the immune response or of the immune system. There is various recombinant, artificial, and natural preparations as immunomodulators. Dietary strategies involving the intake of essential nutrients and promising functional foods are an efficient and successful technique for the modulation of the immune
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system. They provide a more convenient and cost-effective source of specific antibodies. Studies show that both immunosuppression and immunostimulation can indeed be essential in the prevention and control of various pathological states of the organism and that the biopeptides can act against inflammation and autoimmune diseases, prevent transplant refusal, and improve overall health. Cytomodu latory peptides obstruct the growth of cancer cells or promote the development of neonatal intestinal cells and immune cells.
. Antioxidant Activity: Antioxidants can prevent or impede oxida
tion by either inhibiting or inactivating the production of reactive oxygen species (ROS) in the metabolism. Antioxidants are therefore of great significance in the human diet because they can enable the body to minimize oxidative damage. Bioactive proteins and peptides from food sources have emerged as a new source of natural antioxidants. They act as dietary antioxidant supplements and as food preservatives. Their antioxidant activity may be attributed to the radical scavenging and chelation properties with metal ions and inhibition of lipid peroxidation. It has also been proposed that the peptide structure and its sequence of amino acids can affect its anti oxidant properties. Amino acids with aromatic residues strengthen the radical-scavenging abilities of peptide. An increase in peptide hydrophobicity is also believed to increase their lipid solubility and thus improve their antioxidant activity [25, 26].
.4 SOURCES OF BIOLOGICALLY ACTIVE PEPTIDES AND
PROTEINS
Bovine milk and dairy products are considered as the major source of foodderived bioactive proteins and peptides. However, bioactive peptides and proteins are also derived from other sources, including animals and plants. Bioactive proteins are specifically detected or observed in bovine milk, meat, eggs, and diverse fish species such as tuna, herring, sardine, and salmon and also in wheat, maize, rice, mushrooms, soy, pumpkin, and sorghum. Thus, it is known that peptides and proteins extracted from these sources exhibit an overwhelming capacity that can be used in feed and therapeutic sectors. The coming section discusses the major sources of bioactive proteins and peptides and is shown in Figure 7.2.
Bioactive Proteins and Peptides as Functional Foods
FIGURE 7.2 Sources of bioactive proteins and peptides.
7.4.1 ANIMAL PRODUCTS
Milk and dairy products are the main source of health protecting bioactive proteins and peptides. Mammalian milk contains over 60 unique enzymes
including digestive enzymes and antioxidant and antimicrobial enzymes which are essential in terms of milk stability and mammalian defense against pathogenic agents. Fermented dairy products play a functional role either through a direct probiotic effect (action of microorganisms) or through an indirect biogenic effect (action of microbial metabolites formed during the fermentation process). The health-promoting mechanisms of probiotic action are largely focused on the beneficial impact of cytokines and antimicrobial peptides on the immune response due to activation of natural immunity. Whey proteins have anticarcinogenic, immunostimulatory, health-promoting, and antimicrobial functions, which can limit fat accumulation and consequently increase insulin sensitivity [28]. Processes such as fermentation or cheese maturation induce the release of bioactive peptides during the manufacture of milk products. As a result, fermented dairy products have been contained in a wide range of these bioactive peptides. Milk-derived bioactive peptides
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have shown in vivoand in vitrohealth-promoting activities. The milk proteins have gained growing attention as ingredients of health-promoting functional foods which aimed at diet-related chronic diseases such as cardiovascular disease, type two diabetes, and obesity,
Milk protein is associated with reducing the risk of hypertension. Multiple ACE-inhibitory peptides have been found in fermented milk, cheese, and yogurt. Casein, the main milk protein, may generate multiple ACE inhibi tory peptides when hydrolyzed by trypsin in the intestinal tract. This has been a topic of increasing commercial interest with greater awareness and scientific credibility [30]. The presence of the VPP and IPP tripeptides in the milk that was fermented with L. helveticusand Saccharomyces cerevisiae has been identified since 1995. Numerous animal studies have shown that the use of fermented milk containing Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) results in a decrease of blood pressure. These studies are the basement for the production of hypotensive milk-drink products such as Ameal S™ (Calpis Company, Japan) and Evolus® (Valio, Finland) [31, 32].
Milk and dairy products are the main source of antimicrobial activated bioactive proteins and peptides. Fresh milk comprises a unique combination of antimicrobial activity. Many researchers have been paying much atten tion to antimicrobial peptides released from milk which are known to be non-toxic to mammalian cells as they are extracted from a benign origin. Fermented dairy products play a functional role either actively via contact with ingested microbes or passively via the action of microbial metabolites such as proteins, oligosaccharides, peptides, organic acids, and vitamins formed during the fermentation step. Thus, milk-derived antimicrobial peptides have been considered to possess an overwhelming capacity for use in medical industries and feed.
It is worth noting that proteins and peptides extracted from milk have immunoregulatory characteristics. In addition to its function as a growth factor and its antimicrobial activity, lactoferrin is found to exhibit various immunomodulatory effects. Lactoferrin is a very active protein obtained from milk to hinder microbial growth, and it has been suggested that this function is because of its potential to bind iron and get rid of microorgan isms. Bovine lactoferrin (bLF) works well against viral infections. Clinical and animal trials have shown the therapeutic effects of these bLF-containing products. bLF’s antimicrobial and antioxidant properties support its use as a preservative in foods and add in commercial foods include yogurt, milk-based beverages, nutritional supplements, skim milk, and pet foods. In addition, it is used in oral care products and cosmetics. The beneficial
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effect of pet food combined with LF on dermatitis has also been seen in dogs and cats. It is often used as a spray on the surface of raw beef carcasses to minimize microbial contamination and as a component of edible coatings.
K-casein has also been shown to be a class of milk proteins and a significant source to the use of antibacterial peptides in food preservation and health care. This peptide is shown to be inhibitory against bacterium and yeast and is therefore suggested for use in infant nutrition to activate new-born host defense mechanisms. Kappacin (genetic variant of K-casein) shows antimicrobial activity and is used for oral therapy as a pharmaceutical supplement. It has also been commercially available for use for dental care and is a suitable and safe food-grade bio preservative with high potential for use in the food industry.
Whey protein-derived peptides have demonstrated capability to bind calcium, iron, and zinc. Caseinophosphopeptides (CCPs) are bioactive peptides derived from tryptic casein digestion that can bind and solubilize metals such as Ca, Fe, Mg, Zn, Ba, Ni, Co, Cr, and Se. CCPs are known to be additives in functional foods and medicinal formulations and are used in confectionery products. CPPs benefit in reducing anemia, high blood pressure, and osteoporosis [27-29]. Iron peptide complexes are seen as an alternative to reducing iron fortification problems and are considered as an alternative for iron supplements [30, 31].
Milk-derived proteins and peptides with substantial nutritional and therapeutic benefits received growing interest as potential constituents of health-promoting functional foods aimed at diet-related chronic diseases such as obesity [39, 65]. The anti-inflammatory effects of the milk-derived proteins and peptides have been demonstrated on the basis of many in vitro and in vivostudies [40]. Bioactive peptides derived from milk are considered prominent candidates for numerous health-promoting functional foods aimed at the health of the heart, bone, and digestive system, as well as enhancing immune defense, mood, and stress control. The sour milk products CalpisTM and EvolusR, which contain antihypertensive tripeptides are available in the
market and have been clinically proven to reduce blood pressure in human studies.
For centuries, the egg has been known as a high-value source of food for humans, because it is a rich and healthy source of essential amino acids, proteins, minerals, and vitamins. In addition, egg has also found significant applications as additives in functional food preparations as well as in cosmeceutical and pharmaceutical products, due to their gel-forming, emulsifying, and bioactive properties. A number of these properties are
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associated with the respective protein and peptide components found therein. Additionally, eggs have properties that promote health; others are preventive in nature, and others have therapeutic potential. There is constant research into the potential use of particular egg yolk-derived antibodies in healthcare products and clinical medicine, and formulations are being tested against dental caries or gastritis and rotavirus infections [32, 33].
The plentiful essential protein lysozyme in hen’s white egg exerts antibacterial action that is used in semi-hard and hard cheeses to prevent the late blowing defect of Clostridia. It is also used in frozen foods to inhibit the growth of pathogens and in oral health care products to protect against periodontal bacteria and to avoid oral mucosal infections. Eggs are a cheap and low-calorie source of high-quality proteins and other beneficial nutrients. Egg is also abundant in ovalbumin and phosvitin that have multifunctional properties. The phosphoprotein, phosvitin has antimicrobial activity. Ovalbumin and ovotransferrin, derived from egg white, are important sources of ACE inhibitory peptides [45]. The ovalbumin-derived peptide displays antihypertensive activity in vivo. From multiple in vitroand in vivostudies, it is obvious that proteins and peptides extracted from eggs are a healthier option for preserving muscle mass and weight loss and can be considered as a natural nutraceutical [34, 35]. Egg yolk also contains bioactive peptides with anti-inflammatory activities [48-50].
Meat is a highly protein-rich food and contains amino acids, minerals, and vitamins. Some bioactive peptides have also been found to be generated during the processing of meat, such as fermentation and hydrolysis, so the production of these compounds and eventual enhancement in meat products may be advantageous to human health. Meat and its derivatives can also be considered functional foods in so far as they contain a range of bioactive proteins and peptides that are known to work. The meat industry must pursue various possibilities beyond traditional shows, including manipulating the formulation of raw and processed products by attempting to change fatty acid profiles or adding antioxidants to them [36, 37].
7.4.2 PLANT SOURCES
Plants are also potential sources of bioactive proteins and peptides that are produced primarily from peas, wheat, rice, soybeans, pumpkins, oats,
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hemp seeds, canola, and flaxseed and have functional properties. Such functional bioactive proteins and peptides have wide applications in human nutrition includes components in energy drinks, weight management, and sports nutrition products; nutritious sources for elderly people and immunecompromised patients. Protein hydrolysates from agricultural crops like rapeseed, sunflower, soy, barley, and wheat have been explored for their antioxidant property [52, 55-58].
Soybean is a potential source of bioactive proteins and peptides among plant sources. Soy proteins have a high nutritional value, excellent functional features, and relatively inexpensive. In addition to being an excellent source of dietary protein, they do have antihypertensive, anticholesterolemic, antioxidant, antiobesity, and anticancer activity. The precursor of most peptides is glycine and β-conglycinine, the essential soy proteins. Lunasin, a bioactive peptide derived from soy protein shows anticancer, antioxidant, immunomodulatory, anti-inflammatory, and cholesterol-reducing activities. It is used as a dietary supplement in capsules or powder and as an ingredient of soy drinks. Within soy protein, all of the essential amino acids found in animal protein are present. The in vivostudies in rats demonstrate the ACE-inhibitory and blood pressure lowering capacity of soy protein-derived biopeptides [38, 39]. Studies of dietary activity in animals and humans indi cate that proteins soybean play a vital role in loss and maintenance of body weight [60-66, 70]. Soybean is also a source of immunomodulatory peptides [68, 69].
Rice, wheat, maize, and millets are recognized worldwide as essential functional foods and provide good health benefit and health-promoting impact. Cereals are considered as a major source of ACE inhibitors. Bioac tive peptides extracted from rice may be produced by enzymatic hydrolysis from bran and endosperm has functional properties. These can serve as direct scavengers of various free radicals and have beneficial effects, including antihypertensive, immunomodulatory, and anti-inflammatory activities. Functional protein supplements derived from Thai rice have become increasingly common among people who are health-conscious, athletes, and elderly. Brown rice protein fractionation hydrolysate has the efficacy to function as a versatile food component in nutraceutical foods and beverage products that can offer good taste and health benefits [40-43]. Rice dreg hydrolysate inhibitory peptides have shown important antihypertensive action. In vivo studies revealed that kurosu, a product from unpolished rice shows antihypertensive activity [8].
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Studies show that the defatted wheat germ is an important source of protein that can be processed into value-added products like protein hydro lysates or bioactive peptides using suitable processing techniques. In a study, Zohreh et al. demonstrated the antioxidant, ACE-inhibitory, and antitumor activities of bioactive peptides derived from wheat germ protein hydroly sates [25]. In another study, Cian et al. reported the antioxidant and ACE inhibitory activity of wheat gluten hydrolysate peptides [44]. Durum wheat bran protein concentrate contains albumin and globulin proteins enriched in essential amino acids have good functional properties and are recommended in cereal-based foods such as pasta and bread as a fortification ingredient [45]. Bioactive peptides extracted from the rice display anti-inflammatory activities [42, 46].
Corn peptides actively prevent the generation of free radicals and are used as nutritional regulators and stabilizers in drinks, dairy products, and grains. It also acts as antioxidants for functional and medicinal nutritious foods. Corn peptides minimize subcutaneous fat and ingestion of corn peptides ensures weight loss. The experimental results showed that the food enriched with corn peptides generated the highest amount of heat, suggesting that corn peptides have high effect than other proteins on promoting the energy metabolism. Corn peptides are suitable diet for obese people and as a nutritional supplement for weight loss treatment [47].
Nutritional pulses are an important source of protein and have higher lysine, arginine, glutamic, and aspartic acid levels compared with cereals. In addition to their proven nutritional benefits, recent pulse intake has had preventive or therapeutic effects on chronic health conditions, such as CVD, diabetes, and cancer. The use of pulses is also associated with therapeutic or protective effects on health conditions such as overweight and obesity. The potential of pulse seed hydrolysates and BPs for cancer, inflammation, hypertension, cardiovascular disease, and high cholesterol is identified in various in vitro studies. Purified BPs can be used in functional foods as health-enhancing ingredients. Pulses flours are already used for enhancing the functionality and nutritional consistency of baked products and snacks. Pulse-based hydrolysates and bioactive peptides are suggested as suitable sources for the production of new protein-derived products [46, 48, 49].
Oat is a multifunctional crop considered superior to many other unfortified cereals in nutritional terms. Oats are widely used as whole grains, containing essential nutrients such as proteins, vitamins, unsaturated fatty acids, and minerals. Oat protein is high in quality and low in cost. Protein content in oats (11-15% of the grain) can be divided into four fractions. Water-soluble
Bioactive Proteins and Peptides as Functional Foods
albumins (1-12% of the total protein), salt water soluble globulins (80% of total protein), alcohol soluble prolamins or avenins (10 to 15% of total proteins) and acid or base soluble glutelins (5 to 66% of total proteins). Owing to the higher lysine content, which is the key limiting amino acid in cereals, protein contained in oat is considered to be nutritionally superior to that of wheat. With the growing demand for gluten-free foods, oat is seen as a good option for diversifying the diet of patients with celiac disease. Multiple experimental and clinical trials have shown that oat-based products intake can reduce serum cholesterol levels, decrease glucose absorption, and lower plasma insulin response. Such health benefits of oat have drawn the wide interest of scientists and the public. Proper use of oat in food applications can help to prevent cardiovascular disease, obesity, diabetes, and many other diet-related diseases. Oat-based porridge, oat flour, oat bread, biscuits, and cookies, flakes, and infant foods are receiving a lot of interest due to their high nutritional value. Oat proteins have been used in food products such as heatresistant chocolates, due to their emulsifying and viscous properties. Studies have reported using the oat bran as a fat replacement in meatballs. These oat-bran meatballs display high sensory acceptability. Several oats-based probiotic beverages such as Proviva, Yosa, Adavena M40 and Biovessina are launched in the market based on increased awareness of high nutritional value oats and increasing demand for healthy foods [50-54].
7.4.3 MARINE SOURCES
In recent times, the usage of aquatic food has increased globally, owing to a deeper insight of their health benefits and the good outlook of seafood among consumers. Marine organisms have evolved specific properties and bioactive compounds in contrast to terrestrial sources, owing to the large variety of their living environments. They are rich in beneficial nutri ents. Several bioactive proteins and peptides are developed from marine resources, namely fish, oysters, algae, squids, salmon, sea urchins, shrimps, snow crabs, and seahorses. The extraction of functional food ingredients, value-added nutraceuticals, and natural health products from marine sources has been well recognized in conjunction with health promotion, mitigation of infection risk, and cost savings in health care.
Fish protein hydrolysates (FPHs) have become notable over the years as the major source of protein hydrolysates and bioactive peptides. Large variety food formulations may use the various properties of FPH such as
Advances in Nutraceuticals and Functional Foods
good water-holding capability, solubility, emulsion capacity, foaming potential, heat tolerance ability, and gelling potential. They can be used in a wide range of products as stabilizing and emulsifying additives and can help to shape and stabilize foam-based products, including mayonnaise, salad dressings, sausages, beverages, creams, etc. FPHs are also said to have efficacy for pharmaceutical applications. Studies have shown that peptides derived from FPHs display antioxidants, anti-proliferation, antihyperten sion, anti-inflammatory, and antidiabetic efficacy. Fish is also a great source of anticancer peptides [86, 89]. Furthermore, the separation of effective anti cancer components from fish tissue has made the argument for considering fish by-products as sources of chemo-preventive and anticancer components. The nutritional benefit of FPH makes it suitable food that can promote the growth and survival of aquatic life. Due to their efficacy in the prevention and treatment of hypertension, fish derived bioactive peptides have potential as nutraceuticals and pharmaceuticals and are commercially available as a dietary supplement under the brand names of VasotensinR, PeptACER, and
LevenormTM. Fish proteins are also displayed anti-inflammatory effects in
vitro as well as in animal studies [91]. In addition, the food items fortified with Omega-3 oil offer a way of achieving the desired biochemical effects of these nutrients without consumption of nutritional supplements, medica tions, or a significant shift in dietary habits [55-57].
Seaweeds with antibacterial, antiviral, and antifungal properties are known for their abundance in polysaccharides, minerals, and other vita mins, proteins, lipids, and polyphenols. This gives great potential to marine algae as a substitute in functional food. Multiple marine organisms generate biologically active proteins with antimicrobial, anticancer, anticoagulant, hypocholesterolemic, and immunostimulatory activities. Bioactive peptides derived from spirulina, the cyanobacterium (blue-green algae), shows antitumor activity. Most species of seaweed possess all the essential amino acids and are a significant source of acidic amino acids such as glutamic acid and aspartic acid. Numerous studies demonstrated the in vivothera peutical potentials of red, green, and brown seaweeds. Bioactive lectins, carbohydrate-binding proteins of non-immune origin with antibacterial, anti-inflammatory, and anticancer activities are found in macroalgal species. Red seaweed-derived biliproteins are used as fluorescent markers. Phyco biliproteins displayed antioxidant properties that are helpful for the preven tion and treatment of neurological disorders, tumors, and stomach ulcers. As examined by in vitroand in vivoassays, the sulfated heteropolysaccharide compounds, fucoidan found in seaweeds, are able to inhibit the growth of
Bioactive Proteins and Peptides as Functional Foods
different cell lines. Food products supplemented with seaweeds and extracts of seaweed showed beneficial effects on the numerous lifestyle diseases such as obesity, diabetes, and hypertension [23, 94, 99, 100]. Studies on marine organisms such as seaweed and algae revealed their anti-inflammatory effect. Spirulina is a good source of phycobiliprotein that is examined for its anti-inflammatory properties.
The in vitroand in vivostudies performed in acetates Chinensis, a marine shrimp, usually used as a flavoring agent in shrimp sauce demon strated antihypertensive activity. Many marine species, including mollusks and crustaceans, display calcium-binding, antimicrobial, and appetite suppressing activities, thereby encouraging human wellbeing, and avoiding chronic illness. Shrimp-derived peptides have a major effect on cholecys tokinin, a hormone that controls appetite and gastric emptying. Specific foods containing these peptides have the potential to regulate the disorders associated with appetite. For their antioxidant and radical scavenging properties, seaweeds such as alginate, carrageenan, and agar are widely used in food. They have also been introduced to many products, including salad dressings, drinks, and baked goods to boost their protein content, or sold as protein supplements. Given their many possible health benefits, food products, supplements or natural health products that contain marine bioactive are expected to dominate a huge market. CollactiveTM, a marine source of collagen and elastin, may be used as an anti-wrinkle ingredient, and NutripeptinTM, another marine bioactive compound, has been found to be effective in enhancing satiety and weight loss response as examples of commercially available marine-food items. Foods containing FPHs/peptides are believed to be appropriate for consumption by people with mild hyper tension. Examples of two such products include Lapis SupportTM(beverage) and Valtyron®(additive for soft drinks, jelly, powdered soup, dietary supple
ments, etc.), [58-62].
7.4.4 FUNGI
Because of their special properties and nutrient content, fungi have already known applications in the medical and food industry. Mushrooms are a distinct category of edible macrofungi capable of providing good taste and nutritional value with high protein content and low fat. They are known as an alternate source of protein of good quality and are capable of providing the maximum protein amount. Moreover, it contains biologically active
Advances in Nutraceuticals and Functional Foods
compounds having antifungal, anti-inflammatory, antitumor, antiviral, antibacterial, hepatoprotective, antidiabetic, hypolipidemic, antithrombotic, antihypertensive, immunomodulatory, and hypocholesterolemic properties. The protein in the mushrooms comprises the nine essential amino acids that humans require and is particularly rich in lysine and leucine that are deficient in most staple cereal food. In addition, several mushroom proteins demon strate significant pH and thermal stability. ‘Mushroom nutraceuticals’ are the conventional preparations used from older days in the form of extracts, health tonics, fermented drinks, and soups. Due to their high nutritional value, edible items can be fortified with mushrooms, and such food serves as a reservoir of nutrients for undernourished populations. Canned mushrooms are commercialized and used to make soup and pizza. Powdered mushroom has been added to food items such as noodles, pasta, rice porridge, and bakery items. Studies reveal that the high protein content of mushroom help to build a better gluten network and provide better elasticity in bakery products noodles and pasta. The addition of mushrooms also enhances its antioxidant content. Thus, mushroom fortification leads to improve nutritional values, physical properties, and food quality [62-69].
.5 CONCLUSION
Food-derived proteins and peptides have gained significant attention as chronic disease prevention agents because of their exceptional multifunc tional properties related to the maintenance of general health. Such proteins and peptides are a profoundly fascinating commodity in health-promoting foods for future use as active ingredients. They possess multifunctional activities such as antioxidant, antihypertensive, antimicrobial, antimutagenic, antioxidative, anti-inflammatory, immune, and cytomodulatory and mineral binding activity. Depending on their activities, they may be sold as nutraceu tical products or functional ingredients. Fortification with bioactive proteins and peptides leads to beneficial effects in food systems in terms of health implications and functionality. The creation of fully integrated bioprocesses which can be transferred to large-scale operations for the production and purification of these essential biomolecules would help to accelerate their categorization in the major consumer markets. In recent years, the market for functional ingredients and foods has grown astonishingly because of the awareness of consumers and the interest in promoting healthy diets and their lifestyles. It is possible to successfully integrate natural ingredients, such
Bioactive Proteins and Peptides as Functional Foods
as bioactive proteins and peptides, into foods, creating new natural product categories and new business opportunities. In addition, steps need to be taken to incorporate effective and economically viable development methods on an industrial scale.
KEYWORDS
• angiotensin I-enzyme • caseinophosphopeptides • fish protein hydrolysates • functional foods
• Ile-Pro-Pro
• peptides • proteins • Pro-Val-Pro
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65. Mudgil, P., Kamal, H., Yuen, G. C., & Maqsood, S. (2018). Characterization and
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71. Selamassakul, O., Laohakunjit, N., Kerdchoechuen, O., Yang, L., & Maier, C. S., (2020).
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72. Taniguchi, M., & Ochiai, A., (2017). Characterization and production of multifunctional
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74. Alzuwaid, N. T., Sissons, M., Laddomada, B., & Fellows, C. M., (2020). Nutritional and
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75. Lَpez-Barrios, L., Gutiérrez-Uribe, J. A., & Serna-Saldيvar, S. O., (2014). Bioactive peptides and hydrolysates from pulses and their potential use as functional ingredients. Journal of Food Science, 79(3). https://doi.org/10.1111/1750-3841.12365. 76. Li, G., Liu, W., Wang, Y., Jia, F., Wang, Y., Ma, Y., & Lu, J., (2019). Functions and applications of bioactive peptides from corn gluten meal. In: Advances in Food and Nutrition Research(Vol. 87, pp. 1-41). https://doi.org/10.1016/bs.afnr.2018.07.001.
77. Bessada, S. M. F., Barreira, J. C. M., & Oliveira, M. B. P. P., (2019). Pulses and food
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weight management. Journal of Functional Foods, 38, 612-623. https://doi.org/10.1016/j. jff.2017.03.044.
79. Angelov, A., Yaneva-Marinova, T., & Gotcheva, V., (2018). Oats as a matrix of choice
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80. Bekers, M., Marauska, M., Laukevics, J., Grube, M., Vigants, A., Karklina, D., & Viesturs,
U., (2001). Oats and fat-free milk based functional food product. Food Biotechnology, 15(1), 1-12. https://doi.org/10.1081/FBT-100103890.
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advantages of oats and opportunities for its processing as value-added foods: A review. Journal of Food Science and Technology, 52, 662-675. https://doi.org/10.1007/
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82. Sang, S., & Chu, Y. F., (2017). Whole grain oats, more than just a fiber: Role of unique
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83. Zheng, Z., Li, J., & Liu, Y., (2020). Effects of partial hydrolysis on the structural,
functional, and antioxidant properties of oat protein isolate. Food & Function, 11(4), 3144-3155. https://doi.org/10.1039/c9fo01783f.
