(Advances in Food and Nutrition Research Volume 73) Kim, Se-Kwon-Marine Carbohydrates - Fundamentals and Applications, Part B-A PDF

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Brock University, Canada
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Global Harmonization Initiative, The Netherlands
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University of Wisconsin, USA
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Purdue University, USA
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Oregon State University, USA
SERIES EDITORS
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EMIL M. MRAK (19481987)
C. O. CHICHESTER (19591988)
BERNARD S. SCHWEIGERT (19841988)
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STEVE L. TAYLOR (19952011)
JEYAKUMAR HENRY (2011 )
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ISBN: 978-0-12-800268-1
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CONTRIBUTORS
Mohaddeseh Adel
Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur,
Malaysia
Abdul Bakrudeen Ali Ahmed
Malaysia
S. Aisverya
Department of Chemistry, D.K.M. College for Women, Thiruvalluvar University, Vellore,
Tamil Nadu, India
Chong-Su Cho
Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, South
Korea
Peng-Fei Cui
School of Pharmacy, China Pharmaceutical University, Nanjing, PR China
Thandapani Gomathi
Tamil Nadu, India
Hu-Lin Jiang
Department of Pharmaceutics, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, Nanjing, PR China
Fatih Karadeniz
Marine Bioprocess Research Center, Pukyong National University, Busan, South Korea
Pegah Karimi
Malaysia
Se-Kwon Kim
Department of Marine-bio Convergence Science; Marine Bioprocess Research Center, and
Department of Chemistry, Pukyong National University, Busan, South Korea
Eduardo Roberto Morales-Guerrero
Laboratory for Bioactive Compounds and Bioremediation, Department of Marine
Biotechnology, Centro de Investigacion Cientfica y de Educacion Superior de Ensenada
(CICESE), Ensenada, Baja California, Mexico
K. Nasreen
Tamil Nadu, India
Dai-Hung Ngo
ix
x Contributors
R. Nithya
Tamil Nadu, India
Leonel Ochoa
Centro de Innovacion Biotecnologica AC (CENBIOTEC), Ensenada, Baja California,
Mexico
Jorge Olmos-Soto
Laboratory for Molecular Microbiology, Department of Marine Biotechnology, Centro de
Investigacion Cientfica y de Educacion Superior de Ensenada (CICESE), Ensenada, Baja
California, Mexico
Ramjee Pallela
Synthetic Biology and Biofuels Group, International Centre for Genetic Engineering and
Biotechnology, New Delhi, India
Jose de Jesus Paniagua-Michel
Laboratory for Bioactive Compounds and Bioremediation, Department of Marine
Biotechnology, Centro de Investigacion Cientfica y de Educacion Superior de Ensenada
(CICESE), Ensenada, Baja California, Mexico
Mahvash Peidayesh
Malaysia
Prasad N. Sudha
Department of Marine-bio Convergence Science and Marine Bioprocess Research Center,
Pukyong National University, Busan, South Korea, and Department of Chemistry, D.K.M.
College for Women, Thiruvalluvar University, Vellore, Tamil Nadu, India
Jayachandran Venkatesan
Department of Marine-bio Convergence Science and Marine Bioprocess Research Center,
Pukyong National University, Busan, South Korea
K. Vijayalakshmi
Tamil Nadu, India
P. Angelin Vinodhini
Tamil Nadu, India
Thanh-Sang Vo
Rong-Lin Xie
PREFACE
Carbohydrates are large molecules and divided into four main categories,
monosaccharides, disaccharides, oligosaccharides, and polysaccharides.
Polysaccharides from marine source have unique properties due to present
extreme environment and widely used for food application. Biological and
biomedical application of marine-derived polysaccharides has been explored
in small amount. This book aims to collect the material for biological,
biomedical, and industrial application of marine-derived polysaccharides.
This book has been divided into two volumes, each volume contains
12 chapters,
Chapters 15 provide the detailed information about isolation and
characterization techniques of marine polysaccharides (chitin, chitosan,
carrageenan, heparin sulfate, hyaluronic acid, and extracellular polysac-
charides) in detail.
Chapters 615 describe the usage of marine polysaccharides in biological
applications such as matrix metalloproteinase inhibitory effect, antican-
cer, antiallergy, antioxidant, and antidiabetic effects.
Chapters 1618 deal about biomedical application of marine polysaccha-
rides; tissue engineering, drug delivery, and gene delivery applications
are explored well.
Chapters 19 and 23 explain about the usage of marine polysaccharides in
wastewater treatment and industrial application.
Chapters 21, 22, and 24 deal about functional food, nutraceutical, phar-
maceutical, and cosmeceutical value of marine polysaccharides.
This book provides cumulative information about marine polysaccharides
and their biological, biomedical, and industrial applications. Hence, this
book will be important reference for marine biotechnologist, natural product
scientist, and whoever working on marine polysaccharides field.
xi
CHAPTER ONE
Marine-Derived Polysaccharides
for Regulation of Allergic
Responses
Thanh-Sang Vo*, Se-Kwon Kim*,,1
*Marine Bioprocess Research Center, Pukyong National University, Busan, South Korea

1
Corresponding author: e-mail address: sknkim@pknu.ac.kr
Contents
1. Introduction 2
2. Marine Polysaccharides 3
2.1 Alginate 3
2.2 Porphyran 4
2.3 Fucoidans 4
2.4 Chitin and its derivatives 5
3. Pharmacological Properties of Marine Polysaccharides for Modulation of Allergic
Responses 6
3.1 Alginic acid 6
3.2 Porphyran 7
3.3 Fucoidans 7
3.4 Chitin 8
3.5 Chitosan nanoparticles 9
3.6 Chitooligosaccharides 10
4. Conclusion 10
References 11
Abstract
Polysaccharides are macromolecules made up of many monosaccharides joined
together by glycosidic bonds. Polysaccharides from marine sources are widely distrib-
uted as the principle component in cell wall structures of seaweeds or exoskeletons
of crustaceans. So far, marine polysaccharides have been used in many fields of bio-
materials, food, cosmetic, and pharmacology. Especially, numerous pharmaceutical
properties of marine polysaccharides have been revealed such as antioxidant, anti-
inflammatory, antiallergic, antitumor, antiobesity, antidiabetes, anticoagulant,
antiviral, immunomodulatory, cardioprotective, antihepatopathy, antiuropathy, and
antirenalpathy activities. Recently, several marine polysaccharides such alginate,
porphyran, fucoidan, and chitin and its derivatives have been found as modulators
of allergic responses due to enhancing innate immune system, altering Th1/Th2
#
Advances in Food and Nutrition Research, Volume 73 2014 Elsevier Inc. 1
ISSN 1043-4526 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800268-1.00001-9
2 Thanh-Sang Vo and Se-Kwon Kim
balance, inhibiting IgE production, and suppressing mast cell degranulation. This con-
tribution, therefore, focuses specially on the immunomodulatory effect of marine
polysaccharides and emphasizes their potential application as candidates of pharma-
ceuticals as well as nutraceuticals to prevent allergic disorders.
1. INTRODUCTION
Allergy is a disorder of the immune system due to an exaggerated reac-
tion of the immune system to harmless environmental substances, such as
animal dander, house dust mites, foods, pollen, insects, and chemical agents
(Milian & Daz, 2004). It can cause runny nose, sneezing, itching, rashes,
swelling, or asthma (Kay, 2000). It is noteworthy that the allergic diseases
are among the commonest causes of chronic ill-health. The prevalence,
severity, and complexity of these diseases are rapidly rising and considerably
adding to the burden of health-care costs (Kay, 2000). Substantially, allergic
reaction is characterized by the excessive activation of mast cells and baso-
phils by immunoglobulin E (IgE), resulting in an extreme inflammatory
response (Galli, Tsai, & Piliponsky, 2008). Acute allergic sensitization in
individuals is involved in the generation of allergen-specific CD4+ Th2 cells.
These cells secrete various cytokines, including IL-4, IL-5, IL-9, and IL-13,
as well as chemokines such as thymus, leading to further Th2 cell recruit-
ment and the production of allergen-specific IgE by B cells. Subsequently,
IgE circulates and binds surface receptors on mast cells and basophils. Further
exposure to allergen results in crosslinking of IgE on mast cells and basophils
causing cell degranulation, releasing histamine, proteases, chemokines, pros-
taglandins, leukotrienes, and a host of other mediators. This results in
bronchoconstriction and recruitment of activated eosinophils, neutrophils,
lymphocytes, and macrophages (Larche, 2007; Larche, Robinson, & Kay,
2003). These allergic cascades are considered as a source of molecular targets
for regulation of type I allergic reaction and management of allergic diseases.
Recently, the role of marine organisms-derived compounds as antiallergic
agents has been determined in vitro and in vivo by many researchers. Simul-
taneously, numerous marine compounds have been found to be efficient for
antiallergic therapeutics via modulation of Th1/Th2 balance, inhibition of
IgE production, and suppression of mast cell degranulation.
The worlds oceans, covering more than 70% of the earths surface, rep-
resent an enormous resource for the discovery of promising therapeutic
agents. Due to the unusual diversity of chemical structures, marine
Antiallergic Properties of Marine Polysaccharides 3
organisms have received much attention in screening marine natural prod-

ucts for their biomedical potential (Haefner, 2003; Molinski, Dalisay,
Lievens, & Saludes, 2009; Newman & Cragg, 2004). During the last
decades, marine organisms such as algae, tunicates, sponges, soft corals, bryo-
zoans, sea slugs, mollusks, echinoderms, fishes, microorganisms, etc., have
been subjected for isolation of numerous novel compounds. They have sig-
nificant amounts of lipid, protein, peptide, acid amine, polysaccharides,
chlorophyll, carotenoids, vitamins, minerals, and unique pigments (Blunt,
Copp, Munro, Northcote, & Prinsep, 2006; Faulkner, 2001, 2002). Nota-
bly, marine polysaccharides have been known as potential promising mate-
rials for a variety of uses in food, medicine, pharmaceutical, and nutraceutical
industries due to their biological properties and activities. This contribution,
therefore, focuses specially on the immunomodulatory effect of marine
polysaccharides and emphasizes their potential application as candidates of
pharmaceuticals as well as nutraceuticals to prevent allergic disorders.
2. MARINE POLYSACCHARIDES
Polysaccharides from marine sources offer diverse therapeutic func-
tions due to their biocompatible, biodegradable to harmless products, non-
toxic, and physiologically inert. Moreover, most of them are capable of
forming hydrogels because of their remarkable hydrophilicity, which helps
them to bind to proteins and other compounds. Several marine polysaccha-
rides such as alginate, porphyran, fucoidan, and chitin and its derivatives
have been found and extracted from various sources, especially seaweed
and crustacean. In recent years, numerous polysaccharides have been used
in many fields of biomaterials, food, cosmetic, and pharmacology.
2.1. Alginate
Alginates are quite abundant in nature as structural component in marine
brown algae (Phaeophyceae) and as capsular polysaccharides in soil bacteria
(Laurienzo, 2010). The function of alginates in algae is primarily skeletal,
with the gel located in the cell wall and intercellular matrix conferring
the strength and flexibility necessary to withstand the force of water in which
the seaweed grows (DAyala, Malinconico, & Laurienzo, 2008). Alginate is a
linear, anionic block copolymer heteropolysaccharide consisting of -D-
mannuronic acid (M) and -L-guluronic acid (G). The relative amount
and sequential distribution of homogeneous MM segments (M-blocks),
homogeneous GG segments (G-blocks), and alternating MG segments
(MG-blocks), which represent the primary structure of alginate, depend on

the producing species, and for marine sources, on seasonal and geographical
variations (DAyala et al., 2008). Alginates may be prepared with a wide
range of average molecular weights (50100,000 residues) to suit the appli-
cation. The process of the isolation of alginates from brown algae includes
the pre-extraction with hydrochloric acid, followed by washing, filtration,
and neutralization with alkali. Sodium alginate is precipitated from the solu-
tion by alcohol (isopropanol or ethanol) and usually reprecipitated in the
same way (Laurienzo, 2010). Over the last few years, medical and pharma-
ceutical industries have shown an increased interest in alginates due to effi-
cient treatment of esophageal reflux, creates multiquality calcium fibers for
dermatology, and wound healing. Alginate is an effective natural dis-
integrant, tablet binder and offers an attractive alternative for sustained-
release systems. It offers advantages over synthetic polymers as it forms
hydrogels under relatively mild pH and temperature and is generally reg-
arded as nontoxic, biocompatible, biodegradable, less expensive, and abun-
dantly available in nature. Accordingly, alginates are considered to be useful
materials for biomedical applications, especially for controlled delivery of
drugs and other biologically active compounds and for the encapsulation
of cells (DAyala et al., 2008).
2.2. Porphyran
Porphyran is a sulfated polysaccharide isolated from seaweeds of order
Bangiales especially from the genera Porphyra. It is obtained from red algae
of Kingdom Rhodophyta. Chemically, porphyran is related to agarose, con-
sists of linear backbone of alternating 3-linked -D-galactose and 4-linked
3,6-anhydro--L-galactose units. The L residues are mainly composed of
-L-galactosyl 6-sulfate units, and the 3,6-anhydrogalactosyl units are minor.
Porphyran has been reported to possess various pharmaceutical properties
such as antioxidant, antitumor, immunostimulant, hypotensive, antifatigue,
antibacterial, anticoagulant, anticancer, antiviral, antihyperlipidemic, and
hepatoprotective activity (Bhatia et al., 2008).
2.3. Fucoidans
Fucoidans are a complex series of sulfated polysaccharides found widely in
the cell walls of brown seaweeds. In recent years, different brown algae were
analyzed for their content of fucoidans. The low-molecular-weight fractions
of algal fucoidans (less than 30 kDa) obtained by depolymerization have
been shown to exhibit some heparin-like properties, with less side effects
(Karim et al., 2011). Such polysaccharides do not occur in other divisions
of algae and in land plants. However, the related biopolymers were found
in marine invertebrates such as sea cucumbers or sea urchins. These polysac-
charides are simpler than fucoidans derived from marine brown algae and are
referred to as sulfated fucans. The seaweed fucoidans are heterogenic and
represent the mixtures of structurally related polysaccharides with certain
variations of the content of carbohydrate units and noncarbohydrate substit-
uents (Cumashi et al., 2007). Fucoidans are mainly composed of fucose and
sulfate. Besides, they also contain other monosaccharides (mannose, galac-
tose, glucose, xylose, etc.) and uronic acids, even acetyl groups and protein.
The fucoidans of most algae consist of sulfated L-fucose with major fucose
components. However, some fucoidans have minor fucose components and
major other monosaccharides like galactose or uronic acids (Vo & Kim,
2013). According to Cumashi et al. (2007), the polysaccharide backbones
in fucoidans are known as type I or type II chains. The type I chains are
found to contain the repeating (1 ! 3)-linked -L-fucopyranose residues,
whereas type II chains contain the alternating (1 ! 3)- and (1 ! 4)-linked
-L-fucopyranose residues. During the last decades, numerous pharmaceu-
tical properties of fucoidans have been revealed due to their antioxidant,
anti-inflammatory, antiallergic, antitumor, antiobesity, antidiabetes, antico-
agulant, antiviral, antihepatopathy, antiuropathy, and antirenalpathy effects
(Vo & Kim, 2013). These special properties of fucoidans have supported it to
be applied to functional foods for disease prevention and health promotion.
2.4. Chitin and its derivatives

Chitin is a linear polysaccharide consisting of -(14)-N-acetyl
D-glucosamine residues. It is widely distributed in nature and is the second
most abundant polysaccharide in nature after cellulose. It may be regarded as
cellulose with hydroxyl at position C-2 replaced by an acetamino group.
Chitin is a white, hard, inelastic, nitrogenous polysaccharide found in the
cell walls of bacteria and fungi, mushrooms, exoskeleton of crustaceans
and insects, the microfilarial sheath of parasitic nematodes, and the lining
of the digestive tracts of many insect. These organisms use chitin to protect
the invader from the harsh conditions inside the animal or plant host (Elias,
Homer, Hamid, & Lee, 2005). Chitin is highly hydrophobic and it insoluble
in water and most organic solvents. It exists mainly in two forms including
-chitin and -chitin. -Chitin consists of sheets of tightly packed
alternating parallel and antiparallel chains (Minke & Blackwell, 1978).

Meanwhile, -chitin is arranged in parallel (Gardner & Blackwell, 1975),
which occurs less frequently in nature than -chitin. Being nontoxic and
environmentally safe, chitin has become of great interest not only as a
utilized resource but also a new functional biomaterial of high potential
in many fields such as medical, agricultural, and cosmetic applications. It
is readily obtained for commercial use from crustacean shell waste products
generated by the seafood industry (Kumar, 2000; Kurita, 2006). Chitosan, a
partially deacetylated polymer of N-acetylglucosamine, is produced com-
mercially by deacetylation of chitin (Dutta, Dutta, & Tripathi, 2004).
During the past decades, chitosan has received considerable attention
due to its biodegradable, nontoxic, and nonallergenic properties, which
made it possible to be used in many fields including food, cosmetics, bio-
medicine, agriculture, and environmental protection (Kim & Rajapaksea,
2005). Recent studies have focused on the conversion of chitosan to
chitooligosaccharides (COS) since COS are not only water soluble and
possess higher oral absorption but also have various biological effects,
including antimicrobial, antitumor, anticancer, antioxidant, anti-
inflammatory, and antiangiotensin-I-converting enzyme activities
(Kim & Rajapaksea, 2005). Especially, chitin and its derivatives have been
determined to be protective agents against allergic diseases.
3. PHARMACOLOGICAL PROPERTIES OF MARINE

POLYSACCHARIDES FOR MODULATION OF ALLERGIC
RESPONSES
3.1. Alginic acid
Alginic acid, a naturally occurring hydrophilic colloidal polysaccharide
obtained from the several species of brown seaweeds, exhibited different
effects against hyaluronidase activity and histamine release from mast cells
(Asada et al., 1997). In the in vivo conditions, alginic acid inhibited com-
pound 48/80-induced systemic anaphylaxis with doses of 0.251 g/kg
and significantly inhibited passive cutaneous anaphylaxis by 54.8% at
1 g/kg for 1 h pretreatment ( Jeong et al., 2006). Besides, alginic acid was
found to have a maximum suppression rate (60.8%) on histamine release
from rat peritoneal mast cells at concentration of 0.01 g/ml. Furthermore,
the antiallergic activities of alginic acid were also observed due to its suppres-
sive effects on activity and expression of histidine decarboxylase, production
of IL-1 and TNF-, and protein level of nuclear factor (NF)-B/Rel A in
PMA plus A23187-stimulated HMC-1 cells ( Jeong et al., 2006). Notice-

ably, alginic acid oligosaccharide (ALGO), a lyase lysate of alginic acid,
has been revealed to be able to reduce IgE production in the serum of
BALB/c mice immunized with -lactoglobulin (Uno, Hattori, &
Yoshida, 2006; Yoshida, Hirano, Wada, Takahashi, & Hattori, 2004).
Moreover, antigen-induced Th2 development was blocked by ALGO treat-
ment via enhancing the production of IFN- and IL-12, and down-
regulating IL-4 production in splenocytes of mice (Yoshida et al., 2004).
3.2. Porphyran
Porphyran, a sulfated polysaccharide isolated from red seaweeds, has been
recognized to be effective against different allergic responses. According to
Ishihara, Oyamada, Matsushima, Murata, and Muraoka (2005), porphyran
of red algae Porphyra tenera and P. yezoensis were capable to inhibit the con-
tact hypersensitivity reaction induced by 2,4,6-trinitrochlorobenzene via
decreasing the serum level of IgE in Balb/c mice. Moreover, Yoshizawa
and colleagues have revealed that polysaccharide fractions from
P. yezoensis possessed the ability to activate macrophages in vitro and in vivo
via enhancing glucose consumption, the production of nitrite and tumor
necrosis factor (TNF), secretion of IL-1 from macrophages and carbon
clearance activity of phagocytes from mice injected intraperitoneally. It
has been indicated that porphyran is responsible for these effects and its sul-
fate group contributes to the macrophage stimulating activities (Yoshizawa
et al., 1995, Yoshizawa, Enomoto, Todoh, Ametani, & Kaminogawa,
1993). In addition, oral administration of porphyran from Porphyra
vietnamensis evoked a significant increase in weight of the thymus, spleen
and lymphoid organ cellularity, and total leucocyte and lymphocyte
(Bhatia et al., 2013).
3.3. Fucoidans
Recently, algal fucoidans have been found to be effective in suppression of
IgE and Th2 cytokine production in vitro and in vivo. Fucoidan from Undaria
pinnatifida reduced the concentrations of both IL-4 and IL-13 in
bronchoalveolar lavage fluid (BALF) and inhibited the increase of
antigen-specific IgE in OVA-induced mouse airway hypersensitivity
(Maruyama, Tamauchi, Hashimoto, & Nakano, 2005). In the recent study,
Yanase et al. (2009) have reported that the peritoneal injection of fucoidan
caused an alleviative effect of plasma IgE level by suppressing a number of
IgE-expressing and IgE-secreting B cells from OVA-sensitized mice. On the

other hand, the inhibitory effect of fucoidan on IgE production was deter-
mined due to preventing C germline transcription and NF-B p52 trans-
location in B cells (Oomizu, Yanase, Suzuki, Kameyoshi, & Hide, 2006).
Yet, the inhibitory activity of fucoidan has been not observed if B cells were
prestimulated with IL-4 and anti-CD40 antibody before the administration
of fucoidan. Thus, it suggested that fucoidan may not prevent a further
increase of IgE in patients who have already developed allergic diseases
and high levels of serum IgE. However, Iwamoto et al. (2011) have recently
determined that fucoidan effectively reduced IgE production in both
peripheral blood mononuclear cells from atopic dermatitis patients and
healthy donors. These findings indicated that fucoidan suppresses IgE pro-
duction by inhibiting immunoglobulin class-switching to IgE in human
B cells, even after the onset of atopic dermatitis.
3.4. Chitin
Chitin has been evidenced as a potent innate immune stimulator of macro-
phages and other innate immune cells, and thus chitin is able to suppress
allergen-induced type 2 allergic responses. Indeed, Shibata and colleagues
have determined the immunological effects of chitin in vivo and in vitro using
phagocytosable small-sized chitin particles. It has shown that intravenous
administration of fractionated chitin particles into the lung activated alveolar
macrophages to express cytokines such as IL-12, TNF-, and IL-18, leading
to INF- production mainly by NK cells (Shibata, Foster, Metzger, &
Myrvik, 1997). The production of cytokines induced by chitin is identified
to be mediated by a mannose receptor (Shibata, Metzger, & Myrvik, 1997).
In another study, Lee and colleagues have determined that chitin stimulates
macrophages by interacting with different cell surface receptors such as mac-
rophage mannose receptor, toll-like receptor-2, C-type lectin receptor
Dectin-1, and leukotriene 134 receptor (BLT1) (Lee, 2009). These studies
have shown the direct interactions between chitin and its cell surface recep-
tors and thus chitin regulates the specific signaling pathways in immune
responses.
In the further study of Shibata and colleagues, the suppressive effect of
Th2 responses has been confirmed when chitin was given orally in BALB/c
and C57BL/6 mice (Shibata, Foster, Bradfield, & Myrvik, 2000). It was
observed that chitin treatment resulted in decreases of serum IgE levels
and lung eosinophil numbers in both strains. The inhibitory mechanisms
of Th2 responses by chitin was found due to decreases of Th2 cytokines

including IL-4, IL-5, and IL-10 levels and the production of Th1 cytokine
IFN-gamma in spleen cells isolated from the ragweed-immunized mice.
These results indicated that the immune responses were redirected toward
a Th1 response by chitin treatment, and thus downregulating Th2-facilitated
IgE production and lung eosinophilia in the allergic mouse. Moreover, the
Th1 adjuvant role of chitin has been determined via upregulating Th1
immunity induced by heat-killed Mycobacterium bovis and downregulating
Th2 immunity induced by mycobacterial protein (Shibata et al., 2001).
Likewise, Hamajima et al. (2003) has also reported the Th1 adjuvant effect
of chitin microparticles in inducing viral specific immunity.
Notably, the effectiveness of chitin microparticles when given intrana-
sally as a treatment for the symptoms of respiratory allergy and allergy asthma
has been tested in two different mouse models of allergy, namely to
Dermatophagoids pteronyssinus and Aspergillus fumigates (Strong, Clark, &
Reid, 2002). The intranasal application of microgram doses of chitin micro-
particles substantially reduced the allergen-induced serum IgE levels,
peripheral eosinophilia, airway hyperresponsiveness, and lung inflammation
in both allergy models. This effectiveness was found due to the increase
in Th1 cytokines IL-12, IFN-, and TNF- and decrease in IL-4 produc-
tion during allergen challenge. The immunostimulatory properties of chitin
microparticles could offer a novel and natural approach to treating allergic
disease in humans.
3.5. Chitosan nanoparticles

In the most recent study, chitosan nanoparticles have been determined as an
adjuvant agent via promoting immune response in ovalbumin (OVA)-
challenged mice (Wen, Xu, Zou, & Xu, 2011). Mice were immunized sub-
cutaneously with 25 g OVA alone or with 25 g OVA dissolved in saline
containing Quil A (10 g), chitosan (50 g), or chitosan nanoparticles (12.5,
50, or 200 g) on days 1 and 15. It was found that the serum OVA-specific
IgG, IgG1, IgG2a, and IgG2b antibody titers and Con A-, LPS-, and OVA-
induced splenocyte proliferation were significantly enhanced by chitosan
nanoparticles as compared with OVA and chitosan groups. Notably,
chitosan nanoparticles also significantly promoted the production of IL-2
and IFN- cytokines and upregulated the mRNA expression of IL-2,
IFN- cytokines in splenocytes from the immunized mice compared with
OVA and chitosan groups. Besides, chitosan nanoparticles remarkably
increased the killing activities of NK cells activity. The results suggested that
chitosan nanoparticles had a strong potential to increase both cellular and
humoral immune responses.
3.6. Chitooligosaccharides
In the regard of COS on in vitro allergic responses, Vo, Kong, and Kim
(2011) and Vo, Kim, Ngo, Kong, and Kim (2012) have investigated the
inhibitory effect of COS on mast cell activation induced by calcium
ionophore A23187 or antigen. The pretreatment of COS causes signifi-
cant inhibition on mast cell degranulation via reducing histamine and
-hexosaminidase release and intracellular Ca2+ elevation in RBL-2H3
mast cells. Moreover, the inhibitory effects of COS on expression as well
as production of various cytokines such as TNF-, IL-1, IL-4, and IL-6
were also evidenced. Notably, the protective effect of COS (<1 kDa)
against OVA-induced lung inflammation in asthma model mice was also
examined (Chung, Park, & Park, 2012). Oral administration of COS
(16 mg/kg body weight/day) resulted in a significant reduction in both
mRNA and protein levels of interleukin IL-4, IL-5, IL-13, and TNF-
in the lung tissue and BALF. The protein levels of IL-4, IL-13, and
TNF- in BALF were decreased by 5.8-fold, 3.0-fold, and 9.9-fold,
respectively, compared to those in the OVA-sensitized/challenged asthma
control group. Collectively, these results indicate that COS can contribute
to attenuation of allergic reactions and might be a promising candidate for
novel inhibitor of allergic reaction.
4. CONCLUSION
Marine polysaccharides are considered as promising biomaterials that
are the focus of biomedical research today. Notably, many experimental
results clearly indicated that marine polysaccharides such as alginate,
porphyran, fucoidan, and chitin and its derivatives are exciting agents for
modulation of allergic responses via enhance of innate immune system, reg-
ulation of Th1/Th2 balance toward Th1 dominance, decrease in IgE pro-
duction, and inhibition of mast cell degranulation. Accordingly, marine
polysaccharides can be used as safety and efficacy biomaterials for the devel-
opment of food, pharmaceutical, and nutraceutical industries in prevention
and/or treatment of allergic disorders. The possibility of producing a variety
of chemically modified derivatives makes these polysaccharides versatile bio-
materials in almost all fields of biomedical interest.
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CHAPTER TWO
Antioxidant Effects of Chitin,

Chitosan, and Their Derivatives
Dai-Hung Ngo*, Se-Kwon Kim*,,1
*Marine Bioprocess Research Center, Pukyong National University, Busan, South Korea

1
Contents
1. Introduction 15
2. Antioxidants and Oxidative Stress 17
3. Antioxidant Activity of Chitin, Chitosan, and Their Derivatives 17
3.1 Antioxidant activity of chitin and chitosan 17
3.2 Antioxidant activity of chito-oligomers and its derivatives 22
4. Conclusion 26
Acknowledgments 27
References 27
Abstract
Chitin, chitosan, and their derivatives are considered to promote diverse activities,
including antioxidant, antihypertensive, anti-inflammatory, anticoagulant, antitumor
and anticancer, antimicrobial, hypocholesterolemic, and antidiabetic effects, one of
the most crucial of which is the antioxidant effect. By modulating and improving phys-
iological functions, chitin, chitosan, and their derivatives may provide novel therapeutic
applications for the prevention or treatment of chronic diseases. Antioxidant activity of
chitin, chitosan, and their derivatives can be attributed to in vitro and in vivo free radical-
scavenging activities. Antioxidant effect of chitin, chitosan, and their derivatives may be
used as functional ingredients in food formulations to promote consumer health and to
improve the shelf life of food products. This chapter presents an overview of the anti-
oxidant activity of chitin, chitosan, and their derivatives with the potential utilization in
the food and pharmaceutical industries.
1. INTRODUCTION
Chitin is the second most abundant biopolymer on earth after cellulose
and one of the most abundant polysaccharides. It is a glycan of (1 ! 4)-
linked N-acetylglucosamine units, and it is widely distributed in crustaceans
#
http://dx.doi.org/10.1016/B978-0-12-800268-1.00002-0
16 Dai-Hung Ngo and Se-Kwon Kim
and insects as the protective exoskeleton and cell walls of most fungi. Chitin is
usually prepared from the shells of crustaceans such as crab, shrimp, and craw-
fish ( Jayakumara, Prabaharan, Nair, & Tamura, 2010; Muzzarelli, 1997).
Chitosan is a natural nontoxic biopolymer produced by alkaline
deacetylation of chitin. Chitin and chitosan are insoluble in water as well
as in most organic solvents, which is the major limiting factor for their uti-
lization in living systems. Hence, it is important to produce soluble chitin or
chitosan by several methods such as acidic and enzymatic hydrolysis. Chito-
oligomers (COSs) are chitosan derivatives (polycationic polymers comprised
principally of glucosamine units) and can be generated via either chemical or
enzymatic hydrolysis of chitosan. COSs are of great interest in pharmaceu-
tical and medicinal applications due to their noncytotoxic and high water-
soluble properties. Various activities of COSs are affected by degree of
deacetylation (DD), molecular weight (MW), or chain length ( Jayakumar
et al., 2010; Kim, Ngo, & Rajapakse, 2006; Muzzarelli, Stanic, &
Ramos, 1999; Razdan & Pettersson, 1994).
Chitin, chitosan, and their derivatives have important biological proper-
ties in medicinal and pharmaceutical applications such as antioxidative
(Aytekin, Morimura, & Kida, 2011; Kim & Ngo, 2013; Ying, Xiong,
Wang, Sun, & Liu, 2011), antiallergy (Vo, Kim, Ngo, Kong, & Kim,
2012; Vo, Kong, & Kim, 2011; Vo, Ngo, & Kim, 2012), anti-inflammatory
(Lee, Senevirathne, Ahn, Kim, & Je, 2009; Pangestuti, Bak, & Kim, 2011),
antihuman immunodeficiency virus (Vo & Kim, 2010), anticoagulant (Yang
et al., 2012), adipogenesis inhibitory (Cho et al., 2008), antitumor and anti-
cancer (Cho, Park, Seo, & Yoo, 2009; Shen, Chen, Chan, Jeng, & Wang,
2009; Toshkova et al., 2010), antibacterial (Sajomsang, Gonil, & Saesoo,
2009; Xu, Xin, Li, Huang, & Zhou, 2010; Yang et al., 2010; Yang,
Chou, & Li, 2005; Zhong, Li, Xing, & Liu, 2009), antihypertensive
(Ngo, Qian, Je, Kim, & Kim, 2008; Qian, Eom, Ryu, & Kim, 2010), immu-
nostimulant ( Jeon & Kim, 2001), anti-Alzheimers (Cho, Kim, Ahn, & Je,
2011a; Yoon, Ngo, & Kim, 2009), calcium and ferrous binding (Bravo-
Osuna, Millotti, Vauthier, & Ponchel, 2007; Liao, Shieh, Chang, &
Chien, 2007), and hypocholesterolemic (Zhang et al., 2010; Zhou, Xia,
Zhang, & Yu, 2006) properties.
By modulating and improving physiological functions, chitin, chitosan,
and their derivatives may provide novel therapeutic applications for the pre-
vention or treatment of chronic diseases. This chapter centers on chitin,
chitosan, and their derivatives with antioxidant activity relevant to human
health benefits.
Antioxidant Effects of Chitin, Chitosan, and Their Derivatives 17
2. ANTIOXIDANTS AND OXIDATIVE STRESS

Humans are impacted by many free radicals both from inside our body
and surrounding environment, particularly reactive oxygen species (ROS)
generated in living organisms during metabolism. It is produced in the forms
of H2O2, superoxide anion (O2 ) and hydroxyl (OH) radicals. In addition,
oxidative stress may cause inadvertent enzyme activation and oxidative dam-
age to cellular systems. Free radicals attack macromolecules such as DNA,
proteins, and lipids, leading to many health disorders including hypertensive,
cardiovascular, inflammatory, aging, diabetes mellitus, and neurodegenera-
tive and cancer diseases. Antioxidants may have a positive effect on human
health since they can protect human body against deterioration by free rad-
icals (Butterfield et al., 2006; Dhalla, Temsah, & Netticadan, 2000; Fubini &
Hubbard, 2003; Maulik & Das, 2002; Ngo, Wijesekara, Vo, Ta, & Kim,
2011; Seven, Guzel, Aslan, & Hamuryudan, 2008).
Oxidation in foods affects lipids, proteins, and carbohydrates. However,
lipid oxidation is the main cause of deterioration of food quality, leading to
rancidity and shortening of shelf life. Oxidation of proteins in foods is
influenced by lipid oxidation, where lipid oxidation products react with
proteins causing their oxidation. Carbohydrates are also susceptible to oxi-
dation, but they are less sensitive than lipids and proteins (Bernardini et al.,
2011). Therefore, many synthetic commercial antioxidants such as butylated
hydroxytoluene, butylated hydroxyanisole, tert-butylhydroquinone, and
propyl gallate have been used to retard the oxidation and peroxidation pro-
cesses in food and pharmaceutical industries. However, the use of these syn-
thetic antioxidants must be under strict regulation due to potential health
hazards (Blunt, Copp, Munro, Northcote, & Prinsep, 2006). Therefore,
the use of natural antioxidants available in food and other biological sub-
stances has attracted significant interest due to their presumed safety and
nutritional and therapeutic values (Ajila, Naidu, Bhat, & Prasada
Rao, 2007).
3. ANTIOXIDANT ACTIVITY OF CHITIN, CHITOSAN,

AND THEIR DERIVATIVES
3.1. Antioxidant activity of chitin and chitosan
Chitin oligomers (NA-COSs) are hydrolytic products of chitin using chem-
ical, physical, or enzymatic agents and are water soluble. Therefore,
NA-COSs can be used easily both in vitro and in vivo. The cellular antiox-
idant effects of NA-COSs (229.21593.12 Da) produced by acidic hydro-
lysis of crab chitin were determined by Ngo, Kim, and Kim (2008). The
inhibitory effects of NA-COSs on myeloperoxidase (MPO) activity in
human myeloid cells (HL-60) and oxidation of protein and DNA in mouse
macrophages (RAW 264.7) were identified. Furthermore, their direct rad-
ical scavenging effect and intracellular glutathione (GSH) level were signif-
icantly increased in a time-dependent manner. These results suggest that
NA-COSs act as a potent antioxidant in live cells.
In addition, Ngo, Lee, Kim, and Kim (2009) produced two kinds of
NA-COSs with different MWs. Two kinds of NA-COSs with MW of
13 kDa (NA-COS 13 kDa) and below 1 kDa (NA-COS <1 kDa) were
obtained using an ultrafiltration membrane system. They exhibited an inhib-
itory effect against DNA and protein oxidation. Furthermore, intracellular
GSH level and direct intracellular radical scavenging effect were significantly
increased in a time-dependent manner in RAW 264.7 cells. In particular,
NA-COS of 13 kDa was more effective than NA-COS <1 kDa in protein
oxidation and production of intracellular free radicals. These results suggest
that NA-COSs act as potential scavengers against oxidative stress in cells.
The antioxidant effect of chitosan was studied in vitro and in vivo (Liu,
2008). Chitosan at an addition of 0.02% had antioxidant activities in lard
and crude rapeseed oil, but the activity was less than ascorbic acid. When
the addition was increased, chitosan and ascorbic acid had similar activities;
chitosan could significantly reduce serum free fatty acid and
malondialdehyde (MDA) concentrations and increase antioxidant enzymes
activities such as superoxide dismutase (SOD), catalase (CAT), and glutathi-
one peroxidase (GSH-PX), indicating that chitosan regulated the antioxi-
dant enzymes activities and decreased lipid peroxidation. In the food
industry, chitosan (edible chitosan, more than 83% DD) and COSs have
been used as dietary food additives and functional factors for their antimi-
crobial, hypocholesterolemic, and immune-stimulating effects as well as
drug carriers (Xia, Liu, Zhang, & Chen, 2011).
Samar, El-Kalyoubi, Khalaf, and El-Razik (2013) prepared chitosan
from shrimp waste chitin at three particle sizes 20, 40, and 60 mesh by
deacetylating with different concentrations of NaOH solution (30%, 40%,
and 50%) under microwave irradiation for 10 min. The results showed that
the microwave-synthesized chitosan was characterized and the DD
increased with increasing concentration of deacetylation alkali solution.
A DD of 95.19% was achieved after irradiating chitin at 60 mesh with
50% NaOH solution in a microwave for 10 min at 1400 W power.

Microwave-synthesized chitosan showed antioxidant activities of
47.7172.31% at 10 mg/mL and reducing powers of 2.0942.367 at
10 mg/mL. On the other hand, at 10 mg/mL, the scavenging effects of
chitosan on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals ranged from
43.03% to 90.48%.
Yen, Yang, and Mau (2008) reported that chitosan was prepared by alka-
line N-deacetylation of crab chitin for 60 min (C60), 90 min (C90), and
120 min (C120), and their antioxidant effects were determined. Chitosans
exhibited antioxidant effects of 58.370.2% at 1.0 mg/mL and reducing
powers of 0.320.44 at 10 mg/mL. At 10 mg/mL, the scavenging ability
of chitosan C60 on DPPH radical was 28.4%, whereas those of other
chitosans were 46.452.3%. At 0.1 mg/mL, scavenging abilities on OH
radicals were 62.377.6%, whereas at 1 mg/mL, chelating abilities on fer-
rous ions were 82.996.5%. All IC50 values (the effective concentration
at which the antioxidant activity was 50%) of antioxidant activity were
below 1.5 mg/mL. Generally, the effectiveness of chitosans was correlated
with N-acetylation times. Overall, crab chitosan was good in antioxidant
property and may be used as a source of antioxidants, as a possible food sup-
plement or ingredient in the pharmaceutical industry.
Anraku et al. (2008) determined that chitosan (2800 Da) may inhibit
neutrophil activation and oxidation of serum albumin commonly observed
in patients undergoing hemodialysis, resulting in reduction of oxidative
stress associated with uremia. Kim and Thomas (2007) investigated antiox-
idant effect of chitosans with different MWs (30, 90, and 120 kDa) in salmon
where the chitosan with lowest MW (30 kDa) exhibited highest antioxidant
effect. Various chitosan derivatives with high and low MW and variety of
reactive groups have showed significant activities against OH, O2 scav-
enging, and reducing power (Zhong et al., 2007).
Park, Je, and Kim (2004) prepared three kinds of partially deacetylated
hetero-chitosans such as 90%, 75%, and 50% deacetylated chitosan from
crab chitin and investigated their scavenging activities against DPPH, alkyl,
OH and O2 radicals using electron spin resonance (ESR) spectrometer.

The scavenging activities of hetero-chitosans increased from 3% to 69.39%
with increasing concentration from 1.25 to 5 mg/mL of alkyl radical.
In addition, 90% chitosan with relatively high DD showed the highest
radical scavenging effects on the OH, and O2 radicals, and the radical
scavenging activities of these hetero-chitosans depend on their DD and
concentration.
Chitosans with different DD were prepared from crab shell chitin in the
presence of alkali ( Je & Kim, 2006). Aminoderivatized chitosan derivatives
were prepared in addition of amino functional groups at a hydroxyl site in
the chitosan backbone. Six kinds of aminoderivatized chitosan, such as
aminoethyl chitosan, dimethylaminoethyl chitosan, and diethylaminoethyl
chitosan, were prepared from 90% and 50% deacetylated chitosan, and their
ROS scavenging effects were investigated. According to ESR studies,
aminoethyl chitosan from 90% deacetylated chitosan showed the highest
scavenging effects against OH and O2 radicals; the effects were 91.67%
and 65.34% at 0.25 and 5 mg/mL, respectively. In addition,
diethylaminoethyl chitosan from 90% deacetylated chitosan exhibited the
strongest H2O2 scavenging activity. These results suggest that the scavenging
effect depends on their DD and substituted group.
Cho, Kim, Ahn, and Je (2011b) prepared gallic acid-grafted-chitosans
(GA-g-chitosans) with four different grafting ratios by a free radical-induced
grafting reaction in order to improve antioxidant activity and water solubil-
ity. GA-g-chitosan (I) (ratio 1:1) showed 92.26% scavenging activity against
DPPH and 93.15% H2O2 scavenging activity at 50 g/mL. GA-g-chitosan
(I) also showed higher reducing power compared to others. All
GA-g-chitosans showed improved antioxidant activities compared to plain
chitosan treated in the same conditions without GA grafting. In addition,
Cho, Kim, and Je (2011) reported the IC50 value of chitosan gallate against
DPPH was 17.86 g/mL. Chitosan gallate effectively inhibited the gener-
ation of intracellular ROS and lipid peroxidation in RAW 264.7 cells.
Chitosan gallate exhibited the protective effect on OH-induced DNA
damage and upregulated the protein expression of antioxidant enzymes
(SOD-1 and GSH reductase) under H2O2-mediated oxidative stress in
RAW 264.7 cells. Chitosan gallate showed enhanced water solubility com-
pared to plain chitosan and good thermal stability. These results indicated
that chitosan gallate might be the potential antioxidant biomaterial.
Zhang, Geng, Jiang, Li, and Huang (2012) introduced quaternary
amino groups into chitin and chitosan to obtain O-(2-hydroxy-3-
trimethylammonium)propyl chitin (OHT-chitin) and N-(2-hydroxy-3-
trimethylammonium)propyl chitosan (NHT-chitosan). The MW of
OHT-chitin and NHT-chitosan was 8986 and 9723 with polydispersity
of 1.01 and 1.02, respectively. Their antioxidant effects in vitro were deter-
mined. -carotene-linoleic acid values of OHT-chitin and NHT-chitosan
at 0.8 mg/mL were up to 91% and 96%, while that of chitosan was 40%.
Based on photobleaching of DPPH at 326 nm, the DPPH inhibitory activity
of OHT-chitin and NHT-chitosan was 30.9% and 31.9% at 5 mg/mL,

whereas chitosan only gave 4.8%. Furthermore, OHT-chitin and NHT-
chitosan had better antioxidant property than chitosan according to the
reducing power as well as H2O2 scavenging effect.
Kong, Kim, Ahn, Byun, and Kim (2010) investigated antioxidant and
matrix metalloproteinase (MMP)-2 and MMP-9 inhibitory effects in human
fibrosarcoma cells (HT1080) of the water-soluble derivatives of chitosan and
chitin, carboxymethyl chitosan (CM-chitosan), and carboxymethyl chitin
(CM-chitin). Treatment with CM-chitosan and CM-chitin decreased the
formation of intracellular ROS, DNA, protein, and lipid peroxidation.
Moreover, CM-chitosan and CM-chitin reduced the expression levels of
MMP-2 and MMP-9 in gelatin zymography, reverse transcription polymer-
ase chain reaction (RT-PCR), and Western blot analysis without any cyto-
toxic influence. CM-chitin exhibited a higher MMP inhibitory effect than
CM-chitosan through transcriptional downregulations of activator
protein-1 and nuclear factor kappa B (NF-B). Findings from the present
study underscore the nutraceutical value of CM-chitosan and CM-chitin
as a potent antioxidant and MMP inhibitor via alleviations of radical-
induced oxidative damage.
Arancibia et al. (2014) recovered several shrimp active materials (protein
concentrates, protein hydrolysates, and a carotenoid extract) from shrimp
(Litopenaeus vannamei) processing residues (head and exoskeleton) and char-
acterized in terms of chemical composition and antioxidative activities such
as ferric ion reducing power and 2,20 -azino-bis-(3-ethylbenzothiazoline-
6-sulfonic acid) radical scavenging. Different coating solutions were
prepared by solubilizing chitosan, which was obtained from the residual
chitinous material, in lactic acid and blending it with the various recovered
materials. All chitosan-based solutions showed higher antioxidant activity
and reduced viscosity as compared to the base chitosan solution.
Anraku et al. (2011) investigated antioxidant effect of high MW chitosan
supplement (HMCS) in normal volunteers. The use of HMCS for 8 weeks
resulted in a significant decrease in total cholesterol (TC) levels and athero-
genic index and increased levels of high-density lipoprotein cholesterol
(HDLc). HMCS treatment also resulted in a lowered ratio of oxidized to
reduced albumin and an increase in total plasma antioxidant property.
A good correlation between the atherogenic index and oxidized albumin
ratio was found. The results suggest that the ratio of oxidized to reduced
albumin ratio represents a potentially useful marker of the metabolic syn-
drome. In in vitro studies, HMCS slightly decreased the levels of two stable
radicals in a dose- and time-dependent manner. The strong binding capacity

of indoxyl sulfate and low-density lipoprotein cholesterol (LDLc) was
observed with HMCS. These results suggest that HMCS decreased signif-
icant levels of pro-oxidants such as cholesterol and uremic toxins in the gas-
trointestinal tract, thereby inhibiting the subsequent development of
oxidative stress in the systemic circulation in humans.
Song, Yu, Zhang, Yang, and Zhang (2013) evaluated antioxidant effect
of chitosan from the blowfly Chrysomya megacephala larvae through DPPH
scavenging assays. The results showed that the MW of the blowfly chitosan
(501 kDa) was lower than that of the commercial chitosan (989 kDa), and its
DD (87.989.6%) was also higher than that of the commercial chitosan
(83.885.8%). Furthermore, the blowfly chitosan exhibited excellent anti-
oxidant property and its IC50 value was 1.2 mg/mL. Therefore, the blowfly
larvae could be a novel alternative source of chitosan and might be used as a
natural antioxidant.
Ren, Li, Dong, Feng, and Guo (2013) synthesized two kinds of phenolic
antioxidants-functionalized quaternized chitosan in order to develop aque-
ous soluble antioxidant-polymer conjugates. GA-quaternized chitosan (GA-
chitosan) and caffeic acid-quaternized chitosan (CA-chitosan) against OH,
O2 , and DPPH radicals were evaluated in vitro, respectively. The scaveng-
ing effects of the obtained GA-chitosan and CA-chitosan exhibited a
remarkable improvement over those of either chitosan or quaternized
chitosan. And the scavenging effect indices of the products were all higher
than 90% at a concentration of 1 mg/mL. Because GA-chitosan and
CA-chitosan are convenient to prepare and possess improved potential anti-
oxidant activities, these materials may represent an attractive new platform
for utilizations of chitosan.
3.2. Antioxidant activity of chito-oligomers and its derivatives

Radical scavenging activity of COS depends on their DD values and MWs.
Therefore, nine kinds of hetero-COS with relatively higher, medium, and
lower MWs were prepared from partially deacetylated hetero-chitosans
(90%, 75%, and 50% deacetylated chitosan), respectively, and their scaveng-
ing effects were investigated. According to ESR studies, 90% deacetylated
medium MW hetero-COS, which is having relatively medium MWs pre-
pared from 90% deacetylated chitosan, showed the highest free radical scav-
enging activity of all free radicals tested such as DPPH, OH, O2 , and
carbon-centered radicals. In addition, the radical scavenging activity of
hetero-COSs increased with a dose-dependent manner, and it depends on

their DD values and MWs ( Je, Park, & Kim, 2004).
COS can effectively protect human umbilical vein endothelial cells
against H2O2-induced oxidative stress and apoptosis, which might be of
importance in the treatment of cardiovascular diseases (Liu et al., 2010,
2009). COS exerted inhibitory effects on the formation of intracellular
ROS and lipid peroxidation such as MDA, restoring activities of endoge-
nous antioxidants including SOD and GSH-PX, along with the capacity
of increasing levels of NO and NO synthase.
Xu, Ma, et al. (2010) investigated the protective effect of COS against
H2O2-induced oxidative stress on human embryonic hepatocytes (L02 cells)
and its scavenging activity against the DPPH radical in vitro, suggesting that
COS might be useful in a clinical setting during the treatment of oxidative
stress-related liver damages.
Rajapakse, Kim, Mendis, and Kim (2007) identified the cellular antiox-
idant effects of carboxylated COS (CCOS) by assessing oxidation inhibition
potential on cellular biomolecules such as lipids, proteins, and direct scav-
enging of ROS. Radical-mediated oxidation of cell membrane lipids and
proteins was dose-dependently inhibited by CCOS, measured by amount
of lipid hydroperoxides and carbonyl content in RAW 264.7 cells. Further,
CCOS inhibited MPO activity in HL-60 cells suggesting indirect possibility
of inhibiting generation of ROS such as O2 radicals, H2O2, and HOCl.
These results concluded that CCOS can act as a potent radical scavenger
in cells.
GA-grafted COS (GCOS) inhibited intracellular free radical-mediated
oxidation. The free radicals-scavenging activity of GCOS was assessed by
using ESR technique. The inhibitory effects of GCOS on ROS, lipid, pro-
tein, and DNA oxidation were determined in RAW 264.7 and human
chondrosarcoma (SW1353) cells. Furthermore, with the treatment of
GCOS, the expression levels of antioxidant enzymes (SOD and GSH) were
significantly increased according to RT-PCR and Western blot analysis. In
addition, GCOS decreased ROS-induced activation of the NF-B. Collec-
tively, these results suggest that GCOS can be used as a potential natural
compound-based antioxidant in the functional food and pharmaceutical
industries (Ngo, Qian, Ngo, Vo, et al., 2011; Ngo, Qian, Vo, Ryu,
et al., 2011).
Ngo, Kim, and Kim (2012) investigated the inhibitory effects of
aminoethyl-COS (AE-COS) on MPO activity in HL-60 and DNA and
protein oxidation in RAW 264.7 cells. Furthermore, free radical-scavenging
effect of AE-COS was determined by 20 ,70 -dichlorofluorescein intensity and

intracellular GSH level in RAW 264.7 cells. In addition, Ngo et al. (2012)
reported the inhibition of ROS, DNA, protein, and lipid oxidation at the
rates of 77%, 80%, 85%, and 85% at 100 g/mL in murine microglial cells
(BV-2), respectively. Therefore, AE-COS possesses potential antioxidant
activity and can be used as a scavenger in controlling free radicals that lead
to damage to cellular system.
Niu et al. (2013) conducted two trials to evaluate the effects of chitin and
its derivatives (chitosan, COS, and N-acetyl-D-glucosamine) on shrimp,
Penaeus monodon, first on growth performance, second on antioxidant
defenses and oxidative stress status of digestive gland. In the first experiment
(trial 1), shrimp (mean initial wet weight: 1.49 g) were fed with five diets
(control diet, chitin diet, chitosan diet, COS diet, and N-acetyl-D-
glucosamine diet) in triplicate for 70 days. Growth performance (final body
wet weight, weight gain, and biomass gain) of shrimp fed chitosan diet was
higher (P < 0.05) than that of shrimp fed the other diets. Survival of shrimp
in chitosan diet groups was higher (P < 0.05) than that of shrimp in control,
COS, and N-acetyl-D-glucosamine diet groups, but no statistical difference
was found in survival of shrimp in chitin and chitosan diet groups. The total
antioxidant status (TAS) and GSH-PX activity of shrimp fed chitin,
chitosan, and COS diets were higher (P < 0.05) than those of shrimp fed
control and N-acetyl-D-glucosamine diets. The SOD activity of shrimp
fed control diet was higher (P < 0.05) than that of shrimp fed other diets.
Digestive gland MDA and carbonyl protein contents of shrimp fed chitin
and chitosan diets were lower than those of shrimp fed other diets
(P < 0.05). A low dissolved oxygen (DO) stress test was conducted for 7 days
after the rearing trial (trial 2). The antioxidant response was characterized by
higher TAS and higher antioxidant enzyme activities (SOD, phenoloxidase,
and GSH-PX) and higher oxidative stress levels (MDA and carbonyl protein
contents) in the digestive gland compared to levels found in trial 1. Survival
of shrimp fed chitin and chitosan diets was higher (P < 0.05) than that of
shrimp fed other diets after trial 2. The GSH content, TAS, phenoloxidase,
and GSH-PX effects of shrimp fed chitosan diet were higher (P < 0.05) than
those of shrimp fed other diets. The SOD activity of shrimp fed chitosan diet
was lower (P < 0.05) than that of shrimp fed control, COS and N-acetyl-D-
glucosamine diets, but no difference (P > 0.05) was found between shrimp
fed chitin and chitosan diets. Moreover, the oxidative stress levels (MDA and
carbonyl protein contents) recorded in the digestive gland with shrimp sub-
mitted to the chitin and chitosan diets were lower. In conclusion, results
suggested that dietary intake containing chitin or chitosan could enhance the
growth performance of P. monodon and improve its resistance to DO stress.
Eom, Senevirathne, and Kim (2012) prepared eight kinds of phenolic
acids including protocatechuic, 4-hydroxybenzoic, vanillic, syringic,
p-coumaric, caffeic, ferulic, and sinapinic acids conjugated COS with differ-
ent substitution groups by amide coupling reaction. Their antioxidant activ-
ities were evaluated using DPPH, OH, and nitric oxide radicals scavenging
and reducing power assays. CA-grafted COS showed 81.6% and 89.8%
scavenging against DPPH and NO radical formation, respectively.
CA-grafted COS also showed higher reducing power and hydroxyl radical
scavenging activity compared to those of other compounds. Hence,
CA-grafted COS can be a potential antioxidant compound.
Matute et al. (2013) obtained low MW chitosan (LMWC) and COS
derivatives by the introduction of lactobionic acid (LA) through amide for-
mation, obtaining different complexes COS-LA and LMWC-LA (15),
with a degree of substitution (DS) between 3% and 16%. The DPPH radical
scavenging activity of these derivatives was evaluated. COS and COS-LA
presented the best DPPH scavenging effects with IC50 values of 1.29 and
3.45 mg/mL, respectively. LMWC-LA5 (3% DS) showed the highest
DPPH scavenging activity, being higher than LMWC in all the concentra-
tions (10.214.3% and 6.913.7%, respectively). Due to their enhanced
functional effects, these derivatives could be considered as very promising
for their future use as additives in the food.
Katiyar, Singh, Lall, and Haldar (2011) evaluated the effects of COS for
the management of alloxan-induced diabetes in mice. COS lowered about
54.10% and 40.5% blood glucose in diabetic mice treated at the dose of
10 and 5 mg/kg body weight, respectively. As hyperlipidemia and oxidative
stress are the disorders of diabetes, therefore, the serum levels of TC, triglyc-
eride, LDLc, very low-density lipoprotein cholesterol, and HDLc were
determined. For the recovery of oxidative stress, the SOD, MDA, and
CAT in liver as well as glutamate oxaloacetate and glutamate pyruvate trans-
aminases activities in serum were measured. The treatment of COS showed
a significant recovery in the levels of above mentioned biosensors of lipid
profile in diabetic mice. In the hyperglycemic mice, COS exhibited signif-
icant antidiabetic activity at a dose level of 10 mg/kg body weight. In addi-
tion, COSs have no toxic effect in normal healthy mice. These results
enlighted that COSs have antidiabetic, antihyperlipidemic, and
antioxidative effects. COSs seem promising for the development of a
new medicine for diabetes mellitus.
Li, Liu, Xing, Qin, and Li (2013) investigated the antioxidant activities
of two partially acetylated chitotrioses (N-acetylchitotriose and N,N0 -
diacetylchitotriose). Free scavenging activity of N,N0 -diacetylchitotriose
and N-acetylchitotriose with the IC50 values was 1.43 and 1.93 mg/mL
(OH radical) and 0.27 and 0.46 mg/mL (O2 radical), respectively. In
addition, the N,N0 -diacetylchitotriose with high degree of acetylation
exhibited the highest antioxidant activity.
Lu, Guo, and Zhang (2012) obtained sulfated COS (SCOS) with differ-
ent DS by the chlorosulfuric acid/pyridine method. Protective effects of
SCOS against H2O2-induced damage were investigated in pancreatic -cells
MIN6 cells. The cell viability, morphology, insulin contents, MDA inhibi-
tion, lactate dehydrogenase (LDH) release, and the levels of antioxidant
enzymes including CAT, SOD, and GSH-PX were evaluated under oxida-
tive stress by 150 M H2O2 for 6 h. SCOS did not show any harmful or
inhibitory effect on cell growth at concentrations ranging from 0.1 to
0.5 mg/mL. SCOS exhibited antioxidant effect to enhance the cell viability,
attenuate the production of ROS, MDA, and LDH levels in oxidative-
induced MIN6 cells. Protective effects of SCOS were partly attributed to
increasing of antioxidant enzyme activities and reduction of intracellular
ROS, along with the capacity of suppressing MIN6 cell apoptosis subse-
quent to the amelioration of ROS. Furthermore, increased DS could con-
tribute to the defense mechanisms against H2O2-induced oxidative stress in
MIN6 cells. These results indicated that SCOS might contribute to benefi-
cial effects in the treatment of diseases related to oxidative stress through
enhancement of antioxidant defense effects, and the sulfate content of poly-
saccharides made great role in regulating antioxidant activities.
4. CONCLUSION
Recent studies have provided evidence that chitin, chitosan, and their
derivatives play a vital role in human health and nutrition. Furthermore,
the formation of cancer cells in human body can be directly induced by free
radicals and natural anticancer drugs as chemopreventive agents have gained a
positive popularity in treatment of cancer. Hence, antioxidant effect of chitin,
chitosan, and their derivatives can be used indirectly as functional ingredients
to reduce cancer formation in human body. Collectively, the wide range of
biological activities associated with the antioxidant activity of chitin, chitosan,
and their derivatives has the potential to expand their health beneficial value
not only in the functional food but also in the pharmaceutical industry.
ACKNOWLEDGMENTS
This chapter was supported by a grant from Marine Bioprocess Research Center of the
Marine Biotechnology Program funded by the Ministry of Oceans and Fisheries,
Republic of Korea.
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antioxidant activity of the sulfanilamide derivatives of chitosan and chitosan sulfates.
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hydroxylbenzenesulfonailides derivatives of chitosan, chitosan sulfates and carbo-
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by selected chitosan preparations and their physicochemical properties. LWT Food Science
and Technology, 39, 10871092.
CHAPTER THREE
Antidiabetic Activities of Chitosan

and Its Derivatives: A Mini Review
Fatih Karadeniz, Se-Kwon Kim1
1
Contents
1. Introduction 33
2. Derivatization 35
3. Antidiabetics and Antiobesity Applications 36
3.1 Indirect activity 36
3.2 Direct activity 38
4. Conclusion 41
References 41
Abstract
Obesity and diabetes are two important closely related matters to world health with
increasing morbidity and mortality rate. Many recent studies promoted chitosan-based
substances as lead molecules for treatment and prevention of obesity, diabetes, and
related complications due to their easy and potential utilization in the food, pharmaceu-
tical, agricultural, and environmental fields. Although detailed action mechanism and
how chitosan-based molecules act as antidiabetics and antiobesity specifically are
remain to be enlightened, studies exhibited enough evidence to direct our intention
to produce natural therapeutic agents using chitosan and its derivatives as lead sub-
stances. In this chapter, some reported antidiabetics and antiobesity applications of
chitosan and its derivatives have been briefly summarized in regard to acting pathways
and structure-based activity in order to obtain some valuable insights into novel
chitosan-based derivatives and their utilization for antidiabetic and antiobesity
purposes.
1. INTRODUCTION
Chitin is one of the most abundant carbohydrates worldwide and can
be found in varying sources including bacterial cell walls and crustacean
shells. As a derivative of chitin, chitosan is a functional and linear
#
http://dx.doi.org/10.1016/B978-0-12-800268-1.00003-2
34 Fatih Karadeniz and Se-Kwon Kim
carbohydrate prepared by N-deacetylation of chitin in the presence of alka-

line. Generally, deacetylation cannot be completely achieved even under
harsh treatment. The degree of deacetylation usually ranges from 70% to
95%, depending on the method used. Thus, chitosan is available with var-
ious molecular weights and deacetylation degrees. Chitosan is insoluble in
water, alkali, and organic solvents but is soluble in most solutions of organic
acids when the pH of the solution is below 6. The industrial production and
application fields of chitosan have been steadily increasing since 1970s. Early
applications of chitosan were centered on the treatment of wastewater, heavy
metal adsorption, food processing, immobilization of cells and enzymes, resin
for chromatography, functional membrane in biotechnology, animal feed,
and so on. The recent trend is toward producing high-valuable industrial
products such as cosmetics, drug carriers, and pharmaceuticals. Chitin and
chitosan are known to exhibit antitumor, antibacterial, hypocholesterolemic,
and antihypertensive activity (Kim & Rajapakse, 2005). The main motive for
the development of new applications for chitosan lies in the fact that it is a very
abundant polysaccharide, as well as nontoxic and biodegradable. Despite its
functions and importance as a biomaterial, the applications of chitosan in food
and biomedical industries are narrowed owing to its poor solubility, high
molecular weight, and viscosity. There are evidences about the non- or poor
absorption of chitin and chitosan in human intestine due to lack of enzymes to
cleave the -glucosidic linkage in chitosan. Since chitosan is a water-insoluble
large biopolymer, it is difficult to be absorbed by human body. In this respect,
enzymatic hydrolysis of chitosan to obtain oligomers is of great interest
recently ( Jeon & Kim, 2000).
Chitosan oligosaccharides (COS) are hydrolyzed derivatives of chitosan
composed of -(1 ! 4) D-glucosamine units. They have better properties
such as lower viscosity, relatively smaller molecular size in comparison to
chitosan, and short-chain length with free amino groups which makes
COS highly soluble in aqueous solutions. COS are effective agents for low-
ering of blood cholesterol and pressure, controlling arthritis, and enhancing
antitumor properties (Kim & Rajapakse, 2005). Since COS are biodegrad-
able, water-soluble, and nontoxic compounds (Qin et al., 2006), they might
be beneficial biomaterials for diseases such as diabetes and obesity with
increasing morbidity and mortality rates.
As a chronic disease, diabetes that occurs when beta-islet cells (pancreas)
do not secrete enough insulin or the body cannot use insulin efficiently must
be kept under control. Diabetic disorders, especially hyperglycemia can lead
to serious damage to many parts of the bodys especially the nerves and blood
Antidiabetic Activities of Chitosan and Its Derivatives 35
vessels (Vinik, Maser, Mitchell, & Freeman, 2003). The cause of diabetes is
not fully known, although it is clearly shown that both genetic and environ-
mental factors, especially obesity, appear to play important roles. Differen-
tiated adipocytes secrete obesity-related factors called adipokines. Plasma
leptin, tumor necrosis factor , and nonesterified fatty acid levels are all ele-
vated in obesity and play a role in causing insulin resistance (Leong &
Wilding, 1999). Therefore, suppression and regulation of obesity can be
achieved by inhibiting adipocyte differentiation and forcing adipocytes to
lipolysis to reduce accumulated white adipose tissue (Langin, 2006;
Yamauchi et al., 2001). Thus, the increased control of the harmful effects
of the accumulation of adipose tissue and its metabolism contribute to the
search for a better understanding of the prevention of diabetes.
With a long onset and serious complications, which usually result in high
morbidity rate, the treatment of diabetes is a major concern in all countries.
Up to now, many kinds of antidiabetic medicines from natural resources
have been developed for patients (Grover, Yadav, & Vats, 2002; Ivorra,
Paya, & Villar, 1989; Koski, 2004; Li, Zheng, Bukuru, & De Kimpe,
2004), but most of these biochemical agents are not suited for mass produc-
tion to meet to be a pharmaceutical agent. The natural compounds demon-
strated a significant practice and show a bright potential in the treatment of
diabetes and its complications with their naturally occurring structure and
relatively less side-effects. In this respect, chitin, chitosan, and its derivatives
with available large numbers of different chemical structures and bioactiv-
ities offer a great potential to recover and/or preventing obesity and diabetes.
2. DERIVATIZATION
Chitosan and its monomer glucosamine are highly derivatized recently
in order to find new natural compounds with higher bioactivity than their
predecessors (Fenton et al., 2000; Jiang et al., 2007; Prabaharan, 2008). Main
derivatization of chitosan is forming soluble forms of chitin which makes it
more biofriendly and easily absorbed by body after the oral administration
(Hai, Bang Diep, Nagasawa, Yoshii, & Kume, 2003; Ilina & Varlamov,
2004; Kuroiwa, Ichikawa, Hiruta, Sato, & Mukataka, 2002). Since, effective-
ness of the compound, based on its absorption rate by body, this kind of deriv-
atization is opened up new angles for chitin derivatization toward new
bioactive compounds. In this respect, chitosan is the main derivative of chitin.
Rather than the chitosan, COS are also highly bioactive derivatives of chitosan
with significantly high absorption rates and water solubility.
Beside oligomerization, other main derivatization for chitin and its

monomer glucosamine is adding negative and/or positively charged side
chains. In this manner, glucosamine, chitin, chitosan, and COS reformed
under chemical conditions to give sulfated, phosphorylated, car-
boxymethylated, deoxymethylated derivatives and so on (Cho, Lee,
Kim, Ahn, & Je, 2011; Huang, Mendis, & Kim, 2005; Je & Kim,
2006; Kim, Kong, Pyun, & Kim, 2010; Kim et al., 2005; Kochkina &
Chirkov, 2000). This high variety of derivatives comes with high variety
of bioactivities, which changes in the effectiveness of compound in same
bioactivity.
3. ANTIDIABETICS AND ANTIOBESITY APPLICATIONS

