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Munish Puri Editor

Extraction and Biotechnology
Food Bioactives
Munish Puri

Food Bioactives
Extraction and Biotechnology Applications

Munish Puri
Bioprocessing Laboratory, Centre for
Chemistry and Biotechnology
Deakin University
Waurn Ponds, VIC


Centre for Marine Bioproducts

Development, Medical Biotechnology,
Flinders Medical Science and Technology
School of Medicine
Flinders University

ISBN 978-3-319-51637-0 ISBN 978-3-319-51639-4 (eBook)

DOI 10.1007/978-3-319-51639-4
Library of Congress Control Number: 2017930944

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For many years, bioactive compounds have been of great use to humans because
of their applications as flavor compounds and nutritional agents, and for their
medicinal uses as antibiotics, anticancer agents, hypocholesterolemic agents,
immunosuppressants, and therapeutics for pathologies such as cardiovascular,
thrombotic, atherosclerotic inflammatory, and neurodegenerative diseases.
Bioactives include polysaccharides, proteoglycans, peptides, lipids, terpenes,
polyphenols, mussel adhesive proteins, omega-3 fatty acids, carotenoids, and pig-
ments. It has also been discovered that they serve as major defense systems in
plants. In addition, plant food bioactives have shown effectiveness as neurocog-
nitive agents against neurodegenerative disorders such as Alzheimer’s disease.
Bioactive compounds are generally superior to chemically derived products for the
above functions.
This book, edited by Prof. Munish Puri, is an excellent review of bioactive
agents and will be useful to scientists around the world interested in the above
applications. These include microbiologists, chemists, biochemists, and geneticists
in academia and industry, especially in the biotechnology industry. They will profit
by reading about the many beneficial uses of bioactives for humans and plants. In
addition, this book will introduce these amazing developments to scientists unaware
of the occurrence and applications of bioactives. There is no doubt in my mind that
this will result in the development of many more useful products for medicine and
Arnold L. Demain
Research Institute of Scientists Emeritii (RISE)
Drew University, Madison, NJ, USA
Department of Biological Sciences
Massachusetts Institute of Technology (MIT)
Cambridge, MA, USA


This single book volume, titled Food Bioactives: Extraction and Biotechnology
Applications, focuses on the recent cutting edge research advances in the field of
food bioactives, particularly their diverse sources, production and downstream
processing, emerging delivery technologies, and their therapeutic applications.
Contributions from experts in the field provide an overview of current discoveries
and trends in food bioactives research. We are grateful to the contributors for their
generous and timely reflections on current developments in the discipline.
Food bioactives are physiologically active components in foods such as veg-
etables, fruits, and whole grains, which may provide desirable health benefits
beyond basic nutrition to reduce the risk of chronic disease and the process of
carcinogenesis. Bioactives (metabolites synthesised by plants for self-defense) are
obtained selectively from plants as speciality chemicals and can be used as
nutraceuticals. The addition of bioactives to foods, particularly those foods that are
consumed as part of the normal diet of target populations, offers opportunities for
improving the health and well-being of consumers. In this book, we focus on a
number of bioactive compounds that have been associated with health benefits, in
particular in relation to cardiovascular diseases and other chronic diseases. These
are flavonoids, isoprenoids, glucosinolates, long chain n-3 polyunsaturated fatty
acids (PUFAs) and carotenoids. In the introductory chapter (Chap. 1), Kyriaki and
Galanakis discuss the biosynthesis and functionality of glucosinolates from plants.
Studies have shown that diets high in these foods are associated with a reduced risk
of cancer and improved vascular health. The authors of the chapter focus on
emerging technologies (e.g., high pressure processing, ultrasounds and microwaves
extraction, pulsed electric field, supercritical fluids extraction) that promise mild
treatment and preservation of GLs during processing. Chapter 2 by Sanchez deals
with mushroom bioactive compounds such as polysaccharides, proteoglycans,
terpenes, phenolic compounds, lectins, peptides, proteins, and their applications.
Chapter 3 by Sergio Sanchez and Demain is devoted to bioactives from fungi,
especially valuable secondary metabolites, such as antibiotics, anticancer drugs,
hypocholesterolemic agents, immunosuppressants, and others.

viii Preface

Bioactive compounds in plants are present in low concentrations; thus, the

development of an effective and selective method for their production and extrac-
tion is important. Recent advances in “omics” technologies (e.g., genomics,
metagenomics, proteomics), and efficient expression systems have been successful
in generating more yields. Danquah and his group in Chap. 4 describe in detail the
development of a bioprocess for the production of novel bioactive peptides from
food proteins by exploiting fermentative and the proteolytic activities of
Lactobacillus delbrueckii bioactive peptides (BPs). The feasibility of manufacturing
BPs on a large scale is also projected by conducting an economic assessment. In
Chap. 5, Castillo and co-authors describe in detail two production methods of
mussel adhesive proteins (MAPs), natural extraction, and recombinant production.
The authors emphasize low-cost approaches with advantages such as engineering
additional functions. Chapter 6 by Gupta and co-authors describes the production of
natural omega-3 fatty acids and carotenoids through biological (microalgae) path-
ways. This chapter explains the mechanical, chemical, and biological techniques
and the combinations used for the extraction of carotenoids. Chapter 7 by Safarik
and co-authors proposes the use of magnetic materials (nanotechnologies) for
efficient separation of high-value products (antioxidants, vitamins, fatty acids, oils,
polysaccharides, etc.) from algal biomass. Chapter 8 by Singhala and her group
offers an overview of the use of enzymes for efficient extraction of bioactives,
which is a recent and “green” extraction technique. Enzyme-assisted extraction
technique uses specific enzymes to disrupt the cell wall of source material to
improve bioactive extraction yield. This technique can be combined with various
other techniques to enhance the overall recovery of bioactives from source mate-
rials. The authors also provide excellent coverage of the mechanism of
enzyme-assisted extraction and structural modifications of biomolecules during
The delivery of bioactives through food is a major challenge as many bioactives
are prone to degradation. There is therefore a need to protect them throughout their
shelf life in fortified food products, without compromising the sensory properties
of the food. Chapter 9 by Castro and his team discuss the emerging technologies for
bioactive application. Micro- and nanoscale devices introduced in foods will
facilitate the synthesis of novel enriched foods for special purposes.
In Chap. 10, Riberio provides an overview of the emerging technologies of
hydrogels in bioactive compounds delivery with regard to polyphenols. The
effectiveness of polyphenols, which depends on preserving their stability, bioac-
tivity, and bioavailability, limits their pharmaceutical application. The chapter
covers the encapsulation of polyphenols in hydrogels, as well as the classification,
preparation, characterization, and measurements associated with this process.
Beneficial effects of polyphenols in prevention or in therapeutics of important
pathologies, such as cardiovascular, thrombosis, atherosclerosis, inflammation,
cancer, or neurodegenerative diseases, are also outlined in this chapter.
Chapter 11 by Sharma and Puri presents an excellent overview of the use of
multifunctional bioactives for cancer therapy. The authors discuss the novel and
important directions concerning the application of bioactives from plants through
Preface ix

nanotechnology for the improvement of diagnosis and drug delivery, with a par-
ticular focus on cancer therapy. In Chap. 12, Martins and Ferreira describe the role
of plant food bioactives in neurocognitive improvement, which can be used to help
treat the effects of Alzheimer’s disease. The authors provide a systematic overview
of the use of plant food-derived bioactive molecules with evident in vitro and
in vivo neuroprotective and neuroregenerative effects.
Representation of facts and their discussions in each chapter are extensive,
authoritative, and deeply informative; hence, this book serves as a key reference for
recent biotechnological developments of food bioactives and their prospective
applications. The broad interdisciplinary approach of this book will surely make the
work very interesting to researchers, scientists, and postgraduate students deeply
engaged in the research and/or use of food bioactives. We would like to express our
sincere thanks to all the contributors for their excellent reviews in this remarkable
area. It is their participation that made our effort to organize such a book possible.
I am grateful to Dr. Monica Nijhawan Puri (my wife, an analytical chemist) who
assisted in reviewing the contributed chapters. Her chemistry background allowed
her to provide further critical validation of the subject content in this book. This
endeavor would not have been possible without her motivation and constructive
criticism, as well as the cooperation extended by my son Aryan and daughter
Arisha. Most importantly, I am indebted to my parents (Retired Prof. K.K. Puri and
Ms. Anuradha Puri) for inculcating values that made me an academic.
I would also like to express my deep sense of appreciation to all the editorial and
publishing staff members associated with Springer for their keen interest in pub-
lishing this book, as well as their all-around help to ensure that the highest standards
have been maintained in publishing this book.

Geelong, Australia Munish Puri


Part I Bioactive Sources: Plants, Mushrooms and Fungi

1 Glucosinolates and Respective Derivatives (Isothiocyanates)
from Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kyriaki G. Zinoviadou and Charis M. Galanakis
2 Bioactives from Mushroom and Their Application . . . . . . . . . . . . . . 23
Carmen Sánchez
3 Bioactive Products from Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Sergio Sanchez and Arnold L. Demain

Part II Extraction Technologies for Bioactives

4 Process Development for Bioactive Peptide Production . . . . . . . . . . 91
Govind Kumar Gnasegaran, Dominic Agyei, Sharadwata Pan,
Indira P. Sarethy, Caleb Acquah and Michael K. Danquah
5 Comparison of Natural Extraction and Recombinant Mussel
Adhesive Proteins Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
J.J. Castillo, B.K. Shanbhag and L. He
6 Extraction of Lipids and Carotenoids from Algal Sources . . . . . . . . 137
Adarsha Gupta, Avinesh R. Byreddy and Munish Puri
7 Magnetic Particles for Microalgae Separation
and Biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Ivo Safarik, Kristyna Pospiskova, Eva Baldikova
and Mirka Safarikova
8 Enzyme-Assisted Extraction of Bioactives . . . . . . . . . . . . . . . . . . . . . 171
Sandesh J. Marathe, Swati B. Jadhav, Sandip B. Bankar
and Rekha S. Singhal

xii Contents

Part III Techniques Employed for the Bioactives Delivery

9 Emerging Technologies for Bioactive Applications in Foods . . . . . . 205
Liliana G. Santiago, Carlos R. Soccol and Guillermo R. Castro
10 Emerging Technologies of Hydrogels in Bioactive
Compounds Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Maria Henriques L. Ribeiro

Part IV Therapeutic Role in Treating Diseases

11 Neurocognitive Improvement Through Plant Food Bioactives: A
Particular Approach to Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . 267
Natália Martins and Isabel C.F.R. Ferreira
12 Multifunctional Bioactives for Cancer Therapy: Emerging
Nanosized Delivery Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Deepika Sharma, Monica Nijhawan and Munish Puri
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Editor and Contributors

About the Editor

Munish Puri serves as a Deputy Director and Associate
Professor, Flinders Centre for Marine Bioproducts Development,
Medical Biotechnology, School of Medicine, Flinders University,
Adelaide, Australia. Professor Puri research interests include a)
microbial fermentation for producing valuable enzymes and
omega-3 fatty acids, b) nutraceutical extraction for food supple-
mentation, and c) use of nanotechnology for enhancing ther-
mostability of enzymes that has application in bioprocessing.
He holds a Ph.D. in Industrial Biotechnology from Punjabi
University India (1994) and did his post-doctoral training in the area
of protein biotechnology at University of Oxford, UK (2001–02).
He had the privilege of working as a University of New South Wales
Endeavour Fellow, at University of Queensland, Australia (2004–
05). Dr. Puri has worked as a full Professor/Associate Professor,
Department of Biotechnology, Punjabi University, India (2003–09)
where he led the area of enzyme biotechnology before moving to
Deakin University, Australia as a Visiting Professor and Senior
Research Fellow. He has served as the Group head industrial
biotechnology and bioeconomy at the Centre for Chemistry and
Biotechnology (CCB), Deakin University (2009–16) and has sig-
nificantly contributed towards advancing bioprocessing research
where emphasis was laid upon microbial production and enzymatic
processing of various bioactives (enzymes/proteins, sweeteners,
omega-3 fatty acids and carotenoids). He has contributed towards
bioprospecting of novel microorganisms for omega-3, lipids and
enzyme production; transformation of intermediates; expression
and production of peptides and therapeutic proteins; and immobi-
lizing lipases for the concentration of omega-3 acid production. His
group is also engaged in evolving novel technologies for processing
agriculture biomass for producing second/third generation biofuels.
Dr. Puri has published more than 110 scientific articles that
includes 93 peer-reviewed journal papers, 3 patents, 14 book
chapters, mainly in high impact factor biotechnology/applied
microbiology journals. A large number of his publications fall in

xiv Editor and Contributors

the discipline of industrial biotechnology and listed in ISI Web

of Science subject category “Biotechnology and Applied
Microbiology”. He has supervised 16 Ph.D. dissertations, 30 MSc
theses, and about 10 undergraduate projects. In November 2015, he
was admitted as a Fellow of the Royal Society of Chemistry
(FRSC), UK based on his contribution to the discipline.


Caleb Acquah Department of Chemical Engineering, Curtin University of

Technology, Miri, Sarawak, Malaysia
Dominic Agyei Department of Food Science, University of Otago, Dunedin, New
Eva Baldikova Department of Nanobiotechnology, Institute of Nanobiology and
Structural Biology of GCRC, Academy of Sciences, Ceske Budejovice, Czech
Republic; Department of Applied Chemistry, Faculty of Agriculture, University of
South Bohemia, Ceske Budejovice, Czech Republic
Sandip B. Bankar Department of Chemical Engineering, College of Engineering,
Bharati Vidyapeeth University, Pune, India
Avinesh R. Byreddy Bioprocessing Laboratory, Centre for Chemistry and
Biotechnology, Deakin University, Geelong, VIC, Australia
J.J. Castillo Department of Chemical Engineering, Monash University, Clayton,
VIC, Australia
Guillermo R. Castro Laboratorio de Nanobiomateriales, CINDEFI—
Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de
La Plata—CONICET (CCT La Plata), La Plata, Buenos Aires, Argentina
Michael K. Danquah Department of Chemical Engineering, Curtin University of
Technology, Miri, Sarawak, Malaysia
Arnold L. Demain Research Institute for Scientists Emeriti (RISE), Drew
University, Madison, NJ, USA
Isabel C.F.R. Ferreira Mountain Research Centre (CIMO), ESA, Polytechnic
Institute of Bragança, Bragança, Portugal
Charis M. Galanakis Department of Research and Innovation, Galanakis
Laboratories, Chania, Greece
Govind Kumar Gnasegaran Department of Chemical Engineering, Curtin
University of Technology, Miri, Sarawak, Malaysia
Adarsha Gupta Bioprocessing Laboratory, Centre for Chemistry and
Biotechnology, Deakin University, Geelong, VIC, Australia
Editor and Contributors xv

L. He Department of Chemical Engineering, Monash University, Clayton, VIC,

Swati B. Jadhav Food Engineering and Technology Department, Institute of
Chemical Technology, Mumbai, India
Sandesh J. Marathe Food Engineering and Technology Department, Institute of
Chemical Technology, Mumbai, India
Natália Martins Mountain Research Centre (CIMO), ESA, Polytechnic Institute
of Bragança, Bragança, Portugal
Monica Nijhawan Western Heights College, Geelong, VIC, Australia
Sharadwata Pan Department of Chemical Engineering, Indian Institute of
Technology Bombay, Powai, Mumbai, India
Kristyna Pospiskova Regional Centre of Advanced Technologies and Materials,
Palacky University, Olomouc, Czech Republic
Munish Puri Bioprocessing Laboratory, Centre for Chemistry and Biotechnology,
Deakin University, Waurn Ponds, VIC, Australia
Maria Henriques L. Ribeiro Research Institute for Medicines (IMed.ULisboa),
Faculty of Pharmacy, Universidade Lisboa, Lisbon, Portugal
Ivo Safarik Regional Centre of Advanced Technologies and Materials, Palacky
University, Olomouc, Czech Republic; Department of Nanobiotechnology, Institute
of Nanobiology and Structural Biology of GCRC, Academy of Sciences, Ceske
Budejovice, Czech Republic
Mirka Safarikova Department of Nanobiotechnology, Biology Centre, ISB,
Academy of Sciences, Ceske Budejovice, Czech Republic; Department of
Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC,
Academy of Sciences, Ceske Budejovice, Czech Republic
Carmen Sánchez Laboratory of Biotechnology, Research Centre for Biological
Sciences, Universidad Autónoma de Tlaxcala, Ixtacuixtla, Tlaxcala, Mexico
Sergio Sanchez Departamento de Biologia Molecular y Biotecnologia, Instituto de
Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, Mexico
City, CDMX, Mexico
Liliana G. Santiago Área de Biocoloides y Nanotecnología, Instituto de
Tecnología de Alimentos (ITA), Facultad de Ingeniería Química (FIQ),
Universidad Nacional del Litoral (UNL), Santa Fe, Argentina
Indira P. Sarethy Department of Biotechnology, Jaypee Institute of Information
Technology, Noida, Uttar Pradesh, India
B.K. Shanbhag Department of Chemical Engineering, Monash University,
Clayton, VIC, Australia
xvi Editor and Contributors

Deepika Sharma Institute of Nano Science and Technology, Mohali, Punjab,

Rekha S. Singhal Food Engineering and Technology Department, Institute of
Chemical Technology, Mumbai, India
Carlos R. Soccol Department of Bioprocess Engineering and Biotechnology,
Federal University of Paraná, Curitiba, Brazil
Kyriaki G. Zinoviadou Department of Food Science and Technology, Perrotis
College, American Farm School, Thessaloniki, Greece
Part I
Bioactive Sources: Plants,
Mushrooms and Fungi
Chapter 1
Glucosinolates and Respective Derivatives
(Isothiocyanates) from Plants

Kyriaki G. Zinoviadou and Charis M. Galanakis

1 Introduction

Nowadays, the maintenance and the enhancement of good health via dietary habits
have become an important social issue, and in this context, consumption of phy-
tochemicals as a part of a well-balanced diet is noteworthy. Glucosinolates
(GLs) comprise a distinctive group of bioactive compounds exhibiting a wide range
of activities in plants, as their major defense system, as well as in humans in many
ways. Since several studies reported an inverse correlation between the intake of
Brassica vegetables, the most important source of GLs, and the risk for several
types of cancer, these compounds have been on the spotlight. However, GLs can
lose their beneficial properties and transform into antinutrients depending on the
processing conditions. This chapter provides an overview regarding the different
methods that can be applied for the extraction of GLs and the effect of different
processing methods on their stability.

K.G. Zinoviadou
Department of Food Science and Technology, Perrotis College,
American Farm School, 55102 Thessaloniki, Greece
C.M. Galanakis (&)
Department of Research and Innovation, Galanakis Laboratories,
Skalidi 34, Chania 73131, Greece

© Springer International Publishing AG 2017 3

M. Puri (ed.), Food Bioactives, DOI 10.1007/978-3-319-51639-4_1
4 K.G. Zinoviadou and C.M. Galanakis

2 Glucosinolate Structure

Glucosinolates (GLs) are a group of plant secondary metabolites that can be found
only in dicotyledonous plants. Among the different plants, the most studied ones,
since they contain the highest concentration of GLs, are those that belong to the
Brassicaceae (Cruciferae) family such as broccoli, cabbage, kale, Brussels sprouts,
rape, and cauliflower (Angelino and Jeffery 2014; Oerlemans et al. 2006).
Quantities of GLs in these vegetables range from 0.1 to 2.5 g/kg and vary due to
several factors such as the region and cultivation conditions, the plant part, degree
of plant development, and genetic and environmental factors. All GLs have a
similar basic structure that includes:
• A b-D-thioglucose group,
• A sulfonated oxime group,
• A side chain derived from one of the seven protein amino acids.
Based on the structure of the side chain, the GLs can be divided into (i) aliphatic,
(ii) x-methylalkyl, (iii) aromatic, or (iv) heterocyclic (indole) GLs (Smiechowska
et al. 2010). Despite the fact that approximately 120 classes of GLs have been
identified in plants, only a few GLs are found in each plant species in significant
amounts. For instance, sinigrin makes the major contribution of GLs in kales and
glucobrassicin or glucoiberin in cabbage leaves, while the common GLs in broccoli
are glucoraphanin, sinigrin, and progoitrin and the indole GLs glucobrassicin and
neoglucobrassicin (Cartea and Velasco 2008).

3 Biosynthesis, Function, and Enzymatic Degradation

Amino acids are the precursors for the biosynthesis of GLs that proceeds through
three separate steps: (i) the chain elongation of selected amino acids, (ii) glucose
biosynthesis, and iii) modifications of the side chain (Fahey et al. 2001). In plants,
GLs are involved in response to biotic stress; the enzymatically formed
broken-down products of GLs activate the defense system of the plant upon
induction by herbivores or penetration by fungi. In this context, plants with high GL
content could be used as biofumigants in agriculture by incorporating them in
crushed form into the soil (Hanschen et al. 2014). Moreover, GLs are responsible
for the characteristic flavor and odor of the Brassica family. More specifically,
sinigrin and progoitrin have been related to bitterness in Brussels sprouts, while the
bitter taste of boiled cauliflower has been attributed to the presence of neogluco-
brassicin and sinigrin (Oerlemans et al. 2006).
Glucosinolates are not biologically active until they are hydrolyzed by myrosi-
nase. There are two mechanisms for the hydrolysis of GLs: the endogenous
b-thioglucosidase enzymes, commonly known as myrosinase, and certain com-
mensal bacteria. However, which bacteria are involved and the extent of hydrolysis
1 Glucosinolates and Respective Derivatives … 5

pH 4 pH 7

Fig. 1 Degradation products of GLs under different conditions (reproduced from Rask et al.

are still under research (Angelino and Jeffery 2014). In intact plant tissues, GLs and
myrosinase are localized in distinct compartments, since the enzymes are only
located in the vacuoles of myrosin cells. Different processes that may cause tissue
damage such as mastication, cutting, or cooking result in the release of myrosinase
and the hydrolysis of GLs into glucose and the unstable aglycones. Aglycones are
spontaneously converted into isothiocyanates (ITCs) or indoles depending on the
side chain. Based on the several factors such as the pH, the presence of ferrous ions,
the number of double bonds in the side chains as well as different proteins like
epithiospecifier protein further conversion of isothiocyanates and indoles into
epthionitriles, nitriles, thiocyanates, and other compounds occur as illustrated in the
following Fig. 1 (Ghawi et al. 2012; Rask et al. 2000; Terefe et al. 2014).

4 Chemopreventative and Antimicrobial Activity

Glucosinolate hydrolysis products, and in particular ITCs and indoles, have

received a special interest in food research because of their anticarcinogenic
properties. It has been reported that they affect the different stages of cancer
development, including the inhibition of activation enzymes (phase I enzymes) and
the induction of detoxification enzymes (phase II enzymes) (Cartea and Velasco
2008). Despite the fact that for the moment, no direct correlation between specific
ITCs and appearance of cancer has been provided, a large number of studies
demonstrate the link between the total ITC intake in the form of cruciferous veg-
etables and reduced risk of several types of cancer such as lung, breast, gastric,
bladder pancreatic, prostate, and kidney (Gupta et al. 2014). It is worth mentioning
that there are also some studies where the obtained results were opposite to the
6 K.G. Zinoviadou and C.M. Galanakis

expected ones. For instance, Giovannucci et al. (2003) did not observe any sig-
nificant correlation between the short-term intake of cruciferous vegetables and
prostate cancer; however, long-term intake exhibited promising results. Therefore,
it is essential to remember that there might be study limitations that do not allow the
reflection of true associations. For instance, people that consume large amount of
cruciferous vegetables tend to follow a healthier lifestyle and practice more physical
activities. Moreover, the interaction with genes that are involved in ITC metabolism
has not been taken into consideration in the studies, thus leading to inconsistencies
(Herr and Büchler 2010).
It has been demonstrated that ITCs exhibit antibacterial activity against various
food pathogens, but most of the studies are still limited to the microbial inhibitory
concentration (MIC) determination. Their antibacterial activity has been recently
reviewed, and interestingly, it was stated that the MIC of the active ITCs is in the
range or even lower to the compounds commonly used (Dufour et al. 2015). In an
attempt to reduce the amount of conventional preservatives, the use of allyl ITC is
already approved in Japan for food preservation as long as it is derived from natural
plant sources (Nadarajah et al. 2005).

5 GL and ITC Extraction

The presence of GLs in different Brassica vegetables or oilseed crops used for
animal feeding is undesirable due to their toxic effect when they are consumed at
high concentrations (Sun et al. 2008; Tao and He 2004). Consequently, the
recovery of GLs and ITCs from plant materials and wastes and by-products is a
process that can be applied for the detoxification of animal meals and at the same
time can provide us with these bioactive compounds that can be utilized for the
production of functional foods or nutraceuticals. In principle, the recovery of
antioxidants from substrates such as plant material or waste involves the so-called
5-stage universal recovery process that consists of the following steps: (i) macro-
scopic pretreatment, (ii) macro- and micromolecule separation, (iii) extraction by
different methods, (iv) isolation and purification, and (v) product formation
(Galanakis 2012; Galanakis and Schieber 2014).

5.1 Extraction by Conventional Means

Up to now, several conventional methods such as organic solvent extraction, per-

colation, membrane separation, liquid extraction under pressure, and Soxhlet have
been used for the recovery of different compounds of interest from plant material
(Galanakis et al. 2010a, b, 2013a, b, 2014; Patsioura et al. 2011; Heng et al. 2015;
Galanakis 2015). However, their application is limited due to the fact that they are
time-consuming and require large amount of solvents, while there is also the
1 Glucosinolates and Respective Derivatives … 7

possibility of causing molecular changes when high temperatures are applied

(Wang and Weller 2006). It is well established that the extraction from dried
material consists of two stages. Firstly, the material to be extracted is soaked in a
solvent to swell and hydrate it, and then, the soluble components move into the
extraction solvent by the mass transfer actions of diffusion and permeation (Huang
et al. 2013). In Table 1, some representative references regarding conventional
extraction methods are provided.
Due to the fact that there are great differences among GLs and ITCs regarding
their specific biologic effects, it is crucial to develop sensitive and reliable methods
for their extraction and identification. A method for extraction and direct analysis of
GLs was developed by Mohn et al. (2007) by optimizing several parameters such as
solvent composition, particle size, temperature, and number of extraction steps and
has been applied for the analysis of woad (Isatis tinctoria and Isatis indigotica).
Thermal degradation of GLs at temperatures above 50 °C and great losses (>60%)
within 10 min at 100 °C were recorded. Response surface methodology, which is a
widely used method for the optimization of total phenolic extraction, has been
recently used for the evaluation of GL recovery conditions from maca (Lepidium
myenii) (Campos et al. 2013). The extraction parameters investigated in the study
could be ranked in the following order in terms of influence on the total perfor-
mance: ethanol ratio > liquid-to-solid ratio > time > temperature. Moreover, Box–
Behnken design and analysis of variance have been applied in order to optimize the
extraction of twelve intact GLs from broccoli leaves by deactivating myrosinase
using heated water (Ares et al. 2014b). In an attempt to analyze the level of sinigrin
in mustard cv. Centennial (Brassica juncea L.) seed samples, Cools and Terry
(2012) compared four different extraction methods: boiling water, boiling 50%
(v/v) aqueous acetonitrile, boiling 100% methanol, and 70% (v/v) aqueous
methanol at 70 °C. It was found that the 70% (v/v) methanol and the boiling 50%
(v/v) acetonitrile were the most efficacious ones. Similarly, Tsao et al. (2002)
reported the highest sinigrin content in oriental mustard seed and bran when boiling
50% acetonitrile was used as an extraction method. In the study of Cools and Terry
(2012), the same extracts were used for the measurement of total ITCs, by applying
a dichloromethane extraction, and it was found that the water extraction was by far
the most efficient method compared to the others. Recently, the extraction and
chemical characterization of GLs and related compounds from broccoli have been
reviewed (Ares et al. 2013), and it was summarized that the best choice for the
extraction of desulfo-GLs is the use of methanol and water mixtures (70:30, v/v).
The objective of another study was to optimize the batch extraction conditions
(temperature, solvent composition, initial solvent pH, and solid loading) for max-
imum glucoraphanin recovery from Cardaria draba (Powell et al. 2005). At the
determined optimum conditions: 20% ethanol aqueous solvent at 70 °C, initial pH
3, and 50 g/dm3 loading, a threefold increase in glucoraphanin content was
observed compared with the control recovery method.

Table 1 Extraction of glucosinolates (GLs) and isothiocyanates (ITCs) by conventional means adopted from Deng et al. (2015)
Compound Material Sample treatment Yield References
Glucoraphanin Broccoli leaves from six Room temperature extraction (0.1% 12.2–119.4 (mg/100 g fresh Sasaki et al. (2012)
different cultivars formic acid in 80% v/v methanol) leaves)
1-methoxy glucobrassicin followed by ion exchange 3.1–31.1(mg/100 g fresh
solid-phase extraction (SPE) leaves)
Glucoiberin Cabbage leaves from 32 10.0–116.0 (mg/100 g fresh
different cultivars leaves)
Glucoraphanin 0.6–153.9 (mg/100 g fresh
Glucobrassicin Kale from 24 different 0.4–145.4 (mg/100 g fresh
cultivars weight)
Glucoiberin 0–119.8 (mg/100 g fresh
Sulforaphane Broccoli florets Conversion of glucoraphanin to 556 (mg/kg dry weight) Ares et al. (2014a)
Broccoli stems sulforaphane followed by solvent 446 (mg/kg dry weight)
extraction with methyl t-butyl ether
Broccoli leaves 30 (mg/kg dry weight)
and SPE with SI-1 particles
Sulforaphane Fresh cabbage Conventional extraction (30 min) 1.2 (mg/100 g of dry mass) Tanongkankit et al.
using dichloromethane as solvent (2013)
Conventional extraction (30 min) 1.1(mg/100 g of dry mass)
using water as solvent
Glucoraphanin Cardania draba leaves Extraction in stirred baffled vessels, 30 (mg/g dried sample) Powell et al. (2005)
80% ethanol at 70 °C, pH 3,
solid-to-liquid ratio 50 g/dm−3
K.G. Zinoviadou and C.M. Galanakis
Table 1 (continued)
Compound Material Sample treatment Yield References
Isothiocyanates Horseradish (Armoracia Hydrodistillation using 8.03 (% of sample powder) Wu et al. (2009)
Allyl isothiocyanate rusticana) dichloromethane as solvent 6.10 (% of sample powder)
Isothiocyanates Water extraction for 24 h at 20– 4.52 (% of sample powder)
Allyl isothiocyanate 40 °C, ratio of horseradish powder 3.39 (% of sample powder)
to water: 1:12
Progroitin Canola seeds from 5 Anion exchange membrane 2.06–10.16 (lmol/g seed) Szmigielska et al.
Napoleoferin different varieties extraction after heating in boiling 0.04–1.32 (lmol/g seed) (2000)
water for 5 min
Gluconapin 1.15–7.23 (lmol/g seed)
4-hydroxy-glucobrassicin 3.95–5.20 (lmol/g seed)
Glucobrassicin 0.08–4.90 (lmol/g seed)
Sinigrin Three mustard powder No pretreatment. Extraction with 14.35–28.79 (g/100 g Herzallah and
extracts boiling water for 10 min followed freeze-dried extract) Holley (2012)
1 Glucosinolates and Respective Derivatives …

by cooling and staying at 70 °C for

10 K.G. Zinoviadou and C.M. Galanakis

5.2 High-Pressure Food Processing

High-pressure processing (HPP) can be used for the inactivation of pathogens and
enzymes (Galanakis 2013). However, it is expected to be less detrimental than
conventional thermal processes to food components such as flavoring agents, col-
orants, and bioactive compounds since covalent bonds are not affected by pressure
(Butz et al. 2002). Moreover, HPP is a waste-free environment-friendly technology
that is independent of the size and geometry of the sample (Alvarez-Jubete et al.
2014). Since myrosinase is the enzyme responsible for the conversion of GLs to the
biologically active form, the ITCs, the effect of HPP to myrosinase activity is of
great importance. Ludikhuyze et al. (1999) were the first to study the effect of
combined HPP and thermal treatment on isolated broccoli myrosinase. It was found
that the application of pressures lower than 250 MPa at 20 °C did not have any
effect on the enzyme. On the contrary, when pressures between 300 and 500 MPa
were applied, significant inactivation of the enzyme was observed. Recently, the
thermal and pressure stability of myrosinase from the mustard seeds was evaluated,
and it was found that brown and black mustard myrosinase was more resistant than
the one from yellow mustard. In all samples, the enzyme was completely inacti-
vated when combined high-pressure and thermal treatment (up to 70 °C and
800 MPa) was applied (Okunade et al. 2015). Based on the above, it can be
concluded that there are significant differences in processing stability of myrosinase
among the Brassica species.
Studies on the enzyme inactivation in broccoli juice revealed an antagonistic
effect of temperature and pressure at temperatures higher than 50 °C and pressure
up to 200 MPa (Van Eylen et al. 2007). Similar results were found when enzyme
inactivation kinetics in broccoli tissue was studied (Van Eylen et al. 2008). In a
study related to the thermal and high-pressure inactivation of myrosinase from
green cabbage, it was demonstrated that this enzyme is highly susceptible to both
processes and that there was no antagonistic effect of HPP on thermal inactivation
(Ghawi et al. 2012). Moreover, it was shown that the inactivation followed the
first-order kinetics at all the applied combinations (temperature ranging from 35 to
50 °C and pressure ranging from 100 to 400 MPa).
The use of HPP to promote the conversion of GLs to ITCs has been the subject
of several studies. Application of HPP at 500 MPa on Brussels sprouts resulted in a
final concentration of sulforaphane of 1021.8 lmol per kg fresh weight which
corresponded to a 317% increase compared to the control (Koo et al. 2012). Similar
findings have been previously reported for red cabbage (Koo et al. 2011) and white
cabbage (Alvarez-Jubete et al. 2014) treated by HPP. This effect has been attributed
to increased cell membrane disruption induced by the HPP that facilitated the
contact between myrosinase and the substrate. Consequently, HPP could be
employed as a food processing and/or preservation technique to increase the levels
of some of its key phytochemicals such as isothiocyanates.
1 Glucosinolates and Respective Derivatives … 11

5.3 Ultrasound- and Microwave-Assisted Extraction

Ultrasound can be defined as inaudible sound waves at a frequency above 20 kHz.

For food preservation, ultrasound waves of low frequency (18–100 kHz;
k = 145 mm) and high intensity (10–1000 W/cm2) are the most effective (Dujmic
et al. 2013; Barba et al. 2015; Roselló-Soto et al. 2015a, b; Zinoviadou et al. 2015).
Ultrasonic cavitation phenomena create high shear forces that can mechanically
disrupt cell walls and improve material transfer while allowing solvent penetration
into the plant material, thus facilitating the extraction of several compounds
(Szydlowska-Czerniak et al. 2015). Up to now, ultrasonic treatment has been used
for separation processes, and currently, it is developing as a tool to extract natural
bioactive compounds on an industrial scale. A study was conducted in order to
determine the optimum conditions for the extraction of sinigrin from defatted seed
powder of Indian mustard after ultrasound-stimulated treatment. It was shown that
the ultrasound-stimulated solvent extraction improved the productivity by 71% than
that of conventional extraction and this might be attributed to the greater decrease of
the outer pectinous material that was also recorded (Wang et al. 2011). Moreover,
ultrasounds have been applied as a pretreatment step prior to microwave-assisted
extraction (MAE) of bioactive compounds from outer leaves of cabbage. Of all
conditions tested, 30-min sonication was chosen as the suitable time and it was
found that the combination of ultrasound-assisted extraction with MAE led to
higher yield of GLs when compared to the application of the techniques alone.
Results obtained by confocal microcopy suggest that this higher yield could be
attributed to the more extensive cell damage that was recorded when the combi-
nation of the two techniques was applied (Pongmalai et al. 2015).
The use of MAE as a means to extract GLs has been the topic of more studies.
The optimization of a MAE method for the recovery of GLs from Eruca sativa
seeds revealed that the optimum conditions were as follows: methanol extraction at
250 W and 80 °C for 10 min. When comparing the efficiency of MAE with other
methods (the certified ISO and ultrasound-assisted extraction), it was found that
MAE and ISO resulted in the similar yields of approximately 110 lmol/g (Omirou
et al. 2009). Tanongkankit et al. (2013) investigated the optimum conditions for
sulforaphane extraction from white cabbage leaves by studying the effect of solvent
type and partial drying prior to MAE. It was found that MAE was more effective
than conventional extraction since it led to higher sulforaphane yields in a much
shorter extraction time that was decreased as the microwave power increased. No
significant differences were observed when either fresh or semi-dried cabbage
leaves were used, and the results were the same when either water or dichlor-
omethane was used as the solvent. The effect of the drying methods prior to the
MAE was the subject of another study. It was found that the total GLs of the
extracts from cabbage leaves dried by the use of either hot air or low-pressure
superheated steam were significantly lower compared to the extracts from the fresh
sample (Chaisamlitpol et al. 2014).
12 K.G. Zinoviadou and C.M. Galanakis

5.4 Pulsed Electric Field

Pulsed electric field (PEF) is a non-thermal cost-effective preservation technology

that has shown high potentials for microbial reduction and enzyme inactivation
applications (Roselló-Soto et al. 2015a; Sánchez-Vega et al. 2015). Moreover, it has
been used for the extraction of intracellular compounds of commercial interest with
high yields. The underlying mechanism of PEF causes electroporation of the cell
membranes, thus increasing their permeability and the release of the different
compounds (Aguilo-Aguayo et al. 2015). Recently, the effect of PEF treatment
variables (electric field strength from 1 to 4 kV/cm and treatment times from 50 to
1000 ls at 5 Hz) on the Gls content of broccoli stalks and flowers was evaluated.
PEF application seems rather promising for the improvement of phytochemical
availability in broccoli products since it resulted in a significant enhancement (about
twofold) in glucoiberin, glucoraphanin, glucobrassicin, and neoglucobrassicin both
in broccoli flowers and in stalks, while the myrosinase enzyme remained inactive
(Aguilo-Aguayo et al. 2015). On the contrary, in a study regarding PEF disinte-
gration of Brassicaceae tissue composed of cells containing myrosinase and other
cells containing GLs, a decrease of the GL concentration was identified in the
tissue, but no increase was found in the extract. Interestingly, in the same study,
when the same tissue containing no myrosinase was used, an enhanced
extractability and higher levels of GLs were found. It was concluded that due to the
particularities of this raw material, PEF is not an appropriate technology for the
enhancement of GL recovery (Jager 2012). Another study that was recently con-
ducted evaluated the effect of PEF processing on the content of GLs and activities
of myrosinase isoenzymes on broccoli puree and juice. It was found that the
myrosinase activities resulted in nearly total GL transformations autolysis during
sample preparation pointing out the importance of specific focus on the sample
steps preceding the PEF processing (Frandsen et al. 2014).

5.5 Supercritical Carbon Dioxide Technology

Supercritical fluid extraction (SFE) is an operation that takes advantage of the

unique properties of fluids (most commonly CO2) above their critical values in
order to extract the different compounds from a matrix. It is rather advantageous
compared to conventional methods since it is inert and non-toxic and allows faster
extraction at relatively moderate temperatures. Consequently, it can be considered
as an alternative environment-friendly technique (Li et al. 2010; Solana et al. 2014).
Supercritical CO2-extracted meal has been used for the removal of GLs from
canola meals, and it was found that the CO2-extracted meal had lower GL content
when compared to the conventionally hexane-extracted one. Moreover, it was
found that the CO2-extracted meal was superior to commercial meals in terms of
both chemical composition and functionality (Sun et al. 2008). In another study, the
1 Glucosinolates and Respective Derivatives … 13

extraction of allyl isothiocyanate (AIT) from wasabi using supercritical CO2 was
assessed. More specifically, the effect of pressure, temperature, and the moisture
content of wasabi on the yield of AIT was evaluated and it was found that when the
pressure ranged from 15 to 25 MPa and the temperature from 35 to 55 °C, the yield
increased as the pressure raised and/or the temperature dropped. The highest yield
obtained under operating conditions of 25 MPa and 35 °C was 408 mg/100 g of
solid. Much higher yields (930 mg/100 g sample) were reported in a previous study
where the SFE of AIT from freeze-dried wasabi using ethanol as a cosolvent was
evaluated. The differences in yield between the two studies can be attributed to the
solubility enhancement by ethanol. However, it should be pointed out that when
ethanol is used, an additional step is required in order to recover AIT from the
solvent that would inevitably decrease the final amount (Li et al. 2010). Recently,
the use of supercritical CO2 for the extraction of GLs from rocket salad by using
different cosolvents (water, ethanol, methanol, none) was studied. Out of all the
cosolvents, water was the most efficient one and the extractions performed by
supercritical CO2 + water were favored by both higher pressures and temperatures.
When 30 MPa and 75 °C were applied, an extract containing 1.96 mg/g of GLs
was obtained (Solana et al. 2014). In the same study, a sequential extractive
approach was proposed initially using water as a cosolvent for the extraction of
phenols and GLs followed by the use of CO2 and ethanol for lipid extraction.

6 Post-harvesting and Process-Induced Changes in GL


In principle, vegetables undergo a variety of processes prior to consumption

including packaging, storage, preparation, and cooking that can greatly affect the
GL–myrosinase system. It has previously been estimated that a 5- to 10-fold
variation in levels of GLs can be attributed to industrial processing and storage and
a 5- to 10-fold variation household preparation (Dekker et al. 2000).

6.1 Packaging and Storage Conditions

In an attempt to investigate the effect of various post-harvest technologies on the

GL content of fresh-cut vegetables and salads, several studies have evaluated
packaging materials, modified atmosphere packaging (MAP), and storage condi-
tions. When broccoli florets were packed in polyethylene bags with different per-
meability properties and then stored at 4 or 20 °C, it was shown that all MAP
treatments resulted in the reduced degradation of total aliphatic and indole GLs
compared to the control (Jia et al. 2009). Schreiner et al. (2007) studied the changes
in GL content in mixed fresh-cut broccoli and cauliflower florets stored for 7 days
14 K.G. Zinoviadou and C.M. Galanakis

at 8 °C under 2 different MAP conditions and found that both conditions main-
tained aliphatic GLs in cauliflower throughout the whole storage period. On the
contrary, slight decrease was observed regarding the GL levels in the broccoli
florets. Interestingly, storage under certain conditions may even lead to the increase
of certain GLs as previously reported (Hodges et al. 2006). In this study, total GL
levels increased in the samples that were stored in air on day 28 and remained
constant thereafter, while there were no changes observed for the samples stored
under controlled atmosphere between days 14 and 56. These effects may be
attributed to metabolic changes associated with natural or stress-induced senescence
or to the development of a defense system against pathogens.
The effect of time and temperature during storage of the Brassica vegetables has
been previously studied, and different trends have been recorded. When trying to
simulate the storage conditions through the whole supply chain, freshly harvested
broccoli inflorescences were harvested, film-wrapped, and stored for 7 days at 1 °C
followed by storage at 15 °C for 3 days. Major losses (approximately 70%) in total
GL content were observed after the cold storage, and the losses were elevated by
the end of the whole storage period (Vallejo et al. 2003). These results are in
contrast to what has been previously reported by Rodrigues and Rosa (1999) that
recorded only minor losses of total GLs when the broccoli inflorescences were kept
refrigerated at 4 °C for 5 days. However, significant losses, attributed to myrosi-
nase activity, were observed also in this study when the samples were kept at 20 °C
for 5 days. Regarding the effect of storage temperature on the GL level of baby leaf
rocket, in the case of perennial wall rocket higher levels of total GLs were found
when samples were stored at 0 or 7 °C, while for annual garden rocket significantly
higher amounts of total GLs were found in the samples stored at 7 °C. So it was
concluded that any potential health benefits were diminished as a result of storage
temperature over a shelf-life period (Hall et al. 2014). A comparative study on the
effect of storage temperature on four vegetables belonging to the Brassica species
revealed interesting findings. When evaluating the GL content of broccoli, white
cabbage, and turnip after 72 h of storage or directly after harvesting, lower levels
were found after the storage period. On the contrary, total GL content and espe-
cially the amount of two indolyl GLs were increased upon storage of Portuguese
cabbage (Aires et al. 2012). As previously stated, this increase could be explained
by several factors such as the pH presence of cofactors, humidity, and temperature
that may affect the myrosinase activity and consequently the breakdown process
(Verkerk et al. 2001). Recently, the effect of storage on the ITC sulforaphane in two
radish cultivars has been studied, and it was shown that after 4 months of storage at
0C, the level of this ITC was significantly reduced (81 and 40% reduction). This
was attributed to the monitored lower myrosinase activity that resulted in a
decreased formation of sulforaphane (Lim et al. 2015).
1 Glucosinolates and Respective Derivatives … 15

Table 2 Relative importance of the main mechanism related to alterations in the GL content upon
different types of thermal processing (Nugrahedi et al. 2015b)
Mechanism Boiling Steaming Balancing Microwave treatment Stir-frying
Lysis + + + + +
Diffusion in tissue + + + + +
Leaching + ± ± ± −
Enzymatic activity − ± − − −
Myrosinase inactivation + + + + +
Thermal breakdown ± ± ± ± +

6.2 Thermal Processing

During thermal processing, the levels of GLs can be altered because of enzymatic
activity, leaching into cooking water, as well as thermal degradation (Galanakis
et al. 2010c). However, other mechanisms that take place during heating are of great
importance. These include lysis of cells or cellular compartments, diffusion of
different components through the lysed cells, inactivation of myrosinase, and loss of
cofactors such as Fe+2 and ascorbic acid (Nugrahedi et al. 2015b). The importance
of all these mechanisms is presented in the following Table 2.
It is important to point out that in principle, the rate and the level of GL loss are
highly dependent on the plant origin, the initial amount, the cooking type, and the
amount of water used. Moreover, it has been established that the indole GLs are
more heat sensitive than the aliphatic ones; however, the increased losses observed
during preparation are attributed to their higher diffusivity. It can also be stated that
among all cooking methods, steaming of the Brassica vegetables will ensure higher
retention of the GLs (Palermo et al. 2014). Recently, a study has been conducted on
the health perception of different preparation methods in regard to the GL content of
Brassica vegetables. The results revealed that steaming and boiling are perceived
by the food services to have increased beneficial health effects, while as household
respondents are concerned, boiling and stir-frying are perceived as the most ben-
eficial cooking methods (Nugrahedi et al. 2015a). The last years, there has been an
attempt to develop mathematical models that can describe the fate of GLs and
myrosinase in different plant tissues and under different thermal processes (Hennig
et al. 2012; Sarvan et al. 2012, 2014). All this knowledge can be effectively used by
epidemiological studies in order to estimate more effectively the daily GL intake.

6.3 Fermentation

Fermentation is an old processing method that can extend the shelf life of the
products and at the same time result in the formation of several potentially
breakdown products (Galanakis et al. 2015). In the case of Brassica vegetables,
16 K.G. Zinoviadou and C.M. Galanakis

fermentation is achieved by the use of lactic acid bacteria that are either naturally
present or added as starter cultures and the addition of sodium chloride (Nugrahedi
et al. 2015b). Among the fermented Brassica products, sauerkraut is a popular food
made from chopped white cabbage. It has previously been reported that the fer-
mentation of cabbage resulted in a complete degradation of GLs accompanied by an
increase of ITCs (Tolonen et al. 2002). Recently, the changes in GLs and gluco-
brassicin degradation products during the fermentation process of sauerkraut were
evaluated. GL content decreased dramatically between day 2 and 5 and by day 7
there were no detectable amounts. However, fermentation led to the formation of
other bioactive compounds, and the obtained results imply a peak in beneficial
compounds by the end of the fermentation (7–9 days) when compared to fresh
cabbage or stored sauerkraut (Palani et al. 2016). Moreover, the effect of fermen-
tation conditions, cabbage cultivar, and starter culture on the volatile GL hydrolysis
compounds has been assessed (Peñas et al. 2012), as well as the use of
Lactobacillus paracasei LMG-P220432 for the development of a product rich in
phytochemicals and with a high count of live probiotic bacterial cells (Sarvan et al.
2013). In the case of animal feeds such as rapeseed meal, high level of GLs is the
main antinutritional value. Consequently, their degradation is desired, and several
GLs degrading strains have been screened for their ability to improve the quality of
these meals by fermentation (Wang et al. 2012).

7 Future Perspectives

Existing knowledge can be derived from plant sciences and applied in order to
enhance the initial amount of selected bioactive compounds such as GLs in different
plants. The best well-known example is the development of the so-called super
broccoli that contains higher levels of methylsulphonylalkyl GLs, the precursors of
the functional ITCs iberin and sulforaphane. Moreover, the removal of specific GLs
and their breakdown products may reduce bitterness and consequently increase
consumer acceptance. This is an issue of great importance since consumer reports
have indicated that taste and not recognized health value are the key to food
selection (Ishida et al. 2014). However, not only the levels can be elevated by
genetic improvement but also the dynamic behavior of such compounds through the
whole supply chain can be optimized by adapting the plant’s genotype. In this
context, collaboration between food and plant scientists that will lead to the
development of breeding for quantitative food processing traits is a challenging
approach (Hennig et al. 2014). Last but not least, further studies for the optimization
of extraction processes are required and studies on the effect of the different pro-
cesses in order to fully understand the behavior of these bioactive compounds.

Acknowledgements Kyriaki Zinoviadou was supported through the Athinoula A. Martinos

Endowed Professorship, Perrotis College, 2015–2016.
1 Glucosinolates and Respective Derivatives … 17


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Chapter 2
Bioactives from Mushroom and Their

Carmen Sánchez

1 Introduction

This chapter describes the variety and biomedical potential of mushrooms as well as
their bioactive compounds. It starts with a description of the structure, growth, and
composition of mushroom fungi. A description of polyssacharides (e.g., b-glucan)
and polysaccharide–protein complexes was found in different mushrooms, and their
potential medical uses are mentioned. In addition, the immunomodulatory bioac-
tivity of b-glucans is illustrated in this section. Terpene compounds as the largest
group of anti-inflammatory compounds in mushrooms are addressed. The impor-
tance of phenolic compounds acting as free radical inhibitors, peroxide decom-
posers, metal inactivators, or oxygen scavengers in biological systems is described.
Bioactive proteins and peptides, including lectins, which have no enzymatic
activity, as well as those bioactive proteins possessing enzymatic activity such as
fungal immunomodulatory proteins, ribosome-inactivating proteins, and laccases,
are addressed. Finally, other compounds are able to reduce oxidative stress in the
endoplasmic reticulum, demonstrating its potential effect in neurodegenerative
diseases, and others showing antidepressant properties are also mentioned.

2 Mushroom: Structure, Growth, and Composition

Mushrooms are a very large and diversified group of macrofungi belonging to

basidiomycetesand ascomycetes, which have two phases of growth: the reproduc-
tive phase (fruit bodies) and the vegetative phase (mycelia). These organisms are

C. Sánchez (&)
Laboratory of Biotechnology, Research Centre for Biological Sciences, Universidad
Autónoma de Tlaxcala, Ixtacuixtla, Tlaxcala CP. 90062, Mexico

© Springer International Publishing AG 2017 23

M. Puri (ed.), Food Bioactives, DOI 10.1007/978-3-319-51639-4_2
24 C. Sánchez

epigeous (grow above the earth) with the umbrella-shaped fruiting body, where
spores are produced (in lamellae, structures on the underside of the pileus). The
fungal spores for these two groups are located in a special structure called basidium
(for basidiomycetes) or ascus (for ascomycetes). In the fungal growth, after spore
germination (or inoculation of in vitro-grown mycelia), the substrate is invaded by
microscopic filaments called hyphae. The cells in a hypha are separated by a
cross-wall called septum. Hyphae continually grow and branch to form a network
of hyphae or mycelia (mycelial growth). Mycelial growth is generally coupled with
increased enzyme production and respiration. Hyphae absorb digestive products,
penetrating the substrate to some extent. The fungal cell wall can be formed by
mannoproteins, b-D-glucans, and chitin (Fig. 1). From the ecological point of view,
mushroom fungi can be saprotrophs, parasites, and mycorrhiza. There are only few
parasitic mushrooms. Most of the cultivated mushrooms are saprotrophs.
Mycorrhizal mushrooms have a symbiotic relationship with some vegetation,
mainly trees, having a relationship of mutual benefit. Saprotrophs are able to obtain
nutrients from dead organic material, and parasites obtain their food from living
animals and plants, causing harm to the host (Cheung 2008). Mushrooms have been
eaten and appreciated for their exquisite flavor, economic and ecological values,
and medicinal properties for many years. In general, mushrooms contain 90% water
and 10% dry matter (Sánchez 2010). They have a chemical composition, which is
attractive from the nutritional point of view (Dundar et al. 2008). Their nutritional
value can be compared to those of eggs, milk, and meat (Oei 2003). Mushrooms
contain vitamins (thiamine, riboflavin, ascorbic acid, ergosterol, and niacin) as well
as an abundance of essential amino acids. They also have proteins, fats, ash, gly-
cosides, volatile oils, tocopherols, phenolic compounds, flavonoids, carotenoids,
folates, organic acids, etc. (Sánchez 2004; Patel and Goyal 2012). The total ener-
getic value of mushroom caps is between 250 and 350 cal/kg of fresh mushrooms
(Sánchez 2010). Mushrooms can be considered as functional food which provides
health benefits in addition to nutritional value (Rathee et al. 2012). They have been
collected in several countries for hundreds of years, and technological improve-
ments have made possible their cultivation worldwide.

