Professional Documents
Culture Documents
Food
Bioactives
Extraction and Biotechnology
Applications
Food Bioactives
Munish Puri
Editor
Food Bioactives
Extraction and Biotechnology Applications
123
Editor
Munish Puri
Bioprocessing Laboratory, Centre for
Chemistry and Biotechnology
Deakin University
Waurn Ponds, VIC
Australia
and
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
agriculture.
Arnold L. Demain
Research Institute of Scientists Emeritii (RISE)
Drew University, Madison, NJ, USA
and
Ex-Professor
Department of Biological Sciences
Massachusetts Institute of Technology (MIT)
Cambridge, MA, USA
v
Preface
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.
vii
viii Preface
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.
xi
xii Contents
xiii
xiv Editor and Contributors
Contributors
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
e-mail: kzinov@afs.edu.gr
C.M. Galanakis (&)
Department of Research and Innovation, Galanakis Laboratories,
Skalidi 34, Chania 73131, Greece
e-mail: cgalanakis@chemlab.gr
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).
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
ESP
pH 4 pH 7
Fig. 1 Degradation products of GLs under different conditions (reproduced from Rask et al.
2000)
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).
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).
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).
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
leaves)
Glucobrassicin Kale from 24 different 0.4–145.4 (mg/100 g fresh
cultivars weight)
Glucoiberin 0–119.8 (mg/100 g fresh
weight)
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
(continued)
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 …
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
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.
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 ± ± ± ± +
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.
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Chapter 2
Bioactives from Mushroom and Their
Application
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.
C. Sánchez (&)
Laboratory of Biotechnology, Research Centre for Biological Sciences, Universidad
Autónoma de Tlaxcala, Ixtacuixtla, Tlaxcala CP. 90062, Mexico
e-mail: sanher6@hotmail.com
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.
Fig. 1 Schematic representation of mushroom phases of growth and fungal cell wall composition
camphoratus)
(continued)
Table 1 (continued)
Mushroom Common names Phyllum or Bioactive compound Bioactivity Reference
Scientific name group/
Edibility
Auricularia Jew´s ear, B/E Glucan Hyperglycemia, Zhang et al.,
auricula wood ear, Immunomodulatory 2007
jelly ear Anti-tumor
Anti-inflammatory
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
Glucoxylan 2012
Mannogalactofucan
Fucomannogalactan
Agaricoglycerides
Low-molecular weight protein Anti-tumor Kodama et al.,
fraction 2002
(continued)
31
Table 1 (continued)
32
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
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.
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
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).
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
application.
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57(1):43–48
Chapter 3
Bioactive Products from Fungi
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
e-mail: sersan@biomedicas.unam.mx
A.L. Demain
Research Institute for Scientists Emeriti (RISE), Drew University, Madison, NJ, USA
e-mail: ademain@drew.edu
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
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
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
unfeasible.
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.
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
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
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
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
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
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
11 Proteins
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
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
13 Additional Compounds
(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
year.
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
14 Future Perspectives
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Part II
Extraction Technologies
for Bioactives
Chapter 4
Process Development for Bioactive Peptide
Production
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
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.
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
purification.
(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.
E-1 E-2
Water vapour + buffer + nutrients Milk
E-6
E-5 Pure peptide slurries
Lactobacillus cells
Lactobacillus Cell (enzyme)
E-3
E-4
Used Milk
(b) T = 44◦C
pH = 5.7 T = 40◦C
pH = 6.2
Water vapour + buffer + nutrients E-1 Milk
E-2
E-6
E-5 Pure peptide slurries
Lactobacillus cells
Lactobacillus Cell (enzyme)
E-3
E-4
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.
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.
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.
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
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.
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.
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.
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.
1500
1450
1400
1350
1300
1250
1200
1000 1050 1100 1150 1200
Peptide (kg/day)
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.
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
3000
2500
2000 1000 kg/d Peptide
1500 2000 kg/d Peptide
1000
3000 kg/d peptide
500
0
Production of Protein Enzyme
peptide (kg/d) needed
(kg/d) (kg/d)
Requirements
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
demand.
Capital cost is the preliminary cash outflow required to build the process operations.
Essentially, it involves the major plant equipment.
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
subcultivation.
102 G.K. Gnasegaran et al.
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.
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.
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
18,600.
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).
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
price).
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.
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.
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.
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
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
plant.
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.
References
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.
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.
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
plaque.
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.
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
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).
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.
2014).
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
biocompatibility.
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
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).
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
2006b).
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
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.
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
61.5%.
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.
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.
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
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
bioadhesive.
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.
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).
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).
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.
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
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.
References
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5 Comparison of Natural Extraction and Recombinant Mussel … 135
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;
1
2
Lycopene 3
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
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
Cell disruption
Fermentation
Algae (Sonication,
medium Homogenisation etc)
mAU
12
10 Extraction
8
6
Purification
4
2
(Solvent)
0
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
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)
Thraustochytrium
AMCQS5-5
Chlorella vulgaris Ultrasonication 55 dos Santos
et al. (2015)
Scenedesmus sp. Freeze drying + Microwave 29.6 Guldhe et al.
(2014)
Chlorella vulgaris Pressure-assisted ozonation 27 Huang et al.
(2014)
Mixed culture Microwaves 33.7 de Souza Silva
et al. (2014)
Chlorella vulgaris H2O2 + FeSO4 17.34 Steriti et al.
(2014)
Nannochloropsis Microwave 38.3 Wahidin et al.
sp. (2014)
C. vulgaris Ultrasonication 52.5 Araujo et al.
(2013)
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.
(2012)
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)
Chlorella
protothecoides
144 A. Gupta et al.
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).
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).
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
1 Introduction
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.
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
liposomes.
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.
2011).
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.
2008).
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.
2012).
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
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
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Chapter 8
Enzyme-Assisted Extraction of Bioactives
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
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.