Chitosan-based products are known to have many biological activities
such as antitumor, anti-HIV, antifungal, antibiotic, and against oxidative
stress (Artan, Karadeniz, Karagozlu, Kim, & Kim, 2010; Kendra &
Hadwiger, 1984; Kim et al., 2008; Nishimura et al., 1998; Xie, Xu, &
Liu, 2001). Activities can be grouped in two according to use of chitin-based
products. These products are highly used as indirect helping agents to
enhance the effectiveness of other active compounds through chemical
modification or nonchemical linkage against diabetes and obesity. On the
other hand, the main role of chitin-based products, known as therapeutic
nutraceutical agents, is to act directly against diabetes and obesity. In both
cases, these natural product derivatives express high and significant potential
in the light of searching bioactive pharmaceuticals against obesity and
obesity-related diabetes.
3.1. Indirect activity

The preferred route of drug administration for patients is mostly the oral
route on chronic therapy of diseases and complications. However, the oral
delivery of many therapeutic peptides and proteins is still an unsolved prob-
lem basically because of the size, hydrophilicity, and unstable conditions of
these molecules. Thus, several chitosan derivatives have been developed
over the years with improved properties for enhanced applicability
(Fernandez-Urrusuno, Calvo, Remunan-Lopez, Vila-Jato, & Alonso,
1999; Thanou, Verhoef, & Junginger, 2001). Therefore, recent studies
focused on carrier products for administration of insulin efficiently in pre-
or postdiabetic patients and lately one of these products is chitosan deriva-
tives. It has been reported by Portero, Teijeiro-Osorio, Alonso, and
Remunan-Lopez (2007) that chitosan sponges are quite successful for buccal
administration of insulin. Moreover, up-to-date studies presumed that
chitosan-derived particles are highly usable for insulin administration by
orally with their high protective effect and harmless structure (Hari,
Chandy, & Sharma, 1996; Krauland, Guggi, & Bernkop-Schnurch, 2004,
2006). Results of some related studies have suggested that, the observed drug
delivery activity of chitosan is highly promising in the case of insulin. For
example, studies showed chitosaninsulin nanoparticles have strong affinity
to rat intestinal epithelium after 3 h of postoral administration (Ma, Lim, &
Lim, 2005). This suggests that chitosan as a cofactor for drug delivery makes
insulin absorption safe and rapid. Carboxymethyl-hexanoyl chitosan is an
amphiphilic chitosan derivative with important swelling ability and water
solubility under natural conditions and studies showed these hydrogels
can be used for encapsulating the poorly water-soluble drugs for efficient
drug delivery (Liu & Lin, 2010) which lightens up the way for efficient insu-
lin delivery by chitosan derivatives. Also, Mao et al. (2005) showed that
PEGtrimethyl chitosan complexes are efficiently coupled with insulin
and easily taken up by Caco-2 cells.
Beside drug delivery activity for insulin, studies have shown that chitosan
complexes can be efficiently used for gene delivery for gene therapy
(Koping-Hoggard et al., 2001). Therefore, it can be easily adduced that
chitosan complex derivatives are potent gene delivery targets for high prev-
alent diseases such as diabetes. Furthermore, it has been reported that this
chitosan complexes have relatively higher uptake and transfection efficiency
than that of other polysaccharide complexes used for both drug and gene
delivery (Huang, Khor, & Lim, 2004). Several researches prove chitosan
as a nontoxic alternative to other cationic polymers and it gives a high poten-
tial for further studies of chitosan-based gene delivery systems (Sato, Ishii, &
Okahata, 2001). All these results suggest that chitosan and chitosan-based
derivatives are light upon the search of a nontoxic agent for drug and gene
delivery, which is highly crucial for diabetic patients improved life
standards.
Moreover, studies on streptozotocin (STZ)-induced diabetic rats
expressed, beside treatment of diabetic patients, that chitosan-based sponges
are highly effective against diabetic wounds. Wang et al. (2008) suggests that
application of chitosancollagen complex is an ideal wound-healing cover to
enhance recovery of healing of wounds such as diabetic skin wound, which
provides a great potential for chitosan and its derivatives to be used clinically
for diabetic patients.
To conclude, chitosan-based polymers show great potential for treat-

ment of diabetes therapeutically with their high efficient drug and gene
delivery properties as well as effectiveness on diabetic wound healing.
3.2. Direct activity

Overweight and obesity are common health conditions worldwide that
result in diabetes, but there are not so common treatments. Therefore, stud-
ies of chitosan are focused on its fat-lowering and fat-preventing activities.
Several researchers have demonstrated that chitosan tends to bond with the
ingested dietary fat and carry it out in the stool while preventing their
absorption through the gut (Kanauchi, Deuchi, Imasato, Shizukuishi, &
Kobayashi, 1995; Maezaki et al., 1993). Related researches about fat-
lowering activity of chitosan also have shown that chitosan is capable of
absorbing fat up to five times of its weight. In respect of these results, there
are several studies showing chitosan derivatives lower the level of LDL while
increasing the HDL levels. Studies of chitosan and its fat-lowering activity
have expressed that chitosan and its derivatives as highly effective
hypocholesterolemic agents with the ability of decreasing blood cholesterol
level up to as much as 50% ( Jameela, Misra, & Jayakrishnan, 1994). More-
over, diabetic patient-based studies clearly showed that daily administration
of chitosan could drop the blood cholesterol levels by 6% with an increased
level of HDL. In addition to chitosan, COS, oligomerized derivatives of
chitosan, show high activity in regulating blood cholesterol levels. Espe-
cially, studies reported that COS are capable of regulating cholesterol levels
even in liver. COS prevent development of fatty liver caused by the action of
hepatotrope poisons. Despite few studies were carried out for the activities
of COS in regulating blood cholesterol level mechanism, several of them
suggested possible mechanism of COS lowering the LDL levels. As
Remunan-Lopez, Portero, Vila-Jato, and Alonso (1998) suggested, ionic
structure of COS binds bile salts and acids which inhibit lipid digestion
through micelle formation. However, Tanaka et al. (1997) suggest a differ-
ent mechanism of chitosan and COS where lipids and fatty acids are directly
bonded by chitosan.
In addition to fat-lowering mechanisms of chitosan and its derivatives,
studies have also proved that chitosan administration can be lead to increase
the insulin sensitivity in animal models (Neyrinck et al., 2009). It has been
shown that 3-month administration of chitosan significantly increased insu-
lin sensitivity in obese patients and expressed a highly notable decrease in
body weight and triglyceride levels (Hernandez-Gonzalez, Gonzalez-Ortiz,

Martinez-Abundis, & Robles-Cervantes, 2010).
On the other hand, glucosamine and its derivatives are reported to be
highly effective against adipogenesis in vitro. Recent studies showed that
phosphorylated derivative of glucosamine inhibited the adipogenesis of
3T3-L1 cells as well as fat accumulation. Several researches suggested that
acetylated chitin treatment causes adipocytes to break down fats and lower
their fat accumulation as much as half of control cells (Kong, Kim, Bak,
Byun, & Kim, 2011). Kong, Kim, and Kim (2009) demonstrated clearly that
sulfated derivative of glucosamine inhibited the proliferation and
adipogenesis mechanism through AMPK pathways in 3T3-L1 cells. Glucos-
amine, acetylated-, sulfated-, and phosphorylated-glucosamine derivates are
reported as successful adipogenic inhibitors with high potential to prevent
weight gain by adipogenesis in high diabetes-risk patients. As well as, it
has been reported that COS inhibit the fat accumulation and adipogenesis
in 3T3-L1 cell line (Cho et al., 2008). In addition, studies have shown that
treatment with glucosamines reduced the triglyceride content of adipocytes
and enhanced glycerol secretion as a lipid lowering effect. Most of these
studies have expressed the better activity of chitosan-based compounds such
as COS and glucosamines, after derivatization by adding a charged side chain
by phosphorylation and sulfation. Therefore, it can be suggested that cat-
ionic power of glucosamine and COS plays the main role in their antiobesity
effect. Further, a selective synthesis of phosphorylated or sulfated derivatives
of chitosan and glucosamine will open up the way to a better understanding
behind the structuremechanism relation. However, up-to-date researches
have strong proofs that antiobesity effect of chitosans effect through
PPAR- pathway of adipogenic differentiation results in less adipocytes as
adipose tissue and lipid accumulation. Collectively, chitosan and its deriva-
tives such as glucosamines and COS successfully inhibit the differentiation of
cells into adipocytes as well as enhancing adipocytes to hydrolyze the triglyc-
erides which shows a significant effect against lipid accumulation in the
body. This effect of chitosan and its derivatives demonstrates an important
effect against obesity in the way leading to diabetes and shows great amount
of potential to be used as pharmaceutical agents.
Additionally, chitosan and its oligosaccharides act as antidiabetic agents
for treatment of diabetes in manner of protecting pancreatic beta-cells. In
type 2 diabetes, although patients can retain healthy pancreatic -cells for
many years after disease onset, chronic exposure to high glucose will impair
-cell function in later stages. Impaired -cell functionality leads to cellular
damage in type 2 diabetic patients (Ihara et al., 1999). Therefore, the pro-
tection of beta-cells is of high importance for quality life of diabetic patients
and elevated insulin secretion. Recent studies reported that COS as a pro-
tective agent for pancreatic beta-cells against high glucose-dependent cell
deterioration (Karadeniz, Artan, Kong, & Kim, 2010). It is suggested that
at the same time COS could effectively accelerate the proliferation of pan-
creatic islet cells with elevated insulin secretion in the aid of lowering blood
glucose levels. Liu, Liu, Han, and Sun (2007) reported that COS treatment
could improve the general situation and diabetic symptoms of diabetic rats,
decrease the blood glucose levels, and normalize the impaired insulin sen-
sitivity. Moreover, COS were reported as preventive agent in nonobese dia-
betic mice from developing type 1 diabetes, which might be related to
several bioactivities of COS. These results supported the hypothesis that
COS can prevent pancreatic beta-cells of diabetic patients and normalize
the crucial insulin secretion. The mechanism behind this protection is stud-
ied and suggested as related to immunopotentiation and antioxidation activ-
ity of COS.
Renal failure is one of the most common diseases caused by diabetes
mellitus. The metal crosslinked complex of chitosan, chitosaniron(III),
have been recently reported to be highly active for reducing phosphorus
serum levels to treat chronic renal failure (Schoninger, DallOglio, Sandri,
Rodrigues, & Burger, 2010). This relatively new derivative of chitosan is
significantly capable of adsorbing serum phosphorus in alloxan diabetes-
induced rats with symptoms of renal failure progression.
Moreover, recent studies indicate that diabetics may be at higher risk for
blood coagulation than nondiabetics. This life-threatening condition urges
to be treated for diabetic patients. Therefore, sulfated derivative of chitosan
has been shown to possess anticoagulant potency (Vongchan, Sajomsang,
Subyen, & Kongtawelert, 2002). Furthermore, studies have reported that
sulfated chitosan does not show antiplatelet activity unlike heparin which
is an efficient anticoagulant agent. Collectively, results proved that sulfated
chitosan is a more efficient agent than that of heparin, although heparin has
been used for a long time for blood coagulation treatment.
In addition to COS, chitosan also reported to prevent the progression
and symptoms as well as the complications of low-dose STZ-induced slowly
progressive non-insulin-dependent diabetes in rats (Kondo, Nakatani,
Hayashi, & Ito, 2000). Briefly, reports suggest that chitosan-based products
protect pancreatic cells and insulin secretion mechanism in diabetic condi-
tions. Furthermore, these compounds could decrease the progression and
complication rate of diabetes onset in animal models, given that chitosan-

based products have a great potential to be used as a nutraceutical for the
treatment of diabetics.
4. CONCLUSION
High mortality and morbidity of diabetes make diagnosis, preventing,
and treatment more important as more and more patients are diagnosed by
diabetes in the world in recent years. Beside diabetes, factors relating to dia-
betes such as obesity and damaged pancreatic cells must be kept under con-
trol in order to prevent diabetes onset. In this manner, chitosan and its
derivatives possess various biological activities and have a remarkable poten-
tial to be used in several of therapeutic applications. Thus, many of the stud-
ies carried out to search antidiabetic activities of chitosan-based compounds
provide detailed acting mechanisms and activity for prevention and/or treat-
ment of diabetes-based complications. Chitosan and its derivatives such as
COS and glucosamines as monomers express high activity in manner of low-
ering lipid accumulation and cholesterol as well as pancreatic beta-cell pre-
vention. In addition, studies proved that chemical modification of these
compounds could express better activity and understanding of mechanism
lying behind antidiabetic effects. Therefore, future researches should be
directed to enhance the effectiveness of chitosan-based compounds in order
to gain more active and less harmful agents. Collectively in conclusion, these
evidences suggest that chitosan-based agents are highly potent nutraceuticals
for treatment and prevention of diabetes and diabetes-related complications.
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CHAPTER FOUR
Role of Alginate in Bone Tissue

Engineering
Jayachandran Venkatesan*,1, R. Nithya, Prasad N. Sudha,
Se-Kwon Kim*
*Department of Marine-bio Convergence Science and Marine Bioprocess Research Center, Pukyong National
University, Busan, South Korea
Department of Chemistry, D.K.M. College for Women, Thiruvalluvar University, Vellore, Tamil Nadu, India
1
Corresponding author: e-mail address: venkatjchem@pknu.ac.kr
Contents
1. Introduction 46
2. Alginate General Properties 46
2.1 Structure 46
2.2 Molecular weight and solubility 47
2.3 Biocompatibility 48
3. Tissue Engineering 48
3.1 Bone TE 48
4. Alginate in Bone TE 49
4.1 Alginate scaffolds in bone TE 49
4.2 Alginate hydrogels in bone TE 52
5. Future Prospects 53
6. Conclusion 54
Acknowledgments 54
References 54
Abstract
Bone, a typical inorganicorganic biocomposite, is made of approximately 70 wt% inor-
ganic components, mainly hydroxyapatite (HAp,Ca10(PO4)6(OH)2), and 30 wt% of organic
matrix, mainly collagen I. Human organ failure caused by defects, injuries, or other types
of damage is one of the most devastating and costly problems in human health care.
Recently, tissue engineering has emerged as a promising approach for bone repair
and reconstruction. The ultimate goal of bone tissue engineering is the fabrication of
a construct that matches the physical and biological properties of the natural bone tissue.
Biopolymers have some distinct advantages such as their biodegradation rates and
mechanical properties can be tailored to a certain extent for specific applications. Algi-
nate, a natural polysaccharide, is readily processable for applicable three-dimensional
scaffolding materials such as hydrogels, microspheres, microcapsules, sponges, foams,
and fibers. Alginate can be easily modified via chemical and physical reactions to obtain
derivatives having various structures, properties, functions, and applications. The purpose
of this chapter is to review recent research on alginate in bone tissue engineering.
#
http://dx.doi.org/10.1016/B978-0-12-800268-1.00004-4
46 Jayachandran Venkatesan et al.
1. INTRODUCTION
Alginate is the most abundant marine biopolymer and, next to cel-
lulose, the most abundant biopolymer in the world. Alginates comprise a
rather broad family of polysaccharides found in brown seaweeds (Laminaria
sp., Macrocystis sp., Lessonia sp., and others), from which they are produced
industrially. The annual production is estimated to approximately 38,000 t
worldwide (Helgerud, Gaserd, Fjreide, Andresen, & Larsen, 2009). The
major source of alginate is found in the cell walls and in the intracellular
spaces of brown seaweed. The alginate molecules provide the plant with
both flexibility and strength, which are necessary for plant growth in the
sea. The first scientific studies on the extraction of alginates from brown
seaweed were made by the British chemist E.C. Stanford who found that
the extracted substance, which he named algin, possessed several interesting
properties. These included the ability to thicken solutions, to make gels,
and to form films. From these, he proposed several industrial applications.
Alginates are also synthesized by some bacteria (e.g., Azotobacter and
Pseudomonas species). Sodium alginate is the main form of alginate used
(McHugh, 2003). Other types of alginate include alginic acid, calcium,
ammonium and potassium salts, and propylene glycol alginate, an ester
of alginic acid.
2. ALGINATE GENERAL PROPERTIES

2.1. Structure
Alginates are linear unbranched polymers containing -(1 ! 4)-linked
D-mannuronic acid (M) and -(1 ! 4)-linked L-guluronic acid (G) residues.
The blocks are composed of consecutive G residues (GGGGGG), consec-
utive M residues (MMMMMM), and alternating M and G residues
(GMGMGM). Only the G-blocks of alginate are believed to participate
in intermolecular cross-linking with divalent cations (e.g., Ca2+) to form
hydrogels. The composition (i.e., M/G ratio), sequence, G-block length,
and molecular weight are thus critical factors affecting the physical properties
of alginate and its resultant hydrogels (George & Abraham, 2006). The abil-
ity of alginates to form soft hydrogels in the presence of calcium ions forms
the basis for a wide variety of industrial applications, including foods and
pharmaceuticals. The structure of sodium alginate is given next.
Role of Alginate in Bone Tissue Engineering 47
COONa COONa
COONa
OH
O O OH O
HO O OH
OH O
O HO O O
OH
O OH
COONa OH
COONa
OH O
G-C4 G-C4 M-C1 M-C1 G

Alpha(1,4) Alpha(1,4) Beta-1,4 Beta-1,4
Hydrogels are three dimensionally cross-linked networks composed of

hydrophilic polymers with high water content. Hydrogels are often bio-
compatible, as they are structurally similar to the macromolecular-based
components in the body and can often be delivered into the body via min-
imally invasive administration (Sakiyama-Elbert & Hubbell, 2001). In recent
years, research on alginates has largely shifted toward biomedical
applications.
As biomaterials, alginates can easily be formulated into a variety of soft,
elastic gels, fibers, foams, nanoparticles, and multilayers at physiological con-
ditions ensuring the preservation of cell viability and function. Alginate is a
natural biopolymer, which allows efficient cell penetration into matrix and
cell encapsulation. It has been chosen to use this material for two of these
reasons. First, the encapsulation property is very interesting as it is very
difficult to seed the cells homogeneously, using this material should be a
way to ensure that the cells are randomly distributed in the scaffold. The sec-
ond reason is its low price and availability.
2.2. Molecular weight and solubility

The molecular weights of commercially available sodium alginates range
between 32,000 and 4,00,000 g/mol. Sodium alginates is slowly soluble
in cold water, forming viscous, colloidal solution. It is insoluble in alcohol
and hydroalcoholic solutions in which alcohol content is greater than 30%
by weight. It is also insoluble in other organic solvents such as chloroform
and ether and in acids where the pH of the resulting solution falls below 3.0.
Calcium alginate is, however, practically insoluble in water and organic
solvents but soluble in sodium citrate (Shilpa, Agrawal, & Ray, 2003).
2.3. Biocompatibility
Although the biocompatibility of alginate has been extensively evaluated
in vitro as well as in vivo, there is still debate regarding the impact of the algi-
nate composition. Much of this confusion likely relates to varying levels of
purity in the alginate studied in various reports. As long as the alginate is
purified, it is biocompatible (De Vos, De Haan, & Van Schilfgaarde,
1997; Klock et al., 1994; Kl ock et al., 1997). Yet, the alginate with higher
guluronate (G) content produced gels that were not biocompatible. This
confirms that the differences between the M and G residues play some role
in determining biocompatibility, probably via their influence on the physical
properties (stiffness, swellability) of the resulting gels. The following two
factors were more important for biocompatibility: M/G content and molar
mass (Gomez, Perez Lambrecht, Lozano, Rinaudo, & Villar, 2009;
Zimmermann et al., 2001).
3. TISSUE ENGINEERING
Human tissue and organ failure caused by defects, injuries, or other
types of damage is one of the most devastating and costly problem in human
health care. The transferred tissue does not present all the functions of the
native tissue and can involve donor site complication. For these reasons,
stem cells, tissue engineering (TE), and organogenesis have become prom-
ising and important fields of research, which may offer not only tissues and
organs for transplantation but also open new perspectives for treatment of
diseases; TE aims at developing functional substitutes for damaged tissues
and organs (Dvir, Timko, Kohane, & Langer, 2011). The goal of TE is
to surpass the limitations of conventional treatments based on organ trans-
plantation and biomaterial implantation (Langer & Vacanti, 1993). A general
concept in TE is to combine a scaffold or matrix with viable cells to form a
cellbiomaterial construct to be used in promoting the repair and regener-
ation of tissues.
3.1. Bone TE
Bone is a living tissue, which continuously rebuilds its structure. Trauma or
diseases may cause bone defects and bone substitute materials are used for
repair and reconstruction. Conventional treatment approaches involve bone
grafting, where bone from the patient (autograft) at another site (e.g., hips or
ribs) are removed or bone from a human donor (allograft) are used to fill the
defect (Bongio, Van den Beucken, Leeuwenburgh, & Jansen, 2010). The
ultimate goal of bone TE is the fabrication of a construct that matches
the physical and biological properties of the natural bone tissue, without
the drawbacks imposed by autologous or allogenic bone grafts. Bone TE
applications include regeneration of bone after tissue loss due to degenera-
tive, surgical, or traumatic processes, as well as applications such as spinal
arthrodesis. In addition, there is the desire to speed the healing of bone frac-
tures and to treat established nonunion fractures (Hutmacher, 2000). Bone
TE offers new therapeutic strategies to aid musculoskeletal healing, through
the combination of scaffold structures with cells and bioactive molecules.
4. ALGINATE IN BONE TE
4.1. Alginate scaffolds in bone TE
Scaffolds for bone TE must have a highly porous and interconnected pore
structure to ensure a biological environment conducive to cell attachment
and proliferation as well as tissue growth, in addition to providing the pas-
sage of nutrient flow. Scaffolds for bone TE must have enough mechanical
strength to support bone tissue regeneration at the site of implantation and
maintain sufficient integrity during both in vitro and in vivo cell growth (Chu,
Orton, Hollister, Feinberg, & Halloran, 2002; Porter et al., 2000; Thomson,
Yaszemski, Powers, & Mikos, 1995).
The alginate/hydroxyapatite composite scaffolds were prepared by
internal gelation followed by a freeze-drying procedure to obtain a porous
structure. The nanoparticles were prepared in presence of a lactose-modified
chitosan, and this colloidal solution was adsorbed on the scaffolds by exploi-
ting electrostatic interactions and is used as temporary resorbable bone
implants (Marsich et al., 2013). Chitosan/polypyrrole/alginate composite
scaffold can act as a substrate for tissue regeneration, and this can be
employed for bone TE using osteogenic cells by utilizing electrical
stimulation with a bioreactor system and thereby evaluate the role of
conducting substrate in bone regeneration (Sajesh, Jayakumar, Nair, &
Chennazhi, 2013).
Chitosan/alginate hybrid scaffolds displayed improved mechanical
strength and structural stability and were shown to stimulate new bone for-
mation and rapid vascularization (Li, Ramay, Hauch, Xiao, & Zhang, 2005).
Chitosanalginate gel/MSCs (mesenchymal stem cells)/bone morphoge-
netic protein-2 (BMP-2) composites should have become clinically useful
injectable materials to generate new bone (Park et al., 2005). Porous
hydroxyapatite/chitosanalginate composite scaffolds were prepared

through in situ coprecipitation and freeze drying for bone TE ( Jin et al.,
2012). Two different types of polymer scaffolds, such as chitosanalginate
and chitosanalginate with fucoidan, were developed by a freeze-drying
method, and each was characterized as a bone graft substitute by
Venkatesan, Bhatnagar, and Kim (2014).
Stem cell-scaffold approaches hold immense promise for bone TE. An
injectable and mechanically strong stem cell construct, using calcium phos-
phate cement (CPC) paste containing small alginate hydrogel microbeads
was prepared. CPC-containing hUCMSCs (human umbilical cord mesen-
chymal stem cells) encapsulating microbeads are fully injectable and
mechanically strong. hUCMSCs in microbead-CPC constructs can remain
viable and used to synthesize bone minerals (Zhao, Weir, & Xu, 2010). The
controlled biodegradation and enhanced apatite deposition of CS/Alg/
nSiO2 scaffolds by nanosilica by freeze drying would definitely promote
bone formation that is essential in bone TE (Sowjanyaa et al., 2013).
Alginate microparticle and microfiber aggregated scaffolds were pro-
duced, with alginate through aggregation method. Such porous structure
will allow vascularization, oxygenation and cell migration, adhesion and
proliferation, which are biological events that are fundamental for bone tis-
sue regeneration (Valente et al., 2012). Man et al. (2012) hypothesized that a
mixture of platelet-rich plasma (PRP) and adipose-derived stem cells
(ADSCs) could endue the alginate microspheres with osteogenic and angio-
genic potential. PRP sustained cell viability and meanwhile promoted cell
migration from the interior of alginate microspheres to the surface. This
phenomenon indicated that encapsulated cells have the potential to directly
and actively participate into the regeneration process. It also suggested that
the PRP-ADSC-laden microspheres enhance the vascularization and min-
eralization, and this strategy provides a microinvasive therapy for bone
regeneration. Coimmobilization of osteogenic and endothelial cells within
Arginine-glycine-aspartic acid (RGD)-alginate microspheres is a promising
new injectable strategy for bone TE (Grellier et al., 2009).
The in vivo application of a facile polyelectrolyte complexation (PEC)
process employed to condense heparin onto the surfaces of poly-L-ornithine
(PLO), poly-L-arginine (PLA), and DEAE-dextran coated alginate micro-
bead templates that entrap bioactive recombinant human BMP-2
(rhBMP-2). In vivo implantation of PEC shells loaded with rhBMP-2
resulted in new bone formation that could stimulate a mechanically stable
posterolateral spinal fusion in rats (PLO- and PLA-based PEC shells).
This ability to retain/regulate rhBMP-2 delivery and stimulate new bone

formation from heparin incorporated PEC shells could provide a powerful
tool for entrapping and controlling the delivery of several growth factors in a
variety of bone TE (Abbah, Liu, Lam, Goh, & Wong, 2012).
An injectable and biodegradable scaffold based on oxidized alginate
microbeads encapsulating periodontal ligaments (PDLSCs) and gingival
mesenchymal stem cells (GMSCs) was developed by Moshaverinia et al.
(2012). The findings demonstrate that immobilization of PDLSCs and
GMSCs in the alginate microspheres provides a promising strategy for
bone TE.
CPC paste combined with AC (alginatechitosan) microcapsules encap-
sulating MC3T3-E1 cells was studied in vivo by Qiao, Xie, Li, and Xu
(2013). Alginatechitosan microcapsules are better than alginate ones for
seeding cells deep into injectable CPCs, and the novel injectable CPC-
AC-cell construct is promising for bone TE applications. The preparation
of novel porous scaffolds of CPC combined with alginate is used as a
three-dimensional (3D) matrix for drug delivery and TE of bone (Lee,
Park, Shin, & Kim, 2011). 3D-plotted MBG (mesoporous bioactive glass)
incorporated alginate scaffolds with well-ordered nano-pores, controllable
large pores, and significantly improved physicochemical, biological, and
drug-delivery properties is a platform for bone TE (Luo, Wu, Lode, &
Gelinsky, 2013).
Investigations into synthetic and natural inorganic ceramic materials
(e.g., hydroxyapatite and tricalcium phosphate) as candidate scaffold material
have been aimed mostly at bone TE (Burg, Porter, & Kellam, 2000). A high
porosity composite scaffold composing of alginate, chitosan, collagen, and
hydroxyapatite without chemical cross-linking agent was fabricated by
Yu et al. (2013) and this composite mat was used as a scaffold for reg-
enerating bone tissue. A novel herbal scaffold (Alg/O-CMC/Cissus
quadrangularis extract (CQ-E) scaffold) was fabricated by incorporating
medicinal plant CQ-E with natural biopolymers alginate (Alg) and
O-carboxymethyl chitosan (O-CMC) by lyophilization technique. The pri-
mary investigation of physicochemical and biological properties of the
herbal scaffolds suggests that this osteoinductive scaffold could serve as a
potential candidate for bone TE therapeutics (Soumya, Sajesh, Jayakumar,
Nair, & Chennazhi, 2012).
A major challenge for orthopedic surgeons is the reconstruction of lost
bone. Sodium alginate/gelatine scaffold is a promising and efficient bioma-
terial for bone formation and healing, and the scaffold-osteogenic cell
construct is a promising alternative approach for bone regeneration (Xia

et al., 2012). A mixture of chitosanalginate gel, MSCs, and BMP-2
composites has the potential to become a successful injectable material for
inducing new bone formation (Park et al., 2005). A new composite
composed of solid free-form fabricated polycaprolactone, BMP-2, or bone
formation peptide, and alginate is used for bone tissue regeneration (Kim,
Jung, & Kim, 2013).
4.2. Alginate hydrogels in bone TE

The treatment of challenging fractures and large osseous defects presents a
formidable problem for orthopedic surgeons. A hybrid growth factor deliv-
ery system for bone repair that utilizes an injectable alginate hydrogel for
protein delivery and an electrospun nanofiber mesh for guiding bone regen-
eration in the case of fracture nonunions and large bone defects was devel-
oped by Kolambkar et al. (2011). Skeletal muscle satellite cells induced by
PRP encapsulated in alginate gel could produce tissue-engineered bone
in vivo, and this indicates a good potential of PRP enhanced TE in treatment
of bone injury (Huang, Jia, Liu, Fang, & Zhang, 2012).
Comparison of the structural and physicochemical properties of different
concentrations of alginate and high-molecular weight hyaluronic acid (HA)
hydrogels and their biocompatibility and bioactivity after long-term culture
with MC3T3-E1 cells was investigated by Rubert, Alonso-Sande, Monjo,
and Ramis (2012). Unmodified alginate hydrogels supported osteoblast dif-
ferentiation better than HA hydrogels, suggesting that alginates are more
suitable for biomaterial applications in bone TE.
The level of formation of new bone and vascularization in bone TE scaf-
fold implants is considered as a critical factor for clinical application. An
approach using an RGD-grafted oxidized sodium alginate/N-succinyl
chitosan (RGDOSA/NSC) hydrogel as a scaffold and low-intensity pulsed
ultrasound (LIPUS) as mechanical stimulation was proposed to achieve a
high level of formation of new bone and vascularization. These findings sug-
gest the hybrid use of RGD modification and LIPUS might provide one
approach to achieve a high level of formation of new bone and vasculariza-
tion in bone TE scaffold implants (Wang et al., 2014).
An RGD-coupled alginate hydrogel as the scaffold for the simultaneous
encapsulation of hBMMSCs (human bone marrow MSCs) with an anti-
BMP-2 mAb (monoclonal antibodies) to capture endogenous BMP-2
growth factor and hypothesized that this combination of vehicle, MSCs,
and mAbs would provide a three-dimensional, cell delivery scaffold for bone
TE (Moshaverinia et al., 2013). Alginates were modified with peptides
containing RGD (arginineglycineaspartic acid) or PHSRN (proline
histidineserinearginineasparagine) sequences from fibronectin to study
possible additive and synergistic effects on adherent cells. Alginates modified
with each peptide were mixed at different ratios to form gels containing
various concentrations and spacing between the RGD and PHSRN
sequences. Calcium deposition was greater when cells were cultured in
the gels as compared to on the gels, and the results suggest that modifying
this biomaterial to more closely mimic the chemistry of natural cell adhesive
proteins (e.g., fibronectin) may be useful in developing scaffolds for bone TE
(Nakaoka, Hirano, Mooney, Tsuchiya, & Matsuoka, 2013).
Human embryonic stem cells (hESCs) are promising for use in regener-
ative medicine applications because of their strong proliferative ability and
multilineage differentiation capability. Human embryonic stem cells derived
MSCs (hESC-dMSCs) were encapsulated in hydrogel microbeads (alginate
microbeads) in macroporous CPC, showing good cell viability, osteogenic
differentiation, and mineral synthesis for the first time. The hESC-dMSC-
encapsulating macroporous CPC construct is promising for bone regener-
ation in a wide range of orthopedic and maxillofacial applications (Tang,
Chen, Weir, Thein-Han, & Xu, 2012).
The efficacy is increased by using alginate gel as a secondary phase mate-
rial with porous ceramic scaffolds in bone TE applications. Compared to
conventional seeding, alginate gel-assisted seeding (GS), and alginate GS
with BMP-2, alginate GS resulted in greater cell seeding efficiency, greater
cell retention within the scaffold during culture, sustained release of BMP-2,
and improved osteogenic activity in vivo (Florczyk et al., 2012). The inte-
grated bioprocess for generating macroscopic 3D bone formation from
mESCs (murine embryonic stem cells) was prepared, and the bioprocess uti-
lizes encapsulation in alginate hydrogels and culture in rotating cell culture
bioreactors, and it produces bone-like tissue. Such a bioprocess has useful
applications to bone TE (Hwang et al., 2009).
5. FUTURE PROSPECTS
Successful exploitation of alginate-based biomaterials in different
tissues and organs such as skin, cartilage, and bone suggest their pro-
mising future for repair and regeneration applications. However, current
alginate is still unable to meet all the design parameters simultaneously
(e.g., degradation, bioactivities, or mechanical properties). In further studies,

control over these properties can be achieved by chemical or physical mod-
ifications of the polysaccharide itself or the gels formed from alginate and,
thus, support the development of more natural and functional tissues. Despite
all the progress that has been made in these fields, clinical use remains limited
due to some outstanding problems. In many cases, it is a principle that the
scaffolds should be biodegradable replacements for bone. Although some of
them are applied to clinical cases, scaffolds to be clinically completely satisfied
have not being developed. Development of more functional scaffold is needed
so that it may be applied widely. To overcome these limitations, close collab-
oration between the biological sciences and engineering sciences is needed.
6. CONCLUSION
The purpose of this review was to detail the use of alginate in bone TE.
It sought to give an idea of the types of materials available and their unique
properties, allowing reader to the ability to choose a material that would best
suit the bone TE application. In conclusion, alginates are no longer just low
cost commodities for technical applications, but they have expanded rapidly
into the biomedical field and added significantly to the toolbox of bioma-
terial and TE scientists.
ACKNOWLEDGMENTS
The authors are grateful to authorities of D.K.M. College for Women and Thiruvalluvar
University, Vellore, Tamil Nadu, India, for the support. Thanks are also due to the editor
Dr. Se-Kwon Kim, Marine Bio Process Research Center, Pukyoung National University,
South Korea, for the opportunity to review such an innovating field.
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CHAPTER FIVE
Chitin and Chitosan Composites

for Bone Tissue Regeneration
Jayachandran Venkatesan*,1, P. Angelin Vinodhini,
Prasad N. Sudha*, Se-Kwon Kim*
*Department of Marine-bio Convergence Science and Marine Bioprocess Research Center, Pukyong National
University, Busan, South Korea
Department of Chemistry, D.K.M. College for Women, Thiruvalluvar University, Vellore, Tamil Nadu,
India
1
Corresponding author: e-mail address: venkatjchem@pknu.ac.kr
Contents
1. Introduction 60
2. Naturally Occurring Biopolymers 60
2.1 Chitin 60
2.2 Chitosan 61
3. Tissue Engineering Applications of Chitin and Chitosan 61
4. Applications of Chitin and Chitosan for Bone Tissue Engineering 64
6. Conclusions 76
Acknowledgments 76
References 76
Abstract
In the present world, where there is increased obesity and poor physical activity, the
occurrence of bone disorders has also been increased steeply. Therefore, a significant
progress has been made in organ transplantation, surgical reconstruction, and the use
of artificial prostheses to treat the loss or failure of an organ or bone tissue in the recent
years. Bone contains considerable amounts of minerals and proteins. The major com-
ponent of bone is hydroxyapatite [Ca10(PO4)6(OH)2] (6065%) and is one of the most
stable forms of calcium phosphate and it occurs along with other materials including
collagen, chondroitin sulfate, keratin sulfate, and lipids. To remedy bone defects, new
natural and synthetic materials are needed, which will have very similar properties as
that of natural bone. Bone tissue engineering is a relatively new and emerging field,
which paves the way for bone repair or regeneration. Polymers can serve as a matrix
to support cell growth by having various properties such as biocompatibility, biode-
gradability, porosity, charge, mechanical strength, and hydrophobicity. Considerable
attention has been given to chitin and chitosan composite materials and their
#
http://dx.doi.org/10.1016/B978-0-12-800268-1.00005-6
applications in the field of bone tissue engineering in the recent years, which are natural
biopolymers. This chapter reviews the various composites of chitin and chitosan, which
are proved to be potential materials for bone tissue regeneration.
1. INTRODUCTION
A very significant clinical problem, which we face nowadays, is bone
injuries and defects, which are due to trauma, osteoporosis, and tumors.
Most of the people find it difficult to be healed naturally and they have
to undergo multiple surgeries for recovery. Therefore, bone tissue engineer-
ing has become a highly promising tool to tackle the most challenging bone-
related clinical issues. Bone tissue engineering has emerged as a new area of
regenerative medicine and biomaterials have an essential function con-
cerning cell adhesion, spreading, proliferation, differentiation, and tissue for-
mation in three dimensions.
Polymers can serve as a matrix to support cell growth by having various
properties (e.g., biocompatibility, biodegradability, porosity, charge,
mechanical strength, and hydrophobicity) and they can be easily modified
and altered by changing the constituents of monomers in different ratios,
controlling polymerization conditions, or introducing various functional
groups to them (Lee & Yuk, 2007). Because of the biocompatible and bio-
degradable behaviors of natural polymers, much attention has been paid to
the natural polymer-based composites than synthetic polymer composites
for bone tissue engineering applications. The natural-based materials are
biopolymers, which include polysaccharides (starch, alginate, chitin/
chitosan, hyaluronic acid derivatives) or proteins (soy, collagen (Col), fibrin
gels, silk) and a variety of biofibers, such as lignocelluloses (Rezwan, Chen,
Blaker, & Boccaccini, 2006).
2. NATURALLY OCCURRING BIOPOLYMERS

2.1. Chitin
Chitin is a poly(-(1 ! 4)-N-acetyl-D-glucosamine) and is a natural polysac-
charide of which was first identified in 1884. It is the most abundant polymer
after cellulose (CEL) and occurs in nature in the exoskeleton of arthropods or
in the cell walls of fungi and yeast and in crab and shrimp shells (Rinaudo,
2006). Because of its biodegradability, nontoxicity, physiological inertness,
Chitin and Chitosan for Bone Tissue Regeneration 61
antibacterial properties, fungistatic properties, hydrophilicity, gel-forming

properties, and affinity for proteins, chitin has found applications in many areas
other than food such as in biosensors (Krajewska, 2004). The main develop-
ment of chitin film and fiber is in biomedical and pharmaceutical applications
as wound-dressing material (Yusof, Wee, Lim, & Khor, 2003), because it
accelerates wound healing (Hudson & Jenkins, 2003). Another interesting
application is in a hydroxyapatitechitinchitosan composite bone-filling
material, which forms a self-hardening paste for guided tissue regeneration
in the treatment of periodontal bony defects (Ito, Matahira, & Sakai, 1998).
2.2. Chitosan
Chitosan, a deacetylated derivative of chitin, is a linear, semicrystalline polysac-
charide composed of (1 ! 4)-2-acetamido-2-deoxy--D-glucan (N-acetyl
D-glucosamine) and (1 ! 4)-2-amino-2-deoxy--D-glucan (D-glucosamine)
units (Rinaudo, 2006). This unique biopolymer is an outstanding candidate
for biomedical applications especially for tissue engineering because of anti-
bacterial activity (Ong, Wu, Moochhala, Tan, & Lu, 2008; Sudarshan,
Hoover, & Knorr, 1992), antifungal (Aranaz et al., 2009), mucoadhesive
(Lehr, Bouwstra, Schacht, & Junginger, 1992), analgesic (Aranaz et al.,
2009), and hemostatic properties (Yang et al., 2007). Chitosan and its deriva-
tives are very attractive candidates in the scaffold composites, because it is
expected that they degrade as the new tissues are being formed, eventually with-
out inflammatory reactions or toxic degradation (Swethaa et al., 2010). Due to
its controlled biodegradability, biocompatibility, nontoxicity, and biores-
orbable nature, it is used as sutures, wound dressings, bone substitutes, tissue
engineering, and drug- or gene-delivery vehicles (Kim et al., 2008; Martino,
Sittinger, & Risbud, 2005; VandeVord et al., 2002). An interesting application
that concerns self-setting calcium phosphate (CP) cement:chitosan glycero-
phosphate mixed with CP and citric acid forms an injectable self-hardening sys-
tem for bone repair or filling (El Zein, Dabbarh, & Chaput, 2002). Chitosan has
the ability to promote osteogenic progenitor cell recruitment and attachment
thus facilitating bone formation (Kim, Park, Kwon, Baik, & Cho, 2002).
3. TISSUE ENGINEERING APPLICATIONS OF CHITIN

AND CHITOSAN
Tissue engineering has recently emerged as a new interdisciplinary sci-
ence to repair injured body parts and restore their functions using laboratory-
grown tissues, materials, and artificial implants (Suh & Matthew, 2000).
Chitosan is a promising polymer for tissue engineering for its nontoxicity,

biocompatibility, and biodegradability. Moreover, chitosan has structural sim-
ilarity to glucosaminoglycans, which are the major component of the extra-
cellular matrix (ECM) (Girin, Thakur, Alexander, Ajazuddin, & Tripathi,
2012). Chitin has been applied in the form of hydrogels, fibrous scaffolds,
or porous sponges, within which the appropriate cell types are seeded for
in vitro or in vivo culture and evaluation (Wan & Tai, 2013). Collective evi-
dence from the literature suggests that chitin is more suitable than chitosan as a
matrix for cartilage tissue regeneration. Hybrid microspheres of chitin and
polyleucine have also been synthesized via an interfacial polymerization
process based on the ring-opening polymerization of an alpha amino acid
N-carboxyanhydride, with potential utility for drug delivery and tissue engi-
neering (Wang, Liu, & Chi, 2008).
Funakoshi et al. (2006) employed a chitin fabric in the repair of rotator
cuff tendon defects, suggesting two ways by which the fabric could help
direct and improve tissue regeneration. In cartilage tissue engineering,
chitosan has the ability to maintain the round morphology of chondrocytes
that is a normal phenotypic characteristic and preserve their capacity to syn-
thesize cell-specific ECM (Iwasaki, Yamane, & Majima, 2004; Lahiji,
Sohrabi, Hungerford, & Frondoza, 2000; Risbud, Ringe, Bhonde, &
Sittinger, 2001). The porous Col/chitosan/glycosaminoglycan (GAG) scaf-
folds loaded with transforming growth factor b1 was reported to promote car-
tilage regeneration (Kim et al., 2003; Lee et al., 2004). A porous freeze-dried
chitosan scaffold incorporating TGF-b1-loaded microspheres was used for the
treatment of cartilage defects (Kim et al., 2003). The N,N-dicarboxymethyl
chitosan associated with bone morphogenetic protein-7 was also found to
repair femoral articular cartilage lesions in the rabbit, indicative of synergism
of their respective biological properties (Mattioli-Belmonte, Gigante, et al.,
1999; Mattioli-Belmonte, Nicoli-Aldini, et al., 1999).
An injectable hydrogel consisting of methacrylated glycol chitosan
(MeGC) and hyaluronic acid (HA) was created by photocrosslinking with a
riboflavin photoinitiator under visible light (Park, Choi, Hu, & Lee, 2013).
The incorporation of HA in MeGC hydrogels increased the proliferation
and deposition of cartilaginous ECM by encapsulated chondrocytes. The find-
ings demonstrated that MeGC/HA composite hydrogels have the potential for
cartilage repair. Correia et al. (2011) prepared freeze-dried composite scaffolds
of chitosan (CS) and hyaluronic acid (HA) in different weight ratios containing
either no HA (control) or 1%, 5%, or 10% of HA and found that CS/HA com-
posite matrixes have potential use in cartilage repair.
Genipin-cross-linked Col/chitosan biodegradable porous scaffolds were

prepared for articular cartilage regeneration by Yan et al. (2010). The bio-
compatibility of the scaffolds was evaluated by culturing rabbit chondrocytes
in vitro. This study demonstrated that a good viability of the chondrocytes
seeded on the scaffold was achieved. The SEM analysis has revealed that
the chondrocytes adhered well to the surface of the scaffolds and contacted
each other. These results suggested that the genipin-cross-linked Col/
chitosan matrix may be a promising formulation for articular cartilage
scaffolding.
Multilayered constructs were formed by depositing the polyelectrolytes
chitosan and chondroitin sulfate on either bidimensional glass surfaces or 3D
packet of paraffin spheres (Silva et al., 2013). The results clearly showed that
cells attached, proliferated, and were metabolically active over the entire
scaffold. Cartilaginous ECM formation was further assessed and results
showed that GAG secretion occurred indicating the maintenance of the
chondrogenic phenotype and the chondrogenic differentiation of
multipotent bone marrow-derived stromal cells.
Water-soluble chitosan derivatives, chitosan-graft-glycolic acid (GA) and
phloretic acid (PA) (CS-GA/PA), were designed by Jin et al. (2009) to
obtain biodegradable injectable chitosan hydrogels through enzymatic
cross-linking with horseradish peroxidase and H2O2. CS-GA/PA polymers
were synthesized by first conjugating GA to native chitosan to render the
polymer soluble at pH 7.4, and subsequent modification with PA. The
results indicated that CS-GA/PA hydrogels are promising as an artificial
ECM for cartilage tissue engineering.
Chitosan-modified poly(L-lactide-co--caprolactone) scaffolds were fab-
ricated to simulate the main biochemical components of cartilage, as well as
their interaction with the aim to endow them with viscoelasticity similar to
native cartilage. With the fabrication of biomimetic scaffolds, it is possible to
make scaffolds for cartilage tissue engineering, which are not only biocom-
patible but also have mechanical properties similar to native cartilage
(Li et al., 2012).
Chitosan scaffolds having an interconnecting porous structure were
easily fabricated by simple freezing and lyophilization of a chitosan solu-
tion. After rehydration of scaffolds, porcine chondrocytes were seeded
onto scaffolds and cultured for up to 28 days in a rotating-wall bioreactor.
The results suggested that chitosan scaffolds may be a useful alternative to
synthetic cell scaffolds for cartilage tissue engineering (Nettles, Elder, &
Gilbert, 2002). The initial comparison of various mixtures of -chitin
and chitosan as a scaffold for rabbit chondrocyte culture was studied by

Suzuki et al. (2008) and found that only in the pure -chitin sponge scaf-
fold; type II Col was closer to that of normal rabbit cartilage. These results
indicated that pure -chitin sponge scaffold can be used for cartilage tissue
engineering.
Chitosan amine groups were reacted with methacrylic anhydride
resulting in a water-soluble methacrylamide chitosan (MC) that was then
crosslinked by radical polymerization resulting in a scaffold. Biodegradability
by lysozyme and penetrability of the scaffold by rat superior cervical gan-
glion neurons were studied by Laura, Kazazian, and Shoichet (2007) and
the results demonstrated its suitability in neural tissue engineering and its
potential for other engineered tissues, such as cartilage repair, where chitosan
has already demonstrated some utility. Transparent chitin hydrogel tubes
were synthesized, for the first time by Freier, Montenegro, Shan Koh,
and Shoichet (2005), from chitosan solutions using acylation chemistry
and mold-casting techniques to investigate chitin and chitosan as potential
materials for biodegradable nerve guides.
4. APPLICATIONS OF CHITIN AND CHITOSAN FOR BONE