84. Calanche, J., Beltrلn, H., Marquina, P., Roncalés, P., & Beltrلn, J. A., (2019). Eating fish
in another way: Development of functional pasta with added concentrates of farmed sea
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85. Tahergorabi, R., Beamer, S. K., Matak, K. E., & Jaczynski, J., (2012). Functional
food products made from fish protein isolate recovered with isoelectric solubilization/ precipitation. LWT Food Science and Technology, 48(1), 89-95. https://doi.org/10.1016/j. lwt.2012.02.018.
86. Correia-da-Silva, M., Sousa, E., Pinto, M., & Kijjoa, A. (2017). Anticancer and cancer
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CHAPTER 8
News and Trends in the Development of Functional Foods: Probiotic Dairy and Non-Dairy Products
ELIANE MAURحCIO FURTADO MARTINS,
WELLINGTA CRISTINA ALMEIDA DO NASCIMENTO BENEVENUTO,
AURةLIA DORNELAS DE OLIVEIRA MARTINS,
AUGUSTO ALOحSIO BENEVENUTO JUNIOR,
ISABELA CAMPELO DE QUEIROZ,THAINء DE MELO CARLOS DIAS,
DANIELA APARECIDA FERREIRA SOUZA,
DANIELE DE ALMEIDA PAULA,and MAURحLIO LOPES MARTINS
1Federal Institute of Southeast of Minas Gerais, Food Science and Technology Department (DCTA/IF Sudeste MG), Rio Pomba, MG, CEP - 36180-000, Brazil
2Federal University of Viçosa, Food Technology Department, Viçosa,
MG, CEP - 36570-000, Brazil
ABSTRACT
In recent years, there has been an increase in consumer search for healthy eating that promotes health and well-being. This demand has led to the creation of new market niches, consisting of foods with functional appeal. In this context, probiotics stand out, which are live microorganisms that, if consumed in adequate doses, confer health benefits. Lactobacillusand Bifi dobacteriumgenera are more widespread in the market and more used in food industry. With the understanding of consumers about the benefits promoted by functional foods containing probiotics, its use expanded rapidly, with the dairy matrix being the most studied for the transport of these microorgan isms. Dairy products such as cheese, fermented milk, yogurt, ice cream, infant formula and powdered milk are examples of these products. However,
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there is a demand for non-dairy products as a matrix of probiotic bacteria, in order to meet the portion of the population with lactose intolerance and allergy to dairy products, in addition to hypercholesterolemic, vegetarian, and those who do not consume dairy products due to habits cultural. Therefore, non-dairy foods such as fruits and vegetables, chocolates, baked, and meat products are also being studied and gaining notoriety as an alternative matrix to the incorporation of probiotics. The maintenance of these microorganisms during the production process, the product’s useful life and during the passage through the human GIT is a challenge for the industries. Several intrinsic and extrinsic factors can affect the viability of probiotic cells. Thus, tech nological alternatives such as microencapsulation have gained prominence for protecting cells from adverse conditions and, consequently, increasing the viability of microorganisms. The incorporation of probiotics in dairy and non dairy foods points to a promising future, arousing the interest of researchers and industry in the development of new healthy products.
.1 INTRODUCTION
In recent years, there has been an increase in the search by consumers for a healthy diet that promotes health and well-being, less processed, without preservatives and that helps in the prevention of chronic non-transmissible diseases. To meet this demand, researchers and entrepreneurs have been looking for alternatives for the development of differentiated food products, which contributes to the creation of new market niches, consisting of foods with functional appeal.
This scenario is in line with functional and nutraceutical foods. Functional foods are those that have basic nutritional functions in addition to producing beneficial health effects, and nutraceuticals are bioactive compounds present in foods that play an important role in food, and can be consumed in an isolated form from food, in high doses, such as supplements.
In the scope of functional foods, probiotics stand out, which are live microorganisms that, if consumed in adequate doses, are beneficial to health [39, 54]. There are many benefits attributed to the consumption of probiotic bacteria such as regulation of the intestinal microbiota, reduction of intes tinal pathogens, immunostimulation, increased bioavailability of nutrients, elimination of carcinogenic substances, reduction of the incidence of colon tumors, among others.
In order to guarantee consumer health benefits, it is extremely important that as many viable cells as possible are present at the time of ingestion.
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According to the international literature, at least 10-10CFU/g of probiotic
microorganisms should be ingested daily [68].
The probiotic microorganisms most used in food and which are present on the market are those belonging to the genera Lactobacillusand Bifidobac terium[29, 83]. These two genera are predominant inhabitants of the human intestine, with Lactobacillusin the small intestine and Bifidobacterium, in the large intestine [135].
Fermented dairy products comprise the highest percentage of probiotic carrying foods available on supermarket shelves, which are considered good matrices for these microorganisms. However, with the understanding of consumers about the benefits promoted by functional foods containing probiotics, there is a growing demand for other carriers such as ice cream, infant formula, and milk powder. In addition to dairy products, non-dairy foods such as fruits and vegetables products, chocolates, bakery, and meat products have also been studied and gaining notoriety.
.2 PROBIOTIC IN DAIRY PRODUCTS
The most studied matrix for carrying probiotic microorganisms is milk and dairy products [121] and a variety of dairy products have been formulated with the addition of different probiotic bacteria, according to Table 8.1.
TABLE 8.1 Probiotic Bacteria Species and Strains in Dairy Products
Probiotic Products References
Lactobacillus reuteri Infant formula [122]
Lactobacillus casei Zhang Minas frescal cheese [28]
Lactobacillus plantarum Yogurt [143]
Lactobacillus casei Yogurt [15, 24, 33]
Lactobacillus plantarum Milk fermented [92]
Lactobacillus acidophilus Milk fermented [10]
Lactobacillus casei Milk fermented [34, 92, 136]
Lactobacillus rhamnosus GG Pasta filata cheese [27]
Lactobacillus plantarum Feta cheese [103]
Bifidobacterium lactisBB-12 Yogurt [109]
Bifidobacterium animalis subsp. lactis BB-12 Ice cream sweetened [59]
with various polyols
Weissella cibaria D30 and Lactobacillus Cottage cheese [62]
rhamnosus GG
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TABLE 8.1 (Continued)
Probiotic Products References
Lactobacillus bulgaricus Ice cream (milk [90]
powder)
Lactobacillus rhamnosus Cream cheese [94]
Lactobacillus caseiDG, L. paracaseiF19, Mozzarella” and [113]
Lactobacillus paracaseiB21060, Lactobacillus. “scamorza
rhamnosusGG, Lactobacillus rhamnosus
IMC 501 plus; Lactobacillus paracasei ssp.
paracaseiIMC 502
Bacillus coagulansMTCC 5856, Bacillus “Requeijمo cremoso” [125]
coagulansGBI-30 6086, Bacillus subtilisPXN processed cheese
, Bacillus subtilisPB6 and B. flexusHK1
Lactobacillus casei-01 Prato cheese [139]
Lactobacillus acidophilus Yogurt [32, 89]
Lactobacillus rhamnosus B 442 Ice cream (milk with [73, 102]
.2% fat content)
Lactobacillus acidophilus Ice cream [2]
Lactobacillus rhamnosus HN001and Goat milk ice cream [30]
Lactobacillus paracasei LBC82 produced with cajل pulp
Bifidobacterium animalisssp. lactis Yogurt [32]
Lactobacillus rhamnosus Milk fermented [100]
Lactobacillus acidophilusLa-05 Buffalo milk ricotta [119]
cheese
Lactobacillus rhamnosusGG Infant formula [120]
Because to the benefits of probiotic strains to promote health, their use has expanded rapidly in products [143]. In addition to probiotic bacteria provide health benefits to consumers, their use in foods provides different patterns of flavor and texture; each mixture of microorganisms used can result in a specific product [79].
Fermented dairy products have bioactive compounds and metabolites derived from lactic acid bacteria (LAB) produced during fermentation. Due to their special characteristics, fermented dairy products are an excellent matrix for the incorporation of ingredients and/or nutrients that give the final product essential nutritional properties to the human being interested in a healthy diet [46].
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8.2.1 CHEESE
The dairy industries have sought to diversify the product offering in order to meet the demands of the consumer market. For this purpose, the development of cheeses containing probiotic bacteria has shown to be a promising alternative. The viability of probiotic microorganisms, as well as other microorgan isms, is affected by factors intrinsic to food such as pH, oxygen availability, water activity, nutrient availability, by processing conditions such as bino mial applied time and temperature, period, and storage conditions, making it a challenge to maintain adequate doses and to guarantee the functionality of probiotic products [113].
In this context, considering the intrinsic characteristics, cheeses have shown to be a very promising alternative for carrying probiotic microor ganisms, which even stand out over yogurt and fermented milks, such as higher pH values, buffering capacity, solid matrix, which guarantees greater protection during the passage to the gastrointestinal (GI) tract and higher fat content [26, 61].
Figure 8.1 is displayed the characteristics that contribute to preserve the viability of the probiotic in cheeses.
FIGURE 8.1 Characteristics of probiotics cheese.
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For the production of probiotic cheeses, two alternatives can basically be adopted: promoting microbial growth after manufacture, during the maturation of the products, or promoting the maintenance of the inoculated microbial load in high concentration. In addition, it is necessary to evaluate, in addition to the final microbial load, which allows the product to be classi fied as a functional food, the changes caused in the cheeses, which define its acceptability by the consumer [50].
Regarding the form of inoculation, probiotic bacteria can be used as starter cultures or in conjunction with traditional starter cultures, in various combinations of starters and probiotics [99].
Some varieties of cheese, such as ricotta, have characteristics such as high humidity and pH, in addition to reduced salt concentration, which can provide protection to probiotic microorganisms during GI transit. However, their texture and acceptability characteristics may be negatively affected by the cultures. Added probiotics, making it necessary to study alternatives that allow combining the benefits conferred by the matrix and the maintenance of the sensory characteristics essential for the acceptance of cheese by the consumer [119].
Studies seek to evaluate the factors that most affect the development of probiotic cultures added in cheese production.
Reale et al. [119] studied the possibility of large-scale production of two much-appreciated Italian cheese specialties, mozzarella, and scamorza, incorporating commercial probiotic additives by direct inoculum into the vat according to the factory’s standard procedure, and found that the filler step reduces the viability of these microorganisms, but that there is a possibility of their recovery during maturation for a minimum period of 30 days. They confirmed that an easy procedure such as direct-to-vat inoculation of lyophilized adjunct probiotic cultures at a concentration of 10CFU/mL could be a proficient method for the production of func
tional scamorza, guaranteeing with a daily consumption of 100 g of this scamorza cheese a quantity of probiotic bacteria equal to 10CFU/100 g.
Furthermore, the probiotic adjunct did not modify the sensory features of the final product.
Cuffia et al. [26] evaluated the use of two probiotic, Lactobacillus rhamnosusGG and Lactobacillus acidophilusLA5 (either individually or together) to make pasta filata soft cheeses and assessed the effect of the storage temperature (4 and 12°C) on the pH evolution, the viability of both probiotic strains, and the influence on the sensory characteristics of the product. The study showed the storage temperature of probiotic cheeses can
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play a critical role, and this parameter must be considered to maintain cheese quality. The probiotic cheeses maintained at 4°C did not show significant differences in pH, presented a very good overall quality, and maintained the viability of both probiotics at levels higher than 7 log CFU/g after 29 days of storage while Cheeses stored at 12°C, presented lower pH values, significant post acidification with both probiotic strains toward the end of the ripening period, an increase in bitter, acid taste, granularity, and a decrease in overall quality of cheeses.
As shown in Table 8.1, studies with the addition of probiotic bacteria, of different strains and in different types of cheese have been successfully conducted in several countries.
Lactobacillus casei-01 counts in the Prato cheese samples were higher than 8 log CFU/g in a study conducted for Vasconcelos et al. [139], and the addition of the probiotic culture did not influence the starter culture counts, with similar Lactococcus lactiscounts in conventional and probiotic cheeses. The search results showed that probiotic Prato cheese attenuates cigarette smoke-induced injuries in mice.
Prezz et al. [112] also evaluated the inoculation of Lactobacillus rham nosusas a probiotic culture in Minas Frescal without negative effect on physicochemical characteristics evaluated.
In research carried out in Brazil [126], with Requeijمo cremoso, the possibility of using five probiotic strains of the genus Bacilluswas evalu ated, which were inoculated at different stages of manufacture of this melted cheese. The main challenge for the use of probiotic cultures in this type of cheese is heating to 90°C by which the product is submitted during its manu facture. This high temperature causes the elimination of probiotic bacteria from the genera Lactobacillus and Bifidobacterium.
The results obtained in this study were very interesting, indicating that the evaluated strains supported the processing conditions of the Requeijمo cremoso, without generating a negative impact on the proteolytic character istics and on the fatty acid profile of the product.
In addition to nutritional interest, probiotic microorganisms can be added to cheeses for the purpose of bio-preservation, improving the micro biological safety of products by inhibiting the development of undesirable microorganisms.
Moghanjougi et al. [86] evaluated the possibility of using Bifidobacterium animalissubsp. lactisBb-12 and L. acidophilus La-5 encapsulated to produce antifungal agents, seeking out increasing the shelf life of white cheese Feta and demonstrating significantly less results of yeasts during storage.
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In Minas Frescal cheese, the addition of Lactobacillus rhamnosus reduced the Listeria monocytogenescount from the 7thday of storage [112]. It is likely that the inhibitory effect promoted by probiotic bacteria is related to the production of organic acids, bacteriocins or hydrogen peroxide [67], which are natural antimicrobial agents.
8.2.2 FERMENTED MILK AND YOGURT
Probiotics have been used for decades to promote human health benefits and were first known in the forms of yoghurt and fermented milk [115]. Dairy products and especially fermented milk are ideal carriers for the delivery of probiotic bacteria in the gastrointestinal tract (GIT) [109].
Dimitrellou et al. [34] investigated the probiotic Lactobacillus casei ATCC 393 encapsulated in alginates using the extrusion technique. The authors evaluated the survival of the microorganism in simulated GI condi tions during the production of fermented milk and storage for four weeks at a temperature of 4°C. In the simulated GI conditions and after storage for 28 days in fermented milk, it was observed that encapsulated probiotic bacteria showed greater viability when compared to non-encapsulated microorganisms. Additionally, fermented milk showed a better aroma due to compounds produced by L. casei. The authors conclude that economically
the use of encapsulated probiotic microorganisms is a sustainable process for the production of fermented milk.
The study focusing on the selection of probiotic lactobacilli as the main initial culture for the production of fermented milk with probiotics without commercial application concluded that five out 44 isolates showed the highest cholesterol removal capability; one strain (Lactobacillus plantarumDP3) showed the broadest inhibitory range against pathogens and L. plantarum
DP3 and L. caseiDP21 reduced the fermentation time to 10 h. The microbial
count of the final products (> 10CFU/mL) guaranteed its probiotic activity after 24 storage days [92].
Additionally, probiotics could have beneficial effects against carcino genesis [43]. Research was carried out with the probiotic strain Lactoba cillus caseiCRL431 (PFM) to evaluate the immunomodulation exerted by fermented milk when administered to mice in the metastatic stage of breast cancer. The mice that received PFM were compared with the mice that received only milk. It was observed that the administration of PFM reduced the metastasis in the lungs and increased the survival of the mice.
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The results of the research show that the modulation of immune cells in the lungs by PFM can be a strategy to fight cells that cause tumor in metastatic sites [136].
Dairy products have the capacity to carry probiotics, being an alterna tive to reduce the potentially pathogenic microbiota in the oral cavity [121]. Pahumunto et al. [100] evaluated the effect of maltitol and other sugars in milk fermented by Lactobacillus rhamnosus-SD11 on the growth and acid production of Streptococcus mutans. The authors found that fermented milk containing L. rhamnosus-SD11 with maltitol reduced the growth of S. mutans.
Children, 123, were selected who randomly consumed the fermented milk with the probiotic or the control for 4 weeks once a day. The results showed that maltitol showed less acid production than simple sugars. Comparing the probiotic group with the control by means of a clinical trial, it was observed that there was a significant reduction in total salivary streptococci and S. mutans, while there was a significant increase in salivary lactobacilli after the consumption of probiotic fermented milk.
According to Bhalla et al. [13], dairy products such as cheese, yogurt, ice cream, and fermented milk in general when administered with probi otics tend to reduce buccal pH due to the buffering capacity of these foods. In addition, calcium, and calcium lactate that are present in milk can have anticariogenic properties, regardless these products are added with probiotics.
Yogurt can be produced with different combinations of cultures, including starter cultures and probiotic microorganisms [79]. Yogurt produced with probiotic cultures is a consumer trend and a challenge for the industry in relation to the development of functional foods [143].
Symbiotic yogurt was produced with high counts of probiotic Lactoba cillus acidophilus and flaxseed using the response surface methodology. The flaxseed concentration varied from 0 to 4% w/w and the shelf life from 1 to 28 days. The authors found that probiotic yogurt added with flaxseed had a higher L. acidophiluscount (up to 8.82 CFU/mL) compared to the control sample (6.87 CFU/mL). The results indicated that the addition of 4% of flax seed to probiotic yogurt can result in a functional food with a desirability of 76.8%. In addition, the product maintained adequate properties for about 13 days under cold storage [89].
Delgado-Fernلndez et al. [32] evaluated the viability of the probiotic microorganisms Lactobacillus acidophilusand Bifidobacterium animalis ssp. lactisin yogurt produced also with the starter culture of Streptococcus thermophilusand Lactobacillus delbrueckiissp. bulgaricus. In addition to the
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probiotic, symbiotic yogurts were added lactulose and the lactulose-derived oligosaccharide (OsLu). The addition of lactulose to yogurts significantly increased the count of L. delbrueckiissp. bulgaricus, L. acidophilusand B. animalisssp. lactisduring fermentation when compared to the control product.
Wu et al. [143] found that yogurt produced with the probiotic microor ganism L. plantarumhad a higher content of nutrients and better sensory and texture characteristics, being suitable for healthy consumption. Studies have reported that the addition of encapsulated or free cultures tends to reduce post-acidification during storage in addition to changing the product’s texture properties [43]. Pinto et al. [109] evaluated the Greek-style yogurt without lactose as a new matrix to serve spray-dried microcapsules containing the probiotic microorganism Bifidobacterium lactisBB-12. For the formulation of the microcapsules, three different materials were used, gum Arabic, inulin, and maltodextrin. In all formulations, the viability of the probiotic was greater than 8 log CFU g-1throughout 30 days of storage
at 4°C. The addition of microcapsules did not affect the viability of the initial cultures and increased the pH, firmness, and adhesiveness of the product. After 30 days of storage, the viability of the probiotic microorganism was above 6.5 log CFU g-1, indicating that Greek-style yogurt without lactose
may be a good matrix to carry B. lactisBB-12.
8.2.3 ICE CREAM
Frozen dairy products are characterized by containing solid dairy compounds that may or may not include milk fat, and are consumed in a frozen solid state. Within the frozen dairy dessert’s category, the most consumed product is ice cream [49].
Due to the worldwide popularity and its potential as a nutrients carrier, ice cream has aroused the food industry and the academia’s interest in using it as a functional food. For Ferreira et al. [42] functional food is a product that contains nutrients that provide health benefits in addition to basic nutrition.
These foods are a trend of the future, since current science allows the development of processed products that offer health benefits [53].
According to Abdelazez et al. [1], frozen dairy products, such as yogurt and ice cream, maybe the persuasive carriers of probiotics. Probiotic ice cream is a trend in the dairy industry.
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The sensory properties or the general quality of food should not be altered by the incorporation of probiotic cells [20].
For Cruz et al. [25], the incorporation of probiotic bacteria in a frozen dairy dessert formulation must not affect the general characteristics of the product. Therefore, it is essential that the physical-chemical parameters responsible for the quality of this product, such as fusion rate and organo leptic characteristics, present equal or even better characteristics when compared to a conventional ice cream.
The carrying of probiotics has been challenging, since microorganisms must survive technologically and physiologically harmful conditions. In addition, bacteria resistant to technological stresses are not always resistant to the environment of the digestive tract (low stomach pH, bile, digestive juices) and vice versa [48].
The determinants of the survival of microorganisms in ice cream include freezing, components of the mix, presence of oxygen and pH [73]. Cold stress is an important issue in the manufacture of probiotic ice creams, since the growth and viability of these microorganisms are influenced by the environmental temperature [41].
According to Tripathi and Giri [135], probiotic bacteria can survive in frozen products for a longer period of time. However, the freezing process can exert various effects on bacteria, depending on their phenotypic traits and the course of the process [73]. Ice crystals forming inside and outside cells during this process can cause mechanical damage to bacterial cells [102].
In addition to the freezing process, the initial injury caused by the increasing incorporation of oxygen, in addition to the homogenization stage during the ice cream processing, negatively interferes with the probiotic viability [41].
Some strategies can be used to minimize the loss in viability of probiotics added to food products. The use of microencapsulation, products that protect the microbial cell, food inputs that favor growth, packaging of materials that form an oxygen barrier, antioxidant agents, and modification of the storage atmosphere allowed microorganisms to survive better in various processes and formulations [135].
The encapsulation process is one of the strategies that favor the protec tion of microorganisms against technological and physiological agents [48]. Alginate is the most common encapsulating agent to be used due to its mild gelling conditions. Emulsions aiming at encapsulating LAB are generally water-in-oil emulsions, and alginate, pectins, or proteins are mainly used as water phases [20].
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Protective agents commonly used to enhance probiotic viability during processing, storage, and transit through the GIT include dairy-based proteins, hydrocolloids (gelatin, gum Arabic) and polyalcohols. Carbohydrates have been the most widely used protective compounds during dehydration, storage, and exposure to the GIT [20].
Recent studies have been carried out with different probiotic species and strains tested in ice cream. For Muhardina et al. [90], the addition of probiotics to ice cream is one of the methods used to improve the product’s benefit and functional value. The supplementation of foods with probiotic microorganisms makes it essential to adopt adequate measures to maintain their viability during the processing, packaging, and storage of these prod ucts [22].
Calligaris et al. [17] evaluated the potential use of structured emulsions with monoglyceride (anhydrous milk fat or sunflower oil), replacing sour cream, as a delivery system for L. rhamnosusin ice cream. The results obtained highlighted that the emulsions used protected L. rhamnosuscells against tensions and stresses during the ice cream processing and storage.
The bioactive efficiency of probiotics in the human body is usually compromised due to their low stability during storage and digestion processes [141].
Farias et al. [40] investigated the viability and evaluated the survival of L. rhamnosusand L. caseiin yellow mombin ice cream. Tests were carried out comparing the cultures resistance at low temperature, efficiency in the encapsulated form with calcium alginate-chitosan and cell survivability in a simulated GI environment. The ice cream was stored at -18°C for 150 days. The results demonstrated that encapsulated L. rhamnosusASCC 290 showed higher resistance to low temperature, while L. caseiATCC 334 had a higher survival rate during encapsulation and in the GI environ ment. It was observed that the best option for the preparation of functional yellow mombin ice cream, considering all the cellular losses suffered, is the use of L. rhamnosusASCC 290 in free form or encapsulated of L. casei ATCC 334.
In vitroand in vivostudies are performed to test the probiotics survival during passage through the GIT. Afzaal et al. [2] evaluated the survival of free and microencapsulated L. acidophilus(sodium alginate and carra geenan) over a period of 120 days at -20°C. It was also assessed the survival of free and encapsulated probiotic bacteria under GIT conditions. The study results showed that encapsulation significantly improved (p <0.05) the probi otic cells survival in ice cream compared to free cells, and also in in vitro
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conditions. The encapsulation guaranteed the recommended probiotics level (106 to 108 CFU/g) carried in food, offering health benefits.
A study of Akalin et al. [4] used different fibers in the manufacture of ice cream enriched with L. acidophilus and B. lactis. The authors evaluated the viability of probiotics over a period of 180 days in 6 ice cream formula tions one control sample and the others with apple, orange, oat, and bamboo and wheat fibers. At the end of the study, it was concluded that wheat fiber had the potential to enhance rheological and textural characteristics, main tain sensory properties and probiotic viability. On day 120 of storage, the viability of B. lactis was higher in ice cream enriched with wheat fiber when compared to other fibers and the control sample.
8.2.4 INFANT FORMULA AND MILK POWDER
Colonization of the intestine by beneficial microorganisms early in life is essential to establish the barrier of the intestinal mucosa, increase the immune system and prevent infections caused by enteric pathogenic micro organisms. Factors such as mode of delivery, breastfeeding, prematurity, and use of antibiotics influence the beginning of human life. Studies suggest that the use of probiotics in food can prevent disease [12]. In an attempt to establish a microbiota in babies fed formula similar to breastfed ones, infant formula manufacturers are increasingly incorporating probiotics into their products [65].
An observational study was carried out to evaluate the effects of Lactobacillus reuteriDSM 17938 on the composition of the microorgan isms in the GIT of babies. Fecal samples from 30 hospitalized children who received the microorganism and 30 who did not receive the probiotic were analyzed. The groups were different in composition and quantity of intestinal microbial strains. Babies who received the probiotic had a lower gram-negative total anaerobic count (p= 0.03) and a higher gram-positive total anaerobic count (p = 0.02). Enterobacteriaceae and enterococci were significantly higher (p= 0.04) in the group that did not receive the probiotic and lactobacilli and bifidobacteria did not differ between groups. Children who did not consume probiotics had greater coloniza tion by diarrheal E. coli(p= 0.04). The results showed the importance of
probiotics in intestinal health in pediatric patients. The administration of L. reuteriin infancy can reduce colonization of pathogens and improve intestinal health [122].