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
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
extraction:
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
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
extracted
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)
glucoamylase
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)
cellulase
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.
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
products.
4. It reduces the time of extraction and volume of solvent used.
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
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.
4.1.3 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
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)
juice
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.
(2012)
Unripe apples Total phenolics Cellulase – 7.08% Zheng et al. (2009)
181
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
vanilla.
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
polysaccharides.
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.
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
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
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.
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.
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.
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
temperature.
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.
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.
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
dollars.
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
192
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
interest
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.
(2011)
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.
(2013)
Microwave-assisted aqueous Enzyme cocktail (cellulase, pectinase, Oil Pumpkin 64.17% Jiao et al.
enzymatic extraction and proteinase) seeds (2014)
(MAAEE)
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).
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Part III
Techniques Employed
for the Bioactives Delivery
Chapter 9
Emerging Technologies for Bioactive
Applications in Foods
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
e-mail: grcastro@gmail.com
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)
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
mesenteroides)
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
(continued)
213
Table 1 (continued)
214
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
micro-carriers
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
(hexamer)
Beta-lactoglobulin Whey Globular 18.4 4.8–5.1 75
protein
lactoferrin Whey Globular 93.0 *8–9 *60 and
protein *90
Zein Corn Globular 24.0 6.2 –
Modified from Matalanis et al. (2011)
a
Type A gelatin (from pork skin)
b
Type B gelatin (from calf skin)
c
S-type ovalbumin
d
7S soy glycinin fraction
e
11S soy glycinin fraction
216 L.G. Santiago et al.
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.
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
counterparts.
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.
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.
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
1 Introduction
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
veins
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).
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
2011).
230 M.H.L. Ribeiro
2.1.1 Flavonoids
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
2.1.2 Non-Flavonoids
Fig. 3 Phenolic acids present in food: on the left, benzoic acids; on the right cinnamic acids
10 Emerging Technologies of Hydrogels … 233
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
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.
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
Fig. 6 Chemical structure of natural hydrogels from food proteins: gelatin, collagen, casein
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
Fig. 8 Degree of
polymerization
[(mol) = (hydroxyl
groups − n) + (acetate
groups − m)] and degree of
hydrolysis [(mol
%) = (hydroxyl groups − n)/
([hydroxyl
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).
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
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):
not expected that the PVA lenses produced initial have the same diffusion behavior
after been heat treated or exposed to higher temperatures.
3.8 Component
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
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.
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).
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
equation:
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).
Dissolution
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.
Osmosis
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
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
4 Polyphenols in Hydrogels
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.
2008).
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
5 Conclusion
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
applications.
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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1 Introduction
Human life cycle is a complex process, and numerous aspects still remain a mys-
tery. Apart from the birth, growth, and maturation phases, the aging process is also
an important focus among medical community and scientists. Memory decline and
cognitive functions comprise the most evident signals of brain changes and activity
impairment (Alza et al. 2014).
In the last decades, an exponential increase of brain-related diseases, namely
neurodegenerative diseases, has been observed. Taking into account social
and demographic changes, in which older people assume the leadership, it is nor-
mally conceived that age-related diseases tends to increase progressively.
Notwithstanding, and disturbingly, a double prevalence every 20 years has been
counted, increasing nearly to 115 million by 2050 (Vauzour 2014). Among the
neurodegenerative diseases, AD and Parkinson’s (PD) diseases, also dementia,
comprise the most representative and alarming causes of morbidity and autonomy
impairment among worldwide population (Vauzour 2014; Ahmed et al. 2015; Sun
et al. 2015). Genetic and human lifestyle has been pointed as the most pronounced
triggering factors to the development of those complex medical conditions (Sun
et al. 2008; Vauzour 2014; Ahmed et al. 2015). One of the first features in patients
with AD is the presence of brain lesions, commonly known as senile plaques:
deposits of b-amyloid (Ab) protein between neurons (Kumar and Nisha 2014;
Adewusi and Steenkamp 2015). Being a product from sequential proteolytic
cleavage, Ab is abnormally accumulated in older individuals due to a clearance
impairment and/or its overproduction. In particular, to Ab overproduction, genetic
changes/mutations, namely from amyloid precursor protein (APP), are underlying,
being related to early AD manifestation (Yoo and Park 2012). High doses of Ab are
highly toxic, due to its ability of self-aggregation, forming, respectively, fibrillary or
monomeric and then oligomeric forms. Particularly, Ab aggregated as oligomers is
highly harmful once acting as oxidative stress enhancer. Thus, and in association
with free radical overproduction, Ab oligomeric forms largely determine the
magnitude of cognitive damages, leading to synaptic dysfunctions, inflammation,
and, consequently, organic dysfunctions (Yoo and Park 2012; Kumar and Nisha
2014). Ab also induces neuronal death and activates downstream c-Jun N-terminal
kinase signal and N-methyl-D-aspartate-type glutamate receptor (NMDAR), which
leads to synaptic loss and improves neuronal dysfunction (Yoo and Park 2012).
Several studies indicate that the main components present in amyloid plaques of
AD individuals range from 40 and 42 amino acid sequences (Patil et al. 2010;
Kumar and Nisha 2014). Acetylcholine shortage is also a triggering factor for
neuronal decline and AD progression (Yoo and Park 2012). Despite all the phar-
maceutical advances, acetylcholinesterase (AChE) inhibitors still remain the most
popular prescribed drugs for symptomatic intervention, such as donepezil, galan-
tamine, rivastigmine, and tacrine. Memantine is also used but acts as N-methyl-D-
aspartic acid modulator (Ngo and Li 2013). Notwithstanding, those drugs possess
11 Neurocognitive Improvement Through Plant Food Bioactives … 269
numerous side effects and evidence a weak ability to block and even to reverse AD,
acting only on clinical symptoms, but not on causal factors or prevention.