TISSUE ENGINEERING
Chitosan has been extensively used in bone tissue engineering, since it
was shown to promote cell growth and mineral-rich matrix deposition by
osteoblasts cells in culture (Seol et al., 2004).
Hoctor, Killion, Devine, Geever, and Higginbotham (2013) prepared
nanocomposite scaffolds using chitosan (CS) and hydroxyapatite (HA) pow-
der of various known quantities and were subsequently crosslinked. Drying
study, swelling studies, compression testing, contact angle testing, differen-
tial scanning calorimetry (DSC), scanning electron microscopy (SEM), and
energy-dispersive X-ray (EDX) spectrometry were conducted. It was evi-
dent that the samples with a higher ratio of HA, cross-linking agent, and
photoinitiator solution were the most promising samples for use as a
bone-substitute material.
Prabaharan, Rodriguez-Perez, de Saja, and Mano (2007) developed a
novel poly(L-lactic acid) (PLLA)chitosan hybrid scaffolds using PLLA with
different concentrations of chitosan and glutaraldehyde and used as tissue
engineering scaffolds and drug-release carriers. The hybrid scaffolds pre-
pared with the lowest amount of chitosan and cross-linking agent showed
the highest percent of drug release. These preliminary results suggested that
PLLAchitosan hybrid scaffolds could be suitable to release bioactive com-

ponents for stimulating cell differentiation and proliferation or drugs, such as
anti-inflammatories and antibiotics, to induce therapeutic effects in tissue
engineering strategies.
Chitosan has been combined with a variety of materials such as alginate,
hydroxyapatite, CP, poly(methylmethacrylate), PLLA, and growth factors
for potential application in orthopedics; hyaluronan shows morphogenetic
activities suitable for a correct bone architecture (Muzzarelli, 2009).
Tanase, Popa, and Verestiuc (2011) studied the preparation, characteri-
zation, enzymatic degradation, and mechanical properties of composite scaf-
folds containing chitosan (CS) and CPs, obtained by a biomimetic method,
and showed valuable properties that suggest potential application as a mate-
rial for bone regeneration.
A simple process was established by Tsaia, Chena, Liub, and Lai (2011) to
fabricate arginineglycineaspartic acid (RGD)-conjugated and -cross-
linked chitosan scaffolds for bone tissue engineering and found that the
calcium deposition by the osteoblasts in the RGD-incorporated chitosan
scaffolds was almost doubled compared to the control, and thus greatly
increased the mechanical property of the chitosan scaffolds. The
chitosanalginate gel/mesenchymal stem cells (MSCs)/bone morphoge-
netic protein-2 composites were found to stimulate new bone formation
when injected into the mouse (Park et al., 2005).
Yilgor, Tuzlakoglu, Reis, Hasirci, and Hasirci (2009) studied two bone
growth factors, bone morphogenetic proteins BMP-2 and BMP-7, which
were encapsulated in poly(lactic acid-co-GA) and poly(3-
hydroxybutyrate-co-3-hydroxyvalerate) nanocapsules and were then incor-
porated into fibrous chitosan scaffolds. They stated that the sequential
growth factor delivery is a better approach than individual growth factor
use for tissue engineering due to its mimicking the natural process of healing.
The chitosan sponges incorporating platelet-derived growth factor induced
new bone formation in rat calvarial defect (Lee et al., 2000a, 2000b).
Chitinhydroxyapatite composite loaded with MSC-induced osteo-
blasts, when implanted into bone defects of rabbit femur, was able to support
bone regeneration (Ge, Baguenard, Lim, Wee, & Khor, 2004). Three-
dimensional biodegradable chitosannanohydroxyapatite (nHA) composite
scaffolds were prepared and characterized by Thein-Han and Misra (2009)
by superior mechanical, physicochemical, and biological properties com-
pared to pure chitosan scaffolds for bone tissue engineering and found that
the composite as a potential scaffold material for bone regeneration.
In a laboratory experimental study, microdrilled bone defects were cre-

ated in the upper tibia of each leg in 15 adult male rats (Ezoddini-Ardakani
et al., 2012) and the defect in the right leg, filled by the chitosan powder,
was compared with the untreated defect in the left leg in each rat at 1, 2,
and 4 weeks after surgery. Chitosan significantly accelerated the bone
regeneration process in rat tibias and thus, regarding its biocompatibility
and osteoinductivity, it can be studied as a biomaterial in human bone
healing.
The addition of chitosan particles to the media of human bone marrow
stromal cell cultures stimulates osteogenesis by promoting osteoblastic
differentiation and by favoring the release of angiogenic factors in vitro.
The results demonstrated that chitosan particles alone are not sufficient
to promote osteoblast differentiation of BMSCs in vitro and suggested
that chitosan promotes osteogenesis in vivo through indirect mechanisms
(Guzman-Morales et al., 2009).
Different ratios of poly(-caprolactone) (PCL)/hydroxyapatite (HA) and
chitosan/poly(vinyl alcohol) (PVA) were produced by electrospinning and
the PCL/HA electrospun nanocomposite provided a mechanism for con-
trolled release of HA nanoparticles in cell culture studies. Thus, the
nanofibrous composite scaffold of electrospun PCL/HAchitosan/PVA
can potentially be used for the osteogenic differentiation of stem cells
(Shahrooz Zargarian & Haddadi-Asl, 2010).
Hydroxyapatite (HA) in 25%, 50%, and 75% (w/w) fractions was incor-
porated into chitin solutions and processed into air- and freeze-dried mate-
rials and was exposed to cell cultures and implanted into the
intramusculature of a rat model. The HAchitin materials were found to
be noncytotoxic and degraded in vivo and act as a good substrate candidate
for tissue-engineered bone substitute (Ge et al., 2004).
Chitosan (CS) and CEL were codissolved in an ionic liquid and then
regenerated from water. Hydroxyapatite (HA) was subsequently formed
in situ by alternately soaking (CEL + CS) composites in aqueous solutions
of CaCl2 and Na2HPO4. The findings showed the potential use of (CEL
+ CS + HA) composites as scaffolds in bone tissue engineering (Mututuvari,
Harkins, & Tran, 2013).
A series of biocompatible chitosan/hydroxyapatite composites has
been synthesized in an aqueous medium from chitosan solution and soluble
precursor salts by a one-step coprecipitation method and was implanted
into the tibial bones of rats. The porous chitosanhydroxyapatite materials
have shown good osteoconductive properties in in vivo experiments
and the composite materials have proved to be promising for the treatment
of bone defects (Danilchenko et al., 2009).
A nanocomposite scaffold using -chitin hydrogel with bioactive glass
ceramic nanoparticles (nBGC) was developed by lyophilization technique
(Sowmyaa et al., 2011). The -chitin/nBGC composite scaffolds were
found to have enhanced porosity, swelling, bioactivity, and degradation
in comparison to the control scaffolds. The composite scaffolds were non-
toxic to human osteosarcoma (MG63) and human primary osteoblast cells
and supported cell attachment, spreading, and proliferation. These compos-
ite scaffolds were found to be satisfactory in all aspects and these
nanocomposite scaffolds could be promising candidates for the treatment
of periodontal bone defects.
Carbonate apatitechitosan scaffolds (CAChSs) were fabricated using
the lyophilization technique (Ariani et al., 2013) and it had a three-
dimensional interconnected porous structure, good retentive form without
brittleness, and the ability to support the proliferation and differentiation of
osteoblasts. It is suggested that the newly developed CAChSs may be a pos-
sible scaffold material for bone tissue engineering.
A biopolymer-based novel nanocomposite chitosan/montmorillonite/
hydroxyapatite (HA) was developed for biomedical applications (Katti,
Katti, & Dash, 2008) and the cell culture experiments showed that the com-
posite is biocompatible and has a better cell proliferation rate compared to
chitosan (CS)/hydroxyapatite (HA) composites. They represented the
design of a novel claychitosanhydroxyapatite composite with improved
mechanical properties that has potential applications in bone tissue
engineering.
Silk and chitosan were combined to produce a blended scaffold using the
lyophilization technique and were seeded with bone marrow-derived MSCs
and subjected to osteogenic stimulation. It is hoped that the resulting scaffold
would become a highly functional bone graft substitute (Ng, Wong, Goh, &
Toh, 2009). Macroporous CPchitosan composite scaffolds were fabricated
and evaluated for use in bone tissue engineering (Zhang, Ni, Zhang, &
Ratner, 2003). It is indicated that the hydroxyapatitematrix composite
scaffolds might enhance the phenotype expression of MG63 cells, in com-
parison with chitosan matrix scaffolds. Soluble CP glasses should be added to
the scaffolds to prevent chitosan from fast degradation that may affect the
differentiation of osteoblast cells.
Ding, Teng, and Pan (2012) fabricated chitosan/hydroxyapatite (HA)
composite microspheres in a water-in-oil emulsion by in situ generation
of the HA nanoparticles in the chitosan matrix and showed that the

obtained chitosan/HA composite microspheres with uniform microstruc-
tures have more potential for both bone tissue regeneration and drug deliv-
ery applications, when compared to those prepared by the traditional
mixing method.
Biodegradable composite films of chitosanpolyacrylamide
nanohydroxyapatite (CsPAAmnHA) with different concentrations of
chlorotrimethylsilane (CTMS) were fabricated and characterized using Fou-
rier transform infrared (FTIR) spectroscopy and X-ray diffractogram
(XRD) by Kalambettu, Rajangam, and Dharmalingam (2012). They
suggested that the incorporation of CTMS into CsPAAmnHA compos-
ites not only improved their stability but also controlled the degradation of
the composites, which are important for drug delivery applications.
Murugan and Ramakrishna (2004) prepared nanohydroxyapatite (nHA)
composite bone paste with a natural polysaccharide, chitosan, using wet
chemical method at low temperature. The prepared composites were ana-
lyzed by various physicochemical methods and suggested that the nano-HA
crystallites are well intact with the chitosan macromolecules. Also the rate of
bioresorption of nano-HA was improved by the addition of chitosan. These
findings suggested that this kind of composites may have a great impact on
human health care systems and would act as a bioresorbable bone substitute
with superior bioactivity and osteoconductivity in vivo.
Liu et al. (2013) evaluated the effect of hydroxyapatite/chitosan (nHA/
CTS) seeded with bone marrow mesenchymal stem cells (BMSCs) on bone
regeneration and examined the underlying mechanism in vitro and in vivo.
They demonstrated that the nHA/CTS scaffold promotes bone regenera-
tion by supporting the adhesion, proliferation, and activating integrin-
BMP/Smad signaling pathway of BMSCs both in vitro and in vivo. Finally,
they concluded that the nHA/CTS/BMSCs were superior to nCTS/
BMSCs to promote bone regeneration in vivo. This study shows the great
potential of using the HA/CTS nanocomposite nanofibers for bone tissue
engineering applications.
A biomimetic and thermosensitive gel scaffold was prepared from
chitosan (CS), nanohydroxyapatite (nHA), and Col by Huanga et al.
(2011). They suggested that the formed gel acted as a biocompatible sub-
strate for the proliferation of rat bone marrow stem cells in vitro and the
CS/HA/Col system can be injected into body in a minimally invasive
manner providing a biocompatible environment for rBMSCs survival
in vivo. They concluded that the biocompatibility, cytocompatibility,
phase transition process, and rheological property made the CS/HA/Col

system suitable as a delivery vehicle for rBMSCs.
Nanohydroxyapatite/chitosanpectin (nHCP) composite was prepared
via mineralization of a chitosanpectin network and then nHCP/
chitosangelatin (nHCP/CG) scaffolds were prepared via the blending of
nHCP and chitosangelatin solution by Li et al. (2011). The nHCP/CG
scaffolds exhibited high porosity, interconnectivity, water absorption ability,
controllable degradation behaviors, and good mechanical strength. Further-
more, the nHCP/CG scaffolds also showed excellent mechanical stability
and biocompatibility. Therefore, it is concluded that the biodegradable
nHCG/CG scaffold with a desirable physicochemical and biological prop-
erties is a potential biomaterial in bone tissue engineering.
Nikpour, Rabiee, and Jahanshahi (2012) successfully synthesized
nanohydroxyapatite/chitosan composite material with various ratios via
in situ hybridization route. The surface chemical characterization on the
nanocomposite was evaluated by FTIR and XRD. Surface topography,
roughness, and morphology of the samples were observed by atomic force
microscopy and SEM. The characterization results confirmed homogeneity,
interaction, and integration between the hydroxyapatite (HA) and chitosan
matrices. Furthermore, the mechanical compressive testing indicated that
the synthesized potential biodegradable composites have acceptable
mechanical behavior for tissue substitution.
Venkatesan and Kim (2012a, 2012b) prepared certain complexes of
single-walled carbon nanotube (SWCNT) grafted with different molecular
weight polysaccharides, named as SWCNTglucosamine, SWCNT
chitooligosaccharide (<1 kDa), SWCNTchitooligosaccharide (13 kDa),
SWCNTchitosan (310 kDa), and SWCNTchitosan (510 kDa) and sub-
jected to bone tissue engineering application in vitro with MSCs. They
observed that there is no cytotoxic effect in SWCNTglucosamine as well
as higher alkaline phosphatase (ALP) activity and enhanced mineralization.
From the results they proposed that SWCNTpolysaccharide derivatives are
promising biomaterials for bone tissue engineering when administered in
injectable forms.
A novel scaffold (CSHAMSCol) containing chitosan (CS), hydroxy-
apatite (HA) derived from Thunnus obesus bone and marine sponge (Ircinia
fusca) Col (MSCol) was prepared by Pallela, Venkatesan, Janapala, and Kim
(2012) using freeze-drying and lyophilization method. These scaffolds
were characterized by FTIR spectroscopy, thermogravimetric and differ-
ential thermal analyses (TGA and DTA), SEM, XRD, and optical
microscopy. Cell proliferation in composite scaffolds was relatively higher

than pure chitosan when observed by MTT assay and Hoechst staining
in vitro using MG63 cell line. Based on the properties such as good thermal
stability, interconnected porosity, and in vitro cell proliferation, they con-
cluded that CSHAMSCol scaffold is a novel marine sponge collagenous
composite scaffold that will have potential prospects in the field of bone
tissue engineering.
Venkatesan and Kim (2012a, 2012b) developed a novel chitosan/nano-
structured hydroxyapatite (chitosan/nHA) scaffold by freeze-drying method
and subsequently characterized physicochemically for bone graft substitu-
tion. They suggested that the chitosan/nHA scaffold is a novel composite
scaffold that will have great potential applications in the field of bone tissue
engineering.
Chitosan/hydroxyapatite (HA) composite slurries with various HA con-
tents (10%, 20%, 30%, 40%, and 50%) were prepared by Teng et al. (2009) by
a coprecipitation method and then formulated into thin membranes by a
dynamic filtration technique. After the freeze-drying process, the membranes
were crosslinked with genipin. The influences of the HA content of the mem-
branes on their phase and morphology, mechanical properties, and bioactivity
were investigated and they found that as the HA content was increased, the
tensile strength of the membranes exhibited a steady decrease, while the elastic
modulus increased by a factor of 2 when 20% HA was added. The results of
the in vitro cell culture showed that the highest ALP level was achieved at an
HA content of 30%. Finally, it was concluded that the chitosan/HA compos-
ite membranes with an HA content of less than 30% have good potential to be
used as barrier membranes for guided bone regeneration (GBR).
In the study made by Sun, Sun, Huang, and Li (2009), novel composite
scaffolds of nanohydroxyapatite (nHA) and chitosan (CS) were prepared and
seeded on the surface with rhBMP-2 gene-transfecting cells and evaluated its
biocompatibility. The cell line-expressing rhBMP-2 gene was grown, and
the nanometer hydroxyapatite powder was fabricated with the solgel
method and then dispersed completely in 2% chitosan/acetic acid solution.
The porous structure of the composite scaffolds was made by lyophilization.
It was believed that adherence and proliferation of rhBMP-2 gene-
transfecting cells on the surface of nHA/CS were better than those on dense
HA/CS composite. The ALP activity from nHA/CS was approximately the
same as that of dense HA/CS. From the results, it is suggested that the porous
nHA/chitosan composite scaffolds are useful materials for tissue engineering
research.
A novel biodegradable composite scaffold was made using

nanohydroxyapatite and natural-derived polymers of chitosan and
carboxymethyl CEL, namely, nHA/CS/CMC, which was prepared by
freeze-drying method by Liuyun, Yubao, and Chengdong (2009). The
physicochemical properties of nHA/CS/CMC scaffold were tested by
infrared absorption spectra, transmission electron microscope (TEM),
SEM, universal material testing machine, and phosphate buffer
solution-soaking experiment. The biological properties were evaluated
by MG63 cells and MSCs culture experiment in vitro and a short period
implantation study in vivo and confirmed that the scaffold had no feign
body reaction and many blood vessels grew into the porous structure,
while the scaffold was biodegraded gradually. Also it was concluded that
the scaffold had desirable physicochemical properties due to the strong
ionic crosslinking interactions between CS and CMC, such as highly
complicated interconnection irregular porous network structure (the
pore size ranging from 100 to 500 m), and the compressive strength
reached 3.5 MPa with the porosity of 77.8%. These results suggested that
the novel degradable porous nHA/CS/CMC composite scaffold with
desirable physicochemical and biological properties has a great potential
to be used as bone tissue engineering material.
Huang, Tian, Yu, Xu, and Feng (2009) developed a bone-like nano-
hydroxyapatite/Col (nHAC)-loaded chitosan (CS)/-glycerophosphate
injectable scaffold. The feasibility of developing a thermosensitive and
injectable chitosan solution in the presence of nHAC was demonstrated.
Bone marrow-derived MSCs were used to measure the cell proliferation
of CS/GP/nHAC scaffolds based on the cell count kit-8 (CCK-8) assay.
It was found that MSCs proliferated normally with the CS/GP/nHAC
composite scaffolds. The CS/GP/nHAC composite scaffolds developed
in this study exhibited good injectability, thermo-irreversible properties,
and solidified under mild conditions. No more than 0.02 g ml1 of nHAC
filler was required to form a nondecaying hydrogel. Hence, it was concluded
that nHAC-loaded CS/GP composites could be a promising injectable
device for bone tissue engineering.
Karakecili and Arkan (2012) successfully prepared chitosan
nanohydroxyapatite composite scaffolds by a supercritical CO2-assisted pro-
cess. The prepared scaffolds were analyzed using attenuated total reflectance
Fourier transform infrared spectroscopy, XRD, SEM, and energy-dispersive
X-ray analysis. An increase in water uptake and mechanical strength were
observed in composite scaffolds. No cytotoxicity was observed toward
fibroblast cells in vitro. The results obtained from their study indicated that
chitosannanohydroxyapatite scaffolds prepared using supercritical CO2
shall be considered as a potential candidate for bone tissue engineering
applications.
Nanofibrous biocomposite scaffolds of chitosan/PVA/hydroxyapatite
(CS/PVA/HA) were prepared by electrospinning by Yang et al. (2008).
The scaffolds were characterized by FTIR spectroscopy, SEM, TEM, and
XRD techniques. Tensile testing was used for the characterization of
mechanical properties. Mouse fibroblasts (L-929) attachment and prolifer-
ation on the nanofibrous scaffold were investigated by MTT assay and SEM
observation. The attachment and growth of mouse fibroblast was on the sur-
face of nanofibrous structure, and cells morphology characteristics and via-
bility were unaffected. In conclusion, based on their observations, the CS/
PVA/HA biological nanocomposite fiber was considered as a promising
material for bone tissue regeneration.
A novel nanohydroxyapatite (nHA)/chitosan composite scaffold with
high porosity was developed by Kong et al. (2005) and the nano-HA par-
ticles were made in situ through a wet chemical method and dispersed well
on the porous scaffold. They bound to the chitosan scaffolds very well. This
method prevents the migration of nano-HA particles into surrounding tis-
sues to a certain extent. The morphologies, components, and biocompati-
bility of the composite scaffolds were investigated. SEM, porosity
measurement, TGA, XRD, X-ray photoelectron spectroscopy, and FTIR
spectroscopy were used to analyze the physical and chemical properties of
the composite scaffolds. The biocompatibility was assessed by examining
the proliferation and morphology of MC 3T3-E1 cells seeded on the scaf-
folds. The composite scaffolds showed a better biocompatibility than pure
chitosan scaffolds. The results suggested that the newly developed nano-
HA/chitosan composite scaffolds may serve as a good three-dimensional
substrate for cell attachment and migration in bone tissue engineering.
Nanohydroxyapatite (nHA)/chitosan (CS)/konjac glucomannan
(KGM) composite was prepared by coprecipitation method and investigated
by thermal gravitivity/differentiate thermal analysis, FTIR spectroscopy,
XRD, inductively coupled plasma emission spectroscopy, SEM, and
EDX analyzer by Zhou, Li, Zhang, Zuo, and Jansen (2007). The analyses
showed that the three phases of nHA, CS, and KGM combined closely
to each other and in vitro tests were conducted to investigate the degradation
and bioactivity of the composite. From the results, it is concluded that
the biocomposite, which is a combination of nHA with biodegradable
CSKGM polymer, showed potential for controlled drug delivery vehicle

for bone applications.
To combine the high porosity with appropriate mechanical strength,
chitosan (CS)-coated nanohydroxyapatite/polyamide66 (nHA/PA66) scaf-
folds have been prepared by Huang et al. (2012) and investigated the effect of
CS content on the scaffold physical properties and cytological properties.
The results showed that CS coating can reinforce the scaffold effectively.
After coating, the strength and modulus of nHA/PA66 scaffold were signif-
icantly enhanced to five to six times of those of uncoated scaffold, while the
porosity of the coated scaffold slightly reduced to 78% from 84% (uncoated
scaffold). The cytological properties of scaffolds were also studied in vitro by
cocultured with osteoblast-like MG63 cells and it showed good
cytocompatibility, suggesting a positive prospect for bone regeneration or
repair of bone defects.
In the study made by Wang et al. (2009), a dual system of Col and
chitosan (CS) (Col:CS 5:4) was used and the mixture obtained by gelation
of the neutral blend and the intervene effect of in situ hydroxyapatite (HA)
synthesis was investigated and thereby prepared ColCSHA (CCHA)
nanocomposites with varying HA content by a sequential method. The
structural characteristics and biological properties of the CCHA
nanocomposites were evaluated using XRD, FTIR, DSC, TGA, DTA,
and SEM analyses and the osteoblast culture experiment. It is found that
the CCHA nanocomposites along with the pure ColCS mixture had good
cytocompatibility according to the preliminary cellular assay and thus pro-
vide a feasible route for bone grafting nanocomposites.
A zinc oxide (ZnO)-containing nanohydroxyapatite/chitosan (nHA/
CS) cement was developed by Li, Yubao, Zuo, Lan, and Jansen (2010)
and its bone formation ability was investigated in vitro and in vivo. The phys-
icochemical properties of the cement were determined in terms of pH var-
iation during and after setting, injectability, and wettability. The results of
immersion tests in simulated body fluid (SBF) indicated that the cement
exhibited excellent bone-like apatite-forming ability. In vivo studies were
conducted by the installation of the cement of tibial-bone defects in rabbit
tibia. Radiological examination also confirmed that the ZnO-containing
nHA/CS cement significantly induced new bone formation. These results
suggested that the ZnO-containing nHA/CS cement may be beneficial
to enhance bone regeneration in osseous defect sites.
Zhang et al. (2013) fabricated nanohydroxyapatite/chitosan/PLLA
(nHA/CS/PLLA) ternary biocomposites by grafting HA/CS
nanocomposites with D-, L-lactic acid and further blending with the PLLA
matrix. It is characterized using FTIR, 13C NMR spectrum, and TEM.
Time-dependent phase behavior, mechanical properties, and wetting angles
were also observed and the ternary biocomposites with the HA content of
60 and 67 wt.% exhibited high compressive strength of about 160 MPa and
suitable hydrophilicity. The in vitro tests exhibited that the ternary bio-
composites have good biodegradability and bioactivity when immersed in
SBF solutions. All the results suggested that the nano-HA/CS/PLLA ternary
biocomposites are appropriate to application as bone substitute in bone tissue
engineering.
To meet the challenges of designing an in situ-forming scaffold and reg-
enerating bone with complex three-dimensional (3D) structures, an in situ-
forming hydrogel scaffold based on nanohydroxyapatite (nHA), Col, and
chitosan (CS) was synthesized by Chen, Li, et al. (2012). The potential of
ultrasound for the quantitative in vivo evaluation of tissue development in
CS/nHAC scaffold was evaluated and it was injected into rat subcutaneous
tissue and evaluated for 28 days. It was demonstrated that ultrasound can be
used to noninvasively and nondestructively monitor and evaluate the in vivo
characteristics of injectable bone scaffold. In comparison to the CS, the
CS/nHAC scaffold showed a greater stiffness, a less degradation rate, and
a better blood supply in the in vivo biocompatibility evaluation. In conclu-
sion, the diagnostic ultrasound method is a good tool to evaluate the in vivo
formation of injectable bone scaffolds and facilitates the broad use to monitor
tissue development and remodeling in bone tissue engineering.
Fan et al. (2012) used icariin, a plant-derived flavonol glycoside, which
has been proved as an osteoinductive agent for bone regeneration. They
developed an icariin-loaded chitosan/nanosized hydroxyapatite (IC-CS/
HA) system which controls the release kinetics of icariin to enhance bone
repairing. The physical and biological properties of icariin were totally pre-
served in the composite developed without undergoing any chemical
changes during the production and icariin did not remarkably change the
morphology, porosity, and mechanical strength of the CS/HA composite.
The in vitro degradation tests indicated that the icariin-release kinetics from
this IC-CS/HA composite could be governed by degradation of both CS
and HA matrices and could last for more than 90 days. They believed that
this IC-CS/HA system could be a promising biomaterial for bone tissue
engineering.
In order to improve the bioactivity and mechanical properties of
hydroxyapatite (HA), chitosan (CS) was in situ combined into HA to fab-
ricate a composite scaffold by a sublimation-assisted compression method
by Sun, Koh, Ryu, Han, and Lee (2012). A highly porous film with suf-
ficient mechanical strength was prepared and the bioactivity and cytotox-
icity were investigated. The apatite layer was assessed using scanning
electronic microscopy, inductively coupled plasma-optical emission spec-
trometry, and weight measurement. Cell culturing of the composite with
L-929 fibroblastic cells and work-up by WST-8 assay indicated that the
HA/CS scaffold has good in vitro bioactivity and cytocompatibility. Com-
posite analysis showed that a layer of microsized, needle-like crystals was
formed on the surface of the composite film. The results indicated that
HA/CS composites with porous structure have the potential to be applied
in bioapplications, such as bone patching and bone-substitute materials.
In the work by Li et al. (2013), graphene oxide (GO) and chitosan (CS)-
functionalized GO were introduced as templates to fabricate hydroxyapatite
(HA) using a facile solution-based in situ synthesis method, and GOHA and
CS-GOHA nanocomposites were successfully prepared for the first time.
The in vitro cytotoxicity of the prepared nanocomposites was investigated
using CCK-8 assay on murine fibroblast L-929 cell line and human
osteoblast-like MG63 cell line, respectively. The findings may provide
new prospects for utilizing the GO-based hydroxyapatite biocomposites
in bone repair, bone augmentation, as well as coating of biomedical implants
and broaden the application of GO sheets in biological areas.
The bone regeneration potential of nanohydroxyapatite/CS composite
scaffolds was compared with pure chitosan scaffolds when implanted into
segmental bone defects in rabbits (Zhang et al., 2012) and found that the
injectable nanohydroxyapatite/chitosan scaffolds are potential candidate
materials for regeneration of bone loss.
Nanohydroxyapatite (nHA)/chitosan (CS) composite membranes were
prepared by solvent casting and evaporation methods for the function of
GBR. The effect of nHA content and solvent evaporation temperature
on the properties of the composite membranes was studied. The nHA/CS
composite membranes have no negative effect on the cell morphology, via-
bility, and proliferation and possess good biocompatibility. Thus, it makes
the nHA/CS composite membrane a prospective biodegradable GBR
membrane for future applications (Xianmiao et al., 2009).
Verma, Katti, and Katti (2010) investigated osteoblast adhesion, prolif-
eration, and differentiation on nanocomposites of chitosan, polygalacturonic
acid (PgA), and hydroxyapatite. These studies were done on both two- and
three-dimensional (scaffold) samples. This study indicated that chitosan/
PgA/hydroxyapatite nanocomposite films and scaffolds promote cellular
adhesion, proliferation, and differentiation. Venkatesana, BoMi Ryua,
Sudha, and Kim (2012) prepared scaffolds with the use of low- and high-
molecular weight chitosan with 0.0025%, 0.005%, and 0.01% weight of
f-multiwalled carbon nanotube (f-MWCNT) by freezing and lyophilization
method and physiochemically characterized as bone graft substitutes. They
suggested that chitosan/f-MWCNT scaffolds are promising biomaterials for
bone tissue engineering.
5. FUTURE PROSPECTS
Chitin and chitosan have been extensively studied as a biomaterial for
bone tissue engineering applications. New innovative types of chitin and
chitosan and their derivatives in various forms can be prepared and used
for tissue engineering applications in the future more efficiently. It is there-
fore expected that this field will keep growing within the next few years and
efforts should be taken to intensify the working collaboration of researchers
and technologists from different relevant communities.
6. CONCLUSIONS
A new generation of biodegradable natural biomaterials is emerging
due to the significant progress of regenerative medicine with the develop-
ment of modern science and technology. The attractive features of chitin
and chitosan have made it a very promising tool for bone tissue engineering
applications because of the ease of preparing derivatives with new properties.
Thus, this review summarized the potential biomedical applications of
chitin, chitosan, and their derivatives in various forms in the field of bone
tissue engineering.
ACKNOWLEDGMENTS
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CHAPTER SIX
Chemical Modification of Chitosan

for Efficient Gene Therapy
Hu-Lin Jiang*, Peng-Fei Cui, Rong-Lin Xie, Chong-Su Cho{,1
*Department of Pharmaceutics, State Key Laboratory of Natural Medicines, China Pharmaceutical University,
Nanjing, PR China

{
Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, South Korea
1
Corresponding author: e-mail address: chocs@plaza.snu.ac.kr
Contents
1. Introduction 84
2. Ligand Modification for Specific Cell Targeting 85
2.1 Galactose ligand modification 85
2.2 Folate ligand modification 86
2.3 Mannose ligand modification 88
2.4 Hyaluronic acid ligand modification 90
3. Stimuli-Response Modification for Enhancement of Transfection Efficiency 90
3.1 pH-sensitive modification 90
3.2 Thiolated modification 92
3.3 Amino acid modification 94
3.4 Magnetic modification 94
4. Penetrating Modification 95
4.1 Brainblood barrier penetrating modification 95
4.2 Cell penetration peptide modification 96
4.3 Penetration of nuclear membrane 96
5. Conclusion 97
References 97
Abstract
Gene therapy involves the introduction of foreign genetic material into cells in order to
exert a therapeutic effect. Successful gene therapy relies on effective vector system. Viral
vectors are highly efficient in transfecting cells, but the undesirable complications limit
their therapeutic applications. As a natural biopolymer, chitosan has been considered to
be a good gene carrier candidate due to its ideal character which combines biocom-
patibility, low toxicity with high cationic density together. However, the low cell spec-
ificity and low transfection efficiency of chitosan as a gene carrier need to be overcome
before undertaking clinical trials. This chapter is principally on those endeavors such as
Advances in Food and Nutrition Research, Volume 73 # 2014 Elsevier Inc. 83

http://dx.doi.org/10.1016/B978-0-12-800268-1.00006-8
84 Hu-Lin Jiang et al.
chemical modifications using cell-specific ligands and stimuli-response groups as well

as penetrating modifications that have been done to increase the performances of
chitosan in gene therapy.
1. INTRODUCTION
Gene therapy has received much attention in the fields of medicine,
pharmaceutical sciences, and biotechnology because of its potential to cure
genetic disorders and chronic diseases (Park, Jeong, & Kim, 2006). How-
ever, as naked therapeutic genes are rapidly degraded by nucleases and show
poor cellular uptake, a major limiting factor for gene therapy remains the
lack of suitable vectors (Collins, 2006; Rolland, 2005). Therefore, the devel-
opment of safe and efficient gene carriers is a prerequisite for gene therapy
success. Two different approaches, viral and nonviral vectors, have primarily
been used to deliver genes (Merdan, Kopecek, & Kissel, 2002). The vast
majority of approved vectors for current clinical protocols are viral vectors
because of their high transfection efficiency. However, they possess immu-
nogenic properties and the potential ability to cause mutational infection and
toxic side reactions that could limit their doses and frequency of treatment
(Edelstein, Abedi, Wixon, & Edelstein, 2004; Verma & Somia, 1997).
Among nonviral vectors, lipids and polymers are by far the most widely used
gene carriers as alternatives. The cationic lipids such as N-[1-(2,3-
dioleyloxy)propyl]-N,N,N-trimethyl ammonium chloride (Felgner et al.,
1987), alkylammonium (Pinnaduwage, Schmitt, & Huang, 1989), cationic
cholesterol derivatives (Farhood, Bottega, Epand, & Huang, 1992),
gramicidine (Legendre & Szoka, 1993), N-[1-(2,3-dioleoyloxy)propyl]-
N,N,N-trimethyl ammonium ethyl sulfate (DOTAP) (Mansouri et al.,
2004) or LipofectamineTM2000 (Hawley-Nelson, Ciccarone, & Moore,
2008) incorporated with DNA are transferred effectively into cell
membranes. However, DNA/liposome complexes were rapidly cleared
from the bloodstream (Liu & Huang, 2002) and widely distributed in the
body (Song & Liu, 1998) with relatively large in size.
Cationic polymers have been proposed as an alternative approach to the
development of nonviral vectors. Generally, cationic polymers efficiently
form complexes with DNA and interact with negatively charged cell
surfaces. Polymer/DNA complexes are more stable than those involving
cationic lipids, and they protect DNA against nuclease degradation
(Gao & Huang, 1996). Examples include diethylaminoethyl dextran
Chitosan Derivatives as Gene Carriers 85
(Sompayrac & Danna, 1981), poly(L-lysine) (PLL; Wu & Wu, 1987), poly-
ethylenimine (PEI; Boussif et al., 1995), gelatin (Truong-Le, August, &
Leong, 1998), polyamidoamine dendrimer (Haensler & Szoka, 1993),
hexadimethrine bromide (Mumper et al., 1996), poly(vinyl imidazole)
(Ihm et al., 2003), poly(L-histidine)-g-PLL (Benns, Choi, Mahato,
Park, & Kim, 2000), poly(-amino ester) (Anderson, Akinc, Hossain, &
Langer, 2005), and chitosan (Rolland, 1998). Both PEI and the dendrimers
are effective gene carriers, which are synthetic polymers although their
potential toxicity is a concern. Among nonviral vectors, chitosan has been
considered to be a good gene carrier candidate, since it has been reported as a
biocompatible, biodegradable, and low toxic material with high cationic
potential. In spite of the high potential of chitosan as a gene carrier, its poor
solubility and low transfection efficiency have greatly impeded its practical
application. Over the years, methods to boost the transfection efficiency of
chitosan have been studied a great deal and a more general understanding of
parameters that may impact on the efficiency of transfection has been
attained. The objective of this chapter is to summarize the recent progress
in chitosan and its derivatives as gene (DNA or siRNA) carriers for gene
therapy. The roles of chemical modification using cell-specific ligand and
stimuli-response group for the enhancement of cell specificity and transfec-
tion efficiency in vitro and in vivo are also discussed.
2. LIGAND MODIFICATION FOR SPECIFIC CELL

TARGETING
An ideal gene carrier system should efficiently accumulate in specific
target tissues with minimal toxicity to nontarget tissues for increasing the
transfection efficiency of chitosan/DNA complexes. A variety of targeting
ligands conjugated to chitosan backbone are discussed in this part.
2.1. Galactose ligand modification

Mammalian hepatocytes possess asialoglycoprotein receptors (ASGP-R) at a
high density of 500,000 per cell that can recognize and bind exposed galac-
tose (Ashwell & Morell, 1974). Since Pricer and Ashwell first identified the
ASGP-R, introduction of galactose ligand into polymers has been widely
employed.
Trimethylated chitosan having antennary galactose residues was first
studied to get specific targeting to HepG2 cells (Murata, Ohya, & Ouchi,
1997). The modified chitosan had a tendency to increase the cellular
recognition ability with an increase of galactose residues, indicating that gal-

actosylated chitosan (GC) has been specifically internalized via the ASGP-R.
Also, Zhang, Tang, and Yin (2013) developed galactosylated trimethyl
chitosan-cysteine (GTC) nanoparticles as an oral delivery siRNA carrier
to the activated macrophages for treatment of ulcerative colitis. The cellular
uptake of GTC/TPP NPs in activated macrophages was significantly
enhanced compared to trimethyl chitosan-cysteine (TC)/TPP NPs owing
to galactose receptor-mediated endocytosis. Moreover, compared to
TC/TPP NPs, GTC/TPP NPs more efficiently promoted the distribution
of siRNA in ulcerative colon after oral administration.
Kim, Park, Nah, Choi, and Cho (2004) also reported that water-soluble
chitosan (WSC) was used and coupled with lactobionic acid bearing galac-
tose group as the specific ligand to ASGP-R of parenchymal liver cells
because GC could be specifically recognized by primary hepatocytes. Cyto-
toxicity study showed that GC prepared by the WSC had less cytotoxic on
cells. And transfection efficiency into HepG2, which has ASGP-R, was
higher than that into HeLa without ASGP-R. To further increase the bio-
compatibility, an endogenous compound, spermine was introduced into the
delivery system. Kim et al. (2012) synthesized a galactosylated poly(ethylene
glycol)-chitosan-graft-spermine (GPCS) copolymer with low cytotoxicity
and optimal gene delivery to hepatocytes using an amide bond between gal-
actosylated poly(ethylene glycol) and chitosan-graft-spermine, as shown in
Fig. 6.1A. The GPCS copolymer formed nanosized complexes with plasmid
DNA (pDNA), and the GPCS/DNA complexes had well-formed spherical
shapes. Furthermore, GPCS successfully delivered the luciferase reporter
gene through receptor-mediated interactions between galactose and
ASGP-R, as shown in Fig. 6.1B.
2.2. Folate ligand modification

Folic acid (FA) has emerged as an optimal ligand for targeting the cell mem-
brane and allows nanoparticle endocytosis via the folate receptor (FR) for
enhanced transfection efficiency. The FR is known to be overexpressed
on many human cancer cell surfaces, such as the brain, kidney, lung, ovarian,
and breast cancers, but is absent in most normal tissues (Sudimack & Lee,
2000). Lee, Lockey, and Mohapatra (2006) synthesized folate-coupled
chitosan to specifically deliver DNA to FR-overexpressing cancer cells.
They found that FA-conjugated chitosan exhibited significantly enhanced
gene transfer potential in FR-overexpressing cancer cells as compared to
B
a b
108 3000
Relative luciferase activity
107
RLU/mg protein
106
(GPCS/PCS)
2000
5
10
104 1000
***
103
102 0
HepG2 A549
A
20
20
40
40
0
I1
N
D
PE
PC
PC
PC
PC
G
Figure 6.1 (A) Synthesis of galactosylated poly(ethylene glycol)-chitosan-graft-

spermine (GPCS). (B) Transfection studies of GPCS. (a) Transfection efficiency of HepG2
cells by GPCS/DNA (pGL3-control), PCS/DNA, PEI/DNA complexes at various weight
ratios (mean SD, n 3). (b) Luciferase activity of the GPCS/DNA complexes at a weight
ratio of 20 normalized by that of PCS/DNA complexes on HepG2 and A549 cells.
unmodified chitosan. Also, a folate-PEG-coated polymeric liposome (FPL)

formed from octadecyl-quaternized lysine-modified chitosan and choles-
terol was prepared successfully (Wang et al., 2010). In cellular uptake exper-
iment, folate-coated FPLs showed significant higher uptake by MCF-7 cells
as compared to those without folate and traditional liposomes, because of the
folate-receptor-mediated endocytosis. Kim et al. (2013) synthesized folated
poly(ethylene glycol)-chitosan-graft-polyethylenimine (FPCP), as shown in
A Folic acid H2N-PEG-COOH

N N
H
N N O
N H O
O N COOH + O n OH
O COOH
NHS/DCC
N N
H
N N O CH2OH
N O
CH2OH H
H
O N COHN O n OH H O O
H O O
O COOH NHS O OH H
Fol-PEG + H
EDC H
CH2OH NH NH H NH
CH2OH H
PEI PEI CO
H O O
H O O
O OH H PEG
PEG PEG
H H
NH NH H NH2 Fol
Fol Fol
PEI PEI
FPCP
Chitosan-graft-PEI
(CHI-g-PEI)
B 14
**
12 ***
**
*
Over 1 mm tumor number
10
0
Con Pdcd4 FPCP/Pdcd4 PEI 25 kDa/
Pdcd4
Figure 6.2 (A) Proposed reaction scheme for synthesis of FPCP. (B) Intravenous injec-
tion of FPCP/Pdcd4 significantly inhibited the number of liver tumors. Tumor size over
1 mm, tumor numbers (n 4, *P < 0.05; **P < 0.01; ***P < 0.001).
Fig. 6.2A. The FPCP showed low cytotoxicity in various cell lines, and
FPCP/DNA complexes showed good cancer cell specificity as well as good
transfection efficiency in the presence of serum. Further, the FPCP/Pdcd4
complexes reduced tumor numbers and progression more effectively than
PEI 25kDa/Pdcd4 ones in H-ras12V liver cancer model mice after intrave-
nous administration, as shown in Fig. 6.2B.
2.3. Mannose ligand modification

It is well known that antigen-presenting cells (APCs) such as macrophages
and immature dendritic cells express high levels of mannose receptor that are
used for endocytosis and phagocytosis of a variety of antigens that expose
mannose. The APCs can activate the T-cells and the B-cells, which are
essential for the effective therapeutic manipulation of a wide spectrum of
immune functions, such as cancer (Raychaudhuri & Rock, 1998). Kim
prepared mannosylated chitosan (MCS) to induce the mannose receptor-
mediated endocytosis for targeting into APCs (Park et al., 2006). The results
indicated that MCS/DNA complexes showed higher transfection efficiency
on Raw264.7 macrophage cell line than WSC/DNA complexes. Jiang et al.
(2009) prepared mannosylated chitosan-graft-polyethylenimine (Man-
CHI-g-PEI) copolymer for targeting into APCs. They found that the trans-
fection efficiency of Man-CHI-g-PEI/DNA complexes into the Raw 264.7
macrophage cell line, which has mannose receptors, was higher than CHI-g-
PEI itself as well as PEI 25K. Also recently, Yao, Peng, Du, Luo, and Zong
(2013) developed MCS and condensed with preventative anti-GRP DNA
vaccine to evaluate the efficacy on inhibition of the growth of tumor cells.
The MCS/pGRP nanoparticles were intranasally administered in a subcu-
taneous mice prostate carcinoma model. The titers of anti-GRP IgG lasted
for 11 weeks were significantly higher than that for administration of
CS/pGRP nanoparticles and intramuscular administration of a pGRP
solution to mice, as shown in Fig. 6.3.
2.5 IgG1
IgG2a
*#
2 IgG
1.5 *#
OD450 nm
0.5
0
pGRP solution MCS/pGRP CS/pGRP
nanoparticles nanoparticles
Figure 6.3 Antigen-specific IgG1, IgG2a, and IgG levels after intranasal vaccination with
MCS/pGRP nanoparticles and CS/pGRP nanoparticles and intramuscular vaccination
with a pGRP solution (n 6). *P < 0.01 for MCS/pGRP nanoparticles versus CS/pGRP
nanoparticles; #P < 0.05 for MCS/pGRP nanoparticles versus the pGRP solution.
2.4. Hyaluronic acid ligand modification

Hyaluronic acid (HA) is a naturally occurring anionic, noncytotoxic, biode-
gradable polysaccharide. HA of low molecular weight are able to bind clus-
ter determinant 44 (CD44), which is highly expressed in tumor and often
used as a tumor targeting ligand for drug delivery (Qhattal & Liu, 2011).
Ravina et al. (2010) designed HA and chitosan-g-poly(ethylene glycol)
(CS-g-PEG) nanoparticles for a broad range of gene delivery applications.
The resulting nanoparticles were effective carriers for the delivery of both
siRNA and pDNA with minimal cytotoxicity. Moreover, in the HEK-
enhanced green fluorescent protein (EGFP)-Snail1 cell line, the HA/
CS-g-PEG nanoparticles have the ability to silence the expression of the
Snail1 transcription factor, suggestion of great potential for gene therapy
applications. HA of low molecular weight is able to bind toll-like receptor
4 (TLR-4), which are highly expressed in osteoarthritic chondrocytes.
Therefore, Lu et al. used hybrid HA/CS nanoparticles as novel nonviral
gene delivery vectors capable of transferring exogenous genes into primary
chondrocytes for the treatment of joint diseases (Lu, Zhao, Wang, & Lv,
2011). The transfection efficiency of HA/CS/DNA nanoparticles was sig-
nificantly higher than that of CS/DNA nanoparticles under the same con-
ditions and the average viability of cells transfected with HA/CS/DNA
nanoparticles was over 90%. These results suggest that HA/CS/DNA
nanoparticles could be an effective nonviral vector suitable for gene delivery
to chondrocytes.
3. STIMULI-RESPONSE MODIFICATION FOR

ENHANCEMENT OF TRANSFECTION EFFICIENCY
Stimuli response is an important property that affects the ability of sys-
tems to deliver drugs (including nucleic acid) effectively. Drug release could
be triggered by physical or chemical stimuli, such as pH, magnetic field, and
reducing intracellular environment.
3.1. pH-sensitive modification

Although efficient gene transfection includes several main steps, such as
DNA complexation, cellular uptake, endosomal escape, release of DNA,
and transfer into the nucleus, an inefficient release of DNA from endosomes
into the cytoplasm is the primary causes of poor transfection efficiency. The
pH-sensitive chitosan carriers could overcome this problem.
3.1.1 Imidazole modification

Imidazole-containing polymers were reported to have the ability to act as a
proton sponge and enhance release of the complexes into the cytoplasm.
Kim, Ihm, Choi, Nah, and Cho (2003) coupled urocanic acid (UA) as an
imidazole ring bearing molecule with WSC (UAC). The transfection effi-
ciency in 293T cells by UAC/DNA complexes was increased with increasing
the substitution value of UA due to the buffering capacity in the endosomal
compartment, which is similar to that of PEI. Also, wild-type p53 gene was
transfected into HepG2 and into BALB/c nude mice through intratumoral
injection using UAC by Wang et al. (2008). The results indicated that
UAC-mediated efficient p53 gene transfer-induced apoptosis by inhibiting
the growth of HepG2 cells in vitro and in vivo. Shi, Shen, Zhang, Bi, and
Dai (2011) report the synthesis of a novel amphiphilical chitosan derivative,
N-imidazolyl-O-carboxymethyl chitosan (IOCMCS) as shown in Fig. 6.4.
Figure 6.4 Synthesis of imidazole-O-carboxymethyl chitosan (IOCMCS).

The IOCMCS bearing an imidazole ring played the crucial role in endosomal
rupture through proton sponge mechanism. After chemical modification, the
solubility of the resulting polymer is enhanced, and the polymer is soluble in a
wide pH range (pH 410). The transfection in HEK293T cells using the
IOCMCS as gene delivery vector demonstrated the high transfection effi-
ciency although it is dependent on the degree of imidazolyl substitution.
3.1.2 PEI modification

Another more efficient strategy is grafting PEI to chitosan backbone, for PEI
has an excellent buffering capacity and thus facilitates endosomal escape.
Wong et al. (2006) prepared PEI-graft-chitosan by performing cationic poly-
merization of aziridine in the presence of water-soluble oligo-chitosan. The
result is a kind of combination of an oligo-PEI with proton sponge effect,
and chitosan with biocompatible and biodegradable nature. The results indi-
cated that PEI-g-chitosan had a lower cytotoxicity than PEI 25K and PEI-g-
chitosan showed higher transfection efficiency than that of PEI 25K both
in vitro and in vivo.
Jiang et al. (2007) synthesized chitosan-graft-PEI by an imine reaction
between periodate-oxidized chitosan and amine groups of low MW PEI
as shown in Fig. 6.5A. The chitosan-graft-PEI/DNA complexes showed
higher transfection efficiency than PEI 25K in HeLa, 293T and HepG2 cell
lines at high N/P ratio with low cytotoxicity owing to the buffering effect of
PEI and the transfection efficiency of the complexes was comparable to that
of LipofectamineTM 2000, as shown in Fig. 6.5B. Thus, among the chitosan
derivatives studied, the incorporation of low MW PEI into chitosan
enhanced the highest transfection efficiency with low cytotoxicity.
3.2. Thiolated modification

It has been demonstrated that chemically modified disulfide bonds can
improve gene delivery and expression when compared to unmodified poly-
mers because these bonds can be cleaved by cytoplasmic glutathione, thus,
releasing the DNA more efficiently. Thiolated chitosan forms inter-
molecular as well as intramolecular disulfide bonds upon oxidation, allowing
cross-linking of chitosan, which in turn may allow high level of enzyme
inhibitory and permeation enhancing properties. Lee et al. (2007) prepared
thiolated chitosan as a gene delivery system first. It was reported that
thiolated chitosan exhibited a significantly higher gene transfer potential
and sustained gene expression upon cross-linking, indicating their great
potential for gene therapy. Talaei, Azizi, Dinarvand, and Atyabi (2011) used
Figure 6.5 (A) Proposed reaction scheme for synthesis of CHI-g-PEI. (B) Transfection effi-
ciency of copolymer/DNA (pGL3-control) complex at various N/P ratios in 293T cell line.
N-acetyl cysteine-chitosan (NAC-C) and N-acetyl penicillamine-chitosan

(NAP-C) to deliver antisense oligonucleotide (ASOND). Drug release data
after 15 h showed that ASOND were completely released from chitosan-
based particles while a lower and more sustained release of only 22%
8% was released for thiolated particles. Moreover, the ASOND-loaded
thiolated particles significantly suppressed EGFR gene expression in
T47D cells compared with ASOND-loaded chitosan particles and down-
regulated EGFR protein expression in cells.
Glutathione in its reduced form (GSH), a small tripeptide formed by glu-

tamic acid, cysteine, and glycine, widely presenting in a variety of animal
tissues, can role as specific peptide ligand to promote cell adhesion. Mean-
while, free thiol group is able to chelate divalent metal ions, which are essen-
tial to DNase for its activity (Krezel & Bal, 2003). Li et al. (2011) developed a
new GSH and poly[poly(ethylene glycol) methacrylate] (PMPEG) modified
chitosan copolymer. The introduction of brush-liked PMPEG and GSH
groups in CS could prevent self aggregation among cationic polymer and
increase binding ability with cell membrane, thus cellular uptake rate is pro-
moted. CSPMPEGGSH can protect the pDNA from nuclease degrada-
tion during extracellular transit. Disulfide cleavage induced by intracellular
GSH lead to complex dissociation and pDNA release for nuclear transport
and gene transcription. The synthesized CSPMPEGGSH conjugate as a
novel gene delivery vector showed excellent transfection efficiency in
NIH3T3 cells.
3.3. Amino acid modification

Layek & Singh (2013a) developed a series of hydrophobic amino acid grafted
chitosan (AGC) derivatives synthesized by carbodiimide-mediated coupling
reaction. The AGC polymers could effectively bind and condense pDNA to
form polyplexes in the size range of 161263 nm and possessed net cationic
charge. The resultant polyplexes showed 3.45.4-fold greater cellular
uptake and 1330-fold higher transfection efficiency in HEK 293 cells as
compared to unmodified chitosan. Moreover, both cellular uptake and
transfection efficiency were improved with the increasing amino acid
hydrophobicity. Hydrophobic amino acid substitution contributed to the
enhancement of pDNA release at cytosolic pH. These data demonstrate
AGC polymer as a promising novel nonviral gene delivery vector.
3.4. Magnetic modification

Superparamagnetic iron oxide nanoparticles (SPIONs) are highly magne-
tized in a magnetic field, allowing magnetic tumor targeting and biomedical
imaging, but lose their magnetization when the field is switched off, thus
reducing unwanted side effects such as thrombosis due to magnetically
induced aggregation (Allard-Vannier et al., 2012). Development of mag-
netic nanoparticles and techniques for their safe transport and concentration
in specific sites in the body would constitute a powerful tool for gene/drug
therapy in vitro and in vivo. Bhattarai et al. (2008) developed hexanoyl
chloride-modified chitosan (Nac-6) stabilized iron oxide nanoparticles

(Nac-6-IOPs) as magnetic nanoparticles for viral gene (Ad/LacZ) delivery
via magnetofection. This vector, Nac-6-IOPs/Ad/LacZ, bound to K562
cells in the presence of external magnetic fields and resulted in enhanced
expression of the transgene in those cells that do not exhibit the
coxsackie-adenovirus receptor. Kumar et al. combined chitosan with a
superparamagnetic particle (Fe2O3) to synthesize hydrophobic magnetic
nanoparticles. Chitosan-coated magnetic Fe2O3 particles combined with
pDNA expressing EGFP were injected into mice and directed to the lungs,
heart, and kidneys in vivo by means of external magnets of 0.0025 tesla. The
expression of EGFP in these sites was visualized by whole-body fluorescent
imaging (Kumar et al., 2010).
Magnetic resonance imaging (MRI) is another important application for
magnetic modification chitosan. Wang et al. (2012) reported the develop-
ment of dual-purpose chitosan and PEI-coated magnetic micelles (CP-mag-
micelles) to deliver nucleic acid-based therapeutic agents and also to
provide MRI.
4. PENETRATING MODIFICATION
4.1. Brainblood barrier penetrating modification
Delivery of therapeutic molecules to the brain is a challenging task. Malmo,
Sandvig, Varum, and Strand (2013) used chitosan/siRNA nanoparticles to
silence the expression of the P-gp in the blood brain barrier. They showed
that the transfection in rat brain endothelial cells mediated effective knock-
down of P-gp with subsequent decrease in P-gp substrate efflux. This
resulted in increased drug delivery from the bloodstream to the brain.
Malhotra, Tomaro-Duchesneau, and Prakash (2012) synthesized a trans-
activator of transcription-tagged PEGylated chitosan (Chitosan-PEG-
TAT) polymer to deliver siRNA for curing of neurodegenerative diseases
(ND). TAT, comprised of arginine and lysine amino acid residues, having
an important role in the translocation across biological membrane, has been
shown to destabilize the lipid bilayer of the cell membrane through an
energy independent pathway carrying hydrophilic or macromolecules as
large as several hundred nanometers in size across the plasma membrane.
The nanoparticles were tested to deliver a functional siRNA against the
Ataxin-1 gene in an in vitro established model of a ND spinocerebellar ataxia
(SCA1) overexpressing ataxin protein. The results indicated successful
suppression of the SCA1 protein following 48 h of transfection. Result of

this study has potential in ND-like SCA, Parkinsons, Alzheimers, and
others.
4.2. Cell penetration peptide modification

Introduction of cell penetration peptide (CPP) to chitosan could improve its
ability to form complexes with nucleic acid, as well as enhance the cellular
uptake and transfection efficiency of chitosan-based nanoparticles. To this
end, a peptide with nine repeating arginine residues was chemically coupled
to the chitosan backbone, and various characteristics of nonaarginine-
chitosan/siRNA nanoparticles were investigated (Park et al., 2012). The
mean diameter and zeta potential of the nonaarginine-chitosan/siRNA
nanoparticles were dependent on the amount of nonaarginine conjugated
to chitosan. Nonaarginine-modified chitosan/siRNA nanoparticles
demonstrated enhanced cellular association and transfection efficiency
in vitro, while maintaining a low level of cytotoxicity. Penetratin
(RQIKIWFQNRRMKWKKGG), a well-studied CPP derived from the
third -helix of the Drosophila Antennapedia homeodomain protein, was
incorporated into linoleic acid and penetratin dualfunctionalized chitosan
(CS-Lin-Pen) because of its ability to increase cellular uptake of attached
cargos (Layek & Singh, 2013b). The amphiphilic CS-Lin-Pen self-
assembled to form cationic micelles in an aqueous environment and
exhibited excellent hemocompatibility and cell viability. When CS-Lin-
Pen micelles were added to pDNA solution, the electrostatic interaction
between the cationic micelles and anionic pDNA led to the formation of
stable CS-Lin-Pen/pDNA polyplexes. The resultant polyplexes demon-
strated fivefold higher cellular uptake as compared to unmodified chitosan.
4.3. Penetration of nuclear membrane

Nuclear membrane is a serious barrier to the delivery and expression of exog-
enous gene and thus many attempts have been made to overcome this barrier.
Among various approaches, the use of nuclear localization signal (NLS) pep-
tides for nonviral gene transfer has been widely investigated (Dean, Byrd,
James, & Dean, 1999; Prasad & Rao, 2005). NLS, typically consisted of
one or more short sequences of positively charged lysines or arginines, could
help nanoparticles transport into the nucleus through interaction with one or
more of a broad family of NLS receptors. In preparation of CS/DNA com-
plexes containing NLS, peptide with NLS was directly incorporated without
covalent conjugation to pDNA or chitosan (Opanasopit, Rojanarata,

Apirakaramwong, Ngawhirunpat, & Ruktanonchai, 2009). The gene trans-
fer efficiency of CS/DNA complexes with and without NLS was evaluated
in the human cervical carcinoma cell line (HeLa cells). The CS/DNA com-
plexes containing NLS increased transfection efficiencies in a NLS-dose
dependent manner on the HeLa cells, compared to the control (CS/DNA
complexes or NLS). The highest transfection efficiency was significantly
observed in CS/DNA complexes at the weight ratio of 8 with 120 g
NLS and was 74-fold higher than that in the cells transfected with CS/DNA
complexes.
5. CONCLUSION
As described in this chapter, among nonviral vectors, chemical mod-
ifications of chitosan have been attempted in vitro and in vivo for DNA and
siRNA delivery. Specific ligand-coupled chitosan ferried DNA into
targeted cells. Use of environmental stimuli to promote gene delivery is also
a promising strategy. Although the high water solubility, low toxicity, rel-
ative ease of production, and availability of chemical modification of
chitosan for enhancement of transfection efficiency and cell specificity allow
its use in a wide variety of gene therapy, continued development of the
structurefunction relationships and fundamental studies of cellular pro-
cesses in vitro and in vivo should be necessary for future direction of chitosan
as a gene carrier. Also, more in vivo studies need to be carried out for clinical
trials because the majority of studies carried out so far are only in vitro and in
animal models. Ultimately, the optimum use of chitosan will expand the tra-
ditional applications of gene therapy within the clinical trials and will pro-
vide an additional technology for tissue regeneration and gene regulation.
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CHAPTER SEVEN
Marine Carbohydrates of
Wastewater Treatment
Prasad N. Sudha1, Thandapani Gomathi, P. Angelin Vinodhini,
K. Nasreen
1
Corresponding author: e-mail address: drparsu8@gmail.com
Contents
1. Introduction 104
1.1 Sources of wastewater 105
1.2 Composition of wastewater 105
1.3 Wastewater treatment 107
2. Materials Used for Wastewater Treatment 110
2.1 Chitin 111
2.2 Chitosan 112
2.3 Alginate 114
2.4 Agar 116
2.5 Carrageenan 117
3. Application of Marine Polysaccharides in Wastewater Treatment 118
3.1 Chitin 118
3.2 Chitosan 120
3.3 Alginate 123
3.4 Carrageenan and agar 126
4. Advantages and Possible Drawbacks of Using Marine Polysaccharide-Based
Materials for Adsorption 127
4.1 Advantages 127
4.2 Limitations 128
6. Conclusions 129
Acknowledgments 129
References 129
Abstract
Our natural heritage (rivers, seas, and oceans) has been exploited, mistreated, and con-
taminated because of industrialization, globalization, population growth, urbanization
with increased wealth, and more extravagant lifestyles. The scenario gets worse when
the effluents or contaminants are discharged directly. So wastewater treatment is a very
important and necessary in nowadays to purify wastewater before it enters a body of
natural water, or it is applied to the land, or it is reused. Various methods are available for
http://dx.doi.org/10.1016/B978-0-12-800268-1.00007-X
104 Prasad N. Sudha et al.
treating wastewater but with many disadvantages. Recently, numerous approaches

have been studied for the development of cheaper and more effective technologies,
both to decrease the amount of wastewater produced and to improve the quality of
the treated effluent. Biosorption is an emerging technology, which uses natural mate-
rials as adsorbents for wastewater treatment. Low-cost adsorbents of polysaccharide-
based materials obtained from marine, such as chitin, chitosan, alginate, agar, and
carrageenan, are acting as rescue for wastewater treatment. This chapter reviews the
treatment of wastewater up to the present time using marine polysaccharides and
its derivatives. Special attention is paid to the advantages of the natural adsorbents,
which are a wonderful gift for human survival.
1. INTRODUCTION
Water is essential for all known life forms. For many decades, people
were wondering about the source of water, but the answer took long time.
Clean drinking water is important for overall health and plays a substantial
role in infant and child health and survival (Anderson, Romani, Phillips, &
van Zyl, 2002; Fewtrell et al., 2005; Ross, Rich, Molzen, & Pensak, 1988;
Vidyasagar, 2007). Today, we live in an era when all scientists tell us that war
of the future will be for the water. These wars will occur because of the scar-
city of pure water due to the environmental pollution. Therefore, scientists
are searching for the remedy of water pollution.
In recent years, pollution has become one of the most important prob-
lems in the global context as a consequence of industrialization, globaliza-
tion, population growth, urbanization with increased wealth, and more
extravagant lifestyles. When toxic substances enter water bodies like lakes,
streams, rivers, aquifers, and oceans, naturally or through any human activ-
ity, they either get dissolved or lie suspended or deposited on the bed in
water. This results in the contamination of water bodies whereby the quality
of the water deteriorates and can cause diseases, illnesses, epidemics, and
health problem to animals and plants (Coffey, Cummins, Cormican,
Flaherty, & Kelly, 2007; Harris, Miller, & Foster, 2008; Reiff, Roses,
Venczel, Quick, & Witt, 1996). It has been suggested that it is the leading
worldwide cause of deaths and diseases (Pink, 2006) and that it accounts for
the deaths of more than 14,000 people daily (West, 2006). Hence, treating
the wastewater becomes necessary and it is an opportune time, to refocus on
one of the ways to recycle water through the reuse of urban wastewater, for
irrigation and other purposes. The searches for new cost-effective technol-
ogies for the removal of contaminants from wastewater are swiftly increasing
nowadays.
Marine Carbohydrates of Wastewater Treatment 105
1.1. Sources of wastewater