3 Bioactive Compounds in Mushroom

There has been an increasing interest in mushrooms as a source of biologically

active compounds which provide to humans medicinal or health benefits such as the
prevention and treatment of diseases (Rathee et al. 2012). Bioactive compounds can
be found in mushroom as cell wall components such as polysaccharides (e.g.,
b-glucans) and proteins or as secondary metabolites such as phenolic compounds,
terpenes, and steroids. The concentration and efficacy of the bioactive compounds
are varied and depend on the type of mushroom, substrate, fruiting conditions (if
cultivated), stage of development, age of the fresh mushroom, storage conditions,
and cooking procedures (Guillamón et al. 2010). Many studies have reported that
2 Bioactives from Mushroom and Their Application 25

Fig. 1 Schematic representation of mushroom phases of growth and fungal cell wall composition

the medicinal properties of mushrooms include anti-inflammatory, antioxidant,

immunomodulatory, anticarcinogenic, antiviral, antibacterial, antifungal, hepato-
protective, antineurodegenerative, antidiabetic, antiangiogenic, and hypoglycemic,
among others (Badalyan 2012; Elsayed et al. 2014; Xu and Beelman 2015).
Mushrooms’ bioactive compounds on the basis of their chemical structure can be
polysaccharides, phenolic compounds, terpenes and terpenoids, phenols, peptides,
proteins, etc. (Table 1).
Table 1 Biologically active compounds from mushrooms and their medical applications

Mushroom Common names Phyllum or Bioactive compound Bioactivity Reference

Scientific name group/
Agaricus bisporus Champignon, B/E Pyrogallol Anti-inflammatory Moro et al., 2012;
Button mushroom, hydroxybenzoic acid derivatives Ndunguts et al.,
White mushroom, Flavonoids 2015
Agaricus Macro mushroom B/E Agaricoglycerides Anti-inflammatory Han and Cui,
macrosporus 2012
Agaricus Almond B/E Glycoprotein, b-(1, 3)-glucan, Immunomodulatory Firenzuoli et al.,
subrufescens mushroom, with b-(1,6)-glucan branch 2007; Lima, 2008
(= Agaricus blazei, God’s mushroom, Protein fractions and Immunomodulatory Jeurink et al.,
Agaricus Mushroom of life, polysaccharides fractions 2008
brasiliensis, Royal sun
Agaricus Agaricus
Agrocybe Poplar mushroom B/E b-Glucans Anti-oxidant Rathee et al.,
cylindracea Agrocybin (peptide) Hypoglycemic 2012; Zhang
(= Pholiota et al., 2003
aegerita) Gupta et al., 2014
Ngai et al., 2005
Albatrellus ovinus Forest lamb B/E Grifolin and grifolin derivatives Anti-inflammatory Nukata et al.,
(= Polyporus mushroom, sheep Anti-oxidant 2002
ovinus) polypore
Albatrellus Blue albatrellus B/E Phenolic compound Anti-inflammatory Nukata et al.,
caeruleoporus Grifolinones A, B 2002
Quang et al.,
Antrodia Stout camphor B/NE Glycoprotein ACA Immunomodulatory Sheu et al., 2009
camphorata fungus Diterpenes Neuroprotective Chen et al., 2006
C. Sánchez

Table 1 (continued)
Mushroom Common names Phyllum or Bioactive compound Bioactivity Reference
Scientific name group/
Auricularia Jew´s ear, B/E Glucan Hyperglycemia, Zhang et al.,
auricula wood ear, Immunomodulatory 2007
jelly ear Anti-tumor
Boletus edulis Cep, B/E Polysaccharides Anti-inflammatory Moro et al., 2012
penny bun,
king bolete
Boletus spp Gelam mushroom B/E 2,4,6-trimethylacetophenone imine, Anti-oxidant Yuswan et al.,
glutamyl tryptophan, azatadine, 2015
lithocholic acid glycine conjugate
Cantharellus Chanterelle, B/E Pyrogallol Anti-inflammatory Moro et al., 2012;
cibarius golden Dugler et al.,
chanterelle, 2004
2 Bioactives from Mushroom and Their Application

girolle Flavonoids Anti-microbian Palacios et al.,

Polysaccharides 2011
Caffeic acid, catechin Anti-oxidant
Calvatia gigantea Giant puffball B/E Calvacin Anti-tumor Rathee et al.,
Caripia montagnei Pod parachute B/E Polysaccharides (glucans) Anti-inflammatory Queiroz et al.,
Clitocybe maxima B/NE Laccase Anti-tumor Zhang et al.,
Table 1 (continued)

Mushroom Common names Phyllum or Bioactive compound Bioactivity Reference

Scientific name group/
Coprinus Shaggy ink cap, B/E b - 1,3-glucan Immunomodulatory Chan et al., 2009
comatus lawyer’s wig, Protein fractions and Immunomodulatory Jeurink et al.,
shaggy mane polysaccharides fractions 2008
Cordyceps Beldar-mazo, A/E Cordycepin Anti-inflammatory, Won et al., 2005;
militaris deer fungus, Anti- angiogenic Kumar et al.,
caterpillar fungus Anti-cancer 2010
Cordymin Anti-inflammatory Das et al., 2010
Wong et al., 2011
Cordyceps Summer grass, A/E Cordycepin, Anti-oxidant Holliday et al.,
sinensis winter worn 2004
Ciclosporin Immunosuppressive Holliday, 2005
Cordymin (peptide) Anti-inflammatory Wang et al.,
2012; Qian et al.,
Cortinarius Sooty-olive B/NE 6-hydroxyinfractine, Anti-neurodegenerative Brondz et al.
infractus Cortinarius, the infractopicrine 2007; Geissler
bitter webcap et al., 2010
Craterellus Black chanterelle, B/E Myricetin Anti-oxidant Palacios et al.,
cornucopioides horn of plenty, 2011
black trumpet,
trumpet of the
C. Sánchez
Table 1 (continued)
Mushroom Common names Phyllum or Bioactive compound Bioactivity Reference
Scientific name group/
Craterellus Yellow foot, B/E Polysaccharides Anti-inflammatory Tsvetkova et al.,
tubaeformis winter mushroom, 2006
funnel chanterelle
Cyathus africanus Bird’s nest fungi B/NE Diterpenoid (neosarcodonin, Anti-inflammatory Han et al., 2013
cyathatriol, and
Daldinia King Alfred’s A/NE 1-(3,4,5-trimethoxyphenyl) ethanol, Neuroprotective Lee et al., 2002b
concentrica Cake, cramp balls, caruilignan C
coal fungus
Dictyophora Veiled lady B/E Dictyophorine A and B Anti-neurodegenerative Kawagishi et al.,
indusiata mushroom, 1997
(=Phallus bamboo Dictyoquinazol A, B, and C Neuroprotective Lee at al., 2002a
indusiatus) mushroom
2 Bioactives from Mushroom and Their Application

Elaphomyces False Truffle A/NE Syringaldehyde, Anti-inflammatory Wang and

granulatus Syringic acid Marcone., 2011
et al., 2009
Flammulina Golden needle B/E Peptidoglycan Anti-inflammatory, Yin et al., 2010
velutipes mushroom antiviral
Enoki Polysaccharides Anti-inflammatory Wu et al., 2010
Flammulin (protein) Anti-tumor Chen et al., 2003;
Chang et al.,
Fomitopsis Red-belt conk B/NE Polysaccharides Anti-inflammatory Cheng et al.,
pinicola 2008
Table 1 (continued)

Mushroom Common names Phyllum or Bioactive compound Bioactivity Reference

Scientific name group/
Ganoderma Reishi, lingzhi, B/NE Ganoderic acids, Anti-tumor, Xu et al., 2010,
lucidum mannentake ganoderiol, anti-metastasis Walton, 2014; Xu
ganodermanontriol, anti-HIV and Zhong, 2012
Ganoderan A and B Hypoglycemic El-Mekkawy
et al., 1998
Ganopoly Hepatoprotective Rai et al., 2005
Triterpenes Anti-inflammatory Rathee et al.,
Lucidenic acids and ganoderic acids Anti-inflammatory 2012
Gao et al., 2002;
Lanostane-type triterpenic acids Anti-inflammatory
Ling zhi-8 (protein) Immunomodulatory et al., 2009.
Ganodermin (protein) Antifungal Akihisa et al.,
Se-containing protein Anti-tumor 2007;
Iwatsuki et al.
Akihisa et al.
Kino et al., 1989
Wang and Ng
Du et al., 2007
Ganoderma LingZhi (Chinese B/NE Protein GMI Immunomodulatory Lin et al., 2010
microsporum name)
C. Sánchez
Table 1 (continued)
Mushroom Common names Phyllum or Bioactive compound Bioactivity Reference
Scientific name group/
Ganoderma pfeifferi Paksulattakääpä B/NE Sesquiterpenoid hydroquinones Anti-bacterial, Niedermeyer
(Finnish common (lucialdehyde D, ganoderone A, anti-fungal, et al., 2005
name) ganoderone C) anti-viral
Geastrum saccatum Rounded earthstar B/NE Polysaccharides (b-glucans) Anti-inflammatory Guerra-Dore
et al., 2007
Ganoderma tsugae Hemlock varnish Fip-gts (protein) Immunomodulatory Lin et al., 1997
(= Polyporus shelf
Grifola Frondosa Hen-of-the-woods, B/E Grifolan1 Immunomodulatory Yang, 2007;
ram’s head, (1-6-monoglucosyl-branched anti-tumor, Kidd et al., 2000
sheep’s head, b-1,3-glucan) Proteoglycan, Anti-viral,
maitake Heteroglycan, hepatoprotective
Galactomannan, Anti-inflammatory Han and Cui,
2 Bioactives from Mushroom and Their Application

Glucoxylan 2012
Low-molecular weight protein Anti-tumor Kodama et al.,
fraction 2002
Table 1 (continued)

Mushroom Common names Phyllum or Bioactive compound Bioactivity Reference

Scientific name group/
Hericium erinaceus Lion’s mane B/E Phenol-analogous compounds Anti-oxidant Wang et al., 1996
mushroom, (hericenons C, D, E, F, G, H)
bearded tooth, Hericenons Erinacines, hericerins, , Anti-biotic, Mizuno, 1999
Satyr’s Beard, resorcinols, steroids, mono-terpenes, anti-carcinogenic,
pompom diterpenes anti-diabetic,
mushroom, anti-fatigue,
bearded tooth anti-hypertensive,
fungus anti-hyperlipodemic,
Heteroglycan peptide, b-1,3 Hyperglycemia, Lee et al., 2009;
branched-b-1,2-mannan Immunomodulatory Friedman, 2015
neuroprotective, etc.
Lectin (glycoprotein) Anti-tumor, Anti-virus
Hericenones (A-H), Erinacines Anti- neurodegenerative Li et al., 2010b
(A-K, P-Q),
Dilinoleoylphosphatidylethanolamine Anti-neurodegenerative Xu and Beelman,
2015; Phan et al.,
2014 Nagai et al.,
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Table 1 (continued)
Mushroom Common names Phyllum or Bioactive compound Bioactivity Reference
Scientific name group/
Hypsizygus Brown Beech, B/E Ergosterol, Anti-oxidant, Yoshino et al.,
marmoreus buna - shimeji manitol, anti-inflammatory, 2008
(=Hypsizygus Trehalose, Anti-allergic Wong et al., 2008
tessellatus) methionine
Marmorin anti-tumor activity Chowdhury et al.,
Phenolic compounds Anti-bacterial, 2015
flavonoids antifungal,
Inonotus obliquus Chaga, B/E b-D-glucans Anti-oxidant, stomach Rathee et al.,
clinker polypore, diseases, cancer 2012
cinder conk, Mannogalactoglucan Anti-tumor, Wasser, 2010
black mass
Sterols Anti-inflammatory Van et al., 2009;
2 Bioactives from Mushroom and Their Application

Park et al., 2005

Triterpenes Anti-inflammatory, Ma et al., 2013
Lactarius deliciosus Saffron milk cap, B/E Pyrogallol, Anti-inflammatory Moro et al., 2012
(= L. flavidulus) red pine Flavonoids
mushroom Polysaccharides Anti-inflammatory Fijimoto et al.,
Lactarius rufus Rufous milk cap, B/E Polysaccharides: Anti-inflammatory Ruthes et al.,
red hot milk cap (1,3), (1,6)b-D-glucans 2013
Table 1 (continued)

Mushroom Common names Phyllum or Bioactive compound Bioactivity Reference

Scientific name group/
Lentinula edodes Shiitake, B/E Lentinan, glucan, Immunomodulatory Sasaki and
mannoglucan, antitumor, Takasuka, 1976;
Israilides et al.,
Fucomannogalactan Anti-inflammatory Attarat and
Phermthai, 2015
Lentin (protein) Anti-fungal Ngai and Ng,
Catechin (Phenolic compound) Anti-oxidant Chowdhury et al.,
Phenolic compounds Anti-bacterial, 2015
flavonoids antifungal,
Lentinula NA B/NE Catechin Anti-oxidant Attarat and
polychrous Phermthai, 2015
Lentinula NA B/NE Catechin Anti-oxidant Attarat and
squarrosulus Phermthai, 2015
Lenzites betulina Gilled polypore, B/NE Betulinan A Anti-oxidant Rathee et al.,
birch maze gill, 2012
multicolor gill,
Lignosus Tiger milk B/NE Polysaccharides-protein Anti-cancer Gupta et al., 2015
rhinocerus mushroom
Lyophyllum Fried chicken B/E Polysaccharides: Anti-inflammatory Ukawa et al.,
decastes mushroom (1, 3) and (1,6)b-D-glucans 2000
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Table 1 (continued)
Mushroom Common names Phyllum or Bioactive compound Bioactivity Reference
Scientific name group/
Mycoleptodonoides Bunaharitake B/E 3-(hydroxymethyl)-4- Anti-neurodegenerative Choi et al. 2009;
aitchisonii methylfuran-2(5H)-one, Choi et al., 2014
Morchella esculenta Common morel, A/E Heteroglycan Hyperglycemia, Cheung, 2008
Morel, Galactomannan, b-1,3-D-glucan Anti-tumor
Yellow morel,
True morel,
Morel mushroom,
Sponge morel
2 Bioactives from Mushroom and Their Application

Phellinus linteus Black hoof B/NE Glucans Anti-tumor Kim and

mushroom Iwahashi, 2015
Acidic polysaccharides Inmmunomodulatory Hsieh et al.,
2013; Wu et al.,
Hispidin (polyphenol) Anti-oxidant Park et al., 2004
Pholiota adiposa Fatty pholiota, B/E Lectin (glycoprotein) Anti-tumor Zhang et al.,
pineapple Anti-viral 2009
sticky pholiota
Pholiota nameko Nameko, B/E Polysaccharides Anti-inflammatory Li et al., 2008
butters cotch
Table 1 (continued)

Mushroom Common names Phyllum or Bioactive compound Bioactivity Reference

Scientific name group/
Pleurotus Golden oyster B/E Glycoprotein (PCP-3A) Anti-tumor Chen et al., 2009
citrinopileatus mushroom anti-cancer
Pleurotus eryngii King trumpet B/E laccase Anti-viral Wang and Ng,
mushroom, French 2006a.
horn mushroom,
king oyster
Pleurotus florida White oyster B/E b-glucans Anti-oxidant Ganeshpurkar
et al., 2015
Pleurotus ostreatus Oyster mushroom B/E Pleuran (b-1, 3-glucan with galactose Immunomodulatory El Enshasy et al.,
and mannose), Anti-tumor, 2013b
proteoglycan hyperglycemia, Tong et al., 2009
Laccase Ani-viral El Fakharany
et al., 2010
Pleurostrin (peptide) Anti-fungal Chu et al., 2005
Pleurotus Indian Oyster, B/E Polysaccharides Anti-inflammatory Lavi et al., 2012
pulmonarius Italian oyster, b(1,3)-glucopyranosyl
phoenix Polysaccharides (1,3), (1,6)-linked Smirdele et al.,
mushroom, b-glucan 2008
lung oyster
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Table 1 (continued)
Mushroom Common names Phyllum or Bioactive compound Bioactivity Reference
Scientific name group/
Psilocybe species Magic mushroom, B/NE Psilocybin Anti-depressant Mason-Dambrot,
(eg. P.cubensis, shrooms (hallucinogen) (psilocin: 4-hydroxy- (Psychotherapy) 2012;
P. samuiensis, dimethyltryp- Kraehenmann,
P. Mexicana) tamine) 2015;
Grob et al., 2011;
et al., 2012;
Petri et al., 2014
Russula lepida Rosy russula B/NE Lectin (glycoprotein) Anti-tumor Zhang et al.,
(=Russula rosea) 2010a
Schizophyllum Split Gill B/NE Schizophyllan, 1,6- monoglucosyl Immunomodulatory Bae et al., 2004;
commune branched b-1, 3- D-glucan Anti-tumor Hobbs, 2005
Sparassis crispa Rooting B/E b-Glucan Immunomodulatory Ohno et al., 2002;
2 Bioactives from Mushroom and Their Application

cauliflower Takashi, 2013

Termitomyces Termite B/E Termitomycesphins (cerebrosides) Anti- neurodegenerative Qi et al., 2000;
albuminosus mushroom Qu et al., 2012
(=Macrolepiota Termitomycamides (fatty acid amides Anti- neurodegenerative Choi et al., 2010
Trametes versicolor Wild turkey B/E, Krestin (PKS), (PSP) Anti-metastatic Wasser, 2002
(=Coriolus unpalatable) Coriolan Hypoglycemic Rathee at al.,
versicolor (b-glucanprotein complex) 2012
Table 1 (continued)

Mushroom Common names Phyllum or Bioactive compound Bioactivity Reference

Scientific name group/
Tremella aurantia Golden ear B/E Heteroglycan Immunomodulatory Du et al., 2010
Tremella Yellow brain, B/E Glucurono-xylomannan Hypoglycemic Gupta et al., 2014
mesenterica golden jelly polysaccharide Immunomodulatory
yellow trembler,
witches butter
Tricholoma Giant mushroom Trichogin (protein) Antifungal Guo et al., 2005
Tricholoma NA B/E Laccase Anti-viral, Wang et al.,
mongolicum anti-tumor 1996; Li et al.,
Volvariella Paddy straw Fip-vvo Immunomodulatory Hsu et al., 1997
volvacea mushroom, straw
Wolfiporia cocos Hoelen, B/NE Dehydrotrametenolic acid Hypoglycemic Rathee et al.,
(=Poria cocos) poria, 2012
tuckahoe, Lanostane Anti- inflammatory Zheng and Yang,
China root, agents 2008a; 2008b
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Table 1 (continued)
Mushroom Common names Phyllum or Bioactive compound Bioactivity Reference
Scientific name group/
Xylaria hypoxylon Candlestick A/NE Lectin (glycoprotein) Anti-mitogenic Liu et al., 2006
fungus, candle anti-tumor
snuff fungus,
carbon antlers,
stag’s horn fungus
B: Basidiomycota
A: Ascomycota
E: Edible
NE: Non-edible
NA: not avalaible
2 Bioactives from Mushroom and Their Application
40 C. Sánchez

3.1 Polysaccharides

Polysaccharides are the major class of bioactive compounds found in mushroom and
have been reported in most of the edible mushrooms. The general therapeutic effects
of polysaccharides are antioxidant, antidiabetic, antimicrobial, anti-inflammatory,
anticancer, and immunomodulators (Elsayed et al. 2014; Chan et al. 2009).

3.1.1 Glucans

Glucan polysaccharides differ in their primary structure (type of basic sugar, e.g.,
xylose, mannose, galactose, etc.), type of linkage (a or b), degree of branching,
molecular weight, solubility, etc. Fungal glucans can be water soluble, soluble in
alkali or insoluble. Some glucans are intracellular (serve as reserve material), others
are secreted in the medium, and few are present in the cell wall (Ruiz-Herrera
2012). The insoluble fractions are usually structural components of the cell wall and
cross-linked to other polysaccharides like chitin or to proteins (e.g., mannoproteins
and glycoprotein). Soluble glucans correspond to 20–50% of the total glucans, and
insoluble glucans correspond between 50 and 80% (He et al. 2012). The diversity of
glucans results from at least eight different ways in which two glucose units can
link. Formations of a- or b-bond are a result of the condensation reactions. The
diversity of glucans is further increased due to the different length and branches of

Fig. 2 Schematic illustration of the mechanism of immune activation by b-glucan from

2 Bioactives from Mushroom and Their Application 41

chains and substitutions on the sugar rings (Ren et al. 2012). b- and a-glucans can
be present in fungal cell wall. Fruit body extracts of Pleurotus pulmonarius showed
mixed a-linkages and b-anomeric carbon linkages, whereas polysaccharide from
mycelial extracts had mainly a-glucan linkages (Lavi et al. 2010). a(1, 3)-glucan is
present at levels of 9–46% of the cell wall in several basidiomycetes. It can be
present in the cell wall of certain mushrooms such as Agaricus bisporus fruit bodies
(Smiderle et al. 2010). b-glucan is one of the key components of several basid-
iomycete and ascomycete cell wall. It is a long-chain polysaccharide with b-D
glucose as basic subunit linked to one another by 1-3 glycosidic chain with 1-6
glycosidic branches. b-glucans have been reported to have antimicrobial immune
response, acting on several immune receptors such as dectin-1 (major b-glucan
receptor), complement receptor (CR3), and TLR-2/6 (Toll-like receptor-2/6,
receptor of the innate immune) (Chan et al. 2009). Therefore, b-glucans are able to
enhance the immune system and prevent and treat several common diseases to
promote health (Batbayar et al. 2012). In the innate immune system, b-glucan binds
with macrophages that are responsible to detect intruders and coordinate the body
defense system. Macrophages start out as monocytes (white blood cells), which
leave the bloodstream and turn into macrophages. Macrophages are activated by
b-glucan, enhancing their ability to identify and destroy intruders through phago-
cytosis. Macrophages also play an important role in activating the rest of the
immune system (T lymphocyte, B lymphocyte, and NK cells) to destroy invaders.
T lymphocytes (thymus-derived) have a receptor for antigen (T cell receptor) and
are specialized cells trained to kill invaders. B lymphocytes (bone marrow-derived)
make antibody, and their antigen receptor is the antibody on their surface. NK
(natural killer) cells are T lymphocytes, which kill virus or bacterium-infected cells
and tumor cells. In this way, the immune system protects the body from harmful
invaders (Chan et al. 2009; Legentil et al. 2015) (Fig. 2). The bioactive glucans
have been isolated from mushroom fruit bodies and from mycelia produced via
submerged fermentation (Song et al. 2012; Queiroz et al. 2010; Guerra-Dore et al.
2007; Ruthes et al. 2013; Li et al. 2008). Several biologically active fungal
b-glucans have been found in the fruiting bodies from mushrooms. Karácsonyi and
Kuniak (1994) described the isolation of pleuran from Pleurotus ostreatus which is
made of b(1,4)- or b(1,6)-branched for every fourth b(1,3)-glucan backbone (El
Enshasy et al. 2013a). The bioactive glucan, lentinan from Lentinula edodes, is
made of one b(1,6)-branched residue for every three b(1,3) glucose residues with
molecular weight of 400–1000 kDa (Sasaki and Takasuka 1976). It showed
immunomodulatory and antitumor activities (Firenzuoli et al. 2007). Schizophyllan
is the active b-glucan from Schizophyllum commune which is formed by one b(1,6)-
branched residue for every three b(1,3) glucose residues with molecular weight of
450 kDa (Bae et al. 2004). Maitake D-fraction was isolated from Grifola frondosa,
which is made of mixture of b(1,6)-glucan main chain with b(1,4)-branched glucan
and b(1,3)-glucan main chain with b(1,6)-branched glucan (Grifolan) (Kidd 2000).
For example, Agaricus subrufescens extract is rich in b(1,3)-, b(1,4)-, and b(1,6)-
glucans and induces the release of proinflammatory cytokines in human monocytes
42 C. Sánchez

and human vein endothelial cells in vitro (Bernardshaw et al. 2005). Glucans such
as (1,3)-glucopyranosyl from Pleurotus pulmonarius have been reported to exhibit
anti-inflammatory properties (Lavi et al. 2012). Rathee et al. (2012) reported that
ganoderan A and B, glucans from Ganoderma lucidum fruiting bodies, showed
hypoglycemic effects. On the other hand, ganopoly, the polysaccharide-containing
preparation of G. lucidum, exhibited hepatoprotective effects in patients with
chronic hepatitis B (Gao et al. 2002). It has been suggested that glucans from G.
lucidum had immunomodulating properties, as well as enhancement of lymphocyte
proliferation and antibody production. These polysaccharides also showed both
antigenotoxic and antitumor-promoting activities (Bao et al. 2001; Wasser 2002).
The antioxidative and free radical scavenging effects of polysaccharides of G.
lucidum have also been reported (Rathee et al. 2012). A b-glucan (b-1,3-linked
glucose residues, which occasionally branches at O-6) isolated from the fruiting
bodies of P. ostreatus has also been proven to exert antitumor activity against Hela
tumor cell (Tong et al. 2009). Two mechanisms have been proposed to be
responsible for the anticancer effect of b-glucan: (1) via direct cytotoxic effect and
(2) indirectly through immunomodulatory action (Chan et al. 2009). L. edodes has
shown anti-inflammatory activities. The active fraction was made of fucomanno-
galactan with a main chain of (1,6)-linked a-D-galactopyranosyl units, partially
substituted at O-2 (Carbonero et al. 2008). Additionally, glucans such as (1,3)-
D-glucopyranosyl from P. pulmonarius have been reported to exhibit
anti-inflammatory properties (Lavi et al. 2012). Wu et al. (2010) reported that
polysaccharides of Flammulina velutipes are composed of three monosaccharides
(glucose, mannose, and xylose) in a molar ratio of 3.5:0.8:1.4 and have been found
to have anti-inflammatory activities (Wu et al. 2010). Polysaccharides extracted
from mushrooms such as Cantharellus tubaeformis (Tsvetkova et al. 2006),
Lactarius flavidulus (Fujimoto et al. 1993), Lactarius rufus (Ruthes et al. 2013),
Lyophyllum decastes (Ukawa et al. 2000), Pholiota nameko (Li et al. 2008),
Geastrum saccatum (Guerra-Dore et al. 2007), Fomitopsis pinicola (Cheng et al.
2008), Craterellus tubaeformis (Tsvetkova et al. 2006), Auricularia auricula
(Zhang et al. 2007), and Boletus edulis (Moro et al. 2012) have also been reported
‘to exhibit anti-inflammatory properties.

3.1.2 Polysaccharide–Protein Complexes

Some polysaccharides have been identified as polysaccharide–protein complexes,

which have been shown to possess immunomodulatory and antitumor activities. For
example, polysaccharide-K (polysaccharide-Kureha; PSK) also known as krestin,
protein bound with b(1,6) side chain, and b(1,3)-branched b(1,4) main chain glucan
(94–100 kDa) were isolated from Trametes versicolor. Krestin showed antimeta-
static activity (Fisher et al. 2002; Wasser 2002). Coriolan, a b-glucanprotein
complex obtained from submerged grown T. versicolor biomass, exhibited hypo-
glycemic effects and ameliorated the symptoms of diabetes (Rathee et al. 2012).
Chatterjee et al. (2011) isolated calvacin from Calvatia gigantea. It is a moderately
2 Bioactives from Mushroom and Their Application 43

heat stable, nondiffusible, and basic mucoprotein, which showed antitumor activity.
On the other hand, ethanolic extracts and a proteoglycan purified from Phellinus
linteus showed anti-inflammatory properties (Kim et al. 2003, 2004).

3.2 Terpenes

Terpenes are the largest group of anti-inflammatory compounds in mushrooms.

Several terpenes have been isolated from G. lucidum. These are nonpolar
metabolites comprised of the following groups: (1) volatile mono and sesquiter-
penes oils (C10 and C15), (2) less volatile diterpenes (C20), (3) involatile triter-
penoids and sterols (C30), and (4) the carotenoid pigments (C40). Triterpene
chemical structures are based on lanosterol. It is an important intermediate for their
synthesis. Stereochemical rearrangement of this compound among triterpenoids
results in their structural diversity (predominant pairs of C-3 stereoisomers) (Paliya
et al. 2014). Akihisa et al. (2005) and Iwatsuki et al. (2003) isolated nine lucidenic
acids and four ganoderic acids from fruit bodies of G. lucidum. On the other hand,
several lanostane-type triterpenic acids were isolated by Akihisa et al. (2005) and
terpenoids (triterpenes) were also isolated from Reishi mushroom (Dudhgaonkar
et al. 2009). All those terpenes showed anti-inflammatory activity. Some triterpenes
from G. lucidum (ganoderic acid C and derivatives) are able to inhibit the
biosynthesis of cholesterol (Komoda et al. 1989). Other triterpenes (ganoderic acid
F) of this mushroom contribute to atherosclerosis protection (Morigiwa et al. 1986).
The antioxidative and free radical effects of triterpenoids from G. lucidum have also
been shown (Rathee et al. 2012). El-Mekkawy et al. (1998) reported that different
triterpenes from G. lucidum (i.e., ganoderiol, ganodermanontriol, and ganoderic
acid) showed antiviral activity. Sterols and triterpenes (e.g., lucialdehyde D, gan-
oderone A, and ganoderone C) were isolated from the fruiting bodies of
Ganoderma pfeifferi. Antifungal, antibacterial, and antiviral properties were found
for some of such isolated compounds (Niedermeyer et al. 2005). Furthermore,
different sterols with potent anti-inflammatory properties have been also isolated
from Inonotus obliquus (Van et al. 2009; Park et al. 2005). Several triterpenes
(trametenolic acid, ergosterol peroxide, 3b-hydroxy-8,24-dien-21-al, ergosterol,
and inotodiol) were isolated from the sclerotia of I. obliquus, which had
anti-inflammatory and anticancer activities (Ma et al. 2013). Han et al. (2013)
isolated five novel cyathane diterpenes (identified as cyathins DH) and three
diterpenes (neosarcodonin, cyathatriol, and 11-O-acetylcyathatriol) from Cyathus
Africans, which showed potent anti-inflammatory properties. Chen et al. (2006)
reported that several triterpenes (e.g., 19-hydroxylabda-8(17)-en-16,15olide, and
14-deoxy-11,12-didehydroandrographolide) isolated from Antrodia camphorate
showed neuroprotective activity.
44 C. Sánchez

Fig. 3 Schematic representation of antioxidant activity, showing molecules neutralizing free

radicals to prevent cellular and tissue damage

3.3 Phenolic Compounds

Phenolic compounds are aromatic hydroxylated compounds with one or more

aromatic rings and one or more hydroxyl groups. They include phenolic acids,
flavonoids, hydroxybenzoic acids, hydroxycinnamic acids, lignans, tannins, stil-
benes, and oxidized polyphenols (Cote et al. 2010; D’Archivio et al. 2010). It has
been reported that phenolic compounds exhibit antioxidant activity in biological
systems, acting as free radical inhibitors, peroxide decomposers, metal inactivators,
or oxygen scavengers (Dziezak 1986; Yagi 1970). Therefore, the key role played by
antioxidants in the body is their ability to react with free radicals. A free radical is a
chemical compound that contains one or more unpaired electrons. Reactive oxygen
species (ROS) (i.e., superoxide, hydrogen peroxide, hydroxyl radical, hydroxyl ion,
and nitric oxide) are reactive molecules and free radicals derived from molecular
oxygen. These molecules can be produced either by external sources (e.g., cigarette
smoke, ozone, and stress) or as by-products during the mitochondrial electron
transport of aerobic respiration or by oxidoreductase enzymes and metal-catalyzed
oxidation. Because they are reactive, radicals search out ways of pairing up their
electron, so radicals often attack nearby chemical compounds. These chemical
compounds may be involved in important enzyme reactions, may be components of
2 Bioactives from Mushroom and Their Application 45

cell walls (i.e., lipid and protein), or may be part of a DNA molecule. If their
chemical structure is changed, their function in the cell may be lost and the result
can be cellular senescence or apoptosis (chronic diseases in the body). ROS have
the potential to cause several deleterious events, and neutralizing of free radicals or
peroxide radicals by an antioxidant agent may avoid such damage in the cell
(Fig. 3). There are a number of nonenzymatic small molecules that play a role as
antioxidants. Glutathione may be the most important intracellular defense against
the deleterious effects of ROS. It is a tripeptide (glutamyl-cysteinyl-glycine), which
provides an exposed sulfhydryl group as target for attack. Ascorbic acid (vitamin C)
and a-tocopherol (vitamin E), lycopene, and polyphenol are examples of molecules
capable of reducing ROS (Held 2015) (Fig. 3). Palacios et al. (2011) studied the
antioxidant activity of phenolic compounds in Agaricus bisporus, Boletus edulis,
Cantharellus cibarius, Craterellus cornucopioides, Calocybe gambosa,
Hygrophorus marzuolus, and Lactarius deliciosus, and P. ostreatus. C. cibarius,
and C. cornucopioides exhibited the greatest antioxidant effect with respect to the
other species. C. cornucopioides showed the highest myricetin amount, and
C. cibarius presented greater amounts of caffeic acid and catechin. The phenolic
molecule pyrogallol has been extracted from A. bisporus, C. cibarius, and
L. deliciosus (Dugler et al. 2004; Witkowska et al. 2011), which have been found to
exhibit anti-inflammatory activity. Grifolin and grifolin derivatives are farnesyl
phenolic compounds which have been isolated from the edible mushroom
Albatrellus ovinus, which showed anti-inflammatory properties (Nukata et al.
2002). It has been reported that phenol analogous compounds (hericenones C, D, E,
F, G, H) isolated from H. erinaceus had antioxidant activity (Mizuno 1999) and
antineurodegenerative properties (Xu and Beelman 2015). Human trials have been
carried out using H. erinaceus. In this study, 30 subjects were randomized into two
15-person groups, one of which was given H. erinaceus (250 mg tablets containing
96% of this mushroom dry powder) and the other given a placebo. The tablets were
taken for three times a day for 16 weeks. Those subjects whose took H. erinaceus
power showed significantly increased scores on the cognitive function scale com-
pared with the placebo group (Mori et al. 2009). On the other hand, Attarat and
Phermthai (2015) reported that catechin, a major group of phenolic compounds,
was isolated from Lentinula squarrosulus, Lentinula polychrous, and L. edodes,
which exhibited antioxidant activity. Chowdhury et al. (2015) isolated phenolic
compounds and flavonoids from P. ostreatus, L. edodes, and Hypsizygus tessella-
tus, which showed antioxidant, antifungal, and antibacterial properties. On the other
hand, it has been suggested that an increased free radical generation and the con-
sequent elevated oxidative stress in neural system cause neurodegenerative dis-
eases. Mushrooms can potentially reduce the risk of neurodegenerative diseases
attributing to the high antioxidative capacity of bioactive compounds such as
vitamin D and polyphenols (Xu and Beelman 2015). It has been reported that
hericenones (A-H) and erinacines (A-K & P-Q), from fruiting bodies and mycelia
of H. erinaceus, respectively, induced nerve growth factor synthesis (both in vitro
and in vivo) (Kawagishi et al. 2008; Phan et al. 2014). Dai et al. (2010) reported
that hispidin, a class of polyphenols, is an important medicinal metabolite from
46 C. Sánchez

Phellinus spp. Hispidin was isolated from the culture broth of P. linteus, and it has
been shown to be an efficient ROS scavenger (Park et al. 2004).

3.4 Peptides and Proteins

Mushrooms produce many bioactive proteins and peptides, primarily including

lectins, which have not enzymatic activity. Mushrooms also produce bioactive
proteins, which possess enzymatic activity such as fungal immunomodulatory
proteins (FIPs), ribosome-inactivating proteins (RIPs), and laccases. Chu et al.
(2005) isolated an antifungal peptide (pleurostrin) (7 kDa) from P. ostreatus, which
exhibited antifungal activity. Wang et al. (2007) isolated a peptide (SU2) (4.5 kDa)
from Russula paludosa, which showed antiviral properties. Ngai et al. (2005)
isolated an antifungal peptide (agrocybin) (9 kDa) from fresh fruiting bodies of the
mushroom Agrocybe cylindracea. Cordymin, a low molecular weight peptide
(10,906 Da), has been purified from Cordyceps sinensis (a highly prized edible
fungus found in the mountains of Sichuan, Yunnan, and Tibet) (Wang et al. 2012;
Qian et al. 2011) and from Cordyceps militaris (Wong et al. 2011). This peptide
showed anti-inflammatory activity. Lectins are nonimmune proteins or glycopro-
teins that bind specifically to fungal cell wall carbohydrates and have ability to cell
agglutination. Liu et al. (2006) isolated a xylose-specific lectin (28.8 kDa) from
fresh fruiting bodies of Xylaria hypoxylon. It showed potent antimitogenic and
antitumor activities. It has been reported that lectins were isolated from Pholiota
adiposa and from H. erinaceum (16 and 51 kDa, respectively), which exhibited
antiviral and antitumor activities (Zhang et al. 2009; Lin et al. 2010). Zhang et al.
(2010a) isolated a lectin (32 kDa) from Russula lepida, which exhibited antitumor
activity. Ribosome-inactivating proteins (RIPs) are enzymes that inactivate ribo-
somes by eliminating adenosine residues from rRNA. It has been reported that a
ribosome-inactivating protein (9 kDa) (marmorin) was isolated from Hypsizigus
marmoreus and showed antitumor activity (Wong et al. 2008). On the other hand,
laccases are phenol oxidases widely diffused in basidiomycete and ascomycete
fungi. These fungi use laccases to degrade lignocellulosic substrates. However,
laccases with antiviral activity have been isolated from Pleurotus eryngii (Wang
and Ng 2006a) and from P. ostreatus (El Fakharany et al. 2010). Zhang et al.
(2010b) purified a laccase from Clitocybe maxima, which also showed antitumor
activity. Some proteins targeting immune cells known as fungal immunomodula-
tory proteins (FIPs) are a new group bioactive proteins also isolated from mush-
room. Kino et al. (1989) isolated ling zhi-8 (LZ-8), an immunomodulatory protein
from G. lucidum. FIPs have been isolated from the mushrooms F. velutipes
(Fip-fve) (Ko et al. 1995), Ganoderma tsugae (Fip-gts) (Lin et al. 1997), and
Volvariella volvacea (Fip-vvo) (Hsu et al. 1997). It has been reported the potential
application of Fip-fve for tumor immunotherapy (Ding et al. 2009; Chang and Sheu
2006; Chang et al. 2010). A novel immunomodulatory glycoprotein ACA (27 kDa)
was purified from Antrodia camphorata (Sheu et al. 2009). Lin et al. (2010) isolated
2 Bioactives from Mushroom and Their Application 47

an immunomodulatory protein GMI from Ganoderma microsporum, which showed

antimetastasis activity. Du et al. (2007) purified a water-soluble Se-containing
protein Se-GL-P (36 kDa) from the Se-enriched G. lucidum, which exhibited
antitumor activity. The immunomodulatory activity of the isolated protein fractions
and polysaccharide fractions from the mushrooms A. blazei, C. comatus, F. velu-
tipes, G. lucidum, G. frondosa, L. edodes, P. ostreatus, and V. volvacea has been
reported (Jeurink et al. 2008). Maiti et al. (2008) examined the antiproliferative and
immunomodulatory activities of a protein fraction, named Cibacron blue affinity
eluted protein (CBAEP), which was isolated from Astraeus hygrometricus,
Termitomyces clypeatus, Pleurotus florida, Calocybe indica, and V. volvacea.
A glycoprotein (PCP-3A) was purified from Pleurotus citrinopileatus, which
showed antitumor activity (Chen et al. 2009). Kodama et al. (2002) isolated a low
molecular weight protein fraction from G. frondosa, which showed antitumor
activity. Ngai and Ng (2008) isolated a novel and potent antifungal protein lentin
(27.5 kDa) from the fruiting bodies of L. edodes. Guo et al. (2005) also isolated an
antifungal protein (trichogin) from Tricholoma giganteum. Wang and Ng (2006b)
isolated an antifungal protein (15 kDa) (ganodermin) from G. lucidum. Zheng et al.
(2010) isolated a novel antibacterial protein (44 kDa) from dried fruiting bodies of
Clitocybe sinopica.

3.5 Other Compounds

Agaricoglycerides are fungal secondary metabolites that constitute esters of chlori-

nated 4-hydroxy benzoic acid and glycerol, which are produced in the culture of
G. frondosa and Agaricus macrosporus. These compounds showed potent
anti-inflammatory activity (Han and Cui 2012). Nagai et al. (2006) reported that
dilinoleoylphosphatidylethanolamine isolated from fruiting bodies of H. erinaceum
reduces oxidative stress in endoplasmic reticulum, demonstrating its potential effect in
neurodegenerative diseases. It has been reported that termitomycesphins A, B, C, D,
G, and H (cerebrosides) (Qi et al. 2000; Qu et al. 2012) and termitomycamide A, B, C,
D, and E (fatty acid amides) were extracted and identified from dried fruiting bodies of
Termitomyces albuminosus (Choi et al. 2010). These bioactive compounds also
exhibited antineurodegenerative activity, since reduced endoplasmic reticulum
stress-induced. Kawagishi et al. (1997) isolated dictyophorine A and B from
Dictyophora indusiata, which can significantly improve the amount of nerve growth
factor. Lee et al. (2002a) identified dictyoquinazol A, B, and C in D. indusiata,
which showed neuroprotective properties. Choi et al. (2009, 2014) isolated 3-
(hydroxymethyl)-4-methylfuran-2(5H)-one, (3R, 4S, 1’ R)-3-(1’-hydroxyethyl)-
4methyldihydrofuran-2(3H)-one, 5-hydroxy-4-(1-hydroxyethyl)-3-methylfuran-2
(5H)-one, and 5-phenylpentane-1,3,4-triol from Mycoleptodonoides aitchisonii,
which also exhibited activity. It has been reported that Alzheimer’s disease
pathogenesis includes microglial activation associated with neuroinflammation,
increased level of acetyl cholinesterase (AChE) activity, and free radical generation
48 C. Sánchez

(Martorana et al. 2012). Brondz et al. (2007) isolated 6-hydroxyinfractine and

infractopicrine (alkaloids infractine) from Cortinarius infractus, which showed
AChE-inhibiting activity with nondetectable cytotoxicity (Geissler et al. 2010).
Caruilignan C and 1-(3,4,5-trimethoxyphenyl) ethanol were isolated and purified
from Daldinia concentrica, which showed neuroprotective activity (Lee et al. 2002b).
On the other hand, it has been reported that psilocybin from the hallucinogen
Psilocybe species showed antidepressant properties (Mason-Dambrot 2012;
Kraehenmann 2015; Grob et al. 2011; Carhart-Harris et al. 2012; Petri et al. 2014).
Psilocybin is a phosphate derivative of N,N-dimethyltryptamine that is present at
concentrations of 0.1–1.5% in species of the Psilocybe genus. This compound is
considered nonaddictive and rarely abused. In humans, psilocybin converts to psi-
locin, which is a pharmacologically active drug (Norchem 2011). The antioxidant
metabolites, 2,4,6-trimethylacetophenone imine, glutamyl tryptophan, azatadine, and
lithocholic acid glycine conjugate were isolated from Boletus spp, which exhibited
antioxidant activity (Yuswan et al. 2015).

4 Future Trends

Mushrooms are functional food and are a source of biologically valuable compo-
nents that offer great therapeutic potential for the prevention and control of several
diseases. A large number of mushroom-derived bioactive compounds, both cellular
components and secondary metabolites, have been isolated. Some studies about
mushrooms’ bioactivity were assayed using crude mushroom extracts or mixture of
mushroom metabolites. These studies will require the isolation and identification of
the bioactive compounds in order to determine the bioactive effect of each com-
pound. Both the optimization of submerged culture conditions for mycelial growth
and strain improvement by genetic manipulation are crucial in order to overproduce
the desired compound. Further research and clinical trials have to be carried out to
validate that mushrooms are source of bioactive molecules with medicinal


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Chapter 3
Bioactive Products from Fungi

Sergio Sanchez and Arnold L. Demain

1 Introduction

The microbial drug era began back in 1928 when Alexander Fleming discovered in
a petri dish seeded with Staphylococcus aureus that a compound produced by a
contaminating mold killed the bacterium. The active compound, produced by
Penicillium notatum, was named penicillin. By using the same strategy, other
antibiotics such as streptomycin and chloramphenicol were later isolated from
different bacterial and fungal fermentations. Antibiotics can be produced by fer-
mentation, an old technique that was utilized for beer and wine production almost
8000 years ago, during the ancient Egypt and Mesopotamia era. Similarly, cheese
production by Penicillium roqueforti can be traced back for almost 4000 years.
Additional examples of traditional fermentations are soy sauce in Asia and bread
production (Hölker et al. 2004; Seviour et al. 2013). Bread production was common
in Egypt in 4000 BC. Beer production using the non-filamentous fungus
Saccharomyces cerevisiae began in 7000 BC by the Sumerians and Chinese. Wine
was made in Iran in 5000 BC and in Egypt in 3000 BC.
Natural products (NPs) with high commercial value can be produced by
microbial primary or secondary metabolism. Thanks to the technical improvements
in screening programs and techniques for separation and isolation, the number of
natural compounds discovered surpasses one million (Berdy 2005). Among them,
50–60% are produced by plants (alkaloids, flavonoids, terpenoids, steroids,
carbohydrates, etc.), and 5% of these plant products have a microbial origin.

S. Sanchez (&)
Departamento de Biologia Molecular y Biotecnologia, Instituto de Investigaciones
Biomedicas, Universidad Nacional Autonoma de Mexico, Mexico City, CDMX, Mexico
A.L. Demain
Research Institute for Scientists Emeriti (RISE), Drew University, Madison, NJ, USA

© Springer International Publishing AG 2017 59

M. Puri (ed.), Food Bioactives, DOI 10.1007/978-3-319-51639-4_3
60 S. Sanchez and A.L. Demain

About 20–25% of the reported natural products show biological activity and of
these, approximately 10% have been obtained from microbes.
Microorganisms produce many compounds with biological activity. From
22,500 bioactive compounds so far obtained from microorganisms, about 9000 are
produced by fungi (Berdy 2005; Brakhage and Schroekh 2011). Therefore, the role
of fungi in the production of antibiotics and other drugs for treatment of
non-infective diseases has been crucial (Demain et al. 2004).
With less than 5% of the fungal world having been cultured, there have been
significant advances in microbial techniques for growth of uncultured organisms as
a potential source of new chemicals (Kaeberlein et al. 2002). As more genomes are
sequenced, it is found that filamentous fungi grasp the genetic capacity to produce
an arsenal of secondary metabolites. In fungi, biosynthetic genes are present in
clusters coding for large, multidomain, and multimodular enzymes such as
polyketide synthases, prenyltransferases, non-ribosomal peptide synthases, and
terpene cyclases. Genes adjacent to the biosynthetic gene clusters encode regulatory
proteins, oxidases, hydroxylases, and transporters. Aspergilli usually contain 30–40
secondary metabolite gene clusters. Most of these clusters coding for secondary
metabolites are still cryptic or silent under standard culture conditions (Hertweck
2009). Therefore, mining for these cryptic secondary metabolites can be an
excellent source of new drugs by awakening cryptic clusters for secondary meta-
bolism. In addition, recent knowledge on cluster regulation has unlocked many
hidden fungal bioactive compounds. Regulation of fungal secondary metabolism
has been reviewed by Brakhage (2013). Emphasized are the regulatory elements
that control gene transcription, including the targeted activation of silent gene
clusters (Brakhage and Schroekh 2011). A method to predict secondary metabolite
gene clusters in filamentous fungi has been devised (Anderson et al. 2013).
In addition, metagenomics, i.e., the extraction of DNA from soil, plants, and
marine habitats and its incorporation into known organisms, allow access to a vast
untapped reservoir of genetic and metabolic diversity (Colwell 2002; Gaudilliere
et al. 2001). Thus, the potential for discovery of new fungal secondary metabolites
with beneficial use for humans is great.

2 Antibiotics

Of the 12,000 antibiotics known in 1955, filamentous fungi produced 22% (Verdine
1996; Strohl 1997). The beta-lactams are the most important class of antibiotics in
terms of use. They constitute a major part of the antibiotic market. Included are the
penicillins, cephalosporins, clavulanic acid, and the carbapenems. Of these, fungi
are responsible for the production of penicillins and cephalosporins (Fig. 1). The
natural penicillin G and the biosynthetic penicillin V had a market of $4.4 billion by
the late 1990s. Major markets also included semi-synthetic penicillins and cepha-
losporins amounting to $11 billion. In 2006, the market for cephalosporins was $9.4
billion and that for penicillins was $6.7 billion.
3 Bioactive Products from Fungi 61

Fig. 1 Chemical structures

of penicillin (left) and
cephalosporin (right)

Production of all beta-lactams in 2003 had reached over 60,000 tons. The titer of
penicillin is over 100 g L−1 and that for cephalosporin C is more than 35 g L−1
(Masurekar 2008; Yang et al. 2012). Recovery yields are more than 90%. There
have been over 15,000 molecules based on penicillin that have been made by
semi-synthesis or by total synthesis.
Important in penicillin biosynthesis are the regulatory factors. Penicillium
chrysogenum, the producer of penicillin G, contains global regulatory factor
PcRFX1, which positively regulates three beta-lactam biosynthetic genes, i.e.,
pcbAB, pcbC, and penDE (Dominguez-Santos 2012). This regulatory factor not
only controls secondary metabolism but also controls primary metabolism. Related
factor CPCR1 is a global regulator found in the cephalosporin C producer
Acremonium chrysogenum, binding to at least two sequences of the pcbAb-pcbC
intergenic region and regulating cephalosporin C biosynthesis.
1,3-Diaminopropane (1,3-DAP) is secreted by P. chrysogenum and A. chryso-
genum. This and spermidine (which contains 1,3-DAP) increase the transcription
levels of the penicillin biosynthetic genes pcbAB, pcbC, and penDE (Martín et al.
2012). They thus stimulate the production of penicillin G. The mechanism appears
to involve stimulation of the expression of laeA, a global regulator that acts epi-
genetically on the expression of secondary metabolism genes via heterochromatin
reorganization. 1,3-DAP also stimulates the production of cephamycin in
Amycolatopsis lactamdurans. Spermidine’s activity appears to be due to 1,3-DAP.
By the mid-1990s, 160 antibiotics and their derivatives were already on the
market (Strohl 1997; Brown 1996). The market in 2009 was $79 billion dollars.
Despite these impressive figures, more antibiotics are needed to combat evolving
pathogens, naturally resistant microbes, and bacteria and fungi that have developed
resistance to current antibiotics. A new and approved cephalosporin is ceftobiprole,
which is active against methicillin-resistant S. aureus (MRSA) and is not hydro-
lyzed by a number of beta-lactamases from gram-positive bacteria (Shang et al.
2010). Another antibiotic of note is cerulenin, an antifungal agent produced by
Acremonium caerelens. It was the first inhibitor of fatty acid biosynthesis discov-
ered (Vance et al. 1972). It alkylates and inactivates the active-site nucleophilic
cysteine of the ketosynthase enzyme of fatty acid synthetase by epoxide ring
opening. Other properties that are desired in new antibiotics are improved
62 S. Sanchez and A.L. Demain

pharmacological properties, ability to combat viruses and parasites, and improved

potency and safety.
Parafungin from Fusarium lavarum is a recently discovered antifungal agent
inhibiting poly(A) polymerase in Candida albicans as well as in a broad range of
pathogenic fungi (Harvey et al. 2015).

3 Additional Pharmacological Agents

Over the years, non-infectious diseases were mainly treated with synthetic com-
pounds. Despite testing thousands of synthetic chemicals, only a handful of
promising structures was obtained. As new synthetic lead compounds became
extremely difficult to find, microbial products came into play. Since microorgan-
isms are such a prolific source of structurally diverse bioactive metabolites, over the
years, the pharmaceutical industry extended their antibiotic screening programs to
look for additional applications of antibiotics in medicine and agriculture (Cardenas
et al. 1998; Kremer et al. 2000). As a result of this move, some of the most
important products of the pharmaceutical industry were obtained. For example, the
immune suppressants have revolutionized medicine by facilitating organ trans-
plantation (Verdine 1996). Other products include anti-tumor drugs, hypocholes-
terolemic agents, enzyme inhibitors, gastrointestinal motor stimulators, ruminant
growth stimulants, insecticides, herbicides, antiparasitics versus coccidia and hel-
minths, and other pharmacological activities. Stimulated by the use of simple
enzyme assays for screening, prior to testing in intact animals or in the field, further
applications are emerging in various areas of pharmacology and agriculture. In
2013, there were more than 15 secondary metabolites derived from marine fungi in
clinical trials (Bhatnagar and Kim 2013). Many of the new natural products from
marine sources are polyketides.
S. cerevisiae and Pichia pastoris are used for the production of biopharma-
ceuticals (Berlec and Strukelj 2013). Biopharmaceuticals have the fastest growth
rate of products on the market. S. cerevisiae produces 20% of these. Of 211 bio-
pharmaceuticals approved by 2011, 31 were produced by yeasts, 30 by S. cere-
visiae, and one by P. pastoris. The production of biopharmaceuticals by
S. cerevisiae has been reviewed by Nielsen (Nielsen 2013). The yeast is used to
make insulin and insulin analogs. The insulin market was $12 billion in 2011. Other
products are human serum albumin, hepatitis vaccines, and virus-like particles used
for vaccination against human papilloma virus. The advantages of S. cerevisiae
include proper folding of human proteins and their secretion into the extracellular
medium, facilitating purification and proper post-translational modification of the
protein. This includes proteolytic processing of signal peptides, disulfide bond
formation, subunit assembly, acylation, and glycosylation. Human serum albumin
is produced at 3 g L−1.
3 Bioactive Products from Fungi 63

4 Anticancer Drugs

More than 12 million new cases of cancer were diagnosed in the world in 2008; 6.6
million cases were in men and 6.0 million in women, resulting in 7.6 million
cancer-related deaths. The tumor types with the highest incidence were lung
(12.7%), breast (10.9%), and colorectal (9.8%). Some of the anticancer drugs in
clinical use are secondary metabolites derived from plants and fungi. Among the
approved products are taxol and camptothecin.
Taxol (paclitaxel) was first isolated from the Pacific yew tree, Taxus brevifolia
(Wall and Wani 1996), and later found to be a fungal secondary metabolite (Stierle
et al. 1993). It is a steroidal alkaloid diterpenoid that has a characteristic
N -benzoylphenyl isoserine side chain and a tetracycline ring (Fig. 2). It inhibits
rapidly dividing mammalian cancer cells by promoting tubulin polymerization and
interfering with normal microtubule breakdown during cell division. The benzoyl
group of the molecule is particularly crucial for maintaining the strong bioactivity
of taxol. The drug also inhibits several fungi (species of Pythium, Phytophthora,
and Aphanomyces) by the same mechanism. In 1992, taxol was approved for
refractory ovarian cancer and today is used against breast cancer and advanced
forms of Kaposi’s sarcoma (Newman and Cragg 2007). A formulation in which
paclitaxel is bound to albumin is sold under the trademark Abraxane®. Taxol sales
amounted to $1.6 billion in 2006 for Bristol-Myers Squibb, representing 10% of the
company’s pharmaceutical sales and its third largest selling product. It has reached
$3.7 billion annual sales in international markets.
Although synthetic methods for taxol production have been tried, the chemical
molecular structure is so complex that commercial synthetic production is
Currently, Italy, the UK, the Netherlands, and other Western countries are
engaged in the production of taxol by plant cell fermentation technology. Taxol
production by a plant cell culture of Taxus sp. was reported to be at 67 mg L−1
(Sabater-Jara et al. 2010). However, the addition of methyl jasmonate, a plant signal
transducer, increased the production to 110 mg L−1.

Fig. 2 Chemical structure of

taxol. The dotted section
corresponds to the molecule
N-benzophenyl isoserine side
64 S. Sanchez and A.L. Demain

As stated above, taxol has also been found to be a fungal metabolite (Stierle et al.
1993; Jiang et al. 2012). Fungi such as Colletotrichum gloeosporoides,
Colletotrichum capsici, Fusarium maire, Nodulisporium sylviforme, Pestalotiopsis
microspora, Pestalotiopsis versicolor, Phyllosticta citricarpa, Taxomyces andrea-
nae, and Tubercularia sp. are taxol producers (Stierle et al. 1993;
Flores-Bustamante et al. 2010; Gangadevi and Muthumary 2008; Kumaran et al.
2010, 2011; Li et al. 1996; Wang et al. 2000; Xu et al. 2006; Zhao et al. 2004].
C. gloeosporoides produced 163 µg L−1 of taxol (Gangadevi and Muthumary
2008), and the endophyte F. maire made 225 lg L−1 (Xu et al. 2006). The pro-
duction by P. citricarpa amounted to 265 lg L−1 (Kumaran et al. 2008) and was
reported at 417 lg L−1 by submerged fermentation with an engineered strain of the
endophytic fungus Ozonium sp. (EFY-21). The transformed strain overproduced the
rate-limiting enzyme of taxol biosynthesis and taxadiene synthase (Wei et al. 2012).
The endophyte P. versicolor, from the plant Taxus caspodata, produced
478 µg L−1 (Kumaran et al. 2010). C. capsici from Capsicum annuum made
687 lg L−1 (Kumaran et al. 2011). Another endophytic fungus, Phoma betae,
isolated from the medicinal tree Ginkgo biloba produced taxol at 795 lg L−1
(Kumaran et al. 2012). Colletotrichum annutum from Capsium annuum
Cladosporium cladosporoides, an endophyte of the Taxus media tree, produced
800 lg L−1 of taxol (Zhang et al. 2009). Metarhizium anisopiliae H-27, isolated
from the tree Taxus chinensis, yielded 846 lg L−1 (Liu et al. 2009). Although a
review of taxol production by endophytic fungi indicated that strain improvement
had resulted in levels of only 0.4–1.0 mg L−1 (Zhou et al. 2010), it was reported
that another fungus, Alternaria alternate var. monosporus, from the bark of Taxus
yunanensis, after ultraviolet and nitrosoguanidine mutagenesis, could produce taxol
at 227 mg L−1 (Duan et al. 2008).
Another important antitumor agent is camptothecin (Fig. 3), a modified
monoterpene indole alkaloid produced by certain plants (angiosperms) and by the
endophytic fungus, Entrophospora infrequens. The fungus was isolated from the
plant Nathapodytes foetida (Wall and Wani 1996). Recently, it was found that
Trichoderma atroviridi strain LY357, an endophytic fungus from C. acuminata,
was an improved producer of camptothecin. The endophytic fungus produced
142 µg L−1 of camptothecin in the presence of the elicitor methyljasmonate and
XAD adsorbent resin (Pu et al. 2013). In view of the low concentration of camp-
tothecin in tree roots and poor yield from chemical synthesis, the fungal fermen-
tation is very promising for industrial production of camptothecin. It is used for
recurrent colon cancer and has unusual activity against lung, ovarian, and uterine

Fig. 3 Chemical structure of

3 Bioactive Products from Fungi 65

cancer (Amna et al. 2006). Colon cancer is the second-leading cause of cancer
fatalities in the USA and the third most common cancer among the US citizens.
Camptothecin is known commercially as Camptosar and Campto and achieved
sales of $1 billion in 2003 (Lorence and Nessler 2004). Camptothecin’s
water-soluble derivatives irinotecan and topotecan have been approved and are used
clinically. Metastatic colorectal cancer is treated by irinotecan, whereas topotecan
has use for ovarian cancer, cervical cancer, and small-cell lung cancer. A review of
the activities of camptothecin and its many small and macromolecular derivatives
has been published by Venditto and Simanek (2010).
The cellular target of camptothecin is type I DNA topoisomerase. When patients
become resistant to irinotecan, its use can be prolonged by combining it with the
monoclonal antibody Erbitux (Cetuximab). Erbitux blocks a protein that stimulates
tumor growth, and the combination helps metastatic colorectal cancer patients
expressing epidermal growth factor receptor (EGFR). This protein is expressed in
80% of advanced metastatic colorectal cancers. The drug combination reduces
invasion of normal tissues by tumor cells and the spread of tumors to new areas.
Angiogenesis, the recruitment of new blood vessels, is necessary for tumors to
obtain oxygen and nutrients. Tumors actively secrete growth factors that trigger
angiogenesis. Anti-angiogenesis therapy is now known as one of the four cancer
treatments; the other three are surgery, radiotherapy, and chemotherapy. By the end
of 2007, 23 anti-angiogenesis drugs were in Phase III clinical trials and more than
30 were in Phase II. Fumagillin, a secondary metabolite of Aspergillus fumigatus,
was one of the first agents found to act as an anti-angiogenesis compound. Next to
come along were its oxidation product ovalacin and the fumagillin analog TNP-470
(=AGM-1470). TNP-470 binds to and inhibits type 2 methionine aminopeptidase.
This interferes with amino-terminal processing of methionine, which may lead to
inactivation of enzymes essential for the growth of endothelial cells. In animal
models, TNP-470 effectively treated many types of tumors and metastases.
Inhibitors of farnesyltransferase (FTIs) have anticancer activity because farne-
sylation is required for the activation of Ras, a necessary step in cancer progression.
They also induce apoptosis in cancer cells. The fungus Phoma sp. FL-415 produces
an FTI known as TAN-1813 (Bernardes et al. 2010).

5 Immunosuppressant Drugs

An individual’s immune system is capable of distinguishing between native and foreign

antigens and to mount a response only against the latter. Suppressor cells are critical in
the regulation of the normal immune response. The suppression of the immune
response, either by drugs or radiation, in order to prevent the rejection of grafts or
transplants or to control autoimmune diseases, is called immunosuppression.
Microbial compounds capable of suppressing the immune response have been
discovered as fungal secondary metabolites. Cyclosporin A was originally dis-
covered in the 1970s as a narrow-spectrum antifungal peptide produced by the
66 S. Sanchez and A.L. Demain

Fig. 4 Chemical structure of cyclosporin A

mold, Tolypocladium nivenum (previously Tolypocladium inflatum) in an aerobic

fermentation (Borel et al. 1976). Cyclosporins (Fig. 4) are a family of neutral,
highly lipophilic, cyclic undecapeptides containing some unusual amino acids,
synthesized by a non-ribosomal peptide synthetase, cyclosporin synthetase.
Discovery of the immunosuppressive activity of this secondary metabolite led to
use in heart, liver, and kidney transplants and to the overwhelming success of the
organ transplant field (Borel 2002). Cyclosporin was approved for use in 1983. It is
thought to bind to the cytosolic protein cyclophilin (immunophilin) of immuno-
competent lymphocytes, especially T lymphocytes. This complex of cyclosporin
and cyclophilin inhibits calcineurin, which under normal circumstances is respon-
sible for activating the transcription of interleukin-2. It also inhibits lymphokine
production and interleukin release and therefore leads to a reduced function of
effector T cells. Annual world sales of cyclosporin A are approximately $2 billion.
Cyclosporin A also has activity against corona viruses (de Wilde et al. 2011).
Studies on the mode of action of cyclosporin and the later-developed
immunosuppressants from actinomycetes, such as sirolimus (a rapamycin) and
FK-506 (tacrolimus), have markedly expanded current knowledge of T cell acti-
vation and proliferation. These agents act by interacting with an intracellular protein
(an immunophilin), thus forming a novel complex that selectively disrupts the
signal transduction events of lymphocyte activation. Their targets are inhibitors of
signal transduction cascades in microbes and humans. In humans, the signal
transduction pathway is required for the activation of T cells.
Pleuromutilin, a tricyclic terpenoid inhibitor of protein synthesis, was originally
isolated in 1951 from the basidiomycete Pleurotis sp. (Kirst 2012). Although it was
rapidly metabolized and had unfavorable pharmacokinetics, its semi-synthetic
derivatives tiamalin and valnemulin have been successful for control and treatment
3 Bioactive Products from Fungi 67

of swine and poultry diseases. Also, retapamulin (Altabax®) was approved for
topical treatment of human skin diseases.
A very old broad-spectrum antibiotic, actually the first antibiotic ever discov-
ered, is mycophenolic acid, which has an interesting history. Bartolomeo Gosio
(1863–1944), an Italian physician, discovered the compound in 1893 (Bentley
2001). Gosio isolated a fungus from spoiled corn, which he named Penicillium
glaucum, which was later reclassified as P. brevicompactum. He isolated the
crystals of the compound from culture filtrates in 1896 and found it to inhibit the
growth of Bacillus anthracis. This was the first time an antibiotic had been crys-
tallized and the first time that a pure compound had ever been shown to have
antibiotic activity. The work was forgotten, but fortunately the compound was
rediscovered by Alsberg and Black (1913) and given the name mycophenolic acid.
They used a strain originally isolated from spoiled corn in Italy called Penicillium
stoloniferum, a synonym of P. brevi-compactum. The chemical structure was elu-
cidated many years later (1952) by Birkinshaw et al. (1952) in England.
Mycophenolic acid has antibacterial, antifungal, antiviral, antitumor, antipsoriasis,
and immunosuppressive activities. Its antiviral activity is exerted against yellow
fever, dengue virus, and Japanese encephalitis virus (Sebastian et al. 2011). It was
never commercialized as an antibiotic because of its toxicity, but its
2-morpholinoethylester was approved as a new immunosuppressant for kidney
transplantation in 1995 and for heart transplants in 1998 (Lee et al. 1990). The ester
is called mycophenolate mofetil (CellCept) and is a prodrug that is hydrolyzed to
mycophenolic acid in the body. It is sometimes used along with cyclosporin in
kidney, liver, and heart transplants. Mycophenolic acid also appears to have
anti-angiogenic activity (Chong et al. 2006).