Botanical preparations are a complex pool of phytochemicals, being their
ethnopharmacological potential mainly conferred by secondary metabolites
(Spencer et al. 2012). Particularly to AD, a multitude of studies have shown that
they act as prominent brain health improvers, being useful not only to prevent but
also to block and even avert neurological dysfunctions (Marco and Carreiras 2006;
Essa et al. 2012; Smid et al. 2012; Spencer et al. 2012; Konrath et al. 2013; Ahmed
et al. 2015). Furthermore, it is convenient to highlight that the majority of chemical
drugs are plant-derived mimetic; notwithstanding, in the whole plant, different
proportions of phytochemicals, their synergisms, antagonisms, and polyvalent
reactions improve the bioactive potential and also block some harmful substances.
Apart from those aspects, a correct and active lifestyle, which includes physical
and intellectual activity, and balanced diet are also well-established aspects that
provide important benefits to preserve, improve, and even block memory and
cognition impairment (van Praag 2009; Murray and Pizzorno 2012). Regarding
dietary aspects, not only correct food choices, but also nutritional supplementation
confers prominent influences. Functional foods and nutraceuticals have the ability
to act as bioactives by simply enriching daily diet (Murray and Pizzorno 2005,
2012). Overall, the use of plant-derived preparations, as part of a healthy lifestyle,
might have a great impact on life expectancy and health improvement.
Tables 1 and 2 show the major groups of compounds from commercial origin with
reported in vitro neuroprotective effects.
Among phenolic compounds (Table 1), flavan-3-ols, flavones, flavonols, iso-
flavones, lignans, phenolic acids, and stilbenes comprise the most common classes.
Quercetin (a flavonol), followed by myricetin (a flavonol) and kaempferol (a fla-
vonol), and then apigenin (flavone), baicalein (flavone), catechin (flavan-3-ols), and
EGCG (flavan-3-ols) are the most studied phenolic compounds. In particular,
quercetin has shown several neuroprotective effects, being its ability to reduce
and/or to prevent reactive oxygen species (ROS) overproduction amongst to the
most prominent; its active concentrations varied significantly according to the
model used, but interestingly, as described by Vepsäläinen et al. (2013), at 0.5, 2.5,
and 10 µM a, pronounced reduction in ROS overproduction was observed in
11 Neurocognitive Improvement Through Plant Food Bioactives … 271
Table 1 Phenolic compounds (from commercial origin) with reported in vitro neuroprotective
effects
Bioactive Experimental models Active Reference
molecule concentration
Flavan-3-ols
Catechin Mouse cortical neuron cultures 30 µMa Choi et al.
(2014)
Unilamellar vesicles, Ab42-induced 50 µM Gauci et al.
liposome permeabilization (2011)
EGCG Unilamellar vesicles, Ab42-induced 50 µM Gauci et al.
liposome permeabilization (2011)
Mouse cortical neuron cultures 30 µMa Choi et al.
(2014)
Epicatechin Mouse cortical neuron cultures 30 µMa Choi et al.
(2014)
Flavones
Apigenin Unilamellar vesicles, Ab42-induced 50 µM Gauci et al.
liposome permeabilization (2011)
Mouse cortical neuron cultures 30 µMa Choi et al.
(2014)
Baicalein Unilamellar vesicles, Ab42-induced 50 µM Gauci et al.
liposome permeabilization (2011)
Mouse cortical neuron cultures 30 µMa Choi et al.
(2014)
Luteolin Mouse cortical neuron cultures 30 µMa Choi et al.
(2014)
Morin Unilamellar vesicles, Ab42-induced 50 µM Gauci et al.
liposome permeabilization (2011)
Scutellarein Unilamellar vesicles, Ab42-induced 50 µM Gauci et al.
liposome permeabilization (2011)
Flavonols
Fisetin Unilamellar vesicles, Ab42-induced 50 µM Gauci et al.
liposome permeabilization (2011)
Kaempferol PC12 cells 3–30 µM Roth et al.
(1999)
Human T47D breast cancer cells 3–30 µM Roth et al.
(1999)
Mouse cortical neuron cultures 30 µMa Choi et al.
(2014)
Myricetin Th-T assay 1.95 µMb Xie et al.
(2014)
Human SH-SY5Y neuroblastoma cells, 0.5, 2, 5, 10, Vepsäläinen
menadione-induced ROS production 20 µM et al. (2013)
Unilamellar vesicles, Ab42-induced 50 µM Gauci et al.
liposome permeabilization (2011)
(continued)
272 N. Martins and I.C.F.R. Ferreira
Table 1 (continued)
Bioactive Experimental models Active Reference
molecule concentration
Mouse cortical neuron cultures 30 µMa Choi et al.
(2014)
Quercetin Mouse cortical neuron cultures 30 µMa Choi et al.
(2014)
Primary cultures of astrocytes and 8 µMb,c Elmann et al.
microglial cells (2013)
Primary cultures of astrocytes 10 µM Elmann et al.
(2014)
Unilamellar vesicles, Ab42-induced 50 µM Gauci et al.
liposome permeabilization (2011)
Human SH-SY5Y neuroblastoma cells, 0.5, 2, 5, Vepsäläinen
menadione-induced ROS production 10 µM et al. (2013)
Bovine serum albumin, D- 600 lMb Ferchichi
ribose-induced AGE formation et al. (2012)
Isoflavones
Genistein Unilamellar vesicles, Ab42-induced 50 µM Gauci et al.
liposome permeabilization (2011)
Lignans
NDGA Unilamellar vesicles, Ab42-induced 50 µM Gauci et al.
liposome permeabilization (2011)
Phenolic acids
3,5-di-O- Human neuroblastoma clonal SH-SY5Y 20 µM Han et al.
Caffeoylquinic Ab1–42-treated cells (2010)
acid
Caffeic acid Th-T assay 118 µMb Kurisu et al.