Wastewater is not just sewage. All the water used in (a) home, which
includes baths, showers, sinks, dishwashers, washing machines, and toilets;
(b) commercial/service like schools, hospitals, restaurants, offices, hotels,
and small industries; (c) industries; and (d) nonpoint sources that goes down
the drains or into the sewage collection system (Metcalf & Eddy, 1981) is the
wastewater. Small businesses and industries often contribute large amounts
of wastewater to sewage collection systems.
The considerable public concern about the potential risks associated with
pathogens and inorganic and organic contaminants to the environment and
human health (Abdulraheem, 1989). The important source of biological
pollution is domestic sewage and industrial wastes; solid excrete from human
bodies and decomposable organic matter of sewage are the best medium for
the development of bacteria in water (Sharma, 2005). The sources of organic
contaminants are proteins, fats, carbohydrates, etc., as well as synthetic com-
pounds like dyes, pesticides, and herbicides. The synthetic organic deriva-
tion causes more harm to the environment than naturally occurring ones.
The classes of inorganic pollutants are acids, alkalis, heavy metals, and
anionic radicals. Mainly the heavy metal source assessment has divided heavy
metal sources into three main source pathways: domestic wastewater, trade
effluent, and storm runoff. Trade effluents are further divided into commer-
cial effluents, which arise from nonconsented trade activities, and industrial
effluents, which arise from consented trade activities. Major sources of pol-
lution are industries producing metals, paper and pulp, textiles, and food and
beverages. The mining industry is also a significant contributor (Evans,
Hanjra, Jiang, Qadir, & Drechsel, 2012).
1.2. Composition of wastewater

The constituents in wastewater can be divided into main categories
according to Table 7.1. The composition of wastewater may differ from
community to community; all wastewater contains the following groupings
of constituents.
Since all the pollutants are hazardous to the living things, the heavy
metals are highly concern due to its abundance. On the basis of chemical
properties, applications, and physiological effects on life, metals were
divided into four main categories. They are (i) toxic heavy metals,
(ii) strategic metals, (iii) precious metals, and (iv) radionuclides
(Volesky & Holan, 1995). So far, 23 metals are regarded as heavy metals such
as antimony, arsenic, bismuth, copper, cadmium, cerium, cobalt, gallium,
Table 7.1 Composition of wastewater
Composition of wastewater
Physical Chemical Biological
Inorganic Organic
Aggregate organic Typical domestic
constituents waste Individual compounds
Solids, turbidity, Nutrients (nitrogen, Organic constituents 4060% protein, Phenols, pesticides, Microorganisms
color, phosphorus, with similar 2550% detergents, pesticides, pathogenic
temperature, ammonium), metals characteristics that carbohydrate, fat, oil and grease, bacteria, virus,
density, (Hg, Pb, Cd, Cr, Cu, cannot be distinguished 812% oilfat coloring, solvents, and worms eggs
conductivity Ni), gases, chloride, separately phenols, cyanide
sulfur, alkalinity
gold, iron, lead, manganese, mercury, lead, nickel, platinum, silver, tellu-
rium, thallium, tin, uranium, vanadium, and zinc (Glanze, 1996). In that,
the big three toxic heavy metals were mercury, lead, and cadmium by
considering their major impact on environment (Volesky, 1994).
For the survival of living things, pure water is much essential. The con-
stituents in untreated wastewater can be divided into three types: physical,
chemical, and biological. Physical constituents are the particles or solids in
the effluent. Effluent is defined as liquid waste that is untreated, partially
treated, or completely treated. Chemical constituents include nutrients
and heavy metals (Table 7.2). Biological constituents (Table 7.3) include
coliform organisms and other microorganisms such as bacteria, protozoa,
helminthes, and viruses (Crites & Tchobanoglous, 1998).
In order to meet the growing demand for potable water and water of
good quality for industrial use, it has become necessary to treat wastewaters
for renovation, reuse, and pollutant removal before mixing with natural
water bodies containing good quality water.
1.3. Wastewater treatment

There are many types of wastewater including domestic, commercial, indus-
trial, and agricultural (Crites & Tchobanoglous, 1998). The typical waste-
water treatment includes primary treatment, secondary treatment, sludge
treatment, and advanced treatment. The primary treatment includes the
Table 7.2 World Health Organization (WHO) tolerance limits in

drinking water
Heavy metal Maximum acceptable concentration (mg/L)
Zinc 5
Arsenic 0.01
Magnesium 50
Calcium 50
Cadmium 0.003
Lead 0.01
Silver 0.0
Mercury 0.001
Copper 0.5
Chromium 0.1
Table 7.3 Principal constituents of concern in wastewater treatment (Crites &

Tchobanoglous, 1998)
Constituents Reason for concern
Total suspended solids Sludge deposits and anaerobic conditions
Biodegradable organics Depletion of natural oxygen resources and the
development of septic conditions
Dissolved inorganics (e.g., Inorganic constituents added by usage. Recycling
total dissolved solids) and reuse applications
Heavy metals Metallic constituents added by usage. Many metals
are also classified as priority pollutants
Nutrients Excessive growth of undesirable aquatic life,
eutrophication, nitrate contamination of drinking
water
Pathogens Communicable diseases
Priority organic pollutants Suspected carcinogenicity, mutagenicity,
teratogenicity, or high acute toxicity. Many priority
pollutants resist conventional treatment methods
(known as refractory organics)
removal of sludge from the drains and sewers by bar screens and settling. The
secondary treatment follows the primary treatment by aeration and agitation
to treat microorganisms, sedimentation, and lagoons. Tertiary treatment
(advanced treatment) may include processes to remove nutrients, such as
nitrogen and phosphorus, and carbon adsorption to remove chemicals.
These processes can be physical, biological, or chemical. Settled solids
(sludge) from primary treatment and secondary treatment are given further
treatment and undergo several as anaerobic digestion, thickening, heating,
centrifuge, dewatering, and finally for disposal (Water Environment
Federation, 2001).
The main drawback with using conventional sewage treatment is that the
sewage effluent may still contain a very large number of pathogenic organ-
isms after it has passed through the treatment plant. Chlorination of sewage
effluent before discharge to inactivate pathogens is not recommended, even
if there is a serious epidemic in progress. It is expensive, rarely effective, and
furthermore may have a severe negative impact on health and environment.
More number of different treatment techniques for wastewater laden
with heavy metals has been developed in recent years both to decrease
the amount of wastewater produced and to improve the quality of the treated
effluent. Although various treatments such as biological treatments (McMullan

et al., 2001; Pearce, Lloyd, & Guthrie, 2003), chemical precipitation, membrane
processes (Cassano, Molinari, Romano, & Drioli, 2001; Goncharuk,
Kucheruk, Kochkodan, & Badekha, 2002), advanced oxidation processes
(Al-Momani, Touraud, Degorce-Dumas, Roussy, & Thomas, 2002;
Torrades, Perez, Mansilla, & Peral, 2003), coagulationflocculation
(Koprivanac, Bozic, & Papic, 2000; Somasundaran & Runkana, 2005; Zeng,
Yang, Zhang, & Pu, 2007), reverse osmosis, ultrafiltration (UF), electrodialysis
flotation (Chen, Chen, & Yue, 2002; Hu, Lo, & Kuan, 2003; Von Gunten,
2003), ion exchange, phytoremediation, and adsorption (Gupta, Jain, Ali,
Sharma, & Saini, 2003; Netpradit, Thiravetyan, & Towprayoon, 2004;
Rivera-Utrilla, Bautista-Toledo, Ferro-Garcia, & Moreno-Castilla, 2003)
can be employed to remove heavy metals from contaminated wastewater, they
have their inherent advantages and limitations in application. Coagulation and
flocculation are some of the important treatments used for industrial effluents
before discharging them into receiving waters to remove toxic waste (Dos
Santos, Cervantes, & Van Lier, 2007; Gahr et al., 1994).
The disadvantages of the commonly used procedures for removing metal
ions are as below (Ahalya, Ramachandra, & Kanamadi, 2003; Table 7.4).
Adsorption has been considered as possibly the most cost-effective
method for wastewater treatment, because the process is simple, chemical
consumption and waste generation are not significant issues, and adsorbents
can be regenerated and used again. Recently, numerous approaches have
been studied for the development of cheaper and more effective adsorbent
for wastewater treatment using natural biopolymers. Among these, marine
Table 7.4 Some common methods for treating wastewater and their limitations
Common methods Limitations
Reverse osmosis It is expensive
Electrodialysis Results in the formation of metal hydroxides which clog the
membrane
Ultrafiltration Sludge is generated
Ion exchange High cost and partial removal of certain ions
Chemical Sludge containing toxic compounds is produced
precipitation
Phytoremediation Takes long time for the removal and regeneration of the plant
for further biosorption is difficult
organisms still remain a largely unexploited resource on what concerns

its biotechnology application (Da Silva, Bierhalz, & Kieckbusch, 2012).
Polysaccharides such as chitin (Bailey, Olin, Bricka, & Adrian, 1999;
Ravi Kumar, 2000; Varma, Deshpande, & Kennedy, 2004) and their deriv-
atives chitosan (Babel & Kurniawan, 2003), alginate (Da Silva et al., 2012),
agar, and carrageenan are used for wastewater treatment.
2. MATERIALS USED FOR WASTEWATER TREATMENT

In addition to other conventional methods, adsorption is another
effective and widely used method for wastewater remediation. This method
is cost effective and easy to adopt. Today, there is a growing interest in
developing natural low-cost alternatives to synthetic polymers (Crini,
2006). Various adsorption materials have been studied, such as activated car-
bons, clays, polymeric synthetic resins, metal oxides, and some natural mate-
rials. Among them, activated carbon is one of the oldest and most commonly
used adsorbents.
Generally, a suitable adsorbent for adsorption process of pollutants
should meet several requirements such as
a. efficient for removal of a wide variety of target pollutants,
b. high capacity and rate of adsorption,
c. important selectivity for different concentrations,
d. granular type with good surface area,
e. high physical strength,
f . able to be regenerated if required,
g. tolerant for a wide range of wastewater parameters,
h. low cost.
The removal of metals, compounds, and particulates from solution by
biological material is recognized as an extension to adsorption and is named
as biosorption (Boddu, Abburi, Talbott, & Smith, 2003). Sharma, Kaushik,
and Kaushik (2005) reported the various natural and cheaper alternatives
such as wood ash, brick powder, sugarcane bagasse, tea leaves, ash, and
charcoal based on batch adsorption studies for decolorizing the effluent.
Several authors presented review articles on the removal of dyes using
low-cost adsorbents (Singh & Srivastava, 2001). Many biosorbents such
as banana pith (Namasivayam and Kanchana, 1992; Namasivayam,
Prabha, & Kumutha, 1998), coir pith (Namasivayam et al., 2001;
Namasivayam, Jeyakumar, & Yamuna, 1994), bagasse pith (Chen, Hui, &
McKay, 2001), corn cob (Robinson, Chandran, & Nigam, 2002c), saw dust
(Shukla, Zhang, Dubey, Margrave, & Shukla, 2002), apple pomace

(Robinson, Chandran, & Nigam, 2002a), date pits (Banat, Al-Asheh, &
Al-Makhadmeh, 2003), peat (Ho & McKay, 1998, 2003), fruit peel
(Kumar, 2007; Kumar & Porkodi, 2007), rice husk ash (Chandrasekhar &
Pramada, 2006), spent tea leaves (Hameed, 2009), Brazil shells (de Oliveira
Brito, Andrade, Soares, & de Azevedo, 2010), lemon peel (Bhatnagara,
Minocha, & Sillanpaa, 2010), Acacia leucocephala bark powder (Munagapati,
Yarramuthi, & Nadavala, 2010), coconut-based biosorbents (Bhatnagara
et al., 2010), fungi (Acosta, Rodriguez, Guiterrez, & Motctezuma, 2004),
algae (Gupta, Shrivasthava, & Jain, 2001), seaweeds (Elangovan, Philip, &
Chandraraj, 2008; Kratochvil & Volesky, 1998), microorganisms (Fan
et al., 2008; Sahin & Ozturk, 2005), and several biopolymers (Bailey et al.,
1999; Wu, Tseng, & Juang, 2000) have been utilized.
The polysaccharides from the renewable resources are currently being
explored intensively for their applications in water treatment (Gupta &
Ravikumar, 2000). Among the polysaccharide compounds such as chitin
(Ravi Kumar, 2000), starch (Wurzburg, 1986), and their derivatives,
chitosan (Varma et al., 2004) deserves particular attention. These polysac-
charides are abundant, renewable, biodegradable, and low cost and are
the best choice in water treatment and useful tool for protecting the envi-
ronment (Bolto, 1995).
Among the polysaccharides from the renewable resources, the marine
polysaccharides play a vital role in remediating wastewater. They are having
great potential over other biopolymers. The marine polysaccharides such as
chitin and its derivatives chitosan, alginate, agar, and carrageenan are
discussed below.
2.1. Chitin
Chitin consists mainly of -(14)-2-acetamido-2-deoxy-D-glucopyranose
units (Fig. 7.1). Next to cellulose, chitin is the most abundant organic sub-
stance presents on earth. Chitin is a constituent of the natural dissolved and
particulate organics and part of fungi and bacteria. It exists as two different
crystalline forms, -chitin and -chitin, while a third form, -chitin, is a
combination of the and forms (Lotmar & Picken, 1950; Rudall &
Kenchington, 1973). -Chitin chains are the most abundant form that are
arranged in an antiparallel fashion, are very stable, and are present in insect
cuticles, shells of crabs, lobsters, shrimp, and fungal cell walls (Carlstr
om,
1957). -Chitin occurs in diatoms, the pens of squid, the chaetae of annelids,
Figure 7.1 Chemical structure of chitin.
and the tubes secreted by tubeworms of the Siboglinidae family (Annelida).

The distinct molecular packing patterns of the chitin polymorphs confer dif-
ferent physicochemical properties to their corresponding crystalline
structures.
Chitin is rapidly biodegraded (Zobell & Rittenberg, 1983). The annual
worldwide chitin production has been estimated to be 1011 tons, and indus-
trial use has been estimated to be 10,000 metric tons (Kurita, 2006). Chitin
has excellent biocompatibility, nontoxicity, and wound-healing properties,
so it has been widely applied in medical and health-care fields for applica-
tions such as release capsules for drugs, man-made kidney membranes,
anticoagulants, and immunity accelerants (Cho, Cho, Chung, Yoo, &
Ko, 1999; Matsuyama, Kitamura, & Naramura, 1999; Muzzarelli, 1977;
Okamoto et al., 2002; Onishi, Nagai, & Machida, 1997; Tokura et al.,
1990). However, chitin is not soluble in common solvents due to the exis-
tence of intra- and intermolecular hydrogen bonds in chitin and its highly
crystalline structure. This strongly restricts many applications of chitin.
The presence of amino and hydroxyl groups in the chitin repeat
unit provides many opportunities for chemical modification (Kurita,
2001; Kurita et al., 1998). Etherification is one of the most important
modifications used to prepare water-soluble chitin derivatives, including
carboxymethylchitin, hydroxyethylchitin, and hydroxypropylchitin (Hirano,
1988; Qin et al., 2006; Sini, Santhosh, & Mathew, 2005). Alkali treatment
swells and decrystallizes the chitin structure and improves the accessibility of
chitin. From the earlier reports, chitin and its derivatives were used in wide
range of applications.
2.2. Chitosan
Chitosan is one of the natural polymers that have attracted great atten-
tion recently. Chitosan is a heteropolymer with high content of amine
Figure 7.2 Chemical structure of chitosan.
(dNH2) functional group and remarkable availability. It is composed of

both glucosamine and acetylglucosamine units and produced by the alkaline
deacetylation of chitin present in insect cuticles and crustacean shells (Gibbs,
Tobin, & Guibal, 2004; Jin & Bai, 2002). Chitosan is a high molecular
weight polysaccharide composed mainly of -(1,4)-linked D-glucosamine
and partially of -(1,4)-linked N-acetyl-D-glucosamine (Fig. 7.2).
Compared with chitin, chitosan has lower crystallinity. The crystallinity
is decreased during the deacetylation process, due to the removal of a portion
of the acetyl groups from the chitin structure and thus to the greater presence
of primary amine groups (Pillai, Paul, & Sharma, 2009). These facts change
the intra- and intermolecular interactions, conformed mainly by hydrogen
bonds (Ramrez-Coutino, Marn-Cervantes, Huerta, Revah, & Shirai,
2006), and generate amorphous zones in the biopolymer, with swelling
capacity higher than that of crystalline zones, owing in part to the great affin-
ity of primary amine groups to water. The lower crystallinity of chitosan
increases the accessibility to the adsorption sites of the biopolymer.
Though chitosan is a hydrophilic polymer, its dissolution in water only
takes place in dilute acidic solutions, in which organic acids (for example,
formic acid, acetic acid) or mineral acids (for example, HCl, HNO3) are uti-
lized (Galvao Barros, Caldeira Brant, & Catalani, 2011). The amino groups
of the polymer are protonated in acidic medium and a soluble polysaccharide
with positive charge is obtained. The multitude of cationic sites formed due
to the protonation of amino groups by acids along the chitosan chain
increases its solubility by increasing both the polarity and the degree of elec-
trostatic repulsion (Mourya & Inamdar, 2008). Solubilization of chitosan is
an important property for its varied usage. The amino groups are responsible
for several straightforward chemical modifications of chitosan, which predis-
poses its ongoing development for many applications (Alves & Mano, 2008;
Clasen, Wilhelms, & Kulicke, 2006; Kim et al., 2008; Van der Lubben,
Verhoef, Borchard, & Junginger, 2001).
The surface of chitosan contains several positively charged amino groups;
therefore, it can easily react with negatively charged biomolecules like
DNA, proteins, and phospholipids, which makes chitosan bioactive in
nature. And the biocompatibility tendency of chitosan is due to the
monomeric building blocks of glycosaminoglycans or glycoprotein. In addi-

tion to biocompatibility, chitosan exhibits biodegradability, nontoxicity,
nonbacterial, nonallergenic, antifungal, antiacid, antiviral, and antiulcer,
and adsorptive properties of chitosan exhibit the promising biomaterial with
wide range of applications in various fields (Crini, 2005) like textile and
printing, ion-exchange chromatography (Shi et al., 1996), manufacture of
pharmaceuticals (MacQuarrie, 2005), cosmetics (Ravi Kumar, Muzzarelli,
Muzzarelli, Sashiwa, & Domb, 2004), food and packaging industry, wound
healing (Uneno, Haruta, Ishii, & Igarashi, 2001), dental (Muzzarelli, 1989),
biotechnology (Mao et al., 2001), and agricultural sciences.
To improve chitosan properties and further diversify its applications,
various modification strategies have been adopted. This includes (1)
cross-linking (Aly, 1998; Denkbas, Seyyal, & Piskin, 2000; Yamada et al.,
2000), (2) graft copolymerization (Borzacchiello et al., 2001; Huacai,
Wan, & Dengke, 2006; Jenkins & Hudson, 2002; Mahdavinia, Pourjavadi,
Hosseinzadeh, & Zohuriaan, 2004; Yazdani-Pedram, Retuert, & Quijada,
2000), (3) complexation (Blair, Guthrie, Law, & Turkington, 1987; Don,
King, & Chiu, 2002; Kim, Cho, Lee, & Kim, 2000; Sun, Xu, Liu, Xue, &
Xie, 2003; Xie, Xu, Liu, & Xue, 2002; Yazdani-Pedram & Retuert, 1997;
Yazdani-Pedram et al., 2000), (4) chemical modifications (Hall & Yalpani,
1980; Yalpani & Hall, 1984), and (5) blending (Cheung, Wan, & Yu,
2002; Ikejima & Inoue, 2000; Ikejima, Yagi, & Inoue, 1999; Suyatma,
Copinet, Tighzert, & Coma, 2004). In particular, modification of chitosan
by means of blending is an attractive method that has been extensively used
for providing new desirable characters to chitosan (Chanachai et al., 2000; Ma
et al., 2003; Smitha, Dhanuja, & Sridhar, 2006; Smitha, Sridhar, & Khan,
2003; Taravel & Domard, 1996). The chemical modification of chitosan
elaborates its applications in various fields.
2.3. Alginate
Alginate is a naturally occurring anionic and hydrophilic polysaccharide. It is
one of the most abundant biosynthesized materials (Narayanan, Melman,
Letourneau, Mendelson, & Melman, 2012; Skjak-Braerk, Grasdalen, &
Smidsrod, 1989) and is derived primarily from brown algae (seaweed) and
bacteria. Alginate occurs naturally in seaweed mainly in the form of calcium,
magnesium, and sodium salts. It is extracted with dilute alkali from various
species of brown algae. The discovery of bacterial alginates, polymers irreg-
ular structure as composed of D-mannuronic acid and L-guluronic acid,
resulted in several proposals that they could substitute for algal products
(Sutherland, 1990). Alginic acid is the only polysaccharide, which naturally

contains carboxyl groups in each constituent residue, and possesses various
abilities for functional materials (Ikeda, Takemura, & Ono, 2000). The
brown algae are an important assemblage of plants that are classified in about
265 genera with more than 1500 species (Bold & Wynne, 1985).
In molecular terms, it is a family of unbranched binary copoly-
mers of (14)-linked -D-mannuronic acid (M) and -L-guluronic acid
(G) residues of widely varying composition and sequential structure
(King, 1983a, 1983b; Moe, Draget, Skjeak-Brk, & Smidsro, 1995). Typ-
ically, the blocks are composed of three different forms of polymer segments:
consecutive G residues, consecutive M residues, and alternating MG resi-
dues (Fig. 7.3A). The proportion, distribution, and length of these blocks
determine the chemical and physical properties of the alginate molecules.
Figure 7.3 (A) Chemical structure of alginate and (B) mechanism of ionic interaction
between alginate and divalent cations.
While G-blocks provide gel-forming capacity, MM and MG units provide

flexibility to the uronic acid chains, with flexibility increasing in the order
GG < MM < MG.
Alginate has miscellaneous applications of interest such as biopolymer
film or coating component because of its unique colloidal properties, which
include thickening, stabilizing, suspending, film forming, gel producing, and
emulsion stabilizing (Grant, Morris, Rees, Smith, & Thom, 1973; King,
1983a, 1983b; Moe et al., 1995), in biomedical applications due to its out-
standing properties in terms of biocompatibility, biodegradability, non-
antigenicity, and chelating ability as the supporting matrix or delivery
system for tissue repair and regeneration and in some formulations
preventing gastric reflux (Kuo & Ma, 2001; Stevens, Qanadilo, Langer, &
Shastri, 2004). Alginate can produces a gel structure when mixed with cal-
cium ions (Fig. 7.3B), which is expected to be a potential coagulant in water
treatment.
2.4. Agar
Agar is a complex polysaccharide present in the cellular wall of red algae,
namely, agarophytes, including species belonging to the genera Gelidium
and Gracilaria. Commercial agar is mainly extracted from species of
Gelidium, Gracilaria, Acantkopeltis, Ceramium, and Pterocladia. Early
studies of agar showed that it contained galactose, 3,6-anhydro-galactose
(Hands & Peats, 1938; Percival, Somerville, & Forbes, 1938), and inorganic
sulfate bonded to the carbohydrate (Samec & Isajevic, 1922). Agar is now
considered to consist of two fractions, agarose and agaropectin.
It is a typical linear (AB)n copolymer with alternating -(1 ! 3)- and
-(1 ! 4)-linked galactose residues (Fig. 7.4). Agar has a great gelling power
in an aqueous environment, which allows it to form gels that are more resis-
tant (stronger) than those of any other gel-forming agent, assuming the use
of equal concentrations. There is no need to add reagents to produce gela-
tion, such as potassium (or proteins as is necessary with carrageenans) and
Figure 7.4 Structure of agar.

calcium (or other divalent cations as is necessary with alginates). The gel
formed is flavorless and used in food products. When the flavors are mixed
with agar gel, the fragrance elongates for the long time. Thus, agar can act as
a fragrance fixer.
Agar can be used over a wide range of pH, from 5 to 8, and in some cases
beyond these limits. It also withstands thermal treatments very well, even
above 100 C, which allows good sterilization. Agar has an excellent revers-
ibility property when it was melted. The gelling properties of agar are the
origin of its multiple applications; it has the highest natural gel strength of
any gelling agent. In food industries, agar is used to prepare jellies, marsh-
mallows, candies or candy fillers, thickening, and gelling agents, utilized
to prevent dehydration of these confectionery products, to limit fat content
in order to reduce cholesterol. Also it was used in smaller quantities to
increase the viscosity of some alcoholic liquors.
2.5. Carrageenan
Carrageenan is not a single biopolymer but a mixture of water-soluble, lin-
ear, sulfated galactans. Carrageenans are a family of linear-sulfated polysac-
charides, extracted from red seaweeds (Knutsen, Myladobodski, Larsen, &
Usov, 1994). Carrageenan is a sulfated polygalactan with 1540% of ester
sulfate content and an average relative molecular mass well above
100 kDa. Carrageenans consist of alternating copolymers of -(1 ! 3)-D-
galactose and -(1 ! 4)-3,6-anhydro-D- or L-galactose (Fig. 7.5). Several
isomers of carrageenan are known (-, -, and -carrageenans), and they dif-
fer in the number and position of the ester sulfate groups on the repeating
galactose units. -Carrageenan has only one negative charge per disaccharide
and tends to form a strong and rigid gel. The gelling power of -carrageenans
imparts excellent film-forming properties, and -carrageenan forms a firm
gel with the aid of potassium ions.
Figure 7.5 Structure of carrageenan.

Carrageenan gels made with it can be used to carry microbes or immo-

bilize cells. In food and other products, carrageenan works as a thickener,
stabilizer, and emulsifier, meaning that it helps keep mixed ingredients from
separating. Carrageenan is also found in jelly, pie filling, chocolate, salad
dressing, and even as a fat substitute in processed meat. Other nonfood items,
like toothpastes, personal lubricants, and air freshener gels, may also include
carrageenan.
3. APPLICATION OF MARINE POLYSACCHARIDES IN

WASTEWATER TREATMENT
3.1. Chitin
One of the most important properties of chitin is its ability to remove metal
ion (Peiselt da Silva and Pais da Silva, 2004). Numerous studies have dem-
onstrated the effectiveness of chitin and its derived products in the uptake
of metal cations such as lead, chromium, copper, and nickel and the uptake
of oxyanions as well as complexed metal ions (Onsoyen & Skaugrud, 1990;
Sakaguchi, Hirokoshi, & Nakajima, 1981). Their structure allows excel-
lent complexation capacity with metal ions, particularly transition and
posttransition metals (Muzzarelli & Tubertini, 1969). It was supported that
the chelation of a single metal ion by several dNH or NHCOCH3 groups
effectively isolates each metal ion from its neighbors (Lepri, Desideri, &
Tanturli, 1978). Chitin obtained from spider crab shell is discussed as a
potential biosorbent for mercury removal by Wase and Forster (1997)
and Volesky (2003). Much of our knowledge of the interactions
of chitins with metals comes from investigations of their possible uses
to remove heavy metals in waste and natural waters (Muzzarelli, 1973;
Suderb & Wightman, 1977, 1983; Yang & Zall, 1984). However, the
adsorption of chitin toward metal ions is not selective, especially when
alkali and alkaline earth metal ions are also available in the solution in high
level of concentration.
Chitin finds application in the adsorption of Cu(II) and Cr(VI) (Franco
et al., 2004) and Pb(II) ions in an aqueous environment ( Jianlong, Xinmin,
Decai, & Ding, 2001). Chitin phosphate could absorb uranium in the
presence of sodium carbonate solution. Chitosan-based chelating resins have
been developed to absorb mercury (Hakim, Sabarudin, Oshita, Oshima, &
Motomizu, 2008a), Ti(IV), V(V), Mo(VI) (Hakim, Sabarudin, Oshita,
Oshima, & Motomizu, 2008b), W(VI), U(VI) (Oshita et al., 2008), Ag in
aquatic environment (Hosoba et al., 2009), Cd, Ni, V, Ga, Sc, In, and Th
(Hakim, Sabarudin, Oshima, & Motomizu, 2007). Adsorption studies of

iron(III) on chitin were done by Karthikeyan, Rajagopal, and Miranda
(2005), and they showed that the adsorption of Fe3+ ion by chitin is max-
imum with the least particle size and in acidic condition. Chitin has copper
removal capability, which could help to obtain more stable diesel oil (Peiselt
da Silva & Pais da Silva, 2004). It also has been successfully tested for the
adsorption of organic pollutants (Aksu, 2005).
Milhome et al. (2009) focused on phenol adsorption by chitin and
chitosan using batch and column systems for possible use in the treatment
of wastewater from petrochemical industries. The improvement of the qual-
ity of the effluent was evaluated by removal of phenol and the changes of
physicochemical parameters such as pH, conductivity, chemical oxygen
demand (COD), total oil and greases, and dissolved solids.
Dithiazone (diphenyl thiocarbazone) is a suitable ligand for such purpose
because it contains many N donor atoms, NH as well as SH groups, which
are very specific for heavy metal ions such as Pb, Cd, Cu, and Hg
(Maczenko, 1986). Mudasir, Ginanjar, Iqmal, and Wahyuni (2008) investi-
gated sorption capability of diathiazone-immobilized chitin for Cd(II) ion at
a pH 6. Moreover, adsorption of Pb2+ and Cu2+ by cellulose chitin beads
was reported by Zhou, Zhang, Zhou, and Guo (2004) at a pH of 4, and
they showed that the adsorption was selective to be in the order of
Pb2+ > Cd2+ > Cu2+. The capacities of Ni2+ adsorption using vinyl-2-
pyrrolidone-grafted chitin were reported by da Rocha Filho, Bach,
Barrak, and de Queiroz (2001) and indicate that the graft copolymer
vinyl-2-pyrrolidone-grafted chitin is an excellent adsorbent for Ni2+ from
wastewater effluents. Ground water arsenic removal by chitin and chitosan
at pH 7 was studied by Da Sacco and Masotti (2010) and reported that chitin
and chitosan mixture showed a capacity of 0.13 equiv. As/g (pH 7) (Elson,
Davies, & Hayes, 1980).
Several studies have demonstrated that the particle size plays an impor-
tant role in the uptake of metallic ions. Ng, Cheung, and McKay (2002)
found that the uptake of Cr6+ ions depends on the inverse of the particle
sizes. Chitin nanoparticles (between 40 and 110 nm) produced by ionic che-
lation of sorbent were used to remove chromium and copper from aqueous
solution. A decrease in the particle size of the chitin improved the adsorption
capacity for chromium and copper ions. In addition, a decrease in the crys-
tallinity of sorbent was observed (Qi & Xu, 2004) during the decrease in
particle size, which makes the chitin derivatives as effective adsorbents for
wastewater treatment.
3.2. Chitosan
Chitosans functional groups and natural chelating properties make chitosan
useful in wastewater treatment (Kubota & Kikuchi, 1998; Li, Dunn,
Grandmaison, & Goosen, 1992) by allowing for the binding and removal
of metal ions such as copper, lead, mercury, and uranium from wastewater.
It can also be utilized to remove dyes and other negatively charged solids
from wastewater streams and processing outlets.
Chitosan is used as a biosorbent for two reasons. First, it is cheap com-
pared to commercial-activated carbon (chitosan is derived from
deacetylation of the naturally occurring biopolymer chitin, which is the sec-
ond most abundant polysaccharide in the world after cellulose). Second, due
to its outstanding chelation behavior. Amine groups present in chitosan are
strongly reactive with metal ions. One of the major applications of this
amino polymer is based on its ability to tightly bind pollutants, in particular
heavy metal ions (Crini & Badot, 2008). Chitosan chelates five to six times
greater amount of metals than chitin (Yang & Zall, 1984). Indeed, nitrogen
atoms hold free electron doublets that can react with metal cations. Amine
groups are, thus, responsible for the uptake of metal cations by a chelation
mechanism. It chelates strongly with the metal ions and hence forms the
coordination complex. Jha, Iyengar, and Prabhakara (1988) reported
Cd(II) ions from wastewater which were efficiently removed by chitosan.
Adsorption of Cu(II) and Cr(VI) ions by chitosan was documented by
Schmuhl, Krieg, and Keizer (2001) and Taboada, Carbrera, and Cardenas
(2003) that used chitosan to adsorb Cu(II) and Hg(II) ions.
However, the amine groups are easily protonated in acidic solutions.
Hence, the protonation of these amine groups may cause electrostatic attrac-
tion of anionic compounds including metal anions (or) anionic dyes (Gibbs,
Tobin, & Guibal, 2003). Modified chitosan and its derivatives also have
potential application in wastewater treatment (Table 7.5). Shanmugapriya,
Ramya, Venkatachalam, and Sudha (2011) have synthesized graft copoly-
mer of chitosan with acrylic acid polymer using ceric ammonium nitrate,
nitric acid redox system under UV irradiation, which used to remove heavy
metals like copper and chromium from water. Graft copolymers of cross-
linked chitosan with acrylonitrile were prepared by free radical polymeriza-
tion using initiator ceric ammonium nitrate as redox system and used to
remove Cu(II) and Ni(II) from water (Ramya, Sankar, Anbalagan, &
Sudha, 2011).
Table 7.5 Chitosan and its derivatives as adsorbent

Chitosan derivatives Metal ions Authors
Chitosan/PVC beads Cu(II) and Ni(II) Popuri, Vijaya, Boddu, and Abburi
(2009)
Epichlorohydrin Cu(II) Kannamba, Reddy, and AppaRao
cross-linked xanthate (2010)
chitosan
Epichlorohydrin Cu(II) and Ni(II) Tirtom, Dincer, Becerik, Aydemir,
cross-linked chitosan/ and Celik (2012)
clay
Chitosan/magnetite Pb(II), Cu(II), and Liu, Hu, Fang, Zhang, and Zhang
Cd(II) (2009)
Chitin/bentonite Cr(VI) and Cu(II) Saravanan, Hemalatha, and Sudha
polymer blend (2011)
Chitosanbentonite Hg(II) Yang and Chen (2007)
Chitosan/PVA Cu(II), Cd(II) Ngah, Kamari, and Koay (2004)
and Kumar, Bijay, and Vinod
(2009)
Vanillin-modified Cu(II) Cestari, Vieira, and Mattos (2006)
chitosan
Chitosan/GLA beads Cu(II) Ngah and Fatinathan (2008)
Chitosan/ Hg(II), Cd(II), and Monier and Abdel-Latif (2012)
phenylthiourea Zn(II)
Chitosan/clinoptilolite Cu(II), Co(II), and Dinu and Dragan (2010)
Ni(II)
Chitosan/perlite Cu(II), Co(II), and Kalyani, Veera, Siva, and
Ni(II) Krishnaiah (2009)
Chitosan/calcium Ni(II) Vijaya, Popuri, Boddu, and
alginate Krishnaiah (2008)
Chitosan/activated clay Methylene blue and Chang and Juang (2004)
reactive dye (RR22)
Chitosan/cotton fiber Pb(II), Cu(II), Zhang et al. (2008)
Cd(II), and Ni(II)
Chitosan-dust kenaf Cu(II) and Ni(II) Florence, Gomathi, and Sudha
fiber composite (2011)
Continued
Table 7.5 Chitosan and its derivatives as adsorbentcont'd

Chitosan derivatives Metal ions Authors
Chitosan/sand Cu(II) Wan, Kan, Lin, Buenda, and Wu
(2007)
Chitosan/oil palm ash Reactive Blue 19 Hasan, Ahmad, and Hameed
(2008)
Chitosan-coated Cr(VI) and Cd(II) Soundarrajan, Gomathi, and Sudha
carbon (2013)
Chitosan and nylon 6 Cu(II) and Cd(II) Prakash, Sudha, and Renganathan
(2012)
Chitosan binary Cd(II) and Cu(II) Ramya and Sudha (2013)
composite
Chitosan Cr(VI) and Cd(II) Govindarajan et al. (2011a, 2011b)
nanoparticles/CMC
blends
Chitosan/methyl Pb(II) Kanchana, Gomathi, Geetha, and
cellulose/clay Sudha (2012)
composite
Chitosan/starch blends Cr(VI) Ramasubramaniam, Govindarajan,
Gomathi, and Sudha (2012)
Chitosan nanoparticles Cr(VI) Sivakami et al. (2013)
Chitosan gel beads As(III and V) and Dambies, Vincent, and Guibal
Mo(VI) (2002)
Nanomagnetic Cu(II) Budi (2007)
chitosan
Fouling is a problem encountered during filtration process, which

reduces the capabilities of the membrane used. Here, chitosan acts as an anti-
fouling agent by decreasing fouling (le Roux, Krieg, Yeates, & Breytenbach,
2005). Chitosan also serves as the UF membrane for metal ion removal in
past 20 years. Juang and Shiau (2000) examined the removal efficiency of
chitosan UF with the dilute solution, divalent metals.
Removal of dyes from the textile dyeing effluent is one of the main prob-
lems (Lee et al., 2008). Discharge of dyes is objectionable not only for aes-
thetic reasons but also as many dyes and their degradation products are
carcinogenic toward aquatic life and mutagenic for humans (Lee et al.,
2008). The dyes are classified as anionic, cationic, and nonionic dyes
(Mishra & Tripathy, 1993). Here, chitosan with its interesting characteristics
acts as an effective biosorbent for the removal of dyes with outstanding
adsorption capacities. Since the deacetylated amino groups in chitosan
can be protonated, the polycationic properties of the polymer can be
expected to contribute to the charged interaction with anionic dyes
(Wang & Wang, 2008; Wong, Szeto, Cheung, & McKay, 2004).
Fluoride normally enters the environment and human body through
water, food, industrial exposure, drugs, cosmetics, etc. However, drinking
water is the single major source of daily intake (Sarala & Rao, 1993).
Lanthanum chitosan adsorbents show excellent removal of fluoride from
water, which is much better than bare chitosan and chitin (Kamble
et al., 2007).
From the literature, it is concluded that the chitosan and derivatives can
remove many heavy metals like Cu(II), Pb(II), U(VI), Cr(III), Cr(VI), Ni(II),
Cd(II), Zn(II), Co(II), Fe(II), Mn(II), Pt(Iv), Ir(III), Pd(II), V(V), and V(VI)
from the industrial wastewater (Monier, Ayad, & Abdel-Latif, 2012).
3.3. Alginate
Alginate could be effectively used as a biosorbent for the removal of cationic
dyes. Abd El-Latif, El-Kady, Ibrahim, and Ossman (2010) reported the
effective removal of methylene blue (MB) from aqueous solution in a batch
stirred tank reactor using alginate/polyvinyl alcoholkaolin clay composite
material. Three adsorbents, calcium alginate beads, sodium hydroxide-
activated carbon-based coconut shell, and calcium alginate/activated carbon
composite beads, were prepared by Hassan, Abdel-Mohsen, and Fouda
(2008) for the comparative study on the removal of MB. A biohybrid mate-
rial based on SiO2alginate was obtained by Barron Zambrano,
Lopez-Perez, Avila Ortega, Munoz Rodrguez, and Carrera Figueiras
(2013), and its properties of adsorption were evaluated using MB as a model
dye. The potential use of a biopolymer based on grape marc entrapped in
calcium alginate beads for the removal of pigments from an agro-industrial
effluent was evaluated by Perez-Ameneiro, Vecino, Barbosa-Pereira, and
Cruz (2014).
MB adsorption on graphene oxide/calcium alginate composite was
reported by Li et al. (2013). Since graphene oxide has been used as an adsor-
bent in wastewater treatment, its biotoxicity limits its practical application in
environment protection. While when calcium alginate immobilized
graphene oxide, composites overcome the limitations of graphene oxide
alone for wastewater remediation.
Devrimci, Yuksel, and Sanin (2012) used alginate as a potential coagulant

for drinking wastewater treatment. Due to its gelling abilities, calcium algi-
nate has been investigated as a possible coagulant. The removal of phenols
from wastewater with encapsulated horseradish peroxidase in calcium algi-
nate was studied by Alemzadeh and Nejati (2009). They observed that the
removal was highly dependent on pH, contact time, and adsorbent dose.
The adsorption of o-nitrophenol onto nanoiron oxide-loaded calcium algi-
nate beads was studied by Soni, Tiwari, and Bajpal (2012).
Sodium alginate (SA) was also be used as the coagulant aid for synthetic
humic acid (HA) water treatment to investigate the effect on flocs charac-
teristics and UF membrane fouling (Wang et al., 2014). When SA content
was 0.3 mg L1, the treatment of HA by polyaluminum chloride (PAC) plus
SA (PAC/SA) yielded the maximum removal efficiency of 66.5%, which
was higher than the maximum HA removal efficiency of PAC (58%). More-
over, the HA flocs size of PAC would grow from 269 to 367 m after SA
was added. Strength factor and recovery factor of PAC/SA were larger than
those of PAC. These resulted in the improvement in subsequent UF mem-
brane performance. The treatment of tannery wastewater by Ca alginate
beads showed noticeable decrease in color intensity, organic loads, and metal
ions concentration. Alginate showed good metal sorption efficiency
(Araujo & Teixeira, 1997; Veglio, Espoito, & Reverberi, 2002). Among
recent application, alginate has been used as a cell immobilization material
and as a biosorbent material for the removal of divalent heavy metals, such as
Cu2+, Co2+, UO2+, and Zn2+, from aqueous solutions.
Alginate is the most commonly used polymer for microbial cell encapsu-
lation, also called immobilization (Chen & Huang, 1988; de-Bashan & Bashan,
2004; Fenice, Selbman, Federici, & Vassilev, 2000; Paul, Fages, Blanc,
Goma, & Parcilleux, 1993; Smidsrod & Skjak-Braek, 1990; Walker &
Connick, 1983). Calcium alginate (Ca alginate) also has been widely employed
for the immobilization of enzymes or whole cells since it is less toxic than syn-
thetic polymers, easily gelled under mild conditions and inexpensive
(Gonzalez, Herrera, Garcia, & Pena, 2001; Shimomura, Suda, Uchida, &
Yagi, 1997; Wang et al., 2007). Jeon, Park, and Yoo (2002) developed a novel
polyvinyl alcoholboric acid-immobilized alginic acid material for heavy metal
removal. Wilkinson, Goulding, and Robinson (1989) used the alginate
immobilized Chlorella emersonii for mercury removal, while Wojnowska-
baryla, Stolarczyk, and Gretjzinska (1997) investigated cadmium adsorption
onto the immobilized Klebsiella pneumoniae. Klimiuk and Kuczajowska-
Zadrozna (2002) studied the effect of polyvinyl alcohol on cadmium adsorp-
tion and desorption from alginate adsorbents.
The biosorption of Pb(II) ions using Kluyveromyces marxianus

immobilized in alginate beads was carried out by Sai Subhashini, Velan,
and Kaliappan (2013). They suggested that alginate beads can be used as a
biosorbent for an efficient removal of Pb(II) ions from aqueous solution.
Calcium alginate is the most utilized entrapment matrix in environmen-
tal applications (Hill & Khan, 2008; Siripattanakul & Khan, 2010). The
adsorption process of heavy metal ions appears to exchange with calcium
ions to form a metal alginate (Bailey et al., 1999). Alginate in the form of
calcium-cross-linked beads exhibits high uptake capacity for chromium,
cadmium, copper, lead, nickel, iron, and manganese ions depending on their
initial ion concentration with removal efficiency from 43.5% to 74.8%. Ca
alginate beads have been also shown to take up organic loads from tannery
wastewater such as TDS, salinity, COD, BOD, ammonia nitrogen, nitrate,
and phosphorous depend on their initial concentration with removal effi-
ciency from 39.9% to 78.6% (Marwa, EL-Tayieb, El-Shafei, &
Mahmoud, 2013). Biological sludge was immobilized into Ca alginate beads
via entrapment and biosorption of copper, zinc, and chromium was carried
out by Wu, Zhang, and Wang (2004).
Vijaya et al. (2008) studied the removal of nickel(II) from aqueous solu-
tion through modified chitosan and calcium alginate biopolymer sorbents.
The equilibrium batch adsorption and column flow techniques results
showed that comparatively the nickel adsorption was higher in case of cal-
cium alginate than chitosan-coated calcium alginate and chitosan-coated sil-
ica, and biosorbents regeneration with Ni(II) is done using 0.1 M EDTA
solution.
Oil and its derivative from oil manufacturing plants and restaurants have
received attention because of their widespread use. Disposal of this waste-
water into environment causes serious problems due to its high oil content,
COD, and color. Microbial degradation of oil wastewater is a concern in
recent years. Lan, Gang, and Jinbao (2009) suggested that immobilized
Yarrowia lipolytica by Ca alginate might be applicable to a wastewater
treatment system for the removal of oil and COD. (1) The COD degra-
dation was comparatively higher from immobilized cells in Ca alginate
beads than that by free cells; (2) the oil degradation by immobilized and
free cells was similar; and (3) the thermostability, reusability, and storage
ability of immobilized cells for wastewater treatment were retained at a
higher level.
The matrix produced by calcium alginate is likely to break or swell dur-
ing use, affecting lower wastewater treatment performance. Barium alginate
(BA) entrapment was developed and applied to solve the problem (Bajpai &
Sharma, 2004; Chen, Kao, & Chen, 2008; Vogelsang & stgaard, 1996). It
was found that the BA-entrapped cells were well performed and were dura-
ble in environmental pressures. However, most of the previous studies
focused on utilization of the BA-entrapped cells for treating different types
of wastewater (Bajpai & Sharma, 2004; Chen et al., 2008; Vogelsang &
stgaard, 1996). Thus far, there is no publication emphasizing on operating
factors related to performance of the wastewater treatment by the
BA-entrapped cells. Siripattanakul-Ratpukdi and Tongkliang (2012) used
BA-entrapped activated sludge to treat municipal wastewater. The wastewa-
ter treatment kinetics followed the first-order reaction mechanism.
3.4. Carrageenan and agar

Biopolymers of marine algae origin are ubiquitous in surface waters and have
attractive potential. The seaweed extractives of commercial importance fall
into three main groups, two of which (agar and carrageenans) are derived
from red algae and the third (alginates) from brown algae. Use of biopolymer
in place of artificial polymers has been increasing due to stringent environ-
mental regulations (Krishna et al., 2006).
-Carrageenan-g-N-vinyl formamide was synthesized by Mishra,
Yadav, Sand, Tripathy, & Behari (2010) using free radical initiation using
the potassium monopersulfate/malonic acid redox pain in an inert atmo-
sphere as an efficient flocculants for the treatment of coal mine wastewater.
Mahdavinia, Aghaie, Sheykhloie, Vardini, & Etemadi, (2013) developed
K+-cross-linked -carrageenan dried and wet beads to remove the cationic
crystal violet (CV) dye from water. The kinetics and mechanism of CV
adsorption onto both wet and dried carrageenan beads were studied.
Mahdavinia et al. (2013) reported a novel nanocomposite hydrogel syn-
thesized from grafting of acrylamide onto binary mixture of -carrageenan
and SA biopolymers by incorporation of Na-MMt nanoclay. The effect of
pH on the dye removal capacity using nanocomposites was studied, and it
was revealed that the adsorption capacity of nanocomposites was enhanced
at acidic pH as the Na-MMt nanoclay and -carrageenan components were
increased.
In the field of wastewater treatment, entrapped cell systems have been
widely developed over the last several years and become one of the alter-
natives for the treatment of municipal as well as industrial wastewater
(Canizares et al., 1994; Cao, Zhao, Sun, & Zhang, 2004; Chen and Lin,
2007; Chen, Lee, Chin, & Houng, 1998; Degiorgi, Pizarro, Smolko, Lora,
& Carenza, 2002; Hsieh, Huang, Lin, Chen, & Lin, 2008; Kuo and Shu,
2004; Muyima and Cloete, 1995; Pramanik and Khan, 2008, 2009; Song,
Choi, Park, & Yoo, 2005; Vilchez, Garbayo, Markvicheva, Galvan, &
Leon, 2001; Yang, Chen, & Kim, 2003). Elimination of toxic amides from
wastewaters and other industrial wastes has become a very important environ-
mental issue. Many amides and nitriles are extensively manufactured and used
in industry as organic solvents, herbicides, organic feed stocks, extractants, and
precursors in the synthesis of emulsifiers and pharmaceuticals (Hoyle, Bunch,
& Knowles, 1998; Ramakrishna, Dave, & Ravindranathan, 1999). The
removal of acrylamide is of paramount importance because of its deleterious
effects on health and environment. Whereas Chand, Vitzthum, Kumar, &
Bhalla, (2009) found the remedy for the successful degradation of amides in
wastewater by amidohydrolase activity of agar gel-immobilized resting cells
of Nocardia globerula. The degradation process depends on various conditions,
including pH, temperature, and immobilization. Ninety percent of amides
were degraded effectively during treatment of wastewater.
Samiey and Ashoori (2012) investigated adsorption of MB on agar as a
function of temperature (308328 K), different concentrations of NaCl
and HCl, and various weight percentages of binary mixtures of ethanol with
water. The microwave-assisted synthesis of polyacrylamide-grafted agar
(Ag-g-PAM) and its application as flocculant for wastewater treatment were
reported by Mishra, Sen, Rani, & Sinha, (2011). The synthesized
Ag-g-PAM acts as a good flocculant for the reduction of pollutant load of
wastewater.
4. ADVANTAGES AND POSSIBLE DRAWBACKS OF USING

MARINE POLYSACCHARIDE-BASED MATERIALS FOR
ADSORPTION
4.1. Advantages
Today there are many different ways of treating industrial effluent which is
hazardous to the entire world when it is let into the water stream. Industry
is at high competition over the years with price war, and further development
has not progressed due to limitation of funding for R&D. It is competing
today with natural materials. Recently, numerous approaches have been
studied for the development of cheaper and more effective technologies, both
to decrease the amount of wastewater produced and to improve the quality of
the treated effluent (Barakat, 2011). Biosorption is an emerging technology
that attempts to overcome the selectivity disadvantages of adsorption processes
(Crini, 2005).
Among the various synthetic materials available, natural materials are

being used to treat wastewaters. The marine polysaccharides sorbents are
low-cost materials obtained from natural raw resources, and these materials
are versatile in properties with repetitive functional groups. Biopolymers
provide excellent chelating and complexing materials for a wide variety
of pollutants including dyes, heavy metals (Salam, Reiad, & El-Shafei,
2011), and aromatic compounds. It has high capacity and rate of adsorption.
The main advantage of using natural materials is that the regeneration step
is easy.
4.2. Limitations
In spite of potential applications of marine polysaccharides, it is necessary to
establish efficient and appropriate modifications to explore fully the high
potential of these biomacromolecules. Marine polysaccharides have its spe-
cific application as well as inherent advantages and disadvantages in waste-
water treatment. Polysaccharides and their derivatives have some
disadvantages described as low surface area. The disadvantages in using
marine polysaccharides for wastewater treatment can be overcome through
chemical modifications. Chemical modifications are generally difficult in
case of chitin owing to the lack of solubility, and the reactions under het-
erogeneous conditions are accompanied by various problems such as the
poor extent of reaction, difficulty in selective substitutions, structural ambi-
guity of the products, and partial degradation due to severe reaction condi-
tions. Therefore, with regard to developing advanced functions, much
attention had been paid to chitosan rather than chitin (Zohuriaan-Mehr,
2004). On seeing to the drawbacks of chitosan, chitosan has poor mechanical
properties, pH sensitivity, and variability in the polymer characteristics; is
nonporous, not useful to remove cationic dyes, and not much useful in
the native form; and requires derivatization to improve its performance
(Sudha, 2010).
5. FUTURE PROSPECTS
Marine carbohydrates have a great potential in treatment of wastewa-
ter. In addition, marine carbohydrates derivatives can be prepared for
treating wastewater by modifying it with various other bioadsorbents, which
explore its innovative properties against environmental pollution. Also it can
be used in a various fields like food and agriculture, cosmetics, pharma-
ceutics, and tissue-engineering applications.
6. CONCLUSIONS
A brief review of marine polysaccharides for wastewater treatment has
been reported. The review discussed clearly indicates that novel exciting and
promising marine sources of polysaccharides. Further investigations with a
multidisciplinary approach are imperative in order to develop novel marine
carbohydrates as useful as for many fields.
ACKNOWLEDGMENTS
The authors are grateful to authorities of DKM College for Women and Thiruvalluvar
Dr. Se-Kwon Kim, Marine Bio Process Research Center, Pukyong National University,
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CHAPTER EIGHT
Industrial Applications of Marine

Carbohydrates
Prasad N. Sudha1, S. Aisverya, R. Nithya, K. Vijayalakshmi
1
Corresponding author: e-mail address: drparsu8@gmail.com
Contents
1. Introduction 146
1.1 Marine carbohydrates 146
1.2 General structures and terminology 147
1.3 Production of carbohydrates by marine organisms 148
1.4 Analysis of marine carbohydrates 149
1.5 Carbohydrates in sediments 156
2. Applications of Marine Carbohydrates 156
2.1 In cosmetics 157
2.2 In food and agricultural field 157
2.3 In pharmaceutics 160
2.4 In biotechnology and microbiology 166
2.5 In treatment of industrial effluent 167
3. Future Directions for Research 170
4. Conclusion 171
Acknowledgments 171
References 171
Abstract
Biomaterials have been used increasingly in various fields, such as drug delivery, imag-
ing, and tissue engineering. The main reason justifying the widespread use of bioma-
terials relies on its valuable and low-cost source of new drugs. Current research goals are
focused on identifying more potent and specific compounds with antitumor, immuno-
modulatory, antihyperlipidemic, anticoagulant, and antiviral activities. The increasing
knowledge of structural analysis and chemical modifications enables the use of these
marine carbohydrates in a newer way for the human welfare. This chapter focuses on
the recent developments related to industrial and biomedical applications using chitin,
chitosan, alginate, agar, and carrageenan derivatives and reports the main advances
published over the last 1015 years.
http://dx.doi.org/10.1016/B978-0-12-800268-1.00008-1
1. INTRODUCTION
1.1. Marine carbohydrates
Oceans represent vast and exhaustive source of natural products in the globe,
harboring the most diverse groups of flora and fauna. Marine microorgan-
isms have developed unique metabolic and physiological capabilities to
thrive in extreme habitats and produce novel metabolites that are not often
present in microbes of terrestrial origin (Fenical, 1993). Therefore, this rich
marine habitat provides a magnificent opportunity to discover newer com-
pounds such as antibiotics, enzymes, vitamins, drugs, biosurfactant,
bioemulsifier, and other valuable compounds of commercial importance
(Austin, 1989; Jensen & Fenical, 1994; Lang & Wagner, 1993;
Romanenko, Kalinovskaya, & Mikhailov, 2001). Marine carbohydrates
are the polysaccharides which can be extracted from marine plants and ani-
mal organisms or produced by marine bacteria that have been studied for
several decades.
Some of the major components which have been identified in marine
particles over the past decade, including uronic acids (Hung, Guo,
Santschi, Alvarado-Quiroz, & Haye, 2003; Mopper et al., 1995), aldoses
(Skoog & Benner, 1998), neutral sugars (Mopper et al., 1995), and amino
sugars (Muldoon et al., 2001). Recently, the marine-derived proteins are
becoming more popular among consumers because of their numerous
health beneficial effects that can provide equivalents to collagen and gelatin
without the associated risks. Seaweeds are major marine sources having
much kind of applications in various fields. There are three different algae
present in marine water: green, red, and brown algae. Among these, brown
seaweeds are having many types of polysaccharides which play an important
role in medical field. In the year of 1913, Killing first isolated the fucoidan
from marine brown algae and named as fucoidan (Killing, 1913).
Fucoidan are present only in the brown seaweeds with various essential
components. The brown algae, Fucus serratus L., consists of L-fucose, sulfate,
and acetate in a molar proportion of 1:1:0.1 and small amounts of xylose and
galactose (Bilan et al., 2006). The low-molecular-weight fucoidan have
potent anticoagulant and fibrinolytic properties with only minor platelet
activating effects (Diirig et al., 1976). One of the most abundant biopoly-
mers which were found in the marine organic carbon pool is the carbohy-
drates, making up 1070% of organic matter in plankton cells
(Romankevich, 1984). The marine carbohydrates form the basis of a
Industrial Applications of Marine Carbohydrates 147
number of nutraceutical products (from seaweed, microalgae, and shrimp

waste) and also of the long established alginate (from seaweed) industry.
About 200 g carbohydrates per kg wet weight were found to be an appro-
priate substrate concentration for microbial conversion processes (Horn,
Aasen, & stgaard, 2000). The field of natural polysaccharides of marine ori-
gin is already large and expanding.
Marine organisms are constituted by materials with a vast range of prop-
erties and characteristics that may justify their potential application within
the biomedical field. Moreover, assuring the sustainable exploitation of nat-
ural marine resources, the valorization of residues from marine origin, like
those obtained from food processing, constitutes a highly interesting plat-
form for development of novel biomaterials, with both economic and envi-
ronmental benefits. A large number of different types of compounds have
been isolated from aquatic organisms, and these have been transformed into
profitable products for health applications, including controlled drug deliv-
ery and tissue engineering devices. There is a wide range in the relative avail-
ability of different carbohydrates in marine environments. Marine algae
contain large amounts of not only polysaccharides, notably cell wall struc-
tural, but also mycopolysaccharides and storage polysaccharides (Kumar,
Sharma, & Kumar, 2005). Extracellular polysaccharides (EPS) are widely
secreted by various marine organisms including plants, animals, diatoms,
microalgae, and bacteria (Decho, 1990; Gutierrez, Martinez, & Prieto,
1996; Philippis, Margheri, Materassi, & Vincenzini, 1998; Philippis &
Vincenzini, 1998).
This review attempts to explore the plausible industrial and health ben-
efits of modified marine carbohydrates such as chitin, chitosan, alginate,
agar, and carrageenan with particular reference to its nutritional, biocompat-
ibility, and biodegradable properties.
1.2. General structures and terminology