6 Hypocholesterolemic Agents

Only about 30% of cholesterol in humans comes from the diet. The rest is syn-
thesized by the body, predominantly in the liver. Many people cannot control their
level of cholesterol at a healthy level by diet alone and require hypocholesterolemic
agents. High blood cholesterol leads to atherosclerosis, which is a chronic, pro-
gressive disease characterized by continuous accumulation of atheromatous plaque
within the arterial wall, causing stenosis and ischemia. Atherosclerosis is a leading
cause of human death. The last two decades have witnessed the introduction of a
variety of anti-atherosclerotic therapies. The statins form a class of hypo-lipidemic
drugs, formed as secondary metabolites by fungi, and used to lower cholesterol by
inhibiting the rate-limiting enzyme of the mevalonate pathway of cholesterol
biosynthesis, i.e., 3-hydroxymethyl glutaryl-CoA (HMG-CoA) reductase.
Inhibition of this enzyme in the liver stimulates low-density lipoprotein
(LDL) receptors, resulting in an increased clearance of LDL from the bloodstream
and a decrease in blood cholesterol levels. They can reduce total plasma cholesterol
by 20–40%. Through their cholesterol-lowering effect, they reduce the risk of
68 S. Sanchez and A.L. Demain

Fig. 5 Chemical structure of


cardiovascular disease, prevent stroke, and reduce development of peripheral vas-

cular disease (Nicholls et al. 2007).
Currently, there are a number of statins in clinical use. They reached an annual
market of nearly $30 billion before one became a generic pharmaceutical. The
history of the statins has been described by Akira Endo, the discoverer of the first
statin, compactin (mevastatin; ML-236B) (Endo 2010). This first member of the
group was isolated as an antibiotic product of P. brevicompactum (Brown et al.
1976). At about the same time, it was found by Endo and coworkers as a choles-
terolemic product of Penicillium citrinum (Endo et al. 1976). Although compactin
was not of commercial importance, its derivatives achieved strong medical and
commercial success. Lovastatin (monacolin K; mevinolin; Mevacor TM) was iso-
lated in broths of Monascus rubra and Aspergillus terreus (Alberts et al. 1980;
Endo and Monacolin 1979). Lovastatin, developed by Merck & Co. and approved
by the US Food and Drug Administration (FDA) in 1987, was the first commer-
cially marketed statin. In its chemical structure, lovastatin has a hexahydronaph-
thalene skeleton substituted with a p -hydroxy-lactone moiety (Fig. 5).
A semisynthetic derivative of lovastatin is Zocor® (simvastatin), one of the main
hypocholesterolemic drugs, sold for $7 billion per year before becoming generic.
An unexpected effect of simvastatin is its beneficial activity on pulmonary artery
hypertension (Liu et al. 2011). Another surprising effect is its antiviral activity
(Bader et al. 2008). Simvastatin is active against RNA viruses and acts as
monotherapy against chronic hepatitis C virus in humans. It has been shown to act
in vitro against hepatitis B virus (HBV). This virus infects 400 million people and is
the most common infectious disease agent in the world. The virus causes hepato-
cellular cancer, which is the leading cause of cancer.

7 Mycotoxins

Fungi produce poisons called mycotoxins, which, strangely enough, have been har-
nessed as medically useful agents. These agents (e.g., ergot alkaloids) caused fatal
poisoning of humans and animals (ergotism) for centuries by the consumption of bread
made from grain contaminated with species of the fungus Claviceps. However,
mycotoxins later were found useful for angina pectoris, hypertonia, serotonin-related
3 Bioactive Products from Fungi 69

Fig. 6 Chemical structure of


disturbances, inhibition of protein release in agalactorrhea, reduction in bleeding after

childbirth, and prevention of implantation in early pregnancy (Bentley 1997; Vining
and Taber 1979). Their physiological activities include the inhibition of action of
adrenalin, noradrenalin, and serotonin, as well as the contraction of smooth muscles of
the uterus. Antibiotic activity is also possessed by some ergot alkaloids.
Members of the genus Gibberella produce zearelanone and gibberellins.
Zearelanone (Fig. 6) is an estrogen made by Gibbberella zeae (syn. Fusarium
graminearum) (Hidy et al. 1977). Its reduced derivative zeranol is used as an anabolic
agent in sheep and cattle, which increases growth and feed efficiency. Gibberellic acid,
a member of the mycotoxin group known as gibberellins, is a product of Gibberella
fujikuroi and causes “foolish rice seedling” disease in rice (Jefferys 1970). Gibberellins
are employed to speed up the malting of barley, improve the quality of malt, increase
the yield of vegetables, and cut the time in half for obtaining lettuce and sugar beet seed
crops. They are isoprenoid growth regulators, controlling flowering, seed germination,
and stem elongation (Tudzinski 1999). More than 25 tons are produced annually with a
market of over $100 billion.

8 Inhibitors of Enzyme Activity

Enzyme inhibitors have received increased attention as useful tools, not only for the
study of enzyme structures and reaction mechanisms, but also for potential utilization in
medicine and agriculture. Several enzyme inhibitors with various industrial uses have
been isolated from microbes (Umezawa 1972). Among the most important are the
statins and hypocholesterolemic drugs discussed previously. Fungal products are also
used as enzyme inhibitors against cancer, diabetes, poisoning, and Alzheimer’s disease.
The enzymes inhibited include acetylcholinesterase, protein kinase, tyrosine kinase,
glycosidases, and others (Paterson 2008).

9 Pigments

Since 800 AD, Monascus purpurea has been grown on rice to prepare koji or
Angkak (red rice), which is used as a traditional Chinese food and medicine (Ma
et al. 2000). Monascorubramine and rubropunctatin are water-soluble red pigments
70 S. Sanchez and A.L. Demain

formed upon the reaction of the orange pigments monascorubrin and rubropunctatin
with amino acids in fermentation media (Juzlova et al. 1996). The fungus is used to
prepare red rice, wine, soybean cheese, meat, and fish. It is authorized in Japan and
China for food use. There are 54 known Monascus pigments. They have an
amazing number of activities: antimicrobial, anticancer, anti-mutagenesis,
anti-diabetes, anti-obesity, anti-inflammatory, cholesterol-lowering, immunosup-
pressive, and hypotensive (Feng et al. 2012; Lee and Pan 2012). Nutritional control
of the formation of the red pigments has been described in a series of publications
by Lin and Demain (1991, 1993, 1994, 1995).
Carotenoids are tetra-terpenoid pigments which are excellent anti-oxidants. They are
used as nutritional supplements, animal feeds, food additives, pharmaceuticals, food
coloring agents, and in cosmetics. They are composed of hydrocarbons (carotenes and
lycopene) and oxygenated derivatives (xanthophylls) and are used for protection
against cancer, age-related muscular degeneration, and cardiovascular diseases (Roukas
2015). Beta-carotene and lycopene are highly unsaturated isoprene derivatives which
stimulate the immune system and prevent degenerative diseases and cancer. Some are
made microbiologically. They had a 2010 market of $1.2 billion, and their market is
growing by 2.3% per year. Adaptive laboratory evolution was used to increase the
microbial production of carotenoids in a genetically engineered S. cerevisiae strain. It
was carried out by using a periodic hydrogen peroxide shocking strategy. The
improved production was due to up-regulation of genes related to biosynthesis of lipid
and mevalonate (Reyes et al. 2013). The production amounted to 16 mg g−1 dry cell
weight. Beta-carotene, a precursor of vitamin A, has a market of $242 million.
Although most is made chemically, it can be made by Blakeslea trispora at 3 g L−1
(Vachali et al. 2012). Lycopene is another carotenoid.
Phaffia rhodozyma (Xanthophyllomyces dendrorhous) is a heterobasidiomycetous
yeast that has become the most important microbial source for the preparation of the
carotenoid astaxanthin (Andrewes et al. 1976; Rodríguez-Saiz et al. 2010). This oxy-
genated carotenoid pigment (Fig. 7) is used in the feed, food, pharmaceutical,
nutraceutical, and cosmetic industries. It is responsible for the orange to pink color of
salmonid flesh and the reddish color of boiled crustacean shells. Feeding of penreared
salmonids with a diet containing this yeast induces pigmentation of the white muscle
(Johnson et al. 1980). It is a very good antioxidant, 10 times more active than
beta-carotene and 100 times more than alpha-tocopherol. It is the second most
important carotenoid. Astaxanthin enhances the immune system and protects skin from
radiation injury and cancer. It can be produced synthetically as hydroxyl-astaxanthin
from petrochemicals with a selling price of $2500 per kg. However, the natural product
is favored because the synthetic product is a mixture of stereoisomers. X. dendror-hous
produces astaxanthin at 390 mg L−1.
Natural astaxanthin is more stable than the synthetic version and more
bioavailable. The natural product is present in algae and fish as mono- and diesters
of fatty acids. However, it is difficult to hydrolyze the esters from algae, which
limits its usage to trout and salmon. The yeast product is better since it is the 97%
free, non-esterified (3R, 3’R) stereoisomer. The natural product is more expensive
($7000 per kg) than synthetic astaxanthin ($2500 per kg). The astaxanthin market
3 Bioactive Products from Fungi 71

Fig. 7 Chemical structure of astaxanthin

was $219 million in 2007 with 97% being synthetic. Most of the production pro-
cesses with the yeast yield levels of astaxanthin are lower than 100 mg L−1.
However, white light improved production to 420 mg L−1 (de la Fuente et al. 2012)
and mutant strain UBv-AX2 can make 580 mg L−1 (Jacobson et al. 2000).

10 Sweeteners

Thaumatin, a protein produced by the plant Thaumatococcus danielli, can also be

produced by P. roqueforti and Aspergillus niger var. awamori (Faus 2000).
Thaumatin is intensely sweet (i.e., 3000 times sweeter than sucrose) and is
approved as a foodgrade ingredient. The production by A. niger var. awamori was
improved from 2 mg L−1 up to 14 mg L−1 by increasing gene dosage and use of a
strong promoter (Moralejo et al. 1999). The sweetener xylitol, normally produced
by Pichia stipitis, can be produced by recombinant S. cerevisiae in higher con-
centrations by transforming the XYL1 gene of P. stipitis into S. cerevisiae. The
gene encodes a xylose reductase (Hallborn et al. 1991).

11 Proteins

Industrial enzymes include detergent enzymes, technical enzymes, food enzymes,

and feed enzymes (Hellmuth and Bring 2013). Technical enzymes include those
used for textiles, leather, pulp and paper, and fuel ethanol. The largest group is the
food enzymes which include amylases, xylanases, glucose oxidase, hexose oxidase,
pectinases, glucanase, invertase, glucose isomerase, protease, lipase, phosphorylase,
lactase, milk-clotting enzymes, animal rennet, microbial rennet, and chymosin.
Fungal producers are a major source, and the main ones are A. niger and
Klyveromyces lactis.
Advances in the production of biopharmaceutical proteins by metabolic engi-
neering have been reviewed by Nielsen (2013). Yeasts are used to produce
72 S. Sanchez and A.L. Demain

recombinant proteins (Celik and Calik 2012). They rapidly reach high levels of
growth, produce high amounts of recombinant proteins, and do not contain pyro-
gens, pathogens, or viral inclusions. About 20% of the biopharmaceuticals on the
market are made by S. cerevisiae. They include more than 40 different recombinant
proteins. This yeast is important for production of FDA-approved insulin and its
analogs, hepatitis B surface antigen, urate oxidase, glucagons,
granulocyte-macrophage colony stimulating factor (GM-CSF), hirudin and
platelet-derived growth factor. The insulin market was $12 billion in 2011 and is
still on the increase. Human serum albumin, used as a plasma expander in surgery,
is produced by S. cerevisiae at 3 g L−1, and human transferrin, used for anemia, is
produced at 1.8 g L−1.
Yeasts also are used to make human serum albumin, hepatitis vaccines, and
virus-like particles used for vaccination against human papilloma virus. S. cerevisiae
carries out folding of many human proteins, secretes the proteins, and post-
translational modifications, e.g., proteolytic processing of signal peptides, disul-
fide bond formation, subunit assembly, acylation, and glycosylation. However,
S. cerevisiae is not favored today because of plasmid instability, low levels of
produced proteins, lack of secretion due to retention in the proteins in the periplasm,
and hyper-glycosylation of the recombinant proteins including the high-mannose
type of N-glycosylation which shortens the in vivo half-life, reduces efficacy, and
elicits an immunogenic response to the non-human carbohydrate moiety.
The yeasts that are used, having been engineered for more human-type
N-glycosylation, include Pichia pastoris, Hansenula polymorpha, Yarrowia
lipolytica, and Schizosaccharomyces pombe. Titers of P. pastoris have reached 20–
30 g L−1, and it can secrete the proteins. P. pastoris has been engineered to produce
human-like N-glycosylation that includes terminal addition of sialic acid to the
glycoprotein. P. pastoris produces ecallantide, which was approved by FDA in
2009 for hereditary angioedema. It also produces plant-derived hydroxynitrile lyase
at over 20 g L−1 (Hasslacher et al. 1997). H. polymorpha has been used for the
production of hepatitis B vaccine, interferon alpha-2a, hirudin, insulin, phytase,
lipase, hexose oxidase, interleukin-6, serum albumin, glucose oxidase, glycolate
oxidase, and catalase; the first four are on the market. This yeast reaches high
growth density, secretes proteins as large as 150 kDa, and is highly productive. For
example, it produces 13.5 g L−1 of recombinant phytase. Other useful yeasts
include K. lactis for the production of bovine chymosin (rennin), glucoamylase,
human serum albumin, interleukin-1 and interleukin-1 beta, and many other
recombinant proteins. S. pombe has been used to produce human lipocortin I,
human papillomavirus type 16 vaccine, and many others. The beauty of these yeasts
is their ability to perform post-translational modifications similar to those of higher
eukaryotes, e.g., correct folding, disulfide bond formation, N- and O-linked gly-
cosylation, and proteolytic processing of signal sequences. About 70% of all
therapeutic proteins are glycoproteins. The production of recombinant microbial
enzymes by fungi has been reviewed by Liu et al. (2013a, b).
3 Bioactive Products from Fungi 73

12 Biofuels

Systems metabolic engineering for the production of biofuels and chemicals by

Aspergillus and Pichia species has been reviewed by Caspeta and Nielsen (2013).
Ethanol can be used as a fuel by itself or in combination with gasoline (E10, E15,
and E85). It is mainly made in the USA (over 7 billion gallons from corn) and in
Brazil. However, corn can only yield 15 billion gallons, and corn prices are rising.
Cellulose is a possible source of ethanol but instead of containing only glucose,
cellulose also contains C5 sugars such as xylose and arabinose. The best C5 utilizer
is P. stipitis which can produce ethanol and clean up concentrated toxins liberated
from lignocellulose degradation. Its production of ethanol has been reviewed by
Agbogo and Coward-Kelly (2008). It can produce ethanol from pretreated sources
of biomass such as red oaks, wheat straw, sugarcane bagasse, rice straw, corn cobs,
corn stover, aspen wood, pinewood, and poplar wood. From aspen wood such as
Orpinomyces defined medium, 61 g L−1 can be made (Slininger et al. 2006).
Attributes of P. stipitis include consumption of acetic acid, reduction in the furan
ring toxins in HMF, and furfural present in cellulosic biomass conversions.
The production of ethanol via biomass saccharification using fungi has been
discussed by Zhang (2011). Saccharification of biomass involves pretreatment,
fractionation, and enzymatic hydrolysis. Pretreatment may be the most expensive
step, amounting to 40% of total processing costs. Biodelignification of lignocel-
lulose has been carried out by ascomycetes including Trichoderma reesei, basid-
iomycetes such as the white rot fungus Phaenerochaete sp. (Chandel et al. 2015).
The key enzyme in delignification is manganese peroxidase. Biodelignification is
the most expensive step in the conversion of biomass to ethanol mainly due to its
slow rate of action. Protein engineering must be applied to make the delignification
enzymes better suited to the temperature, pH, and reaction conditions of the
industrial process.
Hydrolysis by cellulase is another expensive step costing 50 cents to $1/gallon of
produced ethanol. Nearly 100–200 g of cellulase is used per gallon of ethanol
produced, where specific activities of fungal cellulases are 0.6–1.5 filter paper units
per mg of cellulase. Filamentous fungi can produce native cellulases at levels of
more than 100 g L−1 (Cherry and Fidanstsel 2003). Novozymes, Genencor, and
Iogen produce cellulase from Trichoderma, whereas dyadic uses Chrysosporium
lucknowense. These commercial fungal fermentations produce over 100 g crude
cellulase per liter of broth, much higher than that produced by bacteria. An
important move is to decrease the amount of cellulase used to produce ethanol. The
overall action of T. reesei cellulase on cellulosic biomass is limited by a low content
of beta-glucosidase. The result is an accumulation of cellobiose which limits further
breakdown. By expressing the beta-glucosidase gene of Pericona sp. in T. reesei,
(Dashtban and Qin 2012) were able to increase the level of beta-glucosidase, the
overall cellulase activity, and the action on biomass residues.
During pretreatment of biomass, inhibitors are released such as furfural.
Tolerance to this inhibitor can be achieved by over-expression of S. cerevisiae
74 S. Sanchez and A.L. Demain

genes encoding (a) yeast transcription activator MSN2 (Sasano et al. 2012),
(b) ZWF1 of the pentose phosphate pathway (Gorsich et al. 2006), (c) ADH1
encoding alcohol dehydrogenase 1, and (d) TAL1 encoding transaldolase 1
(Hasunama et al. 2014).
Regulation of cellulolytic and hemi-cellulolytic enzyme production by fila-
mentous fungi involves regulatory transcription factors such as xlnR from
Aspergillus which is involved in D-xylose induction of cellulolytic and xylanolytic
enzymes (Tani et al. 2014). Others include C1R-112 from Neurospora, ManR,
McMA, and C1br from Aspergillus, and Bg1R from Trichoderma which regulate
cellulolytic and/or hemi-cellulolytic enzyme production.
S. cerevisiae is well known for its ability to produce ethanol. Cassava
mash-containing sludge was converted to ethanol at 86 g L−1 by the S. cerevisiae
SSF process, employing continuous fermentation (Moon et al. 2012). Volumetric
productivity was 2.4 g L−1, and the percent yield was 91%. When immobilized on
corn stalks, S. cerevsiae can produce 88 g L−1 of ethanol from food waste (Yan
et al. 2012). Alcohol tolerance in this yeast is increased by adding potassium and
raising the pH of the fermentation with KOH (Lam et al. 2014). Under these
conditions, 127 g L−1 was produced. Using cell cycling of this yeast in very
high-gravity fermentations led to an ethanol titer of 142 g L−1 with a productivity
of 3.5 g L−1 h−1. The strain used (PE-2) was obtained from a distillery in Brazil
producing ethanol from sugarcane (Pereira et al. 2012).
One hundred billion liters of ethanol are produced each year from sugar cane and
corn starch by S. cerevisiae. Production at high temperature (ca 40 °C) reduces
cooling costs, lowers the effects of contamination, and enables more efficient
hydrolysis of feedstocks. This improves the productivity in the simultaneous sac-
charification and fermentation process. Caspeta et al. (2014), using adaptive labo-
ratory evolution, isolated S. cerevisiae strains with improved growth and ethanol
production at 40 °C. These strains grew 1.9 times faster and excreted ethanol 1.6
times faster than the parent strain. They noted a change in sterol composition from
ergosterol to fenosterol due to mutation in the C-5 sterol desaturase gene and
increased expression of sterol biosynthesis genes. Sterols contribute to membrane
fluidity. The thermo-tolerant strains were improved in glucose consumption rate
which increased by 60% at 40 °C and by 300% at 42 °C.
Jerusalem artichokes produce high levels of biomass, grow rapidly, need only
little pesticide, fertilizer, and water, and can grow on marginal land. It could be a
good substrate for the production of important products (Li et al. 2013). Product
titers achieved by fungi growing on Jerusalem artichokes include 154 g L−1 of
ethanol by a mixed culture of S. cerevisiae and A. niger, and 109 g L−1 by
S. cerevisiae alone.
Biodiesel is a monoalkyl ester of long-chain fatty acids made by transesterifi-
cation of feedstocks such as waste animal fats or vegetable oils, e.g., soybean oil. It
is a very good fuel, contains less sulfur than conventional fuel, can be used in diesel
engines without modification, and can be blended in any ratio with petroleum
diesel. It is biodegradable and non-toxic (Lin et al. 2013). The four different
methods of biodiesel production include transesterification, blending,
3 Bioactive Products from Fungi 75

microemulsions, and pyrolysis. Transesterification is the method of choice, the

catalyst being chemical (acid or base) or an enzyme. Favored is transesterification
via enzymes, i.e., lipases. Microbial lipases are excellent since they are stable in
organic solvents, do not need cofactors, have broad substrate specificity and high
enantiospecificity. Candida antartica is a favored lipase producer. Yields of
enzymic transesterification can reach 100%. Maximum enzyme-catalyzed transes-
terification occurs at 55 °C. The cost of lipase is high, but it can be lowered by the
use of enzyme immobilization and recycling of the immobilized enzyme.
Adsorption is the best immobilization procedure due to its simplicity, ease, use of
mild conditions, and low cost. Genetic engineering has been used to convert
S. cerevisiae into a biodiesel producer, i.e., one that is oleaginous, supplying fatty
acids and alcohols, and converting them to biodiesel. Production of intracellular
lipids by yeasts growing on alkali-treated corn stover revealed that Cryptococcus
humicola produces 15 g L−1 lipids in a total biomass weight of 36 g L−1 (Sitepu
et al. 2014).
2,3-Butanediol is a fuel with a high heating value (27,000 J/g) and is used as a
liquid fuel or fuel additive. When compared to acetone, alpha-pinene, 1-butanol,
isobutanol, isopropanol, and fatty alcohols, 2,3-butanediol shows lower toxicity. It
also is used in the preparation of solvents, anti-freeze agents, synthetic rubber, and
plastics. An engineered stain of S. cerevisiae can produce it at 96 g L−1 (Kim et al.

13 Additional Compounds

Metabolic engineering has improved yeasts as producers of important metabolites

(Liu et al. 2013a, b). Important productivities include Y. lipolytica, producing
80 g L−1 erythritol, 154 g L−1 citric acid from glycerol, 63 g L−1 succinic acid, and
27 g L−1 mannitol. S. cerevisiae produces malic acid at 59 g L−1, 2,3-butanediol at
2 g L−1, and the artemisinin precursor amorpha-4,11-diene at 40 g L−1. L-lactic
acid is made by Candida boidini at 86 g L−1.
P. pastoris can covert methanol to formaldehyde in a process responsible for the
production of 6000 tons per year of formaldehyde (Caspeta and Nielsen 2013).
Erythritol can be produced from glycerol by Y. lipolytica at 170 g L−1 (Khanna
et al. 2012). Mannitol is produced from glycerol at 51 g L−1 by Candida magnolia.
Alpha-ketoglutaric acid was produced at 195 g L−1 by Y. lipolytica (Candida
lipolytica) with a yield of 0.9 g g−1 of substrate when grown on n-paraffins
(Weissbrodt et al. 1988). This acid is used industrially in chemical synthesis of
heterocycles or elastomers, as a dietary supplement and as an enhancer of wound
Production of itaconic acid at 90 g L−1 was achieved by A. terreus with a yield
of 0.58 g g−1 glucose and a productivity of 0.29 g L−1 h−1 (Kuenz et al. 2012).
Microbial formation of this compound is more productive than by chemical pro-
cesses. Increasing pH during the production phase was found to increase production
76 S. Sanchez and A.L. Demain

(Hevekerl et al. 2014). A titer of 146 g L−1 was reached by raising pH from 4 to 6
or by raising it to 3 after 2.1 days of cultivation. Itaconic acid is used in the
production of polymers, coatings, adhesives and textiles. About 80,000 tons are
made each year with a selling price of $2 kg−1.
Citric acid production began in England in 1826 by John and Edward Sturge of
the city of Selby. It was made from Italian citrus fruits at that time. In 1893, the
German microbiologist Carl Wehmer discovered that sugar-growing fungi secreted
citric acid. After World War I, the fermentation became the method of choice.
John N. Currie had found that A. niger was an excellent producer of citric acid and,
as a result, the Pfizer company in New York began large-scale fermentation pro-
duction in 1923. Worldwide production is 1.6 million tons per year. About 95% is
used in the food industry. Other uses include chemicals, medicinal, textiles, and
metallurgy. Chemicals include surfactants and synthetic detergents (Morgunov
et al. 2013). In addition to A. niger, another producer is Y. lipolytica. Production by
the latter is favored by limitation of cell growth via limiting levels of nitrogen,
phosphorus, or sulfur with nitrogen limitation being the most useful. This yeast
produces high levels of both citric and isocitric acids from rapeseed oil (Kamzolova
et al. 2013).
Fumaric acid is used as a food acidulent, a beverage ingredient, and an
antibacterial agent in the feed industry (Xu et al. 2012). Its other uses are for the
preparation of biodegradable polymers, plasticizers, polyester resins, and as an
animal feed supplement to reduce methane emissions (Thakker et al. 2015).
Rhizopus arrhizus has been used by Pfizer to produce it at 4000 tons per year
(Roa-Engel et al. 2008). Other species are also good producers, e.g., Rhizopus
nigricans, Rhizopus formosa, and Rhizopus oryzae. R. nigricans produced 121 g
L−1 with a productivity of 1 g L−1 h−1 and a yield of 0.37 (Ling and Ng 1989).
DuPont patented a process using R. arrhizus NRRL-1526 with limited dissolved
oxygen to produce 130 g L−1.
Glycolic acid can be produced by S. cerevisiae and K. lactis (Koivistoinen et al.
2013), although it is currently made chemically. Engineered S. cerevisiae made
only 1 g L−1 but engineered K. lactis produced 15 g L−1 from ethanol plus
D-xylose. It is polymerized to polyglycolic acid which is an excellent packaging
material. Glycolic acid can also be used with lactic acid to make a copolymer
(PLGA) for medical application in drug delivery. The market for glycolic acid was
$93 million for the 40 million kg produced. Glycolic acid is also employed in the
textile industry as a tanning and dyeing agent.
Gluconic acid is used in the construction and in the preparation of chemicals,
pharmaceuticals, foods, beverages, textiles and leather. It is also used to chelate
divalent and trivalent metal ions. About 50,000–60,000 tons are made annually
using glucose as substrate. The price varies from $1.20 to $8.50 kg−1. Usually
glucose or sucrose is used as fermentation substrate. Golden syrup, a by-product of
the process refining sugar cane juice into sugar, or by treating sugar with acid, can
be used for fermentation by A. niger (Purane et al. 2012). About 85 g L−1 was
produced in 44 h with a productivity of 1.94 g L−1 h−1. Previous workers had
obtained 158 g L−1 at 0.238 g L−1 h−1 with A. niger immobilized on cellulose
3 Bioactive Products from Fungi 77

microfibers (Sankpal and Kulkami 2002). Also, Sankpal et al. (1999) reached
135 g L−1 with a productivity of 0.09 g L−1 h−1 using immobilization on cellulose
fibers and surface culture. About 80–100 g L−1 was obtained using immobilization
on waste paper with a productivity of 0.04 g L−1 h−1 (Singh and Kumar 2007).
Brown et al. (2013) described metabolic engineering of Aspergillus oryzae
NRRL 3488 to produce malic acid at 154 g L−1. The result was achieved by
overexpressing (a) the C-4-dicarboxylate transporter and (b) the cytosolic alleles of
pyruvate carboxylase and malate dehydrogenase. The rate was 0.94 g L−1 h−1, and
the yield on glucose was 1.38 mol mol−1. Penicillium viticola 152 produced 168 g
L−1 of calcium malate in a medium containing corn steep liquor (Khan et al. 2014).
The yield was 1.28 g g−1 glucose and productivity was 175 g L−1 h−1. Malic acid
is a C4 dicarboxylic acid produced at 40,000 tons per year. It is used in the food and
beverage industry as an acidulent and taste enhancer/modifier in combination with
artificial sweeteners. Additional uses are for the preparation of polyester resins and
coatings, in foods and feed, and in the pharmaceutical industry. It is sold for
$2–3 kg−1 (Thakker et al. 2015).
Torulopsis glabrata (also called Candida glabrata) can produce pyruvic acid at
94 g L−1 on glucose with a yield of 0.63 g g−1 glucose, a high productivity of
1.15 g L−1h−1 and high glucose tolerance (Liu et al. 2007, 2013). The organism is
an osmotolerant mutant. Production is increased by the use of urea as nitrogen
source (Yang et al. 2014). This yeast is used for commercial production of pyruvic
acid. The process was industrialized in 1992 by Toray Industries at 400 tons per
Erythritol, a polyhydric alcohol, has 60–70% of the sweetness of sucrose and is
used to combat obesity. It is non-carcinogenic and non-caloric since it is not
digested by humans and cannot be fermented by bacteria to cause dental caries.
Repeated batch cultures of Y. lipolytica on crude glycerol yielded 220 g L−1 with a
yield of 0.43 g g−1 glycerol used and a productivity of 0.54 g L−1h−1 (Mironczuk
and Furgala 2014).
Bioconversion of xylose to xylitol by Debaryomyces hansenii amounted to
110 g L−1 from 300 g L−1 xylose (Misra and Raghuwanshi 2012). The yield was
0.48 g g−1. This sugar alcohol is used in food production, has high activity as a
sweetener, is non-cariogenic, and has insulin-independent metabolism properties. It
is commercially produced by chemical reduction of D-xylose, but this is an
expensive process. Its global market is over 125,000 tons per year. The biocon-
version would probably be less expensive than the chemical procedure. Xylitol is an
excellent antioxidant. It can be made from lignocellulosic waste (Lima de
Albuquerque et al. 2014). It is used as a sucrose replacement for cakes, cookies,
chocolate, and chewing gum and in pharmaceuticals to reduce tooth decay. It acts
against oral biofilms produced by bacteria. It is also a contributor to tooth calcifi-
cation and is active against diabetes, anemia, acute otitis media, and osteoporosis.
Candida athensensis converts vegetable waste containing 200 g L−1 xylose to
100 g L−1 xylitol with a yield of 0.81 g g−1 and a productivity of 0.98 g L−1 h−1
(Zhang et al. 2012).
78 S. Sanchez and A.L. Demain

Coenzyme Q (ubiquinone) is an essential part of the respiratory chain producing

ATP. It is composed of a quinonoid nucleus and a side chain of isoprenoids. Best
producers include fungi such as species of Candida, Saitoella, Trichosporon, and
Production of useful products by basidiomycetes includes carotenoids, fragrances,
enzymes, astaxanthin, erythritol, lipids, and oils (Johnson 2013). Trichosporon
sp. produces lipids and is being considered for biodiesel production. Pseudozyma
(Candida) antartica produces lipase for industrial use and is another biodiesel possi-
bility. It also produces 30 g L−1 of itaconic acid. Sporobolomyces carnicolor accu-
mulates 82% of its biomass as intracellular lipids. Cryptococcus species make unique
carotenoids such as the xanthophyll plectaniaxanthin. Some cryptococci utilize glycerol
and accumulate 60% of their biomass as triacylglycerols.
Fungi produce long-chain polyunsaturated fatty acids (PUFAs) (Ratledge 2013).
They include (a) gamma linoleic acid (GLA; 18:3 omega-6) from Mucor
circinelloides, (b) docohexaenoic acid (DHA; 22:6 omega-3) from
Crypthecodinium cohnii spp, (c) arachidonic acid (ARA; 20:4 omega-6) from
Mortierella alpine, and (d) eicosapentaenoic acid (EPA) from genetically modified
Y. lipolytica (Xue et al. 2013). The oil produced has much higher levels of EPA
than natural oils. EPA is important for the anti-inflammatory activity of fish oils,
thus contributing to cardiovascular and joint health. The product is being com-
mercialized by DSM. The yeast was engineered by transformation with 21
heterologous genes encoding five different activities.
PUFAs represent a multibillion dollar industry, mainly ARA and DHA for infant
formulas. They are major components of phospholipids in cell membranes. They
regulate cell fluidity, attachment of specific enzymes to cell membranes, and
mediate signal transduction and other metabolic processes. They are used for the
biosynthesis of eicosanoids, leukotrienes, prostaglandins, and resolvins, which
function as anti-inflammatory, anti-arrhythmic, and anti-aggregatory effectors.
Many improve cardiovascular health, and certain of them improve eye function and
memory in newborn infants and in adults. Microbial oils are produced by 30–40
species of yeast and also by molds. The producers are known as oleaginous
microbes. Fungi can accumulate 70% of their biomass as oils. DHA is produced at
2000 tonnes per year and has a market of $317 million. ARA is blended with DHA
and used in infant formulas. EPA plus DHA can be used to prevent cardiac
Prebiotics have been reviewed by Panesar et al. (2013). They include
fructo-oligosaccharide produced at 116 g L−1 from sucrose by beta-fructofuranosidase
from Aspergillus japonicas. Prebiotics are used in the nutraceutical, pharmaceutical,
animal feed, and aquaculture areas. They stimulate the growth of beneficial intestinal
bacteria and maintain health of humans by suppression of potentially harmful bacteria,
improvement of defecation, eliminating ammonia, preventing colon cancer, stimulating
mineral adsorption, and lowering cholesterol and lipids.
Pullulan is produced at 88 g L−1 by the yeast Aureobasidium pullulans strain
RBF 4A3 (Sharma et al. 2013). It is an exopolysaccharide which has potential
3 Bioactive Products from Fungi 79

application in industries such as medical, food, pharmaceutical, cosmetic, and

Some vitamins are produced by fungi (Ledesmo-Amaro et al. 2013). Although
vitamin D is derived chemically from cholesterol and ergosterol, it can be made by
S. cerevisiae, Saccharomyces uvarum, and Candida utilis at 30 mg g−1 of dry cells.
Riboflavin (vitamin B2) is made by Ashbya gossypii, Eremothecium ashbyii,
Candida flaeri, and Candida famata. A. gossypii produces 14 g L−1 of riboflavin.
The increase in production by A. gossypii as compared to wild-type strain ATCC
10895 is due to (a) a nine percent increase in flux to pentose-5-phosphate via the
pentose phosphate pathway (PPP) and (b) a 16-fold increase in the flux from purine
to riboflavin (Jeong et al. 2015). This is due to increased guanosine triphosphate
flux through the PPP and the purine synthesis pathway.
Resveratrol (trans-3,5,4’-trihydroxystilbene) is a polyphenol found in wine,
grapes, berries, and peanuts which can be made by some fungi It is a phytoalexin,
i.e., a low molecular weight secondary metabolite. It has beneficial effects against
inflammation, carcinogenesis, oxidation, aging, diabetes, and neurodegenerative
disease. Recombinant S. cerevisiae can produce it at 5.8 mg L−1 upon feeding of
coumaric acid or L-tyrosine (Shin et al. 2012). Alternaria sp. 61, isolated from
Merlot cobs, produces 353 µg L−1 (Shi et al. 2012).
An improved process for making the anti-malarial compound artemisinin using
S. cerevisiae was devised by Paddon et al. (2013). The process applies synthetic
biology to a previous S. cerevisiae process and improves the production of arte-
misinic acid which is then chemically converted to artemisinin. Whereas the pre-
vious process yielded only 1.6 g L−1 of artemisinic acid, the new process reaches
25 g L−1.
Genome sequencing of an organism reveals many secondary metabolic path-
ways that are usually silent. Aspergillus nidulans was found to have nearly 50 such
loci encoding polyketide synthases (PKs) or non-ribosomal protein synthases
(NRPs). Using various types of nutritional limitation in continuous chemostat
cultures of A. nidulans, (Sarkar et al. 2012) obtained expression of two PKS genes
encoding synthases of seven phenolic compounds which were not observed pre-
viously under normal growth conditions.
The soil fungus Aspergillus versicolor produces aspergillomarasmine
(AMA) which turns off a bacterial gene that normally leads to antibiotic resistance
(King et al. 2014). The gene encodes New Delhi metallo-beta-lactamase (NDM-1).
Together with a carbapenem antibiotic, AMA inactivates the gene in Escherichia
coli, Acinetobacter, and Pseudomonas. NDM-1 requires zinc, and AMA removes
zinc from the enzymes. The combination of AMA and the carbapenem has shown
its beneficial effect in mice and human cell culture.
Trichoderma species make many valuable secondary metabolites (Keswani et al.
2014) polyketide gliotoxin, an anti-malarial agent and immune system suppressor,
(3) harzianolide, an antifungal agent and plant growth promoter, (4) koninginins,
which are antifungals and plant growth regulators, (5) 6-pentyl-2H-pyran-2-one, a
80 S. Sanchez and A.L. Demain

plant growth promoter, and coconut aroma used commercially in confectionary

products, (6) trichokonins, broad-spectrum antifungals and plant defense inducers,
(7) viridofungins, potential anticancer agents, and bacteriocides, (8) viridian, a
broad-spectrum antifungal agent, anti-neoplastic, and anti-atherosclerosis agent,
and (9) viridiol, a herbicidal and anti-aging agent.
Activation of “silent” gene clusters by genome mining in A. nidulans has
revealed many new secondary metabolites (Yaegashi et al. 2014). The A. nidulans
genome contains 56 potential secondary metabolism core genes including 27
polyketide synthase (PKS) genes, two PKS-like genes, 11 non-ribosomal peptide
synthetase (NRPS) genes, 15 NRPS-like genes, and one hybrid NRPS-PKS gene.

14 Future Perspectives

Microorganisms have greatly contributed for about 85 years to the development of

medicine and agriculture. However, due to different situations, pathogenic microbes
have become resistant to many antibiotics creating a dangerous situation and
therefore the need for new antibiotics is imperative. Unfortunately, most of the large
pharmaceutical companies have abandoned the search for new antimicrobial
compounds. Due to economics, they have concluded that drugs directed against
chronic diseases offer a better revenue stream than do antimicrobial agents, for
which the length of treatment is short and government restriction is likely. Some
small pharmaceutical and biotechnology companies are still developing antibiotics
but most depend on venture capital rather than sales income, and with the present
regulations, face huge barriers to enter into the market. These barriers were raised
with the best intentions of ensuring public safety but they are having the opposite
effect, i.e., termination of antibiotic development while resistance continues to
increase (Livermore 2004). However, there are some new bright possibilities. One
of the more promising is the utilization of uncultivated microorganisms.
Considering that 99% of bacteria and 95% of fungi have not yet been cultivated in
the laboratory, efforts to find means to grow such uncultured microorganisms are
proceeding and succeeding (Kaeberlein et al. 2002). Furthermore, researchers are
now extracting bacterial DNA from soil samples, cloning large fragments into, for
example, bacterial artificial chromosomes, expressing them in a host bacterium and
screening the library for new antibiotics. This metagenomic effort could open up the
exciting possibility of a large untapped pool from which new natural products could
be discovered (Clardy et al. 2006). Another exciting possibility is that of genome
mining (Scheffler et al. 2013). In addition to these relatively new techniques,
chemical and biological modification of old antibiotics could still supply new and
powerful drugs. These comments also apply to non-antibiotics such as antitumor
agents and other microbial products. In addition, natural products must continue to
be tested for desirable therapeutic activities. I believe that significant progress in
identifying new antibiotics, oncology therapeutics, and other useful medicines will
3 Bioactive Products from Fungi 81

be made, probably not by the big pharmaceutical companies, but by biotechnology

companies and small research groups from institutes and universities.


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Part II
Extraction Technologies
for Bioactives
Chapter 4
Process Development for Bioactive Peptide

Govind Kumar Gnasegaran, Dominic Agyei, Sharadwata Pan,

Indira P. Sarethy, Caleb Acquah and Michael K. Danquah

1 Introduction

Bioactive peptides (BPs) are a special category of peptides which, upon introduc-
tion into a living body system, are able to trigger certain desirable physiological
responses and thereby promote the overall health and well-being (Agyei et al. 2015;
Korhonen and Marnila 2013; Korhonen and Pihlanto 2006). The biological activ-
ities triggered by BPs include antioxidant properties, antimicrobial properties,
anticancer properties, anorexic (weight control) properties, anxiolytic
(anxiety-relieving) properties, antihypertensive properties, anticariogenic proper-
ties, opioid activities and immunomodulatory activities (Agyei and Danquah 2011;
Agyei and Danquah 2012b; Aluko 2008; Clare and Swaisgood 2000; Korhonen and
Pihlanto 2003).
Although most BPs are derived as products of protein hydrolysis, they differ
from native proteins in that BPs are usually inactive when within the protein
structure. The bioactivity is expressed only when the peptides have been released
by the action of enzymes. The biological properties of BPs have been attributed to
the unique properties of individual and combinations of amino acids that make up

G.K. Gnasegaran  C. Acquah  M.K. Danquah (&)

Department of Chemical Engineering, Curtin University of Technology,
98009 Miri, Sarawak, Malaysia
D. Agyei
Department of Food Science, University of Otago, Dunedin 9016, New Zealand
S. Pan
Department of Chemical Engineering, Indian Institute of Technology
Bombay, Powai, Mumbai 400076, India
I.P. Sarethy
Department of Biotechnology, Jaypee Institute of Information Technology,
Noida 201307, Uttar Pradesh, India

© Springer International Publishing AG 2017 91

M. Puri (ed.), Food Bioactives, DOI 10.1007/978-3-319-51639-4_4
92 G.K. Gnasegaran et al.

the sequence. For example, peptides containing hydrophobic and sulphydryl groups
usually exhibit antioxidant properties (Park et al. 2010; Ren et al. 2014), while short
and cationic peptides usually show antimicrobial behaviours (Hancock and Sahl
2006). Proline–valine and glycine–histidine combinations promote antihypertensive
and immunomodulatory properties, respectively (Pihlanto-Leppälä 2002). In the-
ory, options for sources of BPs are limitless because almost all hydrolyzable food
proteins have the potential to be converted into BPs if the proteins encrypt amino
acid sequences with bioactive properties. An increasing number of commercial
products that contain BPs or protein hydrolysates with biological properties are
already on the market (Korhonen and Pihlanto 2006).
However, despite these useful properties, there exist some challenges that hinder
the full exploitation of the bioactive properties of BPs in food and pharmaceutical
products. The main challenge concerns the industrial-scale production of bioactive
peptides. To the best of our knowledge, there has not been any report that addresses
the production of purified bioactive peptides from a bioprocess engineering and
economics perspective. Although a number of products that incorporate BPs exist
on the market, most of these products use the BPs in the form of crude unpurified
protein hydrolysates or peptide mixtures without prior purification (Korhonen 2009;
Korhonen and Pihlanto 2006). The development of commercially viable processes
capable of upscaling the production and purification of BPs is crucial to promoting
the use of these peptides as high-value active ingredients that can be used in
nutrition and health products.
We report here a bioprocess for the production of bioactive peptides from food
proteins by exploiting the activity of proteinases from lactic acid bacteria. We have
reported an optimized and scalable process that limits the amount of raw material
used at all stages of the production chain and have also conducted an in-depth
economic assessment of the process to ascertain commercial viability.

2 Methods and Scope

We aimed at designing a complete bioprocess, as shown in Fig. 1, which can be

used to produce antihypertensive peptides at commercial scales by employing a
whole systems approach that looks at the entire bioprocess from raw material
conditioning through processing to final product formulation. Also, we used widely
abundant and most suitable raw materials and processes, respectively, as well as the
optimum conditions and parameters that have been reported in the literature. The
economic analyses have been conducted using medical, pharmacological and sta-
tistical data on hypertension patients in Malaysia. Details of the process develop-
ment and economic feasibility analysis have been given below.
4 Process Development for Bioactive Peptide Production 93

Fig. 1 Whole systems approach to the production of bioactive peptides

2.1 Process Development Method

The step-by-step procedure employed in developing the model for producing BPs
was as follows: (a) comparison and selection of production technique; (b) selection
of protein source and biocatalyst; and (c) peptide production, separation and
(a) Comparison and selection of production technique
As peptides, BPs can be produced by processes such as (a) chemical synthesis,
(b) transgenic expression in suitable hosts, (c) microbial fermentation of proteins
and/or in vitro hydrolysis of proteins by proteolytic enzymes from plants, animals
or microbial origin (Korhonen and Pihlanto 2006). The production of BPs by
employing the hydrolytic action of proteolytic enzymes on food proteins has been
the most widely used approach because it is safe, food-grade and cost-effective.
This approach was therefore chosen for this study.
(b) Selection of protein source and biocatalyst
Putatively, the two key raw materials used (food proteins and proteolytic
enzymes) are widely abundant. In fact, protein wastes from dairy houses and
abattoirs can be valorized and safely converted to bioactive peptides. For this study,
milk was chosen as the protein source, being a very cheap and abundant food
material. From a geographical point of view, Miri in Malaysia is home to several
goat farms, making it easy to obtain milk for the purposes of producing BPs.
Biocatalysts on the other hand can be sourced from whole cells and/or proteolytic
enzymes of lactic acid bacteria (LAB). LAB are ‘generally regarded as safe’, have a
long history of use in food and have high capacity for breaking down milk into
hydrolysates and peptides (Espeche Turbay et al. 2009; Gupta et al. 2002; Kaushik
et al. 2009). Because most LAB are auxotrophic for several amino acids and as such
are only able to grow in milk by utilizing a complex proteolytic system that
comprises of cell envelope proteinases (CEPs), membrane transporters and intra-
cellular peptidases (Axelsson 2004; Khalid and Marth 1990). During growth in
94 G.K. Gnasegaran et al.

milk, LAB utilize the CEPs to degrade the proteins into peptides and this forms the
basis for using these bacteria for bioactive peptide production. Among the LAB,
Lactobacillus delbrueckii subsp. lactis 313 (LDL 313; or Lactobacillus leichmanii;
ATCC 7830) was chosen for this study due to its high CEP production rate (Zaks
and Klibanov 1985), as well as the option of being able to improve CEP yield by
optimizing the cell growth and fermentation parameters (Agyei and Danquah
2012a; Agyei et al. 2013b, c). The conditions and parameters required to ensure the
optimum growth and CEP production by LDL 313 were gleaned from the literature
(Agyei and Danquah 2012a, c; Agyei et al. 2012, 2013a).
(c) Peptide production, separation and purification
This stage is the key in the entire bioprocess, and so the factors affecting the
efficiency of peptide production need monitoring to ensure good peptide yield at
optimum process conditions and cost. Separation methods were also selected and
optimized to remove desired peptide products from other components. This is
usually followed by a final step of peptide purification to guarantee product quality.

(a) T = 44◦C T = 40◦C

pH = 5.7 pH = 6.2

E-1 E-2
Water vapour + buffer + nutrients Milk
E-5 Pure peptide slurries

Lactobacillus cells
Lactobacillus Cell (enzyme)

Crude peptide slurries



Pure peptide powder

Used Milk

(b) T = 44◦C
pH = 5.7 T = 40◦C
pH = 6.2
Water vapour + buffer + nutrients E-1 Milk
E-5 Pure peptide slurries

Lactobacillus cells
Lactobacillus Cell (enzyme)

Crude peptide slurries



Used milk + Crude peptide Pure peptide powder


Lactobacillus cells

Used milk

Fig. 2 Process flow diagram (PFD) of a batch process for bioactive peptide production: a using
the first method and b using the second method
4 Process Development for Bioactive Peptide Production 95

For this study, we employed two processes at this stage for the separation of the
peptides. The first method involved employing a series of membrane filtration
processes to remove the desired peptide product as captured in Fig. 2a, while the
second method involved the use of a single-membrane filtration unit (isolating the
bacterial cells by centrifugation, followed by further filtration for the crude peptide)
as shown in Fig. 2b.

2.2 Economic Analysis Methodology

In order to carry out the economic assessment, it was necessary to set a production
rate to a target set of consumers. As highlighted earlier, the project was carried out
to meet the medical needs of Malaysia’s hypertension patients. A back-calculation
approach was used to work out the amount of raw material required and the eco-
nomic assessment of the entire process in order to determine the net profit per
annum. Also, in order to calculate the amount of enzyme required to break down
the specified amount of protein, the enzyme-to-substrate (E:S) ratio needed to be
recognized. Knowledge of the E:S ratio, the amount of substrate (protein) and the
conversion value enabled us to estimate the enzyme requirement in order to achieve
the targeted production rate of BPs. This in turn assisted us to estimate the microbial
growth rate that can give the required amount of enzyme, as well as the number of
bioreactors needed for the cell growth and peptide production. A schematic of the
quantitative economic analysis conducted is given in Fig. 3.

Setting Production Identifying Amount Identifying Amount Identifying Amount

Rate of Peptide of Protein needed of Enzyme needed of Cell needed

Identifying Amount Costing of

Identifying Amount
` of Bioreactors Equipments and
of Milk needed
needed Utilities

Fig. 3 Procedure for quantitative economic analysis

96 G.K. Gnasegaran et al.

3 Results

As mentioned in the previous section, two different methods were used for batch
production of bioactive peptides. The first method has been demonstrated in Fig. 2a
In the first vessel (E-1), fresh, mature Lactobacilli cells were introduced as feed.
The pH was adjusted to 5.7 by introducing a specified amount of buffer. The
temperature was adjusted to 45 °C (from 44 °C) by adding a vapour stream.
Nutrients were then added to aid the growth of Lactobacilli cells. Once the cell
growth profile and CEP production were optimum (usually at the late exponential
phase), cells were harvested and transferred to the vessel E-2, which is a bioreactor
where the actual peptide production occurred. The CEPs on the Lactobacillus cell
surface interact with milk proteins to produce peptides at optimum process con-
ditions (pH = 6.2 and T = 40 °C). The two procedures, i.e. enzyme cultivation and
protein breakdown, could be operated simultaneously owing to the marginal dif-
ference in their process-operating conditions. Agitation was provided through mixer
to promote the process by enhancing the cell contact with the milk medium. In the
third stage (E-3), the product from the bioreactor E-2 was isolated using ultrafil-
tration (the most suitable separation method), where the milk, peptides and
Lactobacilli cells were fed to the membrane surface in a tangential feed filtration.
The tangential force eventually led the Lactobacilli cells and peptides to be scraped
off from the membrane surface. The spent milk (with most of the proteins fully
utilized) flowed through the membrane and could not be recycled. The Lactobacilli
cells and peptides from E-3 were separated at E-4 in a dead-end filtration step. The
size difference between the bacterial cells (in microns) and the peptides (nanoscale)
was the principal motive behind a physical separation rather than a chemical
approach. The retentate (i.e. Lactobacilli cells) would then be recycled to the feed
stream for the next batch of peptide production. The filtered (or crude) peptide
underwent a series of chromatographic and other purification procedures to give a
purified final product as illustrated in Fig. 4.
The second method is shown in Fig. 2b. Here, E-1 and E-2 are bioreactors that
work exactly the same way as in the previous design. However, E-3 was replaced
with a centrifugal cell separator to extract the Lactobacilli cells as they possessed
the most significant densities at that point. In E-4, ultrafiltration membrane was
used to filter out the peptide from the undesired waste. Furthermore, since the crude
peptides were deemed purer from previous steps, only ion-exchange purification
was used for the final purification stage. Lastly, E-6, a spray dryer, was used to
remove the water, leaving the peptides in a powdered form.

3.1 Production Rate

One of the objectives of the current study has been to meet the medical needs of
hypertensive patients in Malaysia. According to statistics, about 1 out of every 7
4 Process Development for Bioactive Peptide Production 97

Fig. 4 Process steps in

conventional peptide
purification for commercial

Malaysian citizens suffers from high blood pressure. Considering the total
Malaysian population to be 30,257,000, that gives us a total of 4,322,429 hyper-
tensive patients. Now, a single patient suffering high blood pressure needs 300 mg
of peptides per day [provided that a patient must be administered with 2 antihy-
pertensive capsules per day with a peptide content of 150 mg/capsule] (Vertuani
et al. 2011). That gives a daily peptide production rate target of
(4,322,429  300 mg) = 1296.729 kg/day to suffice the needs in Malaysia.

3.2 Protein, Milk and Bioreactor Requirement

Milk is predominantly 80−90% water (reassured by the fact that both milk and
water have similar densities), with the remaining 10% consisting of the major
98 G.K. Gnasegaran et al.

nutrients (fats, carbohydrates, proteins, minerals and vitamins). The protein com-
position of milk varies depending on the source: cow—3.5%, buffalo—3.75% or
goat—4% (Agyei and Danquah 2011). It may be noted that for the current study,
we are aiming at an industrial-scale bioreactor, with a typical size of 10,000 L
(Reboredo-Rodríguez et al. 2014). Considering that usually 75% of the bioreactor is
filled (Cicerale et al. 2011), that gives us a total of 7500 L of milk and cell mixture
that can be processed in a batch. Assuming that 3.5% of milk is made up of protein,
only (0.035  7500 L =) 262.5 L (or 0.262 m3) of protein has to be broken down
into peptides in a single batch. If we take the density of protein to be 1350 kg/m3
(Agyei and Danquah 2011), then the mass of protein present in a batch reactor at
any specific moment is 354.375 kg.
An approximate estimate of the total number of bioreactors required has been
made from the expected daily peptide production rate (as estimated in the previous
section), based on the type of milk (cow/goat/buffalo), and is shown in Table 1. The
results suggest that the number of bioreactors can be reduced from 6 to 5 if goat or
buffalo milk is used instead of the cow milk, owing to the difference in their protein
compositions. Also, if goat or buffalo milk is used as the protein source, it is
possible to obtain the same amount of substrate (protein) using lesser amount of
milk. This results in a significant reduction in the amount of milk that has to be
processed in order to meet the desired peptide production rate. Considering the
price of a single bioreactor to be around USD 200,000 (Mancebo-Campos et al.
2014), the total estimated cost for 5 bioreactors will be USD 1,000,000. Also, since
milk is priced at USD 0.99/L [Bureau of Labor Statistics, US Department of Labor
(2014)] and 39,986.24 L of milk is needed daily, the cost of raw materials to
produce 1 batch of product is USD 39,586.40. Considering that the average market
value of bioactive peptides is USD 88.5/g (Kalogeropoulos and Tsimidou 2014),
the anticipated value of peptide produced on a daily basis is USD
88.5/g  1,296,729 g = USD 114,760,516.5.

Table 1 Number of bioreactors required based on the type of milk

Cow Goat Buffalo
Production of peptide (kg/day) 1296.729 1296.729 1296.729
Before purification (93.5% recovery) kg/day 1386.875 1386.875 1386.875
Before filtration (93% recovery) kg/day 1491.264 1491.264 1491.264
Protein needed (78.93) conversion (kg/day) 1889.35 1889.35 1889.35
Density of protein (kg/m3) 1350 1350 1350
Protein needed in volume (m3) 1.399 1.399 1.399
Percentage of protein in milk (%) 3.5 3.75 4
Milk needed in volume (m3) 39.986 37.32 34.988
Milk needed in volume (L) 39,986.24 37320.49 34,987.96
Total cumulative tank size required (L) 53,314.99 49760.66 46,650.62
Number of reactors needed 5.331 (6) 4.976 (5) 4.665 (5)
4 Process Development for Bioactive Peptide Production 99

3.3 Enzyme Requirement

Guo et al. (2009) have optimized hydrolysis conditions for the production of
angiotensin converting enzyme (ACE)-inhibitory peptides using response surface
methodology from whey protein using crude protease preparation from
Lactobacillus helveticus LB13. Hydrolysis conditions for optimal ACE inhibition
(78.93% inhibition) were found to be at an enzyme-to-substrate ratio of 5 (Ozan
Nazim et al. 2012). Once the enzyme-to-substrate ratio is known, the amount of
enzyme required in order to achieve the target production rate of bioactive peptides
could be estimated from the substrate amount and the conversion factor. In this
case, the specific cumulative yield of the enzyme required from a cluster of cells
was estimated to be 377.87 kg.
The relativity of enzyme requirement and amount of protein is shown in Fig. 5,
which suggests a linear relationship between the amounts of enzyme and protein
required (i.e. 0.2 kg enzyme/kg protein). It may be noted that if the amount of
enzyme is reduced, the conversion will still take place but with a slower projected
rate of conversion. This, in turn, would reduce the number of batches, which would
directly affect the annual rate of peptide production.

3.4 Influence of Protein on Peptide Production

It was interesting to note the difference in the amount of product if all the 3 different
types of milk were to be used in reactors of identical size, as shown in Table 2.

Fig. 5 Enzyme requirement 450

Enzyme Requirement (kg/day)

based on the amount of 400

protein 350
0 500 1000 1500 2000 2500
Amount of Protein (kg/day)

Table 2 Peptide production Cow Goat Buffalo

as function of protein
Total reactor volume (L) 50,000 50,000 50,000
Working volume (L) 37,500 37,500 37,500
Protein (kg/day) 1313 1406.25 1500
Peptide (kg/day) 1036 1109.953 1183.95
100 G.K. Gnasegaran et al.