(2013)
Chlorogenic Bovine serum albumin, D- 2000 lMb Ferchichi
acid ribose-induced AGE formation et al. (2012)
Ferulic acid Mutant human APP-overexpressing 1.563– Mori et al.
murine neuron-like cells 12.5 µM (2013)
p-Coumaric PC12 cells, Ab25−35-induced toxicity 5, 25, 50 µM Yoon et al.
acid (2014)
Rosmarinic PC12 cells, Ab25-35-induced 23.6 µMd Na et al.
acid neurotoxicity (2010)
Unilamellar vesicles, Ab42-induced 50 µM Gauci et al.
liposome permeabilization (2011)
Stilbenes
Resveratrol Unilamellar vesicles, Ab42-induced 50 µM Gauci et al.
liposome permeabilization (2011)
a
Tested concentration to measure the percentage of LDH release after 20 lM Ab25–35 exposure;
b
IC50 values; cvaried according to the used assay; dED50 value
EGCG Epigallocatechin gallate; NDGA nordihydroguaiaretic acid; ROS reactive oxygen species;
Th-T assay Thioflavin-T assay; AGEs advanced glycation end-products
11 Neurocognitive Improvement Through Plant Food Bioactives … 273
Table 2 Non-phenolic compounds (from commercial origin) with reported in vitro neuroprotec-
tive effects
Bioactive molecule Experimental models Active Reference
concentration
Glycosides
Salidroside SH-SY5Y human 10, 50, Zhang
neuroblastoma cells, 100 µM et al.
b-amyloid-induced oxidative (2010)
stress
Iridoid glycosides
Geniposide N2a cell 200 µM Chen et al.
formaldehyde-exposed (2014)
Phenylethanoids
Acteoside-tetramethylether Th-T assay >200 µMa Kurisu
Hydroxytyrosol 96 µMa et al.
(2013)
Oraposide-tetramethylether >200 µMa
Quinones
1,4-Benzoquinone Insulin as amyloid model 50 µM Gong et al.
1,4-Naphthoquinone (2014)
9,10-Anthraquinone
9,10-Phenanthraquinone
Aloe-emodin
Chrysophanol
Emodin
Terpenes
Asiatic acid Primary Sprague–Dawley rat 10 µM Patil et al.
cortical neurons (2010)
Bilobalide Th-T assay 14.84%b Xie et al.
(2014)
Carnosic acid U373MG human astrocytoma 50 µM Yoshida
cells et al.
(2014)
Ginkgolide A Th-T assay 21.10%b Xie et al.
(2014)
Ginkgolide B Th-T assay 13.56%b Xie et al.
(2014)
Unilamellar vesicles, 50 µM Gauci et al.
Ab42-induced liposome (2011)
permeabilization
Ginkgolide C Th-T assay 13.92%b Xie et al.
(2014)
Hyperforin Hippocampal neuron 1 µM Dinamarca
cultures-amyloid fibrils and et al.
Ab oligomer-induced damage (2006)
(continued)
274 N. Martins and I.C.F.R. Ferreira
Table 2 (continued)
Bioactive molecule Experimental models Active Reference
concentration
Ursolic acid PC12 cells, Ab25−35-induced 1, 10, 20 µM Yoon et al.
toxicity (2014)
Withanolide A Primary Sprague–Dawley rat 100 µM Patil et al.
cortical neurons (2010)
Others
Purpurogallin trimethyl Unilamellar vesicles, 50 µM Gauci et al.
ether Ab42-induced liposome (2011)
permeabilization
Rhein Insulin as amyloid model 50 µM Gong et al.
(2014)
a
IC50 values; binhibition percentage of the tested compounds at the concentration of 100 µM
Th-T assay and Thioflavin-T assay
58.7 µMc
Rosmarinus officinalis Whole Luteolin PC12 cells, corticosterone-induced 30, 40, Sasaki et al.
L. plant neurotoxicity 50 µM (2013)
Psoralea corylifolia L. Seeds chromenoflavanone [7,8-dihydro-8- Murine microglial cell line (BV-2), 11.4 µg/mLa Lee et al.
(4-hydroxyphenyl)-2,2-dimethyl-2H,6H- LPS-induced oxidative stress (2005)
benzo-(1,2-b:5,4-b′)dipyran-6-one]
(continued)
275
Table 3 (continued)
276
Phenolic acids
Calophyllum Leaves 3,4-dihydroxybenzoic acid Bovine serum albumin, D-ribose-induced 0.50 mMa Ferchichi
flavoramulum Hend. & AGEs formation et al. (2012)
Wyatt-Sm.
Rosmarinus officinalis Rosmarinic acid PC12 cells, corticosterone-induced 5, 15, 25 µM Sasaki et al.
L. neurotoxicity (2013)
Salvia miltiorrhiza Salvianolic acid B PC12 cells, Ab25–35-induced cytotoxicity 10, 100, Zhou et al.
Bunge 200 µg/mL (2011)
277
(continued)
Table 3 (continued)
278
Valeriana amurensis P. Smirn. Rhizomes Xiecaoside E Ab1–42-induced PC12 cell 5, 12.5, Wang et al.
ex Kom. and roots neurotoxicity 25 µM (2014a)
Quinones
Euclea crispa subsp. Crispa Roots Natalenone HeLa cells 50 µg/mLa Kwon et al.
(2011)
(continued)
279
Table 4 (continued)
280
higher antioxidant potential, namely as free radical scavengers and also as metal
quenchers and hydrogen donators (Heim et al. 2002; Grotewold 2006; Li et al. 2014).