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The prevalence of colic in babies is high and has a significant impact on the lives of children and their families. Study evaluated the effects of probiotic drops in the treatment of colic in children. The 72 children were randomly divided into two groups, the probiotic receiving group (PRR) and the placebo group (PCR). The results showed that there was a significant increase in weight in the PRR group (p < 0.0001) and there was no significant growth in the PCR group (p-value 0.437). Regarding fecal consistency, it was observed that there was no significant difference between the groups [3].
Probiotics have been proposed to be beneficial for the treatment and prevention of food allergy [130], once that for children, food allergy is considered a public health problem [120].
Santos et al. [120], in their studies, selected 18 clinical trials, which predominated babies and children of preschool age, and observed that the most used strain was Lactobacillus rhamnosusGG, alone or in combination. The most used carriers were capsules and infant formulas, and the interven tion period ranged from four weeks to 24 months. Of the 46 evaluations, 27 demonstrated the benefits of using Lactobacillus rhamnosus GG. The
authors observed that the use of probiotics helps to promote immunomodula tion and reduces clinical symptoms.
There are not many powdered milk products on the market that contain probiotics and prebiotics. Although milk is recognized as a nutritionally rich and diverse food, the addition of probiotics is an alternative for improving an individual’s intestinal health [16].
Teanpaisan et al. [131] evaluated the count of Lactobacillus paracasei SD1 in powdered milk by spray drying and observed that the survival of the microorganism varies according to the processing temperature. The authors concluded that spray drying is a process with potential use in large-scale production, in addition to facilitating the transport and storage of strains of the microorganism. Skimmed-milk powder can be used in different applica tions such as instant desserts and confectionery products, so it is possible that the culture contained in the powder can be used in a wide range of functional food applications.
Bradford et al. [16] produced powdered milk containing free and immo bilized cells of Lactobacillus plantarum. The treatments had high viability of the microorganism before and after spray drying. After exposure to simu lated gastric and intestinal conditions, counts of the microorganism greater than 8 log UFC/g were found in samples of powdered milk containing free
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and immobilized cells of Lactobacillus plantarum, which is more than recommended for probiotic products.
.3 PROBIOTICS IN NON-DAIRY PRODUCTS
Probiotic cultures are traditionally carried in dairy products [9, 52], but there is an increasing number of individuals with intolerance and allergy to dairy products, hypercholesterolemic, vegetarians, who need diets with fat restric tion and who do not consume dairy products for habits or cultural reasons. These dietary restrictions justify the search for new non-dairy matrices as a vehicle for probiotic bacteria, in order to serve this portion of the population. The literature highlights that the addition of probiotic cultures in non dairy products represents a challenge, especially with regard to the survival of these microorganisms [34]. Thus, the development of new non-dairy products must take into account the viability of the microorganism during storage and its survival to the GIT when carried in the product.
8.3.1 FRUITS AND VEGETABLES AND DERIVED PRODUCTS
Fruits and vegetables have been presented as an alternative to insert these bacteria in the diet since they have nutrients that help their growth [83]. These foods have micronutrients and fibers, carbohydrates, and are sources of C and B vitamins, pro-vitamin A, phytosterols, minerals, and phytochemicals, which are essential to the diet and microbial metabolism. Therefore, they reveal a potential to act as a vehicle for probiotics due to their intrinsic characteristics, such as, the presence of natural prebiotics that promote growth and act as protectors of probiotic microorganisms during product life and passage through the GIT [56, 82]. Moreover, fermented fruits and vegetables containing prebiotic compounds are sources of probiotics [56].
The most used species in probiotic products of plant origin are L. rham nosus, L. acidophilus, L. casei, L. plantarumand B. lactis[83].
According to Dimitrellou et al. [34] consumers retain their interest and the global market of probiotic foods and beverages is still growing. So, it is believed that these foods have a promising future [9, 81, 117, 144], since there is a great interest from industry and researchers in evaluating new matrices in order to expand the market for fermented or fortified probiotic products [7, 117, 142]. Studies also show that besides foods based on fruits
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and vegetables being carriers of probiotic bacteria, they have good sensory characteristics [45].
Minimally processed products, juices, fermented and non-fermented fruits and vegetables, pickles, dehydrated fruits, ice cream, chocolate, vegetable appetizer, tea, coffee, fruit puree, among others, have been studied as carriers of probiotics (Table 8.2).
TABLE 8.2 Recent Studies on Plant Matrices as a Substrate for Probiotic Bacteria
Product Probiotic Employed Viability (log References
CFU/g or mL)
Minimally processed fruit L. rhamnosusHN001 .49-5 days [81]
salad
Tomato juice L. acidophilus, L. > 8.0-3 days [63]
plantarum, L. casei
Juçara and mango mixed juice L. rhamnosusGG > 8.0-30 days [88]
Pickles L. casei .0-63 days [38]
Coconut fermented beverage L. plantarum DW12 .5-3 days [60]
Cubes of osmotically L. plantarum .0-6 days [37]
dehydrated apples
Strawberry ice cream and L. acidophilus > 7.0-150 days [104]
yacon flour
Chocolate L. paracasei, L. > 6.6-90 days [71]
acidophilus
Apple Snacks L. paracasei > 6.0-28 days [5]
Juçara and pineapple mixed L. rhamnosusGG > 7.2-28 days [18]
juice
Vegetable appetizer L. plantarum LP299v > 7.42 and 8.84, [19]
or L. rhamnosus GG respectively
Tea and coffee B. coagulansMTCC > 8.0-24 [78]
months
Mixed fermented vegetable L. plantarum .34 [144]
juice (purple cabbage, tomato, and carrot)
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TABLE 8.2 (Continued)
Product Probiotic Employed Viability (log References
CFU/g or mL)
Mixed guava and beet L. rhamnosus GG > 7.3-42 days [87]
beverages based on peanut
extract and soy extract
Fruit powder L. plantarum .12-36 days [140]
Mixed jussara and mango L. rhamnosusGG > 5.0-90 days [111]
juice
Carrot juice, apple puree, rice L. rhamnosus LMG > 8.7-1-2 days [14]
cream S-30426
L. acidophilusTISTR 1338 and L. caseiTISTR 390 were added to grape juice jelly in the study of Ampornpat and Leenanon [6]. They found a reduc tion in counts from 9.23 log CFU/g and 8.97 log CFU/g (initial time) to 6.80 log CFU/g and 7.73 log CFU/g respectively, after nine days of storage at 5°C. The survival of bacteria against different harmful factors during product processing and storage depends on the characteristics of each species [135]. Sporulated bacteria can resist high temperatures, including pasteurization and low pH. Therefore, the use of spore-forming probiotic bacteria for the development of probiotic fruit jelly may be an option.
Panda et al. [101] studied prickly pear juice (Opuntiasp.) as a substrate for Lactobacillus fermentumATCC 9338 and the product was well accepted by the panellists. Yang et al. [144] also observed in their work that the blended vegetable beverages were approved by the panelists, demonstrating their potential for commercial production.
According to Vivek et al. [140], probiotic fruit juice powder also can be a suitable alternative for dairy-based probiotic powders. They verified L. plantarumviability of 6.12 log CFU/g at 36 days of storage of the juice. Then, the authors concluded that the juice powder could present a potential application for the industry.
Khodaei et al. [69] studied L. plantarum, L. casei, and Saccharomyces boulardii incorporated in gelatin and low methoxyl pectin (LMP) films. Their viability was monitored during storage at different temperatures, and it was concluded that the material is suitable for safeguard and deliver LAB (>10CFU/g) during storage time.
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Probiotics have the potential to be used for allergies and treatment of various diseases. Lactobacillus probiotics survive the simulated GIT and can inhibiting foodborne microorganisms [117].
Probiotics and their bacteriocin application to fruit and vegetable prod ucts provide an important possibility to chemical compounds to control foodborne pathogens and spoilage microorganisms [56].
In this field, the work of George-Okafor et al. [47] showed that probiotic cultures are used in food products as a bio-preservative. They verified an increase in shelf-life of the home-processed tomato paste, suggesting the possible use of cell-free supernatants contained effective biomolecules of L. plantarumCs and L. acidophilusATCC 314 as bio-preservatives in tomato processing for > 25 days.
Probiotics are often reported to modify the gut microbiota structure of host [110]. So, in vitro,and in vivostudies are very important. In vitrostudies are widely used and, when compared to in vivotesting, they are faster, safer methods and do not have the same ethical restrictions as in vivotesting [18, 145], being an efficient methodology to evaluate the probiotics transport along the TGI (Table 8.3).
These studies employ many enzymes such as α amylase, pepsin, lipase, pancreatin, and bovine bile from different sources, in order to simulate the oral, gastric, enteric I and enteric II phases of the in vitroassay [84].
The viability of L. plantarumLP299v or L. rhamnosusGG in vegetable appetizer were evaluated by Campos et al. [19] and our group found that the appetizer is apt to be considered probiotic.
Marcial-Coba et al. [80] studied B. coagulansBC4 spores embedded in dried date paste and the gastrointestinal resistance in vitroof the bacteria. They found that the product is a suitable vehicle for the delivery of B. coagu lansspores, since the physical properties of this matrix are not conducive to spore germination and consequently do not affect the stability of the probiotic cells during storage.
A pectin coated dehydrated apple snack containing ≥ 9 log colony forming units/20 g portion of L. paracaseiwas developed by Valerio et al. [137]. The probiotic survived during fruit processing and simulated GIT digestion and the apple surface presented a good visual and nutritional quality which could be maintained for 30 days of storage.
Litchi juice was also used for fermentation of L. casei, and it was an effective method that increased the contents of total phenolic, total flavone, and exopolysaccharide [142]. The authors developed an animal experiment, and the results revealed that intake of fermented juice could improve the gut health of mice.
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So, the studies demonstrate that fruits and vegetable bases are a prom ising alternative for add probiotic bacteria being one new potential option for the consumers.
TABLE .3 Studies Involving In Vitro Tests of Gastrointestinal Resistance (GIT) of Probiotic Microorganisms Carried by Fruits, Vegetables, and Derivatives
Matrix Probiotic References
Specie GIT Time
Resistance (Days)
(log CFU/g or
mL)
Osmotically L. plantarum .0 [37]
dehydrated apples
Jabuticaba juice L. rhamnosus GG .0 [97]
Tomato and feijoa Lactobacillus .33 and 5.78, After [138]
juices plantarum respectively fermentation
Mango juice L. acidophilus, L. > 5.0 [44]
plantarum and L.
rhamnosusGG
Mixed fermented L. rhamnosusGG .6 [18]
pineapple and
jussara juice
Vegetable appetizer Lactobacillus .67 and 9.53, [19]
plantarum LP299v respectively
or Lactobacillus
rhamnosus GG
Orange, mango, B. coagulans BC4 > 8.0 [80]
and carrot paste cells and spores
Mixed guava and L. rhamnosusGG .34 and 7.50 [87]
beet beverages in BMPP
based on peanut and BMSP,
extract (MBPP) respectively
or soybean extract (MBSP)
Mango and carrot L. plantarum > 7.0 [98]
mixed juices
Litchi juice L. casei .27 After [142]
fermentation
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8.3.2 CHOCOLATE
Another type of non-dairy and non-fermented food that has been studied as a functional food and specifically as a matrix for probiotic bacteria is chocolate. This product, in addition to being consumed by all age groups worldwide, has shown promising results as a probiotic food [72, 93]. Choco late has an interesting antioxidant capacity and this capacity increases as the concentration of cocoa is higher, as it has a greater amount of phenolic compounds, proanthocyanidins, and flavan-3-ols [74] and this antioxidant capacity can protect probiotic bacteria against oxidative stress. Cocoa butter is also considered a material that can protect probiotics from the action of the GIT [106] and is present in chocolate.
Kemsawasd et al. [64] incorporated L. casei01 and L. acidophilusLA5 in white chocolate, milk chocolate and dark chocolate and stored them at 4 and 25°C for 60 days. As a form of protection for the cultures, the researchers added them to skimmed milk and maltodextrin and carried out spray drying. They found that L. casei01 had better resistance in all types of chocolate and that the cells survived less at 25°C. At 4°C the probiotic survival rates were greater than 6 log CFU/g. The best survival of probiotics in dark chocolate was verified, followed by milk chocolate and finally white chocolate. The authors justify this result due to the greater presence of cocoa in dark chocolate, which contains higher levels of phenolic compounds and, consequently, greater antioxidant activity.
Laličić-Petronijević et al. [76] evaluated the synergistic effect of three probiotic strains microencapsulated with proteins and polysaccharides on quality parameters of milk, semisweet, and dark chocolate at 4 and 20°C for 360 days. The tested bacteria were L. acidophilusLH5; Streptococcus thermophilusST3 and Bifidobacterium breve BR2 (DUOLAC MIX L3). It was evidenced that L. acidophilusLH5 remained at a concentration of 8 log CFU/g regardless of the type of chocolate and the temperature during the 360 days. S. thermophilus ST3 had no significant reduction in milk chocolate, but it did in the other two types of chocolate. B. breve BR2 had a significant reduction at 4°C in milk chocolate after 90 days and after 30 days in the other two types of chocolate, reaching 3 log CFU/g, showing its greater fragility in relation to this matrix in relation to other bacteria. As it was a mixture of probiotic bacteria and their combined concentration was 8 log CFU/g at the end of the storage period, it can be considered that it can bring health benefits. The authors reported the success of the experiment due to the insertion of probiotic bacteria at a lower temperature (32°C) and
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highlighted the possibility of selling the refrigerated product and at room temperature. All chocolates containing probiotics showed excellent general sensory quality throughout the storage period, and furthermore, their rheo logical properties were not affected.
Another study that evaluated microencapsulation as a form of protection against probiotic bacteria was that of Nambiar et al. [91]. The authors devel oped milk chocolate containing L. plantarumHM47 microencapsulated with soft coconut water, Moringa oleiferagum and maltodextrin, storing it at 25°C for 180 days and after this period the cells remained viable in concentration above 8 log CFU/g. The bacteria do not alter the pH of the chocolate, indicating low metabolic activity, due to the microencapsulation and low water activity of this matrix, according to the authors. The product was very well accepted sensorially and also did not cause toxicity in rats. An increase in lactobacilli was also observed in the microbiota intestinal of mice that consumed probiotic chocolate compared to those that consumed only probiotic bacteria without chocolate, indicating that this matrix was able to provide additional protection to the probiotic to the action of the GIT. The incorporation of L. acidophilusNCFM®and B. lactisHN019 and milk and dark chocolate was the object of study by Laličić-Petronijević et al. [76]. L. acidophilusNCFM®had better viability than B. lactisHN019 in
both types of chocolate, mainly at a temperature of 4°C, with values above 8 log CFU/g after 180 days at this temperature, whereas B. lactisHN019 had its concentration above 7 log CFU/g for up to 90 days, which was reduced to below 6 log CFU/g after 150 days, no longer providing adequate quantity for consumption. The rheological properties of chocolates did not change much with the presence of probiotics, only the appearance of granulation, but this did not interfere with the sensory properties of the products.
Mirković et al. [85] developed dark chocolate containing L. plantarum (potential probiotic) and L. plantarum299v (commercial probiotic) and evaluated their influence on volatile compounds and sensory characteristics for 360 days storage at room temperature. The bacteria were washed and then resuspended in skimmed milk, being encapsulated by spray drying, and their addition was carried out at a temperature of about 30°C. The bacteria reached a concentration of 8 log CFU/g after 60 days of storage and 6 log CFU/g after 180 days, demonstrating an ideal shelf life of the product up to 6 months. The authors observed no difference in the volatile profile of chocolates, without affecting the sensory characteristics of the product.
Succi et al. [129] evaluated survival during simulated GI transit of some commercial probiotic formulations in dark chocolate (80% cocoa) after 90
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days of storage. The bacteria in question were L. rhamnosusGG; L. para caseiF19. Chocolate offered bacteria good protection from simulated GI stress conditions.
These studies are encouraging for companies and institutions to invest in the development of probiotic chocolates, enabling a wide variety of people to consume these bacteria that bring health benefits, combined with the pleasure of consuming this type of food.
8.3.3 PROBIOTICS IN BAKERY PRODUCTS
Due to its high daily consumption around de the world, bread is considered an interesting non-dairy food vehicle for probiotics. It is the major type of bakery products and a staple food in most parts of the world. A typical, tradi tional bread-making is largely wheat-flour based, involving dough mixing, proving (i.e., fermentation) and baking.
Bread is identified as a potential food that can be enriched with probiotics due to the presence of a non-digestible carbohydrates like oligosaccharide that have been suggested to promote growth of probiotic bacteria, moreover, high-quality sourdough breads can be found by inoculation LAB [147]. Thus, the study of probiotics in bakery products have increased in recent years [123, 126, 147].
Soukoulis et al. [128] studied a probiotic pan bread constructed by the application of film-forming solutions (sodium alginate or sodium alginate and whey protein concentrate) containing Lactobacillus rhamnosus GG. The bread crust surface containing probiotic did not differ from the control bread. The L. rhamnosus GG viability was reduced during the first 24 h of storage, and viable count losses were low during the 2-3 days of storage, and growth was observed upon the 4-7 days of storage. Based on their calcula tions, an individual 30 e 40 g bread slice can deliver approx. 7.57-8.98 log CFU/portion before in-vitro digestion a 6.55-6.91 log CFU/portion after in-vitrodigestion, meeting the World Health Organization (WHO) recom mended. However, Soares et al. [126] reported that given the complexity and high cost of using edible films to prepare probiotic bread may not be economically viable for the industry.
The incorporation of probiotics in bakery products is challenging due to their viability and sensitivity to the high temperatures during baking. The methodology combining microencapsulation and edible coatings has been successfully.
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In 2016, Lactobacillus acidophilus was encapsulated in the SeyedainArdabili, Sharifan, and Tarzi study [123]. Lactobacillus acidophilus LA-5 and L. casei were encapsulated (with calcium alginate and Hi-maize resistant starch), coated (chitosan) and inoculated (1 g of microencapsulated bacteria per 100 g of final product) into the two types of bread (hamburger buns and white pan bread). The hamburger buns were baked for 15 min and white pan bread baked for 25 min, both at 180°C. The initial cells count before and after encapsulation was approx. 10CFU/g. With an encapsu lated, viable cells survived the baking process and both types of bread met the standard for probiotic products. Using the chitosan coating, a significant increase in probiotic survival was observed. Among the probiotics, L. casei was more resistant to high temperature than L. acidophilus LA-5, so this study showed that bacteria in unfavorable conditions are dependent on species. The type of bread, also, affected the bacteria survival, which was higher in hamburger bun, probably due to the shorter baking time, than in white pan bread. This study, additionally, indicated that the production of symbiotic bread using microencapsulation is possible and increase the viability and thermal resistance of probiotic bacteria in breads [123]. Zhang et al. [147] studied the better impact of different bread sizes, baking temperature and subsequent storage on the survival of probiotic bacteria (Lactobacillus plantarum P8). The viability of bacteria was evalu
ated for both bread crust and crumb. They showed approx. 4-5 log reduction of viable counts of L. plantarumin both the crust and crumb of 5, 30, or 60 g bread after baking at 175, 205, or 235°C for 8 min, while the initial viable count (N0) in dough was 8.8 ± 0.1 log CFU/g. Different bread sizes had little influence on survival of probiotics during 8 min baking, every way, higher residual viability can be obtained in less baking time. The bacteria in the bread crust were found more stable than those in the crumb despite the higher temperature in the crust during baking under certain conditions. The lower moisture content and the dense and glassy microstructure of the crust may have a positive effect on the thermo-stability of bacteria in the crust during baking. After 4-day storage, the population of probiotic bacteria restored to an amount higher than 10CFU/g in the crumb and 10CFU/g in the crust.
These authors suggest that future research should also focus on strategies, such as micro-encapsulation or optimization of processing parameters, to retain high viability of probiotics after baking.
Thus, in the same year, Zhang et al. [148] investigated the survival of dried probiotics subjected to isothermal heating and bread baking by encap sulating. In this study, Lactobacillus plantarum P8 was freeze-dried in four
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different matrices (reconstituted skim milk: RSM, gum Arabic: GA, malto dextrin, and inulin) as protectants. The probiotic powders were added to bread by distributing it on the surface of the dough, and a control group was made without adding this probiotic. The RSM matrix showed the highest protective effect on cells during baking at either 100°C or 175°C, followed by the inulin matrix. However, no protective effect was observed for gum Arabic and maltodextrin matrices during baking.
Another way that can help overcome various technical challenges and expand the possibility of applying probiotic microorganisms in products which are submitted to extremely high temperatures is the use of sporulated bacteria with potential probiotic property.
In the Soares et al. [125] study, the bread dough was separately inocu lated with Bacillus strains (Bacillus subtilis PXN 21, Bacillus coagulans GBI30 6086 and Bacillus coagulans MTCC 5856) and were baked at 180°C for 20 min. The loaves were packaged in plastic bags and stored at room temperature (approximately 25°C) for seven days, and the counts of the strains with probiotic properties were determined throughout the product’s shelf-life. The counts of Bacillus strains with probiotic properties showed reductions below 1 log cycle in the bread samples. The counts of B. subtilis PXN 21 at the end of seven days (4.4 log CFU/g) was lower than that initially observed (5.1 log CFU/g), indicating less resistance or possible germination during the storage. On the other hand, the counts of B. coagulans strains remained practically stable at the same time (7 log CFU/g). These findings indicate that the reduction in the counts of Bacillus strains with probiotic properties should be considered and “corrected” to allow the desired dose of these microorganisms to be achieved in commercial products.
8.3.4 MEAT PRODUCTS
Meat is the source of protein, group B vitamins and minerals important for health. However, they are not sources of carbohydrates and dietary fiber and face negative criticism due to the presence of saturated fats and cholesterol. However, with meat processing, it is possible to introduce these missing nutrients, obtain greater nutritional balance in meat products and produce functional meat products [95].
Studies focused on functional meat products have fermented meat prod ucts as an excellent source of microorganisms with probiotic characteristics
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[146]. According to Ojha et al. [96], consumer demands for high quality meat products including fermented meats.
The bioprotective action on fermented meat products is possible with the use of probiotics and LAB [11, 96].
Target products include kinds of dry sausage that are processed by fermentation without heating or only mildly heated. In fermented sausages, sugar is fermented by bacteria in an anaerobic environment to produce acid, which lowers pH. The ingredients found in a fermented sausage include meat, fat, salt, spices, sugar, and nitrite. De Vuyst et al. [31] alert about the potential negative impact of the fermented sausages environment with high content of curing salt and its low pH and water activity on bacteria viability. Studies with probiotic bacteria in meat are usually carried out in two stages. In a first stage, Kołozyn-Krajewska, and Dolatowski [70] argue about the need to verify whether the probiotic bacterial strains that can be used in the manufacture of dry fermented meat products should be able to survive under the conditions found in fermented products; in addition, they must dominate other microorganisms found in the finished product. Probiotic contribution in food biopreservation act extending shelf life of food and their ability to inhibit spoilage and foodborne pathogens [114]. So, they can act both as a probiotic and bioprotective culture.
Reviews of probiotics in meat products such as De Vuyst et al. [31]; Khan et al. [66]; Kołozyn-Krajewskaa and Dolatowski [70]; Rouhi et al. [118], presented many studies about the potential use of probiotic bacteria in meat products.
Recent studies verified interesting results. Quantities of 8.0 log CFU/g of L. paracaseiLPC02 was inoculated in dry sausage, and at the end of the process, the average count in was 7.59 ± 0.37 log CFU/g, showing that it was able to dominate the endogenous microbiota of the meat batter [23].
Example of probiotic culture like bioprotective was observed by Trabelsi et al. [134], which incorporated probiotic strain in raw minced beef meat during refrigerated storage. The results showed that the incorporation of the probiotic strain can inhibit the proliferation of spoilage microorganisms, such as Listeria monocytogenesand Salmonellaspp., delay the lipid oxida tion, improve texture parameters, extend the shelf life, and then can be used as a biopreservative agent for extending the safety and quality.
In a second stage, is required human clinical studies documenting health promoting effects. In contrast to the successes obtained in the dairy industry in meat industry, human studies using probioticsprobiotics in fermented meats have been very scarce. Jahreis et al. [55] checked the effect of the
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daily consumption probiotic sausage containing L. paracaseiin healthy volunteers for several weeks showed only moderately successful in fecal samples. A statistically significant increase in the numbers of bacteria was observed for some of the volunteers.
Lactobacillus rhamnosusdominated fermented sausages during the ripening process and temporarily colonized the GIT of healthy volunteers, confirming that this strain could be delivered as a potential probiotic [57].
A German producer launched a salami product containing three intestinal LAB strains (L. acidophilus, L. casei, Bifidobacteriumspp.). This product is claimed to have health benefits and is thought to be the first probiotic-like salami product to be marketed. Japanese producers too began to market a new range of meat spread products fermented with probiotic LAB L. rham nosusFERM P-15120 [8]. Despite commercial examples, the commercial application of probiotic microorganisms in dry fermented meat products is not yet common.
Use of different probiotic administration strategies for use in meat prod ucts to ensure the viability of microorganisms mainly for cooked products like frankfurter sausages, have microorganism immobilization techniques such as encapsulation [21]. Pérez-Chabela et al. [107] observed that thermo tolerant LAB encapsulated with acacia gum and sprayed dry were inoculated in cooked meat batters has enhanced initial count.