Peptide Recovery from different source

Protein (kg/day)

1000 1050 1100 1150 1200
Peptide (kg/day)

Cow Goat Buffalo

Fig. 6 Peptide production as function of protein

Table 3 Enzyme requirement based on production rate

Peptide production (kg/day) 1000 2000 3000
Protein (kg/day) 1457.013 2914.025 4371.038
Volume of milk required (m3) 30.836 61.672 92.509
Number of bioreactors required 4.111 8.223 12.334
Enzyme required (kg/day) 291.402 582.805 874.208

Assuming a 75% maximum allowed working volume, it was found that 5 biore-
actors sized at 10,000 L each should be used to produce the desired amount of
peptide using goat and buffalo milk.
Figure 6 supports the previous observation where the type of milk used has a
direct influence on the peptide production. From the figure, a higher rate of peptide
production can be expected when using buffalo milk in 5 bioreactors compared to
the two other milk sources. The gradient of the line in Fig. 6 was found to be
1.267 kg protein/kg peptide. So, in case there is a rise in the market demand of
peptides, the manipulated variable would be the amount of protein.

3.5 Influence of Enzyme on Daily Peptide Production

The enzyme requirement for the daily peptide production rate is presented in
Table 3. When the peptide production rate was varied from 1000 to 3000 kg/day,
there was an increase in the enzyme mass that had to be introduced to achieve the
production target. This was anticipated since the amount of protein increases as well
and this directly correlates with the peptide production.
4 Process Development for Bioactive Peptide Production 101

Enzyme requirement against Production rate

Mass, kg/d

2000 1000 kg/d Peptide
1500 2000 kg/d Peptide
3000 kg/d peptide
Production of Protein Enzyme
peptide (kg/d) needed
(kg/d) (kg/d)

Fig. 7 Enzyme and protein requirement based on the production rate of peptide

From Fig. 7, since the enzyme requirement is linearly dependent on the peptide
production, the rate of enzyme requirement per kilogram of peptide can be esti-
mated from the ratio of enzyme to peptide. The gradient of the linear plot was found
to be 0.291 kg enzyme/kg peptide. However, in response to an increase in the
peptide production rate, the number of bioreactors used would also vary. As a
consequence, an optimal production rate needs to be set based on the consumer

3.6 Capital Cost

Capital cost is the preliminary cash outflow required to build the process operations.
Essentially, it involves the major plant equipment.

3.6.1 Lactobacillus Cell

The lyophilized bacterial strain would be supplied in a glass ampoule and carries a
price tag of EUR 75 (or USD 95) from DSMZ, Germany (2014 price). The pellet
within the ampoule (200 µl aliquot) consists of skimmed milk (protectant) and
vegetative cells of the strain. Therefore, the whole pellet needs to be resuspended
during the initial reactivation of the strain and transferred to a small volume of
medium (5–10 mL), to ensure that the number of cells is sufficient for
102 G.K. Gnasegaran et al.

3.6.2 Centrifugal Cell Separator

The centrifugal cell separator removes the Lactobacillus cells prior to membrane
filtration in order to recover and recycle these enzyme-rich cells into the next batch
of the process. The batch centrifuges could process at most a few litres during a
particular spin cycle and thus are not suitable for industrial-scale protein purifica-
tion, which usually requires processing of several hundreds or thousands of litres of
the crude extract. Industrial-scale centrifugation is normally achieved using
continuous-flow centrifuges. It has been observed that most microbial cells are
sedimented by batch centrifugation by applying a centrifugal force of approxi-
mately 5000 g for 15 min (Mahugo Santana et al. 2009). So, in order to meet our
target, the mixture should leave the bioreactors at a rate of 39,986.24 L/day (or
1666 L/h). However, the largest centrifuge currently used in the industry can only
process at a rate of 200 L/h (Mahugo Santana et al. 2009). The specifications of this
equipment are as follows: process volume: up to 8 L; rotor capacity: 200 L/h; and
speed: 5000 rpm. To achieve separation in a short period of time, more than 1
centrifuge is needed. Using 8 centrifuges at once will increase the capacity to
1600 L/h. Though a further increase in the number of centrifuges would reduce the
cell separation time, it would also drastically increase the capital cost, utility cost
and complicate equipment management. A total of 8 units will cost up to USD
140,000, considering USD 17,500 per unit (Mahugo Santana et al. 2009). The total
time taken to process 1666 L/h was estimated to be 1.04 h.

3.6.3 Membrane (for Lactobacillus Cell Separation)

According to Zacharof et al. (2013), the current separation and purification tech-
niques (such as chemical precipitation, solvent separation and chromatographic
techniques) employed to obtain highly potent and purified Lactobacilli are
expensive, have limited scalability and offer low recovery. Since the maximum
probable cell recovery varies from 64 to 68% using membranes (Singh et al. 2008)
to almost 100% using the cell centrifugation technique, any further cost analysis on
cell recovery method is redundant.

3.6.4 Membrane (for Peptide Separation)

In other research works, the concentration and purification of blue whiting peptide
hydrolysates by membrane processes have been studied, mainly to evaluate the
membrane process performance. Past studies (Gul et al. 2015) have shown that
steady fluxes were reasonable (100 L/h/m2 at 12 bars and 15 °C) using polysul-
phone (PS) membrane of MWCO 20 kDa with a water permeability (Lp) of 351
L/h/m2. These values were satisfactory and could almost be doubled if ultrafiltration
was carried out at 40 °C. Fortunately in our studies, the temperature leaving the
centrifugal cell separator was expected to be within the range of 15–40 °C.
4 Process Development for Bioactive Peptide Production 103

Assuming that the content that needs to be separated loses heat to the surroundings
after leaving the bioreactor, the mixture leaving the bioreactors was expected to
reach a thermal equilibrium with the surroundings. In other words, the mixture was
expected to be at 27 °C (filtration rate 165 L/h/m2; via interpolation) upon mem-
brane filtration. The appropriate membrane thickness is 0.2 mm (Shurtleff and
Aoyagi 2010). Hence, setting a basis of 1 h would allow us to calculate the area,
volume and cost of the total membrane required. The amount of mixture that
needed to be filtered was 39,986.24 L, and the target time for the filtration process
was set as 1 h. Knowing the time requirement and the amount of mixture to be
separated, the area requirement (242.34 m2) was found based on the known fil-
tration rate, and subsequently, the volume (product of the area with the membrane
thickness) of the PS resin could be identified. From the volume and density of
polysulphone (1240 kg/m3), the mass of polysulphone was estimated to be 62 kg.
Since polysulphone is priced at USD 300/kg, the total membrane cost was USD

3.6.5 Ion-Exchange Purification

The required rate of peptide purification was 1386.88 kg/day (57.76 kg/h). The size
of the ion-exchange purification column was picked based on the volumetric flow
rate (46.2 L/h), which is actually the ratio of the mass flow rate (57.76 kg/h) to the
average peptide density (1252 kg/m3). The chosen industrial-scale column had a
capacity of 50 L/h and met the total requirement needed to achieve the target purity.
The specifications for the ion-exchange chromatography column were as follows:
size: small; diameter: 200 mm; maximum bed volume: 10 L; and maximum flow
rate: 50 L/h. This specific model is priced at USD 63,500 by Novasep, France
(2014 price).

3.6.6 Spray Dryer

The daily rate of drying peptide product after purification is 1296.729 kg/day (or
54.03 kg/h). This chosen industrial-scale spray dryer is sized at an operating rate of
150 kg/h and is priced at USD 35,000 by SPX FLOW, Soeborg, Denmark (2014

3.7 Utility Cost

For the entire process, the utilities involved were steam and electricity. The steam
heated up the bioreactors to the required temperature for protein breakdown, while
the electricity was vital in powering up all the process equipment.
104 G.K. Gnasegaran et al.

3.7.1 Steam Requirement

Steam was used to heat up the initial milk mixture from room temperature (25 °C)
to 40 °C in the bioreactor where the bioreaction occurred. A simple heat balance
equation, m1Cp1DT1 = m2Cp2DT2, was used to calculate the steam required to
achieve the target temperature (40 °C) (notations 1 and 2 refer to the milk mixture
and the steam, respectively). The mass of the milk (41,389.65 kg/day) was calcu-
lated from the volume (39.99 m3/day) and density (1035 kg/m3), and the specific
heat capacity of the milk mixture was assumed to be similar to water (the major
milk component). Hence, Cp1 = 4.18 kJ/kg °C, and thus, DT1 = (40 − 25) = 15 °C.
Obtaining m1, Cp1 and DT1 enabled us to calculate the heat load, Q
(2,595,131 kJ/day or 108,130.46 kJ/h), and hence the required mass flow rate of the
steam. Low-pressure steam is available at 200 °C. The expected outlet temperature
of the steam is 120 °C. The average specific heat capacity Cp2 is 2.47 kJ/kg °C.
The mass flow rate of steam was m2 = Q/Cp2 DT2 = 547.22 kg/h. The cost of
steam used is USD 0.105/kg, and since we know the daily mass requirement of the
steam, the total expenditure for steam utility came to USD 1379.

3.7.2 Power Requirement

Centrifugal cell separator and spray dryer require high power consumption. The
power requirement for 1 kg of cells is 0.14 kWh (Celenza 2000). The weight of
Lactobacillus cells was estimated using the weight of protein content in milk.
Multiplying 0.14 with the daily protein content in milk (1889.35 kg/day) would
yield 264.509 kWh/day. Considering that USD 0.9/kWh is the standard price, the
total electricity cost for the centrifuge on a daily basis was (264.509  0.9 =) USD
238.1. For the spray dryer, 40 kWh of power is required to produce 400 kg/h of
powder (APV 2012) or 0.1 kWh for every 1 kg/h. Given that the amount of peptide
desired to be solidified is 1296.729 kg/day (54.03 kg/h), the total power needed to
complete the drying process was 129.67 kWh/day. Hence, the electricity cost for
the drying process was (0.9  129.67 =) USD 116.703. The total electricity usage
on a daily basis was therefore 116.703 + 238.1 = USD 354.8.

3.8 Total Costing and Profit

The total capital cost, taking into account the Lactobacilli cells, bioreactors, cen-
trifuges, membrane, chromatography column and spray dryer, came out to be USD
1,257,195 (Table 4).
The associated cost for steam usage of the plant was USD 1379/day. Assuming
round-the-year (365 days) operation, the total steam utility cost would be
(1379  365 =) USD 503,335. It had also been estimated earlier in this section that
the cost of daily electrical power consumption was USD 354.8. So, assuming an
4 Process Development for Bioactive Peptide Production 105

Table 4 Equipment costing

Equipment Units Unit cost (USD) Total (USD)
Lactobacillus cell 1 95 95
Bioreactor 5 200,000 1,000,000
Centrifuge 8 17,500 140,000
Membrane 1 18,600 18,600
Ion-exchange chromatography 1 63,500 63,500
Spray dryer 1 35, 000 35,000
Total 16 – 1,257,195

operation of 365 days, the total electricity cost would be (354.8  365 =) USD
129,502. Hence, the total annual utility (electrical + steam) cost would be USD
632,837. The raw material cost would only be consisting of milk as it is the only
feed into the bioreactor. Consider 39,986.24 L of milk was used to produce peptide
for 1 batch (5 bioreactors) per day, which would be (39,986.24  365 =)
14,594,977.6 L of milk per year. Taking the milk cost to be USD 0.99/L, the total
cost of milk used in the period of 1 year was (14,594,997.6  0.99 =) USD
14,449,048. The market value of peptide after ion-exchange chromatography (with
74% purity) is USD 88.5/g (Kalogeropoulos and Tsimidou 2014). Since the daily
peptide production rate was 1296.729 kg/day, the plant would be able to produce
(1296.729  365 =) 473,306.085 kg on an annual basis. Therefore, the total value
of peptide produced in a year would be (47,330,608.5  88.5 =) USD
41,858,307,460 (approximately USD 42 billion). The annual revenue can also be
expressed as difference between the peptide value and the total sum of raw materials
and utility costs. According to the estimated revenue, the invested equipment
capital could be recovered quickly with a considerably short payback period for this
process. The total time required for a single batch of production, which is the
summation of time required for bioreaction (15.5 h), cell separation using cen-
trifuge (1.04 h), membrane separation of peptide (1 h), purification using chro-
matography (1 h) and drying (1 h), came out to be 19.54 h. Therefore, it is safe to
assume that (a) only 1 batch can be produced daily and (b) this process is eco-
nomically feasible (based on the annual revenue).

4 Discussion

The production of the biocatalysts (CEP on Lactobacillus cell surface) could pro-
ceed simultaneously with the principal process of protein breakdown only if the
temperature and pH requirements are similar for both stages. Advantageously, this
was the case when Lactobacillus cells were used to produce the enzyme sources.
While a temperature of 45 °C and pH = 6 have been found to be optimal in
promoting the enzyme growth (Hettiarachchy and Kalapathy 1998), a temperature
and pH of 40 °C and 6.2, respectively, have been found to work best for the
106 G.K. Gnasegaran et al.

proteolytic activity of the enzymes (Hughes et al. 2011). This helped in lowering
the cost and reducing the number of unit operations.
Also, both the separation methods in the process designs were expected to work
efficiently. Therefore, only a quantitative analysis could decide the most suitable
separation method that should be used. A quantitative analysis was required to
study the total time taken for both the separation methods and weighing the cost of
centrifugal cell separator against a regular membrane separation unit. The purity of
the final product through both processes should also be evaluated in order to select a
straightforward optimal process design. The purification methods used for the
product (crude peptide) for both the designs varied as the design involving the
centrifugal cell separator was expected to yield a purer crude peptide. This reduced
the initial steps in the series of chromatographic methods for peptide production by
incorporating only ion-exchange chromatography. However, this would have only
been feasible if the economics of a centrifugal cell separator was better than the
series of membranes as previously shown in Fig. 2a, b. Unfortunately, since prior to
the economic assessment, cell recovery through membrane filtration was found to
be only 68%, this design process (i.e. Fig. 2a) was less favourable than the other
process (Fig. 2b) that used centrifugation to recover Lactobacilli cells.
The rate of increase in enzyme requirement was found to be 0.2 kg enzyme/kg
protein, a much anticipated result considering that an increase in substrate would
certainly necessitate the addition of enzyme to break down the additional amount of
protein. The correlation between peptide production and enzyme stood at 0.2914 kg
enzyme/kg peptide. Similarly, an increment in the peptide production rate would
certainly require an addition of enzyme to bioreact with the protein in order to
achieve the expected production rate. The relation between protein requirement and
peptide production was studied, and a gradient of 1.267 kg protein/kg peptide was
obtained when the variables were represented graphically in Fig. 6.
The annual process revenue was found to be significantly larger than the total
combined cost of raw materials and utility, mainly due to the profoundly higher
market value of peptide as compared to the cost of the milk. As a consequence, the
annual profit of the peptide process was overwhelming. There were three principal
reasons for the elevated profit: (a) a significant price/value gap between the raw
material (milk) and the product (peptide), resulting in a noteworthy difference
between the cost and profit; (b) the steep, preset production rate considering the
needs of an entire nation [peptide production rate was set considering that every
desiring citizen is keen to spend on peptides to meet their medical needs, and only a
proper market survey would allow us to set a proper rate of production]; and (c) the
precision in the estimation of profit that may vary in reality, since only the major
equipment involved in the process was considered in the cost and utility calcula-
tions. Also, the economic assessment was not done in detail considering the land
cost, labour cost, salaries for professionals, pipe and fitting including minor
equipment (pumps) and proper waste management system. Reduced and more
logical revenue is expected when these aspects are taken into consideration. In order
to begin operations immediately, purchasing 1 pellet of Lactobacilli cells would not
be sufficient and larger volumes of cells are required to be purchased as part of the
4 Process Development for Bioactive Peptide Production 107

capital cost. Nevertheless, if time is not an issue, there would be sufficient time to
subculture/scale up the cells to bigger volumes during the construction period of the

5 Conclusions and Future Outlook

Bioactive peptide production through microbial fermentation may be a good

alternative to the chemically synthesized, sophisticated and expensive modern
drugs. There is little doubt that the process is feasible in an industrial scale to meet
the quantity demand in the medical field. This process may actually be conceptu-
alized and executed in reality aided by an appropriate economic assessment. An
extensive scale preparation of bioactive peptide processing utilizing microbial
fermentation has not been carried out so far. Considering the unprecedented profits
that could be anticipated as far as well-being is considered, the nutraceutical
business guarantees extraordinary attention. Results from the current study
demonstrate that the production objective might be met at a pace of 1.267 kg
protein/kg peptide. The production rate of enzyme was chosen keeping in mind the
medicinal needs of hypertensive patients in Malaysia. Amounts of protein and
enzyme were controlled by investigating the substrate-to-enzyme ratio. The ACE
restraining rate was utilized to focus on the rate of breakdown of proteins to
peptides, and consequently, the quantity of bioreactors utilized was chosen based
on the amount that must be prepared to meet the production target. The necessary
rate of enzyme expansion was found to be 0.2 kg enzyme/kg protein, and the
connection between peptide production and enzyme was 0.2914 kg enzyme/kg
peptide. The total annual profit of the process was significant and valued at USD
41,858,810,790. This implicates that taking into account probable minor inaccu-
racies during estimations, there is no doubt that the process is feasible and viable
and, more importantly, profitable.
Further research is required to find a cost-effective alternative for the enzyme
source (Lactobacilli cells) as it may lower the cost of production significantly. Also,
the batch process designed in the current work is time-consuming owing to the large
process operation time for each stage. Hence, a continuous production method is
desirable to speed up the bioactive peptide production process. The use of immo-
bilized cells should also be considered since immobilized biocatalysts afford the
option of being recycled, which in the end further reduces cost. The need of the
hour is a laboratory-scale research on microbial fermentation using Lactobacillus
delbrueckii free cells and immobilized forms. This is to ensure a smooth process
flow as expected for an industrial-scale operation. Moreover, the peptides produced
as a result of this laboratory experiment should be closely monitored since there are
several groups of bioactive peptides and different groups of peptides are used for
different medical purposes. There should be a study that would focus on finding
ways to improve the conversion rate of proteins to peptides without compromising
108 G.K. Gnasegaran et al.

on the quality of product. Last but not least, process optimization is another avenue
that would reduce the utility cost and hence needs further attention.


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Chapter 5
Comparison of Natural Extraction
and Recombinant Mussel Adhesive
Proteins Approaches

J.J. Castillo, B.K. Shanbhag and L. He

1 Introduction

Mussels possess strong underwater adhesion on different substrata, and this adhe-
sive property is mainly attributed to mussel adhesive proteins (MAPs) produced by
the mussel byssus (Lee et al. 2011). Early studies and application of mussel
adhesive proteins rely on isolation of MAPs via natural extraction method.
However, the low productivity of this method has prompted researchers to explore
other production approach such as recombinant production. Recombinant produc-
tion of MAPs has been an active field which extends to fusion MAP with
peptide/protein for additional functions. After an overview of mussel proteins, this
chapter focuses on two production methods of MAPs, natural extraction and re-
combinant production.
Modifications and formulations of MAP-based products are essential for their
performance. Particularly, for recombinant MAPs, appropriate modification is
critical to confer adhesive properties (Zhong et al. 2014). In this regard, we will
give a few examples on modification and formulation of MAP, including enzyme
modification, metal-mediated cross-linking, and coacervation. At the end of this
chapter, perspective on two production methods and literature for further reading
are provided.

J.J. Castillo  B.K. Shanbhag  L. He (&)

Department of Chemical Engineering, Monash University, Wellington Road, Clayton, VIC
3800, Australia

© Springer International Publishing AG 2017 111

M. Puri (ed.), Food Bioactives, DOI 10.1007/978-3-319-51639-4_5
112 J.J. Castillo et al.

2 Overview of Mussel Proteins

Mussel byssus, the collection of threads each with plaques at the tip (Fig. 1),
consists of synergistic parts that contribute to its strong adhesive property in a
marine environment. Mussel byssus can be subdivided into four parts: root (base of
mussel foot), stem, threads, and attachment plaques (Fig. 1). Among them,
attachment plaque plays a key role to provide adhesiveness to different strata,
natural or man-made, including rocks, wood, metals, concrete, polyvinylchloride
(PVC), polymethylmethacrylate (PMMA), and polytetrafluoroethylene (PTFE)
(Hongbo Zeng et al. 2015).
Different species of mussels have been studied and compared for their mussel
foot proteins (mfp) or mussel adhesive proteins (MAP) (From here on, the terms
“mfp” and “MAP” will be used interchangeably.). These species include Mytilus
edulis (Blue mussel), Mytilus galloprovincialis (Mediterranean mussel), Mytilus
californianus (California mussel), Mytilus coruscus (Sea Mussel), Perna viridis
(Tropical Green mussel), and Limnoperna fortune (Golden mussel) (Lee et al.
2011). It is estimated that there are 25–30 proteins in Mytilus byssus, and eight
proteins have been identified in byssal plaques (Lee et al. 2011). Among them, five
proteins (mfp-2, mfp-3, mfp-4, mfp-5, and mfp-6) are uniquely located in plaques
and are found in other parts of mussel. Other proteins including mfp-1 are not
confined to plaques.
These mfps have unique amino acid composition. The most abundant amino acid
present in mfp is tyrosine which plays an important role in adhesive property.
Through enzymatic modification, tyrosine is converted to DOPA which is char-
acterized with catechol functionality (Lee et al. 2007). Another amino acid, lysine,
also contributes to adhesiveness. One recent study has suggested a synergistic effect
of adjacent catechol from DOPA and lysine in bioadhesion of mussels (Maier et al.
2015). In salt water, lysine residues displace hydrated cations from the mineral or
oxide-rich surfaces thus leaving the underlying oxides free to bind DOPA.

Fig. 1 Mussel byssus

subdivided into four parts:
foot, stem, threads, and
5 Comparison of Natural Extraction and Recombinant Mussel … 113

Different to other mfps, mfp-1 is the only one located in both byssal thread and
plaque regions of mussels. Four mfp-1 proteins from different species of mussels
are available in UniProt: Mytilus edulis (Q25460), Mytilus galloprovincialis
(Q27409), Mytlilus coruscus (Q25434), and Perna viridis (A1X158). All of them
have repetitive decapeptides and basic isoelectric points (pIs) (10–11). Proline is the
most abundant residue of mfp-1 followed by lysine and tyrosine in Mytilus genus.
Typical post-translational modifications are hydroxylation of proline to
4-hydroxyproline, tyrosine to 3′,4′-dihydroxyphenylalanine (DOPA), and trypto-
phan to 7′-hydroxytryptophan.
Mfp-2 from three species can be found in UniProt: Mytilus edulis (Q1XBT8),
Mytilus galloprovincialis (Q25464), and Limnoperna fortune (A0A024FCK3).
Mfp-2 from Mytilus galloprovincialis is composed of 11 repeats of epidermal
growth factor (EGF)-like sequences, and tyrosine residues near N- and C-termini
provide sufficient exposure for their conversion to DOPA. Cysteine and lysine
dominate the three mfp-2 variants with major post-translational modification,
including the formation of disulfide bonds.
Mfp-3 is the smallest plaque protein with at least 20 variants. Up to date,
sequences of mfp-3 from four mussel species have been listed in Uniprot: Mytilus
edulis (Q9NAV2), Mytilus californianus (Q2TCL0), Mytilus coruscus (D3JVE6),
and Mytilus galloprovincialis (Q9GUX8). All mfp-3 sequences are rich in glycine
and tyrosine and ranked the second highest DOPA-containing protein in byssal
Compared to other types, limited sequence information is available for mfp-4.
Only one sequence from Mytlilus californianus is available in Uniprot (A1XF84). It
is characterized by 36 repeats of a decapeptide and 16 repeats of an undecapeptide.
The sequence is dominated by histidine and valine. Tyrosine is only present in a
small fraction, 2.2% of amino acid composition. Due to the low DOPA percentage,
it has not been extensively studied for mfp adhesion.
Mfp-5 is the second smallest protein of mfps. There are three sequences avail-
able in Uniprot: Mytilus coruscus (C7ENH8), Mytilus californianus (Q1W2I9), and
Mytilus galloprovincialis (Q6QZR3). Post-translational modifications such as for-
mation DOPA and O-phosphoserine are present in more than a third of the residues.
Tyrosine residue dominates (*27 mol%) the sequence, making mfp-5 the highest
DOPA-containing mfp. Mfp-5 from Mytilus californianus has 30 mol% DOPA of
amino acid residues.
Two mfp-6 sequences coming from two species Mytilus californianus (Q0H216)
and Mytilus coruscus (D3JVG0) are listed in Uniprot. Tyrosine and glycine are the
two major residues. However, DOPA content is lower (<5 mol%) compared to
other mfps. Phospho-O-serine is also present as post-translational modification at
around 5 mol%.
114 J.J. Castillo et al.

3 Mussel Adhesive Protein-Based Products and Their


3.1 Products Based on Naturally Extracted MAP

Mytilus edulis or Brown mussel is the most utilized species for extracting mussel
adhesive proteins, and foot protein from Mytilus edulis is denoted as mefp. Among
the companies listed in Table 1, four products are produced by natural extraction.
Most, if not all, of commercially available extracted MAPs are based on mefp-1 due
to its high percentage in the mussel. These products are delivered at a concentration
range of 1–10 mg/mL in acidic buffer solutions either in 5 v/v% acetic acid or 1 v/v
% citric acid. MAPs are only stable at acidic pH as DOPA is spontaneously oxi-
dized to dopaquinone at neutral or alkali conditions.
The commercial products share common applications including cell immobi-
lization, in situ hybridization, immunohistochemistry, immunoassays, and
microinjection. One manufacturer, ACROBiosystems, has suggested two ways to
apply MAPs: hand-spreading and adsorption. Hand-spreading is desirable when
coating only a portion of glass or other substrates. The method involves the use of a
mechanical tool such as glass rod or pipette in order to spread the MAP. Once the
acidic buffer evaporates, a film of MAP remains. Another method, the adsorption
method, allows neutralizing pH of the MAP solution. Adsorption commences and

Table 1 Commercial mussel adhesive protein-based products*

Brand name Company Application MAP Production
component approach
AdheraCell Genex Corp Bioadhesive mefp-1 Recombinant
Cell-Tak™ BD Biosciences Cell or tissue mefp-1 and Natural
Clontech/Corning immobilization mefp-2 extraction
MAPcell Sigma-Aldrich/Biopolymer Cell or tissue mefp-1 Natural
and MEFP-1 Products of Sweden immobilization extraction
MAPcomp Biopolymer Products of Anti-corrosion mefp-1 Natural
Sweden treatment extraction
MAPTrix™ Kolloidis Biosciences, Inc. Bioadhesive mefp-1,2,3,4 Recombinant
Protein extracellular and 5
Products matrix, 3D cell
Mussel abcam® Cell or tissue mefp-1 Recombinant
adhesive full immobilization
Mussel ACROBiosystems Cell or tissue Unspecified Natural
Adhesive immobilization extraction
PureBond™ Columbia Forest Products Wood adhesive DOPA Biomimicry
mefp—Mytilus edulis foot protein
5 Comparison of Natural Extraction and Recombinant Mussel … 115

proceeds spontaneously after adjusting the pH to the optimum pH range of 6.5–8.0.

The final film is thin and more uniform than the film produced by hand-spreading.
Among these different products, MAPcomp is an interesting one (Sababi et al.
2012). This product has a unique application as an anti-corrosion agent for treat-
ment of carbon steel. MAPcomp comprises of a composite film of nanosized cerium
oxide particles and MAP that provides corrosion resistance comparable to stainless
Although several products have been developed by natural extraction method,
the cost of production is high due to low extraction yields. An early study estimated
that 30,000 mussels were required to produce 1 g of pure MAPs (Morgan 1990).
A recent study estimated that around 10,000 mussels were still needed for 1 g
protein with the improvement in extract methods (Cha et al. 2008).

3.2 Recombinant MAP-Based Products

The high cost and low productivity of extraction method of MAPs prompted a few
companies to explore recombinant production approach. AdheraCell from Genex
Corporation was one of the early recombinant MAP products, but this product has
been discontinued. Currently, Abcam® produces recombinant mefp-1 using a full
length amino acid sequence (Uniprot Q25460). Applications and usage of this
recombinant mefp-1 are similar to biomedical applications of naturally extracted
MAPs mentioned above.
Kolloidis BioSciences Inc. markets formulations containing different Mytilus
edulis proteins, including mefp-1, mefp-2, mefp-3, mefp-4, and mefp-5 that are
expressed in E. coli. Products are formulated in different kits, targeting different
applications. MAPTrix™ Adhesive Kit is composed of either recombinant fusion of
mefp-1 and mefp-5 or mefp-1, mefp-3, and mefp-5. MAPTrix™ Coacervate Kit
produces an injectable adhesive when its two components, tyrosinase-pretreated
MAP and hyaluronic acid, are mixed. MAPTrix™ ECM is the first combinatorial
synthetic extracellular matrix library and contains recombinant MAP fused to dif-
ferent extracellular matrix (ECM) peptides (e.g., RGD).

3.3 Application of Mussel Adhesive Proteins

Mussel adhesive proteins are innately biodegradable and have wet adhesive prop-
erties. Unlike commercial chemical-based glues, MAPs function under saline and
hydrated environment, a condition similar to wound sections. The wound sealing
ability of mussel-mimetic adhesives has been tested for repairing punctured fetal
membranes, and it was found that the mussel adhesives has a sealing quality
comparable to the commercial sealant (Kivelio et al. 2013; Haller et al. 2011).
Mehdizadeh et al. (2012) developed an injectable citrate-based mussel-inspired
116 J.J. Castillo et al.

tissue adhesive for wound closure. This injectable mussel glue provided 2.5–8.0
times higher adhesive strength compared to commercial fibrin glue. Remarkably,
the mussel glue was able to stop blood loss without causing inflammation and was
degradable under in vitro tests. Another mussel-inspired glue was used on a porcine
skin (Zhang et al. 2014), and it had 4 times adhesive strength compared to fibrin
while having a fast curing time, no cytotoxicity, and degradability. Replacing metal
amalgam in dental procedures was attempted using MAPs for reconnecting colla-
gens in damaged tissues, and reversible bridging between collagen and mfp-3 was
observed (Martinez Rodriguez et al. 2015).
Commercially available MAP-based products have been used in tissue engi-
neering. The coating ability of mfp-1, the only protein located in the cuticle, has
been utilized for surface functionalization of different substrates for cell immobi-
lization and proliferation. Recent applications include bone tissue engineering
(Hong et al. 2012), fusion of MAPs and a short peptide (RGD) from fibronectin for
better cell attachment (Choi et al. 2010), and MAP-based hydrogels (Kim et al.
Nanoparticles (NP) based on MAPs have been used as drug delivery systems.
Based on complexation reaction of recombinant MAP and Fe3+, NPs were prepared
for drug delivery controlled by pH value (Kim et al. 2015). Hwang et al. explored
the potential of recombinant fp-151 as gene delivery vehicle, showing fp-151 is as
efficient as the widely used transfection agent, Lipofectamine™ 2000 (Hwang et al.
2009). Shin et al. (2015) developed a hyaluronic acid-catecol (HA-CA) hydrogel
for cell therapy without inherent cytotoxicity and with added adhesive property,
which is essential for the localization of transplanted cells. The HA-CA hydrogel
was successfully applied onto liver and heart surfaces with an attractive
Immobilization of cells using MAPs was also used in the development of
biosensors. One study developed a whole cell array biosensor for the detection of
neurotoxic organophosphorus compounds. Recombinant fp-151 efficiently immo-
bilized E. coli cells which remained stable for 28 days with 80% activity retention
(Kim et al. 2013). Another study modified metal electrodes for the detection of
glucose by adsorbing MAP films onto gold, platinum, and glassy carbon electrodes
(Saby and Luong 1998). After MAP oxidation, glucose oxidase was immobilized
on the modified electrodes, and the resulting MAP–metal enzyme electrode was
used to sense glucose.
Wilke et al. examined the coating ability of mefp-1-polymer conjugate on
stainless steel (Wilke and Börner 2012). It was found the Mfp-based coating was
stable and showed an attractive antifouling property. The potential of MAP in
increasing the strength of carbon nanotubes (CNT) was examined by Ryu et al.
(2011), and the mussel-mimetic adhesive increased the CNT tensile strength by
almost 500%.
5 Comparison of Natural Extraction and Recombinant Mussel … 117

4 Natural Extraction of Mussel Proteins

Early studies on mussel proteins relied on natural extraction methods, and a few of
production methods for commercial MAPs are also based on natural extraction. The
extraction process begins with the collection of mussels from rocks and jetties.
Factors such as temperature and pH have to be considered in collecting mussels and
subsequent isolating mussel proteins. For example, low temperature that suits
mussel growth is preferred. Collected mussels are normally transferred to an
enclosed space, usually aquaria or holding tanks, containing seawater at a tem-
perature ranging 4–15 °C (Papov et al. 1995; Zhao et al. 2006). During the process
of protein extraction, auto-oxidation of DOPA in neutral and alkali conditions
should be avoided. The extensive cross-linking of DOPA, as a consequence of its
oxidization to O-dopaquinone and irreversible maturation, not only hinders the
characterization of byssal proteins but also diminishes their adhesive property
(Waite and Qin 2001).

4.1 Secretion, Harvesting, and Dissecting of Plaques

and Threads

Mfp recovery can be sourced either from mussel feet or directly from the plaques.
Following the growth of mussels, secretion of byssal threads and plaques is
achieved either by induced or un-induced methods. Un-induced method allows the
mussels to tether on a substrate such as acrylic, plastic, or glass slide. After
18–24 h, accumulated plaques are harvested using single-edge blade. For extraction
at a small scale, typically 1000–2000 plaques are handled (Zhao et al. 2006; Papov
et al. 1995; Wei et al. 2013; Waite and Qin 2001; Zhao and Waite 2006a). For the
induced method, potassium chloride (KCl) is injected into the mussel feet in order
to get fresh plaques and threads which have minimal cross-linking (Gantayet et al.
2013; Zhao and Waite 2006b). Harvested plaques are rinsed with water before
further processing or being stored at −80 °C for future use. Compared to byssal
plaque, mussel foot is a bank of byssal precursors, and it can be dissected using
single-edge blade until the underlying phenol gland is revealed (Zhao and Waite

4.2 Isolation of mfps

Isolation of mfp from recovered plaques and threads includes extraction and pre-
cipitation. After the addition of solvents to the harvested plaques and threads,
homogenization is carried out using a grinder or homogenizer (Papov et al. 1995;
118 J.J. Castillo et al.

Zhao et al. 2006; Pardo et al. 1990), followed by a few steps of fractionation as
detailed below.
Multiple steps of extraction are typically utilized in order to fractionate mfps. As
shown in Fig. 2, a three-step extraction process is employed to isolate mfps (Hwang
et al. 2010a). Mussel feet are first dissected to obtain the phenol glands which
contain DOPA-rich proteins. First, extraction is done by homogenizing the phenol
glands in 5 v/v% acetic acid with protease inhibitors (leupeptin and pepstatin).
Then, the homogenates are centrifuged and the supernatant (S1) contains mfp-1,
mfp-2, and mfp-6. After removing the supernatant, the pellet (P1) is homogenized
in 5 v/v% acetic acid with 8 M urea for the second extraction. The supernatant (S2)
from the second extraction contains crude mfp-3, which is precipitated by 30%
ammonium sulfate (AS) in the final supernatant (S3). The pellet (P2) undergoes the

Fig. 2 Selective isolation of mussel adhesive proteins from mussel feet, adapted from (Hwang
et al. 2010a). P pellet and S supernatant
5 Comparison of Natural Extraction and Recombinant Mussel … 119

third extraction in 5 v/v% acetic acid with 4 M guanidine hydrochloride. After

centrifuging the homogenates, the supernatant (S4) is treated with 30 w/v%
ammonium sulfate. Dialysis of the supernatant against dilute perchloric acid after
AS precipitation results in pellets (P4) containing mfp-5, while mfp-4 remains in
supernatant (S6).
There are a few patents disclosing methods on natural extraction of mfps. In a
patent filed by Bio Polymer Products (Qvist 2002), mussel feet were treated in
acetic acid at a concentration of 1–10% (preferably 5%). The mixture is then
homogenized, and the collected supernatant is added with salts at a high concen-
tration (preferably 10 w/v%). After cooling for hours, mfps precipitated and thus
isolated. The mfp precipitates can be further purified by re-dissolving in 5 v/v%
acetic acid, leaving unwanted proteins in solid form. The mfp-rich supernatant can
be further processed by adding 15 v/v% perchloric acid to precipitate contaminant
proteins. The recovered mfp-rich supernatant can be further purified by precipi-
tating mfps with the addition of acetone in an acidic solution overnight.
The choice of extraction buffer is a key consideration in order to produce soluble
mfp, avoid auto-oxidation of DOPA, and selectively isolate the target mfp. The
most common extraction buffer is 5 v/v% of acetic acid in the presence of 8 M urea
(Wei et al. 2014; Zhao et al. 2006). The nature of amino acid residues present in the
protein determines the working pH under which the target protein is soluble. In
general, mfps have basic pIs, ranging from pH 8 to pH 11. The abundance of
cationic residues (*20 mol%) in DOPA-rich mfps contributes to this basic pI (Lee
et al. 2011). The low pH (*pH 3) of 5 v/v% acetic acid prevents DOPA residues
from auto-oxidizing. However, if working at a basic pH is unavoidable, borate
which complexes DOPA or an antioxidant such as ascorbate should be added into
the extraction buffer (Waite 1995). A basic borate buffer has been used to extract
Dreissena polymorpha foot protein (Dpfp) because of the unusual acidic pI
(pI = 5.3–6.5) of Dpfp-1 and Dpfp-2 (Gantayet et al. 2013).
Additives such as chaotropic agents are often needed in an extraction buffer. For
example, urea or guanidine hydrochloride is commonly added to extract proteins
that have a lower solubility. The three-step extraction presented in Fig. 2 starts with
an extraction buffer without chaotropic agent to isolate highly soluble mfps.
Subsequently, adding urea and guanidine in the second and third extractions sol-
ubilizes more hydrophobic mfps. Reducing agents such as 2-mercaptoethanol are
also added to break disulfide bonds in mfps, while protease inhibitors such as
phenylmethylsulfonyl fluoride (PMSF) prevents protein degradation (Pardo et al.
1990). Addition of perchloric acid or organic solvents such as acetone precipitates
unwanted materials from the mfp extracts (Pardo et al. 1990).

4.3 Purification of mfps Using Chromatography

Purification and polishing are necessary to achieve a high purity in natural

extraction methods, and the most common technique for purifying mfps is liquid
120 J.J. Castillo et al.

chromatography. Gel filtration chromatography has been used to separate mefp-1

and mefp-2 (Waite 1995). Extracts from 30 g of mussel feet were loaded on a
3  100 cm column of Sephadex G-200. Mefp-1, which is larger than mefp-2,
was eluted first. After lyophilization, the samples were further purified by
reverse-phase HPLC (Waite 1995). A two-step size exclusion chromatography was
used to purify and characterize mfps from Perna viridis (green mussel) (Ohkawa
et al. 2009). Mussel feet extracts were loaded first in a gel filtration column of
Sephacryl S-300 (HiPrep 16/30 column) to produce two fractions. The collected
fractions were dialyzed and then lyophilized before purified by a gel permeation
column of Shodex KW803, which yielded two purified mfp fractions. Ion exchange
chromatography was also applied to purify adhesive proteins from freshwater
mussels (Xu et al. 2014). The mfp extract was first fractionated using gel filtration
chromatography before loading the mfp fractions into a DEAE-Sepharose CL-6B
column, a weak anion exchanger.
In order to minimize purification steps, different types of chromatography matrix
have been combined in one separation medium. One patent has reported a mixed
mode of chromatography which combines three types of adsorption: hydrogen
bonding adsorption, hydrophobic interaction, and electrostatic adsorption (Xing
2009), and the method can significantly improve the purification yield by mini-
mizing steps of chromatography (Gao and Janson 2013).
Affinity chromatography which has a high selective affinity for the target protein
has not yet been broadly used for purification of extracted mfps yet. Phenyl bor-
onate affinity chromatography was tested to purify DOPA-containing proteins, but
precipitation problem occurred for native mefp-1 and mefp-2 because mfps form
complexes with boronate and auto-oxidizes at basic pH (Waite 1995). Improvement
is needed before boronate affinity chromatography can be successfully applied for
mfp purification.

4.4 Yield of Extraction Methods

Yield of natural extraction method is a key factor to consider. To date, it requires a

large number of mussels to prepare a few milligram of mfps. From 1000 accu-
mulated plaques, 0.5 g dry weight of plaques was produced from which four
milligrams of soluble mcfp-3 was purified (Zhao et al. 2006). From 0.2 to 0.3 g wet
weight of phenol gland collections, approximately 0.5 mg of mcfp-4 was extracted
via successive extractions and reverse-phase HPLC (Zhao and Waite 2006b). From
200 g wet weight of blue mussel feet, 0.4 mg of mefp-1 was purified (Taylor et al.
1994). Reported in a patent (Qvist 2002), 1–2 mg of mussel adhesives with 90–
91% purity was exacted from one gram mussel foot using dialysis as the final
purification step, while 0.5–1 mg of mussel adhesive of 95–100% purity was
achieved using acetone precipitation as the final step. The low yield of the extracted
mfp is in part due to a low percentage of mfp in mussels, and partially due to the
low efficiency of the extraction methods.
5 Comparison of Natural Extraction and Recombinant Mussel … 121

5 Recombinant Production Approach

The low productivity of natural extraction method makes the process economically
unattractive, hindering mfps’ broad application. In early 1990s, the feasibility of
recombinant approach was explored for the production of MAPs using yeast as a
host (Filpula et al. 1990; Salerno and Goldberg 1993). Following these early efforts,
several groups have explored the recombinant expression of MAPs using different
expression hosts. The following sections first cover recombinant MAPs with
sequences derived from a single variant of mfp, and then discuss a recombinant
fusion approach where different MAPs are genetically fused with other functional
peptides and proteins.

5.1 Expression Systems and Optimization of Expression

5.1.1 Expression Hosts

The choice of expression host is one key decision in recombinant production of

MAPs. Both eukaryotic and prokaryotic organisms have been explored as
expression hosts for recombinant MAPs (rMAPs) production. In Table 2, the
various hosts used for rMAPs production are summarized. One of the initial
attempts to produce rMAP used yeast strain D8 to express a 24 kDa mefp-1, and
the yield was 3–5% of the total yeast cell proteins (Filpula et al. 1990). Another
yeast strain, Pichia pastoris, was used for the expression of mefp-1 (Zheng et al.
2012). Induction was carried out using different methanol concentrations with the
maximum mefp-1 expression level of 2.1 g/L achieved at 1.5% methanol
In a recent study, recombinant production was combined with in vivo modifi-
cation of tyrosine, serine, and proline, using insect cell as the expression host (Lim
et al. 2011). A fusion construct of mfp-1 and mfp-5, named as fp-151, was
expressed by combining baculovirus and insect Sƒ9 cells. Post-translational mod-
ifications offered by the insect cell host system were evidenced in DOPA, dopa-
quinone, phosphorylated serine, and hydroxylated prolines. Coating abilities of
E. coli- and Sƒ9-derived fp-151 were compared and showed that the latter had a
*2-fold increase in coating ability as a result of successful in vivo modifications,
especially DOPA.
One of the most widely used prokaryotic host systems for rMAPs is Escherichia
coli (E. coli) (Huang et al. 2007; Jiang et al. 2012; Nicklisch et al. 2013; Yang et al.
2013; Zeng et al. 2010) with E. coli BL21 (DE3) being the most commonly used
strain. Depending on expression conditions, the rMAPs can be expressed as soluble
proteins or inclusion bodies (Huang et al. 2007; Jiang et al. 2012; Nicklisch et al. 2013;
Yang et al. 2013; Zeng et al. 2010). Protein yield using E. coli ranges from 10 to 50%
of total protein, which was better than the yield reported in early study using yeast.

Table 2 List of recombinant MAPs with corresponding expression conditions*

MAP Sequence Host Vector Culture parameters Solubility Purification Reference
mefp-1 M-(AKPSYPPTYK)12 E. coli pET-28 1 mM IPTG Inclusion Reversed Zeng et al.
BL21 (+) bodies phase (2010)
pvfp-1 HGHGYGGYGKPGKPGKPGSKGPRGP E. coli pET-32a 0.5 mM IPTG, Soluble IMAC Jiang et al.
mgfp-3A MADYYGPKYGPPRRYGGGNYNRY E. coli pET-23b 1 mM IPTG, Soluble IMAC Yang et al.
GRRYGGYKGWNNGWKRGR BL21 37 °C, 250 rpm (2013)
mgfp-5 SSEEYKGGYYPGNTYHYHSGGSY E. coli pTrcHisA 1 mM IPTG, Soluble Ni-NTA Hwang et al.
HGSGYHGGYKGKYYGKAKKYYYKYKNSG 37 °C, 250 rpm affinity (2004)
mcfp-6 MGSSHHHHHHSSGLVPRGSGGGNYRG E. coli pET-28a 10 lM IPTG, Inclusion IMAC Nicklisch
YCSNKGCRSGYIFYDNRGFCKYGSSSYKYD Rosetta (+) 37 °C, 250 rpm bodies et al. (2013)
mefp-1 KPKPSYPPSYKPKTTYPPTYKPKISYPPTYKAKPS Yeast pUC18 Galactose-induced, Inclusion Ion Filpula et al.
YPATYKAKPSYPPTYKAKPSYPPTYKAKPSYPPT strain and 30 °C bodies exchange (1990),
msfp-1 Not specified Pichia pPIC9 K Methanol-induced, Not Not Zheng et al.
pastoris 30 °C specified specified (2012)
mefp—Mytilus edulis foot protein; pvfp—Perna viridis foot protein; mgfp—Mytilus galloprovincialis foot protein; mcfp—Mytilus californianus foot protein;
msfp—Mytilus sp. JHX-2002 foot protein
J.J. Castillo et al.
5 Comparison of Natural Extraction and Recombinant Mussel … 123

However, rMAPs expressed in E. coli need further modification to convert tyrosine

into DOPA. To simplify purification, histidine tags are either added to the MAP
sequence or inherently built in the plasmid of choice. Using immobilized
metal-affinity chromatography, MAPs tagged with hexahistidine residues are selec-
tively purified in a single step.
Suitable expression conditions depend on the recombinant protein being
expressed and types of host cells. Determining suitable conditions requires different
trials accompanied by monitoring cell growth and estimating protein yield. As
shown in Table 2, most mfps were expressed in E. coli at 37 °C with induction using
low concentrations (10 µM–1 mM) of isopropyl b-D-1-thiogalactopyranoside
(IPTG). On the other hand, a lower temperature, 30 °C, was used in yeast.

5.1.2 Improving Solubility of rMAPs

MAPs are prone to misfolding and frequently expressed as inclusion bodies.

Different strategies can be used to improve solubility rMAP during expression and
prevent formation of inclusion body. One strategy is to express MAPs in
periplasmic space of E. coli. A signal peptide, OmpA, has been used to direct
MAPs to the periplasmic space of E. coli (Lee et al. 2008), producing significant
soluble MAPs.
Co-expression strategy was also used to improve solubility of rMAP. Mfp-151
and tyrosinase were co-expressed with the intention to achieve in vivo
post-translational modification in E. coli (Choi et al. 2012). Most fp-151 was
expressed in soluble form using this strategy, although the specific mechanism
governing this is not known. In contrast, fp-151 expressed without tyrosinase was
obtained as inclusion bodies.

5.1.3 Improving the Expression Yield

The main aim of recombinant approach is to tackle the low productivity of natural
extraction method. Zheng et al. (2012) examined the effect of different methanol
concentrations as inducer and addition of ectoine on the expression level of mfp-1
in Pichia pastoris. Ectoine is a cyclic amino acid that is known to protect cell
membranes as well as proteins and enzymes from harsh environment. The broth
containing ectoine promoted better cell growth (increased by 25.7%) than the plain
broth. Subsequently, the addition of ectoine increased the expression yield by
Co-expression strategy of fp-151 and Vitreoscilla hemoglobin (VHb) was also
used to increase protein production (Kim et al. 2008) because VHb can improve
efficient utilization of oxygen. For both batch and fed-batch modes, co-expression
of VHb increased cell density and resulted in a 1.9-fold increase in fp-151
expression yield.
124 J.J. Castillo et al.

Post-induction nutrient feeding strategy was also explored to improve cell

growth and protein expression using E. coli (Wong et al. 1998). Although the
post-induction cell growth and dry cell weight were found to be independent of the
feeding rate, the amount of protein produced strongly depended on the feeding
strategy. Among six different feeding strategies tested, linear feeding rate of
2.1 mL/h2 gave the maximum protein production of 5.3 g/L (16.2% of the total

5.2 Design of rMAP Fusion Systems

The previous section has reviewed the various strategies for improving expression
of MAP. Although attractive expression level of MAP was achieved using opti-
mized systems and expression conditions, the adhesiveness of the produced rMAP
was low compared to the natural mfps (Dong et al. 2005).
The strong adhesiveness of natural MAPs is largely due to the combined action
of the various types of functional MAPs found in the adhesion plaque of the mussel
foot. However, initial attempts to produce MAP using recombinant technology
focused on the expression of only one type of MAP (e.g., mfp-1), and it is thus not
surprising to have a poor adhesiveness. The fusion of different MAPs or fusion
MAPs with other protein partners have been explored to overcome this limitation.
A fusion approach can improve the characteristics of rMAPs in two aspects: One
objective is to improve the expression levels in a soluble form and allow easy
purification and handling from the production perspective as discussed in the
previous section. Another objective is to improve its functionality as a bioadhesive
from the performance perspective. The following section will focus on improving
the functionality of MAPs, using two different fusion strategies.

5.2.1 Fusion Strategy 1: Hybrid of Different mfps

In this fusion strategy, different mfps were fused with each other to form hybrid
mfps. The amino acid sequences of individual mfp proteins are summarized in
Table 3. Hyung and co-workers have fused mfp-5 with partner protein mfp-1 from
Mytilus galloprovincialis to form a hybrid system mfp-151 (Fig. 3A) (Hwang et al.
2007a). Mfp-5 was chosen as the main protein of the hybrid systems because earlier
work demonstrated its high bioadhesiveness compared to natural mfp Cell-TakTM
(Hwang et al. 2004). Mfp-151 was designed to have six repeats of mfp-1
decapeptide on both the N- and C-termini of mfp-5. The mfp-151 hybrid system
improved soluble expression to yield 1 g of purified protein per liter of culture and
simplified bulk preparation of adhesive solutions at a high concentration of
*330 g/L.
Another hybrid system fused one mfp-3 protein on both N- and C-termini of
mfp-5 to form mfp-353 hybrid system (Fig. 3B) (Gim et al. 2008). Mfp-353 was
5 Comparison of Natural Extraction and Recombinant Mussel … 125

Fig. 3 Hybrid MFPs from Mytilus galloprovincialis. M1: mfp-1; M3: mfp-3; M5: mfp-5. N:
N-terminus; C: C-terminus

expressed as inclusion bodies when expressed in E. coli as opposed to mfp-151

which was expressed as a soluble protein. The advantage conferred by the expres-
sion of mfp-353 as inclusion bodies was preventing the inhibition of cell growth
which was previously encountered when expressing mfp-3 and mfp-5 as individual
proteins. With respect to cell adhesion, the mfp-353 was found to be 2.2-fold
stronger than the commercial Cell-TakTM. After modification using enzyme
tyrosinase, mfp-151 and mfp-353 have 8 and 5.5% DOPA, respectively, lower than
that of natural mfps (13 and 26% for mfp-1 and mfp-5). This low yield of DOPA has
been attributed to the possible steric hindrance caused by the structure of hybrid
mfps, reducing the accessibility of tyrosine residues to enzyme tyrosinase. Although
the DOPA content has been reduced in the hybrid mfps, it has demonstrated rea-
sonable bioadhesiveness. This provides scope to further improve the design of fusion
protein to allow access of tyrosinase for improved bioadhesive property.

5.2.2 Fusion Strategy 2: Fuse mfps with Other Molecules

In an attempt to further improve the functionality of mfps as a bioadhesive, fusion

with other peptides and proteins has been pursued. The fusion of mfp-151 with cell
adhesion RGD peptides was reported by Hwang et al. (2007b). As shown in
Fig. 4B, the cell adhesion hexapeptide GRGDSP was fused into the C-terminus of
mfp-151. The mfp-151-RGD fusion protein had retained special features of
mfp-151, including a high expression level and an adhesive property with a similar
percentage of DOPA of 7.2%. Although the fusion of RGD peptide did not change
the properties of mfp-151, the mfp-151-RGD was expressed as inclusion bodies in
contrast to a soluble form when mfp-151 was expressed alone. The functionality of
RGD peptide in the fusion system was evaluated and demonstrated subsequently
showing that the RGD-fused peptide improved the cell adhesion and spreading
abilities of fibroblast NIH/3T3 cells (Kim et al. 2010).
More recently, RGD peptide was fused to C-terminus of 6 repeats of mfp-1
protein as shown in Fig. 4A (Yoo et al. 2015). Similar to the mfp-151-RGD fusion,
the (mfp-1)6-RGD was also expressed as inclusion bodies in E. coli. The (mfp-1)6-
RGD fusion protein improved cell proliferation of preosteoblast cells when being
126 J.J. Castillo et al.

Fig. 4 MFP fusions with other partner molecules

coated on polystyrene tissue culture surface. The mfp–RGD systems offer the
combined advantages of DOPA-based adhesion by mfp and the cell adhesion by
RGD, showing a better performance than conventional coating methods. Based on
5 Comparison of Natural Extraction and Recombinant Mussel … 127

this fusion approach, fusions of mfp with other extracellular matrix (ECM) peptides
from laminin, type IV collagen, and substance P were also reported (Choi et al.
2010). All ECM peptides were fused into the C-terminus of mfp-151 hybrid
(Fig. 4B) and expressed in E. coli as inclusion bodies. All mfp-ECM peptide
fusions showed enhanced cell adhesion and proliferation making this fusion strat-
egy an attractive platform for tissue engineering.
Improving the bioadhesive strength of rMAPs to match the efficiency of natural
MAPs has always been the priority in this field. A unique fusion strategy has been
developed by Zhong et al. (2014) to improve the underwater adhesiveness of
mgfp-3 and mgfp-5 proteins using an amyloidogenic protein CsgA, a subunit
of E. coli curli fibers. The CsgA protein was fused to the N-terminal and C-terminal
of mgfp-3 and mgfp-5, respectively (Fig. 4C) with a flexible GS linker. This fusion
strategy allowed CsgA of individual fusion monomer to interact and form fibers
while the mfps were exposed on the surface of the fibers. This nanofiber formation
by CsgA mimics the byssal threads of natural mussels that bring the various mfps
together to provide strong adhesion strength. This new material required a shorter
time for fiber formation and provided a synergistic effect by each protein partner
(Zhong et al. 2014).
Other fusions reported for rMAPs focus more on facilitating practical application
of rMAP rather than improving adhesion strength. Typically, these fusions were
designed to incorporate additional features for biomedical-based applications. For
example, the B and C domains of protein A were fused to the N-terminus (Fig. 4D)
of mfp-5, and the fused protein can coat surface of glass, polymeric and metallic
surfaces for antibody immobilization (Kim et al. 2011). Another feature incorpo-
rated into mfp-based biocoating was anti-bacterial activity (Jo et al. 2014). Silver
binding peptides Ag5 and AgP35 were fused into the C-terminus of hybrid mfp-151
(Fig. 4E). Once coated on to the surface, the silver binding peptides allowed silver
nanoparticle formation that showed bactericidal activity against gram-positive and
gram-negative species. These various approaches illustrate that the fusion strategy
offer significant improvement for rMAPs in many aspects. It has not only improved
the adhesive features of rMAPs but also broadened its application as a practical

5.3 Modification and Formulation of rMAPs

Mussels in their natural environment possess a strong adhesive ability due to the
modification of tyrosine residues in their foot proteins into DOPA by co-secretion
of enzyme tyrosinase. Consequently, naturally extracted mussel foot proteins
contain sufficient DOPA. On the contrary, rMAPs are produced to have only tyr-
osine residues when they are expressed in the prokaryotic host systems such as
E. coli. As a result, rMAPs require an additional modification step. Conversion of
the tyrosine residues to may use enzyme tyrosinase either from commercial sources
128 J.J. Castillo et al.

Table 3 Amino acid sequence of MFPs from Mytilus galloprovincialis

MFP type Amino acid sequence

or extraction from mushrooms. Modified rMAPs can be further formulated by

adding different agents to enhance their bioadhesiveness.

5.3.1 Conversion of Tyrosine into DOPA

rMAPs modification using tyrosinase can be performed either in vitro after the
purification step or in vivo within the host cell. For in vitro modification, the first
step is to dissolve the purified mfp into 5 v/v% acetic acid solution containing
25 mM ascorbic acid followed by the addition of 50 µg/mL of tyrosinase. The
enzymatic reaction was carried at basic pH, 25 °C with shaking for few hours
(Dong et al. 2005). Acetic acid is essential to prevent the auto-oxidation of DOPA
to o-quinone at basic pH (Hwang et al. 2004) because tyrosinase catalyzes both
conversions of tyrosine to DOPA and further conversion of DOPA to o-quinone.
Tyrosinase-treated mfp-5 and mfp-3A showed improved adhesion ability of *550
and *230 nN compared to the unmodified forms of *22 and 27 nN, respectively.
For the modification of hybrid mfps such as mfp-151 and mfp-353, 20 mM sodium
borate was added to prevent cross-linking reaction and allow easy handling of
protein sample (Hwang et al. 2007a). Modified mfp-151 showed twice the adhesive
strength (*500 nN) in comparison with commercial Cell-Tak (*246 nN).
Adhesion strength of modified mfp-353 was found to be 2.2-fold higher
(*0.52 MPa) than fibrin glue (*0.24 MPa) (Gim et al. 2008).
In vivo modification of the tyrosine residues in rMAPs can be achieved in two
ways: The first approach is expressing rMAPs in a host system capable of per-
forming post-translation modifications. Lim et al. (2011) reported in vivo-modified
mfp-151 using insect Sf9 cells as the host systems. LC/MS analysis of the in
vivo-modified mfp-151 showed DOPA and dopaquinone at the 5th position, along
with other modifications such as phosphorylation of serine and hydroxylation of
proline residues, and the protein had twofold higher surface coating ability than the
E. coli-derived mfp-151 (Lim et al. 2011). The second approach involves
co-expressing the rMAP along with the tyrosinase enzyme in the same host using a
dual-vector system. mfp-151 co-expressed with tyrosinase in E. coli cells showed a
fourfold higher adhesive strength (*3.01 MPa) compared to the in vitro-modified
mfp-151 (*0.80 MPa) (Choi et al. 2012). The higher adhesive strength was
attributed to the better conversion of tyrosine to in vivo than in vitro where lower
5 Comparison of Natural Extraction and Recombinant Mussel … 129

conversions are observed due to steric hindrance of tyrosinase caused by the rMAPs
aggregation at a neutral pH.
Although the percentage of DOPA and adhesive strength of in vivo-modified
rMAPs was higher than these in vitro-modified, they were still much lower than the
natural mfps. Direct incorporation of DOPA into rMAPs in vivo was performed
using the residue-specific non-canonical amino acid incorporation method.
Tyrosine auxotrophic strain of E. coli with an endogenous tyrosyl-tRNA synthetase
(TyrRS) was used to express mfp-3 while DOPA was supplemented in the growth
media. Since TyrRS also has affinity toward DOPA, depletion of tyrosine in the
media prompts TyrRS to incorporate DOPA from the media into tyrosine positions
during protein translation stage. rmfp-3 produced by this method had 94% DOPA
incorporation resulting in 16.5 mol% of DOPA in the final protein which has
similar DOPA content and adhesive strength as natural mfps (Yang et al. 2014).