Cis-3-(3’4’-dimethoxyphenyl)-4- Trans-3-(3’4’-
[(E)-3’’,4’’- dimethoxyphenyl)-4-[(E)-
dimethoxystyryl]cyclohex-1-ene 3’’,4’’-
dimethoxystyryl]cyclohex-1-ene
Induce neurite Increase neurite Protect against cell Induce neurite Increase neurite Protect against cell
sprouting length and number death sprouting length and number death
(30 µM) (0.3–3 µM) (3-30 µM) (10-30 µM) (0.03–3 µM) (30 µM)
Fig. 2 In vitro neuroregenerative effects of phenylbutanoid dimers obtained from the methanol
extract of Zingiber purpureum Roscoe. roots. Legend Protection against cell death and induction of
neurite sprouting was assessed by using PC12 cells, while the evaluation of neurite length and
number improvement was carried out in cultured primary cortical neurons of rats (Matsui et al.
2012)
Fig. 3 Bioactive molecules from commercial sources with in vitro neuroregenerative effects.
Legend Effects of flavonoids on the mitochondrial function were assessed by using 1 µM of each
one in murine neuroblastoma N2a cells, and then, measure its activity at a level of ROS
production, MMP and ATP levels (Dragicevic et al. 2011); evaluation of Ab production was
assessed by using 50 µM of each one of the tested compounds in CHO 2B7 cells (Chen et al.
2006)
Fig. 4 Plant-origin bioactive molecules with in vivo neuroregenerative effects. Legend Magnolia
officinalis Rhed plant (Matsui et al. 2009) and Zingiber purpureum Roscoe root (Matsui et al.
2012) methanol extracts
Fig. 5 Bioactive molecules (from commercial origin) with in vivo neuroregenerative effects.
1
Dragicevic et al. (2011), 2Chen et al. (2006)
by Ab25–35, being clearly evident its protective effects against cognitive and
memory impairments. 2,3-Dihydroxy-4-methoxyacetophenone (10.94 lM), iso-
lated from the roots of Cynanchum paniculatum (Bunge), also evidenced a pro-
nounced effect against neuronal damage and toxicity, in HT22 cells, induced by
glutamate.
Overall, and comparing the efficacy and efficiency of the studied bioactive
molecules in relation to its sources (commercial vs. plant origin), it is possible to
conclude that plant-origin bioactive compounds possess a doubtless prominent
potential. Although some of them were not studied and then compared from both
sources, several examples should be highlighted. While for luteolin, derived from
commercial (30 lM) and plant (30, 40, and 50 lM) sources, similar active con-
centrations were found, for rosmarinic acid a completely different situation was
observed; the commercial molecule was effective at 23.5–50 lM, while the one
from plant origin was effective at 5, 15, and 25 lM. Similarly, resveratrol isolated
from Vitis vinifera L. was highly effective at 10 and 20 lM, while the commercial
288 N. Martins and I.C.F.R. Ferreira
molecule was active at 50 lM. Moreover, carnosic acid obtained from R. officinalis
was effective at 10, 20, and 30 lM, while the commercial one was active at 50 lM.
In the same line, the phenylethanoid glycosides, acteoside, and oraposide were also
more effective when derived from natural sources (8.9 and 3.6 lM) in comparison
with the commercial molecules (>200 lM). Finally, salidroside isolated from
Rhodiola sachalinensis A. Bor was effective at 1; 5; 10; 50 lg/Ml, while the
commercial molecules were effective at 3; 15; 30 lg/mL.
In summary, plant-food-derived bioactive molecules appear to be more effective
than the commercial ones; notwithstanding, further studies are necessary to confirm
this. Besides, the confirmation of the effective neuroprotective potential needs to be
performed, mainly by in vivo studies, once numerous biochemical parameters
influence the final bioactivity and active concentrations.
In the first case, EGCG was able to improve the activity of a-secretase and to reduce
brain mitochondrial Ab and AbPP levels in AbPP/PS1 double-mutant transgenic
mouse models. Otherwise, ginsenoside Re, Rg1, and Rg3 evidenced a higher potential
to reduce not only Ab42, but also Ab40 brain levels in Tg2576 female transgenic mice
models that overexpress the human APP gene.
Overall, and despite the described effects of the tested bioactive components, it is
difficult to compare their efficacy and also efficiency once different biochemical
parameters were assessed, and also biological antineuronal damage effects.
functioning, once regulates its formation, plasticity and availability, as also from
other signalling molecules (Liang et al. 2014). Furthermore, Zhan et al. (2014)
observed that berberine, a plant alkaloid, was able to reverse synaptic deficits
induced by D-galactose and rescued important intermediates (mRNA, Arc/Arg3.1)
directly involved on normal synaptic plasticity.
Otherwise, and in association with the previous stated effects, it is also important
to point out that hypothalamic neuromodulator systems are also affected/affect daily
energy homeostasis. For example, under specific conditions (such as short-term
fasting and other metabolic state changes), considerable synaptic circuits and
respective (inter)neuronal controllers suffer from restructuration, which leads to
physiological variations on energy homeostasis and may cause synaptic plasticity
impairments (Horvath 2006). Thus, the above-described mechanisms of action of
bioactive molecules can be also extremely useful in other contexts, such as feeding
and appetite controllers. For example, plant-derived cannabinoids (phytocannabi-
noids) and endocannabinoids, in spite of the whole negative connotation attributed
to cannabis, have shown greater contributive properties not only for physiological
appetite and satiety controllers (Berry and Mechoulam 2002), but also for brain
therapeutical purposes (i.e., neuromodulators, neuroprotective, neuroregenerative,
and synapsis plasticity regulators) (Fisar 2009).