Probiotic delivery strategies for use in meat products like encapsulation techniques are promising. The potential use of probiotic bacteria in meat products is a reality, although human studies using probiotic fermented meats have been very scarce. Now, dominated by dairy products, but in future probiotic meat products will become an important part of the meat processing industry.
.4 TECHNOLOGICAL PROBLEMS AND CHALLENGES FOR ADDING
AND MAINTAINING PROBIOTICS IN DAIRY AND NON-DAIRY FOODS: TECHNOLOGIES USED TO MAINTAIN THE VIABILITY OF PROBIOTICS DURING STORAGE
The maintenance of probiotic microorganisms during production processing, the product shelf life, and during passage through the human GIT is a chal lenge for industries. Factors such as temperature, pH, salt content, added of preservatives and oxygen can affect the viability of probiotic cells. Thus,
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technology alternatives can be used aiming to reduce the damage on probi otics, among them, the microencapsulation has gained importance.
The microencapsulation technique is based on the incorporation of probiotic cells into an encapsulating matrix or membrane forming a physical barrier between the microorganism and the external environment [124]. Thus, the main objective of microencapsulation is to protect the probiotic cells from adverse conditions and, consequently, increase the viability of microorganisms. The microencapsulated probiotics are release in appro priate locations (e.g., small intestine), promoting adhesion and colonization in the intestinal epithelium.
Therefore, modern, and innovative methods have been developed over the last decades to creating a wide variety of probiotic microcapsules [105]. Although many industries prefer economic processes thus the balance between cost and benefit must be considered. Some technologies require specific devices or materials affecting production costs. The following are the most important technologies used for probiotics encapsulation:
. Spray-Dryer: The basic principle of the spray drying process is the
atomization of a polymer suspension containing probiotic cells. Thus, the suspension with appropriate viscosity or consistency is sprayed into contact with a hot stream and instantly producing powdered microcapsules. Heat and mass transfer occur simultaneously from air to atomized drops and vice versa, respectively [116].
. Freezing Dryer: The fundamental principle in freeze-drying consists
in the removal of the water present in the material by sublimation. The microencapsulation of probiotics using the freeze-drying stands out when compared to other techniques because drying using low temperatures increases the survival rate of probiotic cells [77].
Besides, freeze-dried products reconstituted very easily and have a long shelf life [58].
. Extrusion: The principle of this technique consists in the extrusion
of a liquid mixture containing the polymers of the wall material and probiotic cells through in the form of droplets into a hardening solu tion or setting bath [35]. For the microencapsulation of probiotics by extrusion, various polymers can be used but alginate, carrageenan, and pectin are the most frequent. The hardening solution contains the divalent salts, generally calcium and potassium, because the poly mers used form gels when they in contact with minerals solutions.
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. Complex Coacervation: It is a liquid-liquid phase separation
process, which occurs spontaneously from mixed solutions of poly mers with opposite charges. In complex coacervation, at a specified pH, occur the electrostatic interaction between polymers with oppo site charges [36]. This causes a formation and aggregation of nearly neutral complexes to reduce the free energy of the system, becoming insoluble. Subsequently, there is a phase separation process; an insoluble polymer-rich phase that contains the coacervate complex stabilized, and a solvent-rich phase [132]. During the encapsulation, the coacervated complex is deposited around the active ingredient (core) leading to sedimentation of encapsulated core [133].
. Emulsification Techniques: The principle of this technique is based
on the formation of an emulsion with a continuous phase (generally vegetable oil) and a dispersed phase where probiotic and hydrocol loid will be present. The mixture containing the continuous phase and the dispersed phase is homogenized. After the homogenization, occur the formation of the water-in-oil (W/O) emulsion and, thus, resulting in aqueous/polymeric droplets containing the probiotic cells. After the formation of the emulsion, the polymer present in the aqueous droplets can be gelled. In this case, the emulsification tech nique can be divided into internal or external ionic gelation process [127]. The coating material used must have some, for example, easy workability during encapsulation; solubility in solvents suitable for use in food; low viscosity at high concentrations; protection of the core against adverse conditions, food-grade status; emulsifying and stabilizing capacity; non-reactivity with the core; they must easily release solvents or other materials used during the process, absence of taste or odor, and low cost [51]. Among the coating material, the following stand out gelatin, whey protein, and various plant proteins, Arabic gum, chitosan, pectin, alginates, xanthan gum, carrageenan, and carboxymethyl cellulose.
Therefore, the use of microencapsulated probiotics is promising and represents an alternative to increase the supply of probiotic products, meet consumer demand and, also allow a wider application of probiotics in the market. However, studies should be executed to select the encapsulation technique, the efficient coating material, and appropriate conditions for the process. Consequently, allowing the formation of microcapsules suitable for applications in food.
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.5 FINAL CONSIDERATIONS
Fortification of foods with probiotics is a tendency. Research on probiotic products with heterogeneous food matrices is necessary in order to meet consumer demand. Thus, it is believed that the functional food industry has a promising future and should focus on research and development of bioforti fied products.
The carrier vehicle is very important for probiotic survival in order to maintain its positive effects. An industry challenge is to ensure that the viability of probiotics is maintained throughout the shelf life of products at an appropriate cost-benefit in order to provide benefits to consumers in general.
In addition, to be used for the health of the host, probiotics have an advantage once they can be used as bioprotective cultures in food in order to avoid spoilage. Our research group works on enriching foods with probiotic bacteria such as L. rhamnosus, L. plantarum, L. acidophilus, L. casei, and
Bacillus coagulansand in vitroand in vivoresistance to TGI, being the results promising.
KEYWORDS
• carrageenan
•gastrointestinal resistance • low methoxyl pectin • oligosaccharide •placebo group
•probiotic receiving group
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CHAPTER 9
Microencapsulation: An Alternative for the Application of Probiotic Cells in the Food and Nutraceuticals Industries
DANIELE DE ALMEIDA PAULA,CARINI APARECIDA LELIS,and
NATALY DE ALMEIDA COSTA
1Federal Institute of Sمo Paulo (IFSP), Campus Avaré - Av. Professor Celso Ferreira da Silva, 1333, Jardim Europa, CEP 18707-150, SP, Brazil
2Federal University of Sمo Carlos (UFSCar), Campus Lagoa do Sino
Rodovia Lauri Simُes de Barros, Aracaçu, Buri, CEP 18290-000, SP, Brazil 3Department of Food Technology, Federal University of Viçosa (UFV),
P.H. Rolfs Avenue, Campus, Viçosa - 36570-900, MG, Brazil
ABSTRACT
In recent years, the use of probiotic microorganisms has increased consid erably. Probiotics are live microorganisms that which when administered in adequate amounts, confer health benefits on the host. Because of the numerous benefits and consumer concern about the importance of healthy diets, a wide variety the products containing probiotics microorganisms are available, represented by food and pharmaceutical formulations. However, maintaining the adequate viability of these microorganisms during processing, shelf life, storage conditions, and during passage through GI conditions is a challenge for industries. Thus, technological alternatives can be used to minimize the damage on probiotics, among them, stands out the microencapsulation. The objective of encapsulation is to create a physical barrier between the microorganism and the external environment, increasing cell viability during processing, storage, and passage through the GIT. Thus, viability will be maintained and the cells released in appropriate locations
Advances in Nutraceuticals and Functional Foods
(for example, small intestine) for adhesion and colonization of the intestinal epithelium to occur.
.1 INTRODUCTION
In recent years, the use of probiotic microorganisms has increased consid erably. The Food and Agriculture Organization (FAO) and World Health Organization (WHO) defined probiotics as: “live microorganisms that which when administered in adequate amounts confer health benefits on the host.” The more important benefits are effects on the gastrointestinal tract (GIT); on the urogenital system; on the immune system modulation, and even on the cardiovascular system. All health benefits are dependent on the strain used, the frequency, and daily dosage.
Because of the numerous benefits and consumer concern about the importance of healthy diets, a wide variety the products containing probi otics microorganisms are available, represented by food and pharmaceutical formulations. The various options aim to attend different publics, such as lactose intolerant, allergic to milk proteins, vegans, vegetarians, and others. Also, the diversity of products allows the consumption of probiotics at various moments of the day and, so, favoring the frequency of ingestion, fundamental factors the provide benefits to the body.
However, maintaining the adequate viability of these microorganisms during processing, shelf life, storage conditions, and during passage through GI conditions is a challenge for industries. Factors such as temperature, pH, oxygen, salt content, and added preservatives can affect the survival of the probiotic’s cells. Thus, technological alternatives can be used to minimize the damage on probiotics, among them, stands out the microencapsulation.
The microencapsulation technique consists of packing the material of interest using polymeric coatings and creating an important barrier between the microorganisms and the extern environment. In the industry, the microencapsulation has solved many limitations, and it is used to protect sensible compounds of external agents, to release of these compounds under controlled conditions, to mask undesirable flavors or odors, and others. Among the materials that can be encapsulated, stand out: acids, bases, oils, vitamins, salts, flavors, dyes, enzymes, and microorganisms.
Therefore, the microencapsulation of probiotic bacteria has been used to protect these organisms from adverse conditions and, so, increase cell viability. The release of the cells occurs at appropriate locations (for example,
Microencapsulation: An Alternative for the Application of Probiotic Cells
small intestine), promoting the adhesion and colonization of the intestinal epithelium. Currently, modern and innovative methods of microencapsu lation have been developed and, consequently, creating a wide variety of probiotic microcapsules. Thus, it represents an alternative for the food and pharmaceutical industry to increase the offer of probiotic products, attend a greater number of consumers and, also allow a varied application of probi otics in the market.
.2 PROBIOTICS
Probiotics are live microorganisms that which when administered in adequate amounts, confer health benefits on the host, act as therapeutic agents [1]. The microorganisms used as probiotics are Lactobacillus: L. acidophilus, L. bulgaricus, L. casei, L. delbrueckii ssp. bulgaricus, L. fermentum, L. johnsonii, L. rispatus, L. salivarius, L. bifidus, L. reuteri, L. plantarum, L. helveticus, L. casei subsp. rhamnosus, L. gallinarum, L. brevis, L. gasseri, L. cellobiosus, L. vitulinus, L. collinoides, L. cremoris, L. ruminis, L. dextranicum, L. lactis, L. rhamnosus, L. curvatus, L. faecium and L. paracasei; Bifidobacteria: B. bifidum, B. adolescentis, B. brevis, B. longum, B. animalis, B. infantis, B. thermophilum, B. breve, B. essencis, and B. lactis; Bacillus-B. coagulans, B. lactis, B. licheniformis, B. subtilis and B. subtilis; Pediococcus: P. acidilactici, P. pentosaceus and P. halophilus;
Lactococcus-L. lactis subspp. lactisand cremoris; Leuconostoc: L. mesen teroides subsp. dextranium, paramesenteroides, or lactis; Streptococcus: S. diacetilactis, S. cremoris, S. lactis, salivarius subsp. thermophilus, S. faeciu and S. equinus, Weissella: W. cibariaand confusa; Yeast: Saccharomyces cerevisiae and S. cerevisiae var. boulardii; Fungi: Aspergillus oryzae and Scytalidium acidophilum[2].
Probiotics belong to the different genres, species, and even phylum, and have been associated with numerous beneficial effects. High numbers of microorganisms are considered probiotics and, currently, research has discovered new species. The development and/or improvement of analyses also reveal and emphasize the beneficial effects derived by the consumption of probiotic microorganisms, as promoters of human health. Lactobacillus and Bifidobacteriumspecies are the most commonly used as probiotics and are normally presented in the GIT in a commensal relationship. Thus, the intestinal microbiota of healthy individuals may represent an important source of these species. Several studies have been demonstrated the isolation
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of probiotics from the oral and vaginal cavity, human milk and other species, colostrum, and various fermented products, indicating the diversity of sources for obtaining probiotics strains [3-5].
However, the selection of microorganisms considered as probiotic candi dates, isolated from these sources, requires a systematic realization of tests to ensure food security and the functional technological aspects. Moreover, the taxonomic identification is very important to avoid the choice of strains antibiotic-resistant, producers of harmful metabolic, infectious, or virulent. Also, microorganisms must have certain characteristics, such as survives physiological stress, acid pH of the stomach, and the presence of bile salts, to be able to adhere to the intestinal epithelium and colonize the colon and ability to compete against pathogenic bacteria. About the functional technological aspects, the probiotics generally need to be suited to the food matrix to maintain their viability, it is necessary to maintain an adequate level of viable cells during the processing and storage of the product and during passage through the human GIT and, besides, they cannot modify the sensory characteristics of food.
Although probiotics have demonstrated their benefits for years only in the last few decades, they have been widely used both in foods and in phar maceutical formulations. Among the beneficial effects caused by consump tion of probiotic microorganisms, there are: prevent the colonization of pathogens; to prevention for different gastrointestinal (GI) disorders (e.g., lactose intolerance, viral, and bacterial gastroenteritis, and inflammatory bowel disease (IBD)); antimicrobial activities due to the production of shortchain fatty acids, production of hydrogen peroxide and bacteriocins, and pH modulation. Moreover, probiotic bacteria can produce important nutrients from foods originating in the diet, for example, vitamin B, vitamin K, pyridoxine (vitamin B), folate, and thiamine, riboflavin, and menaquinone (vitamin K2). Lactobacillusand Bifidobacteriumhave been associated which the production of conjugated linoleic acid (CLA) and conjugated α-linolenic acid (CLNA). These compounds have anti-inflammatory, antioxidant, antiallergic, anticarcinogenic, and anti-obesity properties. Simultaneously, probiotic bacteria can: ferment proteins by producing peptides, amino acids, lactones, indoles, and phenols that collaborate in the energy balance and have antioxidant and anti-inflammatory effects; convert indigestible carbo hydrates into smaller sugars with the production of different compounds such as propionate, acetate, and butyrate; reduce the symptoms of lactose intoler ance due to the presence of β-galactosidase and modification of the intestinal microbiota [6]; make the anaerobic intestinal environment and, therefore,
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unsuitable for pathogenic bacteria; produce exopolysaccharides (EPS) that form biofilms which select beneficial bacteria and have anti-tumor activity in human cells, antioxidant, anti-inflammatory effects, inhibit mutagens and cholesterol reduction [7-9].
However, for probiotics to have beneficial effects on the host, a sufficient number of live cells is necessary during consumption and GI administration. Additionally, the probiotics cells must promote the adhesion and colonization in specific locations. Probiotic microorganisms promote specific benefits and, therefore, it may be necessary to use different strains to obtain all the expected results.
.3 APPLICATION OF PROBIOTICS
The consumption of probiotic microorganisms has increased considerably in recent years due to the numerous beneficial effects. Currently, the commer cialization of probiotics products can occur as pharmaceutical formulations and functional foods.
9.3.1 PHARMACEUTICAL FORMULATIONS
The probiotics are available in pharmaceutical formulations, in the form of powder, capsule, tablets, eye drops, etc., based on the nutraceutical appeal. For the manufacture of probiotic pharmaceutical formulations, the following steps are carried out: (i) production of biomass under pH control; (ii) cell recovery and concentration; (iii) drying of the concentrated; (iv) commercialization in capsule, powers, compressed, etc. These products require dosing and storage under conditions that security the viability of the microorganisms.
Regarding the powder formulation, these are presented in the form of dry and fine solid particles. Thus, these formulations form suspensions when dispersed in the water, easing its administration. The capsules consist of a hard or soft soluble container or “shell” with variable shape, capacity, and properties. Generally, the capsules are unit dosage containers containing the probiotic cells inside. Tablets are solid dosage forms containing the appropriate dose of cells and may be obtained by compressing a volume of particle or extruding, molding, and lyophilizing. The oral administra tion of tablets can be done through chewing, swallowed, or remaining in the mouth until the cells are released [10]. Medicated chewing gums also
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represent pharmaceutical formulations for the delivery of different probi otics and principally indicated for children. These products usually contain a gelatinous base and the active substance is released by chewing [11]. Other pharmaceutical formulas are bioadhesive gels, eye drops, and lozenges.
During all fabrication process is extremely essential to control the processing conditions, to check the dosage and viability of the probi otic’s cells. In conclusion, pharmaceutical formulations are excellent for the delivery of probiotic cells presenting great stability, security, and efficiency.
9.3.2 FUNCTIONAL FOOD
The lactic acid bacteria (LAB) are widely used in fermented products. In the past, these bacteria were identified to cause food spoilage, altering the compo nents, and decreasing the acceptability. Ancient people consumed fermented milk and cheeses obtained by the spontaneous action of these microorganisms. At present, these microorganisms are used to promote health benefits and their use by the food industry has grown. Food matrices have factors that benefit the growth and the survival of probiotic microorganisms, such as water, sugars, fat, proteins, and available peptides. Thus, the consumer benefits from nutri tion and also the benefits related to the consumption of probiotics.
The recommended number of viable cells to be ingested to obtain health benefits associated with probiotics is around 9 log CFU.g-1of the product
[12]. For food applications, a single microorganism, or a combination of them can be used.
Dairy products have the largest probiotic food market share, especially yogurts and fermented milk. Different combinations between initial and probiotics lactic cultures permit the fabrication of products with desirable technological, sensory, nutritional characteristics, and health benefits. The nutritionalcomposition of milkis highly complex, containing many compounds, such as proteins, minerals, fat, sugars. Therefore, it represents a food matrix adequate for the development and viability of probiotic micro organisms. Moreover, foods such as dairy products are known to improve the survival of probiotic microorganisms in gastric juice, probably due to the buffering power.
Studies report that the addition of milk or milk proteins significantly increases the pH of gastric juice and, consequently, improving the survival of some species of Lactobacillusand Bifidobacterium[13]. Recently, probi otic cheeses have been successfully developed for the market. Cheese as
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a food matrix carrying probiotic microorganisms has enormous potential because it is a solid matrix, with high fat content, proteins, higher pH values compared to fermented milk and have a buffer effect against acidic stomach conditions, creating an environment more favorable for the survival of probiotic microorganisms [14].
Ice cream, beyond composition, is a product widely appreciated product representing a good vehicle for probiotic microorganisms. Besides, it has relatively high pH values when compared to fermented products favoring the survival of probiotic cultures [15]. Dairy desserts also represent an interesting option for incorporating probiotic microorganisms. Due to the widespread consumption of these products by all age groups, they can contribute to reaching a wide consumer market.
Recent research reports that chocolate with a high cocoa content may confer to consumer health benefits, principally due to the presence of antioxidant compounds [16, 17]. Thus, because it is a product appreciated worldwide, the incorporation of probiotic microorganisms represents a strategy for the food industries. Succi et al. demonstrated that chocolate with a high cocoa content (80%) and phenols are an excellent environment to carrier probiotic bacteria such as Lactobacillus paracasei and Lactobacillus rhamnosus [18]. Silva et al. also observed that Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactiswere successfully incorporated
in dark chocolate, remaining viable for 120 days at 25°C and maintaining survival after in vitrosimulation of GI conditions [19].
Currently, the food industry has developed new carrier matrices for probiotic microorganisms because of factors such as lactose intolerance, milk protein allergy, the search for products with low fat and no cholesterol, the veganism, and vegetarianism.
Food matrices with potential for application of probiotic microorganisms include juices, meats, cereals, fresh vegetables, and fruits. Probiotic fruit juices are already commercialized and represent an interesting food matrix because of the presence of nutrients such as vitamins, minerals, fibers, and antioxidants. Additionally, they have no starter cultures that compete with probiotic cells for nutrients, and they are widely consumed, without restricting the population allergic to milk protein or lactose intolerant [20]. Besides, vegetable products such as cucumber, sauerkraut, and olives have shown good results as matrices for probiotic bacteria [21, 22]. Fermented meat products have demonstrated to be a viable option for obtaining a probiotic product. The probiotics microorganisms can be naturally present in meat or added as startercultures. Studies have shown satisfactory results compared
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to the growth of probiotic bacteria added to cereal-based products [23, 24]. Cereal grains are rich in proteins, carbohydrates, minerals, vitamins, and fibers that contribute to the metabolism of the probiotic microorganisms. Ribeiro et al. found that the mixed drink composed of banana, strawberry, and juçara has the potential to be a vehicle for Lactobacillus plantarumand Lactobacillus casei. The results obtained indicated high cell viability for 90 days of cold storage, besides, L. plantarumpresented viable populations above 6 log CFU.g-1after in vitro simulation of GI conditions [25]. Santos
et al. found that fermented cocoa juice added with sucralose maintained the viability of Lactobacillus caseifor 42 days (7.05 ± 0.04 log CFU.mL-1)
[26]. Akman et al. impregnated Lactobacillus paracaseiin apple slices and verified that the viability probiotic remained above 6 and 7 log CFU.g-1 after vacuum and oven drying, respectively [27]. Shigematsu et al. coated minimally processed carrots with sodium alginate added of Lactobacillus acidophilus (7.36 log CFU.g-1), followed by immersion in calcium chloride and verified that the cell viability of L. acidophilusremained at 7 log CFU.g-1 [28]. Rubio et al. when evaluating different strains of Lactobacillusduring the manufacture of fermented sausages, observed that L. rhamnosuswas able to grow up to 8 log CFU.g-1, dominating the endogenous population of LAB throughout the process of maturation [29].
.4 LIMITATIONS ON THE APPLICATIONS OF PROBIOTICS
As mentioned before, several beneficial effects have been reported due to the consumption of probiotic microorganisms. In recent years, there has been a significant increase in the variety of pharmaceutical formulas and probiotic foods. New research is constant, aiming to improve cell viability, develop new products, and, consequently, meet the highest consumer demand. The ingestion of probiotic microorganisms by food represents a more natural way when compared to pharmaceutical formulations. However, if we have several food matrices and the health benefits are numerous, why don’t we have an infinite variety of probiotic foods available on the market?
Although studies report that numerous food matrices are promising alternative as a vehicle for probiotic microorganisms, some limitations limit the incorporation. Therefore, the food industry, together with researchers, is trying to find alternatives to overcome these limitations and offer a wider variety of probiotic products at an affordable price. The main limitations in the development of probiotic products will be presented in this section.
Microencapsulation: An Alternative for the Application of Probiotic Cells
9.4.1 CHARACTERISTICS OF THE FOOD MATRIX AND PROCESSING
There are different types of microorganisms with probiotic potential and, there fore, present different characteristics and nutritional requirements. However, some factors are common to all the types of microorganisms used as probiotics, for example, the insertion in a nutrient-rich medium is extremely important for cellular survival. These nutrients may benefit one specifics probiotic, depending on the nutritional needs of each species. Additionally, to nutrients, other dietary properties also affect the viability of probiotic cells, such as water availability, pH, salt content, buffering capacity, presence of antimicrobials, among others. Also, probiotic microorganisms are anaerobic or microaerophilic, therefore, the presence of oxygen is toxic to these microorganisms. Moreover, it should be considered that the addition of probiotic microorganisms can cause changes in the sensory characteristics of foods. For example, some probiotic strains can produce enzymes that act on proteins, lipids, and carbohydrates producing compounds that modify the flavor, aroma, color, and texture.
The different stages of technological processing can expose probiotic microorganisms to adverse conditions, such as high temperatures, freezing, dehydration, presence of gases (CO, O), among other factors, and, conse
quently, significantly reduce the cell viability. Furthermore, in probiotic foods, the fermentation temperature can affect cell viability. The optimum temperature for growth of most probiotics is between 37°C and 43°C. Thus, temperatures above 45°C should be avoided if the probiotic microorganisms participate in the fermentation process, as many LABs. Besides, a vacuum fermentation system is recommended, because the oxygen content formed during the process is harmful to probiotic bacteria.
The storage temperature also influences the viability of probiotics being recommended temperatures between 4°C and 5°C. At freezing temperatures, the formation of ice crystals can cause damage to cells. Also, the crystalliza tion of water during freezing causes the cryogenic concentration of solutes, which can induce osmotic damage and reduce cellular metabolic activities. During thawing, cells are exposed to osmotic stress, hydrogen ions, oxygen, organic acids, etc.
Probiotic cultures usually come in the form of a dry powder, easily stored, and with a long shelf life. For this, bacterial dehydration processes are used, including freeze-drying and spray drying. In spray drying, high tempera tures, around 200°C, are used despite being a fast method, microorganisms are exposed to heat, osmotic, and dehydration stress, among others, which can reduce their viability. In the lyophilization process, in addition to the
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freezing, the dehydration step can modification the protein structure and the physical state of the lipid membranes, reducing the metabolic activity of the microorganisms [30].
During the storage of probiotic product factors such as the thickness of the packaging material, the permeability to light and oxygen, the presence of gases (CO, O, water vapor), among others, can affect the probiotic viability.
Therefore, when developing pharmaceutical formulations or probiotic func tional foods, the selection of appropriate processing steps is an essential factor for the survival of the microorganism.
9.4.2 SURVIVAL DURING PASSAGE THROUGH THE
GASTROINTESTINAL TRACT (GIT)
To promote the various health benefits, probiotic microorganisms must be alive and consumed in adequate quantities. However, the use of probiotic microorganisms is associated with some obstacles, such as the low survival rate during exposure to adverse conditions of the human GIT and the small residence time in the intestine [13, 133] . In the stomach and small intestine, for example, the environment is unfavorable for colonization and bacterial proliferation, due to the low pH of gastric juice, the presence of bile and pancreatic juice, and, also, the peristaltic movements.