One of the most abundant bioactive substances present in the marine organ-
isms is the carbohydrates. The major components of marine organic matter
are the carbohydrates, but few molecular-level carbohydrate analyses in sea-
water have been undertaken owing to the low concentrations of individual
compounds. The term saccharide is derived from the Latin word
saccharum from the sweet taste of sugars. Carbohydrates are hydrates of
carbon having the general formula as Cx(H2O)ywhere x and y may or
may not be equal and range in value from 3 to 12 or more. Another modern
definition of a carbohydrate is that the compounds are polyhydroxy alde-

hydes or ketones that offers potential chemical diversity orders of magnitude
greater than their protein and nucleic acid counterparts (Turnbull &
Field, 2007).
Carbohydrates are major components of marine organic matter. The
largest identified fraction of organic matter in the ocean is the carbohydrates
which accounts for about 2030% of organic matter in marine surface waters
(Benner, Pakulski, McCarty, Hedges, & Hatcher, 1992). It also accounts for
about 330% of the bulk dissolved organic carbon (Gueuen, Guo, Wang,
Tanaka, & Hung, 2006; Hung et al., 2003; Pakulski & Benner, 1994),
whereas, in estuarine and marine surface waters, they are considered to be
the most labile fractions of bulk organic matter. It may play key roles in
the geochemical cycles as reported by several studies (Benner et al., 1992;
Burdige & Zheng, 1998; Middelboe, Borch, & Kirchman, 1995;
Murrell & Hollibaugh, 2000).
Uronic acids (e.g., Mopper et al., 1995), amino sugars (e.g., Kerherve,
Charrieizi, & Gadel, 1995), and neutral sugars (e.g., Borch & Kirchman,
1997; Kerherve et al., 1995; Mopper et al., 1995) are the major classes of
carbohydrates which have been identified in marine waters. Several inves-
tigations of dissolved carbohydrates were done in marine waters (e.g.,
Haniia, 1970; Iitekot, Brockman, & Michafus, 1981; Liebezeit, Bolter,
Brown, & Dawson, 1989), but all of these studies relied on concentration
procedures that fractionate the sample and leave an unknown fraction of car-
bohydrates uncharacterized.
1.3. Production of carbohydrates by marine organisms

The carbohydrate content of marine organisms varies greatly in brown and
red algae. Carbohydrate may comprise up to 74% of the total organic matter
(Romankevich, 1984) while planktonic algae have a carbohydrate generally
ranging from 20% to 40% (Parsons, Stephens, & Stickland, 1961) and zoo-
plankton carbohydrate content is 24 times lower than that of phytoplank-
ton. The phytoplankton serves as the principle source for carbohydrates
found in seawater, particles, and sediments; the photosynthetic conversion
of carbon dioxide to biomass is the basis of carbohydrate production. Phy-
toplankton carbohydrate composition has been surveyed in both field sam-
ples and laboratory monocultures. In general, glucose has been found to be
the most common monosaccharide probably due to the fact that phyto-
plankton storage carbohydrates are primarily composed of glucose.
The major source of carbohydrates to marine sediments is the sinking

particles. Methods of analysis of carbohydrates in sediments usually involve
extraction, separation and, finally, quantitation of the sugars. Carbohydrates
in sediments comprise about 10% of the total organic carbon. Studies of
anoxic surface sediments show that amino acids and carbohydrates are major
constituents of the organic matter (Burdige & Zheng, 1998). Carbohydrates
are remineralized rapidly during epigenetic and diagenetic bacterial activity
and that only less than 10% of these compounds are stable enough to be
incorporated into the sediment a few centimeters below the sediment/water
interface (Degens, Reuter, & Shaw, 1964; Seifert et al., 1990).
1.4. Analysis of marine carbohydrates

Analysis of the structural distinctions of carbohydrates can be difficult, how-
ever, since the isolation of carbohydrates from solution or from organic
matrix is not a trivial problem and determination of carbohydrate structure.
Many microorganisms in the marine environment that can degrade can uti-
lize algal carbohydrates as a carbon source for energy. An interesting source
for a myriad of different bioactive polysaccharides was the microalgae which
can range from various industrial applications to novel food applications.
Polysaccharides are polymers of simple sugars (monosaccharides) linked
together by glycosidic bonds, and they have numerous commercial applica-
tions in products such as stabilizers, thickeners, emulsifiers, food, feed, and
beverages (McHugh, 1987). The most representative polysaccharides in
marine environment are agar, alginate, carrageenan, chitin, and chitosan.
1.4.1 Agar
Agar or agar-agar is a gelatinous substance, obtained from an alga which con-
sists of a mixture of agarose and agaropectin. This was discovered in 1658 by
Minora Tanzaemon in Japan, where it is called Kanten. This can be used as a
laxative, an appetite suppressant, as a vegetarian gelatin substitute, a thick-
ener in soups, in fruit preserves, ice cream, and other desserts, as a clarifying
agent in brewing, and for sizing paper and fabrics (Cregut & Rondags,
2013). The structure of agar was shown in Fig. 8.1.
Agar is a gelling agent that is most commonly used in icings, sugar con-
fectionery, canned meat and fish products, diabetic and health foods, and
dairy products. Its use, however, is declining as more effective and often
cheaper gums become more available. On the opposite cost side, agar is
the most expensive industrially produced hydrocolloid. In 1882, Robert
Koch first introduced it as a gelling agent in culture media for bacteriological
Figure 8.1 Structure of agar.
studies. Since this, agar has become a major product initially used for bio-
logical research or analyses (Cregut & Rondags, 2013).
1.4.2 Alginates
Alginate is a binary linear heteropolysaccharide containing 1,4-linked -L-
guluronic acid and -D-mannuronic acid. This has been widely used in the
field of controlled release, ion exchange, and in the vapor-permeation
membrane-separation technique (Kalyani, Smitha, Sridhat, & Krishnaiah,
2008). The structure of sodium alginate is represented in Fig. 8.2.
Alginate, an algal polysaccharide, is widely used in the food industry as a
stabilizer, or as a thickening or an emulsifying agent. The only alginate
derivative used in food is propylene glycol alginate (PGA). PGA was first
prepared by Steiner (1947). Alginate when mixed with calcium ions is able
to produce a gel structure, which finds use as a thickening agent in the food
industry, in drug release systems during pharmaceutical applications. It is
one of the important biomaterial used in wound healing and cell culture.
Alginates were mainly used in the manufacture of paper and cardboard
pharmaceutical, cosmetic creams, and processed foods (Chapman, 1987).
The graft copolymers of this alginate polysaccharide have find applica-
tions in diverse fields such as pharmaceutical, biomedical, agriculture, and
environmental. Polymers with promising applications in the biomedical
field as delivery systems of therapeutic agents and the bioseparation devices
have been attracting much attention, due to nontoxic nature of alginate. In
literature reported, alginate, a natural anionic polysaccharide obtained by
extraction from brown algae, is composed of (14)-linked -D-mannuronic
acid and -L-guluronic acid blocks.
Due to their gelling ability in the presence of divalent cations, such as
calcium and barium, stabilizing properties and high viscosity in aqueous
solutions, alginate and its derivatives have been extensively utilized in
Figure 8.2 Structure of alginate.
biomedical applications of cell transplantation, drug delivery, and bulk agent

gels. However, as no hydrolytic or enzymatic chain breakages occur within
alginate chains, high-molecular-weight alginate polymers cannot be easily
degraded and may be very slow to clear from the body.
1.4.3 Carrageenan
Carrageenans are too algal hydrocolloids and are among the cheapest hydro-
colloids available with an estimated price of US$10.5 kg1. The main uti-
lization is as a gelling agent for food and cosmetic industries, but
carrageenans role in pharmaceutical industry as an additive is limited by
its variability in structure and properties. However, carrageenans are the
most produced gelling hydrocolloids with approximately 50,000 tons per
year for a global value estimated of US$527 million, in 2009. Carrageenan
is derived from seaweed of the class Rhodophyceae which has no nutritional
value and is used in food preparation for its gelling, thickening, and emul-
sifying properties (Van de Velde, Lourenco, Pinheiro, & Bakkerd, 2002).
Carrageenan is added to processed foods because it can bind water and
improve palatability and appearance through interaction with other sub-
stances in the food (e.g., proteins, sodium or calcium phosphates, starch,
galactomannan, and carboxyl methylcellulose) (Piculell, 2006). The struc-
ture of different forms of carrageenan is represented in Fig. 8.3.
Carrageenans are also used as stabilizers for foams, ice cream, condensed
milk, cream, and salad dressings. Carrageenan is widely used in dairy
products to improve texture, thickness, and solubility (McHugh, 2003).
Carrageenan is also successful in controlling discoloration, maintaining tex-
ture through shelf life, and providing antibacterial protection by coating on
sliced lychee (Plotto, Narciso, Baldwin, & Rattanapanone, 2006), bananas
(Bico, Rapaso, Morais, & Morais, 2009), and mangoes (Plotto et al., 2006).
Figure 8.3 Different forms of carrageenan.
Plain carrageenans, as well as agar, are mainly used as a food additive, but
increasing attention is given to possible biomedical applications, in combi-
nation with synthetic polymers. The synthesis of agar-graft-
polyvinylpyrrolidone (PVP) and -carrageenan-graft-PVP blends by a
microwave irradiation method has been reported (Prasad et al., 2006).
The physicochemical and rheological properties of the corresponding
hydrogels were studied and compared with control agar and -carrageenan
hydrogels. The novel blend hydrogels were found to be not as strong and
showed better spreadability and water-holding capability, so they are poten-
tially useful in moisturizer formulations and active carriers of drugs. The use
of blended PVP with agar in hydrogel dressings has also been reported
(Lugao et al., 1998).
In the biomedical area, the use of modified carrageenans has also been
explored. Porous nanocomposites were prepared by coprecipitation of cal-
cium phosphates into a -carrageenan matrix (Daniel-da-Silva, Lopes,
Gil, & Correia, 2007), whose resulting porosity and morphology were suit-
able for application in bone tissue engineering. The association among car-
rageenan, nanohydroxyapatite, and collagen resulted in an injectable bone
substitute biomaterial, suitable for bone reconstruction surgery (Gan &
Feng, 2006).
1.4.4 Chitin and chitosan

Chitin was first discovered in mushrooms by the French Professor, Henri
Braconnot, in 1811. In 1820s, chitin was also isolated from insects. Chitin
contains 2-acetamido-2-deoxy--D-glucose through a (1 ! 4) linkage.
Chitin is the most abundant natural fiber next to the cellulose and is similar
to cellulose in many respects. The most abundant source of chitin is the shell
of crab and shrimp. Chitosan was discovered in 1859 by Professor
C. Rouget. Chitosan contains 2-acetamido-2-deoxy--D-glucopyranose and
2-amino-2-deoxy--D-glucopyranose residues (Bhatnagar & Sillanpaa, 2009).
Chitin (C8H13O5N)n is a long-chain polymer of a N-acetylglucosamine,
a derivative of glucose which was first identified in 1884 (Gonzalez-Davila,
Santana-Casiano, & Millero, 1990). Chitin can be found in one of the three
crystalline forms -chitin, -chitin, and -chitin, respectively. -Chitin is
mainly present in shells of crabs, lobsters, and shrimps. The -chitin was
obtained from squid pens in which the chains are arranged in a parallel fash-
ion, while -chitin is the form in which the molecules are arranged in both
parallel and antiparallel manners. Based on the intermolecular interactions
and the packing arrangements, it was finally concluded that the -chitin
was found to be more reactive and versatile (Morganti, 2012).
It is the most important inexpensive natural biological polysaccharide
occurring in crustaceous shells or in cell walls of fungi which has gained
much attention for biomedical and industrial applications, due to its bio-
compatibility and restorative properties (Borch & Kirchman, 1997). Exam-
ples of the potential uses of chemically modified chitin in food processing
include the formation of edible films and as an additive to thicken and sta-
bilize foods (Shahidi, Arachchi, & Jeon, 1999) and pharmaceuticals. More-
over, due to its biodegradable nature, chitin has been reported to have some
unusual properties that accelerate healing of wounds in humans (Morganti,
2012), and it is also used as a potential biomaterial for artificial skin, suture,
and drug carrier (Lee, 1996). Up to now, the biotechnologically produced
chitin is not commercially available, but it offers new perspectives for the
production of high-viscosity chitosan, with a promising potential for appli-
cations in biomedicine and pharmacy (Hang, Dunstan, & Dass, 2010). Fer-
mentation of this biowaste using lactic acid bacteria for the production of
chitin has been studied and reported. Fundamental knowledge of the inter-
actions among chitin and proteins, polysaccharides, calcium carbonate,
enzymes, drugs, cells, and synthetic materials is not only important for elu-
cidating biological processes associated with chitin but also for designing
novel chitin-based biomaterials (Wang & Esker, 2014).
Chitosan a unique basic linear polysaccharide obtained from the
deacetylation of chitin which comprises an unbranched chain consisting
of -(1,4)-2-amino-2-deoxy-D-glucopyranose (Muzzarelli, 1993). When
compared to other polysaccharides, chitosan has several excellent properties
such as biocompatibility, biodegradability, nontoxicity, good film-forming
capacity, and excellent chemical-resistant behavior. Due to these advan-
tages, chitosan has been widely used in membranes on ultrafiltration, reverse
osmosis, evaporation, clinics (Badawya & Rabeab, 2009) and surfactants
(Ngimhuang, Furukawa, Satoh, Furuike, & Sakairi, 2004), drug delivery
systems (Sashiwa, Yajima, & Aiba, 2003), and solid polyelectrolyte forma-
tions (Wang, Xu, & Chen, 2007). The diagrammatic representation of for-
mation of chitosan from the chitin is shown in Fig. 8.4.
Chitosan is widely used especially in food industry, pharmaceutical
industry, and biotechnology. In addition, using chitosan as a carrier, many
studies have been conducted with mouse, rat, rabbit, and canine animal
models in order to describe in vivo biocompatibility, biodegradability, drug
delivery, DNA delivery, and wound healing. Chitosan is mainly used in
water purification plants throughout the world to remove oil, grease, heavy
metals, and fine particulate matter that cause turbidity in wastewater streams.
The decolorization by chitosan which is decrystallized by citric acid is effi-
cient, fast, and cost effective and appears to be a promising method for the
treatment of alkaline effluent from textile industry containing mixed dye.
Chitosan has also been approved as a food additive in Korea and Japan since
1995 and 1983, respectively (KFDA, 1995; No, Park, Lee, Hwang, &
Meyers, 2002; Weiner, 1992).
Higher antibacterial activity of chitosan at lower pH suggests that the
addition of chitosan to acidic foods will enhance its effectiveness as a
natural preservative (No et al., 2002). Chitosan has, in the last decades, been
widely used in a variety of applications, both industrially and pharmaceuti-
cally, which has been well described in several comprehensive reviews
Figure 8.4 Chitosan derived from chitin.
(Dodane & Vilivalam, 1998; Felt, Buri, & Gurny, 1998; Illum, 1998;
Prabaharan, 2008; Rinaudo, 2006). A large body of research exists on chem-
ical modification of chitosan through derivatization of the amine and/or
hydroxyl groups (Amidi et al., 2006; Snyman, Govender, & Kotze, 2003;
Thanou et al., 2000). Comparatively, many cellulose derivatives have been
produced in a similar way (Philipp et al., 1996). However, the advantage
of chitosan in comparison with other polysaccharides (such as cellulose,
starch, and galactomannans) is that its chemical structure allows easier
modifications at the C-2 position. Specific groups can be introduced to
achieve novel polymers for selected applications. Attempts to enhance water
solubility of chitosan led to several methods of derivatization. Examples
include sulfonation (Bannikova, Sukhanova, Vikhoreva, Varlamov, &
Galbraikh, 2002), quaternarization (Polnok, Verhoef, Borchard, Sarisuta, &
Junginger, 2004), carboxymethylation (Wongpanit et al., 2005), and N- and
O-hydroxyalkylation (Donges, Reichel, & Kessler, 2000; Richardson &
Gorton, 2003). Furthermore, a variety of graft copolymerization of chitosan,

for example, with lactic acid (Yao et al., 2003), polyacrylic acid (Shim &
Nho, 2003), vinyl pyrrolidone (Yazdani-Pedram & Retuert, 1997), 3-O-
dodecyl-D-glucose (Ngimhuang et al., 2004), and N-isopropylacrylamide
(Lee, Ha, Cho, Kim, & Lee, 2004), have been presented and evaluated as
practical biomedical materials.
Cyclodextrin-linked chitosan is interesting for the viewpoint of pharma-
ceutics, including drug delivery, cosmetics, and analytical chemistry, and
quaternized chitosan has potential as an absorption enhancer across the intes-
tinal epithelium due to its mucoadhesive and permeability enhancing prop-
erties (Sashiwa & Aiba, 2004).
1.5. Carbohydrates in sediments

Carbohydrates are the major organic compounds produced in the biosphere
through autotrophic organisms by photosynthetic methods (Youssef,
El-Said, & Shobier, 2013). Due to its more abundant and ubiquitous nature,
the carbohydrates play an important role in biogeochemical cycles occurring
in the marine water column, sedimentwater interface which accounts for
1085% of the dissolved organic carbon in seawater (Pakulski & Benner,
1994), sediments, and pore waters (Arnosti & Holmer, 1999; Burdige,
Skoog, & Gardner, 2000).
The major source of carbohydrates to marine sediments is the sinking
particles. Methods of analysis of carbohydrates in sediments usually involve
extraction, separation, and, finally, quantitation of the sugars. Studies of
anoxic surface sediments show that amino acids and carbohydrates are major
constituents of the organic matter Carbohydrates are remineralized rapidly
during epigenetic and diagenetic bacterial activity and that only less than
10% of these compounds are stable enough to be incorporated into the
sediment a few centimeters below the sediment/water interface (Degens
et al., 1964).
2. APPLICATIONS OF MARINE CARBOHYDRATES

Carbohydrate polymers have current or potential industrial applica-
tion in many areas such as paper, adhesives, food, textiles, wood, pharma-
ceuticals, biodegradables, and biorefining. It plays an important role in
many biological and biochemical processes such as the maturation, fertiliza-
tion, cell differentiation, protein folding, and degradation. The marine
carbohydrates including glucosamine glycon, chitin, chitosan, fucoidan,
carrageenan, and alginic acid have a host of bioactivities such as antioxidative,

antibacterial, antiviral, antitumor, immunostimulatory, and anticoagulant.
2.1. In cosmetics
Generally, algae are mainly used to produce a wide range of metabolites such
as carbohydrates, carotenoids, proteins, lipids, or vitamins for health, food
and feed additives, and cosmetics and for energy production. The pigment
content in microalgae is a specific feature of each species. Its evaluation is
essential as an indirect measure of cell growth, as well as a parameter to check
the trophic level of waters. Components of algae are frequently used in cos-
metics as thickening agents, water-binding agents, and antioxidants. Some
microalgal species are established in the skin care market, the main ones
being Arthrospira and Chlorella (Stolz & Obermayer, 2005). Microalgae are
the microscopic unicellular organisms which have the capability to convert
solar energy to chemical energy via photosynthesis. The extracts of
microalgae, marine fungi, and bacteria seem to have genuine repair-
and-maintenance effects in skin cosmetics, fighting UV damage, and age
deterioration. Chitosan is used as antiobesity agent, moisturizing agent,
emollient, and film former.
2.2. In food and agricultural field

Marine organisms have been used for centuries as a resource, mainly for sim-
ple fuels, food, and fertilizers. The development of a global high-value
industry based on carotenoids from certain microalgae which provides
medical or health benefits, in food and aquaculture feeds as antioxidants
and colorings for use in nutraceuticals has been seen in the past 40 years.
Only in the recent years, the attention has turned to making use of algae
(seaweeds and microalgae) and invertebrates as sources of platform mole-
cules, building blocks, and processes for a wider range of industries. Another
useful commodity from algae is livestock feed. A large number of algae have
been tested for their biochemical compositions to be used as a livestock feed
supplement or primary livestock feed. Edible seaweeds have reported to be
used as food due to lower calorie, high concentration of minerals, vitamins,
and proteins, and a low fat content (Dhargalkar & Verlecar, 2009).
Algal biomass may well become a significant component of mixed mate-
rials for bioenergy production. The carbohydrates and lipids present in mac-
roalgae and microalgae are raw materials for green fuels, some of which are
finding their way into jet fuel and commercial flights using the biorefinery
approach. Anaerobic digestion of total algal biomass can also provide meth-
ane for combined heat and power. EPS produced by marine organisms also
have a role as inhibitors of crystal formation in frozen foods and sugar syrups
(Colwell, Pariser, & Sindkey, 1986; Sogawa, Kodama, Matsuda, Okutani, &
Shigeta, 1998; Sutherland, 1998).
Spirulina a well-known blue green alga is still used in food supplements
due to its excellent nutrient compounds and digestibility (Kumar et al.,
2005). Besides higher content of protein (6070 wt.%), Spirulina also con-
tains a rich source of vitamins, especially vitamin B12 and provitamin
A (-carotene) and minerals (Thajuddin & Subramanian, 2005). Compared
to other microorganisms, Spirulina can be cultivated in high saline water and
alkaline conditions which give an advantage to function as a feedstock for
livestock feed. In addition, red algae, mainly Porphyra, and brown algae, par-
ticularly Laminaria, Undaria, and Hizikiafusiforme, were directly consumed in
human food (Besada, Andrade, Schultze, & Gonzalez, 2009).
In the human food industry, agar is used mainly as a gelling agent and in a
secondary way as a stabilizing agent and for controlling viscosity. It is used as
an additive, not as a nutrient. The gelling power of agar is so high that it is
used at 1% maximum concentration; for viscosity control and as a stabilizing
agent, the proportion used is 1/100 or less. For this reason, the ingested
quantities are very small and, because agar is not easily digested by the human
body, its calorie contribution is negligible and thus agar can be used in special
diet food. Agar digestion by the human body is imperfect; studies have
shown that less than 10% of the polysaccharide is assimilated. Therefore,
due to the small proportions in which it is used in human food, its impor-
tance as a nutrient is very small (http://www.fao.org/docrep/x5822e/
x5822e03.htm). Agar has been used for many centuries as a high perfor-
mance gelling agent. Its ability to produce clear, colorless, odorless, and nat-
ural gels without the support of other colloids has long been exploited by the
food industry not only as a stabilizer and gelling agent but also in the
manufacturing of confectionery aspics, glazing, icing coatings, piping jellies,
salad dressings, etc. (http://indiaagar.com/Agar.aspx).
The use of chitosan-based edible films is also used to preserve the micro-
bial quality of pork meat hamburger. Tripathi, Mehrotra, and Dutta (2009)
developed a novel antimicrobial coating based on chitosan and PVA and
evaluated its effect on minimally processed tomato. These results indicated
that the film may be a promising material for food packaging applications.
Chitosan is used as a preservative in low-pH foods, either alone or in com-
bination with other preservative systems. The constituents of the food
matrix appear to have an important effect on the antimicrobial efficacy of

chitosan (Rhoades & Roller, 2000). Several workers (Bhale et al., 2003;
Caner, 2005; Lee, 1996) have reported that chitosan coating is effective
in preserving the internal quality of eggs.
Chitosan films as well as chitosan-coated films are used as active packaging,
which can increase shelf life by inhibiting microbial growth (Chen, Yeh, &
Chiang, 1996; Labuza & Breene, 1989). The use of chitin films to preserve
fruits is approved in Canada and in the United States (Shahidi et al., 1999).
Other applications of chitinuous products include enzyme immobilization,
water purification and clarification, deacidification, and as emulsifying, thick-
ening, and stabilizing agents. The search for robust and energy-sparing
enzymes, for food processing and other industrial-scale uses and biocatalytic
conversion for green chemicals, is also finding the marine environment a fruit-
ful source of new candidates. Beyond the well-understood hydrocolloids, the
seaweeds are now being developed for uses that underpin large sectors of
processed foods and drinks, and cosmetics industries.
Coating fruits with semipermeable film has generally been shown to
retard ripening by modifying the endogenous CO2, O2, and ethylene levels
of fruits (El Ghaouth, Arul, Ponnampalam, & Boulet, 1991). Chitosan coat-
ing is used to modify the internal atmosphere without causing anaerobic res-
piration, because chitosan films are selectively permeable to O2 than to CO2
(Bai, Huang, & Jiang, 1988). Therefore, chitosan coating modifies internal
atmosphere in the tissue and fungistatic property and thus has a potential to
prolong storage life and control decay of fruits.
To effectively extend the shelf life of postharvest fruit and vegetable,
chitosan-based coating as a relatively convenient and safe measure, is more
and more concerned in food industry in recent years. Chitosan has strong
antimicrobial and antifungal activities which effectively controls fruit decay
Aider (2010). Chitosan forms a coating on fruit and vegetable, and thus the
respiration rate of fruit and vegetable was reduced by adjusting the perme-
ability of carbon dioxide and oxygen (Elsabee & Abdou, 2013). Chitosan is
also used in many postharvest fruits and vegetables, such as grape, berry,
jujube, and fresh-cut lotus root (Perdones, Sanchez-Gonzalez, Chiralt, &
Vargas, 2012; Yu et al., 2012; Xing et al., 2010). Chitin or chitosan is used
to control postharvest diseases of many fruits such as pear (Yu, Wang, Yin,
Wang, & Zheng, 2008), strawberry (Bhaskara, Belkacemi, Corcuff,
Castaigne, & Arul, 2000; Ge, Zhang, Chen, Ma, & Xu, 2010), table grape
(Mark, Bikales, over Berger, & Menges, 1985), tomato (Badawya & Rabeab,
2009), citrus (Chien, Sheu, & Lin, 2007), and longan (Jiang & Li, 2001).
Though chitosan coating has many advantages for the preservation of

postharvest fruit and vegetable, as for specific fruit or vegetable, single
chitosan coating sometimes demonstrates a certain defect, which includes
limited inhibition to especial microorganism that leads fruit to decay, and
poor coating structure to adjust the permeability of carbon dioxide and oxy-
gen (Ravi, 2000). To overcome this deficiency of single chitosan coating,
there are two main methods to improve the property of chitosan coating.
One method is that the chitosan were combined with organic compounds
such as essential oil, organic acid, or inorganic compound including metal
ions and inorganic nanomaterial, as well as biological control agents.
The other method is that the single chitosan coating was applied with
physical remedies containing heat treatment, hypobaric treatment, gas fumi-
gation, and modified atmosphere packaging. After applying improved
chitosan-based coating, the preserving effects were increased in most of
the cases compared with single chitosan (Jianglian & Shaoying, 2013).
Chitosan with a partial positive charge possess acid-binding properties
(Imeri & Knorr, 1988) and to be effective in aiding the separation of colloidal
and dispersed particles from food processing wastes (Knorr, 1985). These
properties make chitosan an attractive processing aid in fruit juice produc-
tion. Chitosan is also used to improve the quality and shelf life of milk. Ha
and Lee (2001) investigated the effectiveness of water-soluble chitosan to
minimize the microbial (bacterial and yeast) spoilage of processed milk.
Complete inhibition of microbial growth was observed in the banana-
flavored milk containing chitosan. In agriculture, chitosan is used as a coat-
ing for fertilizers, pesticides, herbicides, nematocides, and insecticides for
their controlled release to soil which is done in order to reduce environmen-
tal damage caused by excessive use of these agrochemicals. Chitosan films are
used to coat seeds and leaves to prevent microbial infection (Li, Dunn,
Grand Maison, & Goosen, 1992).
2.3. In pharmaceutics
Algal organisms are rich source of novel and biologically active primary and
secondary metabolites. These metabolites may be potential bioactive com-
pounds of interest in the pharmaceutical industry (Rania & Hala, 2008).
Biopolymers produced by marine organisms are being increasingly investi-
gated for several biomedical applications (dAyala, Malinconico, &
Laurienzo, 2008; Rinaudo, 2008). The polysaccharides derived from marine
materials have been widely used in the development of drug delivery
devices, especially with the shape of spheres of different sizes. The most
probable marine-derived polymers used in the preparation of drug delivery
particles are the chitosan and alginate. These two main polysaccharides (algi-
nate and chitin) extracted from marine plants (algae kelp) and crab shells,
respectively, have an extensive history of use in basic sciences, pharmacy,
and medicine.
The existence of bioactive compounds in algae is to be expected due to
co-occurrence of these organisms in aquatic natural communities, where an
inhibitory interaction occurred between producers and competitors within
the same habitat. Microalgae contain numerous bioactive compounds that
can be harnessed for commercial use. They have emerged as important
sources of proteins and value-added compounds with pharmaceutical and
nutritional importance. The microalgae have a significant attraction as nat-
ural source of bioactive molecules, because they have the potential to pro-
duce bioactive compounds in culture, which are difficult to produce by
chemical synthesis.
Some other novel molecules, which are obtained from the microalgae,
marine fungi, and bacteria, have the exciting potential in medicine for can-
cers, immune disorders, and resistant microbial infections. A toxin named as
holothurin is the earliest biologically active substance of marine origin which
was extracted by Nigrelli from a marine organism, the Actinopyga agassizi
(Nigrelli, Stempien, Ruggirei, Liguori, & Cecil, 1967). Holothurin showed
some antitumor activities in mice. After this invention, the search for drugs
and natural products of interest from marine organisms has continued. Also,
the replacement of the existing polysaccharide polymers such as carrageenan
by the porphyridium polysaccharide was used in biomedical applications.
Chitin and chitosan have been proposed as biomaterials having a range of
biomedical and industrial applications because of their potential beneficial
biological activities (Shigemasa & Minami, 1995), such as antimicrobial
activity and stimulation of healing. Chitosan is a viscous solution and is easily
gelled upon mixing with heparin solution, resulting in an insoluble hydrogel
(Fujita et al., 2004, 2007).
In drug delivery systems, the modification of chitosan is a powerful tool
to control the interaction of the polymer with drugs, enhances the load capa-
bility, and tailors the release profile of the drug carriers. It is an acknowl-
edged polymer for drug delivery in the colonic part, since it can be
degraded by the microflora present in the human colon. Also, it has the
potential of serving as an adsorbent enhancer across intestinal epithelial cells
for its mucoadhesive and permeability enhancing property ( Janes, Calvo, &
Alonso, 2001; Kowapradit et al., 2008). It is also proved to enhance insulin

absorption across human intestinal cell without injuring them.
The performance of chitosan on the cited applications was mainly due to
responsible properties such as excellent chelation behavior, antimicrobial
activity, high adsorption, controllable biological activity, hydrogels forming
ability, film-forming ability, nontoxicity, biodegradability, and biocompat-
ibility (Honarkar & Barikani, 2009; Rinaudo, 2006; Silva, Mano, & Reis,
2010). Polysaccharide-based biomaterials plays an emerging role in several
biomedical fields such as tissue regeneration, particularly for cartilage, drug
delivery devices, and gel entrapment systems for the immobilization of cells.
The ability of chitosan to be processed into porous structure is used in
cell transplantation and tissue regeneration. Three-dimensional biodegrad-
able chitosan nanohydroxyapatite composite scaffolds were reported for
bone tissue engineering application (Thein-Han & Misra, 2009). The
thermogelling chitosan/glycol phosphate solutions prepared by Chenite,
Chaput, and Wang (2000) can form a gel in body temperature and are attrac-
tive as injectable implant system in tissue engineering. These liquid gels are
able to fill any space or shape of a defective site, leaving cells and therapeutic
agents and are incorporated prior to the injection within the solution and the
systems can be implanted in the site without surgery.
In wound healing, an ideal dressing should protect the wound from bac-
terial infection as well as promote healing (Wu et al., 2003). Chitosan-based
materials, produced in varying formulations, have been used in a number of
wound healing applications. Chitosan induces wound healing on its own
and produces less scarring (Azad, Sermsintham, Chandrkrachang, &
Stevens, 2004; Ueno, Mori, & Fujinaga, 2001; Ueno et al., 1999).
When used in wound management, chitosan and its derivatives turn into
gel, when they come into contact with body fluids and reduce friction
between the dressing material and the wound. They also accelerate wound
healing with their hemostatic properties and stimulate macrophage. The
increase in the antimicrobial activity is observed with carboxymethyl
chitosan, which makes the essential transition metal ions unavailable for bac-
teria or binds to the negatively charged bacterial surface to disturb the cell
membranes (Liu, Guan, Yand, Li, & Yao, 2001). In the field of ophthalmol-
ogy, chitosan is used in the making of therapeutic contact lenses and eye
dressing.
Chang, Niu, Huang, and Kuo (2007) proposed the use of polyethylene
glycol (PEG)-g-chitosan for cell adhesion applications, while Wang et al.
(2007) successfully used this copolymer to coat a poly(dimethylsiloxane)
microchip intended for biomolecule (amino acids and proteins) separation,

using an in situ method. In the latter experiment, the coating increased sur-
face hydrophilicity and suppressed adsorption of biomolecules.
Radhakumary, Prabha, Nair, and Suresh (2009) suggested the applica-
tion of chitosan-comb-graft-polyethylene glycol (PEG) monomethacrylate
as a new biomaterial for hemodialysis and immunoisolatory membranes.
This material was found to have discriminating permeability capacities (per-
meable to low-molecular-weight solutes like creatinine, glucose, and urea
and impermeable to high-molecular-weight solutes like albumin). Li
et al. (2009) reported the possibility of making films of mPEG-g-chitosan
by preparing a composite film with suitable hollow and high capacity of
water adsorption, which could have potential application in wound healing
and tissue engineering.
Aly, Abdel-Mohsen, and Hebeish (2010) showed innovative multi-
finishing using O-PEG-g-chitosan/citric acid aqueous system for prepara-
tion of medical textiles. The cotton treated with the copolymer has been
evaluated as healthcare worker uniforms and medical products, acquiring
antimicrobial and anticrease properties.
Usually, chitosan manufactured from squid pen by spin casting technol-
ogy is used in contact lenses. They have good tensile strength, elongation,
modulus, tear strength which are essential in the making of contact lenses.
Also, ocular bandage lenses are made from chitosan-based materials, as they
possess antimicrobial, wound healing, and film capability properties
(Markey, Churchill, & MacDonald, 1989).
Targeted chitosan nanoparticles have been developed to specifically
deliver therapeutics or contrast agents to tumor or metastasis for cancer treat-
ment and diagnosis. So far, several studies have investigated PEGylation of
chitosan to improve its affinity to water and organic solvents. PEG is a neu-
tral, water-soluble, and nontoxic polymer which has been employed for
pharmaceutical and biomedical applications (Harris & Zalipsky, 1997).
PEG is a synthetic polymer approved by the FDA for internal consumption
and injection in a variety of foods, cosmetics, and drug delivery systems
(Cavalla, 2001). Magnetic chitosan nanoparticles, as multifunctional
nanocarriers, were loaded with bleomycin and proved to be a very effective
as targeting system by Kavaz, Odabas, Guven, Demirbilek, and Denkbas
(2010). After systematic administration of these systems into the body, they
circulate in the blood stream and specifically bind to the targeting cancerous
cells or tumor sites due to the incorporation of signaling molecules in their
constituents (Hang et al., 2010).
Recent studies have demonstrated that chewing the chitosan oligomer

containing gum effectively inhibited the growth of carcinogenic bacteria
in the saliva (Hayashi, Ohara, & Ganno, 2007) and also the growth of peri-
odontic bacteria (P. gingivalis) in saliva. The chitosan-containing gum
chewing has a greater antibacterial effect and it also increases salivary secre-
tion (Hayashi, Ohara, Ganno, Ishiazaki, & Yanagiguchi, 2007). Also due to
the antibacterial properties of chitosan, it is an important component in skin
care creams, shampoos, and hair sprays. As chitosan is the only natural cat-
ionic gum that becomes viscous on being neutralized with acids, it is used in
creams, lotions, and as nail lacquers (Mark et al., 1985). Recently, asymmet-
ric chitosan membranes were developed for the guided tissue regeneration
using the two-step phase separation (Kang et al., 2007).
Chitosans functional groups allow it to interact with many materials,
which allow it to be used in conjunction with materials such as hydroxyap-
atite or other calcium-based minerals to form composites that have multiple
applications within the orthopedic and periodontal industries. These cal-
ciumchitosan composites can be used as a coating in conjunction with joint
prostheses. As the chitosan is degraded, new bone can be deposited adjacent
to the prosthesis to stabilize the implant within bone. An additional use for
chitosan in orthopedics includes a direct replacement of bone or hard tissue.
It is also a natural bioadhesive used to improve bone cement which is used to
secure implants as well as to fill bone cavities (Foda, El-Laithy, & Tadros,
2007; Khor & Lim, 2003; Senel & McClure, 2004).
Another recent article shows the finding of the occurrence of silicachitin
fiber composite in skeletons of marine sponges. This is the first report of a sil-
icachitins composite biomaterial found in nature. From this perspective, the
view that silicachitin scaffolds may be key templates for skeleton formation
(Ehrlich et al., 2007). This structural information could be useful in develop-
ing scaffolds for tissue engineering and other applications. In an vitro study on
the degradation and biocompatibility of poly(L-lactic acid)/chitosan (PLLA/
CS) fiber composites, excellent adhesion between osteoblast and PLLA/CS
fabrics was observed, indicating good biocompatibility of the fabrics with
osteoblast and its possible use as supporting materials for chest walls and bones
(Zhang, Hua, Shen, & Yang, 2007). Chitin fiber is also employed to fabricate
novel biomimetic nanostructured bicomponent scaffolds consisting of chitin
and silk fibroin nanofibers by an electrospinning process.
Chitosan is suitable for nerve regeneration based on its biocompatibility and
biodegradability. Haipeng et al. (2000) reported that neurons cultured on the
chitosan membrane can grow well and that chitosan tube can promote repair
of the PNS. Yuan, Zhang, Yang, Wang, and Gu (2004) found that chitosan fibers
supported the adhesion, migration, and proliferation of SCs, which provide a
similar guide for regenerating axons to Bungner bands in the nervous system
(Sudha, Aisverya, Rose, Venkatesan, & Kim, 2013). Some research work, on
Aloe vera gel with the alginate film, to explore its therapeutic properties, which
includes antibacterial, antiseptic, anti-inflammatory, and its ability to stimulate
the fibroblast proliferation and the collagen synthesis (Atiba et al., 2011;
Choi & Chung, 2003; Pellizzoni, Ruzickova, Kalhotka, & Lucini, 2012).
Jayakumar, Menon, Manzoora, Naira, and Tamura (2010) have reported
that the chitosan encapsulated ZnS nanoparticles were further functionalized
with D-mannose to yield mannosylated ZnS of size 120 nm. In vitro cyto-
toxicity of the synthesized nanomaterials assessed using MTT assay suggests
low cytotoxicity of the mannosylated ZnS nanoparticles toward both nor-
mal and cancer cell lines. Active targeting of cancer cells was attempted using
the mannosylated nanoparticles.
Alginate has been used in a number of biomedical applications, such as
wound dressing, tissue engineering, and drug delivery. Several reports have
suggested that certain alginate dressings (e.g., Kaltostat) can enhance wound
healing by stimulating monocytes to produce elevated levels of cytokines
such as interleukin-6 and tumor necrosis factor- Thomas, Harding, and
Moore (2000). Production of these cytokines at wound sites results in
proinflammatory factors that are advantageous to wound healing.
On the other hand, since carrageenan (CG) is negatively charged above
its pKa value, it can spontaneously associate with positively charged polyions
to form polyelectrolyte complexes (Zhang, Du, Wang, & Zhang, 2010).
Tapia and co-workers employed polyelectrolyte complexes of CS and
CG as prolonged drug release matrix. However, the high capacity of
CG to absorb water into tablets induced premature disintegration of tablets
instead of matrix swelling. Recent studies showed that, when CSCGs-
based tablets were transferred from simulated gastric fluid to simulated
intestinal fluid, in situ polyelectrolyte film could be formed on the surface
of tablets, and the polyelectrolyte film could further control drug release
(Li et al., 2013). Thus, CSCG nanoparticles have potential applications
not only in drug delivery but also in tissue engineering and regenerative
medicine (Li, Ni, Shao, & Mao, 2014).
In addition to the aspects described earlier, the utilization of CG in the
pharmaceutical field is being broadened. For instance, besides as a novel pel-
letizing agent, CG can also be used as an efficient drug release modifier for
ethyl cellulose-coated pharmaceutical dosage forms.
2.4. In biotechnology and microbiology

Marine biotechnology has an increasingly important role in the future
bioeconomy. In order to maximize the impact of public funding for research
and innovation in marine biotechnology, the Europe is now at the verge of
creating a higher level of interlinking and aligned national research pro-
grams. Marine biotechnology mainly consists of two aspects. It uses the
products and processes of marine organisms in modern industrial biotech-
nology, and applies the biotechnology in the marine context in areas such
as environmental monitoring and bioremediation.
By controlling the growth conditions of variety of microorganisms in a
bioreactor while tailoring the production of biologically active compounds,
it is possible to obtain different number of polysaccharides using biotechnol-
ogy as the powerful tool. The knowledge of biochemical processes which
was adapted in extreme marine environments is the basis for discoveries
in biotechnology. This was the actual current opinion of most of the scien-
tific community throughout the world in the fields of biotechnology that
could benefit from miming the extremophiles are very broad and cover
the search for new bioactive compounds for pharmaceutical, industrial, agri-
cultural, environmental, and medical uses.
Microalgae are employed in agriculture as biofertilizers and soil condi-
tioners. The majority of cyanobacteria are capable of fixing atmospheric nitro-
gen and are effectively used as biofertilizers. Cyanobacteria play an important
role in maintenance and build-up of soil fertility, consequently increasing rice
growth and yield as a natural biofertilizer (Song, Martensson, Eriksson,
Zheng, & Rasmussen, 2005). The agricultural importance of cyanobacteria
in rice cultivation is directly related with their ability to fix nitrogen and other
positive effects for plants and soil. After water, nitrogen is the second limiting
factor for plant growth in many fields and deficiency of this element is met by
fertilizers (Malik, Ahmed, & Rizki, 2001). Agarose forms an inert matrix
which is principally used in nucleic acid and protein separations. Other very
important applications, besides electrophoresis, are chromatography, gel affin-
ity, ionic exchange, immunodiffusion, biocatalytic support, and its use in solid
culture media and growth of protein crystals (http://www.hispanagar.com/
applications.htm). Thadathil and Velappan (2014) have reported biological
control using microorganisms or its component to repress plant disease offers
an alternative to chemical fungicide and also it is an ecofriendly approach for
controlling agricultural pathogens. Several research groups reported the in vitro
antifungal activity of chitosanases; they can be used to improve the resistance
of plants against different phytopathogenic fungi. A chitosanase from Bacillus

cereus D-11 inhibiting the hyphal growth of Rhizotonia solani 27-kDa chito-
sanase from Amycolatopsis sp. CsO-2 inhibiting hyphal growth of Rhizopus ory-
zae. The major component present in excreted EPS is the glucose and the
galactose, mannose, arabinose being the other components. Various marine
organisms, including plants, animals, diatoms, microalgae, and bacteria widely
secreted the EPS (Decho, 1990; Gutierrez et al., 1996; Philippis & Vincenzini,
1998; Philippis et al., 1998). The EPS produced by the marine organisms have
been explored for various biotechnological applications, such as anticoagulants
(heparin analogs), antitumor agents, and wound dressings for eye and joint
surgery.
Apart from medical applications, EPS are also important as gelling agents
(in cell and enzyme technology and foods), foam stabilizers (in beer and
fire-fighting fluids), emulsion stabilizers (in food and thixotropic paints),
flocculants (in water clarification and ore extraction), and hydrating agents
(in cosmetics and pharmaceuticals).
2.5. In treatment of industrial effluent

Wastewater is actually the water produced by different domestic and indus-
trial activities which contains various inorganic, organic, and biological con-
taminants that are of environmental significance. Nutrients, trace metals, and
gaseous inorganic materials are the major sources of chemical-inorganic pol-
lutants. The source of biological pollutants includes pathogenic bacteria and
the source of physical pollutants includes suspended solids and dissolved
solids. These contaminants can create health hazards if discharged without
proper care and treatment into streams or oceans. Especially the rapid
increase in industries has led to the complexity of toxic effluents.
Industrial wastewater treatment covers the mechanisms and processes
used to treat waters that have been contaminated in some way by anthropo-
genic industrial or commercial activities prior to its release into the environ-
ment or its reuse. Some form of pretreatment was done to the wastewater
obtained from industrial process prior to discharge to a sewer. This form of
pretreatment was mainly done to minimize corrosion and clogging of sewer
lines and to prevent reductions in biological treatment process efficiency by
toxic effects from toxic concentration of organic and inorganic substances.
The efficient removal of toxic metals from wastewater is an important matter
and a number of technologies such as precipitation, reduction, and ion
exchange were developed.
Coagulation or flocculation process was conducted for the treatment of

industrial wastewater to achieve maximum removal of chemical oxygen
demand (COD), total dissolved solids (TDS), and total suspended solids
(TSS). Therefore, the effect of coagulant dose, polyelectrolyte dose, pH of
solution, and addition of polyelectrolyte as coagulant aid and found to be
important parameters for effective treatment of beverage industrial wastewater
was investigated by Amudha and his coworkers. Chitosan, a biological cationic
polymer, was used in treating dairy wastewater, agriculture, food processing,
medicine, cosmetics, wastewater treatment, and biotechnology (Tokura &
Nishi, 1995). Reactive dyes are widely used for textile dyeing, paper printing,
leather dyeing, color photography, and as additives in petroleum products.
Contamination of water resources with dyes is not desirable, since colored
effluents may contain toxic, carcinogenic, and mutagenic chemicals. Approx-
imately, 10,000 different dyes and pigments are used in industry. Chitin and
chitosan both have efficiency in removing dyes from wastewater. Chitosan
chelation is the procedure of choice for dye removal from aqueous solution.
The amine groups on chitosan bind metal cations at pH close to neutral.
At low pH, chitosan is more protonated and therefore it is able to bind
anions by electrostatic attraction (Guibal, 2004). Chitosans functional groups
and natural chelating properties make chitosan useful in wastewater treatment
by allowing for the binding and removal of metal ions such as copper, lead,
mercury, and uranium from wastewater. It can also be utilized to remove dyes
and other negatively charged solids from wastewater streams and processing
outlets. Chitosan grafted with poly(acrylonitrile) has been further modified
to yield amidoximated chitosan (Kang, Choi, & Kweon, 1996), a derivative
having a higher adsorption for Cu2+, Mn2+, and Pb2+, compared to cross-
linked chitosan. The adsorption capacity had a linear dependence on pH in
cases of Cu2+ and Pb2+. However, a slight decrease in the adsorption capacity
was observed in case of Zn2+ and Cd2+ (Kang, Choi, & Kweon, 1999).
Chitosan has been modified with different mono as well as disaccharides.
Yang, Lin, Wu, and Chen (2003) have also reported the metal uptake abilities
of macrocyclic diamine derivative of chitosan. The polymer has high metal
uptake abilities, and the selectivity property for the metal ions was improved
by the incorporation of azacrown ether groups in the chitosan.
Chitosan was successfully coated on PET granules through a dip and
phase inversion process and the coated granules were examined for the per-
formance and mechanism of humic acid removal through a series of batch
adsorption tests. With its positive charge, chitosan can be used for coagula-
tion and recovery of proteinaceous materials present in food processing
operations (Knorr, 1991). Chitin and chitosan are also good adsorbents for
removal of phenol and other pollutants from industrial wastewaters
(Milhome et al., 2009). In addition to this, when chitosan was modified with
some organic compounds, such as aldehydes and organic acids, it was found
that the modified products with salicylaldehyde, cinnamaldehyde, benzalde-
hyde, and carbohydrate showed better Fe and Cu metal ions uptake. Com-
paratively, carbohydrate modified chitosan showed lower metal ion uptake,
which they attributed to the higher ability of carbohydrate to be leached out
of the chitosan matrix due to its higher solubility in water. In yet another
study, chitosan was cross-linked using glutaraldehyde in the presence of
magnetite.
The amino sugars of chitin and chitosan are the major effective binding
sites for metal ions, forming stable complexes by coordination (Chui, Mok,
Ng, Luong, & Ma, 1996). The electrons present in the amino and
N-acetylamino groups form dative bonds with transition metal ions, and some
of the hydroxyl groups in these biopolymers may act as donors. Hence,
deprotonated hydroxyl groups can be involved in the coordination with metal
ions (Lerivrey, Dubois, Decock, Micera, & Kozlowski, 1986). Different
degree of deacetylation (DD) chitosan was prepared in different DD and is
used for the removal of a Reactive Black M-2R (RBM) from aqueous solu-
tion (Li & Ding, 2011). The deacetylated chitosan (HDC) beads, cross-linked
HDC-TPP beads, and chemical cross-linked HDC-ECH were used in the
adsorption behavior of anionic dye (congo red or direct red 28) and cationic
dye (methylene blue or basic blue 9) (Thein Kyaw, Sander Wint, & Myo
Naing, 2011). Chitosancharcoal composite was applied as a media to treat
tannery effluent containing chromium (Siraj et al., 2012). The literature shows
a new composite chitosan biosorbent was prepared by coating chitosan on to
perlite ore and investigated for Cu(II) and Ni(II) removal. Maximum removal
of Cu(II) and Ni(II) on chitosan coated on perlite was at pH 5.0. The max-
imum monolayer adsorption capacity of chitosan coated on perlite was
196.07 mg/g for Cu(II) and 114.94 mg/g for Ni(II).
From the literature, it is clear that chitosan can be used to remove numer-
ous trace metals (Cu(II), Pb(II),U(VI), Cr(III), Cr(VI), Ni(II), Cd(II), Zn(II),
Co(II), Fe(II), Mn(II), Pt(IV), Ir(III), Pd(II), V(V), and V(IV)) from wastewa-
ter. Chitosan has been used in a variety of forms, which include chitosan beads,
flakes, and membranes (Deans & Dixon, 1992; Findon, Mckay, & Blair, 1993;
Mckay, Blair, & Findon, 1989; Onsyen & Skaugrud, 1990).
The removal of Cr(VI) ions from aqueous solutions has been investigated
using chitosan/starch blend. Several metals are preferentially adsorbed in
acidic media, while chitosan can dissolve in acidic condition. To overcome

such a problem, some cross-linking agents such as glutaraldehyde (Ruiz,
Sastre, & Guibal, 2000), epichlorohydrin (Ngah, Endud, & Mayanar,
2002), and ethylene glycol diglycidylether (Li & Bai, 2006) are used to sta-
bilize chitosan in acid solutions. But glutaraldehyde is the most widely used
because it does not have much diminishing adsorption capacity (Ngah et al.,
2002). This method is used to ensure good mechanically and chemically sta-
ble beads, but it has been found to have negative effect on the adsorption
capacity of the chitosan. The main reason for the loss of adsorption capacity
is that amine groups are involved in the cross-linking reaction (Martinez
et al., 2007). Chitosan membranes as sorbents for trace elements determina-
tion in surface waters have been tried by Elisaveta, Mladenova, Grigorova,
and Irina (2011), and the authors have successfully used their membranes as
an efficient sorbents for the preconcentration and they have recommended
their membranes for solid-phase extraction of Cd(II), Eu(II), Ni(II), and
Pd(II) from surface water. In the case of biopolymers, chitin, chitosan,
and nylon 6 are widely employed as biopolymers. As an adsorbent for the
removal of heavy metals from water, biopolymer has been studied. Chitosan
is one of them (Navarro, Guzman, Saucedo, Revilla, & Guibal, 2003).
Chitosan has the ability to form complexes with metals. It has a better
adsorption capacity for metal ion. It has severe limitations in its use in acidic
media because of its solubility in acid (Llorens, Pujola, & Sabate, 2004;
Nomanbhay & Palanisamy, 2005). In order to increase the copper sorption
capacity of raw chitosan beads, Gandhi, Kousalya, Viswanathan, and
Meenakshi (2011) modified chitosan into protonated chitosan beads, car-
boxylated chitosan beads, and grafted chitosan beads (GCB). They showed
a significant sorption capacity of 52, 86, and 126 mg/g, respectively, while
raw chitosan beads displayed only 40 mg/g. They conclude that GCB
showed higher sorption capacity toward Cu(II) ions. The SiCS composite
was used for the chelation of divalent copper and lead from aqueous solu-
tions using batch method (Gandhi & Meenakshi, 2012).
Although the amount of available literature data for chitin/chitosan, algi-
nate, and carrageenan application in water and wastewater treatment is increas-
ing at a tremendous pace, there are still several gaps which need to be filled.
3. FUTURE DIRECTIONS FOR RESEARCH

In future, efforts are made to improve these novel biomaterials for fur-
ther enhancement in their application results. Moreover, designing of new
scaffolds from other natural polymer such as alginate, gelatin would be
possible for soft tissue engineering such as skin. Chitin/chitosan has a great
potential in a variety of biomedical, industrial applications, and chitosan
physicochemical and mechanical properties utilized in fabricating particles
and films can be modulated for specific purposes. Efforts should be made
to prepare nanofibrous scaffolds from other natural polymers including silk
for hard and soft tissue engineering. And the best use of these marine sources
in the field of food, cosmetics industries, in effluent treatment, and in med-
ical field should be made.
4. CONCLUSION
This review summarizes the industrial and biomedical applications of
marine carbohydrates such as chitin-, chitosan-, alginate-, agar-, and
carrageenan-based nanomaterials in tissue engineering, wound dressing,
drug delivery, and cancer diagnosis. In addition, this review also opens up
the novel applications for which these natural biopolymers can be put to
use in a variety of nanostructural forms and sizes. Nanostructured composite
scaffolds can be developed as promising tissue engineered constructs or for
wound healing. Multifunctional use of chitin- and chitosan-based
nanomaterials has been proved to aid simultaneous cancer targeting and drug
delivery. We expect that this chapter provides insights on the use of these
marine carbohydrates for researchers working to discover new materials
with new properties for the valuable applications of these materials.
ACKNOWLEDGMENTS
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CHAPTER NINE
Nutraceutical and
Pharmacological Implications
of Marine Carbohydrates
Ramjee Pallela1
Synthetic Biology and Biofuels Group, International Centre for Genetic Engineering and Biotechnology,
New Delhi, India
1
Corresponding author: e-mail address: rpallela@icgeb.res.in
Contents
1. Introduction 183
2. Marine Carbohydrate Sources 184
3. Marine Carbohydrates as Nutraceuticals 188
4. Marine Carbohydrates as Pharmaceuticals 189
5. Conclusion 191
References 191
Further Reading 195
Abstract
Current day's research has been focusing much on the potential pharmacological or
nutraceutical agents of selective health benefits with less toxicity. As a consequence
of increased demand of nutritional supplements of great medicinal values, develop-
ment of therapeutic agents from natural sources, in particular, marine environment
are being considered much important. A diverse array of marine natural products con-
taining medicinally useful nutritional substances, i.e., marine nutraceuticals have been
focused to the benefit of mankind. Carbohydrates, by being constituted in considerable
amount of many marine organisms display several nutraceutical and pharmaceutical
behavior to defend from various diseases. Moreover, the carbohydrates from algae
as well as from shellfish wastes, like chitosan and its derivatives, showed tremendous
applications in biology and biomedicine. In the current chapter, several of marine
carbohydrates from various marine flora and fauna have been covered with their appli-
cations and prospects in the development of nutraceuticals and pharmaceuticals.
1. INTRODUCTION
Carbohydrates along with protein and lipid molecules play a notice-
able contributory role in nutraceuticals and pharmaceutical development.
http://dx.doi.org/10.1016/B978-0-12-800268-1.00009-3
184 Ramjee Pallela
Because of their possession of moderate barrier properties against diffusion of

oxygen and moisture, most of them exhibit enhanced stability of protein-
based emulsions (Colin et al., 2007). Although several carbohydrate mole-
cules are commercially been used as nutraceuticals, food supplements, or
pharmaceuticals to treat various diseases, natural sources of carbohydrates
like gum-arabic, derivatives from starch, cellulose derivatives, carrageenan,
alginate are gaining tremendous importance keeping in view of traditional
and ancient values of these sources. A great attention has been paid recently
by the consumers toward natural bioactive substances that act as functional
ingredients in the diets, which show several beneficial health effects (Zhang,
Li, & Kim, 2012). Moreover, marine-based medicinal foods like macroalgae
(seaweeds) are identified with several nutraceutically and pharmacologically
important carbohydrate molecules and moieties to combat several health
complications ( Jimenez-Escrig, Gomez-Ordonez, & Ruperez, 2011).
Algae, by being a major constituent in the daily based diet in many of the
Asian countries, play significant role in better health of human beings. This
extensive use of algal foods augmented the interest of many researchers to
find out unique pharmacological and nutraceutical leads that work against
many health complications. The interest in marine nutraceuticals inspired
researchers to concentrate on several bioprocessed marine products as alter-
native sources for synthetic nutritional ingredients, as a part of nutraceuticals
and functional foods via the development of suitable technologies, one for an
example, the ultrafiltration membrane bioreactor system ( Jeon & Kim,
2000; Kim & Senevirathne, 2011). In addition, the polymeric macromole-
cules, e.g., chitosan and its derivatives, are mined from sea to their eventual
application in health and medicine via the extraction of these molecules
from shellfish wastes (Hayes, Carney, Slater, & Bruck, 2008). Chitosan
and chitooligosaccharides (COS) and their derivatives have been proved
for their best implications as nutraceutical and pharmacological moieties
to play major role against some important health complications (Kim,
2010; Xia, Liu, Zhang, & Chen, 2011). The current chapter covers an over-
all nutraceutical and pharmacological potential of marine-based carbohy-
drates from various animals and plants to give a note on the perspectives
and prospects of using marine carbohydrates.
2. MARINE CARBOHYDRATE SOURCES