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Chapter 12
Multifunctional Bioactives for Cancer
Therapy: Emerging Nanosized Delivery
Systems
1 Introduction
D. Sharma (&)
Institute of Nano Science and Technology,
Habitat Centre, Phase 10, Mohali, Punjab, India
e-mail: deepika@inst.ac.in
M. Nijhawan
Western Heights College, Geelong, VIC 3220, Australia
M. Puri
Bioprocessing Laboratory, Centre for Chemistry and Biotechnology, Deakin University,
Waurn Ponds, VIC 3216, Australia
The worldwide burden of cancer continues to intensify largely due to the ageing
and growing global population and a cumulative adoption of cancer-causing
behaviours (particularly smoking) within economically developing countries (Jemal
et al. 2011). In many economically developing countries, female breast, lung, and
colorectal cancers are occurring in high frequencies (Jemal et al. 2011) with cervical
cancer often the most common cancer among women. Nanotechnology at present is
being utilized in cancer for molecular imaging, diagnosis, early detection, targeted
therapy, and cancer bioinformatics. Cancer-related nanodevices include but are not
limited to injectable nanovectors such as liposomes; biologically targeted nanosized
magnetic resonance imaging contrast agents; and novel, nanoparticle-based meth-
ods (Ferrari 2005; Roemeling et al. 2016).
In this write-up, we have collated numerous studies that provide evidence to
support the use of bioactives for cancer therapy through nanotechnology.
3.1 Curcumin
Turmeric is a spice prepared from the root of Curcuma longa, of which curcumin is
one of the constituents. Curcumin has been investigated as both a chemotherapeutic
and chemopreventive agent in many different animal models of carcinogenesis
(Epstein et al. 2010). Its non-toxicity and good tolerability in humans, in con-
junction with encouraging results from in vitro and in vivo studies as well as early
human clinical studies, support the continuing research and development of cur-
cumin as both a preventive and disease-modifying agent (Epstein et al. 2010).
Despite its multifunctional activities in cancer cells, there are some challenges
which need attention. Poor bioavailability of curcumin because of limited solubility
in water is a major limitation (Fig. 1a). Interdisciplinary approaches are being used
to improve its bioavailability. Curcumin has been shown to induce apoptosis in
drug-resistant cancer cells (Roy and Mukherjee 2014). It functionalizes intrinsic
pathway and regulates Bcl-2/Bax ratio in A549 cancer cells (Li et al. 2013).
Curcumin liposomes effectively induced apoptosis, and there was notably
reduced angiogenesis in the LL/2 model (Tang et al. 2013). Another contemporary
study revealed that curcumin-loaded nanoparticles prepared with amphiphilic
methoxy PEG-polycaprolactone (PCL) block copolymers substantially inhibited
cancer growth in mice transplanted with A549 (Yin et al. 2013). Curcumin has also
been shown to exert its cancer-suppressing effects in mice xenografted with HepG2
cells (Dai et al. 2013). Silica nanoparticles conjugated to curcumin have recently
been studied for efficacy in cervical cancer cells (Gangwar et al. 2013).
Curcumin-loaded solid lipid nanoparticles (SLNs) have also been shown to display
302 D. Sharma et al.
Chin b
Chitosan c
d
12 Multifunctional Bioactives for Cancer Therapy: Emerging … 303
Quercen e
Epigallocatechin gallate f
Fig. 1 (continued)
3.3 Resveratrol
3.4 Quercetin
metabolites including lactic acid and glycolic acid. The confluence of information
suggested that delivery of quercetin and tamoxifen encapsulated in PLGA strongly
induced apoptosis in breast cancer cells (Jain et al. 2013). Nanoparticles prepared
from polylactic acid-hyperbranched polyglycerol (HPG-PLA) have also proved to
be novel carriers of drugs. Another drug delivery system, particularly PEG (660)-
12-hydroxystearate (PEG 660-stearate), has also persuasively revealed a consider-
ably improved solubilization of quercetin up to five times (Dora et al. 2012).
Increasingly, it is being realized that nanoemulsion-based delivery systems are
effective in enhancing bioaccessibility of drugs. In agreement with this approach, it
has recently been shown that bioaccessibility of crystalline quercetin was com-
paratively lesser than that of dissolved quercetin (Pool et al. 2013).
Table 1 Examples of various nanoparticles and drug combinations used for cancer therapy
Nanoparticles Functionalization Drug Use Reference
Human serum albumin Amino/acid group Doxorubicin Antineoplastic Dreis et al. (2007)
Trimyristin Sterically stabilized Paclitaxel Ovarian, lung, breast cancer Lee et al. (2007)
PLLA-b-PEG Folate targeted Doxorubicin Solid tumours Lee et al. (2003)
PEG-PE Lipid conjugated Paclitaxel Various cancers Wang et al. (2005)
PEG Lipid conjugated Tamoxifen Lung carcinoma Gao et al. (2002)
Polymer lipid hybrid Lipid conjugated Doxorubicin Solid cancer Wong et al. (2007)
PCL-b-trimethylene carbonate PEG Serum protein Ellipticine Anticancer Liu et al. (2005)
PAMAM dendrimers Folic acid Methotrexate Epithelial cancer Kukowska-Latallo et al. (2005)
PEG Albumin bound Doxorubicin Various cancers Wosikowski et al. (2003)
Micelles Biotin antibody conjugated Daunomycin Brain tumour Schnyder et al. (2005)
PLGA Alendronate Oestrogen Bone-osteoporosis Choi and Kim (2007)
Poly(DEAP-Lys)-b-PEG -b-PLLA Poly(lysine) Doxorubicin pH-sensitive tumour Oh et al. (2009)
PLGA-b-PEG-COOH PSMA Anticancer Prostate cancer Cheng et al. (2007)
PEG or PE particles Transferrin Oligonucleotide Brain-gene Vinogradov et al. (2004)
Multifunctional Bioactives for Cancer Therapy: Emerging …
chemotherapeutic drug were also evaluated for efficacy. The results confirmed that
drug-loaded HDHA NPs were more effective in the delivery of drug to the target
site as compared to drug-loaded HDD NPs. It was further highlighted that instead of
delivering a single therapeutic agent, combinatorial approach using EGCG with
targeted delivery of drug proved to be more efficient in restricting the growth of
tumour cells in mice (Ray et al. 2013).