After ingesting the probiotic cells, they are exposed to the acidic condi tions present in the stomach, the hydrochloric acid (HCl) has pH 0.9. However, the presence of the food can increase this value to pH 3 [31]. Probiotic cells are typically very sensitive to the highly acidic conditions of the human stomach. After passing through the stomach, probiotic cells are released into the small intestine, the decrease in probiotic viability caused by bile salts in the intestine occurs due to cellular disorganization caused by changes in the membrane. Subsequently, the cells reach the large intestine and, in the colon, the microorganisms find favorable conditions for their proliferation due to the absence of intestinal secretions, the slow peristalsis, and the abundant nutritional supply. The colon contains the highest microbial density and the population is known as microflora or intestinal microbiota [32].
To provide beneficial effects to the host, the probiotic must survive passage through the GIT and reach the intestine in adequate quantities. However, the loss of viability of probiotic microorganisms during passage through the GIT has led to the search for new strategies for maintaining viability. In this context, microencapsulation is the most prominent method for the protection of probiotics.
Microencapsulation: An Alternative for the Application of Probiotic Cells
.5 MICROENCAPSULATION
Microencapsulation is an effective technique to protect probiotic cells against adverse conditions and keep them in adequate amounts to confer health benefits on the host. Todd defined microencapsulation as the tech nology of packaging with a thin polymeric coating applied in solid, liquid, and gaseous materials [131]. Thus, forming particles known as capsules that release their contents at controlled rates over prolonged periods and under specific conditions. Arshady in 1993 [33] describes microcapsules as extremely small packages, composed of a polymer as wall material and an active material called core. Currently, the coated material in addition to being called a core is referred to as active material, internal phase, load, and the coating are called a wall material, membrane, shell, matrix, or external phase [34].
The particles formed may have regular or irregular shapes, and classified as mononuclear, polynuclear, or matrix. Mononuclear microcapsules contain the core bypassed by a defined and continuous film of the wall material. The multinucleated have many cores enclosed in the shell material. In the matrix, the microencapsulated material is uniformly distributed in the shell material [35].
One of the main factors that influence the stability of the encapsulated compounds is the type of coating used. Therefore, these materials must have specific characteristics, for example, easy control; low hygroscopicity; low viscosity at high concentrations; ability to disperse or emulsify and stabilize the core material; complete release of the solvent or other materials used during the encapsulation; maximum protection of the core under adverse conditions (light, pH, and oxygen); solubility in commonly used solvents; no taste or odor and low cost [36].
In the industry, the microencapsulation has decreased limitations and is used to protect compounds of external agents; release them in a controlled means, to mask undesirable flavors or odors. Materials that can be encap sulated consist of: acids, bases, oils, vitamins, salts, flavors, dyes, enzymes, and microorganisms.
.6 MICROENCAPSULATION OF PROBIOTICS AND RELEVANCE
INDUSTRIAL
As mentioned before, due to the varied applications, the probiotics market is one of the most promising sectors in the industry. Though, the inclusion of probiotics in products presents challenges and, thus, the microencapsulation
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represents an alternative to face the limitations related to the applications of probiotics. Microencapsulation is considered one of the more efficient technology to protect probiotic cells from adverse conditions.
The objective of encapsulation is to create a physical barrier between the microorganism and the external environment, increasing cell viability during processing, storage, and passage through the GIT. Thus, viability will be maintained and the cells released in appropriate locations (for example, small intestine) for adhesion and colonization of the intestinal epithelium to occur.
The lactic acid bacterium was first immobilized in 1975 on Berl saddles, and Lactobacillus lactiswas encapsulated in alginate gel beads years later [37]. Currently, the encapsulation of probiotic cells is advancing and permitting the development of innovative systems for probiotic products. About the food industry, the use of microencapsulation in probiotics has increased because of the new demands of probiotics products. Numerous food systems containing probiotic encapsulation have been introduced and accepted by consumers [38]. In this context, cellular microencapsulation has gained interest and, thus, increasing the varied application of probiotics in the market.
9.6.1 TECHNIQUES FOR MICROENCAPSULATION OF PROBIOTICS
Probiotics cells are affected by different factors and, thus, the encapsula tion techniques used need to be carried out in mild conditions, for example, low temperature, controlled agitation, low oxygen, moderate pH. Further more, not be used solvents that are toxic for microorganisms, non-GRAS (no generally recognized as safe) encapsulating agents, or that affect the sensory characteristics of the product. The size of the particles obtained must be adequate to protect probiotic cells and not modify the sensory characteristics of the product. The perfect characteristics for a microencapsulated probiotic are in the form of a dry powder, easily stored, and with a long shelf life, or a moist gel with long-term stability [39].
Modern and innovative methods of microencapsulation have been devel oped in recent decades, permitting the creation of a wide variety of probiotic microcapsules. However, it is important to note that industries probably prefer economic processes. Thus, the balance between cost and benefit must be considered. Then, the most important technologies to encapsulate probi otic cells will be highlighted.
Microencapsulation: An Alternative for the Application of Probiotic Cells
9.6.1.1 SPRAY DRYING
Historically, the spray drying process has been extensively used in the food industry. However, there was a rapid expansion of this technique for other sectors such as in pharmaceuticals, cosmetics, and textiles [40].
The main mechanisms involved in the microencapsulation using spray drying include the solubilization of a specific polymer together with the probiotic microorganisms and, then, the sprayed of the solution in contact whit a hot air stream, instantly producing powdered microcapsule [41]. Thus, it is a conversion process, in one step, fluid materials in solid or semi-solid particles [42]. In this technique, heat, and mass transfer occur simultane ously from hot air to atomized drops and vice versa, respectively [43].
The microencapsulation method using spray drying involves the compo nents presented in Figure 9.1. The process is carried out according to the following sequence of operations: Initially, the solution containing the probi otic microorganism and the polymer is prepared; posteriorly, this solution is homogenized and the atomized inside the drying chamber. During the passage through the drying chamber, heat, and mass transfer from the hot air to the atomized droplets occur and vice versa. Finally, the dry material is separated and collected by a cyclone. The process can produce micron- or nano-scale particles, in a short time [40].
FIGURE 9.1 Microencapsulation of probiotics using spray drying.
Spray drying is flexible and produces particles with a moisture content between 4 and 7%, causing better stability during storage [44]. Moreover, spray drying microencapsulation has low operating costs and high produc tion rates, justifying the preference of the industries [45].
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Vanden Braber et al. by microencapsulating Kluyveromyces marxianusand optimizing the spray drying process, observed that the air outlet temperature of 68°C allowed higher encapsulation efficiency (EE) and probiotic viability
of 8.38 CFU.g-1. Besides, microencapsulated microorganisms using chitosan
showed higher tolerance under simulated GI conditions compared to free cells and microencapsulated cells using whey protein concentrate [46]. Rosolen et al. used the spray drying technique for microencapsulation Lactococcus lactis subsp lactis R7. The probiotic cells in the microcapsules presented high viability (13.0 log CFU.g-1) and remained stable for 6 months (> 8.0 log CFU.g-1). After simulation in vitroof human GI conditions, the cells remained viable and, thus, surviving the effects of gastric and intestinal juices [47]. However, maintaining the high cell viability represents a challenge for microencapsulation thus, inlet air temperature, outlet air temperature, and type of the polymer are conditions that can negatively impact the survival of microorganisms [48]. Outlet air temperature above 85-90°C can injury macromolecules, such as proteins, DNA, and RNA, ribosomes, and membranes, being lethal to the probiotic microorganisms [49]. Thus, the factors optimization including the type of polymers and operational drying conditions can minimize damage [50]. Besides, the use of prebiotics can improve the survival of probiotics during processing steps [47, 51, 52]. Generally, the water-soluble polymers are used as wall material in the spray drying techniques, for example, whey proteins, maltodextrin, β-cyclodextrin, and gum Arabic [53].
9.6.1.2 FREEZE-DRYING
For decades, the freeze-drying technique was used for the manufacture of probiotic powders. However, currently, the technique has been associated with encapsulation and used for the production of microencapsulated probi otics [54]. The main application of this technique is the drying of thermo sensitive compounds aiming to improve the conservation of their functional properties [55].
Freeze drying (Figure 9.2) is a low-temperaturedehydration process that involves the removal of water by sublimation [56]. The process depends on heat and mass transfer and can be divided into three main steps: freezing, primary drying (sublimation), and secondary drying (desorption) [57, 58].
Initially, the material is frozen and exposed under vacuum conditions, thus, occur the sublimation process and the water content of dry material
Microencapsulation: An Alternative for the Application of Probiotic Cells
are about 15% [59]. Subsequently, secondary drying occurs by desorption, and the defrosted water absorbed by the material is removed. The residual moisture content of the freeze-dried material is around 1-3% [60] and then it is triturated, resulting in microparticles [61]. Among the steps mentioned, freezing is the most complex stage of freeze-drying due to the development of the structure that previously determines the properties of the dry material [62]. The materials commonly used as coatings are chitosan, gelatin, carrageenan, gum Arabic, gum guar, soy protein, disaccharides, and others [61, 63].
FIGURE 9.2 Encapsulation process by freeze-drying.
The microencapsulation of probiotics by freeze-drying stands out because drying at lower temperatures results in higher survival rates of the probiotic cells [64, 65]. Moreover, the freeze-drying material usually reconstitutes easily and has a long service life [66]. Besides, the selection of the wall material used is extremely important because it can act as a cryoprotectant and contribute to the stabilization of the encapsulated compound [61]. Some cryoprotectants such as sucrose, still, and mannitol are used to minimize the effects of freezing, stabilize the final particle size and prevent aggregation of particles during the process [67].
Li et al. performed the microencapsulation of Lactobacillus caseiby freeze-drying technique. The authors used combinations of whey protein, gellan gum, and cellulose acetate phthalate as wall materials on the microen capsulation. The results obtained showed that microencapsulation protected the probiotic cells and maintained high viability after in vitrosimulation of GI conditions and when exposed to heating [65]. Maleki et al. applied the technique to microencapsulate Lactobacillus rhamnosus.The formulation of the wall materials containing 57.22% whey protein, 25.00% crystalline
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nanocellulose, and 17.78% inulin presented the highest EE (89.60%). Further more, the formulation significantly improved the survival of probiotic bacteria during passage through simulated GI conditions. Thus, it can be a promising alternativefor the production of probiotic microcapsules for use in food and pharmaceutical products [68].
However, the freeze-drying technique has some disadvantages, for example, the need for an extended drying period, resulting in higher energy consumption; drying time may be different according to the sample thick ness and the deficiency of standardization of the particle size [69]; the forma tion of crystals during the freezing can damage the cell membrane and cause stress due to high osmolarity [49].
9.6.1.3 EXTRUSION
The extrusion is commonly applied for microencapsulation of probiotic microorganisms. The technique consists in the extrusion of a mixture containing the polymer and compound that will be encapsulated through an orifice and subsequent formation of droplets at the discharge point of the nozzle. Extrusion technique can be classified into simple drip, electrostatic extrusion, coaxial airflow, jet or vibratory nozzle, jet cutting, and rotary disc atomization. In the simple drip, low speed is applied and droplets with large diameters, around 2 mm, are formed, and production is insufficient for industrial application [70]. Electrostatic extrusion and coaxial airflow allow the production of smaller particles, from 50 µm to 200 µm, with uniform size distribution controlled by variation of the applied potential [49, 71]. Jet cutting, rotating disk atomization, and vibrating jet or nozzle are ideal techniques for mass production [72].
The extrusion technique is relatively simple and consists of the preparation of a solution containing the hydrocolloid and the probiotic microorganism (Figure 9.3). Then the material is forced through a nozzle, for example, a needle of the syringe or spray machine, and the cells suspension is then dripped into a hardening solution resulting in the formation of gelled drops [61].
For the microencapsulation of probiotics by extrusion, some polymers are frequently used as encapsulating material, for example, alginate, starch, agar, carrageenan, gelatin, and pectin [73, 74]. These polymers form a gel in contact with solutions containing minerals, particularly calcium and potas sium [75]. Among polymers, sodium alginate is the most frequently used [39, 74] and has a wide range of applications in the food and pharmaceutical industry [39, 76]. Sodium alginate forms a network structure similar to
Microencapsulation: An Alternative for the Application of Probiotic Cells
an “egg-box,” forming covalent crosslinking between alginate molecules capable of trapping materials such as probiotic cells [77].
FIGURE 9.3 Probiotic encapsulation by extrusion using alginate and calcium chloride.
The extrusion technique is frequently used because of its low cost and the high viability of probiotic cells [78]. Seth et al. evaluated the effect of microencapsulation using the extrusion technique on cell viability during the spray drying of sweetened yogurt. The results obtained showed a 2-log increase in the survival of the encapsulated cells. Besides, it was found that the concentration of sodium alginate significantly affected the size of the microcapsules, the EE, and the survival of the bacteria [79]. Dimitrellou et al. used the technique to microencapsulate Lactobacillus caseiin an alginate capsule for the manufacture of fermented milk. After in vitrosimulation of GI conditions and storage for 28 days of fermented milk, the encapsulation increased significantly the viability of the probiotic microorganism [80].
However, the extrusion technique produces large particles of a large size (500 to 1000 nm) that could influence the sensory characteristics of the product [41, 81]. Another factor is the porosity of the microspheres that allow the exposition of the encapsulated material, principally under acidic condi tions [78]. The addition of more polymer layers during the microcapsule production may be an alternative to decrease the porosity [81].
9.6.1.4 COMPLEX COACERVATION
Historically, the word “coacervate” is derived from the Latin “acervus” meaning aggregation, and the prefix “co” means the colloidal particles. Thus, in colloidal chemistry, the term “Coacervation” is used to denote the process
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of phase separation by modification of the environment (pH, ionic force, temperature, solubility). This process is characterized by the separation of a colloidal dispersion into two immiscible liquid phases: One phase has a high colloid concentration (coacervate phase) and another diluted with low amounts of colloid (equilibrium phase) [82].
In complex coacervation, at a specified pH, the electrostatic interaction between polymers of opposite charge results in the formation of soluble complexes. These complexes aggregate to decrease the free energy of the system, becoming insoluble and, subsequently, there is a separation of phases. In this process, the driving force is electrostatic interaction [83]. However, under conditions in which the electrostatic force is suppressed (for example, high concentration of salt), hydrogen bonds and hydrophobic interactions, among others, can contribute to the formation of coacervates [84]. During the encapsulation procedures based on complex coacervation, the coacervate polymer is deposited around the active ingredient (core), occurring the sedimentation of the encapsulated cores (Figure 9.4).
FIGURE 9.4 Encapsulation process based on complex coacervation.
The coacervation process isaffected by multiplefactors, including, the electrical charge of polymers, polymer concentration, ionic strength, temperature, pH, etc., [85]. These parameters are usually optimized aiming the highest yield and functionality of the complex coacervates. The analysis of zeta potential, absorbance, and yield of the dehydrated coacervates are used to optimize. The complex coacervation technique has varied advan tages compared to other techniques such as versatility, ease of operation, mild conditions, and low costand environmental impact [86]. Besides, the microcapsules produced have an excellent controlled release, modulated by changes in ionic strength, pH, and temperature [18].
Microencapsulation: An Alternative for the Application of Probiotic Cells
Complex coacervation is an old method that has been used in different industrial applications such as pharmaceutical, chemical, cosmetic, and food industries. The polymers generally used for encapsulation by complex coac ervation are gelatin, whey protein, Arabic gum, chitosan, pectin, alginates, xanthan gum, carrageenan, and carboxymethyl cellulose. One disadvantage of the complex coacervation technique is the low mechanical and thermal resistance because of the ionic nature of the interactions, it is necessary to strengthen the structure, for example, to crosslink the polymeric chains [87].
Da Silva et al. produced microcapsules containing B. lactisby complex coacervation using gelatin and gum Arabic as coating materials. Micro encapsulated probiotics-maintained viability after in vitrosimulation GI conditions and, also, the complex coacervation method was efficient in maintaining the viability of probiotics during storage at temperatures of -18°C for 120 days, 7°C for 120 days and 25°C for 90 days [86]. Paula et al. by microencapsulating probiotic cells of Lactobacillus plantarumthrough emulsification followed by complex coacervation using gelatin and gum Arabic. The authors observed that after in vitrosimulation of GI conditions, the viability of encapsulated cells was 80.4%, while for free cells, it was 25%. Additionally, cell viability was maintained during storage at 8°C and -18°C for 45 days. Thus, the results obtained show that the complex coacervation is an appropriate alternative to increase the viability of probiotics [88]. However, although the complex coacervation process is considered as promising for the encapsulation of probiotics, it is little used. Therefore, there is a need to explore more studies on coating materials, the variation of concen trations and association with drying techniques, are desirable to increase the protection of probiotic and allow a more effective application [86].
9.6.1.5 EMULSIFICATION TECHNIQUES
9.6.1.5.1 EMULSIONS
Emulsions are defined as heterogeneous systems and thermodynamically unstable composed of a mixture of two immiscible liquids, in which one liquid (the dispersed phase) is in the form of droplets dispersed in the other (continuous) phase [89]. In general, simple emulsions are categorized according to their dispersed phase. When the oil is the dispersed phase, the emulsion is of the oil-in-water (O/W) type, however, if the water is the dispersed phase, the emulsion is of the water-in-oil (W/O) type. In the food
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industry, products such as milk, sour cream, soups, yogurts, ice cream, butter, and margarine are examples of simple emulsions.
The emulsions do not form spontaneously and require a considerable involvement of energy, usually mechanical [90]. In food processing are used high-speed mixers, colloid mills, homogenizers, and ultrasonic mixers. Moreover, a third component or combination of active and surface agents, often called an emulsifier or emulsifying agent is used [91]. The most used emulsifiers are lecithin; mono and di-glycerides of fatty acids, proteins, phospholipids, and, in certain cases, polysaccharides (hydrocolloids).
Typically, each method has associated advantages and disadvantages. The main disadvantage of the emulsification technique is difficulty in stan dardizing the microcapsule size distribution. Additionally, it also presents difficulties in implementation and the requirement of vigorously agitated which can be potentially injurious to cells, and the inability to sterilize vegetable oil if it is necessary strict aseptic conditions.
The principle of this technique is based on the dispersion of the solu tion of polymer and probiotic cells (dispersed phase), in a large volume of oil, usually vegetable oil (continuous phase). The mixture is homogenized continuously resulting in aqueous/polymer droplets containing the probiotic cells, specifically, the formation of an emulsion water-in-oil (W/O) type. However, after formation, the water-soluble polymer (present in the aqueous droplets) can be insolubilized causing gelation of the aqueous/polymer drop lets. In this case, the emulsification technique can be divided into internal or external ionic gelation.
9.6.1.5.2 EMULSIFICATION/INTERNAL IONIC GELATION
In this method, occur the addition of a solution containing insoluble calcium salt (usually calcium carbonate) in a polymeric solution (for example, sodium alginate) containing probiotic cells, with subsequent dispersion of this mixture in an oily phase containing surfactant for the formation of emulsion W/O (Figure 9.5). Subsequently, for the gelation process to occur, it is neces sary to add an acid solution, that is, reduce the pH and thus release calcium ions (present inside the emulsion droplets), allowing its complexation with the alginate carboxylic groups [92]. In this case, Ca+is crosslinked with
sodium alginate from the inside out of the droplet. The microcapsules are recuperated by filtration or centrifugation.
Microencapsulation: An Alternative for the Application of Probiotic Cells
FIGURE 9.5 Encapsulation of probiotics by internal ionic gelation.
Many studies report that microcapsules prepared by internal ionic gela tion have greater size standardization and better EE [93].
9.6.1.5.3 EMULSIFICATION/EXTERNAL IONIC GELATION
In external ionic gelation, the polymeric solution containing the probiotic cells is initially dispersed in an oily phase containing surfactant for the forma tion of emulsion W/O. Subsequently, a solution containing insoluble calcium salt is incorporated, usually calcium carbonate, with the ion diffusion into the aqueous alginate droplets. Consequently, gel formation occurs from the surface into the droplets (Figure 9.6) [94]. According to King [134], it is possible to manipulate the strength of the gel through changes in processing conditions, such as pH, calcium concentration, concentration, and source of alginate (algae species), etc.
.7 INCREASED RESISTANCE OF MICROENCAPSULATED
PROBIOTICS
In some cases, the use of the encapsulation technique alone does not maintain the satisfactory viability of microorganisms. Thus, technological resources can be used, such as the addition of cryoprotectants, the use of prebiotics, the formation of covalent bonds called crosslinking, and the increase of polymer layers around the microcapsules.
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FIGURE 9.6 Encapsulation of probiotics by external ionic gelation.
The addition of cryoprotectants such as sugars (e.g., trehalose and sucrose) is used to reduce the osmotic difference between the interior of a cell and the exterior environment and can minimize the damage caused by cold. The addition of the prebiotics in the formulation of the microparticles results in a symbiotic combination. Currently identified prebiotics are non-digestible carbohydrates, however, beneficial bacteria can ferment, stimulating the growth and/or activity of bacteria in the colon [135]. The most frequently studied examples are fructans and fructooligosaccharides of the inulin-type. Raddatz et al. using different prebiotics in combination with the microen capsulation of Lactobacillus acidophilusLA-5 found greater cell viability after in vitrosimulation of GI simulation and under storage conditions of 25°C to -18°C [95].
The formation of covalent bonds that hold portions of several polymer chains together is called. The crosslinking can be used to improve the thermal properties of the microcapsules [87]. However, most of the chemical crosslinking such as formaldehyde, glutaraldehyde, glyoxal, diisocyanate, epichlorohydrin have their applications limited due to toxicity and difficulty of complete removal [96]. Consequently, enzymatic crosslinking represents an alternative [97] and transglutaminase is an enzyme widely used to improve the rheological and physical properties of microcapsules [98]. Da Silva et al. evaluated the use of transgluta minase to improve the resistance of microcapsules obtained by complex coacervation. After in vitrosimulation of GI conditions, it was found that microencapsulation together with crosslinking showed good results. Besides, after storage, probiotic viability was maintained for up to 60 days in freezing temperature, with counts of up to 9.59 log CFU.g-1. The
Microencapsulation: An Alternative for the Application of Probiotic Cells
results obtained are innovative and present a promising alternative for the protection of probiotics [99].
The increase of polymer layers around the microcapsules provides extra protection for cells from adverse conditions [39, 100]. According to Grig oriev and Miller for the formation of polymer layers, electrostatic deposition layer by layer can be used. A wide variety of materials have been explored to protect the capsules, including alginate, pectin, starch, and chitosan [101]. Etchepare et al. show that L. acidophilus microencapsulated in calcium alginate particles coated with multilayers showed greater survival under simulated GI conditions, heat treatment, and during storage [102].
.8 BIOPOLYMERS USED IN MICROENCAPSULATION
In the microencapsulation of probiotics, the microcapsules must be able to maintain their structure even under adverse conditions, releasing your content at controlled rates and/or under specific conditions. Generally, the microcapsules release their content because of pH changes, chelating agents, and enzymatic action. Thus, as mentioned before, the coating material must have some characteristics, among them, easy handling; low hygroscopicity; low viscosity at high concentrations; disperse/emulsify and stabilize the core material; good film-forming properties; complete release of the solvent; protection of the core against adverse conditions (for example, light, pH, and oxygen); solubility in commonly used solvents; absence of taste or odor and low cost [36]. The polymers most widely used for this purpose are then highlighted:
. Gelatin:It is a naturally derived polymer that can be obtained
through acidic (Type A) or alkaline (Type B) hydrolysis of collagen. The gelatin represents the main commercial option as wall material due to its excellent water solubility, emulsification, and thickening capacity and high crosslinking activity. Also, the polypeptide struc ture of the molecule facilitates interactions with other polymers of opposite electrical charge, making it an important wall material [103]. However, gelatin solutions, even in low concentrations, have high viscosity and can cause problems of aggregation and agglutina tion during the preparation of the microcapsule process [136].
. Arabic Gum: It is an exudate of acacia trees, of which there are
species distributed over tropical and subtropical regions. In general, the composition of gum Arabic consists basically of two fractions.
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One of the fractions, representing 70% of the gum, is composed of a polysaccharide chain with little or no protein. The other fraction contains molecules of higher molecular mass with proteins as part of their structure [104]. This composition confers an efficient surface property, besides having cold solubility due to the presence of loaded groups and peptide fragments [105]. About the electrical charge, gum Arabic is a weak polyelectrolyte that has a negative charge at a pH greater than 2.2, due to its carboxyl groups [106, 107]. Industrially it is widely used due to its surface activity, low viscosity, emulsifying capacity in aqueous solutions, as well as being non-toxic, odorless, and tasteless.
. Chitosan: It is a biopolymer derived from chitin composed of
N-acetyl-d-glucosamine units, linked by β (1,4)-glycosidic bonds. Chitosan can be obtained by deacetylation of chitin, which, which is the major constituent of exoskeletons of crabs, shrimp, and other crustaceans.
Chemically, chitosan contains groups of free amines in a neutral or alkaline environment, while due to the protonated amine groups (NH+) at pH > 6.3, the polymer becomes soluble in water and posi tively charged. Thus, the characteristics make chitosan suitable for controlled release technologies under specific conditions at the local target. Besides, chitosan is a biocompatible, biodegradable, inexpen sive, and non-toxic polymer, making it attractive for applications in the medical, cosmetics, agriculture, food, and textiles [108].
. Sodium Alginate: It consists of an anionic polysaccharide extracted
from the cell wall of brown algae (Laminaria spp.), it is composed of β-1,4 glycosidic bonds formed between the β-D-mannuronate and α-L-guluronate residues [109]. Alginate molecules can form a struc ture called “egg-box” through crosslinking and exchanging sodium ions for divalent cation such as calcium [110]. Sodium alginate is one of the most used wall materials for microencapsulation [111] due to its biocompatibility, non-toxicity, low cost, and ability to form gel in the presence of calcium ions [112]. However, microcapsules are sensitive in acidic conditions and have a porous structure that affects their stability and efficiency [113].