Over the last few decades, medical and pharmacological industries as
well as academic research institutions have gained increased interest in
Marine Carbohydrates as Nutraceuticals and Pharmaceuticals 185
marine-based carbohydrates. This is because of the wide potentiality of

marine carbohydrates and their application in several biological, biomedical,
and nutritional fields. The molecular weights, structural parameters, and
physiological characteristics of marine carbohydrates are diverse in the sense;
therefore, they behave accordingly to result in specific bioactivities such as
antiproliferative, antitumor, antiviral, anticoagulant, antioxidant, anti-
inflammatory effects, etc.
Majority of marine organisms with potential carbohydrates are macro-
algae or seaweeds that produce vast number of carbohydrates, localized
within the intercellular spaces (Kraan, 2012). The three types of marine algae
viz., green algae (Chlorophyta), red algae (Rhodophyta), and brown algae
(Phaeophyta) contain sulfated polysaccharides (SPs), sulfated galactans,
xylans, alginic acid, fucoidan (sulfated fucose), laminarin (-1, 3 glucan)
and sargassan, agar, carrageenans, xylans, floridean starch (amylopectin-like
glucan), water-soluble sulfated galactan, as well as porphyran as mucopoly-
saccharides. Algal species that produce these important carbohydrates along
with other polysaccharide sugars like rhamnose, arabinose, glucose, galac-
tose, fucose, etc., have been covered in other sections of this book to specify
their biomedicinal properties. However, some important species of algae,
Ecklonia cava, Ecklonia kurome, Laminaria japonica, Laminaria digitata, Lessonia
vadosa, Porphyra haitanensis, Ascophyllum nodosum, Nothogenia fastigiata, Sargas-
sum horneri, Gigartina skottsbergii, Schizymenia binderi, Grateloupia indica, Mon-
ostroma latissimum, Codium fragile, Undaria pinnatifida, Codium pugniformis,
Caulerpa racemosa, Ulva conglobata, Ulva pertusua, Ulva lactuca, Gayralia oxy-
sperma, Cryptonemia crenulata, Nemalion helminthoides are to be exemplified
for their medicinal as well as nutritional interest (Wijesekara,
Pangestuti, & Kim, 2011). Most of these species are with higher biomass
and the possible edibility of them is an attracting characteristic feature to
use them in medicinal foods by direct consumption through diet and indi-
rect consumption through the extracted nutraceutical and functional food
molecules. Polysaccharides, especially from marine red and brown algae,
have been isolated and characterized for their anticoagulant activity.
Macroalgal polysaccharides such as carrageenans (Chondrus, Eucheuma,
and Kappaphycus), alginates (Ascophyllum and Laminaria), algal polysaccha-
rides (Macrocystis), and agars (Gelidium and Gracilaria) are of much industrial
application purpose, which are having much economic potential. Sulfated
galactans and sulfated fucans from Ulva fasciata exhibit high anticoagulant
activity, which may address their use in functional foods and nutraceuticals.
Despite of the much anticoagulant activity from the polysaccharides of
186 Ramjee Pallela
brown and red algae, however, there are very few reports of anticoag-
ulant polysaccharides green algae (Govindan, Thomas, Pratheesh, &
Muraleedhara, 2012). Similarly, polysaccharides are isolated and character-
ized from marine green algae (Ulva) of Kerala coast of India based on the
bioactive and bioeconomic importance of Ulva species (Govindan, 2012).
Despite of the generally available SPs, several other carbohydrate species like
furcellaran, funoran, ascophyllan and sargassan, uronic acid, rhamnose,
xylose, glucose, mannose, mannitol, and galactose as monosaccharide
components were also found in macroalgae. Another combinatorial polysac-
charide, termed phycocolloid, is used to refer to three main products
viz., alginate, carrageenan, and agar, which are extracted from brown and
red seaweeds, respectively.
Even microalgal species of marine environment can be produced at high
biomass to produce highly potential bioactive molecules from lipid, protein,
and carbohydrate molecules. Microalgal biomass, moreover, is not only rich
in lipids but also in carbohydrates and proteins where the lipid-extracted
microalgal debris consists of mainly proteins and carbohydrates used directly
for animal feed. These leftover materials can further be redirected to process
them for the extraction of required carbohydrate molecules of nutraceutical
interest (Pleissner & Lin, 2013). Phytoplanktons, on the other hand, possess
valuable carbohydrates of medicinal value, e.g., chrysolaminaran an abun-
dant type of storage carbohydrate present in marine phytoplanktons like
Phaeocystis and diatoms (Kurita, 2006). The cycling of carbohydrates is
one of the most important processes in the marine carbon cycle to determine
the carbohydrate composition of the biomass of phytoplankton. It is
reported that Phaeocystis is a cosmopolitan in nature and known to produce
copious amounts of carbohydrates majorly, the extracellular mucopolysac-
charides in the colony matrix and storage glucans, of which chrysolaminaran
is the main constituent.
Moreover, the marine microbial carbohydrates have been screened
structurally and functionally for their potential applications in pharmaceuti-
cals, adhesives, and textile industries (Kim, 2013).
Marine animals although contribute in diversified medical and health
applications through the production of various macro- and micromolecules
like proteins, lipids, and carbohydrates, the application of marine polysac-
charides limited majorly to the chitin, chitosan, and its derivatives produced
from shellfish. Properties of chitosan solutions are similar to those of cellu-
lose ethers and thus one may imply the established cellulose applications
to chitosan for its effective conversion to further derivatives like COS
(Xia et al., 2011). Shellfish comprise crustaceans (shrimps, lobsters, and crabs)
and molluscs (such as scallops, cockles, mussels, clams, and oysters). Shellfish
wastes from scallops (Chlamys hastate), cockles (Cerastoderma edule, Clinocardium
nuttalli), whelks (Buccinum undatum), clams and mussels (Mercenaria mercenaria,
Mytilus galloprovincialis, Mytilus edulis), oysters (Crassostrea gryphoides, Crassostrea
gigas), and crustaceans (crabCancer pagurus; lobsterNephrops norvegicus
Homarus americanus; shrimpCrangon crangon) have been redirected toward
the developments of various biopolymers of importance that can be used as
nutritional substances, animal feed, biomedical materials, etc. As the emerging
interest of using animal foods especially of marine origin, the consumption
rate of several shellfish species has been increasing annually. However, the
processing of these shellfish wastes is costly and only a few regions in the world
would be able to produce required quantities of chitin, chitosan, and their
derivatives toward their implication in biological and biomedical fields
(Archer, Watson, Garret, & Large, 2005). In addition, the use and application
of these derivatives in medical and pharmacological sectors is very limited as
shellfish waste is classed as a Category 3 animal by-product, which has to be
handled and approved properly by the suitable legislation authorities
(Mangi & Catchpole, 2012).
According to the simplest process described by Dutta et al. (2004),
obtaining chitosan from marine animals involved four important steps
for producing chitosan from chitin, for instance, from crustacean shells
viz., (i) deproteination, (ii) demineralization, (iii) decoloration, and
(iv) deacetylation; and further the development of COS is possible from
the membrane bioreactor system via enzyme hydrolysis (chtiosanase treat-
ment) method ( Dutta, Dutta, & Tripati, 2004; Jeon & Kim, 2000). Several
stages of this whole processing were demonstrated as a simple flow diagram
in Scheme 9.1.
Further the COSs, which are the degraded products of chitosan or chitin,
have been produced by several methods, such as enzymatic and acidic
Crustacean shells Size reduction Deproteination (NaOH) Washing

Demineralization (HCl) Washing Dehydration Decoloration Chitin
Deacetylation (NaOH) Washing Dehydration Chitosan Enzymatic hydrolysis
(chitosanase) in membrane reactor system or Chemical hydrolysis (acids)
Chitooligosaccharides (COS)
Scheme 9.1 A stepwise process of producing chitosan and chitooligosaccharides from
shellfish wastes.
188 Ramjee Pallela
hydrolysis, to use these derivatives in multiple biological applications (Xia

et al., 2011). Comprehensive studies on the properties and new modification
reactions of chitin, chitosan, COS, and their derivatives are being conducted
to apply these macromolecules in the promising fields of medicine, phar-
macy, cosmetics, toiletries, food processing, agriculture, etc. (Kim, 2010;
Kurita, 2006).
Similarly, clandosan is also a kind of polymeric carbohydrate molecule
from crustacean chitin has been used in fertilizing applications because of
its insecticidal or nematocidal behavior (Spiegel, Chet, Cohn, Galper &
Sharon, 1988). However, the nutritional or pharmacological applications
of clandosan are not well studied.
3. MARINE CARBOHYDRATES AS NUTRACEUTICALS

Most of the Asian countries use macroalgae as food for human con-
sumption, which are the primary source of hydrocolloids such as agar, car-
rageenan, and alginate that possess profuse industrial applications, for
example, gelling, stabilizing, or binding agents. Like other plants, marine
algae contain nutritional elements like proteins, lipids, carbohydrates, vita-
mins, and minerals; the content of these elements varies depending on season
and the area of production (Holdt & Kraan, 2011). Marine algae contain
large amounts of polysaccharides, remarkably the cell wall structural poly-
saccharides, but also the mucopolysaccharides and other storage polysaccha-
rides (Kraan, 2012). The dietary fibers of seaweeds contain valuable
nutritional substances, hence the use of seaweeds as marine medicinal foods
brings much attention to use them in meal, functional foods, and
nutraceuticals for human consumption, because the polysaccharides show
tremendous biological activities like antitumor, antihepatitic, antimicrobial,
as well as antiviral activities (Ghosh et al., 2009; McHugh, 1987). SPs, one of
the best examples of the seaweed polysaccharides, are nontoxic and possess
tremendous antioxidative capacity. These antioxidant principles of marine
algal polysaccharides are an indicative of developing them as potential func-
tional foods or nutraceuticals as well (Wijesekara et al., 2011). In addition,
fucose rich SPs, fucoidans of brown seaweeds have tremendous significance
of their use as nutraceuticals despite of other biological and biomedical
applications (Wijesinghe & Jeon, 2012). It is interesting to note that the
fucoidan-based products are taken up to the level of commercialization,
e.g., Marinova Pty Ltd. supplies commercial volumes of fucoidan extract
and their derivatives, further formulated to the purity levels of up to 95%
(Kraan, 2012). Further, it is noted that several species like Undaria sp.,
Lessonia sp., Macrocystis sp., Cladosiphon sp., Durvillea sp., Laminaria sp.,
Ecklonia sp., Fucus sp., Sargassum sp., Ascophyllum sp., and Alaria sp. are con-
sidered to be Halal and Kosher certified.
Animal carbohydrates like chitosan, COS, and their further derivatives
are also gaining some importance of using as nutraceuticals (Barrow &
Shahidi, 2007; Fernandes et al., 2012). According to the reports of
Fernandes et al. (2012), the modeling studies of COS should also be impor-
tant to evaluate the efficacy in modern therapeutic strategies, as it is in the use
of nutraceuticals. COS has gained much importance by nutritional and func-
tional food experts to use them in daily supplements because of the high sol-
ubility and biological compatibility of COS. Although application of
chitosan as some dietary supplement is biomedically significant to decrease
body weight and serum lipids through gastrointestinal fat binding, its efficacy
remains in dispute (Mhurchu et al., 2004). Hence, possible use of COS is
well advised because of its easy solubility and miscibility in the biological
systems to act at the targeted areas. However, high costing situations and
severe environmental concerns on the disposal and discarding of marine
processing wastes have led the researchers to show an amplified interest in
the identification and extraction and the development of low cost but high
value by-products from shellfish wastes via sustainable technologies (Hayes
et al., 2008).
4. MARINE CARBOHYDRATES AS PHARMACEUTICALS

SPs in the cell walls of marine algae, e.g., fucoidans in brown algae,
carrageenans in red algae, and ulvans in green algae contribute major appli-
cation in the development of nutraceuticals, pharmaceuticals, and cos-
meceuticals (Wijesekara et al., 2011). The antioxidant capabilities of the
SPs are a gained advantage for the researchers to apply them in pharmaceu-
tical industries as well as nutraceutical industries.
Among the animal-derived marine carbohydrates, majorly, chitosan
and its derivatives have been involved in several pharmacological applica-
tions as these carbohydrate macromolecules have unique properties of acting
as excipients to deal with various health complications (Dodane & Vilivalam,
1998; Illum, 1998) (Table 9.1). Moreover, chitin and chitosan-based
nanofibers are contributed in the development of conventional and novel
pharmaceutical products, because of their favorable biological properties
such as nontoxicity, biocompatibility, biodegradability, and antibacterial
190 Ramjee Pallela
Table 9.1 Pharmaceutical applications of chitosan, chitooligosaccharides, and their

derivatives.
Route of
administration Delivery system References
Oral Microparticulate Lorenzo-Lamosa, Remunan-Lopez, Vila-Jato,
and Alonso (1998)
Liposomes Takeuchi, Yamamoto, Niwa, Hino, and
Kawashima (1996)
Buccal disk Remunan-Lopez, Portero, Vila-Jato, and
Alonso (1998)
Solution Lueen et al. (1996)
Vesicle Uchegbu et al. (1998)
Film coating Remunan-Lopez and Bodmeier (1997)
Tablets Acarturk (1989), Sabnis, Rege, and Block,
(1997), Yomota, Miyazaki, and Okada (1994)
Spray-dried Kulvanich, Prugmahachaikul, and Kraisintu
particles (1997)
Capsules Odoriba et al. (1997), Tozaki et al. (1997)
Parenteral Microspheres Jameela, Misra, and Jayakrishnan (1995),
Patashnk, Rabinovich, and Golomb (1997),
Thanoo, Sunny, and Jayakrishnan (1992),
Vasishtha, Barlow, Brashear, and Rastellini
(1997)
Solution Kamiyama, Onishi, and Machida (1996)
Nasal Solution Aspden, Illum, and Skaugrud (1997), Aspden,
Mason, et al. (1997), Illum, Farraj, and Davis
(1994), Tengamnuay, Sahamethapat, Sailasuta,
and Mitra (2000), Sinswat, & Tengamnuay
(2003)
Ocular Suspension Calvo, Vila-Jato, and Alonso (1997), Genta et al.
(1997)
Gene therapy Borchard and Bivas-Benita (2005), Mao et al.
(2001), Mao, Roy, Truong-Le, and Leong
(1997), Murata, Ohya, and Ouchi (1996, 1997)
Other Gel systems Filar and Wirick (1978), Hoffman, Matsura, Wu,
and Gombotz (1997)
Modified from Dodane and Vilivalam (1998)
activity. These nanofibers are further considered as promising material for

the enhancement of absorption of drugs, enzyme immobilization, cell pro-
liferation, and wound healing ( Jayakumar, Prabaharan, Nair & Tamura,
2010). In addition to chitin nanofibers, chitin and chitosan derivatives are
used as excipients and drug carriers in the pharmaceutical field, where
chitosan is used as an excipient in oral dosage form and chitosan tablet
can exhibit a sustained drug release compared to existing commercial prod-
ucts. Films prepared using chitin or chitosan have been developed as wound
dressings, oral mucoadhesive, and water-resisting adhesive by virtue of their
release characteristics and adhesion (Kato, Onishi & Machida, 2003).
Recent efforts by Zhang et al. (2010) focused on the chemical and bio-
logical modification of chitosan in order to increase the solubility of chitosan
in aqueous solutions and absorbability in the in vivo system, thereby better
use of chitosan is possible in the pharmaceutics and medicine (Zhang
et al., 2010).
5. CONCLUSION
There is growing interest in the use of functional foods and
nutraceuticals based on marine-derived carbohydrates. These medical food
sources of traditional value lead the researchers to develop some
nutraceuticals as well as pharmaceuticals from marine flora and fauna to
combat several health complications. Although several carbohydrate mole-
cules of marine origin are researched for their chemicobiological impor-
tance, several gaps have to be filled to bring out efficient nutraceuticals
and pharmaceuticals from these molecules. Since most of the applications
of marine carbohydrates are restricted to the laboratory level, supplementary
studies are very important to confirm the clinical level applications, and fur-
ther commercialization of these macromolecules. The present chapter is a
cumulative review content on various sources of marine carbohydrates
and their possible applications in nutraceutical and pharmaceutical
development.
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FURTHER READING
Abarrategi, A., Gutierrez, M. C., Moreno-Vicente, C., Hortiguela, M. J., Ramos, V., Lopez-
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cells with nanocarbon substrates. Langmuir, 26(4), 22442247.
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nance of hemiround colonies and undifferentiated state of mouse induced pluripotent
stem cells on carbon nanotube-coated dishes. Carbon, 49(7), 22872299.
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sion by single-and multi-wall carbon nanotubes using computational approach. Journal of
Biomedical Nanotechnology, 7(1), 181182.
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single-walled carbon nanotubes synthesized by radio frequency induction thermal plasma
using various catalysts. Journal of Applied Toxicology, 33(10), 11431155.
Allen, B. L., Kichambare, P. D., Gou, P., Vlasova, I. I., Kapralov, A. A., Konduru, N., et al.
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Armentano, I., Marinucci, L., Dottori, M., Balloni, S., Fortunati, E., Pennacchi, M.,
et al. (2011). Novel poly(L-lactide) PLLA/SWNTs nanocomposites for biomedical
applications: Material characterization and biocompatibility evaluation. Journal of
Biomaterials Science, Polymer Edition, 22(46), 541556.
Barrow, C. J., Diepen, C. V., Perrie, C., Curtis, J., Jin, Y., & Zhang, W. (2007). Microen-
capsulation of marine lipids as a vehicle for functional food delivery. In C. J. Barrow, &
F. Shahidi (Eds.), Marine nutraceuticals and functional foods (pp. 115154). Boca Raton,
Florida, USA: CRC Press.
CHAPTER TEN
Pharmaceutical, Cosmeceutical,
and Traditional Applications
of Marine Carbohydrates
Abdul Bakrudeen Ali Ahmed1, Mohaddeseh Adel, Pegah Karimi,
Mahvash Peidayesh
Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia
1
Corresponding author: e-mail address: bakru24@gmail.com
Contents
1. Introduction 198
1.1 Resource of marine carbohydrate 198
1.2 Marine carbohydrate market value 199
1.3 Special areas of conservation 201
2. Pharmaceutical Products and Biological Application 202
2.1 Blood coagulation system 203
2.2 Anticancer activity 204
2.3 Antioxidant activity 205
2.4 Antiviral activity 207
2.5 Antilipidemic activity 208
2.6 Immunomodulating effect 208
3. Cosmeceutical Products and Functional Applications 209
3.1 Fucoidan 209
3.2 Carrageenan 212
3.3 Alginates 213
4. Marine Food and Traditional Application 213
4.1 Marine food carbohydrates and fibers derived as an antioxidants and their
antioxidative activity 213
4.2 Thickeners, stabilizers, and emulsifiers 215
5. Conclusion 215
Acknowledgment 216
References 216
Abstract
Marine carbohydrates are most important organic molecules made by photosynthetic
organisms. It is very essential for humankind: the role in being an energy source for
the organism and they are considered as an important dissolve organic compound
(DOC) in marine environment's sediments. Carbohydrates found in different marine
http://dx.doi.org/10.1016/B978-0-12-800268-1.00010-X
198 Abdul Bakrudeen Ali Ahmed et al.
environments in different concentrations. Polysaccharides of carbohydrates play an

important role in various fields such as pharmaceutical, food production, cosmeceutical,
and so on. Marine organisms are good resources of nutrients, and they are rich carbohy-
drate in sulfated polysaccharide. Seaweeds (marine microalgae) are used in different phar-
maceutical industries, especially in pharmaceutical compound production. Seaweeds
have a significant amount of sulfated polysaccharides, which are used in cosmeceutical
industry, besides based on the biological applications. Since then, traditional people, cos-
metics products, and pharmaceutical applications consider many types of seaweed as an
important organism used in food process. Sulfated polysaccharides containing seaweed
have potential uses in the blood coagulation system, antiviral activity, antioxidant activity,
anticancer activity, immunomodulating activity, antilipidepic activity, etc. Some species of
marine organisms are rich in polysaccharides such as sulfated galactans. Various polysac-
charides such as agar and alginates, which are extracted from marine organisms, have
several applications in food production and cosmeceutical industries. Due to their high
health benefits, compound-derived extracts of marine polysaccharides have various appli-
cations and traditional people were using them since long time ago. In the future, much
attention is supposed to be paid to unraveling the structural, compositional, and sequen-
tial properties of marine carbohydrate as well.
1. INTRODUCTION
1.1. Resource of marine carbohydrate
Carbohydrates are large molecules that are composed of carbon, hydrogen, and
oxygen. Carbohydrates are called saccharides. They are energy transporters
and are structural components in marine organisms. Carbohydrates are classi-
fied as monosaccharides, disaccharides, polysaccharides, and oligosaccharides.
Among these classes, marine and terrestrial organisms contain polysaccharides
that have the storage and structural role. The storage polysaccharides are gly-
cogen and starch, and the structural units are polysaccharides like cellulose and
chitin. Carbohydrates are important due to their activity in the immune system,
fertilization, and food storage. The storage form of carbohydrates are unstable.
It is utilized and degraded by in situ heterotrophic organisms while they deposit
the organic matter from the surface to depths (Handa & Tominaga, 1969).
Besides the polysaccharides, monosaccharides are useful for human and cure
many diseases.
Marine carbohydrates are one of the most important organic compounds
that are produced by photosynthesis in marine living organisms. In marine
environments, carbohydrates are considered as derivative organic compound
(DOC) in seawater and sediments (Arnosti & Holmer, 1999). Dissolved
carbohydrates are the known suitable intermediate for production and
Pharmaceutical, Cosmeceutical, and Traditional Applications 199
consumption during mineralization (Arnosti & Holmer, 1999). Carbohy-

drates are the main source of metabolic energy for organisms, and are hetero-
trophic and produce their energy by using photosynthesis. Structural
polysaccharides are playing a critical role in degrading pathways of organic
matter in marine environments. There is a possibility that carbohydrate poly-
saccharides have pyruvate, sulfate, acetate, and consist of acidic groups such as
amino sugar. These compounds incorporate in various pathways and processes
in the marine environment. Formation of humic acids, detoxifications, bio-
film productions, and extracellular enzyme binding are a few of this incorpo-
ration. Hence, many polysaccharides are produced by aquatic organisms.
Among that, chitin is one of the most abundant compounds. Marine algaes
carbohydrate contents and carbohydrate production is proportional to the
amount of respiration and the light. Respiration and carbohydrate production
significantly decreased when the algae maintained in the dark place.
Marine algae has made of sulfated polysaccharides (SPs) in the cell wall
structure, which is rare to find in marine animals and plants. The hydroxyl
group of simple monosaccharide replaced by a sulfate group. This SP plays
important role in ionic regulation and has various biological activities in
marine organisms. Polysaccharides mostly extract by water. Molecular
weight is an important factor in choosing the temperature of the water.
For example, the high molecular weight, SP usually extract by hot water
and for low molecular weight compounds, cold water used. Molecular
weight of polysaccharides determined by gel permeation method
(Shanmugam & Mody, 2000). In the next step, polysaccharides precipitate
by alcohol from the water extract. The precipitate digests by water and filter
then freeze-dried. The sulfate content of marine organisms carbohydrate
usually measured by one-dimensional and two-dimensional nuclear mag-
netic resonance spectroscopy and sometimes it measured by methylation
analysis (Han, Yao, Yang, Liu, & Gao, 2005).
1.2. Marine carbohydrate market value

Oceans are a major habitat of living organisms as far as supporting the life of
nearly 50 present of all different species on Earth. This is contributing to the
variety of ingredients that are consuming through life cycle of marine
organisms and many more that are producing afterward reaction processes.
Overview of the existing commercial and research activities, which consider
pharmaceutical products and biological functional application of marine car-
bohydrates, is given in this chapter. Wide variety of polysaccharides extract
from marine plants and animal organisms are produced by marine bacteria.
This means that the field of marine polysaccharides is constantly evolving.
There is an enormous amount of bioactivity, mostly on microalgae, with news
about new investments and research programs emerging on an almost daily
basis. Among different organisms such as macroalgae (seaweeds), microalgae
(phytoplankton), and marine animals (as carbohydrates produce or containing
carbohydrates as structural units) algal share is the largest. As shown in
Fig. 10.1, there is a different field of using algae products in the world markets.
Fucoid is the one that helps human life in various aspects among algal
products. Fucoidan is the most abundant SP that extract from marine brown
seaweeds and marine invertebrates such as sea cucumber. Preliminary
research suggests that it may support the human immune system and also
have anticancer activity, antioxidant activity, and antiviral activity. Fucoid
is present in different products such as veggie capsules, liquid beverages,
body creams, and skin serums. Different products from different companies
offer different prices. Base prices for veggie capsules (60 capsule of 70%
fucoidan) from Doctors Best company is from 24.03 $ up to 50.99 $. In
addition, the selling fucoidan content products are around 154 $/50 mg.
Heparin is a sulfated glycosaminoglycan and plays a role as an anticoag-
ulant. It is produced by mast cells and basophils and is a naturally occurring
anticoagulant. In 2011, the global market for heparin is expected to grow at a
compound annual growth rate of 12% from 2012 to 2018. It is widely used as
an injectable anticoagulant and the price of every unit description, dosage
(5000 IU 1 ml 5 ml) is averagely 5 $ from different brands such as Leo
100 /kg
Biomass for energetical use
Market value
10 /kg Proteins, feed
Fine chemicals, food
1 /kg Pharmacutical, cosmetical
Market size
Figure 10.1 Branding paradigm for bottom of the pyramid markets, measuring the
algae products business excellence.
Laboratories Ltd. Alginate isolated from seaweeds, it is mostly found in all

type of marine organism, this can support for the cell walls growth and pri-
mary metabolite production. In 2011, FAO reports alginate market as
10,000 tons for food and pharmaceutical usage and 20,000 tons for all tech-
nical grades. The total market had a value of about US$195 million. Anti-
microbial wound dressings are a product contain alginate. A mixture of
alginate, carboxymethyl cellulose, and silver-coated nylon fiber make a
unique composition that manages exudate effectively in infected or heavily
colonized wounds. This product manufactured by Vygon, offers Silvercel
Non-Adherent Wound Dressing Size 5 5 cm Box of 10 with the price
of US$45.92 in the United Kingdom.
Agar as gelling agent comes from South East Asian seaweed. Agar is used
vastly in scientific purposes especially in biology as filler in paper sizing fabric
and as a clarifying agent in brewing. Also it is used in the food industry to
preserve fruits, ice cream, and other desserts or to thickener for soups. Coast
biologically manufactures agar from the red seaweed, Pterocladia lucida, with
growing in abundance in the clear unpolluted seas that surrounds New
Zealands long coastline. Hence, the researchers are using the agar-agar
and the market demands are reasonable one. Price of agar depends on its
ingredients and brands. For example, there are different types of agar of dif-
ferent companies as a medium for bacterial culture (Table 10.1). As Fig. 10.1
shows, microalgae and microalgae as a source of biomass for energetical uses
have a large size market. Table 10.2 indicates the sections market demands
on the purpose of energetical uses.
1.3. Special areas of conservation

A substantial number of designated marine Special Areas of Conservation
(SACs) have been performed and candidate SACs performed based on rules
and regulations:
Table 10.1 Different types of agar products and their distributor quotation
Agar type Size Price (US$)
Bacteriological agar 1 kg 88
Plant tissue culture low nitrification agar 1 kg 119
Noble agar 500 g 98
Standard grade agar 1 kg 73
Table 10.2 Macro- and microalgae generation status, harvesting methods, and
market value
Biomass Downstream
Resource type generation Harvesting processing Market
Macroalgae Natural Manual Biogas Logistics
(seaweeds) stocks Mechanization Bioethanol Infrastructure
Aquaculture Biorefinery Engines
Near shore Residues
Offshore
Microalgae Natural Filtration Biodiesel Logistics
(phytoplankton) stocks Sedimentation (lipids) Infrastructure
Aquaculture Centrifuge Fermentation Engines
Near shore Flocculation (biomass) Aviation
Offshore Biorefinery
Residues
Existing traditional activities (e.g., seaweed cutting) may be continued

but a minister approval is needed for any substantial increase of
harvesting seaweed and any new conservation process.
Any mechanization of seaweed harvesting within the designated areas
would need the approval of the National Parks and Wildlife Services
(NPWS), Department of the Environment, Heritage and Local
Government.
Seaweed aquaculture has permitted subject to the usual licensing consid-
erations but the NPWS has to be consulted by the Department of Com-
munication, Energy and Natural Resources for approval.
According to the statement, there is no obligatory hindrance as such for the
establishment of seaweed aquaculture in an SAC. Although the applicant for
an aquaculture license may have to prove that the construction of the farm
will not have adverse impacts on the habitat. Therefore, an environmental
survey may need to be conducted before a license issue.
2. PHARMACEUTICAL PRODUCTS AND BIOLOGICAL

APPLICATION
Marine algae are good sources of bioactive compound that used in
pharmaceutical and health applications. These compound polymers vary
due to different marine species (Costa et al., 2010). Among marine algae,
seaweeds have a polysaccharide structure, which belongs to the sulfate
group. The structures are called as SPs. Nowadays, sulfate polysaccharides
that are isolated from marine algae are becoming important in pharmaceu-
tical food and cosmeceutical industry. Biological activity of SPs is widely
dependent on the forming of chains, molecular weight, and structure of
chemicals in the marine organism (Ye, Wang, Zhou, Liu, & Zeng,
2008). Beside the carbohydrate, other components such as enzymes, antiox-
idants, vitamins, and bioactive peptides are available in most marine organ-
isms (Kim & Wijesekara, 2011). These SPs are playing an important role in
various pharmaceutical purposes such as blood coagulation, anticancer activ-
ity, antioxidant activity, antiviral activity, antilipidepic activity, and immu-
nomodulation activity. In this section, these biological activities are studied
in detail.
2.1. Blood coagulation system

Coagulant factors and anticoagulant factors are interfering together and
blood coagulation may happen. Blood coagulation or thrombogenesis
occurs when blood forms clots. Blood vessel cell wall forms fibrin clots to
stop flow of blood and this may help to heal the injury and repairing the
damage. Anticoagulant activity research from marine organism, is the most
applied and important research through the substitute heparin. Iridae
laminarioides (red algae) was the first marine organism that has anticoagulant
purposes studied on it. Anti-blood coagulation compounds were isolated
from brown marine algae and have important roles. After this discovery,
marine algae used to isolate and become an alternative way to produce anti-
coagulant drugs because of their high SPs. The structure of polysaccharides
provides various pharmaceutical probabilities in different marine organisms.
Most of the anticoagulants reported from marine red algae and marine
brown algae. Sulfated groups in SPs enhance the binding of polysaccharides
to biologically active protein. Higher SPs with a higher molecular weight
have a higher anticoagulant activity than lower sulfate content with lower
molecular weight (Shanmugam & Mody, 2000). However, green marine
algae reported to have SP that is contributing to have anticoagulant activity,
but the isolated amount is less comparable to brown marine algae and red
marine algae (Mao et al., 2009). In addition, the determined SPs have the
potential bioactive properties such as anticoagulant activity isolated from
brown algae, red algae, and green algae, the compounds namely fucoidan,
carrageenan, and ulvans, respectively (Kim & Wijesekara, 2011).
Heparin is a bioactive drug that is widely used as blood coagulation from
the glycosaminoglycan group of carbohydrates and it used to isolate from pig
intestine. Nowadays, researchers are looking for another resource of heparin

that can be isolated easier, cheaper, and safer. Marine algae may be a good
source of heparin in blood coagulation therapy. Marine anticoagulant drug
has been isolated from the marine echiuroid worm ( Jo, Jung, & Kim, 2008);
Mytilus edulis ( Jung & Kim, 2007); starfish (Koyama, Noguchi, Aniya, &
Sakanashi, 1998); Ecklonia cava, a brown algae ( Jung et al., 2007); and Mon-
ostroma nitidum, a green algae (Mao et al., 2008). In extraction of anticoag-
ulant SPs from brown algae, after using hot or cold water (depends on its
molecular weight), CaCl2 or mild acid may be used. To monitor heparin
activities in blood coagulation, activated partial thromboplastic time
(APTT) method is used. APTT is the most common method to check hep-
arin activity. Peptide factors of heparin bind closely to the clotting factor and
stop the flow of blood in the body. These factors are nontoxic and used as
ingredients in the pharmaceutical industry.
Sulfated alginate derivatives reported to have anticoagulant activity and
antithrombotic activities among different marine species (Yao et al., 2006).
Since 1998, China isolate propylene glycol mannate sulfate from alginate
and used in pharmaceutical to depress blood viscosity and blood anticoag-
ulants (Changhu, Guangli, Takashi, Junji, & Hong, 1998). Carrageenan iso-
lated from red algae is another anticoagulant that contains 2335% of sulfate
groups and classified into different groups due to its solubility in potassium
chloride (heparin). Carrageenans have anticoagulant activity, which is
15 times less than heparin activity. Anderson and Proc reported the first
polysaccharides isolated from different species such as Eucheuma spinosum
and Gobiodon acicularis have carrageenan contributing anticoagulant activity
(Anderson, 1969). However, carrageenan isolated from kappa has anticoag-
ulant activity (Silva et al., 2010).
2.2. Anticancer activity

Cancer is an unregulated cell growth and division which was induced by
cancer drugs and going under chemopreventive therapy to induce the can-
cer cell formation. Producing anticancer drugs from natural marine products
are becoming important, as the cancer is a popular disease among
populations. Therefore, the novel anticancer drug production becomes
important in the pharmaceutical industry.
In vitro tumor cell lines treated with marine carbohydrate for inhibition of
tumor, antiproliferative activity and antimetastatic activity. SP is an impor-
tant element in all these researches. Porphyra yezoensis (Rhodophyceae) study
of cancer cells showed that SPs in P. yezoensis made an apoptosis in vitro and
the normal cells were not affected (Kwon & Nam, 2006). Polysaccharide
GA3 that is extracted from Gymnodinium sp., is called GA3P (Gymnodinium
sp. A3 phosphate), is an extracellular acidic polysaccharide that has a
D-galactone sulfate and lactic acid and inhibits the growth of human leuke-
mic cell lines. It is shown that GA3P has inhibitory effects on cancer cell lines
(Table 10.3) such as topoisomerase I and topoisomerase II (Umemura et al.,
2003). Besides, fucoidan has an anticancer activity; it contains SPs and rel-
ative molecular weight. By increasing the molecular weight, anticancer
activity might be lower. Hence, many polysaccharides isolated from marine
organisms can only inhibit the growth of human cancer cells (Ahmed,
Vijayakumar, Pallela, Abdullah, & Taha, 2013).
2.3. Antioxidant activity

Natural antioxidant compounds have gained considerable attention in the
past few decades. Oxidation is a chemical reaction that transfers the electrons
or hydrogen to the oxidation processes. Antioxidants block starting of oxi-
dation reaction by using high-energy molecules. Oxidation makes damages
on different parts of macromolecules such as lipid membrane, protein, and
even DNA molecules and all of them may cause tissue injury in organisms
(Butterfield et al., 2002). Antioxidants inhibit the oxidation process such as
vitamins, minerals, and other nutrients that protect the cells from damaging
of oxidation. In marine polysaccharides extractions such as antimicrobial,
anticancer, and antiviral, marine carbohydrates are supposed to have a high
antioxidative effect. Most of the organisms have antioxidant activity to
defense themselves against oxidative damages. The bioactive compounds
and antioxidants that marine organisms produce are important in the phar-
maceutical industry.
Keissleriella sp. is a marine fungus that has antioxidant activity (Wang
et al., 2007). De Souza et al. (2007) reported the fucoidan and lambda car-
rageenan have the highest antioxidant activity among the SP extracts from
brown and red seaweeds. Antioxidant activity mostly analyzes by DPPH
methods, ferric reducing/antioxidant power (FRAP) assay and deoxyribose
assay. DPPH assay is a radical scavenging activity determinant. DPPH is
abbreviated of 2,2-diphenyl-1-picrylhydrazyl that is a chemical organic
compound. It contains of stable free radical molecules. Deoxyribose assay
is a reactivation of tannins toward hydroxyl radicals. FRAP assay determines
the antioxidant power in addition to ferric reducing ability. Lonicera japonica
Table 10.3 Cancer in major organs, involving cell line and growth index differentiation
by GA3P
Origin of cancer Cell line GI50a (g/ml)
Kidney PXF-63IL 9.1
ACHN 8.3
Lung NCI-H23 2.8
NCI-H226 2.2
NCI-H522 1.3
NCI-H460 3.8
A549 11
DMS273 2
DMS114 2.7
Breast HBC-4 5.2
BSY-1 0.67
HBC-5 6.2
MCF-7 2.9
MDA-MB-231 1.5
Stomach St-4 8.4
MKN1 3
MKN7 5.9
MKN28 7
MKN45 2.9
MKN74 4.6
Ovary OVCAR-3 2.2
OVCAR-4 3.2
OVCAR-5 6.8
OVCAR-8 4.1
SK-OV-3 8.1
a
50% growth inhibitory concentration.
is traditional Chinese seafood that contains fucoidan (Ruperez, Ahrazem, &

Leal, 2002). The fucoidan is responsible for hydroxyl radicals and has anti-
oxidant activities. The SP in the cell wall of seaweed has antioxidant activity
that has ionic regulation ability. These SPs, which are available in their cell
walls, do not occur in land plants. Antioxidant compounds play an important
role against various diseases such as aging processes, chronic inflammation,
atherosclerosis, and cardiovascular disorders (Kohen & Nyska, 2002).
2.4. Antiviral activity

Marine polysaccharide protecting embryonic eggs against influenza B.
Gelidium cartilagenium (Rhodophyceae) is an example of antiviral marine
organism. Highly SPs in marine organisms had shown significant antiviral
activity in marine organisms (Huheihel, Ishanu, Tal, & Arad, 2002). The
antiviral activity of marine species determines by few factors such as molec-
ular weight, sulfation degree, and constituent sugar (Adhikari et al., 2006).
Degree of sulfation in algal polysaccharide is important and if the sulfation
degree is low or absent, antiviral activity will be absent. As the natural
bioactive compounds of marine SPs algal derived are important in the phar-
maceutical industry; these products used in natural anti-human immunode-
ficiency virus (HIV) medicine. Various studies have been done on antiviral
activity of marine carbohydrates on HIV. This medicine has fewer side
effects due to the natural therapy. Fucus vesiculosus is brown seaweed that
consists of soluble H2O and showed antiviral activity against HIV. Fucoidan
is the most abundant SP in this alga (Beress, Wassermann, Bruhn, & Beress,
1993). Several species of marine algae reported to have anti-HIV activity.
For example, Grateloupia filicina and Grateloupia longifolia produce sulfated
galactones (Wang et al., 2007), Lobophora variegata produces the sulfated
fucans (Queiroz et al., 2008).
SPs are useful in vaginal antiviral therapy. Drug production from marine
for antiviral activity has potential uses and it is widely acceptable as it is lower
in price with lower cytotoxicity and safer. These days marine-derived drugs
are an important production in pharmaceutical industry. Anti-HIV activity of
SPs has in vitro and in vivo inhibition on flaviviruses such as dengue virus (Ono
et al., 2003). Fucoidans that extracted from marine brown seaweeds contain
SPs. This polysaccharide has an antiviral activity against infectious diseases
(Witvrouw & De Clercq, 1997). Herpes simplex virus type1 causes skin infec-
tions in mucosal epithelia of the oral cavity. It can also make serious problems
in the nervous system. The SP isolated from Sargassum patens showed antiviral
activity against this virus (Zhu, Chiu, Ooi, Chan, & Angjr, 2006).
2.5. Antilipidemic activity

Antilipidemic is an agent that reduces the level of lipid in the serum. These
antilipidemic marine drugs produced to reduce the role of atherosclerosis
process. The high content of dietary soluble fiber in marine makes it as a
good source for antiobesity drug production. Marine fibers slow down cal-
orie absorption and food digestion. Besides, they moderate appetite and
delay gastric emptying (Paxman, Richardson, Dettmar, & Corfe, 2008).
P. yezoensis is a marine red alga that contains porphyran. P. yezoensis can
use as antihyperlipidepic agent and can reduce the lipid synthesis in human
liver cells (Tsuge et al., 2004). Sulfated group makes this antilipedemic fea-
ture to porphyran. Sargassum sp. is brown seaweed that contains high
amount of SPs. Fucoidan and alginate content was high in Sargassum sp.
These species reported to decrease serum total cholesterol and triglyceride
and increase the high-density lipoprotein (Chen, Wang, Liu, Li, &
Liu, 2010).
2.6. Immunomodulating effect

Immunomodulation applies for weakening or modulating the activity of the
immune system. It mostly used for decreasing the immunity of the body for
tissue transplant. Immunomodulation in SPs is mostly about macrophage
modulation. Macrophages are homeostasis modulators by changing the
function of immune cells (Wijesekara, 2011). Polysaccharides isolated from
Enteromorpha intestinalis, have the ability to increase the rate of B and
T lymphocytes production and effect on interleukin-2 and interferon-alpha
production (Xu et al., 2005). SPs that are isolated from marine algae have
anti-inflammatory activities (Abad, Bedoya, & Bermejo, 2008). Anti-
inflammatory effect is important in immunomodulating because of remov-
ing pathogens and cell debris after inflammation. In addition, carrageenan
isolated from red marine algae has an anti-inflammatory activity too. It stud-
ied that the SPs have phagocytosis effect on mouse macrophages (Yoshizawa
et al., 1995). Besides that, some SPs have immune stimulating activity,
which can control macrophage activity and decrease the negative effects
(Schepetkin & Quinn, 2006).
Polysaccharides with higher molecular weight, >90,000 Da, have higher
immunomodulating effect comparing to those with lower molecular
weight. However, the factors such as component of sugar contribute in
immunomodulating activity (Arena et al., 2009). Polysaccharides isolated
from different species of algae and seaweeds have the ability to increase
phagocytes and secretion of macrophages. Many of these SPs used in

immunomodulating effects, taken from algal cell wall. The algae cell wall
contains polysaccharides that contribute in immunomodulating the macro-
phage activity in mammals. Carrageenan has potential uses in stimulating
the immune system and macrophage adjusting (Leiro, Castro, Arranz, &
Lamas, 2007). The high potent activity of marine carbohydrate in
immunomodulating effects such as anti-inflammatory and antiviral activity
makes it important to be studied. Biological application of marine origin
substances isolated from various sources of marine organisms presented in
Table 10.4.
3. COSMECEUTICAL PRODUCTS AND FUNCTIONAL

APPLICATIONS
Cosmetics have become an essential part of human life. Many
researches performed on cosmeceutical applications and production of
newer products that have better quality and cheaper price. Novel cosmeceu-
tical products supposed to have a pharma-like ability beside their main func-
tion. Marine carbohydrates provide a vast area in the production of
cosmetics and applied, since ancient time among the people of different
nationalities. SPs in marine organisms provide a good source of antioxidant
in cosmeceutical industry. Most of these antioxidants are isolated from sea-
weeds. Other biological components of marine organisms are incorporating
to make them as an important source for cosmeceutical industry. Here,
fucoidan, carrageenan, and alginate substances that are used in cosmetic
products are discussed.
3.1. Fucoidan
Fucoidan is the most abundant SPs that is extracted from marine brown sea-
weeds and marine invertebrates such as sea cucumber. Fucoidan is important
in cosmeceutical and food production as it is possible to extract from cheap
resources and used in novel drugs and functional food production. In addi-
tion, it is useful in antiviral, anticoagulant, and antibacterial activities.
Figure 10.2 shows the fucoidan chemical structure and its repeating
dimeric units.
Fucoidan is a cell wall stability enhancer and conservation for
antidehydrating. Fucoidan bioactivity is very important in food and cos-
metic application. It is necessary to know that sulfation content and structure
of fucoidan is important in its biological application. Fucoidan is used as
Table 10.4 The biological application isolated from different sources of marine
organisms
References Organism Source Biological application
Jung and Kim (2007) Mytilus edulis Marine Anticoagulant activity
bivalve
mollusk
Anderson (1969) Eucheuma Red Anticoagulant activity
spinosum seaweed
Anderson (1969) Gobiodon Gobioid Anticoagulant activity
acicularis fish
Jo et al. (2008) Echiuroid Sea worm Anticoagulant activity
Jung et al. (2007) Ecklonia cava Brown Anticoagulant activity
seaweed
Mao et al. (2008) Monostroma Green Anticoagulant activity
nitidum seaweed
Ye et al. (2008) Sargassum Brown Antitumor activity
pallidum seaweed Antioxidant activity
Ruperez et al. (2002) Laminaria Brown Antioxidant activity
japonica seaweed
Ruperez et al. (2002) Focus Brown Antioxidant activity
vesiculosus seaweed
Wang et al. (2007) Keissleriella Marine Antioxidant activity
sp. fungus
Beress et al. (1993) Focus Brown Antiviral activity, anti-HIV
vesiculosus seaweed activity
Ananthi et al. (2010) Turbinaria Brown Antioxidant activity
ornata seaweed Anti-inflammatory
Rodriguez et al. (2005) Callophyllis Brown Antiviral activity
variegata seaweed
Matsuhiro et al. (2005) Schizymenia Red Antiviral activity
binderi seaweed
Melo, Pereira, Foguel, Botryocladia Red Antithrombin activity
and Mourao (2004) occidentalis seaweeds Anticoagulant activity
Zhu et al. (2006) Sargassum Brown Antiviral activity
patens seaweed
Wang et al. (2007) Grateloupia Brown Antiviral activity
filicina seaweed
Table 10.4 The biological application isolated from different sources of marine
organismscont'd
References Organism Source Biological application
Wang et al. (2007) Grateloupia Brown Antiviral activity
longifolia seaweed
Queiroz et al. (2008) Lobophora Brown Antiviral activity
variegata seaweed
Zhu et al. (2006) Sargassum Brown Antiviral activity
patens seaweed
Jiao, Jiang, Zhang, and Enteromorpha Green Immunomodulating
Wu (2010) and Xu et al. intestinalis seaweed activity, antiaging, and
(2005) antitumor activity
Leiro et al. (2007) Ulva rigida Brown Immunomodulating effect
seaweed
Yoshizawa et al. (1995) Porphyra Red Immunomodulating activity
yezoensis seaweed
Kwon and Nam (2006) P. yezoensis Red Anticancer activity
seaweed
Tsuge et al. (2004) P. yezoensis Red Antilipidemic activity
seaweed
Chen et al. (2010) Sargassum sp. Brown Antilipidemic activity
seaweed
Figure 10.2 Fucoidan structure and repeating dimeric unit.

antiulcer in food production and increases the metalloproteinase-1 enzyme

activity in human skin (Moon et al., 2008). This means that fucoidan is used
as antiaging agent to prevent skin photo aging in cosmetic production.
Fucoidan isolated from macroalgae has various uses in cosmetic production.
It is useful in conditioning agents and emollients too. The cheap, healthy,
and novel uses of fucoidan contained carbohydrate substances make this
derivative to have application in cosmeceutical production.
3.2. Carrageenan
Carrageenan is sulfated galactans that are mostly isolated from marine red
algae. Carrageenan is composed of D-galactose units. It is composed of
1,3-linked -D-galactose and 1,4-linked -D-galactose. Carrageenan deriv-
atives and structures present in different chemical structures shown in
Fig. 10.3.
Carrageenan is an important production used in the cosmetic industry
because of its physical and functional ability and antioxidant activity. Most
of the carrageenan derivatives consider for cosmeceutical production and
isolated from red seaweeds. On the other hand, carrageenan utilized in anti-
aging, antioxidants, and anticarcinogenic activity. The gelling ability of car-
rageenan is useful in producing a higher texture with higher consistency in
cosmetic production. Other products such as skin lotions, toothpaste
O
OSO3 OSO3
OSO3 O
OH CH2OH O
CH2OH O OH O
O O
HO O OH
O OH OH
carrageenan carrageenan
O
OSO3 OSO3 OSO3 O
OH CH2OH O
CH2OH O
O OSO3 O O
O HO O OH
OH OSO3
O
OH OSO3 OH O
OH CH2OH O
CH2OH O
O OSO3 O O
O OSO3
O OSO3 HO
OSO3
Figure 10.3 Carrageenan synthesis by the treatment of OH from red algae.

binders, and shaving foams are available from carrageenan isolated from
marine algae (Ahmed & Taha, 2011).
3.3. Alginates
Alginate is found in marine organisms cell walls. It was isolated from sea-
weeds such as kelp. It is made of two units of guluronic and mannuronic
acids, which is highly dependent on pH and temperature modification. Algi-
nates have a wide application in cosmeceutical industry because of their
thickening high stability and gelling agent. The first alginate application
in cosmeceutical field started in 1927. Alginate is applicable in grafting
the skin in plastic surgery. It makes a nice quality cosmetic convenience
for the patients. In addition, it has application in wound healing because
of hydrogel formation and degradability and providing a moist environment
for wound (Pereira et al., 2013). It was studied that biological activity of algi-
nates depends on its molecular weight, sulfated content, and anionic group
that makes it to have antioxidant activity (Xue, Yu, Hirata, Terao, & Lin,
1998). Alginates bioactivity depends on the presence of molecular weights
of sulfated content and anionic group that makes antioxidant activity.
4. MARINE FOOD AND TRADITIONAL APPLICATION

Human being existence depends on responding to his necessity. Need
to eat food is one of this physical necessities. In order to respond his needs,
man discovered the earth to find his food, so that the history of fishing dating
back to 40,000 years. Due to the wide range of environments and organisms
that survive underwater, we can see a large untapped reservoir of bioactive
ingredients that can be used in various applications such as food. Biomole-
cules derived from marine organisms, play an important role in a number of
applications such as food industry, including efficient food production under
unique conditions such as low temperature or high pressure; providing
added nutritional benefits of foods; and using natural pigments, preserva-
tives, or flavors.
4.1. Marine food carbohydrates and fibers derived as an

antioxidants and their antioxidative activity
Edible macroalgae (red, green, and brown seaweed) contain a lot of carbo-
hydrates and dietary fibers. Reduced plasma total cholesterol, LDL choles-
terol, and TAG have been observed due to polysaccharides from edible
seaweed (Amano, Kakinuma, Coury, Ohno, & Hara, 2005) and SPs from
seaweed, have potential use as antioxidants (Ruperez et al., 2002).
4.1.1 Chitooligosaccharide derivatives

Beside cellulose, chitin is the most abundant biopolymer on earth after one
of the most abundant polysaccharides. Chitin is extracted from the shells of
crabs and shrimps.
Recently, chitooligosaccharide (COS) had been in the center of attention
in terms of their pharmaceutical and medicinal applications (Kim &
Rajapakse, 2005) as an antioxidant (Park, Je, & Kim, 2003), antimicrobial
(Park et al., 2003), anticancer ( Jeon & Kim, 2002), antidiabetic (Liu, Liu,
Han, & Sun, 2007), hypocholesterolemic (Kim et al., 2005), anti-Alzheimers
(Yoon, Ngo, & Kim, 2009), and anticoagulant (Park, Lee, & Kim, 2004)
properties and adipogenesis inhibition (Cho et al., 2008). In the food industry,
chitosan (edible chitosan, more than 83% degree of deacetylation) and COS
have been used as dietary food additives and functional factors for their health
beneficial effects as well as drug carriers (Xia, Liu, Zhang, & Chen, 2010).
4.1.2 Sulfated polysaccharides

In recent years, various SPs (the complex group of macromolecules)
extracted from marine algae with a wide range of important biological activ-
ities attracted much attention in the fields of food, pharmaceutical, and cos-
metic industries. Marine algae as the most important source of nonanimal
plus animals such as mammals and invertebrates (Mourao, 2007) contain
these polymers. These SPs demonstrate various health beneficial biological
activities such as anti-HIV-1 (Schaeffer & Krylov, 2000), immuno-
modulation (Leiro et al., 2007), and anticancer (Rocha et al., 2005) activ-
ities. SPs are by-products in the preparation of alginates from edible
brown seaweeds and could be used as a rich source of natural antioxidants
with potential application in the food industry.
4.1.3 Carotenoids
Carotenoids produced by plants, algae, fungi, and microorganisms, but not
animals are a family of pigmented compounds. They are the most important
pigments in nature that are responsible for various colors of different photo-
synthetic organisms (Rao & Rao, 2007). According to studies, carotenoids
are assumed to be responsible for the beneficial properties in preventing
human diseases including cardiovascular diseases, cancer, and other chronic
diseases (Agarwal & Rao, 2000). Marine-derived carotenoids, astaxanthin,
is effective against UVA-induced DNA alteration in human dermal fibro-

blasts, human melanocytes, and human intestinal cells (Lyons & OBrien,
2002). In addition, fucoxanthin (marine-derived carotenoids) and astaxanthin
are bioactive natural functional ingredients that may be important in human
health as potential antioxidants (Sachindra et al., 2007).
4.2. Thickeners, stabilizers, and emulsifiers

Another property of marine carbohydrates such as algins and
exopolysaccharides from cyanobacteria is to be used for the stabilization of
emulsions or as bioflocculants. These potentials allow a wide variety of unique
food products to evolve in order to have a reasonable life. Polysaccharides are
a common approach in food product formulation to achieve a certain texture,
mouthfeel, and body by thickening the food. Most polysaccharides have an
ability, which is viscosity increases or decreases with increasing shear rate,
once they are dispersed in water. This ability is called non-Newtonian.
Some stabilizers result in a solution yield value, i.e., shear stress or applied
force below which the solution will not flow (e.g., ketchup). Because of
the thickening effect and the yield value, addition of suitable polysaccharides
to an aqueous system can stabilize the suspending dispersed phase (could be
solid, liquid, or gas) and prevent it from separating out.
Carrageenan has a unique functional property in its reactivity to protein.
So it is used to stabilize milk protein. Normally, carrageenan is used in com-
bination with other hydrocolloids such as starch, locustbean gum, and car-
boxymethyl cellulose. Furcellaran has a similar function but less extensively
in food. Many functional requirements and various applications such as for-
tification, natural pigments, stabilization, and antimicrobial food coatings are
met by the use of simple and complex carbohydrates derived from marine
food. Thus, development of more efficient and natural food processing tech-
niques can be set in new eras perspective.
5. CONCLUSION
The importance and application of marine carbohydrate made it a
major compound in different industries and productions. SPs in marine
organisms isolated from different species of algae, bacteria, and fungi playing
an important role in pharmaceutical, cosmetics, and food production
because of their bioactive compound. Presence of fucoidan, carrageenan,
agar, and alginate as a SP source in different marine species and their biolog-
ical activities are considerable in production various compounds. Since
ancient time, carbohydrate isolated from marine species used in drug pro-
duction and curing various diseases. Nowadays, marine carbohydrate has
application in novel drug production. Among the carbohydrate group,
SPs are special in the pharmaceutical industry. Besides, carbohydrate isolated
from marine use in the cosmetics industry. Fucoidan, carrageenan, and algi-
nate are the main compounds used in the cosmetic industry. Marine organ-
isms considering as a good source of carbohydrate and their biological
activities such as antioxidants improve them in the food industry. Besides,
food coloring, emulsions, thickeners, and stabilizers are the other role of
marine carbohydrates in the food industry. Studying on marine carbohy-
drates to improve the quality of food, cosmeceutical, and pharmaceutical
industry is one of the ways to improve quality of products. Scientists are still
studying about other biological activities and other applications of marine
carbohydrates to fulfill man demands.
ACKNOWLEDGMENT
The authors would like to thank University of Malaya for the financial support
(RG078-12BIO) and facilities provided to successfully carryout this research.
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CHAPTER ELEVEN
Algal and Microbial

Exopolysaccharides: New
Insights as Biosurfactants
and Bioemulsifiers
Jos de Jess Paniagua-Michel*,1, Jorge Olmos-Soto,
Eduardo Roberto Morales-Guerrero*
*Laboratory for Bioactive Compounds and Bioremediation, Department of Marine Biotechnology, Centro de
Investigacion Cientfica y de Educacion Superior de Ensenada (CICESE), Ensenada, Baja California, Mexico
Laboratory for Molecular Microbiology, Department of Marine Biotechnology, Centro de Investigacion

Cientfica y de Educacion Superior de Ensenada (CICESE), Ensenada, Baja California, Mexico
1
Corresponding author: e-mail address: jpaniagu@cicese.mx
Contents
1. Introduction 222
2. Defining Biosurfactants 224
2.1 Definition and characteristics 224
3. Microalgae: The New and Novel Bioemulsifiers 226
4. Biosynthesis Exemplified in Diatoms and Cyanobacteria 226
5. Cyanobacteria: A Prolific Source of EPS. The Case of Emulcyan 229
6. Diatoms: Photosynthetic Production of Complex EPSs 230
7. EPS: The Genesis in Building Biofilms 233
8. Seaweed Polysaccharides 234
9. Biosurfactants/Bioemulsifiers in Foods. The Marine Alternative 237
9.1 Probiotics EPSs 238
10. Biosurfactants for Sustainable Bioremediation 239
11. The Biosurfactants from Extreme Environments and Deep Sea 242
11.1 EPS from the deep sea 244
11.2 Polysaccharides from marine animals 246
11.3 Marine sources of EPS: The highest source for foods and dietary fibers 247
12. Expectatives and Concluding Remarks 248
References 250
Further Reading 257
Abstract
Currently, efforts are being made to utilize more natural biological systems as alterna-
tives as a way to replace fossil forms of carbon. There is a growing concern at global level
to have nontoxic, nonhazardous surface-active agents; contrary to synthetic surfactants,
their biological counterparts or biosurfactants play a primary function, facilitating
http://dx.doi.org/10.1016/B978-0-12-800268-1.00011-1
222 Jos de Jess Paniagua-Michel et al.
microbial presence in environments dominated by hydrophilichydrophobic interfaces.