Normal tissue vasculatures are lined by tight endothelial cells, which prevent the
nanoparticles from escaping into the tissue, whereas tumour tissue vasculatures are
hyper-permeable with leaky endothelium, which easily allow the nanoparticles to
infiltrate in the tissue. Once the nanoparticles carrying the drugs enter the blood,
they move freely until they reach the tumour tissue where, due to the leaky envi-
ronment, they allow the encapsulated drugs to be released and get accumulated in
the tissue. The nanoparticles could also be conjugated with targeting moieties,
which facilitate the nanoparticles to be delivered only to the tumour cells. The
nanoparticles can be designed to encapsulate single or multiple agents and could
also be conjugated with polyethylene glycol (PEG) to increase the circulation time
of nanoparticles by stabilizing them against opsonization. The concept
nanoparticle-mediated delivery could be implemented for sustained bioavailability
of potentially useful chemopreventive agents. This sustained release and lower dose
requirement could also limit perceived toxicity associated with their repeated use, a
must for human use. To establish the proof of the principle of the concept, the
effectiveness of delivery of a well-known chemopreventive agent,
epigallocatechin-3- gallate (EGCG), the major polyphenol from green tea,
12 Multifunctional Bioactives for Cancer Therapy: Emerging … 311
4.2 Nanoformulations
Gunduz and Oktarc (2014) summarized the existing types of mesoporous silica,
methods of modifying their surfaces, and their main applications for the removal of
the organic and heavy metal ions (Pb, Cd, Zn, Ar, Cr, etc.) from the wastewater. It
is also used as carriers for the controlled release of drugs used in chemotherapy,
such as cisplatin, carboplatin and oxaliplatin, paclitaxel, camptothecin, irinotecan,
rapamycin, and doxorubicin.
4.2.3 Gold
Popescu et al. (2015) presented the applications of gold nanoparticles in the cancer
treatment and immunization by nanovaccines. The interest for this type of
nanoparticles is given by their ability to penetrate blood vessels and tissue barriers
and to be directed to a specific cell by means of specifically functionalized mole-
cules. Moreover, gold nanoparticles (AuNPs) possess special properties which
make them useful in the concomitant cancer diagnostics (medical imaging) and
treatment (tumour ablation by photothermal activation).
The targeted anticancer drug delivery as well as tracking the path of the drug carrier
with a biofriendly heavy metal free quantum dot is a great contribution to cancer
therapy. A heavy-metal-free luminescent quantum dot (QD) based on doped zinc
sulphide (ZnS), conjugated with a cancer-targeting ligand, folic acid (FA), has been
developed as a promising biofriendly system for targeted cancer imaging (Manzoor
et al. 2009). Semiconductor nanocrystals (or quantum dots) are the most promising
fluorescent probes for many biomedical applications. By appropriate bioconjuga-
tion, such nanocrystals can replace the conventional organic fluorescent dyes in
immunostaining and bioimaging of tissues and cancerous cells. However, many of
the quantum dots investigated for this purpose including cadmium sulphide, cad-
mium selenide, and zinc selenide are cytotoxic owing to their heavy metal com-
position (Derfus, Chan and Bhatia 2004).
There are multiple barriers involved in the anatomical and physiological system to
lack the drug efficiency, including enzymatic degradation in the stomach, absorp-
tion across the intestinal epithelium, hepatic clearance, and accumulation in
non-targeted tissues. These barriers also involve a range of complexities from the
tissue to the organelle level along with the time that mismatches the drug potency
in vivo. Collectively, these conditions challenge the active utilization of potent
therapeutic molecules for disease treatment or prevention. Specifically, systemic
delivery of polymeric nanoparticles to the CNS is based largely on their potential
for receptor-mediated transcytosis and adsorptive-mediated transcytosis through the
BBB. This process can be enhanced by the addition of cell-penetrating peptides
and/or targeting ligands to the nanoparticle surface. Folate receptors are
over-expressed in various cancer cells and they provide a receptor-based endocy-
tosis upon interaction with conjugated nanoparticles providing cellular uptake.
Similarly, mannose receptors, which are also studied for their functional applica-
tions, targeted cancer diagnosis (Higuchi et al. 2008). This highlights the role of
nanomedicine in cancer through receptor-mediated imaging via nanoparticles. In
studies to date, the nanoparticle systems described in this section have shown the
most promise for bypassing the BBB.
Poly(butylcyanoacrylate) (PBCA) nanoparticles were the first polymer-based
nanoparticle system used to deliver drugs to the CNS (Kreuter et al. 1995). In this
maiden study, PBCA nanoparticles were loaded with dalargin (a compound with
opioid activity), coated with polysorbate 80, and delivered intravenously, with the
goal of achieving therapeutic drug levels within the CNS. In vivo studies demon-
strated that dalargin-loaded PBCA particles had an antinociceptive effect (Kreuter
et al. 1995). Follow-up studies, using particles that were radiolabelled for sensitive
detection, demonstrated that in the absence of polysorbate-80 coating, there was a
314 D. Sharma et al.
significant decrease in the number of PBCA nanoparticles that crossed the BBB
(Schroeder et al. 2000). As a result of this and other work, polysorbate 80 (also
known as Tween 80 or polyoxyethylene-20 sorbitan monooleate) appears to
enhance the CNS penetration of systemically delivered polymer nanoparticles.
Surface modification with surfactants or ligands can enhance receptor-mediated
endocytosis, while the surface display of positive charges can enhance
adsorptive-mediated endocytosis. Intravenous administration of drugs with transient
physical disruption of the BBB leads to high risk of side effects. A further problem
with evaluating BBB penetration is the difficulty in quantifying BBB transit, and
the risk of artefacts with most experimental techniques that employed. We believe
that radiolabelling techniques are best suited to quantify the delivery efficiency of
systemically administered polymeric nanoparticles. Still, these techniques will need
to be carefully employed to demonstrate that systemically delivered nanoparticles
can accumulate to therapeutic amounts in the brain. Polysorbate-80 used as
emulsifier and surfactant which aids in decreasing nanoparticle clearance by the
reticuloendothelial system (RES) (Kreuter 2004). Some examples of various
nanoparticles with different functionalization and different therapeutic uses based
on the target are shown in Table 1.