5.3.2 Addition of Metal Ions

The catechol side chain of DOPA can participate in interactions such as hydrogen
bonds, metal ion binding, and p–p interaction (Meng et al. 2014) and thus influence
the adhesive binding abilities of the mfps. Native mussels contain high levels of
metal ions such as Fe3+, Cu2+, and Zn2+. These metal ions can act as cross-linking
agents to increase the adhesive strength of mfps. For example, iron complex
coordinated with DOPA molecules, [Fe(DOPA)3], can serve as the key curing agent
(Sever et al. 2004). Note that the concentration of cross-linking agent should be
optimized because a higher concentration reduces adhesion strength (Cha et al.
2009). For example, the addition of Fe3+ ion to a concentration of 10 µM forms bis-
and tris-catecholate iron complex that bridges DOPA proteins and thus increases
adhesive strength of mfp-1. The adhesive strength is hindered at 100 µM Fe3+ ions
due to the formation of mono-catecholate iron complex that no longer forms
connecting bridges for mfps (Zeng et al. 2010).

5.3.3 Formulation of rMAPs by Coacervation

Coacervation is the process of forming macro-ions with oppositely charged ions.

mfps are cationic in nature and can interact with anionic molecules to form coac-
ervates. Hyaluronic acid (HA) is an anionic partner that has been formulated with
mfps to produce coacervated rMAPs. mfp-151 and mfp-131 mixed with HA at 8:2
ratio showed high adhesive strength of 3.17 and 4.00 MPa, respectively. This was
almost twofold higher than the adhesive strengths of mfp-151 and mfp-131 alone
(Lim et al. 2010). Different ratios of mixing of HA with fusion mfp-151–RGD
showed that a ratio of 1:3 was optimal since the coacervate behaved as a shear
thinning fluid at this condition, a feature essential for surface coating and spread
ability. Higher ratios of 1:9 and 2:3 form coacervates that behaved as a shear
thickening fluid (Hwang et al. 2010b). A further understanding of the features of
130 J.J. Castillo et al.

these complex coacervates will not only aid in improving the adhesive strength of
rMAPs but also widen its applications as a bioadhesive for biomedical and
industrial purposes.

5.4 Comparing Adhesive Strengths of rMAPs

A number of studies have compared rMAP with naturally extracted one. An early
study expressed the non-repeating region of Perna viridis foot protein-1 (Pvfp-1) in
E. coli. The recombinant Pvfp-1 had superior adhesiveness compared to commer-
cial Cell-Tak™ when adsorbed on glass and polytetrafluoroethylene (PTFE) (Jiang
et al. 2012). Another group also compared the coating and adhesive ability using
quartz crystal microbalance of recombinant Mytilus coruscus foot protein-3
(rmcofp-3: unmodified and tyrosine-modified) and native mcofp-3. The results
followed a general trend as unmodified rmcofp-3 < modified rcofp-3 < native
mcofp-3 (Li et al. 2011). Meanwhile, the adhesive property of recombinant Mytilus
gallorovincialis foot protein-5 (rmgfp-5) was compared to Cell-Tak™ by Hwang
et al. (2004). Coating experiments using different substrates revealed that rmgfp-5
was better than Cell-Tak™, whereas the latter did not coat antifouling agent-coated
surface. The trend of adhesion strength was unmodified rmgfp-5 < unmodified
Cell-Tak™ < modified Cell-Tak™ < modified rmgfp-5 (Hwang et al. 2004). In a
follow-up study, the adsorption abilities of modified rmgfp-3A were compared with
modified samples of rmgfp-5 and Cell-Tak™. The relative strengths were as fol-
lows: Cell-Tak™ < rmgfp-3A < rmgfp-5 (Dong et al. 2005).

6 Perspective

Commercial applications of bioadhesive proteins are still limited due to bottleneck

in their production. Although native bioadhesive proteins from marine mussels have
been available for many years, there are two fundamental problems associated with
the production from natural sources. First, extraction of mussel proteins from
natural source is labor-intensive and expensive. Second, protein products from
animal sources always carry the risk of viral and animal-related contaminants.
Stringent regulations for safer biological products have forced the industry to
develop alternative production methods.
Recombinant production has recently been explored to overcome the problems
of natural extractions. Different expression systems including yeast, plant cell,
insect cell, and E. coli have been examined for the production of mussel adhesive
proteins (Hwang et al. 2004, 2007a; Jeon et al. 2015; Lim et al. 2011). Despite the
advantage of in situ post-translational modification, tested eukaryotic expression
systems gave a poor production level because a high level of mussel adhesive
proteins is toxic to cells (Cha et al. 2008; Lim et al. 2011). In contrast to the
5 Comparison of Natural Extraction and Recombinant Mussel … 131

eukaryotic systems, the bacterial E. coli system can achieve a high expression level
by the formation of insoluble inclusion bodies which are well tolerated by the cells
(Hwang et al. 2004, 2007a; Jeon et al. 2015). Therefore, microbial fermentation, a
well-established bioprocess, has emerged as a promising method due to its low cost
and high yield (Brubaker and Messersmith 2012). It can not only eliminate the risk
of viral and animal-related contaminants but also make proteins cheaper at a large
scale. Despite these fundamentally attractive advantages, there is an intrinsic lim-
itation of microbial fermentation using E coli: bacteria lack appropriate
post-translation modification of proteins. In vitro enzyme-mediated DOPA modi-
fication of proteins is thus required after the microbial fermentation step. However,
existing enzymatic modification methods using the enzyme tyrosinase have low
modification yield, and the products have not yet delivered the same quality as these
from naturally extracted procedure (Jeon et al. 2015). It remains a challenge to
faithfully modify the recombinant mussel adhesive proteins mimicking the native
post-translation modification. Significant research efforts, particularly on improving
enzymatic modification, are thus required to turn microbial production into an
effective approach that can produce MAPs with a high performance at a low cost.

7 Sources of Further Information

Mechanisms governing mussel adhesion such as surface morphology, chemical

interactions, physiological factors and physical–mechanical interactions are
important to guide the design of engineered adhesive proteins and have been dis-
cussed in a few review articles (Lee et al. 2011; Meng et al. 2014; Palacio and
Bhushan 2012). Catechol reaction pathways and contribution of DOPA to mussel
adhesion were discussed in detail by Meng et al. (2014), and a mini-review dis-
cussed the factors that controls redox of DOPA (Nicklisch and Waite 2012). For
recombinant approaches, recent studies investigated the synergistic effect of lysine
and DOPA on mussel adhesion (Maier et al. 2015) and the role of charge, chain
length, and surface type (Wei et al. 2015), and these studies are valuable references
for optimizing amino acid sequences in engineering adhesive proteins.
Characterization techniques of MAPs were not discussed in this chapter, and this
topic has been covered in a book chapter (Hongbo Zeng et al. 2015). The use of
imaging surface plasmon resonance (iSPR) was introduced to study microscale
bioadhesion in real time (Aldred et al. 2011). To explore broad applications of
MAPs, a chapter on marine biotechnology can serve a good reference (Kim 2015).
The use of mussel proteins as novel wound sealants has been covered in a recent
review (Peng and Shek 2010), and their application as nanoparticles and
nanocomposite can be found in a book chapter (Meng et al. 2014).
132 J.J. Castillo et al.


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Chapter 6
Extraction of Lipids and Carotenoids
from Algal Sources

Adarsha Gupta, Avinesh R. Byreddy and Munish Puri

1 Introduction

Omega-3 fatty acids (FAs) are naturally occurring polyunsaturated fatty acids
(PUFAs) that include mainly alpha-linolenic acids (ALA), eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA) (Adarme-Vega et al. 2012; Delarue and
Guriec 2014; Swanson et al. 2012). The applications of PUFAs for human health
are rapidly expanding, which necessitates exploring alternative sources to fish
(Gupta et al. 2012; Puri et al. 2015). Many microorganisms (marine yeasts, fungi
and bacteria) exhibit the ability to store a significant PUFAs. Marine microalgae are
the primary producer of EPA and DHA in the marine ecosystem (Fig. 1). These
naturally grow under a variety of culture conditions including autotrophic, mixo-
trophic and heterotrophic (Buono et al. 2014). Use of microalgae as a source of
omega-3 FAs production has several advantages over traditional sources. They can
be grown in a controlled environment with readily available substrates in the fer-
mentation process. They do not compete for land and fresh water and also eliminate
the risk of chemical pollutant contamination (Ryckebosch et al. 2012). There are a
number of algal species with higher EPA and DHA contents. Out of all microbes,
thraustochytrids species are considered as good source for producing the elevated
quantities of omega-3 FAs with potential commercial application in infant formulas,
food, cosmetic and pharmaceutical products (Sijtsma and de Swaaf 2004).
Carotenoids are valuable compounds that can be sourced from multiple food
items such as orange juice, spearmint, grains, peppermint and several herbs such as
coriander, basil and parsley. Common sources of lycopene are fruits such as
watermelon, apricot, grapefruit, papaya and guava. b-Carotene is commonly found
in olive oil, leafy green vegetables, red carrots, sweet potato and amaranth;

A. Gupta  A.R. Byreddy  M. Puri (&)

Bioprocessing Laboratory, Centre for Chemistry and Biotechnology,
Deakin University, Waurn Ponds, Geelong, VIC 3217, Australia

© Springer International Publishing AG 2017 137

M. Puri (ed.), Food Bioactives, DOI 10.1007/978-3-319-51639-4_6
138 A. Gupta et al.

Lycopene 3

DHA (Docosahexaenoic Acid) 22:6 ω3

EPA (Eicosapentaenoic Acid) 20:5 ω3

Fig. 1 Chemical structure of some important omega-3 FAs and carotenoids produced by
microalgae (this figure has been reproduced from Yu and Gu 2015 and modified)

however, current yields are not promising. A new source of carotenoids with
enormous commercial potential is algae-derived carotenoids (Mäki‐Arvela et al.
2014). The ease of mass production of carotenoids at high-yield levels endorses
algae as a promising candidate for the production of a carotenoid supplement.
Carotenoids (tetraterpenoids containing 40 carbons), such as lycopene, b-carotene,
zeaxanthin, canthaxinthin and astaxanthin are widely distributed in microorganisms
and have many benefits for human health (Fig. 1). Currently, more than 80% of
b-carotene is synthesized by chemical processes. The chemosynthetic pathway pro-
duces relatively more trans-stereoisomers of b-carotene than from microbial processes.
Trans-stereoisomers are less competent antioxidants and therefore less desirable for
medical applications (Del Campo et al. 2007; Shaish et al. 2006). The halo-tolerant
marine microalga Dunaliella sp. is considered to be a large carotenoid accumulator.
However, high carbon dioxide consumption, low production efficiency, poor control
and the high cost of land have limited the expansion of mass cultures of Dunaliella for
b-carotene production (Ye et al. 2008). Thraustochytrids can be used as an alternative
for carotenoid production. They are a promising source of carotenoids such as as-
taxanthin, zeaxanthin, canthaxanthin, echinenone, phoenicoxanthin and b-carotene
(Fig. 1). Carbon and nitrogen sources are very important nutrient factors for car-
otenoid production. Various methods, using the combination of organic solvents and
ultrasonic baths, have been examined to determine carotenoid content present in
thraustochytrids (Armenta et al. 2006). Semi-fragile cell walls in thraustochytrids
aided the downstream processing of the pigments with effective recovery using a
solvent such as acetone. Mutation strategy has also been implemented to enhance the
astaxanthin productivity of Schizochytrium limacinum by treating the cells with
6 Extraction of Lipids and Carotenoids from Algal Sources 139

Nmethyl-N′-nitro-N-nitroso guanidine (NTG) resulting in intense colour development

in the colonies (Chatdumrong et al. 2007).
The production of valuable compounds, co-products and materials from
microbial sources for use in food, pharmaceuticals and industrial materials, is in
itself a large industry (Borowitzka 2013). Carotenoids are major secondary
metabolites produced by microbial cells and are commonly found in nature.
Currently, the production of the carotenoids through chemical synthesis comprises
the large-scale industries. The chemical synthesis do have the risk of possible
toxicity due to the synthetic by-products. Thus, the need arises to search other
sources of carotenoid. Marine sources such as salmonoids and crustaceans are
known to be good sources of oxygenated carotenoids (Goodwin 1986). Natural
pigments have been extracted from plants, insects, algae, cyanobacteria and fungi
(Mortensen 2006). Various microbes have been reported to accumulate these pig-
ments in their cell bodies, including prodigiosin producing gram-negative bacteria
(Lewis and Corpe 1964), b-carotene-producing yeasts, Rhodotorula sp.,
Rhodosporidium sp., Sporobolomyces sp., Sporidiobolus sp. (Buzzini et al. 2007),
Rhodotorula glutinis (Malisorn and Suntornsuk 2008), different carotenoid pro-
ducing green algae, Chlorococcum humicola (Sivathanu and Palaniswamy 2012),
b-carotene-producing microalgae, Dunaliella sp., (Ye et al. 2008), astaxanthin
producing freshwater algae Haematococcus sp. (Li et al. 2011), many pigment
producing thermo-tolerant microalga Coelastrella sp. (Hu et al. 2013) and fungus
strains such as Blakeslea sp., Monascus sp., Paecilomyces lilacinus and macro-
mycetes (Elbandy et al. 2009; Hajjaj et al. 2012; Mapari et al. 2005; Sun et al. 2012;
Zhou and Liu 2010), and fucoxanthin-producing marine diatom, Phaeodactylum
tricornutum (Kim et al. 2012). A Chlorella sp. was reported to produce zeaxanthin
(Singh et al. 2013). A marine cyanobacterium, Synechococcus sp. producing
b-carotene, zeaxanthin and chlorophyll has been suggested as a suitable candidate
for genetic modification to enhance pigment production (Macias-Sanchez et al.
Thus, microalgae are promising vehicles for the production of omega-3 FAs and
carotenoids which possess advantages such as higher growth rate and productivity,
grow in various environments (fresh, brackish or salt water), do not compete for
land, and have high oil productivity (20–50% by dry weight basis) compared to
conventional crops (Singh et al. 2011).

2 Benefits of Omega-3 Fatty Acids and Carotenoids

In the beginning of the twentieth century, dietary fats were recognized as a good
source of energy and fat soluble vitamins. This view later changed following
various studies, including a study conducted on rats reported that ‘dietary fatty acids
were required to prevent deficiency disease’ (Burr and Burr 1929). The health
benefits of omega-3 FAs have been evaluated by a number of clinical studies.
140 A. Gupta et al.

There is evidence for the efficacy of omega-3 FAs in the prevention of sudden death
from cardiovascular disease and in ameliorating rheumatologic conditions (Barrow
2010; Cicero et al. 2015). An early study on fat consumption and health with
Greenland Inuit indicated the cardiovascular benefits of omega-3 FAs. Further
studies produced more evidence for the cardiovascular benefits associated with
omega-3 FAs, including the reduction of the risk of arrhythmias (Asif 2014),
decreasing platelet aggregation (Gao et al. 2013), lowering plasma triglycerides (Qi
et al. 2008) and decreasing blood pressure (Cabo et al. 2012).
The consumption of dietary omega-3 FAs by ulcerative colitis and Crohn’s
disease patients has been shown to produce beneficial weight gain and significant
improvement in disease activity (Papadia et al. 2010). Omega-3 FAs have been
recently shown to have anti-cancer activity, particularly against colorectal cancer
(Cockbain et al. 2012). Many research investigations have been conducted to
understand the positive effects of omega-3 FAs on growth, learning and behavioural
problems of young children. Supplementation of omega-3 FAs during pregnancy
resulted in an increase in birth size (Makrides et al. 2011). Brain and eye devel-
opment in infants is particularly influenced by DHA, which has led to the addition
of DHA to infant formulae. Learning capacity of school going children is increased
by taking DHA-enriched food supplements (Nunes et al. 2014). A number of
studies have provided evidence for the anti- inflammatory effects of omega-3 FAs.
Omega-3 FAs reduce the production of proinflammatory molecules (eicosanoids)
and increase the production of anti-inflammatory molecules such as resolvins and
protectins (Calder 2012). The combined effect of these multiple health benefits has
been a dramatic increase in the market demand for omega-3 FAs.
Carotenoids may assist in the treatment of cancer and improve vision (Johnson
2002). The carotenoid zeaxanthin is endogenous in humans and is an important
component of eye retina (Roberts et al. 2009). Astaxanthin and canthaxanthin
(chemical structures are shown in Fig. 1) exhibit antioxidant and chemo-protective
properties, making them potential candidates for food additives that may act against
the development of neurodegenerative disorders (Yuan et al. 2011). It helps to
neutralize the free radicals generated as a result of oxidative stress. Certain types of
cancer such as colon and hepatic cancers have been found to be inhibited by
astaxanthin in various studies published before (Nagaraj et al. 2012; Palozza et al.
2009). Astaxanthin also inhibits the age-related macular degeneration (Santocono
et al. 2007). As a photoprotectant, astaxanthin can decrease the damages to the skin
cells due to UV radiation (Guerin et al. 2003). It also has potential for the pre-
vention of cardiovascular disease and cataract formation, helps strengthening the
immune system (Guerin et al. 2003; Higuera-Ciapara et al. 2006). Astaxanthin from
Haematococcus pluvalis has been studied for its antihypertensive and
neuro-protective effects, and dietary astaxanthin has been investigated for its effects
on blood pressure, stroke and vascular dementia in animal models (Hussein et al.
2006). Other carotenoids such as b-carotene acts as a precursor for vitamin A; thus,
its sufficient intake can help to prevent diseases caused by vitamin A deficiency,
including blindness, immune dysfunction and skin disorders (Fierce et al. 2008).
Carotenoids are essential nutrient precursors playing a role in human health
6 Extraction of Lipids and Carotenoids from Algal Sources 141

(Fernandez-Garcia et al. 2012). b-carotene also possesses anti-oxidative and

immune modulatory properties that may assist in the prevention of several diseases
such as cancer, cardiovascular disease, rheumatoid arthritis and neurodegenerative
diseases (Dembinska-Kiec 2005). Microbial astaxanthin is used in the cosmetic,
nutraceutical and aquaculture industries. A recent review discussed numerous
marine microbes known for producing pigments such as carotenes and xanthophylls
(Dewapriya and Kim 2014). Marine microalgae accumulate a variety of carotenoids
such as b-carotene, astaxanthin, canthaxanthin, lutein and violaxanthin (Plaza et al.
2009). In fact, some pigments from marine sources have been commercialized.
b-carotene from Dunaliella salina is used in cosmetics and dietary supplement
preparations (Guedes et al. 2011). Nannochlorpsis gaditan has been demonstrated
as a potential source of pigments (Henriques et al. 2007). Other pigments from
marine bacteria, such as prodiginines, carotenes, violacein and quinones also
have commercial potential (Soliev et al. 2011). Astaxanthin from H. pluvalis has
been approved for commercialization by companies such as Algatechnologies Ltd.
(Israel), AstaReal AB (Sweden), Cyanotech (USA) and U.S. Nutra (USA)
(Campenni et al. 2013). Lutein and zeaxanthin support normal vision and skin
health. They are deposited in the lens of the eye, macula lutea, decreasing the
possibility of cataracts and age-related macular degeneration (Alves-Rodrigues and
Shao 2004; Roberts et al. 2009).

3 Cell Disruption Methods for PUFAs Extraction

Cell disruption is an important downstream processing step as it impacts extraction

yields and therefore the cost of bioactive production (Lee et al. 2012). Various
methods–physical, chemical and mechanical are documented for disrupting the
microbial cells, with mechanical methods generally used for large-scale processing
(Günerken et al. 2015). The selection of a suitable method of cell disruption for
extracting intracellular products depends on cell wall strength, intracellular location
of products, stability and the final use of the recovered products (Klimek-Ochab
et al. 2011).

3.1 Lipid Extraction from Microalgae

Generally, lipid (PUFAs) content differs in microalgae species. Microalgae lipid

composition and fatty acids profile depends on the media composition (type of
carbon source and nitrogen source used), temperature, pH and aeration (Halim et al.
2012a). The process of lipid extraction from microalgae is an energy-intensive
process since organic solvents and multiple recovery methods are applied during
downstream processing (Fig. 2). Selection of efficient microalgae species and
suitable lipid extraction methods is important for commercial production
142 A. Gupta et al.

Cell disruption
Algae (Sonication,
medium Homogenisation etc)

DAD1 A, Sig=470,4 Ref=off (ADARSHA\11K25181.D)



10 Extraction



0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 min

Fig. 2 Flow diagram represents different steps in algal biomass processing for the extraction of
carotenoids. Growth of algae (Thraustochytrium sp.) shown in a flask containing carotenoids.
HPLC chromatogram indicating purity of the extracted compound

(McMillan et al. 2013). Therefore, a suitable extraction technique is a prerequisite

for microalgal lipid extraction. Lipid extraction efficiency is dependent on the
polarity of the solvent and combination of solvent mixture (Lee et al. 2010;
Ryckebosch et al. 2014). The combination of a polar and nonpolar solvent mixture
in some cases has extracted more lipids from microalgae (Ramluckan et al. 2014;
Ryckebosch et al. 2012). For example, the Bligh and Dyer method uses chloroform
and methanol for lipid extraction from a range of biological samples (Ramluckan
et al. 2014). The use of chloroform and ethanol in a 1:1 ratio showed maximum
lipid extraction from Chlorella sp. (Ramluckan et al. 2014), whereas a combination
of dichloromethane and ethanol increased lipid extraction efficiency by 25% in the
same organism (Ranjith et al. 2015).
Microalgae cell walls block the release of lipid present inside the cells. To get
higher product recovery and quality lipids with lower operating costs from
microbial cells, a suitable cell disruption method is required. Cell disruption
enhances the release of intracellular lipids from microalgae by improving the access
of the extracting solvent to fatty acids (Halim et al. 2012b). Cell disruption methods
used in microalgae lipid extraction are classified as chemical and mechanical.
Chemical cell disruption methods involve use of acids, alkalis, enzymes or osmotic
shocks and mechanical methods use microwave, ultrasonication, bead mill, drying
or supercritical fluid extraction. The choice of method influences lipid extraction
yields from a range of microalgae (Lee and Han 2015; Lee et al. 2010; McMillan
et al. 2013; Zheng et al. 2011). Table 1 shows some of the methods used with
various algal species for extracting lipids.
Recently, graphene (graphene oxide) nanosheets have been used to cut through
the cell membrane of E. coli to extract large amounts of phospholipids from the
membranes. This process further caused the degradation of E. coli membranes and
reduced bacterial viability (Ti et al. 2013), thus leading a way for graphene-based
recovery of lipids for biomedical applications.
6 Extraction of Lipids and Carotenoids from Algal Sources 143

Table 1 Some of the lipid extraction methods used with various algal species
Microalgae Cell disruption method Maximum Reference
lipid yield
Schizochytrium Bead mill 49.4 Byreddy et al.
S31 (2016)
Schizochytrium Osmotic shock 48.7 Byreddy et al.
S31 29.1 (2015)
Chlorella vulgaris Ultrasonication 55 dos Santos
et al. (2015)
Scenedesmus sp. Freeze drying + Microwave 29.6 Guldhe et al.
Chlorella vulgaris Pressure-assisted ozonation 27 Huang et al.
Mixed culture Microwaves 33.7 de Souza Silva
et al. (2014)
Chlorella vulgaris H2O2 + FeSO4 17.34 Steriti et al.
Nannochloropsis Microwave 38.3 Wahidin et al.
sp. (2014)
C. vulgaris Ultrasonication 52.5 Araujo et al.
Chlamydomonas Osmotic shock NA Yoo et al.
reinhardtii (2012)
C. vulgaris (SAG Grinding + microwaves + sonication 9.82 Sostaric et al.
211-12) (2012)
Synechocystis Microwave + pulsed electric fields 9–13 Sheng et al.
PCC 6803 (2012)
Chlorella vulgaris Microwaves NA Prommuak
et al. (2012)
Scenedesmus sp. High-pressure homogenization 24.9 Cho et al.
Synechocystis Pulsed electric field 25–75 Sheng et al.
PCC 6803 (2011)
Chlorella sp. Sonication 21 Prabakaran
Nostoc sp. Sonication 18 and Ravindran
Tolypothrix sp. Microwave 16 (2011)
Botryococcus sp. Microwave 28.6 Lee et al.
Chlorella vulgaris 10 (2010)
Scenedesmus sp. 11
Scenedesmus Wet milling 25.3 Shen et al.
dimorphus Bead-beater 18.8 (2009)
144 A. Gupta et al.

4 Extraction Methods for Carotenoids

Algal carotenoids have been investigated in various marine microorganisms such as

Chlorella and thraustochytrids (Gupta et al. 2013; Singh et al. 2013, 2015a). Total
extraction of carotenoids from potential microbes is a huge challenge as this
influences the cost of the production process. Mechanical, chemical or
enzyme-assisted extraction methods have been implemented as a viable option
(Joana Gil-Chávez et al. 2013; Monks et al. 2012; Singh et al. 2015a). Further, the
use of statistical methods such as response surface methodology might assist in
improving the higher yields. Various extraction methods to develop a cost-effective
and higher carotenoid yield are discussed in preceding paragraph. Various extrac-
tion methods used for carotenoid extraction from various algal species are presented
in Table 2.

Table 2 Summary of various methods used for extracting carotenoids

Method used Microorganism/plant Compound of Reference
source interest
Mechanical Thraustochytrids Carotenoids Armenta et al.
methods and SE (2006)
Supercritical fluids Synechococcus sp. Carotenoids and Macias-Sanchez
CO2 chlorophyll et al. (2007)
SE Decane Dunaliella salina b-Carotene Mojaat et al. (2008)
Subcritical carbon Ground red paprika Carotenoids Rutkowska and
dioxide (plant fruits) Stolyhwo (2009)
Supercritical fluids Nannochloropsis Carotenoids Macías-Sánchez
CO2 gaditana, et al. (2009)
Dunaliella salina and
Synecococcus sp.
Supercritical fluids Scenedesmus b-carotene and Macías-Sánchez
CO2 almeriensis lutein et al. (2010)
PLE hexane, Himanthalia elongate; Fatty acids and Plaza et al. (2010)
ethanol, and water Synechocystis sp. carotenoids
Enzyme-assisted Phaffia rhodozyma Carotenoids Michelon et al.
extraction (2012)
UAE, n-heptane Spirulina platensis b-carotene Dey and Rathod
SE, mechanical Thraustochytrids Carotenoids Gupta et al. (2013)
Ultrasonication and Thraustochytrids Carotenoids Singh et al. (2015b)
SE solvent extraction
PLE pressurized liquid extraction
UAE ultrasonic-assisted extraction
6 Extraction of Lipids and Carotenoids from Algal Sources 145

4.1 Cell Disruption Employing Chemicals/ Solvents

Carotenoid extraction using chemicals has been followed as a good replacement for
the physical methods due to its simplicity, minimum extraction time and
cost-effectiveness. Strong acids such as sulphuric acid and hydrochloric acid and
mild acids such as acetic acid, oxalic acid and citric acid including sodium bicar-
bonate and organic solvent DMSO (dimethyl sulfoxide) have been used to disrupt
the cells for extracting colour pigments into acetone (Singh et al. 2013). Other polar
solvents such as methanol and acetone were used to extract canthaxanthinfrom
dried E. coli biomass (Scaife et al. 2012). The selection of the solvents can be
attributed to the type of the microbe and type of carotenoids (Cha et al. 2010),
which suggests that efficiency of the solvent extractability is dependent on the
permeability of the algal cell wall. The determination of the degree of affinity
towards the chemicals to be extracted is really important that is why the solvent
selection is critical. Moreover, the solvent assists in the breakdown of the cells as
well apart from dissolving the chemicals to be extracted. The amounts of extracts
can be estimated by the contact time between the cell extracts and the solvent. Not
using a powerful solvent might avoid the degradation of the cell extracts, and
increasing the time of solvent extraction could elevate the yield of the extraction
process (Henriques et al. 2007). When the cell lysis power of the solvent is not too
high, which can be intentional to avoid damage of the compounds to be extracted, a
longer period of extraction may increase the yield of this process. As Singh et al.
(2013) observed that solvents such as hexane and ethyl acetate in combination
enriched the specific carotenoids (b-carotene over zeaxanthin) after the DMSO
extraction, it can be assumed that each strain and carotenoid type should be tested.
All the cell suspensions were washed with water few times to make sure that there
are no traces of the organic and inorganic acids and solvents. DMSO-assisted cell
disruption has been found effective when compared to other solvents such as
sodium bicarbonate and other strong (hydrochloric acid) and mild acids (acetic
lactic acid) using Phaffia rhodozyma (Michelon et al. 2012), which is a common
analytical technique used before. However, the toxic compounds (in the extract)
derived from DMSO hinders the possibility of its usage in the food industries, thus
limiting its application for industrial production which also does not fulfil the
existing laws for food industries. Further, the problem with the use of acids in cell
disruption is the possibility of the degradation of the carotenoids due to acids. It was
shown by Singh et al. (2013) that total carotenoid production decreased with the use
of strong acid such as HCl and sulphuric acid and increased with weaker acids, but
the carotenoid content was still found to be more than the direct extraction. The
efficient rupturing of the cell wall of Chlorella sp. by the weaker acids and
degradation of carotenoids due to strong acids were assumed when strong acids did
not yield more carotenoids. Although the sodium bicarbonate treatment of the
Chlorella sp. gave highest carotenoid yield, thus assuring different cell walls
behave differently and the optimal chemical treatment has to be performed with
each type of microbial system. Previously, other microbial cells have been
146 A. Gupta et al.

subjected to the HCl treatment to recover more carotenoids before extracting car-
otenoids from them (Michelon et al. 2012). The hydrochloric acid treatment of the
cells resulted into higher carotenoids concentration when compared to lactic and
acetic acids. The reason was described by Ni et al. (2008) by the values of constant
acidity (pKa). The pKa values for hydrochloric acid, lactic acid and acetic acid are
7, 3.83 and 4.74, respectively. Thus, great efficiency was observed in the yeast cell
disintegration with the use of stronger acid (Ni et al. 2008). Moreover, different
types of acids might favour different types of the carotenoids. b-carotene content
was more favoured by strong acids, while zeaxanthin was favoured by milder acids
(Singh et al. 2013).

4.2 Cell Disruption by Mechanical Methods for Carotenoid


The traditional cell disruption methods such as bead milling and high-pressure
homogenization have shortcomings that include higher costs, generation of fine cell
debris, longer extraction process and cleaning of the equipment used (Monks et al.
2012). However, various mechanical means have been applied such as bead beat-
ing, maceration with liquid nitrogen, wet milling, homogenization or sonication to
disrupt the algal cells followed by the extraction in acetone (Henriques et al. 2007;
Singh et al. 2013). Ultrasonication method is easy to use and the solvent treatment
before the sonication process assist the extraction process; however, this has to be
performed in closed vessel with ice bath to prevent any damage from the heat
generated (Henriques et al. 2007). Microwave-assisted cell disruption (MAE) or
with the use of supercritical fluids (carbon dioxide) and pressurized liquid extrac-
tion have also been used for lipids or carotenoid extraction from wet or dry biomass
(Cha et al. 2010; Monks et al. 2012). In general, the generation of heat during the
mechanical disruption of the cells might degrade the carotenoids and can be pre-
vented by using an ice bath during the process. There are several advantages of
using supercritical carbon dioxide (SC-CO2) cell disruption method that includes
the easier separation of the solvent without leaving any residue, cheaper, non-toxic
and non-flammable fluid, easy diffusion into the cell membrane due to its high
diffusivity and non-polarity characteristics. Although the efficiency of the method
depends on the characteristics of the microbial cells such as the cell wall (Egyházi
et al. 2004), Singh et al. (2013) found the ultrasonication method as the best method
to disrupt the cells to obtain the best carotenoid yield with 40-fold increment than
direct solvent extraction. In previous reports using the algal cells or yeast cells, bead
beating method and ultrasonication method was found to be the most suitable
(Michelon et al. 2012; Shen et al. 2009). Michelon et al. (2012) showed that the use
of chemical- or mechanical-mediated cell disruption over direct solvent extraction
improved the total carotenoid yield. It was the same with the thraustochytrids
as well with ultrasonication method yielding higher amount of carotenoids
6 Extraction of Lipids and Carotenoids from Algal Sources 147

(Armenta et al. 2006). The advantage of using ultrasonication to disrupt the cells
comprises of non-toxicity, and no carotenoid degradation issues when compared to
the use of acids and the extracted carotenoids can be directly used in food and feed
industries (Singh et al. 2015a). Moreover, ultrasonication would be a preferable
technique over high energy-intensive methods such as pressurized liquid extraction
or supercritical extractions (Joana Gil-Chávez et al. 2013).

4.3 Enzyme-Mediated Cell Disruption for Carotenoid


Recently, microbial cells have been disrupted by commercially available lytic

enzymes from biological sources. This area has been gaining interest (Michelon
et al. 2012). Michelon and co-workers successfully integrated the mechanical and
enzymatic methods to effectively achieve the highest yield concentration.
Maceration using diatomaceous earth and enzymatic lysis yielded highest specific
concentration of carotenoids and extractability (190.35 µg/g and 122.25%). In
comparison with the cell disruption by DMSO, the combination of maceration with
diatomaceous earth and enzymatic lysis, an increase in 18% in the carotenoid
concentration was observed.
There is limited literature regarding the use of enzymatic lysis in the extraction
of carotenoids. The disintegration of the Phaffia rhodozyma cells using the com-
mercial enzymes from Trichoderma harzanum was reported by Bjerkeng et al.
(2007) when authors used this enzyme to assess the astaxanthin digestibility and its
retention in muscles in the Atlantic salmon (Bjerkeng et al. 2007).

5 Future Perspectives

In recent years, intensive research has been carried on the extraction of omega-3
FAs and carotenoids from algal sources, since these bioactives have promising
health promoting applications. Innovation both at the laboratory and Industry level
should be aimed in devising upstream and downstream strategies for cost-effective
omega-3 fatty acid and carotenoid production. New marine microorganisms that
accumulate high concentration of carotenoids naturally from varied habitats con-
tinue to pose challenge for effective downstream processing. Innovative method-
ologies of enzymatic extraction supported by nanomaterial-based purification will
facilitate improved carotenoid yields, thus facilitating food processing applications.
148 A. Gupta et al.


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Chapter 7
Magnetic Particles for Microalgae
Separation and Biotechnology

Ivo Safarik, Kristyna Pospiskova, Eva Baldikova

and Mirka Safarikova

1 Introduction

Microalgae form a diverse collection of microorganisms that conduct

oxygen-evolving photosynthesis. Both prokaryotic and eukaryotic photosynthetic
microorganisms are covered in this group; they can grow rapidly, live even in harsh
conditions, and are present in almost all ecosystems around the globe. Examples of
prokaryotic microorganisms are cyanobacteria (Cyanophyceae) and eukaryotic
microalgae are, for instance, green algae (Chlorophyta) and diatoms
(Bacillariophyta) (Mata et al. 2010). Large-scale cultivation of microalgae is a
promising way to produce large amounts of biomass containing a wide variety of
high-value products with considerable commercial value, with great potential use in
the aquaculture, food, feed, pharmaceutical, cosmetic, fuel, and other industries.
Species of Chlorella and Arthrospira (also known as Spirulina) are sold as dried
biomass with a high content of proteins, carotenoids, phycobilin pigments, fatty
acids, sterols, polyhydroxyalkanoates, polysaccharides, or vitamins (mainly B12, C,

I. Safarik (&)  M. Safarikova

Department of Nanobiotechnology, Biology Centre, ISB, Academy of Sciences,
Na Sadkach 7, 370 05 Ceske Budejovice, Czech Republic
I. Safarik  K. Pospiskova
Regional Centre of Advanced Technologies and Materials, Palacky University,
Slechtitelu 27, 783 71 Olomouc, Czech Republic
I. Safarik  E. Baldikova  M. Safarikova
Department of Nanobiotechnology, Institute of Nanobiology and Structural
Biology of GCRC, Academy of Sciences, Na Sadkach 7, 370 05 Ceske
Budejovice, Czech Republic
E. Baldikova
Department of Applied Chemistry, Faculty of Agriculture, University of South
Bohemia, Branisovska 1457, 370 05 Ceske Budejovice, Czech Republic

© Springer International Publishing AG 2017 153

M. Puri (ed.), Food Bioactives, DOI 10.1007/978-3-319-51639-4_7
154 I. Safarik et al.

and D2). The green alga Haematococcus pluvialis is the producer of the carotenoid
astaxanthin (a potent antioxidant) used as a food and feed supplements. Microalgal
species, including Anabaena, Botryococcus, Dunaliella, Nostoc, Parietochloris,
Porphyridium, Scenedesmus, and Synechococcus, are exploited for the production
of antioxidants (carotenoids, especially beta-carotene) and important vitamins (e.g.,
retinol, biotin, thiamine, riboflavin, folic acid, L-ascorbic acid, and tocopherol).
Furthermore, the increasing consumer awareness of the therapeutic properties of
omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic and
docosahexaenoic acids, and the expensive procedures for extracting them from fish
oils boost the research toward marine microalgae (e.g., Chrysophyceae,
Eustigmatophyceae, Chlorophyceae, and Cryptophyceae), where some species are
capable to provide high levels of PUFAs (Sastre 2012; Encarnação et al. 2015;
Borowitzka 2013; Lambreva et al. 2015; Bishop and Zubeck 2012; Guarnieri and
Pienkos 2015). Microalgae are also intensively studied as a rich source of
polysaccharides and oils important for biorefinery processes (Gouveia 2011).
Microalgae research and technology employ a wide variety of techniques,
instrumentation, and materials. In this chapter, the attention is focused on the
application of magnetically responsive nano- and microparticles which have already
been used in many biotechnology applications (Borlido et al. 2013; Garcia et al.
2015; Safarik et al. 2012; Safarik and Safarikova 2009). In the microalgae tech-
nology, they can be especially used for magnetic flocculation of microalgae cells
from cultivation media, magnetic modification of microalgae cells, magnetic iso-
lation of target compounds produced by microalgae, magnetic detection of algae
produced toxins, and preparation of magnetically responsive catalysts applicable in
microalgae biotechnology. Despite the fact that there is a real boom of studies
employing magnetic particles for microalgae separation from large volumes, other
areas of microalgae research and technology have not fully employed the potential
offered by magnetically responsive materials. This chapter should stimulate the
microalgae research community in finding new progressive applications of mag-
netically responsive materials.

2 Preparation of Magnetic Materials

Magnetically responsive biocompatible materials have found many important

applications in various areas of biosciences, medicine, biotechnology, and envi-
ronmental technology. Both basic ferro- and ferrimagnetic materials (e.g., mag-
netite, maghemite, and different types of ferrites) and magnetically responsive
composite materials formed by magnetically labeled diamagnetic materials are of
great importance. One of the most significant properties of magnetic materials is
their possibility to be selectively separated (removed) from the complex samples
using an external magnetic field (e.g., by means of an appropriate magnetic sepa-
rator, permanent magnet, or electromagnet). This process enabling efficient selec-
tive separation of magnetic materials is very important for bioapplications due to
7 Magnetic Particles for Microalgae Separation and Biotechnology 155

the fact that absolute majority of biological materials have originally diamagnetic
properties (Safarik and Safarikova 2009).
Various procedures have been used to synthesize magnetic nano- and
microparticles, such as classical coprecipitation, reactions in constrained environ-
ments (e.g., microemulsions), sol-gel syntheses, hydrolysis and thermolysis of
precursors, sonochemical and microwave reactions, hydrothermal reactions, flow
injection syntheses, electrospray syntheses, and mechanochemical processes
(Laurent et al. 2008; Safarik et al. 2011; Wu et al. 2015).
Coprecipitation technique (aging stoichiometric mixture of ferrous and ferric
salts in aqueous alkaline medium) is the simplest procedure to synthesize large
amount of iron oxide nanoparticles, either in the form of magnetite (Fe3O4) or
maghemite (c-Fe2O3). The addition of chelating organic anions (e.g., citric, glu-
conic, or oleic acids) or polymer surface complexing agents (dextran, carboxy-
dextran, starch, or polyvinyl alcohol) during the formation of magnetite can help to
control the size of the nanoparticles (Laurent et al. 2008).
Synthesis of uniform iron oxide nanoparticles can be performed in synthetic and
biological nanoreactors, such as water-swollen reversed micellar structures in
nonpolar solvents, apoferritin protein cages, dendrimers, cyclodextrins, and
Hydrothermal syntheses of magnetite nanoparticles are carried out in aqueous
media in reactors or autoclaves at high pressure and temperature. The sol-gel
process is based on the hydroxylation and condensation of molecular precursors in
solution, originating a “sol” of nanometric particles; further condensation and
inorganic polymerization followed by heat treatments are needed to acquire the final
crystalline state. The polyol process employs, e.g., polyethylene glycol as a solvent
exhibiting high dielectric constants, which can dissolve inorganic compounds.
Polyols also serve as reducing agents as well as stabilizers to control particle growth
and prevent interparticle aggregation (Laurent et al. 2008).
The flow injection synthesis employs continuous or segmented mixing of
reagents under laminar flow regime in a capillary reactor which enables precise
external control of the process. The obtained magnetite nanoparticles had a narrow
size distribution in the range of 2–7 nm. Spray pyrolysis and laser pyrolysis enable
high rate production of nanoparticles. In spray pyrolysis, a solution of ferric salts
and a reducing agent in organic solvent is sprayed into a series of reactors, where
the aerosol solute condenses and the solvent evaporates. Maghemite particles with
size ranging from 5 to 60 nm with different shapes have been obtained using
different iron precursor salts in alcoholic solution (Laurent et al. 2008; Safarik et al.
A wide variety of chemical reactions accelerated by microwave irradiation of
reactants has been observed. Recently, a simple, quick, and cost-effective micro-
wave method to prepare relatively uniform magnetite nanoparticles directly from
Fe2+ salts has been developed; the formation of magnetic nanoparticles using
microwave method requires only a few seconds or minutes (Zheng et al. 2010;
Safarik and Safarikova 2014). Nanosized iron oxide powders can also be synthe-
sized via a mechanochemical reaction. Ball milling of ferrous and ferric chlorides
156 I. Safarik et al.

with sodium hydroxide led to the formation of magnetite (Lin et al. 2006) or
maghemite (Safarik et al. 2014a). To avoid agglomeration, the excess of NaCl is
usually added to the precursor before ball milling.
In order to obtain biocompatible magnetically responsive materials, stabilization
of the prepared iron oxide nano- and microparticles by appropriate modification of
their surface or by their incorporation into appropriate biocompatible matrix is
usually necessary. Compounds with carboxylic and phosphate functional groups
(e.g., citric and oleic acids) can bind to the surface of magnetic particles and
stabilize them. Water-based ferrofluids can also be stabilized by ionic interactions,
using, e.g., perchloric acid or tetramethylammonium hydroxide (Laurent et al.
Biocompatible (bio)polymers are also used for magnetic particles’ stabilization
and modification. Dextran has often been utilized as a polymer coating mostly
because of its excellent biocompatibility. The formation of magnetite in the pres-
ence of dextran 40,000 was reported for the first time in 1980s. Other common
biopolymer coatings are formed, e.g., by carboxymethylated dextran, carboxy-
dextran, starch, chitosan, alginate, arabinogalactan, or glycosaminoglycan, while
polyethylene glycol (PEG) and polyvinyl alcohol (PVA) represent biocompatible
synthetic polymers (Laurent et al. 2008).
Magnetic nanoparticles are often a magnetic component part of magnetically
responsive composite microparticles formed from various synthetic polymers,
biopolymers, inorganic materials, microbial cells, or plant materials (Safarik et al.

3 Magnetic Materials for Microalgae Harvesting

Algal cells can be harvested using various solid–liquid separation steps and
methods such as centrifugation, sedimentation, flocculation, filtration, flotation, or
by a combination of these methods. However, current harvesting methods have
many disadvantages. Therefore, new harvesting procedures employing different
types of magnetic nano- and microparticles have been developed and tested also for
industrial-scale harvesting of algal biomass (Safarik et al. 2016; Wang et al. 2015).
Magnetic flocculation of microalgae enables simple magnetic separation. Naked
magnetite is an efficient flocculation agent for the separation and removal of
microalgal biomass. Magnetic iron oxide particles can be prepared using various
methods (see above) and subsequently applied for microalgae harvesting. Magnetite
particles synthesized by chemical coprecipitation with an average diameter of
approximately 10 nm and an isoelectric point of approximately 7 were efficient in
harvesting Botryococcus braunii, Chlorella ellipsoidea, and Nannochloropsis
maritima (Xu et al. 2011; Hu et al. 2013).
An extremely simple procedure for the magnetic modification of algal cells
which is based on the use of microwave-synthesized magnetic iron oxide nano- and
microparticles has been developed recently. Two very inexpensive precursors are
7 Magnetic Particles for Microalgae Separation and Biotechnology 157

employed (ferrous sulfate heptahydrate and sodium or potassium hydroxide); after

alkalization of ferrous salt and formation of mixed iron hydroxides precipitate, the
suspension underwent microwave treatment (a regular kitchen microwave oven can
be used successfully) and nano- and microparticles of magnetic iron oxides were
formed (Zheng et al. 2010; Safarik and Safarikova 2014). Mixing of magnetic
particles with algal cell (C. vulgaris) suspensions caused cell flocculation and
magnetically responsive cell aggregates (usually ca. 100–300 lm in diameter)
formation. Naked magnetite has ion-exchange characteristics, and the separation is
primarily based on the electrostatic interactions between the magnetite and the algal
cells. In addition, Fe2+/3+ ions released from the magnetic particles’ surface may act
as flocculating agents and benefit the harvesting process (Prochazkova et al. 2013).
Due to the negative surface charge of microalgal cells, a positive charge on the
surface of magnetic particles improves separation. Functionalization of the mag-
netic particles’ surface with cationic groups improves the cells’ magnetic floccu-
lation. Two potential strategies can be used based on the place where a positively
charged polyelectrolyte (polymer) is bound. One strategy consists in coating the
cells with a suitable polymer in the first step and then attaching the naked magnetic
particles; in another approach, the naked magnetic particles are first
surface-functionalized with a polyelectrolyte and then bound to the microalgal cells
(Toh et al. 2014b; Lim et al. 2012).
Recently, several review papers summarizing the application of magnetic
particles for microalgae harvesting have been published (Lee et al. 2015; Safarik
et al. 2016; Wang et al. 2015).

4 Magnetic Modification of Microalgae Cells and Their


Magnetic modification of prokaryotic and eukaryotic cells leads to the formation of

extremely interesting biocomposites. The common characteristics of all magneti-
cally modified cells are their specific interactions with an external magnetic field
(Safarik et al. 2014b). The individual procedures used for magnetic modification of
microalgae cells are given below (Safarik et al. 2016).
Dried Chlorella vulgaris cells were magnetically modified using perchloric
acid-stabilized magnetic fluid in acidic buffer which led to the deposition of
magnetic iron oxide nanoparticles onto the cell surface and formation of magnet-
ically responsive algal cells. Modified cells were applied as a new inexpensive
magnetic adsorbent for the removal of six water-soluble organic dyes. Data from
dye adsorption process were fitted to Langmuir isotherm, and the maximum
adsorption capacities were 24.2 and 257.9 mg of dye per g of dried magnetically
modified cells for Saturn blue LBRR and aniline blue, respectively. Increasing pH
value can positively affect the adsorption capacities of some dyes (e.g., crystal
violet and safranin O) (Safarikova et al. 2008). The same acid magnetic fluid has
158 I. Safarik et al.

been successfully used for magnetic modification of selected diatoms or

chrysomonads (Diadesmis gallica, Mallomonas kalinae); in this case, magnetic
modification can be performed in methanol (Kratosova et al. 2013). Diatom par-
ticles have also been magnetically labeled with human serum albumin (HSA)-
coated iron oxide nanoparticles, prepared from oleic acid/oleylamine-coated
nanoparticles after subsequent surface exchange with dopamine. Diatoms were
contacted with the modified particles in PBS at room temperature for 2 h. Multiple
amine groups on the surface of the MNPs ensured their partial positive charge,
making them appropriate to interact with negatively charged cell surfaces (Todd
et al. 2014).
Cationic polyelectrolyte promoted effective attachment of iron oxide nanopar-
ticles onto microalgal cells through electrostatic attraction. Chitosan and
poly(diallyldimethylammonium chloride) (PDDA)-modified magnetic iron oxide
nanoparticles can easily modify Chlorella sp. cells during simple mixing.
Subsequent magnetophoretic separation of magnetized Chlorella cells was carried
out under low-gradient magnetic separation (LGMS) with a NdFeB permanent
magnet in an inhomogeneous magnetic field with magnetic field gradient
(∇B) < 80 T/m. It was also shown that magnetic nanoparticles can enter the cells.
The internalization of nanoparticles may be through a passive uptake or adhesive
interaction (Toh et al. 2014a, b). PDDA also promoted effective attachment of
IONPs onto other microalgae cells (Lim et al. 2012; Toh et al. 2012).
Another procedure for coating algal cells by magnetite nanoparticles via elec-
trostatic interactions has been described recently. (Poly)allylamine hydrochloride-
stabilized positively charged MNPs (average diameter around 15 nm) were used for
the magnetization of living Chlorella pyrenoidosa cells. The single-step magneti-
zation procedure is very simple and consists of the dropwise introduction of the
aqueous suspension of algal cells into the nanoparticles solution, followed by
intensive shaking for 10 min. TEM images demonstrated the uniform layer of
MNPs on the cell walls with the thickness around 90 ± 20 nm (Fakhrullin et al.
2010). The modified cells have been employed for the construction of a whole-cell
amperometric herbicide biosensor using screen-printed electrodes. The electrode
was connected with supporting tetrafluoroethylene plate with inserted NdFeB
cylindrical magnet (2 mm in diameter) in the cavity below the working electrode
area to produce strong magnetic field in the vicinity of the electrode. The mag-
netically modified cells were mixed with the electron mediator (K3Fe(CN)6) and
electrolyte (Na2SO4), and the drop of this suspension was placed onto the working
area of the electrode. The magnetized cells were immediately assembled above the
magnet in the area of magnetic field. This biosensor was applied for the detection of
triazine herbicides, inhibitors of photosynthetic activity. The biosensor was able to
detect atrazine (from 0.9 to 74 mM) and propazine (from 0.6 to 120 mM) (the
limits of detection 0.7 and 0.4 mM, respectively) (Zamaleeva et al. 2011). The same
magnetic C. pyrenoidosa cells were used as “nanobait,” which was ingested by a
nematode Caenorhabditis elegans as a sole food source. The magnetized cells were
localized inside the digestive tract of the worms. Delivery of modified cells resulted
7 Magnetic Particles for Microalgae Separation and Biotechnology 159

in magnetic labeling of living nematodes, rendering them magnetically responsive

(Daewlaetsina et al. 2013).
A magnetic material based on montmorillonite, a commonly used clay, was
manufactured by the supporting of Cu(II)/Fe(III) oxides on pillared montmorillonite
prepared from Na-montmorillonite using aluminum polycation. This magnetic
material was used for modification and subsequent magnetic removal of the
cyanobacteria Microcystis aeruginosa (Gao et al. 2009).
A specific modification procedure was employed for the magnetization of
cyanobacterium Synechocystis sp. PCC 6803. This photosynthetic microorganism was
immobilized on amine-functionalized MBs (Dynabead M-270 Amine) by 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide (EDC) – N-hydroxysulfosuccinimide (NHS)
coupling chemistry. The carboxylic groups present in the Synechocystis cell wall were
activated with EDC and then subjected to NHS to produce amine-reactive NHS esters
enabling interaction with amine-functionalized MBs to produce covalent amide bonds.
Individual microorganisms immobilized on the magnetic beads (transporting objects)
were directionally manipulated using a magnetic rail track, which was able to
manipulate particles as a result of asymmetric forces from the curved and flat edges of
the pattern on the disk. Transporting objects were then successfully trapped in a
magnetic trapping station pathway (Venu et al. 2013).
Immunomagnetic detection and modification of cells is based on the use of
magnetic nano- or microparticles with immobilized monoclonal- or
polyclonal-specific antibody enabling their selective attachment to target cells.
After incubation, target cells with attached magnetic particles are isolated with the
help of an appropriate magnetic separator. In the direct method, the antibodies
against target surface epitopes are attached to the magnetic particles, which are then
added to the cells containing sample. In the indirect method, the cell suspension is
incubated with free primary antibodies, which bind to the target cells; then, mag-
netic particles with immobilized secondary antibodies, protein A or protein G, are
added, enabling the beads to bind rapidly and firmly to the primary antibodies on
the target cells. Alternatively, primary antibodies can be biotinylated, and magnetic
particles with immobilized streptavidin are used for capturing the target cells
(Safarik and Safarikova 1999; Safarik et al. 2014b). Recently, the cells of toxic
dinoflagellate Alexandrium fundyense have been isolated from natural seawater
plankton samples using Dynabeads with immobilized monoclonal antibody against
the surface antigens. Both direct and indirect procedures were tested as well as three
types of modified magnetic beads (MBs) (streptavidin, and two secondary anti-
bodies: sheep anti-mouse and goat anti-mouse). Optimal indirect bead attachment
protocol enabled the separation of 90% of the labeled A. fundyense cells in unialgal
cultures (with non-specific binding from 5 to 10%). Simpler “direct” technique
(with recovery 80% and non-specific binding ca. 2%) enabled pre-coating of beads
with the specific antibody in bulk before use, which shortened the procedure and
eliminated target cell losses (Aguilera et al. 1996, 2002). Alternatively, purified
polyclonal antibodies against Heterosigma akashiwo were immobilized to
160 I. Safarik et al.

carboxylated MBs Seradyn; immunomagnetic separation enabled to detect an

important species responsible for harmful algal blooms (Huang et al. 2012).
Immobilization of algal cells into different non-magnetic matrices has been
described many times (Mallick 2002). However, only exceptional examples of algal
cell entrapment into magnetic gels can be found in the literature such as the
entrapment of microalgal cells (C. vulgaris) within a non-toxic polyvinylpyrroli-
done (PVP) polymer matrix containing superparamagnetic magnetite nanoparticles
using continuous flow vortex fluidic device. High entrapment efficiency (up to
95%) was obtained. Entrapped cells can be separated from the PVP matrix using
mild sonication (Eroglu et al. 2013).