Algal and microbial biosurfactants/bioemulsifiers from marine and deep-sea environ-
ments are attracting major interest due to their structural and functional diversity as
molecules actives of surface and an alternative biomass to replace fossil forms of carbon.
Algal and microbial surfactants are lipid in nature and classified as glycolipids, phospho-
lipids, lipopeptides, natural lipids, fatty acids, and lipopolysaccharides. These metabolic
bioactive products are applicable in a number of industries and processes, viz., food
processing, pharmacology, and bioremediation of oil-polluted environments. This chap-
ter presents an update of the progress and potentialities of the principal producers of
exopolysaccharide (EPS)-type biosurfactants and bioemulsifiers, viz., macro- and micro-
algae (cyanobacteria and diatoms) and bacteria from marine and extreme environ-
ments. Particular interest is centered into new sources and applications, viz., marine
and deep-sea environments and promissory uses of these EPSs as biosurfactants/
emulsifiers and other polymeric roles. The enormous benefits of these molecules
encourage their discovery, exploitation, and development of new microbial EPSs that
could possess novel industrial importance and corresponding innovations.
1. INTRODUCTION
Anthropogenic damage of the environment has increased environ-
mental concern about the future use of chemical surfactants and the need
of biological compounds derived, mainly surface-active agents due to their
low toxicity and biodegradable nature. Surfactant production shall have to
look for a viable alternative feedstock source that is renewable and less
dependent of the oil hydrocarbon substrates. The increasing demand for
renewable resources places the twenty-first century with a promise to open
up new avenues for sustainable bioactive products. The particular case of
biosurfactants/bioemulsifiers is attracting major interest due to their struc-
tural and functional diversity like surface active molecules and respective
applications in a variety of industrial activities.
The world market of polysaccharides is covered by algae, bacteria, and
higher plants. Annual world production of polysaccharides from marine bio-
mass is approximately 25,00030,000 tons/year (Pichavant, 2009). Bio-
surfactants and bioemulsifiers as amphiphilic compounds containing both
a hydrophilic and a hydrophobic moiety are able to display a variety of sur-
face activities that allow solubilization of hydrophobic substrates (Desai &
Banat, 1997; Satpute, Banat, Dhakephalkar, Banpurkar, & Chopade,
2010; Satpute, Bhuyan, et al., 2010). Advances in new environmental appli-
cations related to emulsification, foaming, detergency, wetting, dispersion,
and solubilization of hydrophobic compounds are already on progress
Algal and Microbial Exopolysaccharides as Biosurfactants 223
(Banat, Makkar, & Cameotra, 2000).Ocean biological and chemical diver-

sities still represent major expectative for novel compounds and surface-
active molecules because marine biosphere has remained mostly untapped
and unexplored. Carbohydrate production by the use of solar energy has
been usually overlooked in marine biomass where the higher yields are
reported and represent a significant source of polysaccharides as a renewable
energy source. Moreover, the current market demand for environmental
alternatives in counterpart to the synthetic surfactants has encouraged the
development for biological production of carbohydrates and polysaccharides
with surface-active properties.
Exopolysaccharides (EPSs) from both prokaryotes and eukaryotes
from marine origin exhibit multiple functions, which are very attractive
to replace the traditionally used of their synthetic counterparts (Fig. 11.1).
Apart from particular exceptions, algal and microbial EPSs represent a huge
variety of chemical structures with high-molecular-weight polysaccharides
(1030 kDa) with homopolymeric and heteropolymeric compositions
(Singha, 2012). In the case of microorganisms, surfactants as amphipathic
molecules exhibit typical molecular weights of 5001500 Da, in different
combinations made up of peptides, saccharides, or lipids (Ward, 2010).
Polysaccharides in algae are important producers of agar, alginate,
Figure 11.1 The versatility of applications of biosurfactants and bioemulsifiers from

marine sources algal and microbial marine exopolysaccharides.
carrageenan, dextran, gellan, pullulan, and xanthan gum (Singha, 2012).

These polysaccharides, have properties as gelling, stabilizing, or thickening
that are important agents as food additives. The unique properties and func-
tionality of biosurfactants at extremes of temperature, pH, and salinity confer
lower toxicity and higher biodegradability. Actually, biosurfactants have
gained importance in many fieldsenvironmental bioremediation, food
processing, pharmacy, biomedicine, petroleum industry, agriculture, and
environment. The different algal and microbial sources and applications
are presented in Fig. 11.1.
Production schemes of polysaccharides are different in organisms
depending on their origin, growth media, and environmental conditions.
In principle, in natural systems, adhesion, desorption, emulsification, and
micellarization are the mechanisms of SACs interactions between microbial
cells and immiscible hydrocarbons (Franzetti, Tamburini, & Banat, 2010).
This review presents an update of the progress and potentialities of the
principal producers of EPS-type biosurfactants and bioemulsifiers, viz.,
macro- and microalgae (cyanobacteria and diatoms) and bacteria from
marine and extreme environments. Particular interest is centered into
new sources and applications, viz., marine and deep-sea environments
and promissory uses of these EPSs as biosurfactants/emulsifiers and other
polymeric roles.
2. DEFINING BIOSURFACTANTS
2.1. Definition and characteristics
The ability of organisms, algae, plant, and bacteria to produce a wide range of
amphipathic compounds, with both hydrophilic and hydrophobic moieties
within the same molecule, enables them to exhibit surface activities at inter-
faces. These compounds are known as biosurfactant or bioemulsifier (Fracchia,
Cavallo, Allegrone, & Martinotti, 2010). They are made up of an acid, peptide
cations, or anions (mono-, di), or polysaccharides and hydrocarbon chains or
fatty acids. These structures confer a wide range of properties, including the
ability to lower surface and interfacial tension of liquids and to form micelles
and microemulsions between two different phases. Figure 11.2 shows the
diverse moieties exhibited by the surfactant structures relative to their
hydrophilic, ionic, and amphoteric compositions. These compounds are
divided into two main classes (Neu, 1996): low-molecular-weight com-
pounds called biosurfactants, such as lipopeptides, glycolipids, and proteins,
Figure 11.2 Different surfactant structures relative to the composition of their moieties:
hydrophilic, ionic, and amphoteric compositions. Modified from Medeiros et al. (2013).
and high-molecular-weight polymers of polysaccharides, lipopolysaccharides

proteins, or lipoproteins that are collectively called emulsans (Rosenberg &
Ron, 1997) or bioemulsifiers (Smyth et al., 2010).
Surfactants as amphiphilic compounds have the ability to reduce surface
and interfacial tensions through the accumulation of immiscible fluids,
which increase the solubility, mobility, bioavailability, and concomitant
biodegradation of insoluble organic compounds (Singh, Kumari, &
Mishra, 2012). The main properties of microbial biosurfactants are their
absence of toxicity, biodegradability, and eco-friendly nature.
Several authors have indicated that microbial products of most of the
marine species exhibit pronounced surface and emulsifying activities. These
surface-active compounds exert their action reducing surface tension at air
water interface, acquiring the name as bioabsorbents, while those involved
in reducing the interfacial tension between immiscible liquids or at the
solidliquid interfaces are called bioemulsifiers. The emulsifying capacity
of biosurfactants not necessarily means that bioemulsifiers may have proper-
ties of biosurfactants. Surfactants exhibit the three major important charac-
teristics of surface-active agents: (i) enrichment at interfaces, (ii) lowering
interfacial tension, and (iii) micelle formation.
Most of the microbial surfactants are lipid in nature and grouped
into glycolipids, phospholipids, lipopeptides, natural lipids, fatty acids, and
lipopolysaccharides. Researches on the replacements for synthetic sur-
factants and emulsifiers are expected to satisfy the needs of new and
modern markets in bioactive compounds and natural products, viz.,
surface-active substances of new generation (effective and ecologically safe)
with many useful industrial and environmental applications such as emulsi-

fication, detergency, wetting, and solubilization of hydrophobic compounds
(Banat et al., 2000).
3. MICROALGAE: THE NEW AND NOVEL

BIOEMULSIFIERS
During recent years, raw material shortages and pollution problems
led to an increase in research and development activities to use microalgae,
transgenic microalgae, protoplast fusion, or macroalgal cell cultures as bio-
technological sources (Cohen, 1999). Algal polysaccharides are also of phar-
macological importance. The results of screening programs to test in vitro
immunologically relevant effects of polysaccharides from microalgae have
shown that certain highly sulfated polysaccharides can trigger either the cel-
lular or the humoral stimulation of the human immune system (Namikoshi
& Rinehart, 1996). Cyanobacteria and diatoms are two representative
microalgal taxa accumulating a prolific amount of EPS with a highly prom-
issory potential. Table 11.1 shows the main representative EPSs from
eukaryotic microalgae and microorganisms.
4. BIOSYNTHESIS EXEMPLIFIED IN DIATOMS AND

CYANOBACTERIA
Polysaccharides exhibit a diversity that is dependent in the structural
variations of monosaccharide content, a feature that generally is under
genetic control. Hence, biosynthesis of EPS differs from one genus to
another. Recently, new properties and applications of polysaccharides in
preservation and protection of food and drugs, gives them the ability to
improving of textures of food colloids and their use as therapeutics. Unfor-
tunately, the use of these synthetic agents to the water or site, increase the
pollution by nondegradable products, which further intensify the problem.
In a number of important cases, the hydrophobic part of a surfactant mol-
ecule is derived from a hydrocarbon containing 820 carbon atoms (e.g.,
fatty acids, paraffin, olefins, and alkyl benzenes); the hydrophilic portion
may either ionize in aqueous solution (anionic or cationic) or remain
nonionic.
The polysaccharides produced by microorganisms can be classified into
three main groups according to their location in the cell. These groups are
Table 11.1 Main exopolysaccharides produced by eukaryotic microalgae and microorganisms
Microbial EPS
Microorganisms Exopolysaccharides strains Substrates (g L 1) References
Cyanobacteria Porphyridium CO2 0.10.3 Rebolloso Fuentes et al. (1999) and Fabregas,
cruentum GarcIa, Morales, Lamela, and Otero (1999)
Microalgae Botryococcus CO2 2.5 Lupi, Fernandes, Tome, Sacorreia, and Novais
braunii (1994)
Diatoms Amphora CO2 0.027 Leandro, Gil, and Delgadillo (2003)
holsatica
Navicula directa CO2 0.026 Leandro et al. (2003)
Yeasts and Pullulan Aureobasidium Sucrose 1.352.5 Duan, Chi, Wang, and Wang (2008), Jiang (2010),
filamentous fungi pullulans Ravella et al. (2010), and Seo et al. (2004)
Scleroglucan Slerotium sp. Sucrose/ 721 Survase, Saudagar, and Singhal (2006) and Survase,
glucose Saudagar, and Singhal (2007)
Schizophyllan Schizophyllum Glucose/ 1.628.03 Kumari, Survase, and Singhal (2008)
commune sucrose
Galactan Sporobolomyces Sucrose 5.63 Pavlova, Koleva, Kratchnova, and Panchev (2004)
sp.
Glucan Rhodotorula sp. Glucose/ 15 Pavlova and Grigorova (1999) and Pavlova,
sucrose Panchev, and Hristozova (2005)
Modified from Donot, Fontana, Baccou, and Schorr-Galindo (2012).
(i) cytosolic polysaccharides, which provide a carbon and energy source

for the cell; (ii) polysaccharides that make up the cell wall, including peptido-
glycans and lipopolysaccharides; and (iii) polysaccharides that are exuded
into the extracellular environment in the form of capsules or biofilm, known
as EPSs. EPSs are divided into two groups: homopolysaccharides and hetero-
polysaccharides. Homopolysaccharides are made up of a single type of mono-
saccharide, like dextran or levan. Heteropolysaccharides are conformed of
several types of monosaccharide like xanthans or gellans, which have complex
structures usually synthesized inside the cell in the form of repeating units
(Bergmaier, Lacroix, & Champagne, 2002; Lahaye, 2006; Roger, 2002).
Moreover, the production pathway and substrate molecule depend also
based on the production of EPS type. Most of the EPS biosynthesis schemes
involve glycosyltransferases that link intracellularly a sugar from nucleotide
sugars to a lipid carrier molecule. The type and availability of sugar nucle-
otides influence greatly the biosynthesis of different EPSs (viz., alginate,
gellan, etc.). Intermediate glycolysis by the interconversion of glucose-6-
phosphate into glucose-1-phosphate (sugar nucleotide precursor), which
is catalyzed by phosphoglucomutase, is a key step in sugar nucleotide bio-
synthesis. In contrast, biosynthesis of other EPSs such as levan, mutan, alter-
nan, reuteran, and dextran is catalyzed extracellularly by levansucrase,
mutansucrase, alternansucrase, reuteransucrase, and dextransucrase, respec-
tively, from sucrose. The active participation of the transfer of D-glucose
moiety of sucrose to an acceptor monosaccharide or oligosaccharide is
the leading mechanism of this process (Patel, Michaud, Singhania,
Soccol, & Pandey, 2010).
Polysaccharides derived from microorganisms, like bacteria, cyano-
bacteria, and diatoms represent an unexplored and unexploited market.
In diatoms, the pathways of carbon flow between photosynthesis and
EPS production has been scarcely studied. In darkness, diatoms EPSs are
proportional to glucan utilization, but diminished its molecular weight of
extracellular carbohydrate (Underwood, Boulcott, & Raines, 2004). Taken
together, these data suggest a pathway between glucan and EPS, and a link
between primary carbon fixation, glucan synthesis, and subsequent carbon
flow into EPS production. Authors suggest that at least two possible routes
by which photosynthetically assimilated carbon can appear in EPS. Hence, a
hypothesis postulates a pathway involving an enzyme that has a high turn-
over rate and catabolizes photosynthesized glucan. Cells growing in optimal
nutrient conditions in culture produced more nonpolymeric extracellular
carbohydrates than EPS, whereas the types of EPS produced by diatoms
changed when cells become nutrient limited. Cells could produce both
types of EPS in darkness by using glucan reserves (Underwood et al., 2004).
Marine microorganisms have developed unique metabolic and physio-
logical capabilities for the production of metabolites, which would not be
observed from terrestrial microorganisms. Bacteria from marine environ-
ments may not have obligatory requirements for sodium and seawater but
have the capability of unique biosynthesis of secondary metabolites
(Nerurkar, Hingurao, & Suthar, 2009). By evolution, bacteria have adapted
themselves to feeding on water-immiscible materials by producing and using
a surface-active products that help bacteria in aqueous phase to adsorb,
emulsify, wet, and disperse or solubilize water-immiscible materials. Among
microorganisms, high- and low-molecular-weight EPSs can be produced.
Low-molecular-weight types are glycolipids (trehalose, tetraesters, fructose,
lipids, sophorolipids, and rhamnolipids) or lipopeptides (surfactin and vis-
cosin). Among these, factors limiting EPS production costs and substrates
as well as the required infrastructures for scaling-up production, bioreactor
equipment, and control of parameters are challenging conditions needed to
be overcome for sustainable production of biosurfactants/bioemulsifiers
(Donot et al., 2012).
5. CYANOBACTERIA: A PROLIFIC SOURCE OF EPS.

THE CASE OF EMULCYAN
Cyanobacteria surpass plants and microbes in efficiently converting solar
energy and carbon dioxide into polysaccharides (Gupta, Ratha, Sood,
Chaudhary, & Prasann, 2013). This is a unique property of the combination
of plant-like photosynthesis and microbial ability to yield high value products
under controlled conditions. The benthic cyanobacteria Phormidium J-1 has
been shown to produce an extracellular sulfated heteropolysaccharide with
the ability to emulsify, termed emulcyan. In principle, this strategy is a way
to increase light availability in water column by flocculating suspended clay
particles. Fattom and Shilo (1984, 1985) suggested that the polymer synthesized
by this strain masks cell surface hydrophobicity, thus causing detachment of the
cells from hydrocarbon interfaces, which could serve cells as a dispersal strategy.
Emulcyan, which appeared to have a molecular weight greater than 100,000,
was able to bring about the detachment of Phormidium from hexadecane drop-
lets or phenyl sepharose beads. Emulsifying activity on emulcyan was reported
a stabilizer of oil-in-water emulsions (Rosenberg & Ron, 1997). Emulcyan
massive accumulation at the start of the stationary phase has registered
Figure 11.3 Left: Atomic force microscopy of released polymer image (500 nm) of the
whole C. closterium cell; right: Light microscopy at 500 microns of exopolysaccharides
surfactant produced by the Cyanobacterium Phormidium sp. Left: Modified after Urbani
et al. (2012). Right: Modified from Rosales-Morales and Paniagua-Michel (2014).
concentrations of 2.5 units/mL after 12 days and 8 units/mL after 28 days.

Cyanobacteria are now included as the potential sources of new polymers, sev-
eral species being characterized by the presence of thick capsules surrounding
the cells and by the ability to release polysaccharide material into culture
medium. Figure 11.3 shows images of atomic force microscopy of released
polymer (500 nm) of whole cells of Cylindrotheca closterium and light micros-
copy at 500 m of EPSs surfactant (right) produced by the cyanobacterium
Phormidium sp..
Carbohydrates such as glucosyl glycerol, trehalose, and sucrose are syn-
thesized by cyanobacteria under different osmotic stresses. The diversity of
molecules of economic importance produced by these organisms is immense
and prospecting extreme habitats and collaboration with industries for scal-
ing up of useful entities can help to exploit their potential in a more focused
manner and conditions. The main EPSs produced by bacteria and cyano-
bacteria are shown in Table 11.2.
6. DIATOMS: PHOTOSYNTHETIC PRODUCTION OF

COMPLEX EPSs
In marine diatoms, the extracellular polysaccharide production plays a
significant route by which photosynthetically produced organic carbon enters
the trophic web and may influence the environment in the sea (Urbani et al.,
2012). EPSs in diatoms contribute to the motility in marine and aquatic envi-
ronments; the presence of EPS plays an ecologically important role as a carbon
source for the inhabiting communities (Underwood et al., 2004). Studies on
C. closterium (Ehrenburg), Navicula perminta (Grun.) in Van Heurck, and
Table 11.2 Main exopolysaccharides produced by bacteria and cyanobacteria

Microbial
strains EPS
Exopolysaccharides Bacteria Substrates (g L 1) References
Cellulose Acetobacter Fructose/ 723.6 Hwang, Yang,
xylinum glucose Hwang, Pyun, and
Kim (1999) and
Naritomi, Kouda,
Yano, and Yoshinaka
(1998)
Acinetobacter sp. Ethanol/ 4.7 Huang, Chen, and
diesel Chen (2008) and
Kang et al. (2009)
Alginate Pseudomonas Xylose 0.4 Celik, Aslim, and
aeruginosa Beyatli (2008)
Azotobacter sp. Glucose/ 1.17.5 Celik et al. (2008)
fructose and Emtiazi,
Etemadifar, and
Tavassoli (2003)
Dextran and Leuconostoc sp. Sucrose 8.17 Santos, Teixeira, and
derivatives Rodrigues (2000)
Curdlan Agrobacterium Glucose/ 5.0276 Shih, Yu, Hsieh, and
sucrose Wu, 2009, Liu and
Shen (2008)
Alcaligenes Glucose 3072 Wu, Zhan, Liu, and
faecalis Zheng (2008)
Gellan Sphingomonas Starch 13.235.7 Nampoothiri,
Singhania,
Sabarinath, and
Pandey (2003)
Hyaluronic acid Streptococcus sp. Glucose 5.010.0 Jagannath and
Ramachandran
(2010)
Xanthan Xanthomonas Molasse 53 Kalogiannis,
campestris Iakovidou,
Liakopoulou-
Kyriakides,
Kyriakidis, and
Skaracis (2003)
Continued
Table 11.2 Main exopolysaccharides produced by bacteria and cyanobacteriacont'd

Microbial
strains EPS
Exopolysaccharides Bacteria Substrates (g L 1) References
Levan Erwinia sp. Sucrose 15 Shih, Chen, and Wu
(2010)
Bacillus spp. Glucose/ 0.3286.3 Liu and Shen (2008)
sucrose and Shih et al. (2010)
Zymomonas Sucrose 2250 Bekers et al. (2005)
mobilis and de Oliveira, da
Silva, Buzato, and
Celligoi (2007)
Other EPS Enterobacter sp. Glycerol/ 618 Alves et al. (2010)
glucose and Prasertsan,
Wichienchot,
Doelle, and Kennedy
(2008)
Halomonas sp. Sucrose/ 1.64.5 Bejar, Llamas, Calvo,
glucose and Quesada (1998)
and Poli et al. (2009)
Synechocystis sp. CO2 0.350.55 Ozturk, Aslim, and
(cyanobacteria) Suludere (2009)
Emulcyan Phormidium J-1 Calcium 2.58 Fattom and Shilo
(cyanobacteria) (1984, 1985)
Modified from Donot et al. (2012).
Amphora exigua Greg revealed that the proportion of EPSs in the extracellular
carbohydrate pool increased significantly (to 4469%) as cells became nutrient
limited. In general, microalgae show diverse types of EPSs differing in sugar
composition and production patterns. C. closterium produced two types of
nutrient-replete cells in a complex EPS containing rhamnose, fucose, xylose,
mannose, galactose, glucose, and uronic acids. Nutrient-limited cells pro-
duced an additional EPS containing mannose, galactose, glucose, and uronic
acids. In algae, both EPS types are produced under illuminated and darkened
conditions. Among the proposed hypothesis on the benefits of enhanced EPS
production for benthic diatoms, we can mention stimulation of bacterial
assemblages resulting in increased nutrient cycling, protection from desicca-
tion, or providing a substrate for subsequent heterotrophic utilization by the
diatoms themselves (Leandro et al., 2003; Urbani et al., 2012).
7. EPS: THE GENESIS IN BUILDING BIOFILMS

Biofilms are high cell density associations of microorganisms. The struc-
tural conformation of EPS in biofilms is responsible of the glycocalyx, which is
the essential factor for the formation of a biofilm. Moreover, EPSs allow the
microbial population to attach to a biological support, as a substrate for the
microorganism growth (Donot et al., 2012). It seems that EPSs do not nec-
essarily function as energy reserves, considering that microorganisms are
unable to catabolize the EPSs produced. EPSs play the main role (Cerning,
1995) to protect the cell in its environment. The anionic charge of the exterior
polysaccharide envelope is associated to capture essential minerals and nutri-
ents from media. EPS envelope also interacts with certain molecules to reg-
ulate the diffusion between the extracellular and intracellular environments,
which allows bacteria to spread and to resist surfactants and antibiotics
(Donot et al., 2012; OToole, Kaplan, & Kolter, 2000). One of the main
properties of biofilms is that the metabolic products of one species can act
as substrates for metabolic processes of another species. The data base from
literature records accounts for productions of EPS in average of 50 g L 1 from
six microorganisms surveyed for levan pullulan, curdlan, and xanthan. Higher
figures were in the range from 50 to 86.3 g L 1 (Donot et al., 2012).
Biofilms as a complex association of cells from many natural environ-
ments combine extracellular products and detritus either trapped within
the biofilm or released from lysed cells as the biofilm matures. The mixture
of polysaccharides secreted by the cells within a biofilm is the main attach-
ment substance, which confers grouping and adhesion among cells
established within the biofilm (Sutherland, 2001). The structures of EPSs
synthesized by microbial cells vary greatly in their composition and hence
in their chemical and physical properties. The majority are polyanionic
due to the presence of either uronic acids (D-glucuronic acid being the com-
monest, although D-galacturonic and D-mannuronic acids are also found) or
ketal-linked pyruvate. A few are neutral macromolecules. Excess or limita-
tions of carbon substrates and nutrients determine the biosynthesis of EPS
within the biofilm. The heteropolysaccharides are composed of regular
repeat units of 2 8 monosaccharides, while bacterial and algae alginates
are structures from interrupted sequences of D-mannuronic acid and
L-guluronic acid residues. This acetylation of mannuronosyl residues makes
the difference from algae. EPSs are the main molecules that determine the
properties of biofilms. In the case of hyaluronic acid can bind up to 1 kg
water (g polysaccharide) (Sutherland, 2001). The issue of biosurfactants and

bioemulsifiers from biofilms remains a challenge. The bioscreening of a mix-
ture of EPS from microbial consortia should have an expected potential for
the control of different antagonists. Hence, the yield of EPS production
from monospecific biofilms could differ from that produced from consortia
or polymicrobial communities. Definitely, food and environmental bio-
technology could benefit from the production and control of polymeric
hydrogels retaining water molecules or specific secondary metabolites.
Potential and promissory applications can be expected as wetting agents,
lubricants, and solubilizers.
Actually, antimicrobial hydrogels are studied as an auto defense mecha-
nism for combating drug-resistant infections, because polymeric gels exhibit
many properties avoiding the freely dissolved condition, which enable them
to remain in place while maintaining antimicrobial activity (Li et al., 2013).
These characteristics confer attributes for wound healing, implant/catheter
coatings, and skin infections. The development of antimicrobial hydrogels
from and for biodegradable materials has encouraged research and develop-
ment initiatives in biotechnological applications (Li et al., 2013). Antimicro-
bial hydrogels not only have the ability to remain stable and active but also
are biodegradable, for long-term applications in bioremediation and natural
attenuation. Table 11.3 shows specific examples of marine microalgae pro-
ducers of EPS.
8. SEAWEED POLYSACCHARIDES
Seaweed polysaccharides, like agar, alginates, and carrageenans, are
economically the most important products from macroalgae or seaweeds.
Other chapters of this book provide detailed information of this subject.
They are used in diverse fields of industry because of their rheological gelling
or thickening properties (Pulz & Gross, 2004). The principal polysaccharides
from red algae (Rhodophyceae) and brown algae (Phaeophyceae) are the
following classes: carrageenans, alginates, and agar (De Ruiter &
Rudoplh, 1999).The total polysaccharide concentrations in the seaweed
species represent up to 476% of their dry weight. The highest contents
are found in species such as Ascophyllum, Porphyra, and Palmaria. Green sea-
weed species such as Ulva also have a high content, up to 65% of dry weight.
Figure 11.4 shows the representative structures of biosurfactants and
bioemulsifiers from algae (macro and micro) and bacteria.
Table 11.3 Marine microalgae producing EPS

Microalgae/
cyanobacteria/ Type of
diatoms Group polysaccharide Main sugars References
Cylindrotheca Diatoms sPS Xylose, Pletikapic et al.
closterium glucose (2011)
Navicula sPS Glucose,
salinarum xylose
Phaeodactylum EPS Glucose, Chen et al. (2011)
tricornutum (sulfated) mannose
Haslea ostrearia EPS Rince, Lebeau,
and Robert
(1999)
Nitzschia EPS Penna et al. (1999)
closterium
Skeletonema EPS
costatum
Chaetoceros sp. EPS
Amphora sp. EPS
Chlorella Chlorophytes PS Glucose, Guzman, Gato,
stigmatophora (sulfated) xylose Lamela, Freire-
Garabal, & Calleja
(2003)
Chlorella sp. sPS Yingying and
Changhai (2009)
Tetraselmis sp. Prasinophyte sPS
(Chlorophyta)
Isochrysis sp. Prymnesiophyte/ sPS
haptophyte
Porphyridium Rhodophytes sPS Xylose, Geresh and Arad
sp. galactose (1991) and Arad
(1988)
P. cruentum sPS Xylose, Garcia, Morales,
galactose Dominguez, &
Fabregas (1996)
P. purpureum sPS Radonic et al.
(2010)
Continued
Table 11.3 Marine microalgae producing EPScont'd

Microalgae/
cyanobacteria/ Type of
diatoms Group polysaccharide Main sugars References
Cochlodinium Dinoflagellates sPS Mannose, Hasui, Matsuda,
polykrikoides galactose Okutani, &
Shigeta (1995)
Aphanothece Cyanophytes EPS Glucose,
halophytica fucose
Arthrospira Cyanophyte Sulfated Rhamnose, Radonic et al.
platensis calcium fructose (2010) and
Hayashi, Hayashi,
Spirulan
Maeda, & Kojima
(CaSp)
(1996)
Anabaena, Aphanocapsa, Cyanothece sPS
Gloeothece, Nostoc, Phormidium, Senni et al. (2011)
Synechocystis
EPS, exo- or extracellular polysaccharide; sPS, sulfate-containing exopolysaccharide; PS, polysaccharide.
Modified from Raposo, de Morais, Santos, de Morais, and Miranda (2013).
Figure 11.4 Representative structures of biosurfactants and bioemulsifers from algae

(macro and micro) and bacteria. (A) Ulvane (left: glucuronic or right: iduronic acid);
(B) fucoidan; (C) alginic acid (left: D-manuronic acid; right: L-guluronic acid);
(D) emulsan; (E) rhamnolipids; (F) surfactin.
9. BIOSURFACTANTS/BIOEMULSIFIERS IN FOODS.
THE MARINE ALTERNATIVE
According to their properties, EPSs can be used as viscosifying
agents, stabilizers, emulsifiers, gelling agents, or water-binding agents in
food (Singha, 2012). An important number of EPSs have been engineered
(chemically, metabolically, or enzymatically) to improve their rheological
properties, e.g., cellulose, starch, alginate, and carrageenan. Because of the
pseudoplastic nature of microorganisms, EPS confers unique rheological
properties because of their capability of forming and controlling the vis-
cosity of solutions. In the case of dextran, its water-soluble properties
enable its use in a great number of applications. The use of dextran in
confectionary to improve moisture retention and viscosity and inhibit
sugar crystallization is an important application being actually diversified.
A quite different application is in gum and jelly candies, where it acts as a
gelling agent. Its inhibitory role of crystal formation in ice cream and in
commercial pudding preparations fits the current demand by consumers
(Singh et al., 2012).
Recently, the effect of salt in various processes of fermentation can be
catalyzed by halophilic and halotolerant microorganisms, which play an
essential role in various fermentation processes that occur in the presence
of salt. As a consequence, different particular characteristics of marine-
derived compounds are developed in the products, viz, taste, flavor, texture,
and humidifying conditions. Among the properties and value added of
marine origin biosurfactants, the following have been reported: production
of pickles (fermented cucumbers); brine strength is increased gradually up to
15.9% (w/v) NaCl. Lactobacillus plantarum also exert other equally important
applications, as in the case of pickled cabbage preparation where the salt con-
centration is prepared up to 2.5%. Polymers from halophilic EPSs from
marine organisms exert a salt attribute with promissory benefits in food sci-
ence applications, viz., polymers with appropriate properties acting as emul-
sifiers and controllers of pollutants and additives mobility. Strains and species
where these active emulsifications of petroleum properties have been
reported include Halobacterium salinarum, Haloferax volcani, and Halobacterium
distributum (Kulichevskaya, Milekhina, Borzenkov, Zvyagintseva, &
Belyaev, 1992). Among the respective properties of these polysaccharides
of halophilic origin, high viscosity, high temperatures, pseudoplasticity,
and resistance to salt and thermal degradation have been reported. In the case
of the halophilic Halomonas sp., a production of emulsified up to 2.8 g EPS/l

has been registered as more effective in hydrocarbons removal more effi-
ciently than synthetic surfactants (Bouchotroch, Quesada, Izquierdo,
Rodriguez, & Bejar, 2000). Sulfated EPSs also have been reported as inhib-
itors of virus infestation into host cells.
9.1. Probiotics EPSs

The interest to obtain healthy nutraceuticals with valuable bioactive prop-
erties has increased attention to probiotic EPSs as a strategy of bacteria to
obtain beneficial influence on health of the host. The growing interest
for obtaining benefits in foods is growing steadily in the food industry world-
wide as functional foods and feeds which assert health-related benefits
beyond their dietetic constituents, as in the case of probiotic EPSs. Probiotic
bacteria are recognized by their ability to produce exocellular polymers
called EPSs, which are the responsible of the health benefit exerted by these
bacteria. As in the case of secondary metabolites, the composition, structure,
and biological functions of EPS are susceptible to vary depending on the
type of microorganism and environmental conditions (Ciszek-Lenda,
2011). Recent reports indicate that EPS production is a process under con-
trol of quorum sensing and regulated by gene expression for proteins
involved in EPS biosynthesis (Vu, Chen, Crawford, & Ivanova, 2009).
Members of Lactobacillus and Bifidobacterium are common probiotics used
for human consumption, in pharmaceuticals and biomedicine. Other strains
and genera have been applied in agriculture, veterinary, and most recently
marine aquacultural biotechnology (Ochoa-Solano & Olmos Soto, 2006).
The recent development of biofactories based on probiotic bacteria has been
utilized for the production of various polysaccharides for novel application
in food processing industry. Selected biological activities exhibited by bio-
surfactants/bioemulsifiers from marine origin are shown in Table 11.4. The
enhancement of carbon metabolism in algae and microorganisms play a
clever role in determining the sugar nucleotide as an effect directly corre-
lated with EPS production. During the past couple of decades, important
advance in probiotic bacteria dealing with EPS production has led to the
identification and characterization of interesting and functional genes.
Recent findings have reported the importance of specific enzymes for the
desig of metabolic engineering strategies to increase the EPS production
level by probiotic organisms. Still robust studies are needed in order to pre-
dict EPS function as well as molecular mass or chain length, to determine
their effect on a product property like viscosity and other related
Table 11.4 Selected biological activities exhibited by marine biosurfactants/

bioemulsifiers
Biosurfactant/
bioemulsifier Organism Biological activity References
Glycolipid Nocardioides sp. Induce hemolysis and inhibits Vasileva-
Bacillus subtilis cells Tonkovar and
Gesheva (2005)
Lipopeptide Pseudomonas sp. Antimicrobial action against Gerard et al.
(MK90e85; Mycobacterium tuberculosis and (1997)
MK91CC8) Mycobacterium avium
Lipopeptide Bacillus pumilus Ovicidal and cytotoxic effect Kalinovskaya
KMM 150 against cells of the ova of echinus et al. (1995)
in early stages of development
Lipopeptide Brevibacillus Antimicrobial action against Desjardine et al.
laterosporus multidrug-resistant (2007)
PNG-276 Staphylococcus aureus (MRSA)
and Gram-positive human
pathogen Enterococcus sp.
Modified from Das, Mukherjee, Sivapathasekaran, and Sen (2010).
characteristics. The studies on EPS biosynthesis shall lead to construct

engineered probiotic strains that could produce designer EPSs at an
increased level (Patel et al., 2010).
10. BIOSURFACTANTS FOR SUSTAINABLE

BIOREMEDIATION
A common characteristic of biosurfactants is to relax or decrease sur-
face tension, which increases solubility. This condition enhances degrada-
tion of hydrocarbons as a consequence of the mobility of hydrocarbons,
as exemplified in Fig. 11.5, which exhibit the different mechanisms of
hydrocarbon removal by biosurfactants depending on their molecular mass
and concentration. In oil-polluted saline environments, biosurfactants from
halophilic/halotolerant microorganisms play a significant role in the accel-
erated remediation of these environments. In marine environments, Banat
et al. (2010) reported an effective remediation of these environments with
the action of EPS. Several studies have indicated potential for pollution
treatment in marine environments or coastal areas (Banat et al., 2000).
Microbial production of a surfactant derived from trehalose by marine spe-
cies as the case of Rhodococcus surveyed proved successful for n-alkanes
Biosurfactants
Molecular mass High

Low
Critical micelle concentration

Emulsification
Above Solubilization
Below
Mobilization
Figure 11.5 Mechanisms of hydrocarbon removal by biosurfactants depending on their
molecular mass and concentration. Modified from Pacwa-Pociniczak, Paza, Piotrowska-
Seget, and Cameotra (2011).
removal (Yakimov, Abraham, Meyer, Giuliano, & Golyshin, 1999). These

kinds of applications could have important roles as surface-active agents for
in situ bioremediation of fragile and sensitive environments anchoring a high
biodiversity as in the case of marine environments, including cold marine
environments.
Bioremediation of coastal and marine environments is one of the greatly
benefited branches of biotechnology with the use of biosurfactants/
bioemulsifiers. Hydrocarbon oxidation in the presence of salt performed
by halophiles/halotolerant organisms is a unique and useful biological treat-
ment of saline ecosystems contaminated by petroleum derivatives. In Arctic
and Antarctic environments, bioremediation of oil spills has been success-
fully applied (Delille et al., 1998; Margesin & Schinner, 2001). The strain
Streptomyces albaxialis was reported as able to degrade crude oil and petro-
leum products even in the presence of 30% (w/v) NaCl (Kuznetsov
et al., 1992), while the extremely halophilic Haloferax mediterranei was capa-
ble to metabolize oil as the sole carbon source when grown at 25% (w/v).
Strains able to degrade n-alkanes (C10C30) in the presence of 30% (w/v)
NaCl, is a feasible property (Zvyagintseva et al., 1995). The coastal and marine
effect of pollutants, viz., hydrocarbons, may benefit also these approaches by
bioremediation of marine bioprospected strains, as a friendly method of sen-
sitive and fragile environments. Moreover, brine removal from agricultural
activities can be successful with the use of EPS produced and supported by
marine organisms.
A similar application can be obtained with biofilm reactors loaded with
heterotrophic, halophilic bacteria for the treatment of hypersaline
wastewaters. A main and surrogated role of EPS in cyanobacteria and certain

microbial consortia and organisms from coastal to marine environments has
been reported as able to metabolizing oil hydrocarbons, a condition that is
enhanced by pollutant substrate as hexadecane and diesel oil (Rosales-
Morales & Paniagua-Michel, 2014). Surfactant activity in most
hydrocarbon-degrading and pathogenic bacteria is attributed to several cell
surface components, an advantage that can be overexploited for eco-
sustainable bioremediation of sensitive marine environments (Table 11.5).
Table 11.5 Environmental remediation potentials and industrial application potential

of various marine biosurfactants/bioemulsifiers
Environmental
remediation/
Biosurfactant/ Producer industrial
bioemulsifier organism potentials References
Ex polysaccharide Planococcus Emulsifying Suresh Kumar, Mody,
maitriensis property and oil and Jha, 2007
Anita I dispersion
potential
Ex polysaccharide Antarctobacter sp. Emulsification of Gutierrez, Mulloy,
food oils and as Bavington, Black, and
metal adsorbent Green (2007), Gutierrez,
Mulloy, Black, and
Green (2007)
Ex polysaccharide Halomonas sp. Emulsification of Gutierrez, Mulloy,
TG39. TG 67 food oils and in Bavington, et al. (2007),
emulsion Gutierrez, Mulloy,
stabilization Black, et al. (2007)
Glycolipopeptide Corynebacterium Emulsification and Thavasi, Jayalakshmi,
kutscheri degradation of Balasubramanian, and
hydrocarbons Banat (2007)
Glycolipopeptide Yarrowia Emulsification of Amaral et al. (2006)
lipolytica aromatic
hydrocarbons
Glycolipopeptide Yarrowia Thermostable Zinjarde,
lipolytica emulsifier of Chinnathambi, Lachke,
aromatic and Pant (1997)
hydrocarbons
Continued
Table 11.5 Environmental remediation potentials and industrial application potential

of various marine biosurfactants/bioemulsifierscont'd
Environmental
remediation/
Biosurfactant/ Producer industrial
bioemulsifier organism potentials References
Glycolipopeptide Halomonas sp. Remediation of Pepi, Cesaro, Liut, and
ANT-3b the oil spills in Baldi (2005)
cold environments
Glycolipid Pseudomonas Enhanced oil Thaniyavarn et al. (2006)
aeruginosa A41 recovery
Glycolipid Bacterial strain Removal of Passeri et al. (1992)
MM1 marine oil
pollution
Glycolipid Aeromonas sp. Thermostable Ilori, Amobi, and
emulsifier of Odocha (2005)
various
hydrocarbons
Lipopeptide Bacillus circulans Polyaromatic Das, Mukherjee, and
hydrocarbon Sen (2008)
solubilization
Aminolipid Myroides sp. Emulsification of Maneerat et al. (2006)
strain SM1 weathered crude
oil
Ex, extracellular.
Modified from Das et al. (2010).
11. THE BIOSURFACTANTS FROM EXTREME

ENVIRONMENTS AND DEEP SEA
There is growing interest in isolating new EPS-producing microor-
ganisms from marine environments, particularly from extreme marine envi-
ronments such as deep-sea hydrothermal vents (Poli, Gianluca, & Nicolaus,
2010). Other representative environments for the production of EPSs in
marine environments include several extreme niches such as the cold marine
environments typically of Arctic and Antarctic sea ice. These niches and
environments are characterized by low temperature and low nutrient con-
centration and the hypersaline conditions of marine environment found in a
wide variety biotopes and ecosystems (salt lakes, salterns, brines). Most of the
EPSs from these environments are heteropolysaccharides containing three
or four different monosaccharides arranged in groups of 10 or less to form

the repeating units (Poli et al., 2010). These polymers are often linear with
an average molecular weight ranging from 1 105 to 3 105 Da. Some
EPSs are neutral macromolecules, but the majority of them are polyanionic
for the presence of uronic acids or ketal-linked pyruvate or inorganic resi-
dues such as phosphate and sulfate. EPSs provide the required properties and
protection for organisms to survive in a high salinity, predators, complex,
and competitive media as the seawater and marine ecosystems. The protec-
tion and other assumed functions emerge by forming a layer surrounding the
cell. The properties of these molecules based on their structural novelty, ver-
satility, and diversity are presently proposed as useful for many therapeutic
applications (Gudina, Rangarajan, Ramkrishna, & Rodrigues, 2013) and
allow envisioning into their commercial application as biosurfactants/emul-
sifiers. Figure 11.6 shows the diverse uses of food surfactants and their
respective combinations with lipids, proteins, and carbohydrates. The pres-
ence of exopolymers in the Antarctic marine environment has been associ-
ated to cryoprotection in high-salinity, low-temperature, and abundant
brines. A convincing hypothesis of the presence of algae in hydrothermal
vent environments of the deep sea is related to the biochemical production
of particular exopolymers to face and adapt to physical stresses such as
extremes of temperature and pressure.
One of the major advantages of EPSs in the marine environments as
high-molecular-weight carbohydrate polymers is their ability to make up
a substantial component of the extracellular polymers surrounding most
Usesproperties of food surfactants

Surface active and respective interactions (lipids, proteins, carbohydrates)
Emulsifier
Solubilizer
Thickener
Wetting agent
Lubricant
Foaming
Protection
Defoaming agent
Figure 11.6 The different uses of foods surfactants and their respective combinations
with lipids, proteins, and carbohydrates.
microbial cells. EPS is the largest reserve of the reduced carbon reservoir in
the ocean. That EPS plays an important role in enhancing and allowing the
survival of marine bacteria as a consequence of modifying the physicochem-
ical environment around the bacterial cell.
Antarctic ecosystems are environments relatively unexplored. In the
Arctic marine environment, microbial EPSs are abundant, which could assist
microbial communities to endure extremes of temperature, salinity, and
nutrient availability (Mancuso et al., 2005). Large amounts of microbially
produced exopolymeric substances occur in sea ice and at the icewater
interface, which could explain the diatoms and bacterial abundances in these
environments. The high polyhydroxyl content and concentrations of
exopolymer would decrease the freezing point of water in the low temper-
ature, and high salinity especially closer to the cell, where massive accumu-
lation of EPS occurs (Krembs, Eicken, Junge, & Deming, 2002; Mancuao
et al., 2005). It has been hypothesized that EPSs in brines provide buffering
capacity against high salinity as well as cryoprotecting the microbes living
there against ice crystal formation by depressing the ice nucleation temper-
ature of water (Krembs et al., 2002; Mancuso et al., 2005). Mancuso et al.
(2005) reported that EPSs produced by sea-ice isolates were shown to be
between 5 and 50 times larger than the average observed for other marine
EPSs (13 105 Da; Decho, Visscherb, & Reid, 2005). The cryoprotective
role of EPS has been assigned as controller of marine extreme environments
of high salinity and low temperature (Krembs et al., 2002).
11.1. EPS from the deep sea

Recent discoveries in deep-sea hydrothermal vent environments are associ-
ated with high pressure, extreme temperature, and heavy metals, common
characteristics of deep-sea environments (Mancuso et al., 2005). It is until
recently that the commercial value of microbial EPSs from these habitats
has been recognized offering a huge variety of promissory possibilities for
industrial applications. Nevertheless, the exploitation of these biopolymers
from hydrothermal vent environments remains largely unaltered which
confers huge potentialities for their biotechnological exploitation. Deep seas
(>1000 m), including submarine hydrothermal systems are a variety of
environments at depths greater than 2200 m where hydrostatic pressure
averages 25 106 P, and temperatures can range from 380 C within the
fumarole to 2 C in the surrounding seawater (Yayanos, 1998). Hot anaer-
obic waters rich in hydrogen sulfide and heavy metals escape the vents and
blend with cold oxygenated seawater. Despite these environmental extre-

mes, a complex food web based on chemosynthesis, including a unique
and original genetic and metabolic diversity of organisms (invertebrate
and microbials) with heterotrophic and autotrophic bacteria, was found near
the vents (Antoine, Guezennec, Meunier, Lesongueur, & Barbier, 1995).
The promissory expectative for innovations in many processes associated
to the study of these extremophiles environments should lead to the dis-
covery of new microbial biochemical processes and microbial by-products
used for growth and survival in these hydrothermal vent environments
(Deming, 1998).
The 25 years of the discovery of the deep-sea hydrothermal vents is an
excellent opportunity to ponder the new source of a variety of fascinating
microorganisms well adapted to these extreme environments. Over that time,
an increasing number of new genera and species have been isolated from these
vent communities. One interesting feature of these bacteria from deep-sea
hydrothermal vent communities was their ability to produce unusual extracel-
lular polymers in an aerobic carbohydrate-based medium (Pseudoalteromonas,
Alteromonas, and Vibrio). Studies on chemical composition and their role in the
deep-sea vent ecosystem of these polymers denote a commercial potential
remaining to be studied and expanded innovation initiatives.
Actually, abundant and diverse types of polysaccharides have been iden-
tified from different marine organisms (from marine bacteria to marine ani-
mals) and several of them have attracted attention as promising anticancer
substances. Some of these compounds are already used in clinical practice.
Among the useful properties and mechanisms of action of these anticancer
and cancer preventive substances, the following have been mentioned:
induction of apoptosis, inhibition of tumor cell proliferation, inhibition
of angiogenesis, etc. Other assigned functions of these biopolymers and their
derivatives are radical scavenging, antiviral, and immunostimulatory prop-
erties. The increasing exploration of marine biological sources will help
to identify the most promising of these compounds.
Recently, allelopathy has been discovered in green microalgae, Micro-
cystis aeruginosa, as the mechanisms involving secondary metabolites are pro-
duced by algae, plants, and microorganisms that influence the growth and
development of biological systems. Allelopathic compounds act as natural
toxins inhibiting the presence of competitors by the release of compounds.
This process has been hypothesized to play a role in the formation and con-
trol of harmful algal blooms in coastalmarine environments (El-Sheekh,
Khairy, & El-Shenody, 2012).
In the marine environment, algal EPSs are ubiquitous and abundant mol-
ecules where they play essential functions that enhance adaptation and survival.
Macroalgae such as brown, red, and green algae have been part of the traditional
food ingredients for people inhabiting populating in sea coasts areas. Among
algae, brown species belonging to Laminaria, Saccharina, Fucus, Alaria, Sargassum,
Undaria, and Pelvetia genera; green algae such as Ulva spp. and Caulerpa
lentillifera; and red algae such as Gracilaria spp. have been worldwide recognized
as constituents of dietary supplements due to their antimutagenic, anticoagu-
lant, and antitumor properties, and food and feed with the high content of die-
tary fiber (Fedorov, Ermakova, Zvyagintseva, & Stonik, 2013).
Apart from algae, the abundance of polysaccharides in many other
marine organisms is known to exhibit unusual structures and useful proper-
ties with promising natural products for medicinal and dietary applications.
Marine polysaccharides enable their utilization for cell therapy and tissue
engineering (Fedorov et al., 2013).
Bioscreening of many polysaccharides and/or their derivatives including
semisynthetic compounds has demonstrated anticancer and cancer preven-
tive properties. The inhibitory action of cancer cells on carcinogenesis and
tumor development, may recover the broken balance between proliferation
and programmed cell death (apoptosis). These are useful properties of
polysaccharides for cancer prophylactics (Fedorov et al., 2013). Particular
attributes of these marine natural products are due to their ubiquitous pres-
ence, low toxicity, suitability for oral application, and having a great variety
of mechanisms of action (Fedorov et al., 2013).
In the case of alginic acids, molecular masses ranged between 10 and
600 kDa are used in the pharmaceutical industry and in biotechnology, par-
ticularly for cell immobilization and encapsulation. Alginic acid-coated
chitosan nanoparticles have been constructed as an oral delivery carrier
for the legumain-based DNA vaccine. The positive bioconversion of alginic
acids probably has some cancer preventive properties because of the ability
of polysaccharides to bind toxins and heavy metals in the intestines and trans-
form these dangerous compounds into less harmful forms.
11.2. Polysaccharides from marine animals

The high content of polysaccharide compounds in sea cucumbers, sea urchins,
sponges, starfish, ascidians, etc., as glycosaminoglycans, fucans, and galactans
has demonstrated diverse biological properties, including anticoagulant and
antithrombotic, antioxidative, neuroprotective, and antiviral activities. How-
ever, scarce studies on the anticancer and cancer preventive activities of the
polysaccharides from marine animals have promoted more research in this

group of marine and coastal organisms (Fedorov et al., 2013). Sulfated poly-
saccharide obtained from the sea cucumber Cucumaria frondosa was reported as
interfering the maturation of monocyte-derived dendritic cells. Oral admin-
istration of sea cucumber fucoidan (100 mg/kg body weight) for 5 days from
Acaudina molpadioides can significantly prevent the formation of gastric ulcer in
rats. Chondroitin sulfate isolated from ascidian Styela clava inhibited phorbol
ester- and TNF--induced expression of inflammatory factors VCAM-1,
COX-2, and iNOS by blocking Akt/NF-B activation in mouse skin.
11.3. Marine sources of EPS: The highest source for foods and
dietary fibers
Traditionally, dietary fiber polysaccharides and derived molecules have been
applied and commercialized as food and pharmaceuticals in several biotech-
nological and industrial applications. Foods with dietary fibers include algi-
nates, agars, carrageenans, ulvanes, and fucoidans. Recent discoveries such as
immunomodulatory, antitumor, antithrombotic, anticoagulant, anti-
inflammatory, antimicrobial, and antiviral activities including anti-HIV
infection, herpes, and hepatitis viruses have been reported by sulfate poly-
saccharides (Misurcova, Skrovankova, Samek, Ambrozova, & Machu,
2012). The sulfation properties of algal polysaccharides display different
compositions and effects of structural compounds. These characteristics have
been assigned as the responsible of the several applications in food science
and industry (Misurcova et al., 2012). Table 11.6 shows selected marine
Table 11.6 Selected marine microbial emulsifiers

Bioemulsifier Microorganism Source
BD4 emulsan Acinetobacter Kaplan, Zosim, and Rosenberg
calcoaceticus BD4 (1987)
Biodispersan Acinetobacter Desai and Banat (1997)
calcoaceticus A2
Lipid- Yarrowia lipolytica Zinjarde and Pant (2002)
Carbohydrates-protein
HE39 Halomonas TG39 Gutierrez, Mulloy, Bavington, et al.
(2007) and Gutierrez, Mulloy, Black,
et al. (2007)
Yansan Yarrowia lipolitica Trindale et al. (2008)
IMUFRJ
Modified after Nerurkar et al. (2009)
microbial emulsifiers. A renewed interest in these marine and freshwater

polysaccharides has been recently focused for developing functional food
and bioremediation initiatives.
12. EXPECTATIVES AND CONCLUDING REMARKS

Glycolipid biosurfactants produced by marine organisms offer exciting
expectative and are promissory for future sustainable biotechnology of large-
scale commercial products. Advances in recognized old challenges of surfac-
tants are now dominating the field of biosurfactants such as yield, cost,
downstream processing, and tailoring of biosurfactant molecules to fulfill
specific roles in product formulations. The presence of commercial bio-
surfactants will demand in the near future innovations and improvements
in order to further exploitation of the goodness and potentialities of this
exciting area of biotechnology. Leading branches are food and feed biotech-
nology, bioremediation, pharmaceuticals, aquaculture, marine biotechnol-
ogy, and nanomaterials.
Lipopeptide, glycolipid, and other types of biosurfactants, owing to their
structural novelty and diverse biophysical properties, have recently emerged
as possible broad-spectrum agents for cancer chemotherapy/biotherapy and
as safe vehicles or ingredients in drug delivery formulations. Many new
applications of these biomolecules have been suggested, mainly owing to
their significant surface-active properties that enable them to interact with
cell membranes or surrounding environments to bring about the desired
effect as a therapeutic molecule or as a part of a DDS. The search for safe
and biocompatible biosurfactants for such applications will drive this field
of research in the coming years. The abilities of these molecules to selectively
inhibit the proliferation of cancer cell lines and disrupt cell membranes caus-
ing cellular lysis by operating apoptotic machineries may provide the clues
for the mechanism and mode of their actions. There are several other areas
where drug delivery has a promissory potential, viz., the use of biosurfactants
in therapeutic application and immunization, apart from the use of bio-
surfactants as adjuvants in microemulsion formulations. The biopolymers
from algae already in the market appear quite adequate for most applications
and industrial demand. Polysaccharides from marine red microalgae and
from other microalgae have also exhibited in vitro antiviral activity against
human immunodeficiency virus and the absence of a cytotoxic effect
directed against the host cells. Therefore, the look for new sources and
greener technologies as the marine environment shall increase the use of

algal EPS for industrial applications.
Capsular polysaccharide might promote the adherence of bacteria to bio-
logical surfaces, thereby facilitating the colonization of various ecological
niches. EPSs are particularly important because they confer adherence of
biofilms to a variety of substrate. In principle, the surface activity properties
of these molecules interact with cell membranes of several organisms and/or
with the surrounding environments and thus can be viewed as potential cancer
therapeutics or as constituents of drug delivery systems (Gudina et al., 2013).
Studies pointed out the fact that some types of microbial surfactants
(lipopeptides and glycolipids) have been shown to selectively inhibit the
proliferation of cancer cells and to disrupt cell membranes causing their lysis
through apoptosis pathways (Gudina et al., 2013). Most recently, the uses of
biosurfactants as drug delivery vehicles offer enormous attractive as novel
applications in the many fields of application of these molecules, as in the
case of passive immunization.
EPSs of marine Vibrio and Pseudomonas have been reported as antitumor,
antiviral, and immune stimulant activities of polysaccharides. In the case of
organisms obtained from deep-sea hydrothermal vents, such as Alteromonas
infernus, a low-molecular-weight heparin-like EPS exhibiting anticoagu-
lant property has been identified. L-Fucose containing polysaccharide has
a potential role in preventing tumor cell colonization of the lung, in regu-
lating the formation of white blood cells, and in the synthesis of antigens for
antibody production as well as cosmeceuticals as skin moisturizing agent.
An important application issue of the use of EPS in environmental bio-
remediation mediated by biofilms has been reported as more efficient and
safer alternative to bioremediation than free or planktonic bacteria or con-
sortia. The advantages of growing in biofilms are the enormous possibilities
of adaptation to different environmental conditions and their subsequent
survival. The improvements of mineralization processes by biofilms have
been related to their ability to maintain optimal chemical and physiological
conditions, localized solute concentrations, and redox potential. Actually,
the use of reactors packed with biofilms is a reality to treat hydrocarbons,
heavy metals, and large volumes of dilute aqueous solutions such as industrial
and municipal wastewater. The role of EPS in biofilms is also related to
its ability for the removal of heavy metals from the environment, a property
related to their involvement in flocculation and ability to bind metal ions
from solutions. Bacteria with this property are specifically related to
Enterobacter and Pseudomonas species.
In recent years, there has been a growing interest in the isolation and
identification of new microbial polysaccharides that may have novel appli-
cations such as gelling agents, emulsifiers, stabilizers, and texture enhancers
among others. Extremophilic microorganisms are recognized not only as an
important source and resource for exploitation in biotechnological processes
but also as models for investigating new properties on biomolecules, such as
its ability to face and stabilize under extreme conditions. The case of deep-
sea hydrothermal vents and Arctic environments is the most exemplifying
example of new source of biodiversity of novel organisms as producers of
exopolymers with biotechnological potential for the low cost and sustainable
production of biosurfactants and bioemulsifiers. Are these environments that
surely will provide innovation solutions to face challenges of the inter-
connected world crises and future demands?
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CHAPTER TWELVE
Complex Carbohydrates as a
Possible Source of High Energy
to Formulate Functional Feeds
Leonel Ochoa*, Jos de Jess Paniagua Michel, Jorge Olmos-Soto{,1
*Centro de Innovacion Biotecnologica AC (CENBIOTEC), Ensenada, Baja California, Mexico
Bioactive Compounds and Biorremediation Laboratory, Department of Marine Biotechnology, Centro de