Local delivery of therapeutic agents in the CNS has a long history of clinical
success (Kunwar et al. 2010). Local delivery of therapeutics bypasses the BBB
altogether. Initial work in this field focused on the implantation of drug-loaded
biodegradable polymer wafers (Gliadel), which are able to release drugs in a
controlled fashion over a prolonged period of time and resulted in modest
improvements in patient survival (Fung and Saltzman 1997). Although capable of
delivering large doses of a drug to the site of tumour resection over a sustained
period, the drug released from the implants had limited penetration beyond the
tumour margin, which could limit overall efficacy (Fung et al. 1996).
Convection-enhanced delivery (CED) of drug-loaded polymer nanoparticles offers
a solution to this problem. In CED, an external pressure gradient is established,
typically through a syringe pump, and agents are infused continuously into the brain
tissue via bulk fluid flow (Bobo et al. 1994). This can lead to the distribution of
therapeutics over large volumes in the brain. Although the investigation of
nanoparticles for local CNS delivery has, thus far, focused largely on liposomal
preparations (Saito et al. 2006), it is now possible to design polymer nanoparticles
that can be delivered by CED (Sawyer et al. 2011).
The first particulate systems that were used for direct drug delivery to the brain
were microspheres. Polymer microspheres have been fabricated from a variety of
materials for the purposes of local delivery including PLGA, poly(methylidene
malonate) (PMM), poly(epsilon-caprolactone), and chitosan. These systems have
been used to deliver a range of therapeutics, including cyclosporine, paclitaxel,
imatinib, mitoxantrone, phenytoin, and nerve growth factor (Turkoglu et al. 2010).
One advantage of microparticles, over earlier implant systems such as Gliadel, is
that the particles can be introduced without surgery. The particles larger than 1
12 Multifunctional Bioactives for Cancer Therapy: Emerging … 315
micron in diameter do not move readily through the BBB or the brain interstitium
(Thorne and Nicholson 2006); thus, it is difficult for microparticles to distribute
through large volumes of brain tissue.
For most intracranial applications, this implies achieving a volume of distribu-
tion that is >3 times the volume of infused therapeutic agent (Chen et al. 2005). The
drug-loaded polymer nanoparticles can be used effectively to treat disease in the
brain when delivered via CED. Specifically, camptothecin-loaded PLGA
nanoparticles were delivered locally and demonstrated to be effective for treatment
of an intracranial tumour model (Sawyer et al. 2011). Further optimization of
polymer nanoparticle design, by control of nanoparticle size, charge, and surface
coatings, promises to make this delivery strategy even more effective (Kunwar et al.
2010).
5 Conclusion
6 Future Perspectives
Many food constituents have been found to possess antimutagenic and anticar-
cinogenic properties. However, to successfully convert a potent food bioactive to a
clinically viable drug will require detailed consideration of in vivo pharmacoki-
netics, how they are taken up by the body, and how do they modify process in the
body. More research effort is desirable to understand the underlying molecular
mechanism of action of food bioactives.
With the rapid advancements in medical research, diagnostic technology and
increased public health initiative and awareness, overall cancer death rates in
western societies are declining following the trend of eating at least five portions of
fruits and vegetables to maintain good health. There is an opportunity for the
316 D. Sharma et al.
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A E
Alzheimer’s disease (AD), 267–269, 289, 291, Enzymes, 171, 172, 174–194
292 Extraction, 171–193
Anti-depressant, 23, 48
Anti-microbial, 23, 40, 41 F
Anti-neurodegenerative, 23, 25, 45, 47 Fortified foods, 205, 206
Anti-oxidant, 23, 25, 40, 45 Fusion partners, 124
Ascomycetes, 23, 41, 46 Fusion protein, 125
Astaxanthin, 138–141, 147
G
B Gelation, 211, 212, 219
Basidiomycetes, 23, 24, 41, 46 Glucosinolates, 3–5
Bioactive peptides (BPs), 91–93, 95, 96, 98,
99, 107 H
Bioactives, 171–176, 178, 179, 184–189, 191, Heat set gelation, 210, 212
192 High-pressure processing, 3, 10
Bioadhesive, 124, 125, 127, 130 Hybrid matrices, 215
Biodiesel, 162–165 Hydrogels, 227, 237–242, 244, 245, 247–251,
Biologically active compounds, 23, 24 253, 256
Bioprocess development, 91
Blood–brain barrier (BBB), 305, 311–315 I
Byssal plaques, 112, 113, 117 Immobilized lipase, 163
Immunomodulatory activity, 23, 42, 46, 47
C Isothiocyanates, 5, 10
Cancer therapy, 299, 300, 311, 313
Canthaxanthin, 138, 140, 141, 145 L
Carotene, 137–141, 146 Lactobacillus delbrueckii, 91, 94, 107
Cell adhesion, 125, 127
Coacervation, 111, 129 M
Cold-set gelation, 210, 212 Magnetic biocatalysts, 164
Magnetic modification, 153, 154, 156–158
D Magnetic particles, 154, 156, 157, 159, 161,
Delivery, 227, 237, 238, 241, 242, 244, 250, 165
251, 253, 254, 256 Magnetic separation, 153, 156, 158, 160, 162,
Dihydroxyphenylalanine (DOPA), 111–114, 163, 165
117–121, 123, 125–129, 131 Magnetic techniques, 161, 165
Downstream purification, 91 Mechanism of action, 172, 174
Drug delivery, 299, 304–306, 308, 310, 311, Metastatic cancer, 306, 316
313, 314, 316 Micro/Nano encapsulation, 227, 253