5 Magnetic Separation of Microalgae Biologically Active


Isolation, separation, and purification of various types of biologically active com-

pounds, including proteins, peptides, (poly)saccharides, nucleic acids, oligonu-
cleotides, lipids, as well as of other specific molecules, are used in almost all
branches of biosciences and biotechnologies. Recently, separation techniques,
capable of treating dilute solutions or solutions containing only minute amounts of
target molecules, in the presence of vast amounts of accompanying compounds,
even in the presence of particulate matter, have become available for both small-
and large-scale processes. Specific group of separation processes is based on the use
of magnetically responsive materials, which can be applied for magnetic affinity,
ion exchange, hydrophobic or adsorption batch separation processes, applications
of magnetically stabilized fluidized beds or magnetically modified two-phase sys-
tems (Safarik and Safarikova 2004; Franzreb et al. 2006).
The basic principle of batch magnetic separation is very simple. Magnetically
responsive carriers bearing an immobilized affinity or hydrophobic ligand or
ion-exchange groups, or magnetic biopolymer particles having affinity to the target
compound, are mixed with appropriate source of biomolecules and incubated in
order to capture the target compound(s). Then, the whole magnetic complex is
easily and rapidly removed from the suspension using an appropriate magnetic
separator. After washing out the contaminants, the isolated target compound(s) can
be eluted and used for further work (Safarik and Safarikova 2004).
Large amounts of biologically active compounds have already been isolated
using magnetic separation techniques (Franzreb et al. 2006; Safarik and Safarikova
2004). Surprisingly, this advanced technology has not found many applications in
microalgae research and technology. The few existing examples should stimulate
the interest of the broad microalgae research community.
Microalgae cells have to be disintegrated in order to enable efficient separation
of intracellular components. Mild but efficient cell disruption is necessary. Various
mechanical procedures (e.g., bead milling, high-pressure homogenization,
7 Magnetic Particles for Microalgae Separation and Biotechnology 161

high-speed homogenization, ultrasonication, microwave treatment, explosive

decompression, and pulsed electric field treatment), chemical cell disruption, and
enzymatic cell lysis can be successfully used. A review paper summarizing the
microalgae disintegration procedures has appeared recently (Gunerken et al. 2015).
Magnetic particles have been successfully used for the isolation of algal cells
that derived biologically active compounds and toxins. Genomic DNA from fixed
cultures of Alexandrium minutum cultures was separated using silica-magnetite
beads (Taylor et al. 2000); in this case, the silica-magnetite beads adsorb DNA
under high salt conditions and release it when salt concentration decreases.
Alternatively, DNA was separated using DEAE-agarose-magnetite microparticles
(Bruce et al. 1996), and the ionically bound DNA was subsequently eluted via ion
competition. Among the DNA extraction techniques compared, the magnetic par-
ticle-based methods provided the best results (Bertozzini et al. 2005).
A magnetic bead-based system for DNA isolation utilizing monodisperse beads
(Dynabeads DNA DIRECT; originally designed for isolating PCR-ready DNA
from human whole blood) was employed to produce a general approach for
PCR-ready DNA. Different types of algae (Gyrodinium aureolum, Heterocapsa
triquetra, Scrippsiella trochoidea, Chlorella vulgaris, Chlamydomonas reinhardtii,
Caulacanthus ustulatus, Chrysochromulina polylepis, and Ceramium strictum)
were used; all algae tested gave DNA yields in the range 100–200 ng/105 cells (50–
100% relative to phenol-/chloroform-based isolations). Also, potential
PCR-inhibitory polysaccharides as well as other inhibitory compounds, such as
polyphenols, were removed (Rudi et al. 1997).
Silica-coated, superparamagnetic nanoparticles conjugated to a DNA capture
sequence (probe) complementary to a specific region of 5.8S rDNA of the genus
Alexandrium have been used for the specific purification of microalgal DNA from
the cells of Alexandrium catenella in cultured or environmental samples. Then, a
PCR assay was performed with primers specific for the genus Alexandrium to
assess the specificity and sensitivity of the nucleic acid extraction method. In both
cultured and field samples, the detection limit was one A. catenella cell (Galluzzi
et al. 2006).
Bacterial magnetic particles (BMPs) were used for the identification of
cyanobacterial DNA. Genus-specific oligonucleotide probes for the detection
of Anabaena spp., Microcystis spp., Nostoc spp., Oscillatoria spp., and
Synechococcus spp. were designed from the variable region of the cyanobacterial
16S rDNA of 148 strains. These oligonucleotide probes were immobilized on
BMPs via streptavidin-biotin conjugation and employed for magnetic capture
hybridization against digoxigenin-labeled cyanobacterial 16S rDNA. Bacterial
magnetic particles were magnetically concentrated, spotted in 100-lm-size
microwell on MAG-microarray, followed by the fluorescent detection. The entire
process of hybridization and detection was automatically performed using a mag-
netic separation robot, and all five cyanobacterial genera were successfully dis-
criminated (Matsunaga et al. 2001).
Paralytic shellfish poisoning (PSP) toxins from a toxic strain of the marine
dinoflagellate Alexandrium tamarense CCMP-1493 have been isolated after cells’
162 I. Safarik et al.

disintegration using glutaraldehyde-activated amine-coated hollow glass magnetic

microspheres (Ferrospheres-N) with immobilized monoclonal antibody. High toxin
recovery (up to 96.2%) was obtained using optimized separation conditions (Devlin
et al. 2011b). Alternatively, these coated microspheres were utilized for extracting
PSP toxins from naturally contaminated mussel samples; it was shown that mag-
netic separation could be a convenient alternative to conventional extraction pro-
cedures used in toxin purification prior to sample analysis (Devlin et al. 2011a).
Recently, magnetic solid phase extraction (Safarikova and Safarik 1999)
employing C18-functionalized magnetic silica nanoparticles (Fe3O4@SiO2@C18
MNPs) was successfully used for the determination of hepatic toxin microcystin-LR
(MC-LR) in reservoir water samples, followed by high-performance liquid chro-
matography–diode array detection (HPLC–DAD). After the extraction, the adsor-
bent can be conveniently and rapidly separated from aqueous samples by an
external magnet. High enrichment factor 500 was attained. The calibration curve of
MC-LR was linear in the range of 0.1–10.0 mg/L; limit of detection was
0.056 mg/L. The developed method was successfully applied to the determination
of MC-LR in reservoir water samples (Ma et al. 2015).

6 Magnetically Responsive Catalysts in Microalgae Oil


Biodiesel is a renewable, clean-burning diesel replacement; currently, mainly veg-

etables and seed oils are used as its precursor. In addition, the microalgae with high
lipid content could also be employed as an oil source. The biodiesel synthesis is
based on the triacylglycerol transesterification with a short-chain alcohol (e.g.,
methanol and ethanol); such a conversion is usually performed in the presence of the
catalyst. Both enzymes (lipases) and solid acid/base catalysts have been successfully
used (Mata et al. 2010; Takisawa et al. 2014). In the following part, only examples of
magnetically responsive (bio)catalyst utilized for biodiesel synthesis and some other
organic reactions using microalgae oil as a substrate will be presented. Magnetically
responsive catalysts can be reused after their simple recovery by a magnetic field,
thus avoiding the filtration or centrifugation separation processes.

6.1 Magnetic Lipases in Microalgae Biotechnology

An extracellular halo-thermo-tolerant, solvent stable lipase was purified from a

bacterial strain isolated from brackish water. The purified enzyme showed optimum
activity at pH 7 and temperature 30 °C. The enzyme was found to be stable at broad
ranges of pH (5.0–9.0), temperature (10–60 °C), and salinity (up to 30%).
Interestingly, the enzyme showed stability in different polar solvents of log P < 2 at
7 Magnetic Particles for Microalgae Separation and Biotechnology 163

a concentration of 75% (v/v) for 7 days. Lipase was immobilized on magnetite

particles modified with [3-(2-aminoethylamino)propyl]trimethoxysilane after acti-
vation by glutaraldehyde. The recyclability of the enzyme was shown practically
unchanged for seven consecutive cycles. The lipase-catalyzed hydrolysis of
Chlorella oil showed selective enrichment of oleic acid content with a substantial
threefold increase in concentration (Jain and Mishra 2015).
Lipase from Candida rugosa (CRL) was immobilized and stabilized on mag-
netically separable, large pore mesostructured magnetic hollow mesoporous silica
microspheres (MHMSS) by means of multiple-mode adsorption based on both
hydrophobic and strong cation-exchange interactions. Benefiting from the hollow
large mesoporous structure, ultrafast enzyme immobilization could be realized in
5 min, with a high loading of CRL (95.2 mg g−1). Stabilized CRL@MHMSS was
successfully used for the ultrafast transesterification of phytosterol with refined and
bleached algae oil and other triglycerides in a solvent-free system, which reached
high conversions (≧90.9%) within 15 min at 55 °C. Magnetic separation of
MHMSS facilitated the repeated usage of CRL@MHMSS for more than 50 suc-
cessive reactions without negative effect on its catalytic activity. Its high activity
and stability make the MHMSS-immobilized enzyme an attractive catalyst for
green synthesis in a solvent-free system (Zheng et al. 2015).
An indigenous microalga Chlorella vulgaris ESP-31 grown in an outdoor
tubular photobioreactor with CO2 aeration obtained a high oil content of up to
63.2%. The microalgal oil was then converted to biodiesel by enzymatic transes-
terification using a lipase from Burkholderia sp. C20. Lipase was immobilized on
commercial magnetic nanoparticles modified with tetraethyl orthosilicate and
dimethyloctadecyl[3-(trimethoxysilyl)propyl] ammonium chloride (Tran et al.
2012a). The conversion of the microalgae oil to biodiesel was conducted by
transesterification of the extracted microalgal oil (M-I) and by transesterification
directly using disrupted microalgal biomass (M-II). The results showed that M-II
achieved higher biodiesel conversion (97.3 wt% oil) than M-I (72.1 wt% oil). The
immobilized lipase worked well when using wet microalgal biomass (up to 71%
water content) as the oil substrate. The immobilized lipase also tolerated a high
methanol to oil molar ratio (>67.93) when using the M-II approach and can be
repeatedly used for six cycles (or 288 h) without significant loss of its original
activity (Tran et al. 2012b).
In other experiments, Chlorella vulgaris ESP-31 containing 22.7% lipid was
harvested by coagulation (using chitosan and polyaluminium chloride (PACl) as the
coagulants) and centrifugation. The harvested cells were directly employed as the
oil source for biodiesel production via transesterification catalyzed by Burkholderia
lipase immobilized as described above. The enzymatic transesterification was sig-
nificantly inhibited in the presence of PACl, while the immobilized lipase worked
well with wet chitosan-coagulated cells, giving a high biodiesel conversion of
97.6% w/w oil, which is at a level comparable to that of biodiesel conversion from
centrifugation-harvested microalgae (97.1% w/w oil). The immobilized lipase can
be repeatedly used for three cycles without significant loss of its activity (Tran et al.
164 I. Safarik et al.

Wet oil-bearing microalgal biomass of Chlorella vulgaris ESP-31 was directly

converted into biodiesel using Burkholderia lipase immobilized on magnetic carrier
as the catalyst. The microalgal biomass (water content of 86–91%; oil content 14–
63%) was pre-treated by sonication to disrupt the cell walls and then directly mixed
with methanol and solvent to carry out the enzymatic transesterification. Addition
of a sufficient amount of solvent (hexane is most preferable) is required for the
direct transesterification of wet microalgal biomass. The biodiesel synthesis process
was more efficient and economic when the lipid content of the microalgal biomass
was higher (Tran et al. 2013a).
Experiments employing a mixture of free fatty acids which simulates the com-
position of fatty acids coming from microalgae (Scenedesmus sp.) to synthesize
biodiesel were carried out using commercial lipase B of Candida antarctica (EC; Lipozyme, Novozymes, Denmark). Lipase was immobilized on magnetite
nanoparticles functionalized by 3-aminopropyltriethoxysilane and activated by
glutaraldehyde. Alternatively, magnetic cross-linked enzyme aggregates were pre-
pared using amino-modified magnetite nanoparticles and glutaraldehyde. Both
robust magnetically separable biocatalysts formed by lipase showed higher stability
and better performance for biodiesel formation than the soluble enzyme (López
et al. 2014).

6.2 Magnetic Solid Acid/Base Catalysts

Green approach to biodiesel production has stimulated the application of sustain-

able solid acid catalysts as replacements for liquid acid catalysts so that the use of
harmful substances and generation of toxic wastes are avoided; meanwhile, the easy
way of catalyst separation after the reactions can be realized. Recent studies have
proven the technical feasibility and the environmental and economical benefits of
biodiesel production via heterogeneous acid-catalyzed esterification and transes-
terification. In this perspective, various solid acids including sulfated metal oxides,
H-form zeolites, sulfonic ion-exchange resins, sulfonic-modified mesostructured
silica materials, sulfonated carbon-based catalysts, heteropolyacids, and acidic ionic
liquids can be used as catalysts for esterification and transesterification (Su and Guo
2014; Lee et al. 2014).
Recently, a new magnetic solid base catalyst has been employed in microalgae
technology. Core–shell Fe3O4@silica magnetic nanoparticles functionalized with a
strong base, triazabicyclodecene (TBD), were successfully synthesized for har-
vesting microalgae and for one-pot microalgae-to-fatty acid methyl ester (biodiesel)
conversion. Three types of algae oil sources (i.e., dried algae, algae oil, and algae
concentrate) were used, and the reaction conditions were optimized to achieve the
maximum biodiesel yield. The results obtained in this study show that
TBD-functionalized Fe3O4@silica nanoparticles could effectively convert algae oil
to biodiesel with a maximum yield of 97.1%. Additionally, this material acts as an
efficient algae harvester because of its adsorption and magnetic properties. This
7 Magnetic Particles for Microalgae Separation and Biotechnology 165

method demonstrates the wide scope for the use of covalently functionalized core–
shell magnetic nanoparticles for the production of biodiesel from algal biomass
(Chiang et al. 2015).

7 Future Trends

Magnetically responsive nano- and microparticles or their composites with dia-

magnetic materials have increasing potential for applications in many fields of
biosciences, biotechnology, and environmental technology. A relatively novel
approach to the application of magnetic materials and techniques in the field of
microalgae flocculation can significantly simplify the harvesting processes even in
industrial scale. Appropriate interaction of magnetic particles with microalgae
biomass is an extremely simple step leading to the flocculation of algal cells with
subsequent efficient magnetic separation. Future attention in this field should be
focused on further development of simple and inexpensive methods for the
preparation of magnetic materials or on the improvement of large-scale magnetic
separation processes. Microalgae cells (or cell walls after disintegration and target
compounds separation), magnetically modified by various types of magnetic par-
ticles, could be further applied for the adsorption of harmful xenobiotics from
wastewaters. Equally interesting is the possibility of harmful algae detection in the
environment after their interaction with immunomagnetic particles. Very promising
is also the utilization of magnetically modified microalgae in bioanalytics, where
the cells can act as a sensitive biorecognition element of biosensors for monitoring
the inhibitors of algae photosynthetic activity, especially herbicides.
Despite the fact that microalgae magnetic harvesting is currently experiencing a
real boom, other applications of magnetic materials in microalgae research are still
in infancy. Magnetic separation of microalgae biologically active compounds using
appropriate magnetic adsorbents has several advantages in comparison with
“standard” separation techniques; they can be separated in a single step even from
complex mixtures, often bypassing other more complicated isolation and purifica-
tion procedures. Few examples of recently published articles focused on the sep-
aration of genomic DNA or microalgae toxins should stimulate the microalgae
research community to develop magnetic separation protocols using magnetic
affinity or ion-exchange particles to isolate efficiently the target compounds. Such a
development should lead to the construction of industrial-scale separation processes
employing relatively inexpensive magnetic adsorbents and relatively simple
magnetic separators.
Applications of immobilized enzymes have become a routine in many
biotechnological processes. Immobilization of enzymes (specifically lipases) on
magnetic carriers for biodiesel production from microalgae oils is only the first step
which should be followed by the application of other relevant enzymes
(e.g., polysaccharide hydrolases or proteinases) immobilization and application.
166 I. Safarik et al.

Magnetically responsive solid/base and enzyme-like catalysts represent another

promising direction leading to cost-effective transformation of microalgae raw
materials and metabolites.


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Chapter 8
Enzyme-Assisted Extraction of Bioactives

Sandesh J. Marathe, Swati B. Jadhav, Sandip B. Bankar

and Rekha S. Singhal

1 Introduction

Among the vast array of biomolecules present in the living organisms, some of
them are essential for survival and are called primary metabolites. However, other
biomolecules called as secondary metabolites are also naturally produced. These are
extra nutritional in nature and may have a beneficial or adverse effect on living
organisms (Amsath 2013). Secondary metabolites have limited distribution in
nature and present only in the specific group of organisms. Biomolecules from plant
and microorganisms have been used for centuries, and their demands have
increased in food, medicinal, and chemical industries due to their unique biological
activities. Bioactive compounds are constituents other than nutrients that generally
occur in small quantities of foods, and whose intake has been associated with
protective effects against adverse health or physiological disorders, for instance,
cardiovascular, diabetes, or cancer. These bioactives have a diverse range of
chemical structures varying from phenolic compounds to phytoestrogens to car-
otenoids to terpenoids to organosulphur compounds among many others. The
efficacy of bioactives has been established from epidemiological studies as well as
in vitro and in vivo studies in both animals and humans. The discovery and efficacy
of bioactives are now the basis of a billion dollar nutraceutical industry globally.
One of the main challenges is to extract these biomolecules from their respective
natural sources. Different techniques have been reported on these aspects in the
literature, each having their own pros and cons. The choice of technique mainly

S.J. Marathe  S.B. Jadhav  R.S. Singhal (&)

Food Engineering and Technology Department, Institute of Chemical Technology,
Matunga, Mumbai 400 019, India
S.B. Bankar
Department of Chemical Engineering, College of Engineering, Bharati Vidyapeeth
University, Pune-Satara Road, Pune, India

© Springer International Publishing AG 2017 171

M. Puri (ed.), Food Bioactives, DOI 10.1007/978-3-319-51639-4_8
172 S.J. Marathe et al.

depends on the type of raw material, environmental concerns, process conditions,

and future applications of the bioactives.
Enzyme-assisted extraction offers safe, green, and novel approach for the
extraction of bioactives. Several researchers have reported this method as a best
choice for extraction of bioactives from various sources. Enzyme-assisted extrac-
tion has been reported for the extraction of lipids (Jin et al. 2012), oils (Huo et al.
2015), and various other bioactives from plants (Puri et al. 2012).
This chapter covers a brief reference to conventional extraction technique and
the importance of enzyme-assisted extraction of biomolecules. It also covers the
mechanism of action and various hyphenated technologies using enzymatic
extraction of different types of bioactives.

2 Conventional Extraction Techniques

Over the years, different extraction techniques have been reported and used for the
extraction and purification of the biomolecules from the natural resources. The low
content of active molecules in the source material and the complexity of raw
material makes it necessary to find alternative methods of effective extraction. It is
worthwhile to understand the conventional methods of extraction before discussing
enzyme-aided extractions.

2.1 Solvent Extraction Methods

Solvent extraction is the oldest and traditional method of extraction which mainly
depends on factors such as nature of the solvent, energy input, and agitation to
improve the chemical solubility and efficiency of mass transfer (Awika et al. 2003).
The selection of solvent for the extraction depends on the raw material to be used
and the product of interest. Lipophilic compounds can be well extracted using
nonpolar organic solvents such as hexane or dichloromethane, whereas hydrophilic
compounds can be extracted using polar solvents such as acetone, methanol, or
ethanol. Mixture of acetone and water has been used for the extraction of antiox-
idant (Awika et al. 2003). The recovery of polar compound such as lignin glyco-
sides can be further enhanced by adding polar solvents such as water to the sample
(Cacace and Mazza 2006). The use of solvents such as dichloromethane, dichlor-
oethane, acetone, hexane, and alcohol is very common for the extraction of aroma
principles from various raw materials either in hot or in cold conditions (Ravindran
and Madhusoodanan 2002). Xu and Chang (2007) have demonstrated the influence
of solvent type on the rate and amount of extraction of polyphenols. Polyphenols
are commonly extracted using solvents such as methanol, ethanol, acetone, ethyl
acetate, and their combinations with different proportions of water. The lower
molecular weight polyphenols can be extracted effectively using methanol, while
8 Enzyme-Assisted Extraction of Bioactives 173

higher molecular weight polyphenols have shown preference toward aqueous

acetone (Metivier et al. 1980). However, ethanol has also been considered as a good
solvent for polyphenol extraction which is safe for human consumption (Shi et al.
The addition of organic acids such as formic acid, acetic acid, citric acid, and
phosphoric acid in the solvent is also practiced to increase the yield of anthocyanins
by denaturing the cell membranes and stabilizing the anthocyanins (Nicoué et al.
2007). Further, sulfured water can also be used to reduce the use of organic solvents
as well as the cost of extraction (Cacace and Mazza 2002).
Solvent extraction has many disadvantages such as (i) consumption of large
amount of organic solvents or water during extraction step, (ii) separation of solute
that needs high energy, (iii) coextraction of impurities, and (iv) chances of degra-
dation of thermosensitive compounds such as carotenoids.

2.2 Physical Extraction Methods

Essential oils from spices such as pepper, ginger, and cardamom can be obtained
using steam distillation. Aroma-rich raw materials are subjected to steam distilla-
tion, where the volatile compounds condense and separate from water (Ravindran
and Madhusoodanan 2002). In the process of hydrodistillation, the raw material is
boiled with water. The steam carries the volatile aroma compounds and condenses
it. Distillation method is not generally useful for industrial purposes as it requires
longer time. However, supercritical fluid extraction (SCFE) is one of the recent
techniques which uses the gases above its critical temperature and pressure. In the
supercritical state, the physicochemical property of the gas is intermediate between
liquid and gas. This state of the gas can effectively extract flavors and bioactive
compounds from plant materials. The use of carbon dioxide gas is very common for
this technique. SCFE has many advantages including the absence of solvents and
high concentration of compound of interest in extract (Mukhopadhyay 2000).
However, this method adds cost in the process.
Following are the limitations of using physical and solvent methods of
1. The necessity of pretreatment to raw material;
2. The use of chemicals and solvent. These solvents are usually not recycled and
hence increase the cost of process. The cost of process also increases by adding
removal step for hazardous waste;
3. Non-specificity of the methods;
4. Variation in the quality of product might be due to remaining unwanted
materials; and
5. Low extraction yield.
174 S.J. Marathe et al.

3 Enzyme-Assisted Extraction

Enzyme-assisted extraction can be considered as a boon to overcome the limitations

of the conventional methods. It is a green approach and helps to reduce the issues of
environmental concerns. The most commonly used enzymes for the extraction of
bioactives are cellulases, hemicellulases, and pectinases. The major sources of
enzymes are from bacteria and fungi but can also be obtained from animal organs
and/or vegetable/fruit extracts.

3.1 Mechanism of Action of Enzyme

The mechanism of enzymatic action is the heart of enzyme-assisted extraction

process because of its selectivity. Enzyme-aided extraction is primarily dependent
on the ability of enzymes to hydrolyze the cell wall components and to disrupt the
structural complexity of the cell wall. These processes allow the easy release of
compound of interest in the bulk solution (Gardossi et al. 2010). The mechanism by
which enzyme hydrolyzes the cell wall is by forming an enzyme–substrate binding
complex. The enzyme binds to substrate with the change in conformation of the
enzyme that enables better interaction with the substrate. These changes cause
stress and strain on the substrate which in turn promotes the hydrolytic reactions
(Sowbhagya and Chitra 2010). In the enzyme-aided extraction process, the oper-
ational conditions such as temperature of reaction, pH of system, enzyme con-
centration, particle size of substrate, and time of extraction are important. The use of
appropriate enzyme complex breaks down the bonding of unwanted material and
releases the compound of interest in the aqueous media, which in turn enhances the
quality of product. Some enzymes used for the extraction of bioactives are compiled
in Table 1.
The particle size of a raw material decides the extent of hydrolytic action of the
enzymes. It is worthwhile to understand the catalytic property, mode of action, and
optimal operational conditions of the enzymes before considering its application for
the process. The types and combination of enzymes depend on the nature of raw
material (Puri et al. 2012). For instance, if a source raw material is plant, then one
needs to know the complex arrangement of polysaccharides in the cell wall.
Primary cell walls of a variety of higher plants have common structure, which
consists mainly of cellulose fibers with hemicelluloses attached to it (Talmadge
et al. 1973). These fibers are buried in a matrix made up of pectic substances which
all together are linked to a structural protein. This basic understanding of cell wall
structure gives a clue that the enzyme preparation that can be used for the
hydrolysis of cell wall must contain a mixture of cellulases, hemicellulases,
pectinases, and proteases (Christensen 1989). In the mixture of enzymes, the role of
carbohydrases (pectinase, cellulase, and hemicellulase) is different from those of
proteolytic enzymes. For example, both types of enzymes play their individual roles
8 Enzyme-Assisted Extraction of Bioactives 175

Table 1 Different enzymes used in the process of enzyme-assisted extraction of bioactives from
variety of sources
Enzyme used Bioactive Source material Conditions used Reference
Cellulase Polysaccharides Garlic Temperature Pan and Wu
45 °C, pH 5.0, (2014)
time 80 min
a-Amylase Oleoresin Turmeric – Kurmudle et al.
and (2013)
Cellulase, Polysaccharides Alfalfa Temperature Wang et al. (2013)
papain, and 52.7 °C, pH
pectinase 3.87, time
2.73 h
Cellulase, Seed oil Pumpkin Temperature Jiao et al. (2014)
pectinase, and 44 °C, time
protease 66 min
Alginate lyase Fucoxanthin Undaria Temperature Billakanti et al.
and lipids pinnatifida 37 °C, pH 6.2 (2013)
a-Amylase Polysaccharides Panax ginseng – Sun et al. (2015)
Pectinase and Carotenoids Tomato waste – Strati et al. (2014)
Lipase and Proteins Olive pulp and Temperature Vergara-Barberán
phospholipase stone 30–40 °C, pH et al. (2014)
7.0, time
15 min
Papain, Fatty acids Strongylocentrotus Temperature Zhu et al. (2010)
protease, and nudus 40–55 °C, pH
trypsin 7.8–8.5, time
180 min

during the extraction process of oils from oil seeds. Carbohydrases hydrolyzes the
cell wall and enables higher release of oil in aqueous media, whereas proteolytic
enzymes improve the yield of oil by hydrolyzing the structural fibrous protein in
which fat globules are embedded (Yoon et al. 1991). Moreover, the proteolytic
enzyme modifies the emulsifying capacity of protein released in aqueous media.
The proteolytic enzymes used in the process of extraction have a huge impact on
the emulsifying capacity of the released proteins. The emulsifying capacity of the
protein increases during enzymatic proteolysis until certain degree of hydrolysis is
achieved, and thereafter, it starts decreasing. However, the reverse happens to the
stability of resulting emulsion (Puski 1975). The proteolytic enzymes used in
the extraction process can have positive or negative effect on process depending on
the degree of hydrolysis of the protein. The proteolytic enzyme releases the oil from
lipid bodies, but at the same time, the increased emulsifying capacity can lower the
extraction of free oil. This makes it necessary to control the process and extent of
proteolytic action to obtain a higher oil yield.
176 S.J. Marathe et al.

The cell walls and cuticles of marine algae are made up of chemically complex
and heterogeneous biomolecules. It needs carbohydrases and proteases to break
down the cell walls of natural matrices to release the cell content (Grosso et al.
2015). Some commonly used enzymes for these applications are xylanase, arabi-
nase, cellulase, amylase, protease, and glucanase (Kadam et al. 2013). Brown
seaweed Undaria pinnatifida contains alginate polysaccharide in abundance as a
part of cell wall and intracellular materials. In this case, alginate lyase was used to
increase the extraction of fucoxanthin from the seaweeds. Alginate lyase degrades
the alginate by b-elimination mechanism targeting glycosidic linkages between
monomers (Billakanti et al. 2013).
Presently, it appears that the aqueous extraction process using enzymes are
gaining attraction majorly for oils with high commercial value such as olive oil and
avocado oil. However, the cost of enzyme-assisted processes needs to be taken into
consideration which is mainly due to the separation steps, water and enzyme recycle
process, and reutilization.

3.2 Advantages of Enzyme-Assisted Extraction

1. This technology gives higher yields by breaking down the complex structure of
raw material.
2. It removes unwanted components of raw material selectively.
3. It shows high catalytic efficiency and preserves the original efficacy of natural
4. It reduces the time of extraction and volume of solvent used.

4 Enzyme-Assisted Extraction of Bioactives

from Natural Sources

Bioactives such as oils, proteins, carbohydrates, and phenolics can be obtained from
variety of sources such as plants, bacteria, fungi, algae, and animals. The specific
details of enzyme-assisted extractions vary with the biomolecules of interest and its
source material.

4.1 Oils

Plant oils are commonly used in food, detergent, and paint industries. Plant oils
with higher content of polyunsaturated fatty acids (PUFAs) are important in food
industries. Conventionally, plant oils have been extracted using solvent extraction
8 Enzyme-Assisted Extraction of Bioactives 177

(Bernardini 1973) where hexane is a commonly and commercially used solvent

(Rosenthal, Pyle and Niranjan 1996). However, hexane causes many environmental
concerns. Hence, aqueous extraction methods are better alternatives to organic
solvent extraction. Although aqueous extraction is an environmentally cleaner
technique, it is not successful due to the lower oil yields (Rosenthal et al. 1996).
This limitation can be overcome using enzymatic treatment during aqueous ex-
traction of oils (Badr and Sitohy 1992). Moreover, it is also beneficial to simul-
taneous extraction of oils and proteins (Jiang et al. 2010; Hanmoungjai et al. 2002).

4.1.1 Structure of Oleaginous Plant Seeds

The major oil and protein content of oilseeds is found in discrete cellular organelles
called lipid and protein bodies or oleosomes and aleurone grains, respectively.
Scanning electron microscope (SEM) analysis reveals the oleosomes from soybean
(Wolf and Baker 1975) and peanuts (Young and Schadel 1990) to be embedded in a
cytoplasmic network composed of proteins. The oleosomes and cytoplasmic net-
work fill up the spaces between protein bodies (Young and Schadel 1990). The
cytoplasmic network thus contains proteins and lipids. On the other hand, the walls
surrounding the cells are predominantly made up of cellulose, hemicellulose, lignin,
and pectin. It is significantly notable that the oleosomes contain high amount of
proteins called oleosins that form a membrane around the oleosomes, which play a
role in stability of these bodies.

4.1.2 Process of Enzymatic Extraction of Oils

Extraction of oils using enzymatic extraction process includes preliminary steps

similar to the conventional extraction method such as cleaning, cracking, flaking,
and pressing. The pressed oleaginous material can be treated with enzymes before
further step. Enzymes such as cellulases and pectinases are used either individually
or in combination. The use of combination of hydrolytic enzymes is however
preferred due to better yields so obtained. The enzymatic pretreatment is followed
by the conventional solvent extraction method to obtain higher yield of oils
(Domínguez et al. 1994).
Mechanical pretreatment processes such as flaking and extrusion impact on both
the aqueous oil process (AEP) and enzyme-assisted aqueous oil extraction process
(EAEP) (Lamsal et al. 2006). The oil extraction is reportedly increased from 46 to
71% in AEP and 56 to 88% in EAEP after including extrusion step in the extraction
process. However, extrusion denatures the protein and poses a challenge for the
simultaneous recovery of proteins (Caine et al. 1998). Enzyme-assisted extraction
processes for oils have been well reported in the literature (Moura et al. 2008;
Domínguez et al. 1994; Latif et al. 2008). However, there are some stringent
conditions that need to be maintained for enzyme-assisted aqueous extraction
process, which include the following:
178 S.J. Marathe et al.

1. Reduction in particle size of the source material to facilitate proper enzymatic

action and
2. The control over moisture content for effective enzyme action. Enzymes such as
cellulase need control over the moisture content as water activity plays a sig-
nificant role in the swelling and expansion of fiber and also in increasing the
surface area for activity of the enzyme (Domínguez et al. 1994).
Latif and Anwar (2011) used enzyme-assisted aqueous extraction to obtain oil
and protein from sesame seeds using a mixture of enzymes including protease and
carbohydrases (cellulase, hemicellulase, xylanase, b-glucanase and arabinase). This
extraction technique not only enhanced the yield of oil but also improved the
quality of oils extracted. Oxidative stability, antioxidant activity, and tocopherol
profile of sesame seed oil obtained after enzymatic extraction were better than that
obtained after hexane extraction. Though enzyme-assisted extraction is commonly
carried out using aqueous extraction, some attempts have been made using methods
other than aqueous extraction. The seeds of rose hip have been used to extract oil
using enzyme-assisted cold pressing (Concha et al. 2004). Enzymatic pretreatment
using pectinase, cellulase, and glucanase enhanced the oil extraction rate as well as
the yield of oil. In the aqueous extraction method, enzyme degrades the seed cell
wall and ruptures the polysaccharide–protein colloids. It forms an emulsion that
lowers the yield. However, in the absence of protein–polysaccharide colloids as is
the case in cold pressing, enzyme facilitates only hydrolysis of seed cell wall. The
enzyme-assisted cold pressing has also been reported to extract hemp seed oil (Latif
and Anwar 2009) with significant improvement in the oil yield. Apart from higher
oil yields, the method stabilizes the other seed constituents such as proteins and
fiber. In addition, the color intensity, tocopherol levels, rancimat profile, and sen-
sory score are better than conventionally extracted control samples.
Enzymatic pretreatment has also been used to assist the alkaline extraction
method to obtain oil from rapeseeds (Zhang et al. 2007). In this process, rapeseed
slurry is first treated with pectinase, cellulase, and b-glucanase and then subjected to
alkaline extraction and alkaline protease hydrolysis to produce protein hydrolyzate
and de-emulsify the oil. Many similar reports are available in the literature showing
extraction of oil from plant sources using enzyme-assisted extraction process.
Table 2 shows the oils extracted from various plant sources using enzyme-assisted

4.1.3 Phenolics

Phenolics are secondary metabolites possessing one or more aromatic rings with
one or more hydroxyl groups. A vast variety of phenolics are known till date which
are structurally as simple as phenolic acids or as complex as highly polymerized
tannins (Dai and Mumper 2010). Researchers and food manufacturers have special
interest in this class of bioactives due to their potent antioxidant properties,
abundance in the diet, and ability to prevent various oxidative stress-associated
8 Enzyme-Assisted Extraction of Bioactives 179

Table 2 Enzyme-assisted extraction of oil from different plant sources

Source (oilseed) Enzyme used Concentration of Yield Reference
enzyme/activity (%)
Soybean Protease 0.2% 86.0 Yoon et al.
Soybean Protease 0.5% 97 Campbell
Rapeseed Pectinase + cellulase 0.4:0.1% 80.2 Deng et al.
Peanut Protease 3% 78.0 Lanzani et al.,
Peanut Protease 1.5% 79.32 Jiang et al.
Onion Amylase + cellulase 1:1% 18.38 Salina et al.
Sesame Protease 2% 57.4 Latif and
Anwar (2011)
Canola (Brassica napus Carbohydrase – 26.0 Latif et al.
L.) (2008)
Goldenberry(Physalis Cellulase + pectinase 1:1 v/v 20.9 Ramadan
peruviana L.) pomace et al. (2008)
Rice bran Protease 1% 79 Hanmoungjai
et al. (2002)

diseases. It shows preventive action in case of many diseases such as cardiovas-

cular, neurodegenerative diseases, and cancer (Manach et al. 2004). In order to
utilize these bioactive for the preparation of dietary supplements or nutraceuticals,
food ingredients, pharmaceutical, and cosmetic products, they are required to be
efficiently extracted from raw materials.
Conventionally, solvent extraction method is common for the extraction of
phenolics from plants using solvents such as methanol, ethanol, acetone, and ethyl
acetate, either individually or in combination with different proportions of water.
Moreover, phenolics are generally attached to other plant components such as
carbohydrates and proteins which do not allow the development of a universal
protocol for the phenolic extraction. An additional step needs to be performed to
remove the unwanted components from the mixture after solvent extraction (Dai
and Mumper 2010). The bound phenolics can be extracted using pectinolytic
enzymes and cell wall-degrading enzyme preparation.
Continued research is being carried out using water as a sole extraction solvent.
However, the problem of lower yield persists in extraction of phenolics when
compared with that obtained by using solvents such as acetone (Meyer et al. 1998).
Solubility of phenolic compounds increases with an increase in the polarity of
solvent used (Naczk and Shahidi 2006). However, too high water content in
extraction solvent causes collateral extraction of other phytochemicals, thereby
lowering the concentration of phenolics (Spigno et al. 2007).
180 S.J. Marathe et al.

The mode of action of hydrolytic enzymes on extraction of phenolics is by

cleaving the cell wall components, thus favoring the exposure of phenolics to the
extraction. Li et al. (2006) observed a 28% increase in concentration of phenolic
compounds extracted using commercial enzyme Cellzyme MX, as compared to the
control (without enzyme). Also, Hansen and Laroze (2009) observed a 35%
increase in the yield of phenolics, while evaluating the effect of enzymatic treatment
during hydroethanolic extraction from raspberry residue.
As explained in earlier sections, the extraction efficiency may vary with several
processing variables such as type of enzymes used, substrates, solvents, and
operating temperature (Meyer et al. 1998). The extraction yield depends signifi-
cantly on particle size of mashed samples. The yield of phenolic compounds can be
increased by lowering the particle size (Pinelo et al. 2005). Salina et al. (2013)
studied enzyme-assisted extraction of phenolics from onion using different enzymes
(cellulase and amylase individually and in combination). The maximum phenolic
content of 5.9 mg/ml was obtained using cellulase individually for 16 h at 35 °C.
Cellulase has also been used for the extraction of total phenolics from citrus peels
(Li et al. 2006). A 65.5% recovery of phenolics with potential antioxidant activity
was observed. Commercial enzymes, Viscozyme L (carbohydrases including ara-
binase, cellulase, b-glucanase, hemicellulase, and xylanase) and Rapidase, had been
used to extract the phenolic compounds from cauliflower harvest and showed the
combination of both the enzymes to result in better efficiency in recovery of phe-
nolics (Huynh et al. 2014). Furthermore, pectinase was more useful to extract
phenolic antioxidants from raspberry wastes (Laroze et al. 2010). Experimental
results showed that the enzyme-assisted extraction using hydroethanolic mixture for
18 h at 50 °C could efficiently increase the phenolic content to 35%.
One of the most crucial properties of plant phenolics is to retain the antioxidant
activity. In vitro assays have shown plant phenolics to be a more potent antioxidant
than vitamin C, vitamin E, and carotenoids (Rice-Evans et al. 1995, 1996). This
makes the extraction of phenolics from plant material important. Several reports are
available in the literatures (Table 3) on extraction of phenolics from a variety of
sources using enzyme-assisted extraction.

4.1.4 Flavorings and Colorants

The use of colorants and flavorings in food industry is increasing significantly since
the last few decades. The quality of food is majorly affected by color and flavor
which decides the appearance and acceptance of the product. The growing demand
for colors is met by synthetic colorants. However, the synthetic colorants are prone
to have adverse health effect including carcinogenicity. Hence, these are being
phased out by regulatory bodies in many countries. This in turn has led to a growing
interest to find natural food colorants and flavorings (Chandrasekaran 2012).
Methods such as solvent extraction, hydrodistillation, steam distillation, and
supercritical carbon dioxide extraction are common for the extraction of flavorings
and colorants. Generally, the extraction of flavorings and colorants is incomplete
Table 3 Enzyme-assisted extraction of plant phenolics from various plant sources
Source Phenolic Enzyme Enzyme Yield of Reference
extracted concentration phenolics
Blackberry Anthocyanins Pectinase 0.2% 639 g/l Hankun et al. (2014)
8 Enzyme-Assisted Extraction of Bioactives

Saffron tepals Anthocyanin Cellulase + hemicellulase + pectinase 5% 6.7 mg/g Lotfi et al. (2015)
Grape pomace Phenolic acids Pectinase + cellulase 2:1 91.9% Maier et al. (2007)
Grape pomace Total phenolics Pectinase 10% 3072 mg/l Meyer et al. (1998)
Apple skin Total phenolics Cellulase + pectinase + protease 1:1:1 104.94 mg/l Pinelo et al. (2008)
Grapes Total phenolics Pectinase – 90 ± 0.37% Gómez-García et al.
Unripe apples Total phenolics Cellulase – 7.08% Zheng et al. (2009)
182 S.J. Marathe et al.

because cellulose is responsible for its sequestration in the cell. Enzymes cause
partial destruction of plant cell wall and help in separation of intracellular com-
pounds (Waliszewski et al. 2007).
Cellulolytic enzyme has been used to extract vanillin from vanilla beans by
Waliszewski et al. (2007). They found the hydration process in 5% ethanol for 48 h
and enzymatic pretreatment with cellulase for 12 h to double the vanillin content in
the extract with excellent sensory quality as compared to control treatment without
enzymes. Zhang et al. (2014) also attempted to extract vanillin from green vanilla
pods using enzyme-assisted extraction combined with prefreezing and thawing.
Cellular compartmentalization of vanilla green pods was destroyed by prefreezing
and thawing. It was followed by treatment of pectinase to hydrolyze the pectin
between glucovanillin substrate and b-glucosidase. This method could successfully
transform the glucovanillin to vanillin and produce natural vanillin from green
The extracted anthocyanins find wide range of applications in various industries.
They have been used as natural food colorants and antioxidants in pharmaceutical
products. Extraction of anthocyanins using different enzymatic and non-enzymatic
extraction methods has been studied by various researchers (Chandrasekhar et al.
2012; Vanini et al. 2009; Hankun et al. 2014). Lotfi et al. (2015) used pectinex
(containing cellulase, hemicellulase, and pectinase) at varying concentrations to
extract anthocyanins from saffron tepals and also compared the yield with con-
ventional ethanol extraction method. They observed a 40% increase in the yield of
total anthocyanins.
Barzana et al. (2002) extracted carotenoids from marigold flowers using enzy-
matic extraction with hexane as a solvent. Under optimal conditions, they obtained
a recovery yield of 97%. Lenucci et al. (2015) performed studies on enzymatic of
lycopene, a carotenoid red pigment, synthesized and stored in tomato berry chro-
moplasts using glycodiase. The results showed that the enzymatic extraction could
increase the yield of lycopene by 153% as compared to solvent extraction.

4.1.5 Carbohydrates

Carbohydrates play an important role in cell signaling, cell adhesion, and molecular
recognition in the immune system (Dwek 1996). Researchers have shown various
biological activities of carbohydrates such as wound healing, stimulation of
immune system, and treatment of tumor (Schmidgall et al. 2000). Hence, studies are
being performed to isolate and identify carbohydrates from different plant sources
and also to test their pharmacological activities. Carbohydrates are ubiquitously
present in plants and can be isolated from different parts of plant such as leaf, seed,
and root, each of which may give carbohydrates with differential bioactivity.
The conventional method for extraction of carbohydrates from plants involves
steps such as size reduction, extraction, and filtration. However, aqueous extraction
is a safe and economical method which gives better yield of carbohydrates than
conventional solvent extraction methods (Hu et al. 2013). Apart from various
8 Enzyme-Assisted Extraction of Bioactives 183

advantages of aqueous extraction method listed in earlier sections, there are other
benefits of using water such as easy penetration in the plant tissue resulting in high
yield and stability of carbohydrates extracted (Hu et al. 2013). Hence, recent studies
have focused on enzyme-assisted extraction of carbohydrates from plants. The
general method for enzyme-assisted extraction of carbohydrates is similar to the
conventional extraction method. The dried plant material is powdered and extracted
using water in the presence of enzyme. Proteins can be removed with lead acetate,
and excess lead can be removed with potassium oxalate. Filtration can then be
carried out to eliminate residual material (Weinmann 1947).
Smith, Paulsen and Raguse (1964) studied the method of enzyme-assisted
extraction to obtain carbohydrates from grass and legume tissues using takadiastase
and obtained yields better than the aqueous extraction. Takadiastase hydrolyzes
starch (insoluble in water) into maltose residues (soluble in water), which increases
the total carbohydrate yield. Bahramian et al. (2011) showed the significance of
enzyme dosage during the study of optimization of enzyme-assisted extraction of
sugars from kabkab date fruit using pectinase and cellulase. Furthermore, Patindol
et al. (2007) used cellulase to extract oligosaccharides by enzyme-assisted extrac-
tion method. They could improve the total carbohydrate yield from 69.2 to 87.2%.
Ng et al. (2014) used enzyme-assisted extraction method to obtain cellobiose,
glucose, and fructose by hydrolysis of agricultural waste grapefruit peel and orange
peel using cellulase. Besides cellulase, pectinase is also common for
enzyme-assisted extraction method to obtain carbohydrate of interest. This enzyme
has been explored by Dzogbefia et al. (2008) to extract starch from cassava with
high yield. Pectinolytic enzymes are used in the treatment of plant materials for cell
wall disintegration, de-pectinization, reducing viscosity to increase the flow rate,
and release cell components, thus increasing the ultimate yield (Demir et al. 2001;
Rai et al. 2004).
The method of extraction can be decided based on intention of application of
polysaccharides. This has been explained well from the experiment done by Pan
et al. (2015). They found that the polysaccharide obtained from Dendrobium
chrysotoxum using cellulase-assisted extraction had higher immunomodulatory
activity compared to that of hot water extraction method which showed better foam
stabilization activity. The improvement in the yield of polysaccharide extraction by
enzyme-assisted extraction method compared to hot water extraction method has
also been shown by Zhu et al. (2014) during the extraction of Hericium erinaceus
Although cellulase, pectinase, hemicellulase, and glucanase are commonly used
enzymes in enzymatic extraction of carbohydrates, other uncommon enzymes have
also been explored by some researchers based on the nature of raw materials used.
Chen et al. (2014) have used glucose oxidase for the extraction of polysaccharides
from Astragalus membranaceus. They have optimized the process by response
surface methodology and obtained a 250% increase in the yield and better
antioxidant activity as compared to control process. The same enzyme was also
used for the extraction of Fructus mori polysaccharides (Deng et al. 2014) and
184 S.J. Marathe et al.

found to increase in the yield of the polysaccharide by 160% using enzyme-assisted

extraction method.

4.1.6 Proteins

Among all the bioactives, proteins are most important as a nutritional and dietary
supplement. Proteins and peptides together contribute major constituents of regular
food and can be obtained from plant as well as animal sources. Various methods of
extraction and fractionation of protein and peptides are available, but the choice of
method depends on several factors such as solubility, hydrophobicity, molecular
weight, and isoelectric point (pI). Efficient and optimized techniques must be used
to remove interfering compounds such as lipids, phenolics, carbohydrates, oxidative
enzymes, and pigments without protein degradation or modification.
The presence of indigenous proteases in plant tissue makes the extraction of
proteins complicated (Wang et al. 2008). Proteins are usually found in protein
bodies (also called as aleurone grains) inside the cells. Hence, the complete solu-
bilization and extraction of proteins depends on cell disruption. The method used
for cell disruption depends on the types of plant material used (leaf, fruit, root, seed,
etc.) or even on the stage of development of plant. A number of chemical methods
(using solvents such as ether, acidified alcohol, and chloroform) and physical
methods (such as bead-beating, sonication, and mortar–pestle) are known to be used
for the disruption of cells. However, due to the differences in the nature and
proportion of components, the choice of method may vary.
Commercially produced protein concentrates usually consist of aqueous solu-
bilization of protein, thus making water as a solvent of choice for extraction. The
extraction yield of protein can further be increased by using enzyme-assisted
aqueous extraction of proteins. Different carbohydrases can be used to release
proteins from raw materials. Guan and Yao (2008) used viscozyme L to hydrolyze
cell wall by cleaving the linkages within polysaccharides that effectively release
intracellular protein from oat bran. Jung et al. (2006) showed successful use of
pectinase to improve extractability of soy protein without protein degradation. The
protein recovery was increased by 50% as compared to control with improved foam
stability. Further, Vergara-Barberán et al. (2015) explored the use of cellulase to
improve the protein extraction from olive leaves. They optimized the process of
enzyme-assisted extraction of proteins from olive leaves and found that the method
was faster with higher recovery and reduced solvent usage. Recently, the focus has
been shifted to use proteases to hydrolyze the proteins partially and convert them to
peptides. This increases the solubility of peptides making their extraction effective.
Oil seed meal that is obtained as by-product after meal production is a potential
source of protein. Proteases have been used to extract proteins from oilseed meals
such as rapeseed, soybeans, and microalgae meals (Sari et al. 2013). The addition of
proteases enhances the yield of protein extraction to 90% from soybean meals and
50–80% from rapeseeds and microalgae meals. The use of proteases has also been
8 Enzyme-Assisted Extraction of Bioactives 185

Table 4 Enzyme-assisted extraction of proteins from different plant sources

Source Enzyme used Concentration of Yield Reference
enzyme/activity (%) (%)
Peanut Protease 1.5 71.38% Jiang et al. (2010)
Sesame Protease 2 87.1% Latif and Anwar
Soybean Protease 1 87% Campbell (2010)
Rice Protease 2 68% Hanmoungjai et al.
bran (2002)
Soybean Protease 0.5 85% Moura et al. (2008)
Rapeseed Protease 1.5 83% Zhang et al. (2007)
Rice Phytase + xylanase – 74.6% Wang et al. (1999)

explored for the extraction of peptides of interest for various pharmacological

actions. Furthermore, antioxidant and a-amylase inhibitory peptides have been
extracted from pinto beans by enzyme-assisted extraction using enzyme protamex
(Ngoh and Gan 2016). Similarly, antioxidative and antihypertensive bioactive
peptides have been successfully extracted from Parkia speciosa seed using alcalase
(Siow and Gan 2013).
Simultaneous recovery of protein and lipids using enzyme-assisted extraction
method is gaining attraction due to dual benefits. Protease has been used for
simultaneous recovery of protein and oil from extruded soybean flakes using
enzyme-assisted aqueous extraction method (Moura et al. 2008). The yield of oil
was 96%, whereas that of protein was 85%. Niu et al. (2012) used the same
technique for extraction of rapeseed oil and protein from dehulled cold-pressed
double-low rapeseed cake. They obtained 82.10% yield of protein and 71.89%
yield of oil using enzymes Viscozyme L (carbohydrases) and Alcalase (proteases).
The reports available on enzyme-assisted extraction of protein from various
plant sources are compiled in Table 4.

5 Bioactives from Non-plant Sources

Although plants are the most important sources of bioactives, other sources such as
bacteria, algae, fungi, and animals have also been explored to obtain bioactive of
interest. This reduces the dependency on plant sources. The bioactives obtained
from different sources using enzyme-assisted extraction method are detailed in the
subsequent sections.
186 S.J. Marathe et al.

5.1 Oils

Since centuries, the major sources of oils are from plants and used mainly for
human consumption, which then extended in recent times for biodiesel production.
Currently, oils derived from microbial sources (single cell oils) are gaining
importance as an alternative to vegetable oil for biodiesel production mainly due to
their comparable chemical properties (Christophe et al. 2012). All the oils that are
presently being produced as single cell oils are high in PUFA content and are
intended to be used mainly for human consumption as nutraceuticals. Some other
oils are also being used as feed for animals and farmed fish (Ratledge 2013).
The growing demands for energy resources have caused an adversity, and hence,
the current research on biodiesel production focuses mainly on microbial oils (Xia
et al. 2011). Microalgae are also gaining attention due to their ability to produce
high amounts of oil and fast growth, by fixing large amounts of CO2 (Demirbas and
Demirbas 2010; Huo et al. 2011). Conventional methods used for the extraction of
oils require the algal or fungal biomass to be in dry form. This needs an additional
step of dehydration during extraction which further increases the process cost.
Moreover, conventional extraction methods also use chemical solvents which are
hazardous to the environment and also give inadequate recovery, thus making their
use undesirable. As detailed in earlier sections, the use of green extraction methods
such as enzyme-assisted extraction overcomes these drawbacks for better end
application. Enzyme-assisted extraction is not only environmentally friendly but
also economically efficient method, as it avoids the additional drying process and
also reduces the use of hazardous solvents during extraction process (Liu et al.
2013). Moreover, it does not affect the quality of value-added biomass
(Gómez-García et al. 2012) making it desirable.
Algal and fungal cell walls resemble plant cell wall and also act as a barrier to
the extraction of bioactives using enzyme-assisted extraction. For efficient extrac-
tion, a combination of enzymes can be used which effectively breaks the microbial
cell walls. Several researchers have studied the effect of combination of enzymes on
extraction of oil from algae (Huo et al. 2015; Liang et al. 2012). Algal cell walls are
made up of either polysaccharides such as cellulose or a variety of glycoproteins, or
both. This makes the cellulases and proteases good candidates for lysis of algal cell
walls. Huo et al. (2015) studied the effect of different quantities of enzymes (cel-
lulase, pectinase, and hemicellulase) taken in combination with each other, along
with the process parameters (temperature, pH, algal biomass concentration) on the
extraction of oil from wet microalga Scenedesmus sp. G4. They observed the
extraction yield reaches 86.1% under optimal conditions. Liang et al. (2012) also
carried out enzyme-assisted extraction of lipid from microalgae using enzymes
cellulase, snailase, protease (neutral and alkaline), and trypsin. They observed a
highest lipid recovery of 49.82% by using enzyme treatment along with sonication
at pH 4 with enzymes, snailase and trypsin being better than cellulase and proteases.
Zuorro et al. (2015) evaluated enzyme-assisted extraction of lipids from microalgal
8 Enzyme-Assisted Extraction of Bioactives 187

cells using 1,4-ß cellobiosidase, galactomannanase, and ß-glucosidase and recov-

ered 70% of the lipids.
The concentration of enzyme(s) and their types may vary depending upon the
microbial species. After the cell lysis using hydrolytic enzymes, hexane is added to
the enzymatically hydrolyzed algal biomass as an extraction solvent, and the
mixture is then centrifuged. This gives two phases, viz. a light phase, which pre-
dominantly contains the extracted oil in solvent, and a heavy phase that contains
water and algal residue (along with oil and solvent carryover). The two phases can
be separated, and oil can be isolated from light phase, thus leaving solvent (hexane)
behind. The solvent can further be reused for next extraction cycle. Subsequently,
the residual material can be sent for anaerobic digestion. Water can also be used as
an alternative to hexane, thus making the process cheaper and environmentally
friendly. It forms two phases comprising of extracted oil in one phase and the water
with the residual biomass in another (Davis et al. 2013).
Several fungal species have the capacity to produce and accumulate high
amounts of lipid (up to 70%). These oleaginous fungi produce oils that contain
triacylglycerol (TAG) and PUFA and hence are desirable for applications in the fuel
and food industries. In fungal biomass, typically, the extraction of oil is carried out
using Soxhlet extraction and pressurized liquid extraction (Cescut et al. 2011).
Methods for extraction of lipids using wet cell mass by organic solvents (Bligh and
Dyer 1959) and supercritical fluids (Halim et al. 2011) have also been established.
Fungal cell walls are made up of components such as mannoproteins, ß-glucan, and
chitin. Fungal chitin is difficult to degrade as compared to plant cellulose. Thus, the
choice of enzyme here is different as compared to enzymes used during the
extraction of bioactives from plant/algal cells. Hydrolytic enzymes such as chitinase
and ß-1,3-glucomannanase can be used for breaking fungal cell walls. Breaking the
cell wall makes lipids accessible to the extraction solvent that improves the
extraction efficiency.
Jin et al. (2012) extracted lipids from the culture of yeast Rhodosporidium
toruloides. They used the recombinant ß-1,3-glucomannanase for this purpose; then
carried out the extraction in ethyl acetate; and found 96.6% lipid to extracted
directly from culture without dewatering. Thus, the process was deemed as

5.2 Polysaccharides

Several algal, fungal, and bacterial species have also been proven to be a potential
source of polysaccharides. Extensive research has been done to study the extraction
methods and biotechnological applications of these polysaccharides (Costa et al.
2010; Rioux et al. 2007; Wijesinghe et al. 2011). Fu et al. (2010) studied the
enzymatic hydrolysis of microalgae cell walls using immobilized cellulase under
the optimized conditions to isolate reducing sugars. Wijesinghe et al. (2011) also
188 S.J. Marathe et al.

carried out enzyme-assisted extraction of sulfated polysaccharides from the brown

seaweed Ecklonia cava.

5.3 Phenolics

Few attempts have been made to extract phenolics from other than plant source.
Machu et al. (2015) performed experimental studies on the extraction of phenolics
such as gallic acid, 4-hydroxybenzoic acid, catechin hydrate, epicatechin, catechin
gallate, epicatechin gallate, and pyrocatechol from commercial algal foods such as
brown and red algae, green freshwater algae, and blue green algae using different
extraction solvents. The lysis of microbial cells can be carried out using hydrolytic
enzymes to improve the extraction of phenolics. The effect of hydrolytic enzymes
such as proteases and carbohydrases on the lysis of algal cells and improvement in
the extraction yield of polyphenols and other antioxidant ingredients from red algae
Palmaria palmate were studied by Wang et al. (2010). The protease could enhance
the extraction of polyphenols and other active components as compared to carbo-
hydrases and water extraction. Heo et al. (2005) also showed the antioxidant
activities of brown seaweed extracts obtained after enzymatic process to be higher
than the commercial antioxidants.

6 Combination of Enzyme-Assisted and Other

Techniques to Extract Bioactives

With the rising importance of enzyme-assisted extraction of bioactives from plant

and microbial sources, researchers are now interested in studying the effect of
combination of enzyme-assisted extraction and other non-conventional extraction
techniques on the extraction of bioactives. Non-conventional techniques such as
three-phase partitioning, microwave-assisted extraction, ultrasound-assisted
extraction, and supercritical fluid extraction have already been studied (Gupta
et al. 2012). The combination of enzyme-assisted extraction and these techniques
can be considered economical as well as an efficient method to extract bioactives.

6.1 Enzyme-Assisted Three-Phase Partitioning (EATPP)

Three-phase partitioning (TPP) is a new technique used to separate proteins by

precipitating them using t-butanol and ammonium sulfate. The method usually used
for separation of proteins is now being studied for its use in extraction of bioactives
such as oil and carbohydrates primarily from plant sources. In TPP, the proteins are
8 Enzyme-Assisted Extraction of Bioactives 189

separated from aqueous phase by adding t-butanol and ammonium sulfate which
forms two immiscible liquid phases to precipitate the proteins at the interface of two
layers (Gaur et al. 2007).
TPP offers several advantages over conventional protein extraction methods,
which includes the use of mild operational conditions and structural stability of
proteins in their native form. TPP can further be used to scale-down or scale-up of
processes. Moreover, it can be used directly on crude plant materials, thus reducing
the process cost. The use of inexpensive chemicals such as t-butanol and ammo-
nium sulfate also makes the process economical (Rachana and Lyju Jose 2014).
Enzyme-assisted three-phase partitioning (EATPP) is an advanced technique
which is a combination of enzyme-assisted extraction and TPP. The plant material
is pretreated with enzyme preparations followed by regular TPP process. Sharma
et al. (2002) performed TPP for the extraction of oil from soybean and obtained a
yield of 82% within 1 h. Similar experiments were carried out using EATPP with
the help of Protizyme (a protease) to extract oil from soybean and obtained a yield
of 98% (Gaur et al. 2007). This shows the better efficiency of EATPP over the
TPP. Kurmudle et al. (2011) carried out EATPP for the extraction of turmeric
oleoresin by pretreating the turmeric slurry with a commercial preparation of en-
zymes such as a-amylase and/or glucoamylase. Oleoresins were extracted in less
time as compared to conventional acetone extraction. Harde and Singhal (2012)
also used this method for the extraction of forskolin (diterpene) from Coleus for-
skohlii roots. The extraction was found to be increased from 30.83% by TPP to
83.85% by EATPP.

6.2 Microwave-Assisted Enzymatic Extraction

Microwaves are electromagnetic radiations with their frequencies ranging from

300 MHz to 300 GHz. These are non-ionizing radiations and can cause molecular
motion on contact with matter without changing the molecular structure. Also, they
can heat the target material, and the amount of heat generated greatly depends on
their frequencies and on the applied power. Microwaves can be used in the
extraction of bioactive compounds, and the yield of extraction depends on several
factors such as the power of microwaves, time for which the material is exposed to
microwaves, size of sample, extractant (solvent), and temperature. The choice of
solvent affects the extraction process due to the solubility of compound in solvent
and the ability of a solvent (extractant) to absorb microwave energy. Higher
absorption might generate high heat leading to effective extraction.
Microwave-assisted extraction (MAE) has been studied extensively by various
researchers for the extraction of different bioactives (Pan et al. 2003; Lianfu and
Zelong 2008; Chen et al. 2007). Moreover, microwave-assisted enzymatic extrac-
tion (MAEE) was used by Yang et al. (2010) for the extraction of corilagin and
geraniin from Geranium sibiricum Linne. The increased yield of bioactives with
good potential for natural antioxidant was found in the extract. Similar technique
190 S.J. Marathe et al.

was explored by Zhang et al. (2013) to enhance the extraction of polyphenols from
the waste peanut shells. The yield of polyphenol obtained was higher than other
methods such as heat-refluxing extraction, ultrasonic-assisted extraction, and
enzyme-assisted extraction. Cellulase is a common enzyme used in MAEE where it
makes the process efficient and environmentally friendly. Recently, the method has
also been used for the extraction of polysaccharides from the fruits of Schisandra
chinensis Baill (Cheng et al. 2015) where the yield obtained was higher at low

6.3 Ultrasound-Assisted Enzymatic Extraction

Ultrasonic waves are sound waves with high frequencies (20 kHz–100 MHz) and
are not audible to humans. Ultrasonic waves have been used for several purposes
such as cleaning, atomization, and extraction. Ultrasonic waves cause cavitation
that results in disintegration of material; this property of ultrasonic waves has been
utilized for extraction procedures. It displays several advantages over other
extraction methods such as reduced processing time, higher extraction rate, and
better extract quality (Cravotto et al. 2004).
Ultrasonic waves cause vibrations in the extractant leading to the formation of
bubbles which collapse near the cells and cause a shock wave. This leads to
breakage of cells and release of cell contents in the extractant. Ultrasound-assisted
extraction (UAE) has already been proven to be better than other methods such as
microwave-assisted extraction and simple aqueous extraction (Gu and Pan 2014).
UAE technique can further be improved by combining it with enzyme-assisted
extraction (EAE). Ultrasonic-assisted enzymatic extraction (UAEE) is a perfect
combination of enzymolysis and ultrasonication which shows efficient extraction of
polysaccharides from Cucurbita moschata and arabinoxylan, a major dietary
component from wheat bran (Wang et al. 2014). The method has been optimized
with respect to temperature, pH, ultrasonic power, liquid-to-material ratio, enzyme
dose, and time of extraction. Recently, Pu et al. (2015) have optimized the UAEE
method for the extraction of polysaccharides from Atratylodes macrocephala using
response surface methodology and have recommended this method as appropriate
and efficient.