. Maltodextrin:It is composed of multiple glucose units linked by α-(1,4) glycosidic bonds and obtained from the partial hydrolysis of starch with acid or enzymes [114]. They are classified according to the degree of hydrolysis, expressed in dextrose equivalent (DE)
Microencapsulation: An Alternative for the Application of Probiotic Cells
which is the percentage of reducing sugar calculated as dextrose on a dry-weight basis [115]. They are commonly used as wall material in microencapsulation technique because they have low density, low viscosity when used at high temperature, good solubility in water, do not alter the characteristics of the product, and have a low cost [116]. However, they have some limitations, such as low emulsifying capacity and retention of volatile compounds, and thus are used in mixtures with other wall materials [117].
. Gellan Gum:It is an anionic polysaccharide with a linear structure of
a repetitive tetrasaccharide sequence consisting of two β-D-glucose residues, one of β-D-glucuronate and one of α-L-rhamnose [74]. Obtained by microbial fermentation from the bacteria Sphingomonas elodea or Pseudomonas elodea [118]. Commercially, gellan gum is available in two forms, being high acyl (acylated) and low acyl (deac ylated) and each type has individual properties [119]. In the presence of cations, high-acyl gelatin gum forms soft, flexible hydrogels after cooling to 65°C, while low-acyl gum forms rigid and brittle hydrogels after cooling to 40°C [120]. The deacylated form has been used successfully as a coating material for probiotic microencapsulation [74], and when mixed with other types of gum, such as xanthan gum, it has a high resistance to acidic conditions [121].
. Xanthan Gum: Among the gums used in microencapsulation,
xanthan gum is the most used. It consists of a high molecular weight extracellular polysaccharide that is produced by fermenting the bacteria Xanthomonas campestris[122]. Xanthan gum has some characteristics such as tasteless, odorless, has low viscosity, stability at high temperatures, good solubility in water, and can be widely used [123].
. Carrageenans Gum: These are neutral polysaccharides extracted
from red algae (Rhodophyta) and commonly used in the food industry. Red algae are capable of producing three distinct types of commer cial carrageenan (κ-, ι-e λ-carrageenan) that differ in their structures and chemical properties [124]. They are extremely compatible with microbial cells, ensuring high viability during microencapsulation techniques, however, the gel formed presents physical instability in adverse conditions [41, 125].
. Milk Proteins: These are divided into two groups, caseins (80%) and whey proteins (20%). Caseins are relatively hydrophobic, but they also have polar and charged residues, thus representing natural
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and viable polymers for the encapsulation of compounds. The physic-chemical properties and structural enable its functionality, with emphasis on the binding of ions and small molecules, surfactant properties, emulsifying, and water-binding capacity. Moreover, if the ability to protect or compose its structure, allows it to control its bioavailability [126]. In addition to low cost, it is considered non-toxic, heat-stable, and GRAS. The main whey proteins are β-lactoglobulin, ɑ-lactoalbumin, bovine serum albumin, lactoferrin, and immunoglobulins, which can vary in size, molar mass, and func tion [127]. Whey proteins are also recognized as GRAS, low cost, high nutritional value, have techno-functional properties (ability to form gels, foams, and emulsions), good sensory properties, and structural properties making them suitable for the transport of other molecules. However, they are globular proteins with a high level of structural organization, being susceptible to denaturation. Depending on the conditions of the environment, such as pH, strength, and temperature, these substances can exist in individual or agglomer ated form, these characteristics being important for the development of distribution systems.
.9 FINAL CONSIDERATIONS
The growing number of benefits presented by regular consumption of probi otics has attracted the attention of the pharmaceutical and food industry in recent years. The development of different probiotic formulations possibility the optimization of the delivery of microorganisms. The commercialization of a wide variety of probiotic foods is a market trend. The different options such as juices, chocolates, cereals, and yogurts, reach different audiences, with different dietary restrictions, providing an improvement in health from children to the elderly. Besides to Lactobacillusand Bifidobacteria, others have been reported as probiotic species and thus allowing the industry to use the microorganism with the greatest potential for processing and manufac tured product. Besides, the use of more than one microbial species permits a wide range of benefits provided to human health by probiotics. Although numerous pharmaceutical formulas and food products
containing probiotic microorganisms are already available on the market, the new product development still represents a true challenge. Among all factors, the main problem is to maintain the viability of probiotic microorganisms.
Microencapsulation: An Alternative for the Application of Probiotic Cells
Besides¸ the nutritional requirements, the processing steps, the characteris tics of the food matrix, and the conditions during the passage through the GIT can affect the survival of the cells and, therefore, the health benefits of consumers.
In this way, the development of innovative technologies is an important area to optimize the manufacture of probiotic products. Among the tech nologies used, microencapsulation is considered one of the most efficient to protect probiotic cells from adverse conditions. The objective of micro encapsulation is to create a physical barrier between the microorganism and the external environment, increasing cell viability during processing, storage, and passage through the GIT. Thus, viability will be maintained and cells released at appropriate sites (for example, small intestine) for adhesion and colonization of the intestinal epithelium.
However, encapsulation systems must be economically viable, relatively easy to handle, efficient, and safe. These characteristics depend on the technology and the polymers used as a coating. The search for biocompat ible, non-toxic, biodegradable, and naturally sourced materials represents a preferred solution. Thus, making it possible to expand the application of probiotics in different foods, the development of different pharmaceutical formulas, and, consequently, reach a larger target audience.
KEYWORDS
• conjugated linoleic acid •dextrose equivalent •exopolysaccharides •gastrointestinal tract •generally recognized as safe • lactic acid bacteria
• oil-in-water
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127. Fox, P. F., McSweeney, P. L. H., Cogan, T. M., & Guinee, T. P., (2000). Fundamentals
of Cheese Science (1stedn.). Springer US.
. Cruz, A. G., Buriti, F. C. A., Souza, C. H. B., Faria, J. A. F., & Saad, S. M. I., (2009).
Probiotic cheese: Health benefits, technological and stability aspects. Trends in Food Science & Technology, 20(8), 344-354. https://doi.org/10.1016/j.tifs.2009.05.001.
129. Leuenberger, H., (2002). Spray freeze-drying-the process of choice for low water
soluble drugs. Journal of Nanoparticle Research, 4(1, 2), 111-119.
. Succi, M., Tremonte, P., Pannella, G., Tipaldi, L., Cozzolino, A., Coppola, R., &
Sorrentino, E., (2017). Survival of commercial probiotic strains in dark chocolate with high cocoa and phenols content during the storage and in a static in vitrodigestion model. Journal of Functional Foods, 35, 60-67. https://doi.org/10.1016/j.jff.2017.05.019.
131. Todd, R. D., (1970). Microencapsulation and flavor industry. Flavor Industry, 1(11),
768-771.
132. Tripathi, M. K., & Giri, S. K., (2014). Probiotic functional foods: Survival of probiotics
during processing and storage. Journal of Functional Foods, 9, 225-241. https://doi. org/10.1016/j.jff.2014.04.030.
133. Gandomi, H., Abbaszadeh, S., Misaghi, A., Bokaie, S., & Noori, N. (2016). Effect of
chitosan-alginate encapsulation with inulin on survival of Lactobacillus rhamnosusGG during apple juice storage and under simulated gastrointestinal conditions. LebensmittelWissenschaft + Technologie, 69, 365-371. http://dx.doi.org/10.1016/j.lwt.2016.01.064 (accessed 10 August 2021).
134. King, A. H. (1988). Flavor encapsulation with alginates. ACS Symposium Series, 370,
122-125.
. Vieira da Silva, B., Barreira, J. C. M., & Oliveira, M. B. P. P. (2016). Natural
phytochemicals and probiotics as bioactive ingredients for functional foods: Extraction, biochemistry and protected-delivery technologies. Trends in Food Science & Technology, 50, 144-158.
136. Xu, C., Lei, C., Meng, L., Wang, C., & Song, Y. (2012). Chitosan as a barrier membrane
material in periodontal tissue regeneration. J Biomed Mater Res Part B Appl Biomater 100, 1435-1443.
CHAPTER 10
Nutraceuticals-Based NanoFormulations: An Overview Through Clinical Validations
SHELLY SINGH and SHILPA SHARMA
Department of Biological Sciences and Engineering, Netaji Subhas University of Technology, Dwarka, New Delhi, India
ABSTRACT
Due to surge in diseases and awareness about health, the requirement for modified food products, known as “nutraceuticals” has also increased. In order to formulate nutraceuticals, food products are fortified with nutrients and essential elements to treat various disorders and diseases. But due to certain disadvantages like poor solubility, low bioavailability, poor adsorp tion, low stability, low permeability in vivo, etc., the potential of nutraceu ticals has not been utilized fully. Nanotechnology is increasingly being used to address such issues. Nanotechnology has been used to enhance the quality of nutraceuticals, for detection and sensing of chemical and biological contaminants and for preservation and packaging of nutraceuticals, thereby increasing their shelf life. Nanotechnology has also been used to encapsu late nutraceuticals to form nano-nutraceuticals which have enhanced thera peutic activities, better solubility and increased bioavailability. The chapter discusses various nano-nutraceuticals with their applications and therapeutic outcomes, commercially available nano-nutraceuticals and nutraceuticals based nano- delivery systems through their clinical validations.
.1 INTRODUCTION
Nutraceuticals are defined as “designer food” products, fortified by nutrients such as essential elements, minerals, amino acids, vitamins, etc., which
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not only act as dietary supplements but are also used in the prevention and treatment of various diseases [1]. The word ‘nutraceutical’ was coined in 1989 by Stephen L. DeFelice, Founder and Chairman of the Foundation of Innovative Medicine (New York), by combining two words viz.‘nutrition’ and ‘pharmaceutical’ and hence is defined as hybrid of food and drug [2]. The nutraceuticals may function as immunomodulators and provide health benefits against various diseases and disorders like cancer, neurological diseases, cardiovascular disorders, respiratory disorders, diabetes, obesity, etc. Nutraceutical products have historical aspect as well because in the ancient times, many civilizations and indigenous tribes used to depend on natural herbs and minerals. In today’s world, much of the information is acquired from plants and herbs used during ancient times. Some examples of different nutraceuticals and their therapeutic outcomes are presented in Table 10.1.
TABLE 10.1 Some Examples of Nutraceuticals and Their Therapeutic Outcomes
Nutraceuticals Therapeutic Outcomes References
Carotenoids Antioxidant, pro-vitamin A activity, cholesterol, [3]
cataract, and other chronic diseases.
Flavonoids Antioxidant, prevent enzymatic oxidation of [4]
ascorbic acid
Omega 3-PUFA Cardiovascular diseases, prevention of [5]
atherosclerosis
Anthocyanins Neuro-protective effects, liver health improvement, [6]
and anti-inflammatory effect
Theobromine Antioxidant and psychoactive effects [7]
Terpenoids Therapeutic agent for liver cancer and [8]
chemopreventive agent
Caffeine Sleep therapeutics [9]
Eucalyptol Antioxidant, anti-inflammatory, and [10]
gastroprotective
Curcumin Anti-bacterial, anti-inflammatory, anti-diabetic, [11]
anti-viral, antioxidant, anti-venom, anti-obesity,
anti-arthritis, anti-depressant, and wound healing
Resveratrol Antioxidant, anti-inflammatory, immunomodulatory, [12]
glucose, and lipid regulatory, neuroprotective, and
cardio-vascular protective effect
Quercetin Anti-diabetic, anti-inflammatory, and anti-cancerous [13]
Lutein Effective against age-related macular degeneration, [14]
cardiovascular diseases, cataracts, and certain types of cancers
Nutraceuticals-Based Nano-Formulations
TABLE 10.1 (Continued)
Nutraceuticals Therapeutic Outcomes References
Co-enzyme Q10 Anti-inflammatory, antioxidant, anti- [15]
hyperlipidemic, anti-hyperglycemic, cardioprotective, and neuroprotective
Lycopene Effective against certain cancers like colon, skin, [16]
and prostate cancer; cardiovascular diseases and is a strong antioxidant
Phytosterols Lowers ratio of low density to high density [17]
lipoprotein bound cholesterol in serum and lowers blood cholesterol
Gallic acid Antimicrobial, antioxidant, anti-cancerous, anti- [18]
hypertensive, anti-inflammatory, anticoagulant,
hypolipidemic, and hypoglycemic
Polyphenols Anti-diabetic, modulate lipid and carbohydrate [19]
metabolism, improve adipose tissue metabolism,
cardiovascular diseases, neuropathy, and retinopathy
Caffeine Antioxidant and Central nervous system stimuli [20]
Tangeretin Anti-inflammatory and anti-cancerous [21]
Phosphatidyl Improves brain metabolism, memory, and brain [22, 23]
Serine activity in early stages of Alzheimer’s disease
Capsanthin Chemopreventive effects [24]
Β Lapachone Targets colon and lung cancer cells [25]
Toxifolin For liver health [26]
Gambogic acid Acts against lymphoma cells [27]
Probiotics Gastrointestinal diseases [28]
Nucleic acid Anti-cancerous [29]
Functional yogurt Anti-cancerous [30]
Piperine Gastrointestinal diseases [31]
Source: Adapted from Ref. [74].
In 2014, the global market of nutraceuticals was at US $165.62 billion and according to the report published by transparency market research in September 2015, the market is growing at a compound annual growth rate (CAGR) of 7.3% from 2015 to 2021 and by the end of 2021 the market is expected to reach US $278.96 billion [32]. Some examples of companies operating in the global nutraceuticals market are Royal DSM N.V., Archer Daniels Midland Company, BASF SE, Cargill, Incorporated, Groupe Danone S.A., E. I. du Pont de Nemours, Nestle S.A., and Company, General Mills, Inc, etc.
TABLE 10.2 Some Commercial Examples of Nano-Nutraceutical Products
Nano- Active Component Manufacturer Nanomaterial Used Benefits
Nutraceutical
Product
Lipimed Lactosorb complex Lactonova® Encapsulated in liposomes Regulates cholesterol level
Glutasolve Glutamine Nestle healthcare Delivery using gold Treats deficiency of glutamine caused
nutrition nanoparticles because of injury or illness
Nano curcumin Curcumin Neurvana® Polymeric nanoparticles Wound healing
Nano resveratrol Resveratrol Neurvana ® Solid lipid and polymeric Anti-cancer, anti-inflammatory, and
nanoparticles anti-diabetes.
Mi-omega NF Omega-3 polysaturated Midlothian Nanoemulsions Reduces risks of heart diseases and
fatty acid, Folic acid laboratories promotes healthy skin
Glutagut powder L-glutamine Bionova® Encapsulated in nanocarriers Dietary supplement which promotes gut
and brain health
Nevical forte Calcium carbonate, Folic Bionova® Nano calcium Treatment of osteoporosis, joint
soft gel acid inflammation, and arthritis
Casein Casein Chaitanya Casein nanoparticles Used in production of minimal media for
hydrolysate Agrobiotech sporulation by resuspension
Promilk Calcium Chaitanya Nano calcium In bone health and body cell functioning.
Agrobiotech
Soya concentrate Soy protein Chaitanya Nano aggregates Protein supplement
Agrobiotech
Cinnamon Essential oil Lactonova ® Encapsulation in chitosan Lowering of blood sugar level and
extract nanoparticles reduces heart disease risks.
Mulberry leaf Chlorogenic acid Lactonova® Nano crosslinking particles Has an antioxidant property.
extract
TABLE 10.2 (Continued)
Nano- Active Component Manufacturer Nanomaterial Used Benefits
Nutraceutical
Product
Chondroitin Glucosamine Lactonova ® Chondroitin sulfate Helps in the treatment of osteoporosis.
sulfate nanoparticles
Sydlife-D Lozenges Sydler ® Nanoencapsulation Helps in weight loss.
Nutrisyd Biotin Sydler ® Nanoemulsions For skin and bone health.
Vitabuz Multivitamin Zeon Lipid-based nanoparticles Dietary supplement (multivitamin)
Lifesciences Ltd.
Biotrex Biotin Zeon Lipid nanoparticles Helps to form red blood cells, has
Lifesciences Ltd. antioxidant property, and maintains
energy level.
Co-enzyme Ubiquinone Agati Healthcare Carried by lipid nanoparticles For growth and maintenance of cells.
Q-10 Ltd.
Fracpro Cissus quadrangularis Neiss Wellness® Lipid nanoparticles Prevents chronic diseases, anti-aging,
and improves health.
Nano tea Selenium antioxidant Qinhuangdao Nanoparticles Absorbs cholesterol, fat, viruses, and
Taiji Ring free radicals and good supplement of
Nanoproducts selenium
Company Ltd.
Nanoceuticals™ Artichoke RBC Nanoclusters Immunity booster, balance body pH and
artichoke Lifesciences® provides hydration
nanoclusters
Canola active oil Phytosterols Shemen Nanodrops (nanosized lipid Inhibits uptake and transportation of
Industries, Israel micelles) cholesterol
TABLE 10.2 (Continued)
Nano- Active Component Manufacturer Nanomaterial Used Benefits
Nutraceutical
Product
OilFresh® Oil conditioning device OilFresh Nanoparticles Enhances heat conductivity of oil for
Corporation, faster cooking at low temperature.
USA
LifePak® Vitamins, minerals, and Pharmanex® Nanoparticles Boost’s immunity, helps in
fatty acids cardiovascular and brain health
Novasol® Coenzyme Q 10, Vitamin Aquanova® Nano micelles Provides stability to nanomicelles with
ADEK-Q10 A, D, K, and E respect to pH and temperature
Nano C Vitamin C Neurvana® Nanoparticles Enhances bioavailability of quercetin and
α-lipoic acid
Source: Adapted from Ref. [74].
Nutraceuticals-Based Nano-Formulations
The use of majority of nutraceuticals is limited by poor bioavailability, poor adsorption, low stability, low solubility, safety, ineffective targeting, and low permeability in vivo. Therefore, efforts are underway to increase the efficacy, metabolism, and prevent the physical and chemical degradation of nutraceutical products in order to achieve improved therapeutic effects. In this context, nanotechnology is increasingly being used to target the abovementioned drawbacks. Nanotechnology is defined as the understanding and control of matter at dimensions of roughly 1-100 nm, where unique phenomena enable novel applications [33]. The properties of nanoparticles (NPs) change drastically from their bulk counterparts as the surface-to volume ratio increases tremendously. The nutraceuticals formulated using nanotechnology are called nano-nutraceuticals. Table 10.2 represents some examples of commercial nano-nutraceuticals. They have improved pharma cokinetic and physicochemical properties. The nano-dimension and large surface area per unit mass of nanomaterials lead to higher mucoadhesive possibility within the small intestine and also higher chances of interac tion with the enzymes and metabolic factors in gastrointestinal tract (GIT) thereby leading to enhancement in the biological activity, bioavailability, and solubility of encapsulated nutraceuticals [34, 35]. This enables increased therapeutic effect of nutraceuticals at low dose and hence reduced possible risk of associated toxicity as compared to nutraceuticals alone. However, safety, and quality of nano-nutraceuticals need to be tested before they reach the market. This chapter gives an overview of nutraceuticals being formu lated using nanotechnology through their clinical validations.
.2 APPLICATIONS OF NANOTECHNOLOGY IN THE
NUTRACEUTICAL INDUSTRY
Over the years, nanotechnology is being increasingly used in the food industry. Figure 10.1 showcases various applications of nanotechnology in the nutraceutical industry. Table 10.3 represents some patents of nano formulations of nutraceuticals.
10.2.1 NANOTECHNOLOGY FOR ENHANCING QUALITY OF
NUTRACEUTICALS
With increasing population literacy and improved lifestyle of people, demand for nutritive products is escalating day by day. People are becoming aware
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about their health and the need to maintain it. There is an increasing demand for products which have positive outcome in wellbeing and are able to provide extra nourishment. For this to happen, nanotechnology is being used to develop novel nutraceutical products which can provide nourishment, fight diseases and also are low in toxicity. Nanotechnology is being used to improve the structure, texture, flavor, and fat content of the already existing product. For example, companies like Unilever and Nestle have report edly developed nanoemulsion-based ice creams with low-fat content with retention of fatty texture and flavor like their full-fat alternatives, thereby providing a healthier option to the consumer [46]. In one study, paprika oleoresin NPs were used for increasing the marinating performance, i.e., color of the surface, color penetration, saltiness, paprika flavor, toughness, and juiciness of poultry meat, suggesting use of NPs for improvement in mari nating performance and sensory acceptability of marinated meat products [47]. Similarly, a German company, Aquanova has employed 30 nm micelles named as “NovaSol” to encapsulate nutraceuticals such as vitamin E, vitamin C and fatty acids that have improved potency and bioavailability of active ingredients [48]. Products such as breads, beverages, dairy products, and cereals are fortified with NPs of probiotics, minerals, vitamins, plant sterols, antioxidants, and bioactive peptides [49].
FIGURE 10.1 Applications of nanotechnology in the nutraceutical industry.
TABLE 10.3 Some Patents on Nano-Nutraceuticals
Patent Title Patent Number Nutraceutical Nanoformulation Nutraceutical Year References
Active Ingredients
Composition comprising WO20181359 12A2 Curcumin was encapsulated Curcumin [36]
curcumin captured ginsenoside within controlled ginsenoside or
and phospholipid-based lipid phospholipid-based nanoparticles.
nanoparticle as effective ingredient
for preventing or treating
Helicobacter pylori infection
Novel nutraceutical compositions WO2004/041257 A3 Epigallocatechin gallate (EGCG) EGCG [37]
comprising epigallocatechin comprising Co-enzyme Q 10,
gallate phytanic acid, lipoic acid, etc.
Formulations containing omega-3 US201802432 53A1 The active ingredients were Omega-3 fatty acids [38]
fatty acids or esters thereof loaded into soft gel capsules. or esters Maqui
and maqui berry extract and Additionally, it was claimed that berry extract
therapeutic uses thereof the active ingredients could be
loaded into nanoparticles and
other sustained released, novel
formulations
Novel nutraceutical compositions AU2008333570B2 A composition comprising 5 Stevia extract [39]
containing Stevia extract or Stevia Stevia extract enhance cognitive
extract constituents and uses function
thereof
TABLE 10.3 (Continued)
Patent Title Patent Number Nutraceutical Nanoformulation Nutraceutical Year References
Active Ingredients
Diabetes preventing and treating CN106174011 A The natural ingredients were Chinese yams, [39]
nutritional formula nanoparticles loaded into nanoparticles. Radix astragal,
and preparing and processing Rhizoma
method thereof anemarrhenae,
chicken’s gizzard membranes, Radix puerariae, raw gypsum, Rhizoma alismatisand/or others.
Pharmaceutical, cosmetic or food WO2020019043A1 The present invention relates Vitamin D3 [41]
products and use of nanoparticles to pharmaceutical, cosmetic or
containing vitamin d food products containing vitamin
D in nanoparticles, particularly
vitamin D3.
Molecular particle superior US20190289895A1 A nano solid-liquid HO Encapsulation [42]
delivery system concentrate and method, the of particles, e.g.,
concentrate containing molecules foodstuff additives
of HO each encapsulating a composite nanoparticle including selected nutrient particles of nanoscale
TABLE 10.3 (Continued)
Patent Title Patent Number Nutraceutical Nanoformulation Nutraceutical Year References
Active Ingredients
Bioactive substance or JP6426288B2 Intimate drug-carrier mixtures Protein [43]
composition for protein delivery characterized by the carrier, (Immunoglobulins,
and use thereof e.g., ordered mixtures, immune serum,
adsorbates, solid solutions, cyclodextrins, etc.)
co-dried, co-solubilized,
co-kneaded, co-milled, co-ground products, co-precipitates; drug nanoparticles with adsorbed surface modifiers with organic compounds
Combination of bioenergy and WO20172134 86A2 Nanoparticles were used for Amino acids [44]
nutra-epigenetic metabolic formulation. (glycine, arginine,
regulators, nutraceutical and cysteine)
compounds in conventional resveratrol.
and nanotechnology-based combinations, for reversing and preventing cellular senescenceaccelerated by chronic damage caused by diabetes and other complex chronic degenerative diseases
Modified resveratrol composition WO20180423 24A1 Resveratrol nanoparticles were Resveratrol [45]
and use thereof coated with tree fat.
Source: Adapted by Ref. [1].
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The quality of nutraceuticals can be increased by increasing the shelf life of the product so that its freshness is maintained. Nanotechnology has been used to increase the shelf life of nutraceutical containing products. For instance, shelf life of tomato was increased by encapsulating quercetin in biodegradable poly-D,L-lactide NPs [34]. Similarly, the shelf life of guava was increased by application of zinc oxide NPs -containing nano structured coatings of chitosan and alginate [50].
Additionally, a lot of research is being done to increase the freshness of nutraceuticals by incorporating them within smart and biodegradable nano packagings. The nano-packagings are made using polymers like starch, polylactic acid (PLA), polyhydroxybutyrate, and polycaprolactone (PCL). These formulated biodegradable nanopackagings are highly compatible with various food products such as dairy, fresh meats, and beverages; prevents oxidation of products, thereby maintaining their freshness for a longer dura tion [51]. Omega-3 unsaturated fatty acids which are naturally found in seed oils, fish oil, and some plants have been incorporated into a wide range of products such as breads, milk, fruit juices, meat, etc., using microencapsula tion technology that prevents oxidative deterioration of unsaturated fatty acids and also extends their shelf life [52]. One of the bakeries in Western Australia has a successful top-selling product ‘Tip-Top’ Up bread which has tuna fish oil (a source of omega-3 fatty acids) incorporated in nanocapsules which break open only in stomach, thus avoiding the unpleasant taste of the fish oil [53].