6.4 Enzyme-Assisted Supercritical Fluid Extraction

Supercritical fluid extraction has been widely used for the extraction of alkaloids,
flavonoid (Giannuzzo et al. 2003), catechin, and epicatechin (Ashraf-Khorassani and
Taylor 2004) from different sources. This is a relatively new technique in the field of
extraction. In recent times, enzyme-assisted supercritical fluid extraction (EASFE)
has started gaining attention. The source raw materials are pretreated enzymatically,
8 Enzyme-Assisted Extraction of Bioactives 191

and the bioactives of interest can be extracted using supercritical fluid extraction
technique. Mushtaq et al. (2015) have extensively studied this method for the ex-
traction of antioxidant phenolics from pomegranate peels. EASFE could produce
crude extract of double recovery with increased level of phenolic constituents,
improved radical scavenging capacity, trolox equivalent antioxidant capacity, and
inhibition of linoleic acid peroxidation. Further, Dutta and Bhattacharjee (2015)
have used a-amylase in the process of EASFE to extract black paper oleoresin. This
method not only enhanced the yield of the oleoresin but also improved the phyto-
chemical properties of oleoresins. The extraction was studied comprehensively by
using batch and continuous mode where the most significant results of yield were
obtained with batch mode of operation. Table 5 shows the extraction of bioactives
using combination of enzyme-assisted and other techniques.

7 Large-Scale Enzymatic Processes

As evident from foregoing review, several enzymes are being used for extraction of
biomolecules and now traded as commodity products globally. Although the cost of
enzymes for use at the research scale is often very high, the increased production
and multiple use of enzymes reduce the cost dramatically. Enzymes are currently
involved in industrial processes with annual turnovers totaling many billions of
Cell wall-degrading enzymes can be used to extract oil by solubilizing the
structural cell wall components of the oilseed. This concept has already been
commercialized for the production of olive oil and has also been investigated for
other oil-bearing materials (Christensen 1989). The enzyme cocktail works syner-
gistically to give better results than individual enzymes. Many enzymes have been
commercialized for the industrial enzymatic extraction processes and have been
reported well in the literature as explained in different sections of this chapter.
Enzymatic treatment also destabilizes the lipophilic extractives in the filtrates and
facilitates their attachment to thermomechanical pulping fibers. The enzymes are
also used in the preparation of easily biodegradable cardboard (Buchert et al. 1998),
manufacturing of soft paper including paper towels and sanitary paper (Salonen
1990; Hsu and Lakhani 2002), and removal of adhered paper (Sharyo et al. 2002).
In recent years, extraction of olive oil has attracted the interest of international
market because of its numerous health claims. To produce high-quality olive oil,
freshly picked, clean, and slightly immature fruits are used under cold pressing
conditions (Galante et al. 1998; De Faveri et al. 2008). Although high yields are
obtained with fully ripened fruit, when processed at higher than ambient temper-
atures, these process conditions result in poor oil quality with high acidity, ran-
cidity, and poor aroma (Galante et al. 1998). Hence, an improved method for the
extraction of high-quality olive oil was needed to meet the growing consumer
demand. The commercial enzyme preparation, Olivex (a pectinase preparation with
cellulase and hemicellulase from Aspergillus aculeatus), was the first enzyme

Table 5 Combination of enzyme-assisted extraction and other techniques for the extraction of different bioactives from various sources
Technique Enzyme(s) used Bioactive(s) of Source Yield Reference
Enzyme-assisted three-phase ProtizymeTM Oil Soybean 98% Gaur et al.
partitioning (EATPP) (2007)
Enzyme-assisted three-phase a-Amylase Oleoresin Turmeric 8.96% Kurmudle
partitioning (EATPP) et al.
Enzyme-assisted supercritical Enzyme cocktail (acid cellulase, Phenolics Pomegranate 32.19 ± 1.26% Mushtaq
fluid extraction pectinase, viscozyme, kemzyme, et al.
alcalase) (2015)
Microwave-assisted enzymatic Cellulase Phenolics Geranium 6.79 mg/g (corilagin) Yang et al.
extraction (MAEE) (corilagin and sibiricum and 19.82 mg/g (2010)
geraniin) (geraniin)
Microwave-assisted enzymatic Cellulase Polyphenols Waste 1.75 ± 0.06% Zhang
extraction (MAEE) peanut shells et al.
Microwave-assisted aqueous Enzyme cocktail (cellulase, pectinase, Oil Pumpkin 64.17% Jiao et al.
enzymatic extraction and proteinase) seeds (2014)
Ultrasonic-assisted enzymatic Pectinase Flavonoids– Celery 42.5 mg/g (Luteolin) Zhang
extraction (UAEE) luteolin and and 25.3 mg/g et al.
apigenin (apigenin) (2011)
Ultrasonic-assisted enzymatic Papain Polysaccharide Zizyphus 21.95% Sun et al.
extraction (UAEE) jujuba (2011)
Ultrasonic-assisted enzymatic Enzyme cocktail (papain, pectase, Crude Epimedium 5.98% Chen et al.
extraction (UAEE) cellulase, and a-amylase) polysaccharides leaves (2012)
S.J. Marathe et al.
8 Enzyme-Assisted Extraction of Bioactives 193

mixture being used to improve the extraction of olive oil (Fantozzi et al. 1977).
Furthermore, the use of macerating enzymes increased the antioxidants in
extravirgin olive oil and reduced the induction of rancidity (Galante et al. 1998).
The main advantages of using macerating enzymes during olive oil extraction are as
follows: (i) increased extraction (up to 2 kg oil per 100 kg olives) under cold
processing conditions; (ii) better centrifugal fractionation of the oily must; (iii) oil
with high levels of antioxidants and vitamin E; (iv) slow induction of rancidity;
(v) overall improvement in plant efficiency; and (vi) low oil content in the
wastewater (Galante et al. 1998). Likewise, the macerating enzymes could play a
prominent role in the extraction of oils from other agricultural oilseed crops.
In wine production, enzymes such as pectinases, glucanases, and hemicellulases
play an important role by improving color extraction, skin maceration, must clar-
ification, filtration, and finally the wine quality and stability (Singh et al. 2007;
Galante et al. 1998). A number of commercial enzyme preparations are now
available for use by the wine industry. The main benefits of using these enzymes
during wine making include better maceration, improved color extraction, easy
clarification, easy filtration, improved wine quality, and improved stability (Galante
et al. 1998).
Cellulases have a wide range of potential applications in food biotechnology.
The production of fruit and vegetable juices requires improved methods for
extraction, clarification, and stabilization. Cellulases also have an important
application as a part of macerating enzymes complex (cellulases, xylanases, and
pectinases) used for the extraction and clarification of fruit and vegetable juices to
increase the yield of juices (Minussi et al. 2002; De Carvalho et al. 2008). Enzyme
mixtures containing pectinases, cellulases, and hemicellulases are also used for the
improved extraction of olive oil. Thus, the macerating enzymes, composed of
mainly cellulase and pectinase, play a key role in food biotechnology, and their
demand will likely to increase for the extraction of juice from a wide range of fruits
and vegetables (Dourado et al. 2002).

8 Challenges and Future Perspectives

Extensive research has been carried out in large-scale application of enzyme-

assisted extraction of biomolecules. However, there are various challenges asso-
ciated with cost-effective applications in current commercial processes. The pos-
sible solutions for further commercialization of enzymes in extraction industry
include the following: (a) reduction in the cost of enzyme production, (b) im-
provement in the performance of enzymes by using protein engineering and genetic
engineering, and (c) repeated use of enzymes with the help of improved enzyme
immobilization techniques. In the past decade, commercial enzyme companies have
made significant progress in producing new generations of enzymes with higher
specific activities and lower cost using different biotechnology and process engi-
neering approaches. However, a technoeconomic analysis suggests further progress
194 S.J. Marathe et al.

be made for better commercial applications. Another novel approach is expressing

enzymes in plants that could be extracted and used after pretreating the extracted
biomass (Egelkrout et al. 2012). Enzymes could also be produced directly in
biorefineries rather than producing them in a centralized location. Producing
enzymes on-site at biorefineries would eliminate the need for concentration, stor-
age, and shipping and could reduce the production costs by using pretreated sub-
strates already available at the biorefinery (Culbertson et al. 2013).


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Part III
Techniques Employed
for the Bioactives Delivery
Chapter 9
Emerging Technologies for Bioactive
Applications in Foods

Liliana G. Santiago, Carlos R. Soccol and Guillermo R. Castro

1 Introduction

The world population is close to 7.4 billion people with an annual growth rate of
1.1%, equivalent to 75 million people yearly, and it is expected to reach 8.4 billion
people in the middle of 2030 (The World Bank 2015). Under this frame, the
increase in food demand on production and quality is creating high pressure for the
natural resources, producers, food processing, and worldwide distribution. In
addition, it is required to change dietary habits for at least to two large and extreme
populations: the undernourished and the obese—overweighed people. Most of
undernourished people are living in developing countries. On the other side of the
spectrum, more than 1.9 billion people were overweight with 600 million obese,
and with 42 million kids less than five years old were obese or overweight in 2013
(The World Health Organization 2015). Those people are mostly living in countries
with medium- to high-income range. Both groups, about 20% of world population,
are requiring different food strategies including special designed foods. In addition,
the elderly population is rising globally which are requiring special care, including
fortified foods to provide all nutrients that contribute to their biological and

L.G. Santiago
Área de Biocoloides y Nanotecnología, Instituto de Tecnología de Alimentos (ITA),
Facultad de Ingeniería Química (FIQ), Universidad Nacional del Litoral (UNL),
1 de Mayo 3250, 3000 Santa Fe, Argentina
C.R. Soccol
Department of Bioprocess Engineering and Biotechnology,
Federal University of Paraná, Curitiba, Brazil
G.R. Castro (&)
Laboratorio de Nanobiomateriales, CINDEFI—Departamento de Química,
Facultad de Ciencias Exactas, Universidad Nacional de La Plata—CONICET (CCT La Plata),
Calles 47 y 115, B1900AJI La Plata, Buenos Aires, Argentina

© Springer International Publishing AG 2017 205

M. Puri (ed.), Food Bioactives, DOI 10.1007/978-3-319-51639-4_9
206 L.G. Santiago et al.

physiological well-being. In the present context, the development of modified,

fortified, and special type foods with properties over and above the standard
nutritional role of regular one is strongly desirable to be developed and available
during the next decade at large scale. The main options are functional and fortified
foods, and also foods for special purposes containing molecules generically defined
as nutraceuticals, e.g., antioxidants, micronutrients, vitamins, cofactors, and pep-
tides to be incorporated for specific health requirements. Nutraceutical was coined
from the combination of two words: nutrient (from food) and pharmaceutical (i.e.,
drugs) which provides therapeutic and/or health benefits (Andlauer and Fürst 2002).
There are many different types of healthy molecules that consider nutraceuticals
such as antioxidants, fatty acids, lutein, lycopene, minerals, phytosterols, and
vitamins and also probiotics such as living cells are considered beneficial for human
health. However, before incorporating nutraceuticals in foods, some tasks must be
addressed previously to reach massive markets such as sensorial issues and tech-
nological and economic barriers. The main problems of nutraceuticals are associ-
ated with the physicochemical and biological properties of the molecules related to
the environment. Incorporation of nutraceuticals in natural or processed foods
requires a deep knowledge of the food matrices involving physical structure, sta-
bility and storage conditions, chemical composition and interaction between the
components among others. On the other side, the digestive tract is harsh environ-
ment for nutraceuticals and must be protected from hydrolytic enzymes (e.g.,
amylases, proteases, and lipases), surfactants, pHs, temperature, and in some cases
also from the intestinal microbiota. In addition, the main challenge of the
nutraceutical molecules from structural point of view can be summarized in high
hydrophobicity, variable molecular weight, and chemical composition, sensitive to
environmental stresses (e.g., pH, ionic strength, and oxygen) and low bioavail-
ability (McClements 2015).
In the last decade, several novel strategies were described for controlled release
of pharmaceuticals, particularly for drugs with high undesirable side effects in
where encapsulation plays a key role. The encapsulation technology developed in
the pharma arena is now being followed by the food technologist in order to
develop and enhance the quality and stability of nutraceuticals in foods
(Kontogiorgos et al. 2015). The main properties required for an oral matrix design
is to keep enough amount of load keeping high chemical and biological stability
until the target is reached, not to show matrix toxicity and/or their individual
components and also of a result of any biotransformation process during the
digestive transit, their ability to reach the target with a minimal loss of the load,
extend and/or protect the food during storage and prevent the interaction of the load
with the food matrix and/or the environment. However, the food complexity, i.e.,
complex and diverse composition, microstructure, interfaces among other intrinsic
properties, is challenging the pharmaceutical strategies for controlled release of
molecules. Food engineering entails large knowledge of molecular structure and
arrangements of the food components involving chemical entities and their inter-
actions, i.e., hydrogen bonds, dipole–dipole, van der Waals, p–p interaction, and
9 Emerging Technologies for Bioactive Applications in Foods 207

hydrophobic and dispersion forces that could lead to the development of matrices
with effective delivery of loads.
Recent advances in micro- and nanotechnologies are providing novel platforms
to modify and improve food quality. The large availability of molecules well
characterized and with high purity plus novel and available advanced analytical
instrumentation is providing a myriad of options and platforms for the development
of nutraceuticals. Many different micro- and nanoparticles using several compo-
nents were developed in recent years for specific applications, but they are still in
their infancy (Joye et al. 2014).
The common platforms for the matrix synthesis are made of three main com-
ponents: lipids and its derivatives, polymers, and proteins. Gel matrices can be
employed to transform food properties such as foam stabilization, gelling and
thickening, improvement in organoleptic properties, preventing flocculation, ice
formation, and crystallization among others. Alternatively, the combination of the
mentioned molecules plus other inorganic or organic compounds is providing
complex matrices with novel properties, commonly named as composites.
The manufacture of nanodevices is produced by two different approaches named
“top-down” and “bottom-up.” The top-down approach involves the miniaturization
of objects based on the principles of solid-state physics (e.g., semiconductors),
meanwhile the “botton-up” involves the molecular self-assembly following
physicochemical principles (Fig. 1). By the classic definition, nanotechnology is
defined as any object with dimension between 1 and 100 nm. However, the defi-
nition was established by physics, far away from the biology domain in which the
interaction of objects and cells is in between the microworld and the nanoworld.
In the present work, some of the most popular micro- and nanodevices for
different food applications are reviewed.

Fig. 1 Interface of living organisms with the manufacture of nanodevices produced by top-down
and bottom-up approaches
208 L.G. Santiago et al.

2 Lipidic Carriers

Lipidic carriers as liposomes and emulsions were developed during the 1950s, and
more recently with the advent of nanotechnology novel formulations such as
nanoliposomes, nanoemulsions, solid lipid nanoparticles (SLNs), and nanostruc-
tured lipid carriers (NLCs) were developed (Fig. 2) (Martins et al. 2007; Das and
Chaudhury 2011; Fathi et al. 2014).

(a) (b)

(c) (d)

Fig. 2 Cartoon of common lipidic carriers

9 Emerging Technologies for Bioactive Applications in Foods 209

Nanoliposomes are the “nano” version of liposomes means submicron bilayer

lipid vesicles made of lipids and/or phospholipids like the cellular membranes. The
lipidic molecules contain a hydrophilic “head” and a hydrophobic acyl chains
named “tails” giving bifunctional physicochemical properties useful for interacting
with hydrophilic and hydrophobic molecules. The hydrophobic character of the
“tails” can be tailored by changing the length of the acyl residues and also the
unsaturation degree of the carbon chains, making very versatile structures with
different transition phase temperatures. Additionally, the lipidic bilayers can be
modified by the insertion of sterols and/or other molecules. For example, choles-
terol enhances the lipid bilayer stability and regulates the fluidity of the structure
typically by steric hindrance. The choice of many molecules to make the lipid
bilayers with specific properties, e.g., target, loads, environments, is one of the main
advantages of this type of formulations.
Nanoemulsions are colloidal nanodroplets physically dispersed made by mixing
two immiscible liquid phases: organic (oily) and aqueous. The main quantitative
phase ratio generally ranges from 5 to 20%, which is establishing the major
properties of the nanoemulsions, such as oil dispersed in water (O/W) or water
dispersed in oil (W/O). Also, the composition of the nanoemulsion determines
oily-watery microdomains coexisting in the media. Besides, the nanoemulsions
requires the use of additional molecules such as surfactants and/or co-surfactants in
order to stabilize the formulations in order to get a particle size of about 500 nm
(Martins et al. 2007; Neves et al. 2015).
The most recent nanoscale lipid matrices can be classified based on their
structure and composition into solid lipid nanoparticles (SLNs) and nanostructured
lipid carriers (NLCs) (Fig. 2c, d). SLNs are generally made using lipids with
melting points generally higher than body and/or room temperatures and dispersed
in an aqueous phase in the presence of surfactants by high energy, e.g., sonication.
After synthesis, the particles remain solid and during the oral administration the
dissolution rate depends on their chemical composition. In this way, the SLNs
protect active labile loads from environmental conditions such as oxidation, light
exposure, and moisture. Besides, SLNs synthesis requires lipids of high purity to
crystalize in a perfect lattice, which reduces space for the load, limiting the amount
of cargo, and the compatibility within the matrix (Muller et al. 2002).
The other lipidic nanostructure is named as nanostructured lipid carriers (NLCs)
and is considered the evolution of SLNs. The NLCs were developed using a variety
of lipids with different physiochemical properties instead of only one lipid as in
SLNs (Fig. 2b). In consequence, the lipid mixture can display unstructured matrix
without crystallization allowing to reduce the loading problems and potential
extrusion of the cargo from the matrix during the synthesis (Fig. 2d).
In general, the lipidic carriers can be grouped based on their stability under
standard environmental conditions. The nanoemulsions and nanoliposomes are
dynamic structures and consequently unstable along the time depending on
210 L.G. Santiago et al.

environmental factors. On the other side, SLNs and NLCs are solid
nanoformulations under room temperature, and their stability is dependent more on
chemical composition rather than the environmental conditions.

3 Biopolymeric Particles

Biopolymeric particles (BP) synthesized of proteins and/or polysaccharides and

their hybrid structures are commonly employed for the development of modified
foods (Burey et al. 2008). BP can be classified based on biophysical and structural
properties, and their components (Joye and McClements 2014).
The main advantages of biopolymeric carriers are the large amount of available
molecules with different properties combined with many easy techniques producing
matrices for different purposes.
BP components are selected considering several parameters such as chemical
structures and properties, the characteristics of the loads, and the environmental
conditions of the release such as ionic strength, temperature, and pH. The strategies
of BP synthesis are based on the physical or chemical methods or the combination
of both for the synthesis of complex particles.
The physical methods for BP synthesis involve the use of extreme temperatures
for molecular cross-linking. Cold- and heat-set gelation procedures allow to syn-
thesize BPs with different properties and taking into account the functional prop-
erties of the load and the site of cargo release.
The cold-set gelationfor BP synthesis usually requires the dissolution of the
biopolymer until colloidal state is reached, which depends on the physiochemical
properties, e.g., solubility, viscosity, molecular weights, functional and ionic
groups, and hydrophobic areas, tertiary structures, among others, of biopolymers
and solvents. The procedure involves fast decrease in colloidal suspensions tem-
perature to 0 °C or by the freeze (−18 °C) and thawing (room temperature) to
increase the strength of inter- of intra-hydrogen bridges between the polymer
chains. The cold-set technique is relevant to the sensitive molecules to environ-
mental changes and/or exposure to redox conditions (Lee et al. 2002).
Heat-set gelation is generally performed by raising the biopolymers above the
melting points of the molecules. However, it is expected to observe severe con-
formational changes in molecules including random associations and partial or total
denaturation of quaternary and tertiary structures and consequently aggregation of
the biopolymers. This technique is particularly sensitive to proteins, which probably
lose their biological activities.
On the other side, chemical cross-linking involves two different approaches:
covalent or/and ionotropic synthesis of particles. The main differences among them
are reversibility of both procedures and the environmental stability of the particles.
The chemistry of covalent techniques can involve the use of bifunctional reagents,
9 Emerging Technologies for Bioactive Applications in Foods 211

Fig. 3 Cartoon of alginate gel matrices made by ionotropic gelation

which are specific of the functional groups to be cross-linked, e.g., amine–amine,

carboxyl–amine, in which most of them are commercially available. The bifunc-
tional reagents could display toxicity such as formaldehyde, which requires further
purification step; meanwhile, hydrolysis rates of succinic anhydride are low in
aqueous media. The main advantages of covalent cross-linking are the synthesis of
particles with an average of low pore diameter on particle surface and the strict
control of cross-linking methodology. Besides the rigid cross-linked surface of the
BP, the particle core remains in solgel state, depending on the environmental
conditions of the procedure, which is producing a burst size release of the load after
surface break or erosion.
On the other hand, the main procedure for reversible cross-linking of biopoly-
mers is based on ionotropic gelation. This procedure is based on the presence of
multivalent ions in the solutions, e.g., calcium, magnesium, zinc, and the presence
of free carboxylate residues in the biopolymer. The interaction of multivalent ions
and the carboxyl residues is making hierarchy structures of biopolymer interchains
producing 3D gel network structures. The typical example is alginate which in the
presence of calcium is making gel structures commonly named as egg-box (Fig. 3).
The advantages of ionotropic gelations are the simplicity of the technique without
any further purification required, the synthesis of more homogenous matrices
(which by a diffusional mechanism can be also controlled), the preservation of the
native structure of the biopolymers, and the reversibility of the cross-linking which
can modify the particle stability but also could be disadvantageous depending on
the environmental physicochemical conditions (Cauerhff et al. 2014).
In the present chapter, encapsulation and/or entrapment of bioactive molecules
are discussed in materials only reported as GRAS up today: lipids and biopolymers.
Additionally, other potential uses of micro- and nanotechnologies in the develop-
ment of coatings, films, and sensors were out of the scope of the chapter.
212 L.G. Santiago et al.

4 Carbohydrate Polymers as Nanocarriers of Food

Bioactive Compounds

Carbohydrate polymers are hydrocolloids means hydrophilic molecules containing

mainly hydroxyl groups and some other polar groups able to form solgels in
aqueous media. The main advantage of using polysaccharides as carriers is to
provide dual-bioactive tridimensional matrices delivering simultaneously the load
and dietary fibers (prebiotics). Polysaccharides are present in almost all biological
kingdoms from algae, plants to crustaceans and produced by many microorganisms
in large quantities and with high purity (Table 1). The high diversity in natural
non-degradable polysaccharides is based on their huge diversity in chemical
composition (e.g., functional groups, residual charges, stereoisomery, selectivity)
and their wide physicochemical properties (e.g., molecular weight, water solubility,
hydrophobicity, viscosity) that can provide a wide range of applications and
bioactivities. The major bioactivities reported for polysaccharides are associated
with the prebiotic effects, inhibition of cancers, and prevention of constipation,
cholesterol decline, and modulation of glucose quantities in the blood (Cushen et al.
2012). However, the polysaccharides employed to develop food matrices must be
qualified as GRAS, Generally Recognized As Safe by regulatory agencies (e.g.,
European Food Safety Authority and Food and Drug Administration, US).
The ability of soluble polysaccharides to form gel matrices, solgel transition, is
contingent on the nature of polymer and the environmental physicochemical con-
ditions. The main mechanisms reported for polysaccharide gelation are as follows:
ionotropic gelation, cold- and heat-set gelations. The main advantage of ionotropic
and cold-set gelations is the preservation of the component quality; meanwhile,
heat-set gelation could increase the denaturation of molecules, loosing partially or
totally their original biological activities.
Particularly, hydrophobic and hydrophilic motifs of polymers must be consid-
ered to determine the interaction between the cargoes and the polymer chains. Most
of the polysaccharides contain anionic residual charges attributed to the carboxy-
late, sulfonate groups (e.g., alginates, carrageenans, pectins, xanthans), others are
neutral but with polarizable groups like hydroxyls (e.g., amylose, amylopectin,
cellulose), and only one natural biopolymer is cationic, i.e., chitosan, because of the
amine groups of glucosamine units. Besides, polar polysaccharide gelation is
generally made by ionotropic methods between the chemical components of the
system (e.g., alginates and multivalent cations), which makes the gel structure
stability depending on environmental conditions and the thermodynamic equilib-
rium constants. Therefore, an increase in gel nanoparticle stability can be achieved
by enhancing the cross-linking among the components by the use of biocatalysts,
chemical reagents, or physical procedures (Coviello et al. 2007; Matalanis et al.
In addition, covalent cyclic amyloses linked by b-1,4 glucose units commonly
named as cyclodextrins of different chain lengths from C6 to C8 were recently
reported in food applications because of their capacity to complexes with
Table 1 Summary of relevant molecular characteristics of food-grade polysaccharides for assembling biopolymer particles
Name Source Main structure Major monomer/s Gelation mechanism
Alginate Algae Linear D-manuronic and Multivalent cations
guluronic acids
Amylose Plants (e.g. potato tubers) Linear a-1,4-b-D-glucose Organic solvents
(from starch)
Amylopectin Plants (e.g. potato tubers) Branched a-1,4-b-D-glucose and Acid
(from starch) a-1,6-b-D-glucose
Arabic gum Acacia spp. Branched 3,6-b-D-galactopyranose High concentration, organic solvents
and other sugars
Carrageenan Algae Linear/helical Sulfated D-galactose and Cooling (K1+ and Ca2+)
3.6 anhydrogalactose
Cellulose Bacterial Fibrils b-D-glucose –
(Gluconacetobacter spp.)
Cyclodextrins Bacterial (Bacillus spp.) Cyclic Cyclic a-1,4 amyloses Poor soluble (C7) to soluble (C6 and C8) in aqueous
(C6 to C8) media. Organic-aqueous solvent mixtures
Chitosan Crustaceans/invertebrates Linear 2-amino-2-desoxy-b-D- Weak acid solutions
Dextran Bacteria (Leuconostoc Linear a-1,6 glucose Partially soluble in water
9 Emerging Technologies for Bioactive Applications in Foods

Gellan gum Bacteria (Sphingomonas Linear D-glucose, D-rhamnose Organic solvents
elodea) and glucuronic
Guar gum Guar beans Short branched b-1,4-mannose and Ca+2 and/or borax
(GG) a-1,6-galactose
Inulin Plants or bacteria Linear b-2,1-D-Fructose Conc. dependent
Kefiran Bacteria Branched Glucose and kefirose Organic solvents
Locust bean Plants Short branched b-1,4-D-mannose and Partially soluble in water, insoluble in organic solvents
Gum (LBG) a-1,6-D-galactose
Table 1 (continued)

Name Source Main structure Major monomer/s Gelation mechanism

Pectin Plant cell walls Branched/coiled Methoxylated Multivalent cations (LM and MM), solutes and acids
galacturonic acids (HM)
Pululan Fungal (Aureobasidium Linear a-1,4 maltotriose Organic solvents
Scleroglucan Fungal (Sclerotium Branched b-1,3- and b-1,6-D- Organic solvents
Rolsfsii) glucose
Xantan Bacterial (Xanthomonas Linear/helical Glucose, mannose, and High concentration
campestris) (at high MW) glucuronic acid (2:2:1)
Modified from Matalanis et al. (2011)
L.G. Santiago et al.
9 Emerging Technologies for Bioactive Applications in Foods 215

hydrophobic compounds (Cravotto et al. 2006). Cyclodextrins are capable of

encapsulating hydrophobic molecules in the central cavity but are not degradable by
enzymes produced in mammalian cells but can be hydrolyzed by microorganisms of
the colon.
Besides, the development of bionanocomposites (multiphase biomaterials) and
hybrid materials based on polysaccharides is providing superior advantages in the
matrix structure and functionalities such as mechanical resistance, decrease in gases
exchange (e.g., oxidation), extension of shelf life, avoidance of microbial con-
tamination, and in some cases polysaccharides are degradable by the intestinal flora
but not by mammalian cells.

5 Proteins as Nanocarriers of Bioactive Compounds

Food-grade proteins from animal or plant sources exist with different molecular,
physicochemical, and functional properties (Table 2). The selection of the most
suitable protein for a specific nanocarrier application depends on the desired particle

Table 2 Relevant properties of food grade proteins commonly used in the synthesis nano- and
Protein Source Conformation MW (kDa) pI Tm (°C)
Bovine Serum Albumin Bovine Globular 66.5 4.7 70–90
Alpha-Casein Milk Rheomorphic 23.0 4.1 125–140
Beta-Casein 24.0 4.5
Gamma-Casein 19.0 4.1
Kappa-Casein – 5.8–6.0
Gelatin (from collagen Animal or Linear *100 7.0–9.4a; 35–40
type III) fish (monomer) 4.7–5.4b
Ovoalbumin Egg-white Globular 45.0 4.5–4.7 74, 82c
Soy glycinin Soybean Globular 320 *5.0 67d, 87e
Beta-lactoglobulin Whey Globular 18.4 4.8–5.1 75
lactoferrin Whey Globular 93.0 *8–9 *60 and
protein *90
Zein Corn Globular 24.0 6.2 –
Modified from Matalanis et al. (2011)
Type A gelatin (from pork skin)
Type B gelatin (from calf skin)
S-type ovalbumin
7S soy glycinin fraction
11S soy glycinin fraction
216 L.G. Santiago et al.

properties (e.g., size, charge, surface characteristics, permeability, and degradability),

on the properties of the bioactive compound to be encapsulated (e.g., polarity,
solubility, and stability), and on environmental conditions (e.g., pH, ionic strength,
solvent quality, and temperature) (Table 3).

Table 3 Loaded molecules in carriers of protein origin

Nanocarrier Bioactive (Ref.) Ref.
Animal proteins
Beta-lactoglobulin Folic acid 33
Beta-lactoglobulin (variants A, B, C) Retinol and EGCG 34
Beta-lactoglobulin Curcumin 35
Beta-lactoglobulin Polyphenol extracts of teas, coffee 36
and cocoa
Beta-lactoglobulin Oleic and linoleic acid 37
Beta-lactoglobulin Oleic and linoleic acid 38
Pre-heated Beta-lactoglobulin Narangin and naringenin 39
Beta-lactoglobulin Naringenin 40
Casein Catechin 41
Beta-lactoglobulin, bovine serum albumin Folic acid 42
(BSA) and a-lactalbumin
Ovalbumin Caffeine, theophylline and 43
Ovalbumin and lysozyme Tea polyphenol 44
Plant proteins
b-conglycinin Vitamin D 45
b-conglycinin Curcumin 46
Canola protein and Pea protein isolates Ketones 47
Gliadin and zein Resveratrol 48
Modified proteins
Pre-treated Beta-lactoglobulin Linolenic acid 49
Hydrolyzed Beta-lactoglobulin Linolenic acid 30
Isostatic high-pressure Beta-lactoglobulin Retinol 51
Pre heated Ovalbumine Linolenic acid 50
Pre-heated Ovalbumine Linolenic acid 30
Pre-treated Beta-lactoglobulin/high metoxil Linolenic acid 49
Beta-lactoglobulin/sodium alginate b-carotene, folic acid, curcumin 52
and ergocalciferol
Sodium caseinate–gum arabic EPA/HDA 53
9 Emerging Technologies for Bioactive Applications in Foods 217

6 Animal Proteins

Proteins from animal sources, such as casein, whey, egg, and gelatin are widely
used for nanoparticles formation in the food industry. Among all of them, milk
proteins are interesting encapsulation agents because of their ability to bind dif-
ferent bioactives or to entrap them. Caseins and whey proteins, proteins isolated
from milk, are one of the most commonly used with this objective. Although the
same strategies apply for both, different approaches based on protein specificities
have been used to increase the encapsulation potential of each type of protein. The
use of milk proteins as agents of encapsulation and for the transport of bioactives,
and highlighting the strategies developed to explore the potential of these proteins
has been reviewed recently (Tavares et al. 2015). The functional properties of milk
whey proteins could be explained in terms of b-Lactoglobulin (BLG), which
constitutes the main fraction of whey proteins (Pérez et al. 2012a, b). Currently,
BLG binding properties have received considerable attention due to their potential
uses in bioactive compound delivery strategies because it is well known that BLG
has two lipophilic ligand binding sites: a central hydrophobic b-barrel (or calyx)
and a superficial pocket. Usually, fatty acids bind at BLG pocket, while other
molecules (as retinol) bind to BLG calyx. The design of BLG particles with tailored
properties continues receiving an increased attention. The interaction between
beta-lactoglobulin with resveratrol and its biological implications was reported by
Liang et al. (2013a, b). Synthesis, characterization, and biological implication of
ß-lactoglobulin/folic acid complexes for the binding properties of naringenin
(NG) to BLG were studied by different authors (Liang et al. 2013a, b; Pérez et al.
2014; Gholami and Bordbar 2014). Analysis of spectrofluorimetric titration data
indicated the formation of 1:1 complex, significant binding affinity of the load,
negative values of entropy and enthalpy changes and demonstrated the essential
role of hydrogen bonding and van der Waals interactions in binding of NG to BLG.
Shpigelman et al. (2014) used native and preheated BLG-based nanovehicles to
narangin and naringenin. Naringenin forms complexes with preheated and
non-preheated BLG. No binding was found between naringin and BLG, probably
due to the lower hydrophobicity of naringin and the steric interference of its sugar.
Thermally induced protein–polyphenol co-assemblies of beta-lactoglobulin-
based nanocomplexes as protective nanovehicles for EGCG were reported
(Shpigelman et al. 2010). Also it was reported that the genetic BLG variants A, B,
and C have different numbers of binding sites for retinol (almost completely
incorporated into the calyx), as well as for EGCG (exclusively bound on the sur-
face) (Keppler et al. 2014). Recently, Li et al. investigated the binding of curcumin
(CCM) to BLG (Li et al. 2013). The antioxidant activity of CCM might be
improved by binding with BLG. Stojadinovic and collaborators studied the
non-covalent interactions between BLG and polyphenol extracts of teas, coffee, and
cocoa (Stojadinovic et al. 2013). The polyphenol–BLG systems were stable at pH
values of the gastrointestinal tract. Fang and collaborators demonstrated that BLG
can bind to fatty acids such as oleic acid (OA) and linoleic acid (LA) and evaluated
218 L.G. Santiago et al.

the complexes in terms of antitumor activity and thermostability. Cell viability

results revealed that BLG-LA and BLG-OA exhibited similar antitumor activities.
There were more binding sites for OA than for LA on BLG, although the binding
constants of the 2 fatty acids were similar, with both acids interacting with the
protein through van der Waals and hydrogen bonding interactions (Fang et al.
Therefore, it is broadly demonstrated that BLG-binding properties could be
applied for the development of nano- and microparticles to encapsulate lipophilic
and hydrophilic bioactive compounds in order to protect them from deterioration
factors, such as oxygen, UV radiation, moisture, temperature, and to increase the
Of particular interest are the complexes that exhibited unexpected new func-
tionalities that were not predictable from those of isolated molecules. This was the
case for the complex formed between apo-alfa-lactalbumin and oleic acid known as
HAMLET/BAMLET (human/bovine a-lactalbumin made lethal to tumor cells),
which induces apoptosis of tumor cell. Besides, heat-denatured alfa-lactalbumin
was also able to form a complex with oleic acid that induced apoptosis in cancer
cells (Lišková et al. 2010). In addition, a complex formed by BLG and oleate was
shown to induce apoptosis in cancer cells comparable to the activity of BAMLET
(Lišková et al. 2011). The improvement in fatty acid solubility through its bonding
to these proteins is probably a driving mechanism behind the observed apoptotic
Other important milk proteins are caseins; they form micelles and can be con-
sidered an example of a biomimetic approach of an encapsulation system for
bioactive compounds. Due to its sponge-like structure consisting of internal cavities
connected to each other and to the porous surface by channels, casein micelles were
presumed for a long time to protect and transport molecules. Casein micelles consist
of submicellar structures that naturally encapsulate calcium phosphate. The core of
the casein micelles is primarily formed by as1-, as2-, and b-caseins, while the outer
layer consists of j-casein, which helps stabilize them by generating a strong steric
repulsion. The ability of “native” casein micelles, different casein fractions, and
caseinates to interact with hydrophobic molecules and minerals has been the subject
of extensive investigations. Casein micelles were produced to encapsulate
hydrophobic compounds such as vitamin D (Haham et al. 2012), flavonoids,
beta-carotene (Gutiérrez et al. 2013), curcumin, and phenolic components such as
epigallocatechin-3-gallate (EGCG). Casein micelles were also tested for their ability
to stabilize minerals such as iron; compared to whey proteins, caseins exhibit a
higher ability to stabilize iron. Lastly, Liang and his team investigated the response
of the synthetic form of folates (B group vitamin) known as folic acid to UV
irradiation in the presence of BLG, bovine serum albumin (BSA), and
a-lactalbumin (a-LA). Photodecomposition of folic acid was delayed in the pres-
ence of the proteins, which ranked in the order BLG > BSA > a-LA in terms of
effectiveness (Liang et al. 2013a, b). A recent study focused on the association of
tea polyphenols with casein micelles and the consequences of the interactions on
the renneting behavior of skim milk (Haratifar and Corredig 2014). The formation
9 Emerging Technologies for Bioactive Applications in Foods 219

of catechin (epigallocatechin-gallate, EGCG)–casein micelles complexes affected

the rennet-induced gelation of milk, and the effect was concentration-dependent.
Another animal protein with important physicochemical and functional proper-
ties is ovalbumin (OVA), the main protein of egg white proteins (EWP). It has been
demonstrated that OVA has the ability to bind hydrophilic compounds such as
caffeine, theophylline, and diprophylline, forming complexes (Wang et al. 2013).
Results showed that the formation of complexes gave rise to the fluorescence
quenching of OVA by caffeine, theophylline, and diprophylline. Thermodynamic
results showed that diprophylline was the strongest quencher and bound to OVA
with the highest affinity among three compounds. The influence of molecular
structure on the binding aspects was reported. Shen and collaborators examined the
promotion or inhibition of gastrointestinal digestion of tea polyphenol (TP) toward
ovalbumin (OVA) and lysozyme (LYZ) (Shen et al. 2014).

7 Plant Proteins

Plant proteins are also useful for producing protein particles, e.g., soy, wheat, corn,
and peas (Levinson et al. 2014; David et al. 2015). Plant proteins offer some
advantages over animal proteins for the formation of particle-based delivery sys-
tems as the risk of contamination and infection is reduced, they are not subjected to
cultural dietary limitations, and they are often cheaper than their animal
Recently, composition of soybean proteins, their functionalities, and the effects
of temperature, pH, ionic strength, processing conditions such as high pressure,
ultrasonic treatment, and utilisation of enzyme, chemical modification were
extensively reviewed (Nishinari et al. 2014). Soy proteins isolates has been used for
the encapsulation of fish oil and curcumin. Recently, the formation of nanoparticles
using soybean b-conglycinin (b-CG) to encapsulate vitamin D (VD) was reported
(Levinson et al. 2014). Formulating VD3 in these nanoparticles offers several
important benefits: nanoparticles are small (50–250 nm), and their solutions are
optically clear. The entrapment of VD3 in b-CG nanoparticles provides protection
both at pH 6.8 and pH 2.5, decreases vitamin degradation during pasteurization and
by gastric digestion, and increases product shelf life. Furthermore, other researchers
found that the b-CG–curcumin complexes system were significantly smaller and
colloidally stable nanoparticles compared to the dispersion of curcumin alone.
During an accelerated shelf life stress test, significant protection conferred by b-CG
to curcumin was observed against exposure to light and oxygen-induced degra-
dation, representing a ten-fold slower degradation rate (David et al. 2015).
Wang and Arntfield observed a competitive binding when homologous ketones
were added to canola protein isolates (CPIs) and pea protein isolates (PPIs) and
when homologous aldehydes were mixed with CPIs. The effect of heat treatment
over the binding process was also studied (Wang and Arntfield 2015).
220 L.G. Santiago et al.

Other plant proteins of particular interest for the formation of food-grade protein
particles are zein and gliadin, protein fractions derived from maize and wheat
gluten. They are highly hydrophobic, unlike many other food proteins; so their
preparation not only requires adaptations of the particle production process (such as
the solvents used), but also it is possible to encapsulate hydrophobic components
without the need for oil phases. Indeed, these proteins have already been widely
studied for the fabrication of particles to encapsulate bioactive compounds because
they do not need hardening to ensure their integrity in aqueous media. Nevertheless,
their aggregation stability is usually limited; consequently, stabilization of these
hydrophobic protein particles by the addition of surfactants or copolymers is often
required (Li et al. 2013). Recently, the encapsulation of resveratrol in protein
nanoparticles (gliadin and zein) in order to overcome its low water solubility,
chemical stability, and bioavailability was reported (Joye et al. 2015). Although,
resveratrol accomplishes with both proteins, binding constant was higher for zein
than for gliadin at 35 °C. Furthermore, binding between resveratrol and gliadin
increased at higher temperatures, which was not observed for zein.

8 Modified Proteins as Nanocarriers of Bioactive


On the other hand, it is well known that protein structural characteristics and
functional properties are easily modified by different processes including heating,
high pressure, and enzymatic hydrolysis. Therefore, the induction of a suitable
structural modification could be used in the development of delivery systems with
improved encapsulation efficiency, changes in binding affinity, and/or repercussions
in the release of lipophilic bioactive molecules (Fioramonti et al. 2014; Sponton
et al. 2014, 2015a, b; Pérez et al. 2014b; Le Maux et al. 2013).
Fioramonti and collaborators have shown that a controlled heat treatment could
promote conformational changes in the tertiary structure of whey proteins affecting
their functional properties and interactions with other molecules (Fioramonti et al.
2014). Structural modifications were attributed to exposition of reactive and
hydrophobic amino acidic residues buried into the protein
According to the literature, formation and characteristics of BLG aggregates
strongly depend on heating temperature and on bulk conditions, such as pH, ionic
strength, and salt type. Around neutrality, it was reported the formation of protein
filamentous aggregates, whereas at pH values close to pI (4.8–5.2), spherical or
particulate aggregates were obtained. BLG aggregates would be more hydrophobic
than native BLG because heat treatment could promote a greater exposition of
buried hydrophobic domains. These changes in protein tertiary structure could be
irreversible (Pérez et al. 2014a).
The assumption that heat-induced BLG aggregates with improved hydrophobic
properties could have a greater ability to bind linolenic acid (LA) than native BLG
9 Emerging Technologies for Bioactive Applications in Foods 221

was reported (Pérez et al. 2014a). They also studied the effect of pH and concluded
that BLG aggregates formed at pH 6.5 were the most hydrophobic. The binding
mechanism of BLG aggregates-LA depends on aggregate formation conditions (pH,
heating time and/or combination of both), besides conjugation should require the
preservation of LA binding site. The formation of complexes between linoleate and
native or aggregated beta-lactoglobulin: interaction parameters, and in vitro cyto-
toxic effect was investigated recently (Le Maux et al. 2012, 2013).
On the other hand, enzymatic structural modifications of BLG could mainly
include a decrease in protein molecular weight and an increased exposition of
buried hydrophobic sites (Pérez et al. 2012a, b). The effect of limited enzymatic
hydrolysis on the BLG ability to bind linoleic acid (LA) was reported (Sponton
et al. 2014). BLG enzymatic hydrolysis produced a major exposure of Trp19
(buried hydrophobic aminoacid in BLG calyx) to the aqueous medium. However,
the structure (spatial disposition) of hydrophobic domains, especially at BLG
pocket, could be broken as consequence of a-chymotrypsin cleavage. This effect
could produce the deterioration of BLG properties to bind LA.
Alternatively, the effects of isostatic high-pressure up to 350–400 MPa and
temperature on the stability and the retinol-binding capacity of BLG or phospho-
casein (PC) was compared at pH 6.5–6.6 (Blayo et al. 2014).
The effect of heat treatment on OVA molecular structure has been extensively
studied. Heating promotes protein unfolding, in which hydrophobic amino acids are
exposed. This phenomenon depends on environmental conditions, pH, ionic
strength, and protein concentration. Moreover, under determinate ionic strength and
pH conditions, heated OVA dispersions can produce protein aggregates with dif-
ferent sizes and morphologies (Nyemb et al. 2014).
Ovalbumin nanoparticles (OVAn) were produced by heat treatment (75, 80, and
85 °C from 0 to 25 min) with size around 100 nm, higher surface hydrophobicity
and higher LA binding ability than native OVA (Sponton et al. 2015a, b).
Fluorescence and Z-potential results suggested that LA binds to OVAn by mean of
hydrophobic interactions. Moreover, it is important to remark that OVAn prepared
at 85 °C during 5 min produced clear solutions due to OVAn–LA nanocomplexes
formation. In another study, OVA nanoparticle size and fluorescence (both intrinsic
and extrinsic) characteristics heat-induced aggregates produced in a range of tem-
perature below to the OVA denaturation temperature (80.1 °C) and at different pH
and protein concentrations were reported (Sponton et al. 2014). They found
experimental conditions for which OVA heat-induced aggregates had nanometric
particle size (OVAn), a suitable surface hydrophobicity and greater LA binding
ability than native OVA. Differences in LA binding ability OVAn could be
attributed not only to the differences in surface hydrophobicity but also to the
differences in their size and so their specific surface area. In general, the highest
binding ability was for OVAn with the lowest size.
222 L.G. Santiago et al.

9 Particles Formed Using a Protein and Other


An interesting strategy to prepare nanoparticles is primarily based on the utilization

of attractive forces between a protein and another biopolymer (“aggregative” par-
ticle formation), while the other strategy is based on the utilization of repulsive
forces (“segregative” particle formation). For the purpose of this review, we will
focus on the aggregative approach. This strategy could use biopolymer nanopar-
ticles (BNPs) as matrices or vehicles for the transport, protection and controlled
release of bioactive compounds as reported by many authors (Joye et al. 2015;
Mohammad et al. 2015; Perez et al. 2015). These particles could be obtained under
aqueous medium conditions (pH, ionic strength, biopolymer concentration), in
which protein–polysaccharide self-assembly takes place (Fioramonti et al. 2014).
The design of BNPs should involve the systematic study of the individual
biopolymers functional properties, molecular interactions between biopolymers and
bioactive compounds, and the process variables that govern such interactions. Other
important aspects, such factors involved in phase behavior and colloidal stability
(particle size and electrical properties), should be considered in BNPs design. It is
well established that lower particle size and higher electrical potential are
requirements for a high colloidal stability in food aqueous mediums
(Davidov-Pardo et al. 2015). Lately, information about the design of biopolymer
nanoparticles (BNPs) for polyunsaturated fatty acid (PUFA) vehiculization also was
recently published (Perez et al. 2015). The ability of linolenic acid (LA) to bind
BLG was applied to obtain BLG–LA complexes. BNPs were obtained by elec-
trostatic deposition of high methoxyl pectin (HMP) onto the BLG–LA complex
surface. In terms of the HMP protective role, a 2:1 Prot: HMP ratio could favor
high-protective effects to LA in BNPs, turning the most suitable condition for the
design of LA vehiculization systems to be used in acidic mediums. Mohammad and
collaborators explored the intrinsic transporting property of BLG and four
nutraceutical models including b-carotene, folic acid, curcumin, and ergocalciferol
(Mohammad et al. 2015). The transporting occurred under all conditions but varied
as a function of pH and nutraceutical type. The stability experiments demonstrated
that the nutraceuticals of low solubility in water were successfully entrapped within
electrostatically stable nanocomplexes arising from BLG-sodium alginate interac-
tions. The electrophoretic mobility analysis showed that soluble nanocomplexes
had good stability against aggregation. The nanoencapsulation of EPA/DHA with
sodium caseinate–gum arabic complex and its usage in the enrichment of fruit juice
(Ilyasoglu and El 2014). Also, high stability of cardamom oil was preserved in
hybrid microcapsules formed of whey protein, guar gum, and carragenins (Mehyar
et al. 2014).
Recently, cells and phages were encapsulated for oral administration. Novel
approach for encapsulation of bioactives such as probiotic, i.e., live microorganisms
members of lactic acid bacteria, using whey proteins–guar gum microparticles was
reported (Bosnea et al. 2014). Similarly, alginate-pectin coacervates were
9 Emerging Technologies for Bioactive Applications in Foods 223

emulsionated with oleic acid to support the encapsulation of phages against

enterohemorrhagic Escherichia coli (EHEC) and support extremely acid stomach
conditions in bovines (Dini et al. 2012).
Control of particle size, morphology, surface and internal properties is crucial for
obtaining protein particles with the necessary properties for emerging applications.
The latter include not only the use of protein particles in foods, where they can
improve the stability of foods at high protein content, but also as food-grade par-
ticles for the delivery of bioactives. By tuning the morphology and size of protein
particles, protection or controlled release of various bioactive components may be
obtained (Sağlam et al. 2012).

10 Future Trends

Several micro- and nanosystems able to encapsulate and entrap nutraceuticals and
micronutrients for food applications were successfully developed in recent years
mainly based on natural components. The interaction between the loads, environ-
ment, and the matrix components in the devices showed beneficial properties such
as improved stability under harsh environmental conditions, extended shelf life,
enhanced functionality, and increased bioavailability. However, it is required to
establish clearly regulatory aspects at a global scale regarding public and envi-
ronment safety issues in order to be adopted by the food industry. In this sense, it is
necessary to develop novel analytical tools and to purify technique and normalize
the technologies principally for synthetic and semi-synthetic molecules under strict
protocols, and properly label all modified foods.


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Chapter 10
Emerging Technologies of Hydrogels
in Bioactive Compounds Delivery

Maria Henriques L. Ribeiro

1 Introduction

Polyphenols are bioactive compounds with important therapeutic properties. In recent

years, particular interest in polyphenols have been demonstrated by researchers from
food and pharmaceutical industries. The main reasons are related to the biological
activity of these compounds. In fact, preclinical, human clinical trials and epidemi-
ological studies have shown that polyphenols reduce a range of pathologies associ-
ated with cardiovascular disease including thrombosis, atherosclerosis, and
inflammation, as well as displaying anticancer and neuroprotective properties. One
problem associated with the biological activity is that polyphenols display poor
bioavailability (only a proportion of ingested amounts are absorbed and mainly with
rapid excretion), complex pharmacodynamics and metabolism.
A useful approach to improve delivery of bioactive compounds, e.g. polyphe-
nols, probiotics, minerals, vitamins, fatty acids, into pharmaceuticals, foods and
supplements, is (micro/nano) encapsulation and entrapment into hydrogels. Their
uses in the food and supplements industries led to improvement in the production
of functional foods and health benefits.

2 Bioactive Compounds: Polyphenols

Polyphenols are secondary metabolites present in plants, which constitute a large

family of compounds from simple molecules to complex structures (Munin and
Edwards-Lévy 2011) (Table 1). These natural substances have in common the

M.H.L. Ribeiro (&)

Research Institute for Medicines (IMed.ULisboa), Faculty of Pharmacy,
Universidade Lisboa, Av. Gama Pinto, 1649-003 Lisbon, Portugal

© Springer International Publishing AG 2017 227

M. Puri (ed.), Food Bioactives, DOI 10.1007/978-3-319-51639-4_10

Table 1 Classes and subclasses of polyphenols, examples of compounds, sources, and biological properties
Classes Subclasses Compounds Sources Biological properties
Flavonoids Flavonols Quercetin, kaempferol, Onions, cabbage, broccoli, apples, Anti-inflammatory, anti-allergenic,
myricetin tea, berries anti-viral, antispasmodic, antibacterial,
Flavones Lutein, apigenine Celery, parsley, thyme, pepper antioxidant and anticarcinogenic activities
Hepatoprotector, enzymatic inhibitors,
Flavanones Hesperidin, naringin, Citrus (orange, lemon, grapefruit)
vitamin P factor protector of capillaries and
naringenin, eriodictyol
Isoflavones Daidzein, genistein, Soy products, vegetables
glycitein soy
Flavanols Catechin, epicatechin Fruit (apricot, cherry, grape, peach,
apple), green and black tea, red
wine and cider
Anthocyanidins Catechins, cyanidin, Green tea, chocolate, blue berries,
pelargonidin, peonidin apples, red and purple, red grapes,
red wine, radish, eggplant
Non-flavonoids Hydroxybenzoic Gallic acid, vanillic acid, Tea, red fruit (raspberry, black Antimicrobial activity and fungi toxicity,
acids protocatechuic acid, currant, strawberry) anti-inflammatory limited therapeutic
p-hydroxybenzoic acid interest
Hydroxycinnamic Caffeic acid, p-coumaric Fruit (kiwis, blueberries, apples)
acids acid, sinapic acid, ferulic Cereal grains (wheat, rice, oat,
acid flours)
Coumarins Umbelliferone, Tonka bean, bark (chestnut), sweet Anti-inflammatory and antiviral activities
aesculetin, scopoletin woodruff, meadowsweet, sweet Limited pharmacological applications:
clover hepatotoxicity
Stilbenes Resveratrol Skin of grapes, blueberries, Anti-inflammatory activity and
raspberries, mulberries anticarcinogenic effects
M.H.L. Ribeiro
10 Emerging Technologies of Hydrogels … 229

presence of one or several phenolic rings bearing one or several hydroxy functions,
derived from the metabolism of shikimic acid and/or polyacetate (Bruneton 2009).
The phenolic compounds have a wide range of essential functions in the
development, defense, and reproduction of plants. They are a protection for the
plants, providing protection against ultraviolet, resistance against pathogens and
predators, increasing the astringency of the food (Leopoldini et al. 2011), scav-
enging free radicals, which derive their antioxidant properties.
The main sources of polyphenol compounds in the human diet are fruits and
vegetables and can be found in large concentrations in beer, wine, olive oil,
chocolate, cocoa, peanuts, pomegranates, corn, berries, tea, and coffee (Cadenas
and Davies 2000; Grassi et al. 2009; Ravichandran 2010; Leopoldini et al. 2011).
The biological activities of these compounds are achieved via a range of
mechanisms including well-characterized antioxidant effects (Scalbert et al. 2005;
Pandey and Rizvi 2009; Bogdan 2016), inhibition of intracellular kinase activity
(Wright et al. 2010), binding to cell surface receptors (Ruoslahti et al. 2010), and
disruption of the integrity of cell plasma membranes (Wright et al. 2010).

2.1 Characterization, Chemical Structure, and Main


To date, more than 8000 polyphenolic compounds have been characterized in plants
and grouped together in various classes. Although the polyphenols are chemically
characterized as phenolic compounds, this group is highly diverse and contains
several subclasses as a function of the number of phenol rings containing and
structural elements that connect the rings together, and therefore structures may
range from a simple phenolic molecule to complex molecules of high molecular
weight (Garcıa-Lafuente et al. 2009). Inside each of the subclasses, the variations
around the basic chemical skeleton are related to the degrees of oxidation,
hydroxylation, methylation, glycosylation, and the eventual connections to other
molecules (e.g., primary metabolites such as carbohydrates, lipids, proteins, or
phenolic secondary metabolites).
Two main groups can be distinguished in polyphenols: flavonoids and
non-flavonoids (phenolic acids, stilbenes, and tannins) (Table 1).
Polyphenol compounds have substantial economic importance, namely to
numerous sectors of the food-processing industry as natural additives (natural
coloring agents, conservative agents, natural antioxidants, nutritional additives) and
to the most important in the field of human health. Nowadays, many plant extracts
rich in phenolic molecules of interest are used as food complements or can be
integrated into cosmetic or pharmaceutical formulations (Munin and Edwards-Lévy
230 M.H.L. Ribeiro

2.1.1 Flavonoids

Flavonoids, known as nature’s tender drugs, present various biological/

pharmacological activities including antioxidant, anti-inflammatory, anticancer,
antimicrobial, antiviral, and neuroprotective. Flavonoids are commonly present in
plants conjugated to glucosides. The glucosidic residue is important for their
activity and in some cases deglycosylation improves the biological activity.
Moreover, the glucosidic residue may influence not only the pharmacokinetics but
also the pharmacodynamics of the bioactive compound. Therefore, the biological
activity of the compound with or without this residue can differ substantially,
depending also on the global molecular structure.
Flavonoids are the most structurally diverse group of polyphenols and the most
abundant in nature (Rahman et al. 2006). They can be divided into six different
subclasses, depending on the groups linked to the phenolic ring: flavonols, flavones,
flavanones, isoflavones, and anthocyanidins (Table 1; Fig. 1).
The flavonols in foods are mainly represented by quercetin and kaempferol. The
principal sources include the onion, cabbage, garlic, broccoli, tea, blueberries, and
red wine. The biosynthesis of this type of phenolic compounds is stimulated by
light and therefore accumulated in the outer and aerial tissues (skin and leaves) of
plants (Manach et al. 2004).
The flavones are less common than flavonols in fruits and vegetables. They
consist mainly of glycosides lutein and apigenin. The plant sources identified to
date are parsley, celery, thyme, and pepper (Manach et al. 2004; Munin and
Edwards-Lévy 2011).
Flavanones can be found in low concentrations in plants such as tomatoes and
certain herbs, as mint. Moreover, these flavonoids are present in high concentrations

Fig. 1 The molecular structure of each subclass of flavonoids

10 Emerging Technologies of Hydrogels … 231

in citrus and more abundantly in the membrane parts (Manach et al. 2004; Ribeiro
et al. 2008; Vila-Real et al. 2011a).
Flavanols exist in two distinct forms: monomers (catechin) and polymers
(proanthocyanidins). Catechins are mainly found in apricot, red wine, green tea, and
chocolate are by far the richest sources of flavanols. Proanthocyanidins are known as
condensed tannins (polymers of catechins). The formation of complexes between
tannins and salivary proteins gives the character of astringent and bitterness, in some
cases. The astringency changes throughout the maturation and often disappears
when the fruit reaches the mature state (Duffy et al. 2001; Manach et al. 2004).
Isoflavones exist in the form of three molecules: genistein, daidzein, and gly-
citein. They have structural similarities to estrogen. They have the ability to bind to
estrogen receptors, being designated as phytoestrogens. The main sources of iso-
flavones in the human diet are vegetables, soybeans, and its processed products
(Manach et al. 2004; Droke et al. 2007).
Anthocyanins are pigments, which are dissolved in the vacuole of the epidermal
tissues of flowers and fruits. The coloration of these pigments varies from pink to
red, blue, and purple (Pojer et al. 2013). Anthocyanidin is the most common in
food, particularly in red wine, in a variety of grains, leaves, roots, vegetables
(eggplant, cauliflower, beans, onion, and radish) (Manach et al. 2004).
Recently, attention has been given to isolated flavonoids, namely those from
citrus (Fig. 2), as potential anti-inflammatory agents. Acute inflammation is typi-
cally characterized by increased permeability of endothelial tissue and leukocyte
leakage into the interstitium resulting in edema. Many different biological mediators

Fig. 2 Molecular structures of the flavanones: naringin, prunin, and naringenin, and the flavonols:
rutin, isoquercetin, and quercetin
232 M.H.L. Ribeiro

influence the various steps of the inflammatory process, and typically,

anti-inflammatory agents exhibit therapeutic properties by blocking the actions or
synthesis of these mediators.
The antioxidant activity exhibited by flavonoids seems to be related with the
number of hydroxyl groups in the B ring, responsible for part of the
anti-inflammatory properties of these compounds (Ribeiro et al. 2008; Sikora et al.
2010; Hamaguchi et al. 2010). Besides being related with free radicals scavenging
and inhibition of lipid peroxidation, anti-inflammatory activity of flavonoids is also
associated with the inhibition of cyclooxygenase and 5-lipoxygenase pathways
involved in the arachidonate metabolism (Ribeiro et al. 2008; Amaro et al. 2009).