10.2.2 NANOTECHNOLOGY FOR DETECTION AND SENSING OF
CHEMICAL AND BIOLOGICAL CONTAMINANTS
Nanotechnology has immense applications in the fabrication of biosen sors for the quantification of food constituents, detection of pathogens in the processing plants and also to alert consumers, manufacturers, and distributors about the safety standards of the product. Numerous studies have reported the development of nanosensors using NPs (gold, iron oxide), nanofibers, nanotubes, nanorods, and nanowires and their applications in detection of pathogens, pesticides, contaminants, adulterants, toxins, and nutrients with high sensitivity and quick response [54-61]. The nanosensors also act as indicators that give response when the environmental conditions such as temperature, humidity, microbial contamination are changed or there is degradation of products [62]. Highly sensitive nanotechnology-based
Nutraceuticals-Based Nano-Formulations
immunosensors where specific antibodies, antigens, proteins, etc., are used for the detection of microbial cells or substances in food have been developed. For example, a hybrid nanosensor based on magnetic resonance and fluorescence for detection of E. coli O157:H7 could sense different concentrations of bacteria in milk in less than an hour [63]. Similarly, in a recent report, zeolitic imidazolate framework encapsulated cadmium sulfide (CdS) quantum dots were used for sandwich-type electrochemical immu nodetection of E. coli O157:H7 in milk samples using anti-E. coliO157:H7 antibody, with the detection limit of 3 CFU mL-1[64].
10.2.3 NANOTECHNOLOGY FOR PRESERVATION AND PACKAGING
OF NUTRACEUTICALS
Nanotechnology has gained widespread attention in the preservation and packaging of nutraceuticals. The nanotechnology-based food packaging has been classified as active packaging and smart/intelligent packaging systems. Active packaging means the use of nanomaterials that are moisture-regulating agents, carbon dioxide scavengers and emitters, oxygen scavengers and antimicrobials, for providing protection and hence increasing the shelf life of the food product. Silver NPs and nanocompos ites have been widely used as antimicrobials in the food industry [65]. In a study, various deposition processes and chemical modifications were explored for attaching silver NPs on the surface of plastic substrates, which facilitated the slow release of silver ions to inhibit their inclusion in food [66]. Besides, iron, silver, carbon, zinc oxides, titanium oxides, magne sium oxides, and silicon dioxide NPs have also been employed as effec tive antimicrobial agents in packaging. Natural antimicrobial substances encapsulated in nanoemulsions (NEs) can be adhered to via covalent, electrostatic, and hydrogen bonding interactions to develop antimicrobial packaging systems. Chemical giant Bayer (Leverkusen, Germany) has developed a transparent film in which clay NPs are dispersed uniformly on a plastic film that prevents oxygen, carbon dioxide, and moisture from reaching fresh meats and other foods [67]. A number of patents on the utili zation of nanoclays and nanosilver in food packaging have been filed in the USA, Europe, and Asia [68]. Nanocomposites containing NPs of silicon dioxide, clay, titanium dioxide, nanocellulose, nanofibrillated cellulose, carbon nanotubes, etc., in polymer matrix enhance their mechanical and gas barrier properties [69].
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The smart packaging system is designed to detect and alert the consumer of any biochemical or microbial changes in the food products. Nanosensors have been incorporated in packaging material to detect chemicals, toxins, gases, aromas, food pathogens, products of microbial metabolism, etc., during storage and transport. They are being increasingly used as they can provide real-time status regarding freshness of food, thereby eradicating the need of estimated expiration dates in the consumables. For instance, Timestrip developed a detection system based on gold nanoparticles (AuNPs) for chilled foods [70]. The system appeared red above freezing temperature, but when accidental freezing occurred, the AuNPs aggregated, leading to loss of red color.
Nanolaminates are another category of modification where nanotech nology is used to enhance the quality and preservative value in nutraceuticals. Edible nanofilms that protect nutraceuticals from lipids, gases, and moisture, improve their texture and serve as carriers for nutrients, antioxidants, anti microbials, colors, and flavors come under the category of nanolaminates. Nano-laminates are basically nano dimensional thin food-grade films of two or more layers of a material, i.e., 1 nm to 100 nm each of every layer bonded chemically or physically with each other [71]. Besides protecting foods from gases and humidity, the nanolaminates can also improve the texture of food and serve as carriers of nutrients, colors, antioxidants, flavors, antimicro bials, etc. Nano laminates are used in products such as fruits, vegetables, meats, baked goods, chocolates, and candies to increase their quality as well as their shelf life [72].
10.2.4 NANOTECHNOLOGY FOR ENCAPSULATION OF
NUTRACEUTICALS
A majority of nutraceuticals have low aqueous solubility and low perme ability across membrane, short half-life, low stability, and fast metabolism thereby making them poorly bioavailable. For example, curcumin, which has anti-inflammatory, antioxidant, chemopreventive and anti-neoplastic properties is lipophilic and hence water-insoluble. Some nutraceuticals have stability issues in vivo, they get degraded or oxidized in the GIT. Others may form complexes with gastrointestinal (GI) fluid constituents (like bile salts, phospholipids, proteins, dietary fibers, surfactants, etc.), thereby decreasing their availability in systemic circulation. For instance, lutein, a xanthophyll known to be effective at retarding the development of age-related macular
Nutraceuticals-Based Nano-Formulations
degeneration, gets degraded in the acidic environment and by enzymes present in GI fluid. Similarly, lipids, which are major constituent of vitamins are susceptible to oxidation, leading to bad taste and degradation. Hence, the full therapeutic potential of nutraceutical product is not utilized. High dosage may be associated with toxicity. Therefore, achieving maximum therapeutic outcome of nutraceutical at a dose which causes minimal/negligible toxicity is a challenge. In this context, nano-encapsulation has emerged as an effec tive delivery system of nutraceuticals.
Nano-encapsulation is a process of encapsulation of bioactive compounds such as vitamins, antioxidants, proteins, lipids, carbohydrates, aromas, etc., inside a material at the size of nanoscale in order to provide more stability and thus increase shelf life of the nutraceutical formulation [73]. It also increases bio-availability of encapsulated nutraceuticals. Nanoencapsulation increases protection against high temperatures during processing of nutra ceutical products so that they are able to retain their nutritional properties. Additionally, nano-encapsulated nutraceuticals can be easily incorporated in clear and transparent foods, because of their size, which is much smaller than the wavelength of light, without causing problems of colors. Liposomes, NEs, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), polysaccharide, and protein-based NPs, nanosuspensions, etc., are different nanoencapsulation technologies used. These nanocarriers release a controlled amount of bioactive compounds at the right time and at right place.
NutraLease, an Israel based nutraceutical company has commercial beverages and food products in the market which contain encapsulated functional compounds like omega-3, β-carotene, lutein, lycopene, coenzyme Q10, phytosterols, isoflavones, and vitamins A, D, and E in self-assembled NEs [74]. The NEs increase the encapsulation rate and bioavailability of these nutraceuticals which have poor water solubility and low oral bioavail ability. Similarly, nanoencapsulation is employed for the delivery of probi otics as it provides a protective coating on the probiotic bacteria separating it from the surrounding environment, thereby enhancing the viability rate of probiotic bacteria. Novel nano-formulations of nutraceuticals having thera peutic properties are under development to target cancer and cardiovascular diseases (CVD). For instance, using antioxidants in cherry extract for treat ment of CVD has issue of low bioavailability because of oxidation and less absorption in GIT. This issue was addressed by encapsulating cherry extract in NPs based on chitosan derivatives that increases the residence time in GI lumen, and improves the intestinal absorption of cherry antioxidants, thereby enhancing their antioxidant and anti-inflammatory activity [75].
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10.2.4.1 AN OVERVIEW OF NANOTECHNOLOGY-BASED DELIVERY
SYSTEMS FOR NUTRACEUTICALS
The nutraceuticals exist naturally in a range of different molecular structures having a range of polarities, conformations, and molecular weights leading to distinct physicochemical properties such as solubility, chemical, and physical stabilities. These parameters influence their bioavailability and absorption in GIT. Therefore, a number of different types of nanotechnologybased delivery systems (Figure 10.2) have been developed with different physicochemical properties and functional attributes that improve the factors influencing the bioavailability of nutraceuticals. Depending on the specific physicochemical requirement for a specific nutraceutical, specific nanocar rier can be employed for developing nutraceutical nano-formulation. The advantages of using nanotechnology for nutraceuticals are:
FIGURE 10.2 Some nanotechnology-based delivery systems for nutraceuticals.
Nutraceuticals-Based Nano-Formulations
• Efficient encapsulation;
• Protection of labile nutraceuticals from degradation due to environ
mental stresses;
• Better physicochemical stability in GIT; • Increased gastric retention time; • Controlled release;
• Improved pharmacokinetic properties (aqueous solubility, stability,
etc.);
• Enhanced bioavailability and hence better therapeutic benefits; • Reduced dose, hence minimal side effects.
Some of the design considerations for development of nano-nutraceuticals are as under:
• The formulation should use legally approved ingredients and
processing methods.
• It should be stable, able to withstand different types of environmental
stresses (pH changes, cooling, heating, dehydration, and mechanical agitation, etc.), during production, storage, and transportation.
• It should protect the encapsulated nutraceutical from chemical
degradation as well as in the human digestive system and maintain its bioaccessibility.
• For nutraceuticals that are insoluble or less soluble in aqueous media,
the nano delivery system should be able to increase its solubility in aqueous solution.
• It should not compromise with the quality of food product such as
texture, appearance, flavor, rheology, and shelf life, i.e., must be compatible with the food medium.
• Cost of development should not be high.
In the following section, different nanotechnology-based delivery vehicles for nutraceuticals shall be discussed:
. Nanoliposomes: Liposomes are spherical phospholipid vesicles formed by folding of lipid bilayer(s) with an aqueous core and hydro phobic tails facing each other. They can be used for encapsulating both lipophilic and hydrophilic nutraceuticals either simultaneously or alone. They are used widely for the administration of nutritional ingredients and drugs into the tissues. They are made artificially from non-toxic phospholipids for the sole purpose of transportation
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of nutritional ingredients into the system. “Second-generation lipo somes” are also under research which are obtained by manipulation of lipid composition, size, and charge of the vesicle. Using various molecules like sialic acid and glycolipids, surfaces of the liposomes can be modified. The release of ingredient depends upon the rigidity and permeability of the liposome, which can be modified by modi fying the composition of its bilayer. For example, components such as dipalmitoylphosphatidylcholine forms a rigid bilayer, whereas egg and soybean phosphatidylcholines form a more permeable bilayer [76]. The advantages and disadvantages of liposomes in the nano-delivery system of nutraceutical ingredients are listed in Table 10.4.
TABLE 10.4 Advantages and Disadvantages of Nanoliposome as Delivery System
Advantages Disadvantages
High stability Low solubility
Biodegradable and biocompatible Sometimes its phospholipids undergo oxidation
Non-toxic Might undergo leakage
High efficiency and accuracy High production cost
Useful for encapsulation Short half-life
Nanoliposomes have been used as delivery vehicles for nutrients, food additives, enzymes, and antimicrobials. In a very recent report, β-carotene containing liposomes of sizes about 90-150 nm was prepared using supercritical carbon dioxide with ultrasonication, the liposomes obtained had improved stability, an important factor in nutraceuticals [77]. In another recent study, garlic extract and nisin were encapsulated in phosphatidylcholine liposomes using oleic acid and cholesterol as stabilizers for membranes, and it was shown that oleic acid stabilized liposomes showed the highest antimicrobial activity against Salmonella enteric and Listeria monocytogenes [78].
Nanoliposomes loaded with nutraceuticals have also been used for the treatment of skin diseases. Quercetin loaded-vitamin C-based aspasomes (ascorbyl palmitate vesicles), of sizes in the range of 125-184 nm was shown to have beneficial effects in the treatment of acne [79]. In a recent study, ammonium glycyrrhizinate which is a derivative of glycyrrhizic acid found in plant Glycyrrhiza glabraand is known to have anti-inflammatory and anti-allergic properties was
Nutraceuticals-Based Nano-Formulations
entrapped in ultradeformable liposomes (transfersomes) which were demonstrated to cause decrease in skin inflammation on the human volunteers, thereby making them a potential topical drug delivery system for anti-inflammatory therapy [80].
. Nanoemulsions (NEs): A mixture of two or more immiscible
liquids such as oil and water stabilized by surfactants or other types of stabilizing agents to form a solution, in which one of the liquids is dispersed as spherical droplets in the other liquid is known as an emulsion [81]. An oil-in-water emulsion (O/W) has organic phase (oil) in the form of droplets in the aqueous continuous phase and vice versa. An emulsion is said to be a nanoemulsion when the size of dispersed droplets is in the range of 10-100 nm. The lipophilic nutraceuticals are encapsulated within the droplets of NEs, which serve as an effective delivery system with improved properties like the ability to modulate product texture, high optical clarity, better stability to droplet aggregation and gravitational separation, and increased bioavailability of lipophilic components in comparison to conventional emulsions [82]. Besides, the bioavailability of encapsu lated nutraceutical in droplets of NEs is increased. The large surface area owing to the small size of droplets in nanoemulsion, makes their digestion rate higher, releasing their contents which are absorbed more easily. The absorption is also increased owing to increased residence time due to penetration of small droplets into mucous layer coating the epithelial cells in small intestine, and increased aqueous solubility of lipophilic components as the droplet size is decreased. Another advantage of using NEs is that they can be incorporated into clear or slightly turbid products without changing their visual appearance.
In a very recent report, capsanthin, a nutraceutical with poor aqueous solubility, poor stability, and low/variable oral bioavailability was encapsulated in nanoemulsion (with size <50 nm) to increase its
solubility without compromising its physical and chemical stability and retention of its antioxidant properties [83]. Another hydrophobic nutraceutical namely benzyl isothiocyanate having antitumor and antimicrobial properties but with low bioavailability was successfully encapsulated in rhamnolipid based nanoemulsion which provided solution to low solubility, poor stability, and diminished bioavail ability of benzyl isothiocyanate [84]. The developed nanoemulsion was effective against bacterial strains E. coli and S. aureus. The
Advances in Nutraceuticals and Functional Foods
cinnamon essential oil nanoemulsion incorporated in the pullulan coating on strawberries lowered the loss in fruit mass, firmness, total soluble solids, and titratable acidity of strawberries after six days of storage [85]. The developed nanoemulsion also exhibited antimicro bial activity, thereby prolonging the shelf life of strawberries during room storage.
. Lipid Nanoparticles (NPs): These are used for encapsulating water
insoluble nutraceuticals. The lipid NPs are classified as SLNs and NLCs. SLNs are made of lipids that are solid at room temperature like paraffins, triacylglycerols. The aqueous dispersions of SLNs usually have 0.1% and 30% w/w solid lipids and are stabilized by a 0.5%-5% w/w surfactant. They are in the size range of 50-1000 nm and can be prepared via hot homogenization and cold homog enization methods depending on the thermostability of nutraceutical encapsulated. The SLNs used for encapsulating nutraceuticals that are heat stable are prepared by hot homogenization technique, and the ones used for entrapping nutraceuticals that are thermolabile, are prepared by cold homogenization method [86]. They are easy to synthesize, their small size gives them high surface area that improves bioavailability of encapsulated nutraceutical and have higher loading capacity. In a very recent report, α-tocopherol acetate was successfully loaded on SLN, which was prepared using stearic acid as solid lipid, phosphatidylcholine as stabilizer and coated by chitosan [87]. The nanoformulation was stable with high entrapment efficiency of 90.58 ± 1.38% with a no-burst slow release up to 10 days tested, indicating its potential as a promising drug delivery system for vitamin E. In another report, α-bisabolol loaded SLNs were synthesized through hot homogenization method and exhibited improved therapeutic efficacy and bioavailability of α-bisabolol for combating Alzheimer’s disease [88].
NLCs are another type of lipid NPs which use liquid lipid or a mixture of liquid lipids to form NPs. They have greater stability that SLNs. They have been used for delivering nutraceuticals with slow-release profile and also provide protection to encapsulated nutraceutical from degradation. Many nutraceuticals like lutein, quercetin, etc., have been incorporated into NLCs to achieve their slow-release pattern and enhanced bioavailability [89, 90].
. Polysaccharide and Protein-based Nanoparticles (NPs): These have also been explored for encapsulation/entrapment of bioactive
Nutraceuticals-Based Nano-Formulations
ingredients for formulation of novel nano-nutraceuticals. The advan tages of using polysaccharide NPs are enhanced bioavailability, efficiency, sustained release, and a higher control of drug targeting [91]. Polymeric NPs provide protection to the entrapped nutraceuti cals from degradation in the GIT. Diffusion, swelling, and erosion are some of the mechanisms of release of active ingredients from polymeric NPs into the gastrointestinal tract [92]. Biodegradable and smart polymers are good option for encapsulation as they can be degraded in the body by biological or chemical processes and can release encapsulated bioactive ingredient in response to particular environmental conditions, respectively. Polysaccharide NPs are composed of polymeric matrices which can be synthetic or natural in nature. Natural polysaccharides include chitosan, alginate, dextran, etc. Synthetic polymers hold distinctive properties because of their chemical structure, the method of synthesis, type of functional groups present in the molecule and the degree of polymerization. Most explored synthetic polymers are aliphatic polyesters like poly-lactic acid, poly-ε-caprolactone and their copolymers [93]. In one study, nutraceutical lycopene was encapsulated in thermosensi tive PNIPAAM-PEG-based co-polymeric NPs that demonstrated stronger antioxidant and anti-cancerous activity as compared to free lycopene in vivo[94]. In another study, resveratrol was loaded on to poly (dl-lactide-co-glycolide) (PLGA) NPs, which showed improved bioavailability in male Wistar rats as compared to pure drug and marketed product [95].
Nutraceuticals have also been incorporated in protein NPs. The proteins commonly used are from animal origin like casein, gelatin, whey proteins and albumin (serum albumin and ovalbumin). Nano carriers arising from self-assembly of some milk proteins have been successfully used for the delivery of hydrophobic nutraceuticals such as ω-3 polyunsaturated fatty acids (PUFAs) and vitamin D in casein micelles, resveratrol, and curcumin in β-lactoglobulin nano delivery systems [96-99]. They have also been used for the delivery of hydro philic nutraceuticals such as tea polyphenols [100]. The polyphenols (catechin and epicatechin) were nanoencapsulated in BSA NPs in tea to enhance their stability and bioavailability [101]. In a very recent report, curcumin was encapsulated in insect mealworm protein NPs that were uncoated or coated with chitosan [102]. Curcumin bound to the hydrophobic core of the insect protein NPs was more stabilized in
Advances in Nutraceuticals and Functional Foods
the coated nano-complexes and around 90% of it was released after exposure to model GI conditions. Plant proteins, namely zein, soy proteins, wheat gliadins, and barley proteins, are also increasingly being used as delivery vehicles for nutraceuticals [103].
. Nanocrystals/Nanosuspensions: These are colloidal dispersions
(nanoparticles) of pure bioactive/nutraceutical compounds which are carrier-free and contain very little amount of surfactant and/or polymer for its stabilization [104]. Nanocrystals/nanosuspensions can be synthesized using a combination of top-down and bottom-up techniques like supercritical fluid methods, aerosol solvent extrac tion method, precipitation-lyophilization-homogenization technique, spray freezing into liquids and solution enhanced dispersion by the supercritical fluids [105]. Nanosupsension of nutraceuticals
like α-tocopherol, quercetin, curcumin, β-amyrin, etc., have been reported which had increased solubility, stability, dissolution rate and bioavailability [106-108]. In a very recent report, the technique rapid expansion of supercritical solution into air (RESS) process was successfully used for the production of nanosuspensions of nutra ceutical antioxidants namely α-tocopherol and β-amyrin to increase their efficacy and bioavailability [109].
.3 CLINICAL VALIDATION OF NUTRACEUTICALS
With rising preference for foods with high content of nutraceuticals, it becomes imperative to evaluate the efficiency, safety, and toxicity of nutraceuticals. Also, due to increasing customer awareness, the demand for information regarding the biological efficiency of nutraceutical products is growing. Therefore, many companies are now investing in the research and development to pass the clinical trials of their product. According to Jay Udani, MD, CEO, and medical director of Medicus research at Northridge CA, though the clinical trials conducted by the nutraceuticals industry has grown over the last few years, it is still less than the products launched into the market. Due to the repositioning of dietary supplements into drugs, companies are taking care of clinical as well as pre-clinical research as there are more regulations for the drug development industries to follow. This trend has increased the standard and quality of the nutraceutical product because companies want to create their USPs (unique selling products), USPs can be in the terms of nutrition provided, flavor, image improvement,
Nutraceuticals-Based Nano-Formulations
health benefits or health claims. For a clinical trial, the first step is data collection, i.e., assembling all the existing data about the product or its ingredients. Sometimes enough data is obtained to jump into the clinical trial, but when data is unavailable, in-vitrotesting is done, which is followed by a pilot study or a proof-of-concept study. Good extraction of basic data helps in maximizing the chances of getting robust data which is later used for marketing and regulatory purposes. After this in-vivostudies are done to determine the safety of the product. AIBMR is a United States (US) based company which performs a thorough literature survey to get the information regarding the product and its ingredients. AIBMR is known to maintain one of the biggest nutraceutical research libraries in the world. After analyzing all the required data, the product is then sent for clinical validation; after which the product is launched into the market.
.4 SAFETY AND REGULATIONS
Inclusion of nanotechnology into the nutraceutical and food industry poses arrays of risks. There is no denial in the fact that NPs and nano-foods can cause serious health issues. Research is being done to identify the problems caused by nano products and the ways to tackle them. Reactive oxygen species (ROS) generation leading to oxidative stress, which causes degenera tion of mitochondria and induces apoptosis is one of the crucial mechanisms of toxicity by nanomaterials. There have been reports according to which these products can be labeled as toxic and dangerous to health. This could be due to the fact that these products in nano form have higher chemical reactivity than their larger counterparts; they have higher bioavailability which may lead to their toxic behavior; they readily cross the membrane barriers and capillaries leading to different toxicokinetic and toxicodynamic properties; they may undergo changes in the body and may not be same as originally administered; our immune system can be compromised by them and its possible that they might have long term pathological effects. They can also cause oxidative cell damage by translocating into the skin, liver, and brain cells. They are also linked with escalating levels of immune dysfunc tion and inflammation in the gastrointestinal tract, causing inflammatory bowel disease (IBD), known as Crohn’s disease (CD) [40]. They can also cause lesions in kidneys and liver, clots, cancer, and granulomas due to build up toxicity when used in access. They can be taken up by damaged skin and brain cells due to their minute size. Impairment of DNA replication and
Advances in Nutraceuticals and Functional Foods
transcription can also occur in some cases because particles having size less than 70 nm are able to enter cell nuclei [40]. The use of new nanotechnologybased food products is still a challenge as they need to undergo safety assess ment before being commercialized for human use. There are a number of regulatory bodies working currently such as Food and Drug Administration (FDA), Environmental Protection Agency (EPA), European Food and Safety Authority (EFSA), National Institute for Occupational Safety and Health (NIOSH), Consumer Product Safety Commission (CPSC), US Patent and Trademark Office (USPTO), US Department of Agriculture (USDA) and Occupational Safety and Health Administration (OSHA). The safety regula tions are not clear for nanofoods because the fate and toxicity of the NPs is still less understood by the researchers. There needs to be a widely accepted international regulatory framework for the regulation of the use of nano materials in the food industry. Proper government guidelines and directives and rigorous toxicological screening methods are the need of hour for the commercialization of nanofoods.
.5 CONCLUSION
The fortification of food products with nutraceuticals has gained increasing significance for preventing and improving the health of people suffering from various diseases. Nanotechnology has a wide potential in addressing challenges presently faced by nutraceuticals like limited solubility, stability, shelf life and bioavailability, which compromise their therapeutic effective ness. Nanotechnology has been used to improve the quality of nutraceuti cals, for detection and sensing of chemical and biological contaminants, in preservation and packaging of nutraceuticals and for nanoencapsulation of nutraceuticals. Several nanosystems like nanoliposomes, SLNs, NLCs, polysaccharide NPs, protein-based NPs, NEs, and nanocrystals/nanosuspen sions have been used for effective delivery of nutraceuticals in vivo and also to enhance their efficiency. There are a number of nanotechnology-based nutraceutical products commercially available in the market. However, proper evaluation of the efficacy and safety of nutraceutical nano-formu lations is still needed. Incomplete knowledge about the fate of NPs once they enter organs, tissues, and cells and associated toxicity of nanomate rials is still a concern while using these commercial nano-formulations of nutraceuticals.
Nutraceuticals-Based Nano-Formulations
KEYWORDS
• nutraceuticals • nano-formulation • nanocarrier
• nanostructured lipid carriers • nanosuspension
• solid lipid nanoparticles
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An Electrochemical immunobiosensor for ultrasensitive detection of Escherichia coli O157:H7 using CdS quantum dots-encapsulated metal-organic frameworks as signalamplifying tags. Biosensors and Bioelectronics, 126, 493-500. https://doi.org/10.1016/j. bios.2018.11.001.