2.1.2 Non-Flavonoids

Phenolic compound non-flavonoids correspond to substances, such as phenolic

acids, hydrolyzable tannins and other phenolic derivatives important as stilbenes
(Table 1; Fig. 3). Many of these compounds, as stilbenes, are produced under stress
conditions, which may explain the biological properties (e.g., antioxidant) observed
in vitro or in vivo. They are involved in defense against infection and confer
protective effects to the plants against stress, such as ultraviolet radiation, patho-
gens, and physical damages (Curin and Andriantsitohaina 2005).
Phenolic acids are found abundantly in the plant kingdom. They can be divided
in two subgroups: (i) derived from benzoic acid and (ii) cinnamic acid derivatives
(Table 1). The hydroxybenzoic acid concentrations in plants are generally low, with
the exception of red fruits, radish, and onions (Vita 2005). The hydroxycinnamic
acid is more abundant and can be classified in coumarin acid, caffeic acid, and
ferulic acid. Among them, caffeic acid, either in free form or in esterified form, is
generally the most abundant phenolic acid and represents 75–100% of the existing
hydroxycinnamic acid in the fruits (Manach et al. 2004).
The tannins are high molecular weight phenols found in complex alkaloids,
polysaccharides, and proteins, being embodied as a group of phenolic compounds

Fig. 3 Phenolic acids present in food: on the left, benzoic acids; on the right cinnamic acids
10 Emerging Technologies of Hydrogels … 233

Fig. 4 Chemical structure of


soluble in water (Tsao 2010). These compounds can be classified as hydrolyzable

and condensed. The hydrolyzable tannins are linked oligomers in the form of esters
of gallic acid or ellagic acid with glucose or other sugar (Tsao 2010). They are at
greater abundance in grapes, apple juice, strawberries, peaches, olives, wine,
chocolate, vinegar, and lentils, among others.
Stilbenes, such as benzoic acid, constitute another class least distributed in the
plant kingdom. The naturally occurring stilbene is resveratrol (Fig. 4). It is found
abundantly in grapes in response to fungal attacks. The products from grapes, such
as wine, have high concentrations of resveratrol (Tsao 2010).

2.2 Biological Activity

The increase in scientific knowledge about polyphenols led to the identification of

numerous biological properties (Cassidy et al. 2015). Many studies have shown the
importance of these compounds as antioxidants because of their activity as free
radical scavengers, induction of endogenous antioxidants, chelating metals, and
modeling enzymes acting against oxidative stress (Vita 2005). The antioxidant
properties of these molecules are due to the presence of hydroxyl (OH) group, the
conjugated system of double bonds and the facility to accept electrons to form
relatively stable radicals, thus interrupting possible oxidation reactions in cellular
components (Vita 2005).
They are also currently recognized as modulators of cell signaling and modu-
lation of inflammatory mediators and genes related to survival or cell death
(Mukamal et al. 2002; Pandey and Rizvi 2009; Romier et al. 2009; González et al.
2011; Mileo and Miccadei 2016).
The biological properties allow the existence of cardioprotective, antitumor, and
antidiabetic and neuroprotective effects; therefore, a diet rich in polyphenols may
prevent cardiovascular, cancer, diabetes, inflammatory bowel diseases, and
degenerative diseases (Hodgson et al. 2002; Weggemans and Trautwein
2003; Mandel and Youdim 2004; Lau et al. 2007; Vauzour et al. 2008; Wendeburg
et al. 2009; Queen and Tollefsbol 2010; Kawaguchi et al. 2011; Sahil et al. 2015;
Shay et al. 2015; Derek et al. 2015; Olejnik et al. 2016).
234 M.H.L. Ribeiro

In fact, the consumption of polyphenols limits the incidence of coronary heart

disease (Osiecki 2004; Garcıa-Lafuente et al. 2009). Atherosclerosis is a chronic
inflammation developed in the wall of the arteries. Polyphenols are potent inhibitors
of low-density lipoprotein cholesterol (LDL) oxidation, which appears to be the key
mechanism in the development of atherosclerosis (Miura et al. 2001; Sun et al.
2010; Vita 2005; Mukamal et al. 2002; Suzuki et al. 2009).
Polyphenols have a predominantly protective effect in cancer cell lines and
significantly reduce the number of growing tumors (Mileo and Miccadei 2016).
Among the flavonoids, quercetin and resveratrol are those that demonstrated the
most significant protective effects, both in terms of anticancer properties and in the
prevention of various stages of tumor development, respectively (Wang et al. 2002;
Yoshida et al. 2007; Vingtdeux et al. 2008; Garcıa-Lafuente et al. 2009; Mileo and
Miccadei 2016).
Emerging evidence indicates that polyphenols may also behave as prooxidants to
initiate a reactive oxygen species-mediated cellular DNA breakage and consequent
cell death (Spencer et al. 2008). It has been reported that such a prooxidant
mechanism is a result of redox-active microenvironment in cancer cells due to the
elevated levels in copper (Scalbert and Williamson 2000).
The metabolism of glucose leads to a physiological imbalance—the develop-
ment of hyperglycemia and hence the onset of diabetes. The polyphenols, in par-
ticular quercetin, have a strong antidiabetic activity since it gives greater protection
to the cells against oxidative stress, thus preventing the various long-term physi-
ological changes such as blindness, nephropathy, ulcers, and limb amputation
(Visioli et al. 2011).
At the cerebral level, the phenolic compounds have a strong neuroprotective
activity. The neurodegenerative disease is due to oxidative stress and consequently
the occurrence of brain damage. Alzheimer’s disease is the most common ailment,
affecting 18 million people worldwide (Rossi et al. 2008). The consumption of
polyphenols provides protection against the onset of degenerative diseases. These
compounds show great potential in regard to neuroprotection, by the ability of
modulating processes such as cell signaling, proliferation, differentiation, and
apoptosis. Moreover, phenol consumption also produces neuroprotective effects in
Parkinson’s disease (PD) (Aquilano et al. 2008). This disease is characterized by
several abnormalities including inflammation, mitochondrial dysfunction, oxidative
stress, and iron accumulation. Due to the high ability to chelate iron, polyphenols
are an alternative in PD therapy (Mounsey and Teismann 2012).
In recent years, the interest in bioactive compounds of fruits and vegetables has
increased due to their health benefits, particularly protection against a variety of
diseases as cardiovascular and some types of cancers (Wright 2013). Flavonoids
and their metabolites have demonstrated to act as free radical scavengers to mod-
ulate enzymatic activities and to inhibit cellular proliferation as well as to possess
another several biological activities such as anti-ulcer, anti-allergenic,
immunomodulatory, anti-diarrhea, analgesic, antibiotic, and antithrombotic with
inhibition of platelet aggregation. Citrus flavonoids and their metabolites, as potent
antioxidants, are able to restrain many of the inflammatory and tumorigenic events
10 Emerging Technologies of Hydrogels … 235

through mechanisms mediated by reactive oxygen species (Cadenas and Davies

2000). Free radicals are well known to play an important part in the inflammatory
process. They are involved in inflammation and tissue destruction and also impli-
cated in the biosynthesis of prostaglandins (Grassi et al. 2010).
Nowadays, it is known that a large number of bacteria colonize the large
intestine and form a complex with intestinal ecosystem called microbiome. The
intestinal microbiome plays a key role in the metabolism of chemical compounds
present in foods. It is estimated that the bacterial flora of a human adult is composed
of approximately 1014 bacterial cells, very diverse in the human population and
each person has their own profile of microbial species, closely related to age and the
health of the host (Duda Chodak et al. 2015). The composition of this ecosystem is
influenced by several factors such as the origin, age, environment, diet, and
antibiotics (Duda Chodak et al. 2015). If the equilibrium is disturbed even in
host-microbiome relationship can progress to the development of diseases. Any diet
that is too selective or incomplete in relation to the content of nutrients leads to
disruption of the host-microbiome balance. This can promote the overgrowth of
pathogenic agents and weaken the body’s defenses against the development of
infections and chronic inflammation (Duda Chodak et al. 2015). Many studies have
been examining the potential effects of polyphenols against the development of
pathogens. However, there are few studies investigating the influence of phenolic
compounds on the composition and activity of microbial gut community. Potent
inhibitors of growth of pathogenic microorganisms are polyphenolic compounds of
green tea and black tea (Duda Chodak et al. 2015). Other studies have shown that
polyphenols have the ability to stimulate the growth of beneficial bacteria com-
mensal whereas the pathogenic strains are inhibited. A number of in vitro and
in vivo studies showed a high modeling capability of the intestinal barrier by the
polyphenols, and thus, further research must be carried out for understanding of the
therapeutic mechanism of these compounds (Duda Chodak et al. 2015).

2.3 Bioavailability

The most common polyphenols in the human diet are not necessarily those with
higher activity in the body, and it can be due to the low intrinsic activity, low
absorption at the intestinal level, or high metabolization and rapid elimination. The
metabolites found in the blood and target tissues, resulting from digestive or liver
action, may differ from native substances in terms of biological activity. For this
reason, a more detailed knowledge about the bioavailability of polyphenols is need
allowing the evaluation of the various biological effects on health (D’Archivio
et al. 2007).
Bioavailability varies considerably depending on the type of phenolic com-
pounds involved. The absorption is mainly determined by the chemical structure of
polyphenols and depends on factors such as glycosylation/acylation conjunction
with other phenols, molecular weight, degree of polymerization, and solubility.
236 M.H.L. Ribeiro

The chemical structure of the compound more than the concentration influences the
amount and extent of absorption, as well as the nature of the metabolites circulating
in the plasma.
Generally, aglycones can be absorbed directly into the small intestine. However,
most of the polyphenols present in the food are in the form of esters, glycosides, or
polymers, which cannot be absorbed in its native form. These compounds should be
hydrolyzed by intestinal enzymes or by colonic microflora prior to absorption.
When the hydrolysis occurs at the level of the intestinal microflora, the absorption
efficiency is lower, since it also degrades releasing producing aglycones and other
aromatic acids.
During the absorption process, the polyphenols are subjected to various bio-
transformations, primarily in conjunction with the level of the small intestine and
later submitted to methylation, sulfation, or glucuronidation. This set of biotrans-
formations is part of an efficient metabolic detoxification process by increasing
hydrophilicity, restricting potential toxic effects, and facilitating the elimination via
the urine or bile.
The penetration ability of the polyphenols is quite high mainly in the tissues in
which they are metabolized, small intestine, and the liver. The administration of a
given dose of polyphenols radiolabelled, to rats or mice, allowed the detection of
the same blood-level radiolabelled and digestive tissue. This study proves the
accumulation capacity in tissues which are the main sites of metabolism.
The metabolites of polyphenols can follow two routes of elimination: i) urinary
or ii) biliary. Given that the polyphenols are normally absorbed through the
intestinal mucosa, it should be noted that the amount of polyphenols in urine intact
depends on the structure of the phenolic compound. Typically, the metabolites of
higher and lower molecular weight are eliminated by the biliary and urinary path-
ways, respectively (Manach et al. 2004; D’Archivio et al. 2007; Bilia et al. 2014).
Although these compounds display poor bioavailability (only a proportion of
ingested amounts are absorbed and excretion is rapid) and complex pharmacody-
namics and metabolism (Manach et al. 2005), they present therapeutic properties.
A substantial body of evidence (epidemiological studies, animal studies, and human
clinical trials) indicates that polyphenols reduce a range of pathologies associated
with cardiovascular disease including thrombosis (Navarro-Nuñez et al. 2008),
atherosclerosis (Chiva-Blanch et al. 2012), and inflammation (Rieder et al. 2012),
as well as displaying anticancer (Gali et al. 1991) and neuroprotective (Gatson et al.
2013) properties.
Some studies take into account confounding effects of age, sex, and lifestyle on
polyphenol effects, but complete information on how these may disrupt the impact
of these compounds on health is also lacking. Therefore, at present, methodology
for therapeutic application of these compounds is not entirely clear, but what is
clear is that dietary supplementation is not enough. An understanding of the sig-
nificance of individual functional groups on the inhibitory activity/potency of these
compounds may allow more potent and selective analogues to be made using
polyphenols as a basis. Information from structure-activity studies has allowed the
construction of more selective analogues, for example, quercetin-3-O-amino acid
10 Emerging Technologies of Hydrogels … 237

esters were more selective for Src tyrosine kinase than epidermal growth factor
(EGF) receptor kinase (Huang 2009). Rieder et al. (2012) suggested an alternative
strategy involving an unmodified polyphenol (resveratrol) as viable treatment for
lung injury.
The anti-inflammatory properties of resveratrol present this small molecule as a
potentially important therapeutic agent against a number of degenerative conditions
(e.g., coronary artery disease, brain injury) that manifest acute inflammation as a
major pathological factor. A key recent study described the preclinical application
of a grape extract containing resveratrol to a group of coronary artery disease
patients and demonstrated an increase in anti-inflammatory factors (Tomé-Carneiro
et al. 2013).
Network/systems pharmacology may allow the development of methodology to
direct the clinical use of resveratrol, through the use of enhanced pharmacodynamic
models that analyzes regulatory networks involved in drug action. These can
account for multiple targets of a drug as well as the effects of genomic, epigenomic,
and posttranslational changes on drug efficacy and, therefore, may allow the
development of resveratrol or resveratrol analogues into precision therapy.
The anti-inflammatory properties of resveratrol may render this compound as an
immunosuppressant. This property may confound the beneficial effects exerted by
this compound. The therapeutic effects of resveratrol may be directed through local
delivery of the stilbene using biomaterial devices. This strategy has already been
investigated using nanomaterial devices that allow controlled release of polyphe-
nols (including resveratrol), concentrating the compound at predetermined amounts
over specified time periods in the physiological region of interest.

3 Hydrogels

Hydrogels are currently subject of substantial scientific research due to their

potential applications in high technology fields, as biotechnology, biomedical,
pharmaceutical, cosmetics, food, agriculture, biosensor, bioseparation, and oil
recovery (Peppas et al. 2000a, b; Syed et al. 2011).
Over the past few decades, advances in hydrogel technologies have spurred
development in such applications, including controlled delivery systems.
Controlled release or controlled delivery systems are intended to provide the
bioactive compound or a drug at a predetermined temporal and/or spatial way to
accomplish a specific goal or therapeutic needs within the body.
Controlled delivery systems can be used to achieve some goals, namely:
(i) maintain constant concentration of therapeutically active compounds in the
blood; (ii) predictable and reproducible release rates over a long period of time;
(iii) protection of bioactive compounds having a very short half-time; (iv) elimi-
nation of side effects, waste of drug and frequent dosing; (v) optimized therapy and
better patient compliance; and (vi) solution for drug stability problems (Davis and
Anseth 2002).
238 M.H.L. Ribeiro

Hydrogels have unique characteristics combination and particular properties,

which make them to be potentially considered as one of the future drug delivery
applications and controlled release systems. Many novel hydrogel-based delivery
matrices have been designed and fabricated to fulfill the ever-increasing needs
(Wright 2013).
Hydrogel enfolds three-dimensional network structures obtained from different
polymers, with high degree of flexibility due to large water content, as they can
absorb and retain significant amount of water.
Hydrogels are polymeric materials with capability to swell and retain large
amounts of water without dissolving in it. The network structure of a hydrogel
determines the properties.
In the last years, due to specific characteristics as non-toxic swelling, biocom-
patible, and mechanical, the uses of hydrogels include bioactive compounds de-
livery (Wright 2013), drug carrier systems, gene transfection (Gojgini et al. 2011),
tissue engineering scaffolds (Hou et al. 2011), wound dressings, contact lenses
(Bühler et al. 1999), biosensors (Frisk et al. 2007), and dye removal materials
(Abdel-Halim 2013).
Some of the important methods to prepare and characterize hydrogels are in
Table 2 highlighted as well as some techniques of measurements used (Ahmadi
et al. 2015).
The physical behavior of hydrogels is dependent on their equilibrium and
dynamic swelling behavior in water; since upon preparation, they must be brought
into contact with water to yield the final, swollen network structure. The most
important parameters that define the structure and properties of swollen hydrogels
are the polymer volume fraction in the swollen state, the effective molecular weight
of the polymer chain between cross-linking points, and the correlation distance
between two adjacent cross-links.
There are a number of methods for estimating the relative amounts of free and
bound water, as fractions of the total water content. The use of small molecular
probes, differential scanning calorimetry (DSC), and nuclear magnetic resonance
(NMR) are the three major important methods.
Some of the techniques used for determining the surface property include
electron spectroscopy (ES), secondary ion mass spectrometry (SIMS), scanning
electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR),
scanning tunneling microscopy (STM), and atomic force microscopy
(AFM) (Table 2). FTIR is a useful technique for identifying the chemical structure
of a compound and is widely used to investigate the structural arrangement in a
hydrogel by comparison with the starting materials. SEM can be used to provide
information about the sample surface topography, composition, and other properties
such as electrical conductivity. This is a powerful technique widely used to visu-
alize the characteristic “network” structure in hydrogels. Atomic force microscopy
(AFM) can be used not only for imaging the topography of surfaces but also for
measuring forces on a molecular level. The information obtained through these
methods can be used to monitor contamination, ensure surface reproducibility, and
explore the interaction of the hydrogels with living systems (Ebara et al. 2014).
10 Emerging Technologies of Hydrogels … 239

Table 2 Methods of hydrogel preparation, parameters characterization, and techniques of

Preparation Characterization Techniques of measurement
methods parameter
Isostatic ultra-high Morphology/network Quasi-elastic laser light scattering
pressure (IUHP) pore size Electron microscopy
Rubber elasticity measurements
Use of Degree of swelling Dimensional changes with time
cross-linkers Aqueous medium or medium-specific pH
Volume or mass degree of swelling
Use of water critical conditions of drying equilibrium water content
Use of gelling Cross-linking Ultimate compressive strength
agents and mechanical strength Change in polymer solubility
Use of nucleophilic Bioactive compounds Membrane permeability
substitution diffusion Controlled strength experiments
reaction Nuclear magnetic resonance
Fourier transform infrared spectroscopy
Scanning electron microscopy
Quasi-elastic laser light scattering
Use of irradiation Bioactive compounds Fourier transform infrared spectroscopy
and freeze-thawing distribution Scanning electron microscopy

Hydrogels can be classified using different criteria, as shown in Tables 2 and 3.

3.1 Origin

According to the origin of the polymeric material, hydrogels are natural, synthetic,
or hybrid (Peppas et al. 2000a, b). The natural hydrogels include alginate,
k-carrageenan, chitosan, dextran, gelatin, casein, collagen, elastin, fibrin, hyaluronic
acid, and xanthan. Examples of synthetic hydrogels are hydroxyethyl methacrylate,
polyvinyl alcohol, N-2-hydroxypropyl methacrylate, N-vinyl-2-pyrrolidone,
N-isopropyl acrylamide, vinyl acetate, acrylic acid, methacrylic acid, polyethylene
glycol acrylate/methacrylate, and polyethylene glycol diacrylate/dimethacrylate.
Hybrid hydrogels include gelatin methacrylamide and PEG, or PEG cross-linked
with chitosan, or fibrin and polyurethane.

3.2 Natural Hydrogels

Hydrogels made from natural polymers show advantageous properties such as

biocompatibility, biodegradability, and biologically recognizable moieties that
240 M.H.L. Ribeiro

Table 3 Classification of hydrogels

Origin Natural
Cross-linking method Chemical and physical
Network electrical charge Nonionic (neutral)
Ionic (anionic or cationic)
Amphoteric (ampholytic)
Zwitterionic (polybetaines)
Degree of swelling Low swelling
Medium swelling
High swelling
Network Structure Non-porous
Network morphology Amorphous
Hydrogen-bonded structures
Supermolecular structures
Component Homopolymer
Function Biodegradable
Stimuli responsive
Mechanism controlling the compounds release Diffusion-controlled release systems
Swelling-controlled release systems
Chemically controlled release systems
Environment-responsive systems

support cellular activities, after they may not provide sufficient mechanical prop-
erties, evoke immune or inflammatory responses, and eventually contain pathogens.
Polymers, such as alginate, chitosan, k-carrageenan, collagen, dextran, gelatin,
hyaluronic acid, are natural hydrogels. Conditions for fabricating hydrogels are
relatively mild. Gel formation usually takes place in situ at ambient temperature
without the requirement of organic solvents (Chien-Chi and Metters 2006).
Alginate, a naturally occurring polysaccharide, from brown algae, forms gels by
ionotropic gelation. An example is the formation of calcium alginate particles
(Fig. 5) from sodium alginate after gelling in calcium chloride (Ribeiro et al. 2010;
Furtado et al. 2012a). The mechanical strength of the gel generally increases with
an increase in the Ca2+ concentration during the solidification process. It is possible
10 Emerging Technologies of Hydrogels … 241

Fig. 5 Chemical structure of natural hydrogels from polysaccharides: chitosan, alginate, calcium
alginate, and k-carrageenan

to postulate that gelation of alginate in a suitable concentration of Ca2+ is effective

for the promotion of mass transfer characteristics in beads (Furtado et al. 2012a).
K-carrageenan, another naturally occurring polysaccharide, from algae, forms
gels with K+ ions (Fig. 5) (Ribeiro and Ribeiro 2008).
Chitosan is a polysaccharide, from shrimp and other crustacean, composed of
randomly distributed b-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine.
Chitosan particles can be prepared by different methods, namely precipitation or
cross-linking method (Ahmadi et al. 2015). Glutaraldehyde, sodium sulfate
(Furtado et al. 2012a), or trypolyphosphate are cross-linkers used.
These natural hydrogels, calcium alginate, k-carrageenan, chitosan, considered
GRAS (generally recognized as safe) material by the FDA (Food and Drug
Administration) had been used for protein entrapment in the deglycosylation of
some flavonoids (e.g., naringin to prunin and naringenin, hesperidin to hesperitin)
in order to improve their biological activity, namely anti-inflammatory (Ribeiro
et al. 2008; Amaro et al. 2009), neuroprotective (Ribeiro et al. 2009), anticancer,
and bioavailability (Furtado et al. 2012b).
Some examples of food proteins used as natural hydrogels include gelatin
(Fig. 6), obtained from collagen by acid and alkaline hydrolysis and considered as
GRAS material by FDA (Ahmed 2015). The functional groups are accessible for
various chemical modifications, which may be especially useful in developing
targeted bioactive or drug delivery vehicles. As it is denatured, it shows low
antigenicity. Gelatin has both cationic and anionic groups along with hydrophobic
Collagen (Fig. 6) is the most abundant mammalian protein, about 20–30% of
total body proteins. Some disadvantages of collagen-based systems arose from their
poor mechanical strength and the difficulty of ensuring adequate supplies. Due to
their small size with a large surface area, high adsorptive capacity, and ability to
disperse in water to form a clear colloidal solution, collagen-based nanoparticles
have been used as a sustained release formulation for antimicrobial agents (Ahmed
et al. 2012; Belitz et al. 2007).
Casein (Fig. 6), the major milk protein, is inexpensive, available, non-toxic, and
highly functional. As a natural food product, this GRAS protein is biocompatible
242 M.H.L. Ribeiro

Fig. 6 Chemical structure of natural hydrogels from food proteins: gelatin, collagen, casein

and biodegradable. Many of the structural and physicochemical properties of

caseins facilitate their functionality in drug delivery systems including binding of
ions and molecules, exceptional surface-active stabilizing properties, excellent
emulsification, and self-assembly properties together with supergelation and
water-binding capacities (Ahmed et al. 2012). A major advantage is that caseins are
not sensitive to temperature (Livney 2010).
Caseins are amphiphilic proteins that can be thought as block copolymers with
high levels of hydrophobic or hydrophilic amino acid residues. Therefore, caseins
exhibit a strong tendency to self-assemble into spherical casein micelles 50–500 nm
in diameter (Livney 2010). Casein-based nanoparticles as bioactive compounds
delivery systems have been developed, namely casein micelles protected vitamin
D2 and x-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) against
UV-light-induced degradation and oxidation, respectively (Ahmed et al. 2012).
Proteins as natural polymers are heterogeneous mixtures of different sizes with a
wide range of molecular weights. Protein nanoparticles successfully controlled the
release rate of bioactive compounds/drugs for prolonged periods. Protein-based
nanoparticles offer various possibilities for surface modification due to the presence
of functional groups (i.e., carboxylic and amino groups) on the surface of the
nanoparticles, thus enabling specific targeting to the site of action (Ahmed et al.
2012). Proteins are less expensive compared to other functional carriers.

3.2.1 Synthetic Hydrogels

Synthetic hydrogels, on the other hand, do not present the inherent bioactive
properties of natural ones. In fact, they usually have well-defined structures that can
be modified to yield tailorable, degradability, and functionality. Examples of syn-
thetic polymers include polyvinyl alcohol (PVA), hydroxyethyl methacrylate
(HEMA), N-2-hydroxypropylmethacrylate (HPMA), N-vinyl-2-pyrrolidone (NVP),
N-isopropyl acrylamide (NIPAAm), vinyl acetate (VAc), acrylic acid (AA),
10 Emerging Technologies of Hydrogels … 243

methacrylic acid (MAA), polyethylene glycol acrylate/methacrylate

(PEGA/PEGMA), and polyethylene glycol diacrylate/dimethacrylate
Polyvinyl alcohol (PVA) is a biodegradable synthetic polymer, with the fol-
lowing properties: odorless and tasteless, translucent, white or cream-colored
granular powder. It is soluble in water, slightly soluble in ethanol, but insoluble in
other organic solvents. Typically, a 5% solution of PVA exhibits a pH in the range
of 5.0–6.5 (Ku and Lin 2005). The solubility of the different series depends pri-
marily on the degrees of hydrolysis and of polymerization. At the same tempera-
ture, the solubility increases along with the reduction of the degree of hydrolysis
and decreases with the increase of the viscosity; for the PVA series with the same
degree of polymerization and with a low degree of hydrolysis, the temperature to
complete dissolution decreases, and vice versa; for the PVA with the same
hydrolysis degree, the higher the degree of polymerization, the higher the disso-
lution temperatures, and vice versa (Nunes et al. 2010, 2012). PVA is produced in a
two-step process. In the first step, polymerization of vinyl acetate (VAc) to poly-
vinyl acetate (PVAc) is carried out; in the second step, partial or complete
hydrolysis of PVAc is performed, in order to remove acetate groups (Fig. 7).
The PVA membrane strength, aqueous solution viscosity, and other properties
vary greatly depending on the degree of polymerization. It is also possible to
produce PVA with different characteristics by copolymerizing other monomers
during this process.
In the second step, the hydrolysis process using an alkaline catalyst, viz. sodium
hydroxide or sodium methoxide, is used in the presence of methanol to convert the
acetate groups of the polyvinyl acetate to hydroxyl groups, leading to the formation
of PVA (Fig. 8). This process, called the degree of hydrolysis, controls the amount
of hydroxyl groups (Nassan and Peppas 2000).
The properties of PVA (e.g., solubility, viscosity of aqueous solution, film
strength, and moisture absorption property) are affected by the degree of poly-
merization and the degree of hydrolysis.
Partially hydrolyzed grades contain residual acetate groups, which reduce the
overall degree of crystallinity. Their formulations generally have lower melting

Polyvinyl Alcohol (PVA)

Vinyl Acetate

Fig. 7 Production of polyvinyl alcohol

244 M.H.L. Ribeiro

Fig. 8 Degree of
[(mol) = (hydroxyl
groups − n) + (acetate
groups − m)] and degree of
hydrolysis [(mol
%) = (hydroxyl groups − n)/
groups − n] + [acetate
groups − m])  100

points, lower strength, and lower water dissolution temperatures and are easier to
process, than those based on fully hydrolyzed polymers (Tang and Alavi 2011).
Advantages of PVA hydrogels include non-toxic, non-carcinogenic, and
bioadhesive characteristics, with an easy processing. Additionally, PVA has a
straightforward chemical structure, which makes possible modifications by simple
chemical reaction (e.g., glutaraldehyde, boric acid, and boronic acids) (Nunes et al.
2012). PVA gels exhibit a high degree of swelling in water and a rubbery and
elastic nature. Specific applications of PVA include membranes, bioactive com-
pounds, and drug delivery applications (Peppas et al. 1999; Nassan and Peppas
2000; Nunes et al. 2014); in contact lenses (Bühler et al. 1999); and in articular
cartilage (Yusong et al. 2007), catheters, artificial skin, and tendons (Schmedlen
et al. 2002; Kobayashy et al. 2001), wound dressings, and biodegradable scaffolds.
PVA has been used in biomedical, pharmaceutical, and food industries (Peppas
1986). In addition, they have potential application in biotechnology industries
(Bolto et al. 2009), as support networks for protein (Caramori et al. 2011; Grosová
et al. 2008) and cell entrapment/encapsulation (Baia et al. 2010; Takei et al. 2011).

3.2.2 Hybrid Hydrogels

Natural (c.f. 3.1.1) and synthetic (c.f. 3.1.2) hydrogels have been combined to
obtain materials that are both mechanically strong and bioactive. Hybrid hydrogels
result from that combination. Initially, blends of these polymers were formed
non-chemically cross-linked, showing enhanced cell adhesion and high tenability
over mechanical properties (Buwalda et al. 2014). Nowadays, covalently
cross-linked hybrid hydrogels have been designed by introduction of functional
groups in both polymers (natural and synthetic) (Buwalda et al. 2014). Examples of
natural–synthetic hydrogels include combinations of gelatin methacrylamide and
PEG (Daniele et al. 2014), fibrin and polyurethane (Huang et al. 2013), and PEG
cross-linked with chitosan (Tan et al. 2013).
10 Emerging Technologies of Hydrogels … 245

Alternate deposition of thermoplastic polymer fibers and hydrogels leads to the

formation of a mechanically strong hydrogel (Schuurman et al. 2011). A hydrogel–
electrospun composite showed significantly decreased burst release of albumin
(BSA) from 20 to 7% due to the incorporation of hydrophobic poly(e-capro-
lactone)-based fiber mats (Han et al. 2012). An hybrid hydrogel constructs with
alginate and 3D Ormocomp framework increased the cell viability and successfully
released dopamine (Kang et al. 2014).

3.3 Cross-Linking Method

Gelation refers to the linking of macromolecular chains together which initially

leads to progressively larger branched soluble polymers depending on the structure
of the starting material (Ullah et al. 2015). The gelation method can be physical (or
non-covalent) or chemical cross-linking (or covalent) (Table 4). Different types of
gelation mechanism are summarized in Table 4.
Physical gels can be subcategorized as strong and weak gels. In the first case,
strong physical bonds between polymer chains are effectively permanent at a given
set of experimental conditions (Syed et al. 2011; Ahmed 2015). Physically
cross-linked hydrogels are generally obtained from multiblock copolymers or graft
copolymers. These can be composed of a water-soluble polymer backbone, e.g., a
polysaccharide, to which hydrophobic units are attached, or hydrophobic chains
containing water-soluble grafts. The most commonly used thermogelling polymers
are Pluronics® and Tetronics® (Ullah et al. 2015). At low concentrations in water
are formed micelles and at higher concentrations formed thermoreversible gels.
Some of them have been approved by the Food and Drug Administration
(FDA) and Environmental Protection Agency (EPA) for applications in food
additives, pharmaceutical ingredients, and agricultural products.
Examples are lamellar microcrystals, glassy nodules, or double and triple helices
(Ebara et al. 2014). Weak physical gels have reversible links formed from tem-
porary associations between the chains. Examples are hydrogen bond (e.g., PAAc),
hydrophobic interactions (e.g., PEO–PPO–PEO), block copolymer micelles (e.g.,
enantiomeric lactic acid), ionic associations (e.g., alginate), and supramolecular
chemistry (e.g., inclusion complex) (Peresin et al. 2010; Syed et al. 2011; Vila-Real
et al. 2010b; Vila-Real et al. 2011a).
On the other hand, chemical gelation involves formation of covalent bonds and
always results in strong gels (Vila-Real et al. 2010a, b). The three main chemical
gelation processes include condensation (e.g., polymer–polymer), addition poly-
merization (e.g., acryloyl group), radiation (e.g., c-ray), and cross-linking (e.g.,
glutaraldehyde) (Table 4).

Table 4 Classification of gelation mechanism and some examples

Gelation mechanism Examples
Hydrogels Physical Strong Lamellar microcrystals Block polymers, elastomers, gelatin
Glassy nodules
Double/triple helices
Weak Hydrogen bonds Xanthan, Polymer–polymer complexes, acacia gum
Ionic bonds
Hydrophobic interactions
Chemical Condensation Critical percolation Polyester gel
Addition polymerization Kinetic growth Polydivinylbenzene, CMC-acrylic acid
Cross-linking End-linking Polydimethylsiloxane, cis-polyisoprene
Random cross-linking
M.H.L. Ribeiro
10 Emerging Technologies of Hydrogels … 247

3.4 Network Electrical Charge

The classification of hydrogels, based on the network electrical charge, dependent

on the presence or absence of charge located on the cross-linked chains, such as
nonionic (neutral), ionic (anionic, cationic), amphoteric electrolyte (ampholytic)
containing both acidic and basic groups, and zwitterionic (polybetaines) containing
both anionic and cationic groups in each structural repeating unit (Ahmed 2015).

3.5 Water Content or Degree of Swelling

Depending on the water content, hydrogels can be classified as low swelling,

medium swelling, high swelling, and superabsorbent.
The swelling behavior of hydrogel systems is an important parameter, especially
in their applications in food, pharmaceutical, ophthalmology, and tissue
The polymer chains in a hydrogel interact with the solvent molecule and tend to
expand to the fully solvated state, while the cross-linked structure applies a
retractive force to pull the chains inside. Equilibrium is achieved when these
expanding and retracting forces counterbalance each other.
The equilibrium swelling ratio or water content is generally used to describe the
swelling behavior of hydrogels and evaluated as follows:

Equilibrium swelling ratio ¼ Wswollen =Wdry

where Wswollen is the weight of the swollen gel and Wdry is the weight of the dry gel.
The kinetic studies of swelling in water of pure PVA and PVA-alginate hy-
drogels showed that equilibrium was attained after 4 h (Nunes et al. 2010).
A correlation was observed between the swelling behavior in acetate buffer at pH
4.0 and the equilibrium properties of alginic acid gels (Nunes et al. 2012). High
contents of cross-link gel [long -guluronic acid blocks (G-blocks)], known to give a
high acid gel strength, reduced the rate of swelling and also the amount of solu-
bilized alginate molecules leaching out of the gel beads (Nizam et al. 2007).
The swelling of hydrogels can be determined from the swelling kinetic curves.
First, the weight of the dry gel (W0) is determined. After that, the dried gel is
immersed in an excess amount of water until the swelling equilibrium is attained.
The surface water is removed and the weight of the wet gel (Wt) is determined. The
swelling ratio (SR) is calculated, at a given temperature, with the following
equation (Kim et al. 2008; Li et al. 2007):

SRð%Þ ¼ ðWt  W0 Þ=W0  100

248 M.H.L. Ribeiro

Swelling measurements are relatively simple means to characterize cross-linked

polymer networks being helpful in the interpretation of diffusion transport processes
through the macromolecular material (Braze and Peppas 2000) and of drug release.
Penetration of solvent into the polymer leads to its swelling which is involved
with diffusion of solvent molecules through the polymer matrix, and local relax-
ation of polymer segments. For rapid relaxation rates, penetration speed is limited
by diffusion process. In this type of swelling mechanism, diffusion of water
molecules inside the polymer is a rate-limiting step (Braze and Peppas 2000).
Polyvinyl alcohol has been proved to be very useful as a membrane material as it
has high water permeation and film-forming characteristics. It has been observed
that the degree of swelling of the hydrogel membrane has a great influence on the
permeability. A high swelling leads to an open structure and the resultant mem-
branes show low salt rejection (Gohil et al. 2006).
The swelling ratio decreased with increasing alginate content, indicating that it
possibly contributes to low mass transfer in PVA beads (Nunes et al. 2010, 2012).
Swelling capacity, rate of swelling and solubility of alginic acid seemed to
depend on a balance between the tendency of homopolymeric blocks in alginate to
form intermolecular junction zones, and the tendency of alginate to reduce the
chemical potential of water. As expected, swelling rate increased with decreasing
bead size (Nunes et al. 2010).
Different ratios of boric acid/PVA led to significant difference in water uptake by
the beads (Nunes et al. 2012, 2014). Higher ratios led to lower swelling ratio. The
presence of dimethyl sulfoxide DMSO in the membranes decreases considerably
the swelling ratio of the PVA particles, probably related to the membrane porosity
(Nunes et al. 2012). Chain entanglement along with increase in cross-linking agent
concentration would result in a decreased network expansion (Braze and Peppas
High porosity favors the diffusion into the PVA matrix, because large pores are
easily occupied by water molecules and with increasing water content, the degree of
swelling of the PVA membranes also increased.
The hydrogels have a porous structure, and according to Kita and co-workers
(Kita et al. 1990), the pore diameter of hydrogels becomes smaller with increasing
concentrations of DMSO, until no clearly porous PVA structures are obtained at
DMSO concentrations of 60 and 80% (Kita et al. 1990). Still, based on the positive
swelling ratio and high residual activity values obtained in the present study, it is
suggested that 60% DMSO-PVA membrane cross-linked with glutaraldehyde has
some kind of porosity.
The degree of cross-linking is not always specified. Good cross-linking is crucial
for performance as highly cross-linked membranes will have a higher salt rejection,
but lower water permeability and permeate flux rate. This arises because of the
tighter network, resulting in lower swelling ratios (Bolto et al. 2009). After swel-
ling, the membranes appear to be less hydrated; such results may be due to the
presence of excess of water when the gel is initially prepared; after heat treatment,
water is expelled, leading to a retraction and consolidation of the PVA network,
which is unable to absorb the same amount of water loss before swelling. It is thus
10 Emerging Technologies of Hydrogels … 249

not expected that the PVA lenses produced initial have the same diffusion behavior
after been heat treated or exposed to higher temperatures.

3.6 Network Structure

The classification of hydrogels, based on the network structure dependent on

porosity, is non-porous, microporous, macroporous, and superporous.

3.7 Network Morphology

The classification of hydrogels, based on the network morphology, is amorphous,

semicrystalline, hydrogen-bonded structures, supermolecular structures, and

3.8 Component

The classification of hydrogels, based on the component, dependent on the method

of preparation, is homopolymer, copolymer, multipolymer, and interpenetrating.
Homopolymer hydrogels are cross-linked networks of one type of hydrophilic
monomer unit, whereas copolymer hydrogels are produced by the cross-linking of
two comonomer units, at least one of which must be hydrophilic to render them
swellable. Interpenetrating polymeric hydrogels are produced by preparing a first
network that is then swollen in a monomer. This later reacts to form a second
intermeshing network structure.
Homopolymers refer to polymer networks derived from single species of
monomer. It is the basic structural unit, comprising of any polymer network (Ullah
et al. 2015). They may have a cross-linked skeletal structure depending on the
nature of the monomer and polymerization technique. An example is polyethylene
glycol (PEG)-based hydrogels, a suitable biomaterial for the efficient and controlled
release of drugs, proteins, biomolecules, and growth factors. Chemically
cross-linked PEG hydrogels are used as scaffolds for protein recombination and
functional tissue production.

3.9 Function

The function of the hydrogel is based on the organization of the monomers. They
can be biodegradable, non-biodegradable, stimuli responsive, and superabsorbent.
250 M.H.L. Ribeiro

3.9.1 Biodegradable and Non-Biodegradable

Depending on the nature and composition of the hydrogel, the next step is the
disintegration and/or dissolution if the network chain or cross-links are degradable.
Biodegradable hydrogels, containing labile bonds, are therefore advantageous in
applications such as bioactive compounds/drug delivery, tissue engineering, and
wound healing. These bonds can be present either in the polymer backbone or in the
cross-links used to prepare the hydrogel. The labile bonds can be broken under
physiological conditions either enzymatically or chemically, in most of the cases by
hydrolysis (Hennink and Nostrum 2002).
Biocompatibility is the third most important characteristic property required by
the hydrogel. Generally, hydrogels possess a good biocompatibility since their
hydrophilic surface has a low interfacial free energy when in contact with body
fluids, which results in a low tendency for proteins and cells to adhere to these
surfaces. Hydrogels, due to their significant water content, possess a degree of
flexibility similar to natural tissue. It is possible to change the chemistry of the
hydrogel by controlling their polarity, surface properties, mechanical properties,
and swelling behavior.
An understanding of compound transport processes is the first step toward
developing a suitable mathematical model. Mass transport governs the translocation
of drug from the interior of hydrogels to the surrounding environments. Multiple
factors affect the mass transport of encapsulated molecules including the network
cross-linking density, extent of swelling, gel degradation, the size, and charge of the
encapsulated molecules, and the physical interactions these molecules exhibit for
themselves and for the polymer matrix. If specific drug-binding motifs are present
within the hydrogels, the kinetics and/or thermodynamics of drug–ligand binding
must also be understood and quantified to predict the controlled release of the
encapsulated molecules.

3.9.2 Stimuli Responsive and Superabsorbent

The stimuli responsive or environment-sensitive polymers have the ability to

answer concerning to small physical or chemical changes. The hydrogels are
designed to change their configuration in response to stimuli based on different
available mechanisms (Ullah et al. 2015). In response to internal or external stimuli,
hydrogels can exhibit changes in their swelling behavior, network structure, per-
meability, or mechanical strength (Ullah et al. 2015). Various stimuli have been
explored for modulating compounds (e.g., drug) delivery. These stimuli include
physical factors such as temperature, ultrasonic, magnetic or electric fields, light
radiation, pressure, sound, or chemical factors such as pH, ionic strength, solvent
composition, and molecular species such as metal, glucose, antibody, or
10 Emerging Technologies of Hydrogels … 251

The external stimuli are generated using adequate devices, whereas internal
stimuli are produced within the body to control the structural changes in the
polymer network and to exhibit the desired drug release (Ullah et al. 2015).
The ability of these systems to exhibit rapid changes in their swelling behavior
and pore structure in response to changes in environmental conditions lend to these
materials favorable characteristics as carriers for delivery of bioactive agents,
including peptides and proteins. Many responsive hydrogels show an entirely
reversible mechanism of the network structural changes. An example of this
behavior is for pH or temperature changes. Responsive hydrogels are highly sen-
sitive to changes in the environment and have been used in several applications
(e.g., biosensors, superabsorbent polymers, site specific drug delivery systems, bio-
and mucoadhesive drug delivery systems, emerging nanoscale technologies, and
tissue engineering).

3.10 Mechanism Controlling Bioactive Compounds Release

Hydrogels have characteristics that make them useful in compounds’ delivery

applications. Hydrogels can absorb large amounts of water (>90%, w/v), due to
their hydrophilicity. The physicochemical properties of the hydrogel network and
the selection of bioactive compound-loading method determine the mechanism(s)
by which the loaded compound is released from the cross-linked matrix.
The mechanism of controlling bioactive compounds’ release systems from
hydrogels microcapsules is diffusion, dissolution, osmosis, and erosion. Other
mechanisms may be involved in bioactive compounds’ release from hydrogels,
namely swelling and chemically or environment-responsive systems (Chien-Chi
and Metters 2006).
Some common models which can predict and describe the release rate are as
follows (Siepmann 2012; Peppas et al. 2006): release of core material depends
mainly on
Applied release models
Zero order: Co − Ct = K.t;
First order: ln Co − ln Ct = K.t;
Hixson–Crowell: (Co)1/3 − (Ct)1/3 = K.t; and
Peppas: Ct/Ca =
where Co is the initial concentration, Ct is the concentration after time t, K is the rate
release constant; Ca is the final concentration of compound release, k is a
structural/geometric constant for a particular system, and n is designated as release
exponent representing the release mechanism.
Diffusion is the most common mechanism of bioactive compounds release. The
dissolution fluid penetrates the particles, then the core material comes into the
252 M.H.L. Ribeiro

contact with the dissolution fluid, and the compound material leaks out through the
interstitial channels or pores.
The release of core material depends mainly on (Siepmann 2012): (i) the rate of
bioactive compounds dissolution in the dissolution fluid; (ii) the rate of penetration
of dissolution fluid to the microcapsules; and (iii) the rate at which the dissolved
bioactive compounds escape from the microcapsule.
The kinetics of bioactive compounds release, mainly, follows the Higuchi’s
Q ¼ ½D/J ð2A  e. CsÞ  Cs t 1=2

where Q is the amount of bioactive compounds released per unit area of exposed
surface in time t, J is the tortuosity of the capillary system in the wall, D is the
diffusion coefficient of the solute in the solution, A is the total amount of bioactive
compounds per unit volume, e is the porosity of the wall material, and Cs is the
solubility of bioactive compounds (Siepmann 2012).
The release rate of bioactive compounds from microcapsule depends on the dis-
solution rate of polymer membrane. The solubility in the dissolution fluid and
thickness of membrane influence the release rate.
Another method of bioactive compounds release is through osmosis. The essential
requirement of osmosis is semipermeable membrane and in microcapsule polymer
coat serves the purpose. As the process progress, an osmotic pressure is created
between the outside and inside membranes of microcapsule, which result in release
of bioactive compounds through small pores (Jamileh and Lakkis 2007).
Erosion of membrane generally occurs due to pH or enzymatic hydrolysis and
causes bioactive compounds release with certain membrane materials such as
stearyl alcohol and glyceryl monostearate.
Controlled release mechanisms
Delayed release is a mechanism whereby the release of an active substance is
delayed from a finite “lag time” up to a point when/where its release is favored and
is no longer hindered. Examples include encapsulating probiotic bacteria for their
protection from gastric acidity and further release in the lower intestine, flavor
release upon microwave heating of ready meals, or the release of encapsulated
sodium bicarbonate upon baking of a dough or cake batter (Peppas et al. 2006;
Siepmann 2012).
Sustained release is a mechanism designed to maintain constant concentration of
an active at its target site (Jamileh and Lakkis 2007). Examples of this release
pattern include encapsulating flavors and sweeteners for chewing gum applications
so that their rate of release is reduced to maintain a desired flavor effect throughout
the time of chewing (Jamileh and Lakkis 2007).
10 Emerging Technologies of Hydrogels … 253

Burst release is simply described by a high initial delivery of an entrapped

bioactive compound, before the release reaches a stable profile, thus reducing the
system’s effective lifetime and complicating the release control (Jamileh and Lakkis
2007). Burst release may be preferred for flavor high-impact applications.

4 Polyphenols in Hydrogels

Hydrogel matrices with intrinsic porosity, particularly biocompatible alginate gels

(Wright et al. 2013), are suitable for the release of polyphenols to localized areas.
Encapsulation technologies (micro/nano) have gained increased interest to the
food industry as they are applied to sustain stability of the bioactive compounds
during processing and storage also to prevent undesirable interactions with the food
matrix (Bilia et al. 2014). Important benefits of encapsulation are due to controlled
release of the incorporated ingredients and deliver them to a specific target in a
suitable time (Huang 2009; Nedovic et al. 2011).
Microencapsulation is a useful approach to improve delivery of bioactive
compounds into foods, particularly probiotics, minerals, vitamins, fatty acids, and
antioxidants. Several microencapsulation techniques have been discussed (e.g.,
extrusion, emulsion, and spray drying) for use in the food industry which have
shown a promotion for the production of functional foods (Ahmed 2015). In
addition to the matrix material, the chosen encapsulation technique determines the
physical characteristics of the resulting particles. While spray drying processes are
relatively cheap and comparably small capsules are created, as a disadvantage they
are mostly water soluble, extrusion method allowed the formation of predominantly
large and water-insoluble (micro)capsules. In comparison, the emulsion technique
has the advantage that smaller capsules can be created (Ahmed 2015). The in situ
polymerization process is mainly used for the synthesis of nanocomposites and
consists of emulsification of the monomer component, mostly vinylic and acrylic
compounds (e.g., styrene or methyl methacrylate), in an aqueous phase added with
an appropriate surfactant (Munin and Edwards-Lévy 2011).
Protein–lipid systems showed an important encapsulation efficiency of
polyphenolic compounds (Munin and Edwards-Lévy 2011).
Moreover, these technologies could improve the successful delivery of bioactive
ingredients to the gastrointestinal tract. In Table 5 are summarized some examples
of different types of hydrogels used for delivery of bioactive compounds/drugs in
the gastrointestinal tract ranging from the oral cavity to the colon.
Briefly some examples are described about polyphenols micro-/nanoencapsu-
lated in hydrogels.
A method for coupling resveratrol through a hydrolyzable covalent bond to the
carboxylic acid groups in porous poly-e-caprolactone surface grafted with acrylic
acid was described and used to construct for in vivo bone regeneration (Li et al.
2011). Resveratrol-loaded nanoparticles of polycaprolactone and poly(ethylene
254 M.H.L. Ribeiro

Table 5 Tissue localization of some hydrogel-based bioactive compounds/drug delivery systems

Hydrogel-based bioactive compounds/drug delivery systems Tissue localization
Thermosensitive hydrogels Ocular cavity
Hydrogel contact lens
Bioadhesive systems
Tablet superdisintegrants Oral cavity
Mucoadhesive hydrogels
Injectable hydrogels Subcutaneous
Thermosensitive hydrogels
pH-sensitive hydrogels Stomach
Mucoadhesive hydrogels
Superporous hydrogels
Gastric retention devices
Gastric floating systems
Hydrotropic hydrogels Small intestine
pH-sensitive hydrogels
Tablet superdisintegrantes Large intestine
Mucoadhesive hydrogels
pH-sensitive hydrogels Vaginal/rectal
Mucoadhesive hydrogels

glycol) showed a protective effect of PC12 cells against superoxide-induced dam-

age during the phenomenon of oxidative stress (Munin and Edwards-Lévy 2011).
Furthermore, sodium deoxycholate elastic liposomes loaded with resveratrol
were shown to be stable when delivered subcutaneously (Cadena et al. 2013).
Encapsulation and delivery of a red grape skins antioxidant extract (polyphenols)
in superparamagnetic composite matrices was obtained by in situ precipitation of
magnetite and calcium alginate and calcium alginate–chitosan (Bădescu et al.
Quercetin was encapsulated in nanocomposites by in situ polymerization
(Bernardy et al. 2010). Quercetin was shown to be 100 times more soluble when
encapsulated on lipid-coated nanoparticles and stable for more than ten weeks, no
degradation product being detected (Barras et al. 2009).
Red grape skins lyophilized extracts were encapsulated with two different sys-
tems: calcium alginate and calcium alginate–chitosan. The interaction of the two
polysaccharides was evaluated in FTIR spectra, and the DLS measurements
allowed the determination of the average size and size distribution of the polymeric
nanoparticles encapsulating polyphenols.
Proteins (sodium caseinate and gelatin), hydrocolloids (gum arabic), and
hydrolyzed starch (starch, lactose, and maltodextrin) were tested as wall materials.
A mixture of maltodextrin (60%) and gum arabic (40%) has been used for
encapsulation of procyanidins from grape seeds.
A soybean extract rich in polyphenols was immobilized within a matrix com-
posed of maltodextrin, starch, or silica (Tixosil® 333) (Georgetti et al. 2008).
10 Emerging Technologies of Hydrogels … 255

The results show that the Tixosil 333 reduced the degradation of the encapsulated
polyphenol and protected its antioxidant activity.
Procyanidins and epigallocatechin gallate (EGCG) were encapsulated within a
carbohydrate matrix. These particles were able to inhibit steps of the tumorigenesis
process (Rocha et al. 2011).
Epigallocatechin gallate (EGCG) was immobilized on lipid-coated nanoparticles
(Smith et al. 2010) keeping up to 90% of its capacity to stimulate the a-secretase
in vitro, and the EGCG bioavailability after encapsulation was increased twofold
compared to that of the free form in vivo.
Chitosan was used as a coating material for the encapsulation of olive tree leaves
extract (Kosaraju et al. 2006). Chitosan nanoparticles (carboxymethyl and chitosan
hydrochloride) immobilizing a tea polyphenol extract (Liang et al. 2011) were
prepared by this method. Particles showed to be good nanosystems for slow drug
release by diffusion, the polyphenolic material being maintained active.
Carrageenan showed to be an interesting material as a means of conservation of
the antioxidant activity for the encapsulation of diverse natural polyphenol-rich
extracts (Krishnaiah et al. 2009).
Grape seed extract, apple extract, and olive tree leaf extract, rich in oleuropein,
were immobilized within a sodium caseinate—soy lecithin matrix (Kosaraju et al.
The encapsulation of an extract of oak (Quercus resinosa), very rich in
polyphenols, was recently realized by means of a high-pressure homogenization
(Rocha-Guzmán et al. 2010). This extract presents instability, bad taste, and strong
astringency, which requires its encapsulation before its incorporation in foodstuffs.
Within a matrix consisting of sodium caseinate and lactose, a high antioxidant
activity was measured even at very low phenolic concentrations.
Chitosan nanoparticles (carboxymethyl and chitosan hydrochloride) immobi-
lizing a tea polyphenol extract (Liang et al. 2011) and an Elsholtzia splendens
extract (Lee et al. 2010) were studied. Particles showed to be good nanosystems for
slow drug release by diffusion, the polyphenolic material being maintained active.
Catechins are powerful natural antioxidants, but a major drawback is that they
are very unstable in alkaline conditions encountered in biological fluids, and in
some experimental protocols. Catechin and (-)-epigallocatechin were immobilized
within chitosan tripolyphosphate nanoparticles, maintaining an antioxidant activity,
respectively, 88.3 and 73.4%, after 24 h.
Protein/polyphenol microcapsules with (-)-epigallocatechin gallate as a phenolic
compound and type A gelatin as a protein source were obtained by this method
(Shutava et al. 2009). The core of these particles was manganese carbonate, the
shell being formed by polyelectrolytes assembled in successive layers (polystyrene
sulfonate/polyallylamine hydrochloride, polyglutamic acid/poly-L-lysine, dextran
sulfate/protamine sulfate, and carboxymethyl cellulose/gelatin A) into which the
EGCG was inserted. Synthesis, characterization, and release studies of polyphenols
from these particles revealed that EGCG inside the membrane preserved its
antioxidant activity and blocked the production of hepatocyte growth factor
(HGF) from cancer cell lines MBA-MD-231 as effectively as free EGCG.
256 M.H.L. Ribeiro

In the future, polyphenols may be administered as a form of personalized

medicine that involves tailoring polyphenol/analogue doses or dietary regimen
dependent on the medical history, lifestyle, and genetic makeup of the individual as
well as the condition being treated. Routine medical use of polyphenols may be a
goal for the distant future.

5 Conclusion

Polyphenols are among the most powerful bioactive compounds synthesized by

plants, showing a unique combination of chemical, biological, and physiological
activities. Their limited stability and/or solubility, often combined with a poor
bioavailability, has to be improved in order to make these compounds able to
answer growing demands in nutrition, health, and cosmetics. The use of hydrogels
is an interesting approach to potentialize the activity of polyphenolic compounds.
Hydrogel properties (composition, particle size and density, release mechanism and
kinetics, degradation mechanism and kinetics, and final physical form) must be
clearly established in order to select the best process and protect polyphenols
against drastic conditions such as oxidation and thermal degradation, thereby
contributing to increase the shelf life of the encapsulated bioactive compound.

6 Future Trends

The future of polyphenol epigenomic therapy has several challenges ahead and is a
promising field for clinical interventions.
In the age of nanofabrication, there is a need for miniaturization of hydrogels
with enhanced durability, mechanical properties, and biocompatibility for new
The (bio)synthesis of new polymers and cross-linkers with high biocompatibility
and better biodegradability is essential for successful applications. Also, protein
engineering might contribute to the development of hydrogel systems with very
precise control over their microstructure and thus their properties.
Research will accelerate the reasoned biotechnological production and use of
natural polyphenols compounds, not only as food additives or as nutritional sup-
plements, but also as active drugs or cosmetics.
Although mathematical simulations have been performed to predict and design
better hydrogel systems, there are still many challenges associated with the mod-
eling of bioactive compounds/drug delivery phenomena and accurate prediction of
release profiles from complex hydrogel systems. Boosted by recent remarkable
scientific advances, human clinical trials are in progress. Therefore, realizing the
clinical requirements and simultaneously limiting the complexity of the hydrogel
formulation will be the main goals for the coming decades.
10 Emerging Technologies of Hydrogels … 257


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