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75. Beconcini, D., Felice, F., Fabiano, A., Sarmento, B., Zambito, Y., & Di Stefano, R.,
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Goto, M., (2020). Preparation of liposomes encapsulating β-carotene using supercritical carbon dioxide with ultrasonication. The Journal of Supercritical Fluids, 161, 104848. https://doi.org/10.1016/j.supflu.2020.104848.
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E., Mamdouh, W., & Sammour, O., (2020). Cosm-nutraceutical nanovesicles for acne treatment: Physicochemical characterization and exploratory clinical experimentation. International Journal of Pharmaceutics, 577, 119092. https://doi.org/10.1016/j.
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nutraceuticals: Role of the food matrix, processing conditions, the gastrointestinal tract,
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with resveratrol and its biological implications. Biomacromolecules, 9(1), 50-56.
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and epicatechin on BSA NPS improved their stability and antioxidant potential. EXCLI J., 13, 331-346.
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CHAPTER 11
Growth Patterns, Emerging Opportunities, and Future Trends in Nutraceuticals and Functional Foods
ASAD UR REHMAN,,2SALMAN AKRAM,and THIERRY VANDAMME
1University of Strasbourg, CNRS 7199, Faculty of Pharmacy, 74 Route du Rhin, CS - 60024, 67401 ILLKIRCH CEDEX, France
2University of Paris Descartes, UTCBS CNRS UMR 8258-INSERM U1267,
Faculty of Pharmacy, 4 Avenue de l’Observatoire, Paris - 75006, France
ABSTRACT
This chapter will first provide information about the background of the nutraceuticals. This background includes the historical perspective of using food items for healing purposes, gap in literature for stringent regulatory definitions for these products. Then, growing market trends in the domain of nutraceuticals and functional foods and their potential use in the prevention and treatment of diseases are discussed. In later parts, recent developments in their industrial manufacturing, novel dosage forms for their delivery into the body, including nanotechnology-based dosage forms, will be discussed in detail. Finally, some of the prominent clinical trials both completed and in the process will be discussed in detail to throw light on progress in their use as a potential alternative treatment.
.1 BACKGROUND AND GROWTH PATTERNS IN
NUTRACEUTICALS AND FUNCTIONAL FOODS OVER THE YEARS
Historically for a very long time, there was no distinction between the food and drugs in terms of their use for medical purposes. The drugs were presumed to
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be some sort of special food items needed to heal injury, to replenish energy and to antagonize the effects of accidental or intentional poisoning. During this era of human history, there were no scientific methods established to check the authenticity of the reports of medicinal use of different food items in different cultures. Despite this history accounts various statements on the importance of the food items for health purpose. The famous Greek physician Hippocrates mentioned “…difference of diseases depends on the nutriment” [1]. From that time to dawn of modern science the idea of use of food elements for medical purpose was popular in all cultures.
Dr. Stephen DeFelice was the first person to use the term “nutraceutical” from the combination of words “nutrition” and “pharmaceutical” in 1989 [2]. He described them as food or part of food that provides medicinal and health effects. FDA covers most of the nutraceuticals under the definition of “Food supplements” [3]. According to European Nutritional Association, the term nutraceutical is defined as “Nutritional products that provide health and medical benefits, including the prevention and treatment of disease” (ENA 2016). Whereas the “Functional food” term is defined by Hardy as “Any food or ingredient that has a positive impact on an individual’s health, physical performance, or state of mind, in addition to its nutritive value” [4]. A lot of people have tried to define these terms for clarification of their meaning and scope. According to literature, the definition of these terms overlaps with each other and there is no strict regulatory definition which can differentiate between the terms “food supplements,” “functional foods” and “nutraceuticals” [2, 3]. For the nutraceuticals there is debate in literature either to set same safety and efficacy parameters for them as pharmaceuti cals or establish special safety protocols for them. The work on establishing proper regulatory guidelines on their use is in progress and clinical trials of the many nutraceuticals have been done to demonstrate the safety and efficacy of these products.
The global market of nutraceutical was $106 billion in 2004 [5] and $128 in 2008 [6]. The global market of nutraceuticals was projected to cross $171.8 billion in 2014 and $241.1 billion by 2019 [7]. It is difficult to estimate current global market value of the nutraceutical due to overlapping between nutra ceuticals and functional foods and other simple food supplements as they are marketed under the umbrella of the same group name [8]. This substantial growth in the global market is attributed to the following factors [9]:
• Recent developments in science and technology; • Higher proportion of aging population;
Growth Patterns, Emerging Opportunities, and Future Trends
• Increase in lifestyle related diseases;
• Awareness on the medical benefits of the food items; • Exploration of alternative treatment options;
• Biotechnology and genetic engineering modified plants and food
products.
In addition to above-mentioned factors, this recent growth in the market is also influenced by various other factors, for instance, in Canada whey waste in millions of tons is produced each year. This whey was considered as waste byproduct produced during the industrial processing [10]. Recently, the industry has developed processes to process this whey waste to produce essential healthy nutrients. Important proteins [11] obtained by biotech nology from the whey proteins are now on market. Another factor driving the growth in this domain is lesser regulatory constraints and less product development time required [12].
.2 EMERGING OPPORTUNITIES AND FUTURE TRENDS IN
NUTRACEUTICALS AND FUNCTIONAL FOODS
11.2.1 PREVENTION AND TREATMENT OF DISEASES
The nutraceuticals are emerging as an alternative option in prevention and therapy of number of diseases. By each passing day, their scope is getting broader and they are finding their applications in new pharmacotherapy areas. Following is the some of the key fields in pharmacotherapy in which they are finding their applications.
11.2.1.1 NEURODEGENERATIVE DISEASES
Neurogenerative diseases is a term used mainly for number of diseases related to peripheral or central nervous systems such as Parkinson’s diseases, Alzheimer’s disease, and such others. In these diseases, the neuronal integ rity or neuronal transmission is compromised, which ultimately leads to the loss of sensory or motor activities [13, 14]. This leads to life-threatening conditions, death, and a huge financial burden on public health systems [15, 16]. They also lead to the psychological and physical stress of the affected families [17]. Below are mentioned some of the representative nutraceuticals
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currently under investigation and use for the management and cure of these group of diseases:
. Curcumin: The active ingredient of turmeric known as curcumin
is one of the most widely used nutraceutical used owing to its beneficial therapeutic effects [18]. Historically it has been reported for its use as flavoring and coloring agent and as traditional remedy for various diseases [19]. Besides its effects as anti-inflammatory and antioxidant effects it is well reported for its use for neuro protective functions. Curcumin can cross the blood brain barrier and so can be exploited for treatment of various neurodegenera tive disorders [20]. The major limitation in its use as therapeutic agent is its poor bioavailability due to its limited absorption and immediate clearance from body [21]. Clinical trial results indicate its beneficial effects for improving cognitive functions and as antidepressant [22, 23].
. Coenzyme Q10: The role of Coenzyme Q10 in ATP cycle and as anti
oxidant makes it one of essential micronutrients for the human body. Despite its many other functions as antidiabetic, antihyperlipidemic, cardioprotective agent it can also cross the blood-brain barrier and has positive impact on different neurodegenerative diseases [24, 25]. It exhibits its neuroprotective effects by inhibiting ACE activity and through improving activity of endogenous antioxidant system [26]. This suffers from low bioavailability due to its high lipophilicity. Clinical trial data supports its beneficial role in depression and Parkinson’s disease [27, 28].
. Resveratrol: This polyphenol is present mostly in red grapes, cher
ries, and berries. It has multiple beneficial effects and its anticancer, antidiabetic, and anti-inflammatory effects are frequently reported in literature. It can effectively cross the blood brain barrier and also shows neuroprotective effects [29, 30]. Its safety is well established as every age group has well tolerance for it. By upregulating Nrf2/ HO-1 and PI3K/Akt signaling pathway it exhibits its antioxidant and hence neuroprotective properties [31, 32]. However, its major neuroprotective function is due to its ability to act as an anti-protein aggregation agent which overall improves cognitive function. This also suffers from low bioavailability due to its rapid metabolization rate and rapid clearance from body [33]. Clinical trial data suggests its beneficial role in improving cognitive functions [34].
Growth Patterns, Emerging Opportunities, and Future Trends
. Polyunsaturated Fatty Acids (PUFAs): These are group of essen
tial fats as they cannot be synthesized by human body. The most important of these fatty acids are normally found in salmon fish and walnuts. These important nutritional components show antioxidant, antihyperlipidemic, and cardioprotective properties. They perform their neuroprotective function by blocking microglia/astrocytes via JNK and PPAR-y signaling pathway [35, 36]. It also enhances neurotransmission and neurogenesis and hence improve overall brain function activity [36-38]. Clinical trial data shows its benefi cial effects for its use for treatment of Parkinson’s and Alzheimer’s diseases [38, 39].
11.2.1.2 DIABETES
Diabetes is non-communicable, multifactorial, lifestyle chronic disorder characterized by hyperglycemia [39]. In diabetes the metabolism of several nutritional components is impaired due to inadequate amount or inactivity of insulin [40]. Due to its prevalence as pandemic state and enormous burden on the public health system by causing lifetime morbidity and mortality diabetes is major challenge of today’s world [41]. The diabetes can cause number of secondary health problems or aggravate them and hence complicate the therapy of the individual elements. Several various therapeutic options have been proposed for the prevention and cure of this public health problem but still there are huge gaps in its effective treatment. This gap leads to poor patient compliance and hence decreases the chances of the effectiveness of overall pharmacotherapy. Various nutraceuticals have been explored in traditional medicine for the prevention and cure of this metabolic disorder as an alternative option for prevention, management, or therapy of the diabetes [42]:
. Antioxidants: These can potentially play a vital role in the manage ment of diabetes. Various animal studies suggest that antioxidants can delay the onset of the diabetes induced complications by relieving oxidative stress [43]. Vitamin C reduces protein glycation, sorbitol accumulation and lipid peroxides [44]. All these impacts on reduction of diabetes induced complications. Similarly, vitamin E also leads to the reduction of peroxides and aldehydes as well as increases the platelets activation [44, 45]. Similarly, vitamin D intake by infants helps in reduction in development of type 1 diabetes [46].
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While in adult patients the intake of vitamin D led to the decrease of the amount of insulin needed for the diabetes management [47]. α-Lipoic acid is also an important antioxidant that protects the retina against ischemic injury [43]. Ischemic injury occurs in diabetes and is one of the most important causes of vision loss. Clinical trial data suggests that supplementation by α-Lipoic acid helps in the treat ment of diabetic neuropathy [48].
. Minerals: Chromium in an important trace element needed for the
healthy function of the body. It has been shown that chromium intake leads to the insulin sensitivity and glucose tolerance [48]. Another important mineral that increases the insulin sensitivity is magnesium [49]. Similarly, zinc supplementation reduced the oxidative stress over the retina and decreases the chances of the age-related eye diseases [50].
. Plant Derived Nutraceuticals: The plant-based nutraceuticals act
by inhibiting the key enzymes involved in the starch degradation into the smaller carbohydrate units and ultimately their absorption into the blood circulation [51, 52]. The two key enzymes involved in the glucose metabolism in the body are α-amylase and α-glucosidase [52]. Limonoids from A. indica(Neem), myricetin from the guava, aqueous, and ethanolic extracts from brown seeds and several other plant extracts are reported in literature to effectively inhibit the activity α-amylase and α-glucosidase [53-56]. In addition to that, plant-based nutraceuticals are also effective against glucose regu latory enzymes such as DPP4 (dipeptidyl peptidase-IV), GLP-1 glucagon-like peptide-1 (GLP-1), and GIP (glucose-dependent insulinotropic polypeptide). Essential oil from Cymbopogon citratus, active ingredients from Inonotus obliquus, Rhizophora mucronate, ethanolic extract of Urena lobate are the prominent plant nutraceuticals inhibiting the activity of these enzymes and hence aids in diabetes management [57-59]. In addition to this many phenolic derivatives are also responsible for aldose reduc tase enzyme which is responsible for the sorbitol accumulation and hence aggravation of diabetic complications [60]. The peroxisome proliferator-activated receptors (PPARγ), whom activation leads to the increase of cellular glucose uptake and reduces plasma glucose level is an important target for diabetes treatment. Catechin from green tea, activates them and hence decreases the chances of the diabetes type 2 [61].
Growth Patterns, Emerging Opportunities, and Future Trends
11.2.2 TECHNOLOGY FOR DESIGN AND DEVELOPMENT OF
NUTRACEUTICALS AND FUNCTIONAL FOODS
There has been a lot of innovation in design and development of nutraceuti cals and functional foods over the past few decades. Modern pharmaceutical technology, biotechnology, and genetic engineering techniques have been employed to manufacture these products. Below is a summary of production of nutraceuticals by two different key techniques over the past few years, i.e., biotechnology and pharmaceutical nanotechnology:
. Biotechnology: It is playing a major role in the development of
this industry. Biologically active non-nutritive components of food and food items are being utilized to manufacture nutraceuticals by biotechnology approaches [12]. To produce enzymes and recombinant microorganism used in nutraceuticals there is immense need to explore new food elements. The biotechnology is key for exploration of these new food items for production of novel nutraceuticals [62]. Although the biotechnology is playing a vital role in development of this new industry, but the nutraceuticals manufactured by this way suffer from technology constraints and consumer distrust [63] and regulatory limitations [64]. Despite this, there are some incentives for companies exploiting biotechnology to produce the nutraceuticals. For instance, a successful food product life cycle is around 21 years [12]. By employing the biotechnology and genetic engineering exploring new advantages of this food product and mentioning some health claims to it can increase the market value and life cycle of this food product [12].
. Pharmaceutical Nanotechnology: It is one of keyway used in recent
times to produce nutraceuticals and functional foods for research lab scale level and at industrial scale. Various nanoscale dosage forms have been developed over the years, such as nanoemulsions, lipo somes, solid lipid nanoparticles (SLNs), and others. Details on these dosage forms and their contribution in field of nutraceuticals will be provided in the next sections of this chapter. Here, we will briefly use different strategies employed to produce these dosage forms. The technology has huge impact on the properties of the final delivery system produced and on its capacity to effectively encapsulate and release the nutraceuticals [65]. The amount of energy supplied has impact on the final particle size of the formulation which ultimately defines the stability of the product. The comparison of two different
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high energy methods, i.e., mircrofluidization, and high-pressure homogenization were compared in a study. Results showed that low particle size produced in mircrofluidization due to higher energy employed in mircrofluidization have a long stability [66]. Other studies compared the ultrasonication with mircrofluidization and mircrofluidization and have reported better results with mircrofluidi zation [67, 68]. Although these high energy methods are very effec tive, but they suffer from limitations of easy scale up and use with sensitive food components in some nutraceuticals such as proteins. The excessive high energy can have negative impact on the stability of the formulation [68, 69]. The low energy methods are interesting for sensitive components [70]. The two most important methods in this domain are spontaneous emulsification and phase inversion method [71]. There are other various methods used in domain for production of nano scale dosage forms and although they are mainly affected by process parameters, formulation parameters also play an important part in determining the final particle size, stability, and effectiveness of the nutraceuticals produced [72, 73].
11.2.3 DEVELOPMENT OF VARIOUS DELIVERY SYSTEMS FOR
NUTRACEUTICALS AND FUNCTIONAL FOODS
Nutraceuticals include a wide range of compounds such as carotenoids, bioactive peptides, lipids, phenolic compounds, vitamins, essential minerals, etc., and provide physiological or therapeutic benefits beyond the basic nutritional needs. In the recent years, the incorporation of nutraceuticals in food products has provided a very simple and efficient way of developing novel functional foods. However, the nutraceuticals often show low aqueous solubility, instability during food processing or storage, chemical transfor mation, or/and poor absorption within the gastrointestinal tract (GIT), thus resulting in low bioavailability and reduced health benefits of the nutraceu ticals [74]. Some of the nutraceuticals (e.g., carotenoids, Vitamins), when directly added into the food products, can show unwanted interactions with other food components, which may affect the texture, appearance, bioavail ability, and stability of those components. All these factors limit their direct incorporation into the food products. Over the past couple of decades, the efforts to overcome these limitations and an increased research interest in the formulation of food products enriched with nutraceuticals, have led to the development of the delivery systems for the nutraceuticals and functional
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foods. The major classes of the nutraceuticals and functional food compo nents, which have shown great therapeutic potential and can benefit from their encapsulation into the delivery systems, are shown in Table 11.1. However, the development of a delivery system for nutraceuticals is very challenging process because the components which are used to form delivery systems must be of food grade (so only a limited number of approved materials can be used) as well as the method to be used for the development of delivery system must be very economical, robust, and reproducible [75].
11.2.3.1 IDEAL PROPERTIES OF NUTRACEUTICAL DELIVERY SYSTEMS
The development of nutraceutical delivery system is a very challenging process and it requires a thorough knowledge of the properties of nutraceuticals to be encapsulated as well as the use of the suitable materials and techniques. The knowledge of the properties of an ideal delivery system significantly assist to understand the main parameters to be considered during the selection or development of a delivery system for nutraceuticals. Following are the main properties of an ideal delivery system for nutraceuticals,
11.2.3.1.1 HIGH ENCAPSULATION EFFICIENCY (EE)
The ideal deliver system should provide a very high encapsulation of the nutraceuticals, and effectively retain the encapsulated nutraceutical until it reaches the desired site of action, providing a high bioavailability [75, 76]. The lipophilic/hydrophobic nature of the delivery system also helps to increase the bioavailability of encapsulated lipophilic active ingredients (e.g., nutrients, vitamins, and other bioactive agents). However, the selection of the encapsulation technique is very important to keep the nutraceuticals in their bioactive form, e.g., use of heat producing encapsulation techniques for the nutraceuticals which are sensitive to heat can result in the loss of the bioactivity of the nutraceuticals.
11.2.3.1.2 PROTECTION OF THE LOADED NUTRACEUTICALS
The delivery system should provide protection to the encapsulated nutra ceuticals against the chemical degradation (e.g., degradation resulting from oxidation or hydrolysis) as well as protection from the adverse physiological factors (e.g., harsh gastrointestinal (GI) pH, digestive enzymes) while it is
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carrying the loaded components to the site of action. It should also provide protection to nutraceuticals during production, storage, and transport against the factors such as temperature, light, pH, etc. As a result, the nutraceuticals are safely delivered at the desired site of action in their bioactive form and provide maximum health benefits.
11.2.3.1.3 TARGETED AND CONTROLLED DELIVERY OF THE
FUNCTIONAL COMPONENTS
The delivery system should carry the loaded nutraceuticals to the desired site of action and provide either a controlled release or a release under the influ ence of an environmental trigger (e.g., temperature, pH, enzyme activity, ionic strength) of the loaded functional components. The components to be used to develop a delivery system are selected based on the environment at the site of action as well as the mechanism of the delivery or release of the loaded components.
11.2.3.1.4 COMPATIBLE WITH THE COMPONENTS OF THE FOOD
PRODUCT
The delivery system should be compatible with the other components of the food product (or matrix) and it should not affect the texture, appearance, flavor, aroma, and stability of the final product. The food matrix may be composed of single or multiple components (e.g., water, lipids, proteins, surfactants, polysaccharides, etc.). Delivery systems can be incorporated into different kinds of food matrices, e.g., trapped inside a biopolymer matrix (sauces, yogurts) or a solid matrix (cereal products) or it can be dispersed in an aqueous solution (drinks, beverages) [77].
11.2.3.1.5 MASKING THE BITTER TASTE OR OFF-FLAVORS
The bitter taste and unwanted flavors are major contributors of non-compli ance in the consumers. However, this bitter taste as well as the off-flavor can be masked by inhibiting the direct interaction of bioactive molecule with the oral mucosal surface. The lipophilic delivery systems prevent the encapsu lated bitter components and unwanted flavors, from interacting directly with the taste receptors, thus improving the consumer compliance.
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Table 11.1 Different Classes of Nutraceuticals which can Benefit from Encapsulation
Class Health Benefits Encapsulation Benefits
Bioactive proteins Have antimicrobial, anticancer, No interactions with other food
and peptide (e.g., antioxidant, antihypertensive, components
immunoglobulin, and cholesterol lowering
Resistance against denaturation
lactoferrin, casein properties
and conformational changes phosphopeptides)
Immune system mediators
Improved bioavailability
Vitamins (e.g., Antioxidant properties Easy incorporation into food
Vitamin A, B products without losing their
Play vital role in the cellular
complex, C, D, E, bioactivity
functions, e.g.: and K)
Masking the off-flavors o The production of red
blood cell and insulin Enhanced chemical stability
o The metabolism of proteins,
fats, and carbohydrates
o The formation of teeth and
bones
o Clotting and wound healing
Prevention and treatment of cardiovascular diseases, cancer, and diabetes
Carotenoids (e.g., Antioxidant activity Protection from the
β-carotene and lutein) environmental factors, e.g.,
good vision and
oxygen, temperature, and light protect from eye disease
Ideal solution for their Treatment of Cancer and
limitations to be used directly, coronary heart disease
e.g., low aqueous solubility, crystalline structure, etc.
Phytosterols (e.g., Treatment of coronary heart Protection from the external
Stigmasterol, disease factors, e.g., oxygen,
β-Sitosterol) temperature, and light
Regulate the cholesterol level
Potential solution for their low
aqueous solubility
Essential minerals Play an important role in the Protection of the food products
(e.g., zinc, iron, cellular functions, e.g., from adverse effects
chromium, calcium)
o Protein function Improved bioavailability
o Growth and development Masking the unpleasant taste
o Keep the bones healthy o Enhance insulin activity
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Table 11.1 (Continued)
Class Health Benefits Encapsulation Benefits
Fatty acids (e.g., Provide protection against Enhance their bioavailability
butyric acid, ω-3 fatty various chronic diseases, e.g.,
Easy incorporation into food
acids, conjugated arthritis, coronary heart disease
products without losing their
linoleic acid) and cancer
bioactivity
the bones and brain
Mask the unpleasant taste healthy
Polyphenols (e.g., Possess antioxidant, anticancer, Easy incorporation into food
curcumin) antimicrobial, and anti- products without losing their
inflammatory properties bioactivity
Masking the unpleasant taste
Enhanced chemical stability
Fibers (e.g., pectin, Regulation of cholesterol and Easy incorporation into food
cellulose) blood glucose products without affecting the
texture and flavor of the food Prebiotic effects
products
To treat constipation
Source:Refs. [74, 75, 78-83].
11.2.3.1.6 FACILITATE THE LYMPHATIC UPTAKE
The food components and nutraceuticals which suffer from hepatic first pass metabolism can be benefitted by their encapsulation into a lipidbased delivery system. The lymphatic uptake is an efficient alternative for systematic transport of lipophilic compounds which helps them to bypass the hepatic metabolism and results in an increase in the bioavailability of those nutraceuticals [84]. As the extent of lymphatic uptake is directly related to the ability of the bioactive compounds to associate with the lipoproteins within the enterocyte [85], the lipid-based delivery systems (having nano-sized particles) provide an effective way to improve the direct intestinal lymphatic uptake of the lipophilic bioactive compounds.
11.2.3.2 THE EMERGING NUTRACEUTICAL DELIVERY SYSTEMS
Different types of delivery systems have been developed to encapsulate and deliver the nutraceuticals, depending upon the molecular and physicochem ical properties of the nutraceuticals and food components to be encapsulated. These delivery systems usually differ from one another in terms of their cost, biocompatibility, ease of development, ease of use, biodegradability,
Growth Patterns, Emerging Opportunities, and Future Trends
and their capacity to encapsulate, protect, and deliver the nutraceuticals. As mentioned earlier, the materials which are used for the development of these delivery systems to encapsulate nutraceuticals and food components should be of food grade. That is why the bio-based materials are usually used as the encapsulating materials for nutraceuticals because of their biocompat ibility, biodegradability, and non-toxicity, e.g., proteins, lipids, surfactants, polysaccharides, etc. The most important types of delivery systems which are currently used as well as the ones having potential to be used as delivery systems for nutraceuticals are summarized here.
11.2.3.2.1 LIPOSOMES
The liposomes are the spherical structures consisting of an internal aqueous compartment enclosed by one or more phospholipidic bilayers, known as lamellae. Liposomes were first described by Bangham et al. as spherical bilayer structures composed of phospholipid and cholesterol [86], known as the classical or conventional liposomes. When the phospholipids are hydrated, they form bilayers (by self-association) surrounding an aqueous interior. The cholesterol improves the fluidity and stability of the bilayers, which prevents the leakage of the active payload. However, incorporation of cholesterol in food products should be avoided in certain health condi tions (e.g., hypercholesterolemia), in that case some other compounds can be used instead of cholesterol to help maintain the integrity of the liposome membrane (e.g., phytosterols). Among all the emerging systems developed to encapsulate nutraceuticals, liposomes have gained much importance due to their unique bilayer structure which enables them to encapsulate both lipo philic and hydrophilic nutraceuticals. The general structure of the classical liposomes is shown in Figure 11.1.
Based on the lamellarity and size, liposomes can be classified into the following types:
• Small unilamellar vesicles (SUVs) with a size < 100 nm; • Large unilamellar vesicles (LUVs) with a size > 100 nm; • Multilamellar vesicles (MLVs) with a size > 0.5 µm.
The classical liposomes are generally cleared from the blood very rapidly, that’s why scientists have developed the advanced liposomes to deal with this problem. The advanced liposomes can be categorized into the following main types, stealth liposomes, cationic liposomes, fusogenic liposomes, and ligand targeted liposomes, having a wide range of applications in food, pharmaceutical, and cosmetic industry.
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