Investigacion Cientfica y de Educacion Superior de Ensenada (CICESE), Ensenada, Baja California, Mexico
{
Laboratory for Molecular Microbiology, Department of Marine Biotechnology, Centro de Investigacion
Cientfica y de Educacion Superior de Ensenada (CICESE), Ensenada, Baja California, Mexico
1
Corresponding author: e-mail address: jolmos@cicese.mx
Contents
1. Introduction 260
2. Carbohydrates 261
3. Complex Carbohydrates 261
4. Oligosaccharides and NSP 263
5. Polysaccharides 266
5.1 Terrestrial polysaccharides 267
5.2 Marine polysaccharides 268
6. Enzymes and Digestibility 270
7. Prebiotic Ingredients 274
8. Probiotic Bacteria 276
9. Functional Feeds 277
9.1 Definition 278
9.2 Study cases, functional benefits, and usage suggestions 279
References 283
Further Reading 288
Abstract
Carbohydrates (CHOs) are the most abundant organic compounds found in living
organisms and are a great source of metabolic energy, both for plants and animals.
Besides of CHOs great potential to solve animal's energy requirements and diminishing
high feed cost, we first must to understand its digestibility and assimilation to avoid
several inconvenients. Today, CHOs feed animal inclusions are of great concern about
costbenefits, animal's health status, and environmental pollution. In this chapter, we
make a brief description about sugars (DP12), oligosaccharides (DP39), polysaccha-
rides (DP 10), and their essential characteristics to understand the role of marine
and terrestrial CHOs in animal nutrition. Subsequently, we talk about basic concepts,
CHOs functional benefits, suggestions about their application and successful cases.
http://dx.doi.org/10.1016/B978-0-12-800268-1.00012-3
260 Leonel Ochoa et al.
This information will contribute to produce a new generation of high-quality and energetic
functional feed formulations for livestock and aquaculture farms; which must be of low
cost, healthy, and environmentally friendly, with the inclusion of prebiotics and probiotics.
1. INTRODUCTION
Carbohydrates classification is based principally on their monomers
composition, degree of polymerization (DP), and nature of chemical bonds,
as agreed by the Food and Agriculture Organization/World Health Orga-
nization Expert Consultation in 1997. This classification distributes carbo-
hydrates into three mayor groups; sugars (DP12), oligosaccharides
(DP39), and polysaccharides (DP 10). Within this classification, terms
such as mono- and disaccharides, polyols, oligosaccharides, starch, modified
starch, nonstarch polysaccharides (NSPs), total carbohydrate, and sugars, are
been used. Carbohydrates functionality is not always related with their pri-
mary chemistry and neither their physical properties: water solubility, hydra-
tion, gel formation, and crystalline state. Instead, CHO in animals
functionality is most related with the molecule structure and its bonds
(Arellano & Olmos, 2002; Olmos, Ochoa, Paniagua-Michel, &
Contreras, 2011). Carbohydrates could be in association with other mole-
cules such as proteins, lipids, divalent cations, and in aggregation within
complex structures such as cell walls and other specialized tissues.
A classification based on their chemistry is essential for its measurement sys-
tem, prediction of properties, and estimation of intakes, but does not allow a
simple translation into nutritional effects, since most carbohydrate has over-
lapping physiological, energetic, and health properties. This dichotomy has
led to the use of a number of terms to describe food carbohydrates: intrinsic
and extrinsic sugars, prebiotic, resistant starch, dietary fiber, available and
unavailable carbohydrate, complex carbohydrate, glycemic, and whole grain
(Cummings & Stephen, 2007).
Among marine resources, algae are valuable source of huge and diverse
bioactive compounds including carbohydrates. The cell wall of marine algae
is rich in polysaccharides such as fucoidans in brown algae, carrageenans
in red algae, and ulvans in green algae. These compounds exhibit many
bioactivities such as anticoagulant, antiviral, antioxidants, anticancer, and
immune system induction (Berteau & Mulloy, 2003). In addition, marine
algae have great potential for further development of products like
nutraceuticals, pharmaceuticals, and as a possible source of high energy in
functional feeds (FFs).
Complex Carbohydrates as a Possible Source of High Energy 261
Actually, a great deal of interest remains to isolate novel bioactive com-

pounds from marine resources, because of their health and nutritional ben-
efit effects. Finfish, shellfish, seaweeds, microalgae, and a diverse group of
microorganisms are all sources of several life-supporting compounds such as
vitamins, carotenoids, proteins, polyunsaturated fatty acids (omega-3, 6),
and complex carbohydrates (Venugopal, 2008).
The high-molecular-weight of marine carbohydrates has found a variety
of applications in biotechnology, pharmaceutical, and others industries such
as food technology, with great potential to formulate FFs for human and
animal nutrition.
Additionally, terrestrial complex carbohydrates will be analyzed, and also
their application in animal-food technology development will be discussed.
2. CARBOHYDRATES
Carbohydrates are a diverse group of molecules with a range of chem-
ical, physiological, metabolic, and energetic properties. Sugars comprise
(a) monosaccharides, (b) disaccharides, and (c) polyols (sugar alcohols).
Oligosaccharides are (a) malt-oligosaccharides (-glucans), principally produced
by hydrolysis of starch, (b) non--glucans such as raffinose and stachyose,
known as -galactosides, and (c) fructo- and galactooligosaccharides. Polysac-
charides may be divided into (a) starch (-1:4 and 1:6 glucans) and (b) NSPs,
of which the most abundant are the polysaccharides of plant cell wall such as
cellulose, hemicellulose, and pectin. But also includes plant gums, mucilages,
and hydrocolloids (Table 12.1; Cummings & Stephen, 2007).
CHOs are the main and less expensive ingredient of animal feeds, they
are a great source of high energy, however their digestion and assimilation
are difficult in animals generating several economic, health, and environ-
mental problems. Starch is second only to cellulose in abundance in terms
of polysaccharides synthesized by plants and represents the primary feed
source of high energy for many monogastric species, including humans.
Animal feed less-included carbohydrates are NSP and free sugars
(Burger, 1993).
3. COMPLEX CARBOHYDRATES
This term was first used in the McGovern report, Dietary Goals for
the United States in 1977 (Select Committee on Nutrition and Human
Needs, 1977). It was coined largely to distinguish sugars from other carbo-
hydrates, and in the report denotes fruit, vegetables, and whole grains.
Table 12.1 The major dietary carbohydrates (Cummings & Stephen, 2007)
Class (DPa) Subgroup Principal components
Sugars (12) Monosaccharides Glucose, fructose, galactose
Disaccharides Sucrose, lactose, maltose, trehalose
Polyols (sugar Sorbitol, mannitol, lactitol, xylitol,
alcohols) erythritol, isomalt, maltitol
Oligosaccharides (39) Malto- Maltodextrins
(short-chain oligosaccharides
carbohydrates) (-glucans)
Non--glucan Raffinose, stachyose, fructo- and
oligosaccharides galactooligosaccharides, polydextrose,
inulin
Polysaccharides (10) Starch Amylose, amylopectin, modified
(-glucans) starches
Nonstarch Cellulose, hemicellulose, pectin,
polysaccharides arabinoxylans, -glucan,
(NSPs) glucomannans, plant gums and
mucilages, hydrocolloids
a
Degree of polymerization or number of monomeric (single sugar) units. Based on Food and Agriculture
Organization/World Health Organization, Carbohydrates in Human Nutrition report (1998) and
Cummings et al. (1997).
The term has never been formally defined and has since come to be used to
describe either starch alone or the combination of all polysaccharides (British
Nutrition Foundation, 1990). It was used to encourage the consumption of
what are considered to be healthy foods such as whole-grain cereals, but
becomes meaningless when used to describe fruits and vegetables, which
are low in starch. As a substitute term for starch, it would seem to have little
merit and, in principle, it is better to discuss carbohydrate components by
using their common chemical names (Cummings & Stephen, 2007).
Complex carbohydrates consist of a chemical structure that is made up of
three or more sugars, which are usually linked together to form a chain. Due
to their complexity, they take a little longer to digest, and they do not raise
the sugar levels in the blood as quickly as simple carbohydrates. Complex
carbohydrates act as the bodys fuel, and they contribute significantly to
energy production (Foster-Powell, Holt, & Brand-Miller, 2002). Similar
to simple carbohydrates, complex carbohydrates are divided into two cate-
gories: oligosaccharides and polysaccharides. Oligosaccharides consist of a
small number of monosaccharides, which does not exceed 10. Polysaccha-

rides are often made up of a large number of monosaccharides and disaccha-
rides. Complex carbohydrates have a higher nutritional value than simple
carbohydrates. It may be confusing to differentiate simple and complex car-
bohydrates due to the fact that complex ones contain certain elements of
simple ones. Nevertheless, differentiating both should not be a problem
since their chemical structures are very different, and therefore, they can
be distinguished by their nutritional properties and energy released
(Table 12.2; Bush, Martin-Pastor, & Imbery, 1999).
4. OLIGOSACCHARIDES AND NSP

An oligosaccharide is a saccharide polymer containing typically three
to nine of simple sugars. Oligosaccharides can have many functions, for
example, they are commonly found on the plasma membrane of animal cells
where they can play a role in cellcell recognition. In general, they are found
either O- or 0 f-linked to compatible amino acid side-chains in proteins or to
lipid moieties. Oligosaccharides are food products with interesting nutri-
tional properties (Delzenne, 2003). They have recently been recognized
as components of dietary fiber because of their interesting physiological
effects, which are similar to those of well-known soluble fibers
(Flamm, Glinsmann, Kristchevsky, Prosky, & Roberfroid, 2001). The avail-
ability of feed products containing nondigestible oligosaccharides is increas-
ing. They may now be documented as being carbohydrates with interesting
functional effects. Nondigestible oligosaccharides can be considered benefi-
cial molecules when added to feed because they enhance the probiotic bac-
teria development, including those that contain fructose, raffinose,
melibiose, xylose, galactose, and maltose (Rumney & Rowland, 1995).
Diets for monogastric animals always contain ingredients that are either
poorly digested or completely undigested due to lack of endogenous
enzymes. The most common of these antinutritional factors included in diets
are complex carbohydrates, phytate, trypsin inhibitors, lectins, alkaloids,
polyphenols, lignin, cellulose, hemicellulose, and NSP (Swick, 2002).
NSPs are macromolecules consisting of a large number of monosaccha-
ride (glucose) residues joined to each other by glycosidic linkages, and are
principally found in the plant cell wall (IUB-IUPAC, 1982). NSPs are
the most diverse form from all the carbohydrate groups and comprise a mix-
ture of many molecular forms, of which cellulose is the most widely distrib-
uted. NSPs present in plant materials reduce the performance of animals due
Table 12.2 Principal physiological properties of dietary carbohydrates (Cummings & Stephen, 2007)
Increase Source Alter balance Increase
Provide Increase Cholesterol calcium of of microflora stool
energy satiety Glycemica lowering absorption SCFAb (prebiotic) output Immunomodulatory
Monosaccharides
Disaccharides
Polyols c
Maltodextrins
Oligosaccharides
(non--glucan)
Starch d d
NSP e
a
Provides carbohydrate for metabolism (FAO, 1998).
b
Short chain fatty acids.
c
Except erythritol.
d
Resistant starch.
e
Some forms of nonstarch polysaccharide (NSP) only.
to their antinutritional effects are because of the increased viscosity in the

intestine and the enclosure of nutrients, making them unavailable to diges-
tion. NSPs consist of an extensive range of plant polymers, including
-glucans, pentosans, cellulose, hemicellulose, pectic substances, and
-galactosides (raffinose, stachyose, verbascose) (Fig. 12.1; Choct, 1997).
Figure 12.1 Oligomers of six major types of complex polysaccharides (Roman

numerals), their component sugars and enzymes (capital letters) that act on specific
bonds within the molecules (Weiner et al., 2008).
Table 12.3 Substrates used for the evaluation of different enzymatic activities
(Ochoa-Solano & Olmos-Soto, 2006)
Enzymatic activity Substrate
Proteases Skim milk, soybean meal, texturized soybean meal
Carbohydrolases Wheat flour, corn flour, and algae meal
-1,4-Glucosidases Amylose
-1,6-Glucosidases Amylopectin
-Galactosidases Melibiose and raffinose
Lipases Olive oil, fish oil, soybean lecithin, squid oil
Most feedstuffs contain some antinutritional factors (NSP) such as raffi-

nose, stachyose, and/or verbascose. The raffinose families of NSPs are insol-
uble, and monogastric animals are unable to digest them without the help of
a specific exogenous -galactosidase enzyme that hydrolyze these to galac-
tose and sucrose (Wenk, 1993). Since the animals lack the intestinal enzymes
for the degradation of NSPs, the supplementation of degradative enzymes in
the diet will break down these antinutritional factors and result in better feed
assimilation. Such an approach has been successfully used in fish and shrimp
diets (Olmos, Paniagua-Michel, Lopez, & Ochoa, 2014, Olmos et al., 2011).
Pigs and poultry lack -galactosidase enzyme needed to hydrolyze NSP
to galactose and sucrose; hence, there has been interest in the use of probiotic
microbial -galactosidase enzymes in diets as a means of increasing the nutri-
tional value of feeds. Furthermore, their addition to low-cost diets as a diges-
tive supplement should lead to better assimilation of diets, lower production
cost, faster animals growth, less pollution and diseases in livestock. The iden-
tification of -galactosidase and specific glycosidase producing probiotic
bacteria is very important. In order to find out the type of carbohydrolases
present in the strains, specific substrates have been used to identify alpha gly-
cosidase and alpha galactosidase enzymes (Table 12.3; Arellano & Olmos,
2002; Ochoa-Solano & Olmos-Soto, 2006).
5. POLYSACCHARIDES
Polysaccharides are the most complex carbohydrates and the most
abundant organic compounds, constituting about half of the organic carbon
on Earth. Agar, alginates, arabic gum, carrageenan, and methyl cellulose are
some examples (Phillips & Williams, 2000). Polysaccharides are macromol-
ecules made up of many monosaccharides joined together by glycosidic
bonds; hence, they are very large and branched. Polysaccharides differ not
only in the nature of their component monosaccharide but also in the length
of the chains and in the amount of chain branching (Aspinall, 1970). Poly-
saccharides are the major hydrocolloids also referred to as gums utilized in
food industry for different applications; they can be used as thickeners, emul-
sifiers, gels, water retention, and texture enhancers. Polysaccharides from
some seaweed have been reported to possess biological activity with poten-
tial therapeutic properties. Furthermore, polysaccharides have become very
important products in the food industry (Van de Velde, Knutsen, Usov,
Rollema, & Cerezo, 2002). In view of their high potential nutritional values
and functional properties, polysaccharides can also be used as additives in the
food/feed industry. However, only a small group of animals like ruminants,
with cellulolytic microbial flora can digest and assimilate some of them. In
this sense, most work must be done to improve polysaccharides digestion
assimilation in monogastric animals (Ochoa-Solano & Olmos-Soto, 2006;
Ochoa, 2012; Olmos et al., 2011). For instance, polysaccharides are excel-
lent feed agglutinants improving texture and water stability of pellets and as
attractants increasing feed intake.
5.1. Terrestrial polysaccharides

Terrestrial compounds are dominated by polysaccharides such as cellulose,
starch, mannan, and xylans. The main functions played by polysaccharides
are either storage or structural functions. By far, the most common storage
polysaccharide in plants is starch. Structural polysaccharides exhibit proper-
ties that are dramatically different from those of the storage polysaccharides,
even though their monosaccharides compositions are similar. Cellulose is
the most abundant structural polysaccharide produced in nature. Found
in the cell walls of nearly all plants, included marine algae, cellulose is
one of the principal components, providing physical structure and strength
(Holland et al., 1992).
Starch, the principal carbohydrate in most diets, is the storage carbohy-
drate of plants such as cereals, root vegetables, and legumes, and comprises
only glucose molecules. It occurs in a partially crystalline form of granules
and contains two polymers: -amylose and amylopectin. Most common
cereal starches contain 1530% amylose, which is a nonbranching helical
chain of glucose residues linked by -1,4 glucosidic bonds. Amylopectin
is a high-molecular-weight, highly branched polymer containing both
-1,4 and -1,6 linkages. Some starches from maize, rice, sorghum, and bar-
ley contain largely amylopectin and are known as waxy. The crystalline
structure of amylose and amylopectin in the starch granules confers on them

distinctive X-ray diffraction patterns, A, B, and C. The A type is character-
istic of cereals like rice, wheat, and maize. The B type is found in potato,
banana, and high amylose starches, while the C type is between A and
B and is found in legumes. In their native (raw) form, the B starches are resis-
tant to digestion by pancreatic amylase. The crystalline structure is lost when
starch is heated in water (gelatinization), thus allowing digestion to take
place (Galliard, 1987; Hoover & Sosulski, 1991). The limited capacity of
carnivorous fish (<12%), shrimp (<20%), and in general all animals
(<20%) to digest starch is of great concern about feed high cost, animals
health status, and water pollution (Olmos et al., 2014). Aforementioned is
due to the animals limited capacity of -amylases (-1,4 and/or -1,6)
enzymes production. In this sense, alternatives for increasing digestibility
of starch feed formulations exist, by the inclusion of Bacillus strains isolated
from marine and terrestrial environments (Table 12.4). Bacillus strains pro-
duce large amounts of extracellular thermostable -glucosidase enzymes,
inducing growth and health on highly formulated starch diets (Arellano &
Olmos, 2002; Olmos et al., 2011).
5.2. Marine polysaccharides

Seaweeds are commonly classified into three main groups based on their pig-
mentation: (a) Chlorophyta, or green seaweeds; (b) Rhodophyta, or red
Table 12.4 Ability of each strain isolated to utilize vegetable flours (Ochoa-Solano &
Olmos-Soto, 2006)
Strain Algae meal Corn flour Wheat flour
33 ++ + ++
9b +++ ++ +++
34 +++ ++ ++
3 + +++ +
21b + +++ +
9a ++ + ++
46 +++ +++ ++
42 + + +
31 ++ ++ +
Area (cm ) of degradation: +++ (0.71), ++ (0.50.7), + (0.10.5), (0).
2
seaweeds; and (c) Phaeophyta, or brown seaweeds (OSullivan et al., 2010).

The principal cell-wall polysaccharides in green seaweeds are ulvans, those
in red seaweeds are carrageenans and agars, whereas those in brown seaweeds
are alginates and fucans as well as the storage polysaccharide laminarin ( Jiao,
Yu, Zhang, & Ewart, 2011; Rioux, Turgeon, & Beaulieu, 2007).
Seaweeds are traditionally used in human and animal nutrition. In some
green seaweed, such as the species belonging to the genus Ulva, the protein
content can represent between 10% and 26% (dry weight) of the plant
(Fleurence, 1999). For instance, the species Ulva pertusa, which is frequently
consumed by the Japanese people, has a high protein level between 20% and
26% (dry product) (Fujiwara-Arasaki, Mino, & Kuroda, 1984). The macro-
algae Ulva reticulata can be cultured successfully for the production of useful
algal biomass and be used for animal production feeds. But, specially, this
genus has innumerable applications in nutrients removal in contaminated
waters of shrimp, fish, and shellfish ponds effluents.
Carrageenan is a generic name for a family of linear, sulfated galactans
obtained from certain species of marine red algae. The backbone of
the polysaccharide is composed of D-galactose units, linked alternately to
-(1 ! 3) and -(1 ! 4) linkages (Norziah, Foo, & Abd, 2006). Carra-
geenans are polysaccharides used in the food processing industry for their
gelling, thickening, and stabilizing properties (DAyala, Malinconico, &
Laurienzo, 2008).
Agar is an unbranched polysaccharide obtained from the cell membranes
of some species of red algae, primarily from the genuses Gelidium and
Gracilaria, largely used as gelatin and thickener in food industry, and as a
gel for electrophoresis in microbiology. Chemically, it is constituted by
galactose sugar molecules; it is the primary structural support for the algaes
cell walls (DAyala et al., 2008).
Brown seaweeds are known to produce different polysaccharides, like
alginates, fucoidans, and laminarans. Alginic acid or alginate is the common
name given to a family of linear polysaccharides containing 1,4-linked -D-
mannuronic and -L-guluronic acid residues arranged in a nonregular, block
wise order along the chain (Andrade et al., 2004). Alginate produced by
brown seaweed, especially in the form of sodium and calcium alginate, is
widely used in the food and pharmaceutical industries due to their ability
to chelate metal ions and to form highly viscous solutions (Gupta &
Abu-Ghannam, 2011). Fucoidans, which are homopolysaccharides and
the class of heteropolysaccharides known as fucans, are metabolic products
of sulfated fucose found in brown seaweed. Fucoidan is a complex sulfated
polysaccharide, derived from Fucus vesiculosus, that mediates a variety of sig-

nificant biological effects (Patankar, Oehninger, Barnett, Williams, & Clark,
1993). In addition, their products are involved in the preparation of products
for food and cosmetic industries (Berteau & Mulloy, 2003). The major sugar
of Laminaria species is laminaran whose structure and composition vary
according to algae species. Laminaran was first discovered in Laminaria spe-
cies and appears to be the food reserve of all brown algae (Gupta & Abu-
Ghannam, 2011). Laminarans are the main water-soluble polysaccharides
of brown algae (Shanmugam & Mody, 2000). Most laminarans form com-
plex structures that are stabilized by interchain hydrogen bonds and are
therefore resistant to hydrolysis in the upper gastrointestinal tract (GIT)
and are considered as dietary fibers (Neyrinck, Mouson, & Delzenne,
2007), and as prebiotics they have also been reported to possess antibacterial
and antitumor activities (Nelson & Lewis, 1974).
6. ENZYMES AND DIGESTIBILITY

Despite their similar chemical composition, carbohydrates can arrange
an enormous number of combinations through the stereochemical variety of
the hydroxyl groups that they carry, the possibilities to assemble monosac-
charides one to another, and through the wealth of noncarbohydrate sub-
stituents that can decorate the resulting oligo- and polysaccharides.
Complex carbohydrates are widely distributed in nature, where they medi-
ate a multitude of biological functions, from carbon reserve to structural
molecules, or as the mediators of intra- and intercellular recognition within
one organism or between organisms. The diversity of complex carbohy-
drates is controlled by a panel of enzymes involved in their assembly
(glycosyltransferases) and their breakdown (glycoside hydrolases, polysac-
charide lyases, carbohydrate esterases), collectively designated as
Carbohydrate-Active enZymes (Cantarel et al., 2009). In this sense, due
to the extreme variety of monosaccharide structures, to the variety inter-
sugar linkages and to the fact that virtually all types of molecules can be gly-
cosylated (from sugars themselves, to proteins, lipids, nucleic acids,
antibiotics, etc.), the large variety of enzymes acting on these
glycoconjugates, oligo- and polysaccharides probably constitute one of
the most structurally diverse set of substrates on Earth (Lombard,
Ramulu, Drula, Coutinho, & Henrissat, 2014). The diversity of oligo-
and polysaccharides provides an abundance of biological roles for these car-
bohydrates. The enzymes hydrolyzing these compounds, the glycoside
hydrolases, therefore mediate a variety of biological functions. Glycoside

hydrolases fall into a number of sequence-based families. The recent analysis
of these families, coupled with the burgeoning number of 3D structures,
provides a detailed insight into the structure, function, and catalytic mech-
anism of these enzymes. The enzymatic breakdown of complex polysaccha-
rides requires complex, multienzyme systems with diverse activities and
substrate specificity (Table 12.5; Henrissat & Davies, 1997).
The enzyme degradation of insoluble polysaccharides is one of the most
important reactions on earth. Despite this, glycoside hydrolases attack such
polysaccharides inefficiently as their target glycosidic bonds are often inac-
cessible to the active site of the appropriate enzymes (Bayer, Belaich,
Shoham, & Lamed, 2004). In this sense, the starches of cereal grains are inef-
ficiently digested by the amylolytic enzymes of monogastric animals. Apart
from starch, cereal grains contain between 10% and 30% NSP (xylans,
-glucans, and cellulose). Corn and sorghum contain low levels of NSP,
in particular the soluble fraction, whereas wheat, rye, and triticale contain
substantial amounts of both soluble and insoluble NSP. The main soluble
NSP in these grains are arabinoxylans, whereas in barley and oats they are
-glucans (Choct, 1997). Feed enzymes such as carbohydrases will play a sig-
nificant role in diets to increasing the utilization of NSP as energy sources
and improving the use of poorly digested nutrients for better animal
Table 12.5 Different substrates with specific indicators used to assess enzyme
specificity (Ochoa-Solano & Olmos-Soto, 2006).
Amylopectin and Melibiose and Raffinose and
Strain Amylose and X--glu X--glu X--gal X--gal
33 + + + +
9b + +
34
3
21b
9a + +
46 + +
42 + +
31 + + + +
Category: + (positive), (not detected).
production. The enzymes cleave the large molecules of NSP into smaller
polymers, thereby reducing the thickness of the gut content and increasing
the nutritive value of the feed (Fig. 12.2; Bedford & Morgan, 1995). Some
enzymes, including xylanases, -glucanases, and cellulases, have been used to
enhance the nutritive value of cereal by-products for pigs and poultry
(Choct, Dersjant-Li, McLeish, & Peisker, 2010).
Figure 12.2 Schematic organization of a typical glycosyl hydrolase catalyzing endo-

cleavage of a polysaccharide shown within a generic cellulase system pathway
(Weiner et al., 2008).
Feed ingredients from plants sources like wheat, oats, barley, and ener-
getic moieties (starch, complex carbohydrates, and oligosaccharides) contain
some compounds that most farmed animals cannot digest or that hinder its
digestive system because they cannot produce a specific enzyme. For this
reason, supplementation with enzymes with the capacity to digest these
compounds can help to improve the utilization of dietary energy, resulting
in improved performance of livestock, fish, and shrimps (Ochoa, 2012;
Olmos et al., 2011). In addition, these enzymes can increase nutrient utili-
zation, reduce feed cost, and diminish excretion of undigested nutrients into
the environment. In humans and animals, digestion of feed is carried out by
the digestive system and by microorganisms that inhabit intestinal tract.
However, with the addition of high levels of complex carbohydrates in diets
(prebiotics), the inclusion of live microorganisms (probiotic) to produce spe-
cific enzymes to digest them, is recommended to formulate economical and
safety FFs (Olmos et al., 2014).
Concerning its carbohydrate composition, soybean meal contains
approximately 32%, from which 12% are soluble sugars, distributed as
follow; 45% sucrose, 12% raffinose, 3:54:5% stachyose, as well as small
amounts of melibiose and verbascose. 20% the remaining fraction which
is difficult to digest are known as anti-nutritional compounds, due to
the toxic effects they induce in monogastric animals. In addition, soy
protein concentrate is difficult to produce and expensive to shrimp feed
formulation. On the other hand, high levels of complex carbohydrates
also represent an important obstacle to shrimp aquaculture, due to
shrimps limited carbohydrates digestion capabilities (Arellano & Olmos,
2002; Le Chevalier & Van Wormhoudt, 1998; Samocha, Davis,
Saoud, & DeBault, 2004).
Some enzymes have been used in fish/shrimp feed formulations over
the past several years, which include cellulases (-glucanases), xylanases,
and associated enzymes like phytases, proteases, lipases, and galactosidases.
Enzymes in the feed industry have mostly been used in cultured animals, to
neutralize the effects of the viscosity of NSPs, produced by cereals and
other food grains (Dierick & Decuypere, 1994). The genus Bacillus secrets
a significant amount of enzymes, among which are carbohydrases, prote-
ases, and lipases, which can aid in the digestion of carbohydrates, proteins,
and lipids, respectively (Ochoa-Solano & Olmos-Soto, 2006). Maximizing
the breakdown and assimilation of starch and complex carbohydrates with
probiotic bacteria represents a safe solution of economic importance in ani-
mal feeding, since probiotics facilitate the assimilation of monosaccharides,
eliminate the toxic effects of antinutritional ingredients, and at the same
Alpha (a) glycosidase enzymes a-1,6 and a-1,4 Glycosidase enzymes
Starch substrate Amilopectin and amilose substrates
OH OH
CH2 CH2
O O
OH OH a-1,6-Glycosidase
O O
OH OH a-1,4-Glycosidase
O
OH OH OH
CH2 CH2 CH2 CH2
O O O O
OH OH OH OH
O O O O O
OH OH OH OH
Figure 12.3 Glucosidase enzyme activity with specific substrates.
time diminish the amount of fish meal and/or animal protein added to
commercial formulas (Ochoa-Solano & Olmos-Soto, 2006). Bacillus enzymes
are very efficient in breaking down a large variety of carbohydrates, lipids,
and proteins into smaller units. In previous studies, glucosidase enzyme
activity was tested using the chromogenic substrate X--Glu (5-
bromo-4-chloro-3-indolyl--D-glycopyranoside), from strains that grew on
glucose/starch medium (Fig. 12.3) (Arellano & Olmos, 2002). Mineral
medium agar plates with melibiose and raffinose 0.3% (w/v) (Sambrook,
Fritsch, & Maniatis, 1989) and the chromogenic substrate -X-Gal (5-
bromo-4-chloro-3-indolyl--D-galactoside) were used to identify and to
analyze -galactosidase enzymes activity on Bacillus (Fig. 12.4) (Ochoa-
Solano & Olmos-Soto, 2006).
7. PREBIOTIC INGREDIENTS
The marine environment is a great source of bioactive compounds.
Specifically, marine macroalgae (SW) are rich in polysaccharides and
Alpha galactosidase enzymes
Raffinose substrate
a-Galactosidase
OH
O CH2 CH2OH
OH O OH
O O
OH OH
CH2OH O CH2OH
OH
OH OH
Figure 12.4 -Galactosidase enzymes activity with specific substrates.
complex carbohydrates, which are not digested by intestinal enzymes. These

seaweeds carbohydrates are an important source of dietary fibers and can be
exploited as prebiotic functional ingredients for both human and animal
health applications. A prebiotic is a nondigestible food ingredient that ben-
eficially affects the host by selective stimulation of a limited number of bac-
teria in the colon, improving host health (Gibson, Probert, Van Loo,
Rastall, & Roberfroid, 2004; Gibson & Roberfroid, 1995). Specific dietary
carbohydrates also function as prebiotics to increase the numbers of benefi-
cial bacteria (probiotics) within the colon, in a selective manner. This results
in a wide range of physiological benefits for host, including reduced gut
infections and constipation, improved lipid metabolism, higher mineral
absorption, enhanced immunomodulation, and reduced risk of carcinogen-
esis (Roberfroid, 2000).
Many polysaccharides from several sources have displayed prebiotic
activity both in vitro and in vivo. The principal substrates for bacterial devel-
opment are dietary carbohydrates which have escaped digestion in the upper
GIT. These may be starches, dietary fibers, other nonabsorbable sugars,
sugar alcohols, and oligosaccharides. Prebiotic agents are food ingredients
that are potentially beneficial to the health of consumers. The main
commercial prebiotic agents consist of oligosaccharides and dietary fibers

(mainly inulin). They are essentially obtained by one of three processes: (1)
the direct extraction of natural polysaccharides from plants; (2) the controlled
hydrolysis of such natural polysaccharides; and (3) enzymatic synthesis, using
hydrolases and/or glycosyl transferases. However, to date, only three carbohy-
drates are accepted as true prebiotics: inulin:oligofructose, galacto-
oligosaccharides, and lactulose (OSullivan et al., 2010).
8. PROBIOTIC BACTERIA
Probiotic-supplemented functional food/feed could contain single or
mixed cultures of microorganisms capable of improving the health of the
host (Fuller, 1992). A probiotic is defined as a living microbial supplement
that (a) positively affects hosts by modifying the host-associated microbial
community and immune system, (b) secrete a variety of enzymes to improve
food/feed degradation enhancing its nutritional values, and (c) improves
quality of environmental parameters (Farzanfar, 2006; Gatesoupe, 1999;
Olmos et al., 2011; Verschuere, Rombaut, Sorgeloos, & Verstraete, 2000).
Probiotic bacteria are frequently used as feed additives to farm and pet
animals and recently have been utilized to improve nutrient assimilation
and to enhance growth performance and health of monogastric animals
(Cheeke, 1991; Fuller, 1992; Ochoa-Solano & Olmos-Soto, 2006). Probi-
otic bacteria feed inclusion has emerged as a new field with huge applications
in the aquaculture industry (Farzanfar, 2006; Gatesoupe, 1999). In shrimp
feeds, the probiotic bacteria can facilitate digestion of protein constituents
and carbohydrates, because the enzymes produced by the bacteria can com-
plement the shrimp enzyme activity, increasing food digestibility. Addition-
ally, pollution of shrimp effluent ponds also has been treated with
microorganisms (Olmos et al., 2011).
Actually, there has been an increased interest of using several species
of Bacillus as probiotics. The genus Bacillus is one of the most diverse and
abundant; species are found on the land and/or associated with water sources
such as rivers, coastal waters, and estuaries. Bacillus species presents a high
metabolic diversity, containing species that degrade cellulose, starch, pro-
tein, agar, complex CHO, and other substrates derived from plant and
animal sources (Ochoa-Solano & Olmos-Soto, 2006; Slepecky, 1992).
Furthermore, Bacillus species are the most investigated bacteria for animal
probiotic development due to its growth nutrients versatility, high enzyme
levels production, and secretion of antimicrobial peptides (Bechard,
Eastwell, Sholberg, Mazza, & Skura, 1998; Ochoa-Solano & Olmos-Soto,

2006; Olmos et al., 2011).
The genus Bacillus constitutes a diverse group of rod-shaped, Gram-
positive bacteria, characterized by their ability to produce a robust spore.
Most Bacillus species are not harmful to mammalians, including humans
and are commercially important as producers of a high and diverse amount
of secondary metabolites: antibiotics, heterologous proteins, fine chemicals,
and enzymes (Olmos-Soto & Contreras-Flores, 2003; Sonnenschein,
Losick, & Hoch, 1993). Bacillus enzymes are very efficient in breaking
down a large variety of proteins, carbohydrates, and lipids into smaller units.
Bacillus species grow efficiently with very low-cost carbon and nitrogen
sources (Sonnenschein et al., 1993). Taking into account, the advantageous
characteristics of Bacillus strains, these bacteria are good candidates for con-
sideration as probiotics in crustaceans and fishes diets (Ochoa, 2012; Olmos
et al., 2011).
Bacillus subtilis is one of the best candidates for expression of heterologous
proteins, because it is capable of producing and secreting functional proteins
directly to the culture medium, is nonpathogenic, and has no significant bias
in codon usage. Likewise, there is a great deal of vital information con-
cerning its mechanisms of transcription and translation, genetic manipula-
tion, and large-scale fermentation (Wong, 1995); and its genome had
been completely sequenced (Kunst et al., 1997). B. subtilis secretes a variety
of enzymes responsible for breaking down complex carbohydrates, some of
which are of economic importance (Debabov, 1982; Mountain, 1989;
Sonnenschein et al., 1993). In addition, B. subtilis is general recognized as
safe by the food and drug administration, meaning it is harmless to animal
and humans (Olmos-Soto & Contreras-Flores, 2003).
9. FUNCTIONAL FEEDS
The main role of diets is to provide enough nutrients to meet the
requirements of the host, while giving to the consumer a feeling of satisfac-
tion and well-being. The most recent knowledge in bioscience supports the
hypothesis that nutrients also control and modulate some functions in the
body, and, in doing so, contribute to a health status necessary to reduce
the risk of some diseases. This hypothesis gave origin to both concepts
functional food and the development of a new scientific discipline of
functional food science (Roberfroid, 1998).
FF development represents one of the greatest areas of opportunity in the

nutritional world. FFs have the characteristic of generating benefits beyond
those of conventional feeds. FFs must be harmless to animals and human
beings. FF must promote growth and health, improve immune system,
and induce physiological benefits beyond traditional feeds (IFIC, 2004;
Olmos et al., 2014). In FFs, the probiotic bacteria can facilitate digestion
of protein constituents and complex carbohydrates, because the enzymes
produced by the bacteria can complement the animals enzyme activity,
increasing feed digestibility, and assimilation. In addition, the probiotic
enzymes have a broader pH, temperature, and target range extending diges-
tion time (Ochoa-Solano & Olmos-Soto, 2006). In livestock and aquacul-
ture industry, this functionality must be reflected in an improvement in the
percentage of survival, greater growth, and weight gain among the animals
which consume it (Ochoa, 2012). This means that carbohydrates, proteins,
and lipids are well assimilated and directed to their corresponding primary
functions. In addition, it must be economically attractive and friendly with
the environment (Olmos et al., 2014). FF development represents one of the
greatest areas of opportunity in the nutritional world. FF must promote
growth and health of the cultivated organisms (Kureshy & Davis, 2002),
improve immune system, and induce physiological benefits beyond tradi-
tional feeds (Hong, Duc le, & Cutting, 2005).
9.1. Definition
Today, the focus of nutritional science is shifting toward the concept of
optimal nutrition, the objective of which is to optimize the daily diet in
terms of nutrients and nonnutrients, as well as other food properties that
favor the maintenance of health status (Ashwell, 2003). A large number
of definitions exist worldwide for FFs. Some definitions maintain that
only fortified, enriched, or enhanced foods with a component having a
health benefit beyond basic nutrition can be considered (Center for
Science in the Public Interest, 1999). Some definitions indicate that if a
health claim can be made, a food is functional (Iowa State University,
2000; Westrate, Van Poppel, & Verschuren, 2002). While some definitions
are quite simple: feeds with dietary ingredients that provide health and eco-
nomic benefits beyond basic nutrition (Labrecque, Doyon, Bellavance, &
Kolodinsky, 2006). In this sense, complex carbohydrates and probiotic bac-
teria supplemented in diets could transform livestock and aquaculture in a
sustainable, competitive, and profitable industry through FFs utilization
(Fig. 12.5; Olmos et al., 2014).
Figure 12.5 Functional feed particle formulated with SBM, CHOs, vegetable oil, and
Bacillus subtilis multifunctional probiotic strain (Olmos et al., 2011).
9.2. Study cases, functional benefits, and usage suggestions

The seaweeds have the potential to be used as a functional food ingredient.
The human consumption of algal fiber has been proven to be health-
promoting and its benefits are well documented in the scientific literature.
The consumption of this dietary fiber has been related to the following
health-promoting effects: (1) promotes the growth and protection of the
beneficial intestinal flora, (2) reduces the overall glycemic response, (3)
greatly increases stool volume, and (4) reduces the risk of colon cancer.
In addition to some of the components which have potential benefits for
the human body, the presence of dietary fibers provides some technological
advantages for the use of marine algae as ingredients in food products such as
meat products. The presence of these prebiotics can also be used to support
the growth of lactic acid bacteria, using seaweed broth as a sole carbon
source of nutrition (Gupta & Abu-Ghannam, 2011), with the subsequent
addition of the probiotics that can benefit human health.
9.2.1 Research in pigs

The prebiotic effects of feeding pigs with laminaran, fucoidan, and selected
microflora on intestinal fermentation resulted with a reduction of intestinal
Enterobacteria and an increased Lactobacilli sp., suggesting that feeding of sea-
weeds can act as a dietary means to improve gut health in pigs (Lynch,
Sweeney, Callan, OSullivan, & ODoherty, 2010). Reilly et al. (2008) also
demonstrated the positive effects of dietary supplementation with extracts
containing laminarin and fucoidan from different varieties of brown sea-

weeds on gut morphology and intestinal microbial populations in pigs.
The inclusion resulted in an inhibitory effect on the Enterobacteria population
within the caecum and colon of weaned pigs. ODoherty, Dillon, Figat,
Callan, and Sweeney (2010) showed that feeding laminarin resulted in
the reduction in fecal Escherichia coli population and an increase in daily gain
and gain to feed ratio to improve gut health in postweaning pigs. However, a
combination of laminarin and fucoidan was reported to be more effective at
reducing diarrhea postweaning. Dillon, Sweeney, Figat, Callan, and
ODoherty (2010) have also reported that the inclusion of a combination
of laminarin and fucoidan, increased daily gain and gain to feed ratio of pos-
tweaned piglets. According to the authors, this was mainly due to an increase
in nutrient digestibility and decreased E. coli populations in the guts. Most
carbohydrates in pig diets are enzymatically hydrolyzed to absorbable mono-
mers as glucose (from starch). However, some carbohydrates escape enzy-
matic digestion in the small intestine and become a substrate for the
gastrointestinal microflora of the large intestine. These include complex car-
bohydrates like nondigestible oligosaccharides, which are found in various
feedstuffs. -Galactooligosaccharides can be found in soybeans, lupines, and
peas (Saini, 1989) and are -linked galactose units to the glucose moiety of
sucrose. Fructooligosaccharides can be found in most cereals, including bar-
ley, wheat, and rye (Henry & Saini, 1989), and are -linked fructose units to
the fructose moiety of sucrose. -Galactooligosaccharides have long been
known as antinutritional factors; they may reduce dietary metabolizable
energy (Leske, Jevne, & Coon, 1993) and can induce diarrhea, extensive
flatulence, and discomfort in nonruminants (Saini, 1989). However, probi-
otic bacteria and specific enzymes included in the diet can help to a better
CHOs feed digestionassimilation for the production of energy and growth
improvement (Ochoa, 2012).
9.2.2 Research in poultry

An alternative approach to subtherapeutic antibiotics in livestock is the use
of probiotic microorganisms, prebiotic substrates that enrich certain bacte-
rial populations, or synbiotic combinations of prebiotics and probiotics.
Research is focused on identifying beneficial bacterial strains and substrates
along with the conditions under which they are effective (Patterson &
Burkholder, 2003). In poultry, Carre, Gomez, and Chagneau (1995)
reported very low digestibility of NSP in both broiler chickens and
adult cockerels, when both SBM (325 g/kg) and peas (475 g/kg) were
included in diets. These authors reported total tract digestibility of NSP of

around 6% or less (range 2.86.1%). Total tract digestibility of the
-galactooligosaccharides was much higher, with a mean value of 0.867
recorded for broilers and 0.99 for adult birds. These data suggest that there
is considerable microbial degradation of these compounds in the digestive
tract of birds, although no measurements were made of the extent of hydro-
lysis of -galactooligosaccharides prior to their entry into the ceca. This is
likely to be important given the possibility of digestive upsets caused by
the passage of -galactooligosaccharides into the ceca, as well as the ener-
getic implications of digestion in the small intestine and absorption as glu-
cose versus production of volatile fatty acids and their subsequent absorption
(Muller, Kirchgessner, & Roth, 1989). Here, in addition to reducing excreta
organic matter and moisture content, the use of an exogenous
-galactosidase may be appropriate. The use of enzymes in poultry diets
is now almost universal. The reasons why they are used are manifold and
include: (1) to increase the feeding value of raw materials. (2) To reduce
the variation in nutrient quality of ingredients. (3) To reduce the incidence
of wet litter. Evidently, these benefits are realized by the poultry industry and
the consumer (Bedford, 2000).
9.2.3 Research in fish

SW are rich in bioactive compounds that could potentially be exploited as
functional ingredients for both human and animal health applications.
Despite the intensive efforts that are being made to isolate and identify
new compounds with potential medicinal, health, or pharmaceutical
activities, very few compounds with real potency are available. Bioactive
compounds that are most extensively researched include sulfated polysac-
charides, phlorotannins, and diterpenes. These compounds have been
reported to possess strong antiviral, antitumor, and anticancer properties.
Several studies have proved that addition of small amount of SW in
aquafeeds resulted in considerable positive effect on growth performance
and feed utilization efficiency, carcass quality, physiological activity, intes-
tinal microbiota, disease resistance, and stress response (Valente et al.,
2006). Nonetheless, it has also been noted in other publications that high
SW inclusion reduces fish growth and feed efficiency. Moreover, certain
substances with antinutritive activity may be present in SW, like lectins, tan-
nins, phytic acid, and protease and amylase inhibitors (Oliveira et al., 2009).
Such antinutritional factors might interfere with bioavailability and/or
digestibility of nutrients special emphasis should be focused on protease
inhibitors. Binding of protease inhibitors to proteolytic enzymes causes the

pancreas to secrete larger amounts of digestive enzymes to overcome the
negative effects of inhibitors on the digestion of dietary protein. From the
literature available, it can be deducted that the response of animals to SW
seems to be dose dependent and species specific. Furthermore, discussions
of data from both in vitro and in vivo studies that have examined the prebiotic
potential of seaweed polysaccharides are essential.
On the other hand, starch is limited to 10% in carnivorous fishes due seri-
ous health and growth problems occurs when elevated amounts of this kind
of complex carbohydrates are included. Additionally, SBM inclusion is also
restricted to 20% to avoid health and growth problems, due to the presence
of complex carbohydrates and antinutritional factors in this ingredient. Lim-
itation in starch and SBM feed utilization in fish farms increases feed prices,
the pollution in pounds, and diseases proliferation. However, with the addi-
tion of specifically selected B. subtilis probiotic strain, both starch- and
SBM-induced problems disappeared and more surprisingly, the animals
tested gained weight and growth, better than the control animals without
the probiotic bacteria (Ochoa, 2012; Olmos et al., 2014).
9.2.4 Research in shrimps

Cruz-Suarez, Ricque, Pinal-Mansilla, and Wesche-Ebelling (1994) and
Shiau (1998) summarize the studies on carbohydrate utilization by crusta-
ceans and Penaeid shrimps, respectively. In general, complex carbohydrates
like starch or glycogen have a higher nutritional value for Penaeid shrimps,
than simple sugars. Shiau and Peng (1992) investigated the utilization of dif-
ferent carbohydrate sources and the possible sparing effect of dietary protein
by carbohydrate in Penaeus monodon reared in seawater. Results indicated
that shrimp fed starch had significantly higher weight gain, feed efficiency
ratio, protein efficiency ratio, and higher survival than those fed with glu-
cose. For this reason, many researchers have suggested the use of complex
carbohydrates such as starch, which undergoes enzymatic hydrolysis before
assimilation, in shrimp diets. In this case, glucose from starch appears at gut
absorption sites at a rate slower than free glucose (Alava & Pascual, 1987;
Shiau, 1998; Shiau & Peng, 1992).
The concept that carbohydrate can substitute proteins as energy source in
shrimp feed, is already documented (Cruz-Suarez et al., 1994). However,
many authors have demonstrated that marine shrimp have severe restrictions
for the utilization of dietary CHO (Le Chevalier, Sellos, & Van
Wormhoudt, 2000; Shiau & Peng, 1992). Furthermore, the inability of
the shrimps -1,4-glucosidase to cleave the -1,6-bonds of amylopectin

inhibit assimilation of starch, resulting in low growth rates (Arellano &
Olmos, 2002; Le Chevalier & Van Wormhoudt, 1998). In this sense, low
levels of shrimps carbohydrolases is a conditional deficiency from using
high amounts of complex carbohydrates like starch and SBM (Arellano &
Olmos, 2002; Rosas et al., 2000). However, inclusion of selected pro-
biotics strains producing -1,4 and -1,6-glucosidases to be used in feeds,
besides with the objective to produce -galactosidases to degrade anti-
nutritional carbohydrates contained in SBMs, was successfully tested
(Olmos et al., 2011). With the inclusion of these bacteria in shrimp diets,
all the energy stored as carbohydrates in starch and SBM was assimilated
and incorporated to the animal bodies and activities. Furthermore, carbohy-
drate utilization diminishes food costs by a better protein-sparing effect and
consequently leads to a decrease in the amount of nitrogen waste and less
pollution (Olmos et al., 2011). In this sense, FFs must be economically
attractive to the aquaculture industry and friendly with the environment.
In this respect, animal products feed inclusion must be totally or partially
eliminated from feed formulations and, alternative-economical vegetable
protein and carbohydrates sources inclusion needs to be increased
(Fig. 12.5). Furthermore, type and amounts of carbohydrate feed inclusion
its a great concern for economic, environmental, and health issues too
(Olmos et al., 2014).
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dietary ingredients in European sea bass (Dicentrarchus labrax) juveniles. Aquaculture,
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FURTHER READING
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Cambridge University Press.
INDEX
Note: Page numbers followed by f indicate figures and t indicate tables.
A molecular weight and solubility, 47

Activated partial thromboplastic time structure of, 4647, 150, 151f
(APTT), 203204 Antigen-presenting cells (APCs), 8889
Agars, 269
food products, 149150 B
market value, 201, 201t Bacillus subtilis, 277
structure of, 116f, 149, 150f Barium alginate (BA), 125126
viscosity control and stabilizing agent, 158 Bioemulsifiers, 223f, 226, 247t
wastewater treatment biological activities, 239t
gelling properties, 117 definition, 225
marine polysaccharides, 126127 environmental remediation and industrial
Alginates, 269270 application, 241t
annual production, 46 in food, 237239
anticoagulant activity, 204 structures of, 236f
biocompatibility, 48 Biofilms, 233234
biomedical applications, 165 Biomimetic method, 65
bone tissue engineering Biopolymers
chitosan/alginate hybrid chitin, 6061
scaffolds, 4950 chitosan, 61
CPC, 51 Bioremediation, 249
future aspects, 5354 hydrocarbon removal, 239240, 240f
herbal scaffold, 51 industrial application, 241t
hydrogels, 5253 oil spills, 240
injectable and biodegradable scaffold, Biosurfactants, 223f, 242248
51 biological activities, 239t
physical and biological properties, bioremediation
4849 hydrocarbon removal, 239240, 240f
platelet-rich plasma (PRP), 50 industrial application, 241t
polyelectrolyte complexation (PEC), oil spills, 240
5051 definition and characteristics, 224226
sodium alginate/gelatine scaffold, in food, 237239
5152 properties and functionality, 223224
stem cell-scaffold approaches, 50 structures of, 236f
therapeutic strategies, 4849 Bone tissue (BT) engineering
brown seaweed, 46 alginate
in cosmeceutical industry, 213 chitosan/alginate hybrid scaffolds,
food industry, 150 4950
marine polysaccharides CPC, 51
for biomedical applications, 34 future aspects, 5354
functions, 34 herbal scaffold, 51
sodium alginate, 34 hydrogels, 5253
market value, 200201 injectable and biodegradable scaffold, 51
289
290 Index
Bone tissue (BT) engineering (Continued ) in rat tibias, 66

physical and biological properties, rhBMP-2 gene, 70
4849 single-walled carbon nanotube, 69
platelet-rich plasma (PRP), 50 water-soluble methacrylamide
polyelectrolyte complexation (PEC), chitosan, 64
5051 ZnO-containing nHA/CS
sodium alginate/gelatine scaffold, 5152 cement, 73
stem cell-scaffold approaches, 50
therapeutic strategies, 4849 C
chitin and chitosan Carbohydrates (CHOs). See also Marine
-chitin/nBGC composite scaffolds, 67 carbohydrates
biocompatible chitosan/ in animals functionality, 260
hydroxyapatite composites, 6667 dietary carbohydrates, 262t, 264t
biomimetic and thermosensitive gel enzymes and digestibility, 270274, 271t,
scaffold, 6869 272f
biomimetic method, 65 oligosaccharides (see Oligosaccharides)
biopolymers, 6061 polysaccharides (see Polysaccharides)
bone morphogenetic proteins, 65 prebiotic ingredients, 274276
carbonate apatitechitosan scaffolds, 67 probiotic bacteria, 276277
and CEL, 66 sugars, 261
C/GP/nHAC composite scaffolds, 71 Carotenoids, 214215
ChiHApMSCol scaffolds, 6970 Carrageenans, 269
chitin fabric, 62 and agar, 126127
chitosanpolyacrylamide anticoagulant activity, 204
nanohydroxyapatite, 68 in cosmetic industry, 212213
chondrocytes, 62 dairy products, 151
extracellular matrix, 6162 drug release modifier, 165
future aspects, 76 forms of, 151, 152f
GBR, 75 in pharmaceutical industry, 151
genipin-cross-linked Col/chitosan structure of, 212, 212f
matrix, 63 in wastewater treatment
graphene oxide (GO), 75 -carrageenan, 117
human bone marrow stromal cell, 66 marine polysaccharides, 126127
hybrid microspheres, 6162 structure of, 117f
hydrogels, 6162 Cell penetration peptide (CPP), 96
hydroxyapatite composites, 66, 67, Chitin
7475 -chitin, 56, 153
icariin-loaded chitosan/nanosized antioxidant activity
hydroxyapatite, 74 chitin oligomers, 1718
konjac glucomannan composite, 7273 crab chitin, 19
methacrylated glycol chitosan, 62 novel therapeutic applications, 16
nanohydroxyapatite/chitosanpectin, and oxidative stress, 17
69 -chitin, 56, 153
nanohydroxyapatite/polyamide66 COS, 56
(nHA/PA66) scaffolds, 73 -chitin, 153
nanostructured hydroxyapatite, 70 matrix metalloproteinase, 21
poly(L-lactic acid), 6465 pharmacological properties, 89
pure -chitin sponge scaffold, 6364 quaternary amino groups, 2021
Index 291
Chito-oligomers, antioxidant activity brainblood barrier penetrating

alloxan-induced diabetes, 25 modification, 9596
aminoethyl-COS, 2324 cationic polymers, 8485
carboxylated COS, 23 cell penetration peptide (CPP), 96
ESR studies, 2223 folate ligand modification, 8688
GA-grafted COS, 23 galactose ligand modification, 8586
shrimp SOD activity, 2425 hyaluronic acid ligand
sulfated COS (SCOS), 26 modification, 90
Chitooligosaccharides (COS), 10, 214 magnetic modification, 9495
antidiabetic activities, 39 mannose ligand modification,
medicinal applications, 214 8889
pharmaceutical applications, 190t, 214 nonviral vector, 8485
shellfish wastes, 187s PEI and dendrimers, 8485
Chitosan penetrate nuclear membrane, 9697
advantage of, 154156 pH-sensitive modification, 9092
in agriculture, 160 thiolated modification, 9294
antibacterial activity, 154156 viral vector, 84
antidiabetic activities marine carbohydrate, 187
anticoagulant potency, 40 in pharmaceutics
chitosan oligosaccharides, 34 cancer treatment and diagnosis, 163
COS, 39 chewing gum, 164
derivatization, 3536 contact lenses, 163
diabetic wounds, 37 drug carriers, 161162
drug delivery activity, 3637 nerve regeneration, 164165
fat-lowering mechanisms, 3839 orthopedics, calciumchitosan
gene therapy, 37 composites, 164
genetic and environmental factors, skeleton formation, silicachitin
3435 scaffolds, 164
glucosamine, 39 wound healing, 162
mortality and morbidity, 41 postharvest fruits and vegetables, 159, 160
pancreatic beta-cells, 3940 in wastewater treatment
renal failure, 40 adsorption capacity, 168, 170
antimicrobial coating, 158159 amine groups, 120
antioxidant activity amino groups, 113
aminoderivatized chitosan, 20 biosorbent, 120
crab chitosan, 19 chemical structure, 113f
gallic acid-grafted-chitosans, 20 chitosancharcoal composite, 169
HMCS treatment, 2122 crystallinity, 115f
low MW chitosan (LMWC), 25 dilute acidic solutions, 113
matrix metalloproteinase, 21 fouling, 122
nontoxic biopolymer, 16 glutaraldehyde, 169170
novel therapeutic applications, 16 graft copolymer, 120
and oxidative stress, 17 heteropolymer, 112113
quaternary amino groups, 2021 PET granules, 168169
in vitro and in vivo studies, 18 removal of dyes, 122123
decolorization, 154 water purification plants, 154
gene therapy water-soluble chitosan, 160
amino acid modification, 94 Clandosan, 188
292 Index
Complex carbohydrates, 261263 halophilic EPSs, 237238

functional feeds marine microalgae producers, 234, 235t
definition, 278 probiotics EPSs, 238239
fish research, 281282 quorum sensing, control of, 238239
pig research, 279280 seaweed polysaccharides, 274276
poultry research, 280281
probiotic bacteria, 278
F
Ferric reducing/antioxidant power (FRAP)
shrimp research, 282283
assay, 205207
polysaccharides, 266270, 274275
FFs. See Functional feeds (FFs)
COS. See Chitooligosaccharides (COS)
Folate receptor (FR), 8688
Cosmeceuticals, 157
Fucoidans, 200
alginates, 213
chemical structure, 209, 211f
carrageenan, 212213
food and cosmetic application, 209212
fucoidan, 209212
marine polysaccharides
CPP. See Cell penetration peptide (CPP)
low-molecular-weight fractions, 45
Cyanobacteria
pharmacological properties, 78
exopolysaccharides, 228230, 231t
seaweed fucoidans, 45
soil fertility, 166167
sulfated L-fucose, 45
type I/type II chains, 45
D
Fucus serratus, 146147
2,2-Diphenyl-1-picrylhydrazyl (DPPH),
Functional feeds (FFs)
205207
definition, 278
functional benefits, and uses
E
fish research, 281282
Exopolysaccharides (EPSs)
pig research, 279280
advantages of, 243244
poultry research, 280281
Arctic marine environment, 244
probiotic bacteria, 278
as bioemulsifiers (see Bioemulsifiers)
shrimp research, 282283
biofilms, 233234
as biosurfactants (see Biosurfactants) G
biosynthesis Gene therapy, chitosan
glycosyltransferases, 228 amino acid modification, 94
heteropolysaccharides, 226228 antidiabetic activities, 37
homopolysaccharides, 226228 brainblood barrier penetrating
properties and applications, 226 modification, 9596
carbon metabolism, 238239 cationic polymers, 8485
cryoprotective role, 244 cell penetration peptide (CPP), 96
cyanobacteria, 228230, 231t folate ligand modification, 8688
deep-sea environment, 244246 galactose ligand modification, 8586
dextran, use of, 237 hyaluronic acid ligand modification, 90
diatoms magnetic modification, 9495
glucan utilization, 228229 mannose ligand modification, 8889
photosynthetic production, 230232 nonviral vector, 8485
eukaryotic microalgae and PEI and dendrimers, 8485
microorganisms, 226, 227t penetrate nuclear membrane, 9697
fermentation processes, 237238 pH-sensitive modification, 9092
food and dietary fibers, source for, thiolated modification, 9294
247248, 247t viral vector, 84
Index 293
Grafted chitosan beads (GCB), 170 thickeners, stabilizers, and emulsifiers,

Gymnodinium sp. A3 phosphate (GA3P), 215
204205, 206t market value, 199201, 200f, 201t, 202t
nutraceuticals implications, 188189
H in pharmaceutics (see Pharmaceuticals)
Heparin production of, 148149
blood coagulation, 203204 resource of, 198199
global market for, 200201 sources
Heteropolysaccharides, 226228, 233234, biomass, 186
242243 clandosan, 188
Holothurin, 161 COS, 187188
Homopolysaccharides, 226228 macroalgal polysaccharides, 185186
Human bone marrow stromal cell (BMSC), shellfish, 186187
66 Spirulina, 158
Human embryonic stem cells (hESCs), 53 structures and terminology, 147148
Hyaluronic acid ligand modification, 90 of wastewater treatment (see Wastewater
treatment)
I Marine polysaccharides, 268270
Iridae laminarioides, 203 alginate
for biomedical applications, 34
K functions, 34
Keissleriella sp., 205207 sodium alginate, 34
alginic acid, 67
L chitin and derivatives
Lonicera japonica, 205207 -chitin, 56
-chitin, 56
M COS, 56
Magnetic resonance imaging (MRI), 95 pharmacological properties, 89
Marine carbohydrates chitooligosaccharides, 10
agar (see Agars) chitosan nanoparticles, 910
algal biomass, 157158 fucoidans
alginates (see Alginates) low-molecular-weight
in biotechnology and microbiology, fractions, 45
166167 pharmacological properties, 78
carrageenans (see Carrageenans) seaweed fucoidans, 45
chitin, 153156, 155f sulfated L-fucose, 45
chitosan, 153156, 155f, 187 type I/type II chains, 45
components, 146 porphyran
in cosmetics (see Cosmeceuticals) characteristics, 4
edible seaweeds, 157 pharmacological properties, 7
harvesting methods, 202t wastewater treatment
macro- and microalgae generation status, advantages, 127128
202t agar, 126127
marine food and traditional application alginate, 123126
carotenoids, 214215 carrageenan, 126127
chitooligosaccharide derivatives, 214 chitin, 118119
sulfated polysaccharides (see Sulfated chitosan, 120123
polysaccharides (SPs)) limitations, 128
294 Index
Marine sponge (Ircinia fusca) Col (MSCol), marine polysaccharides

6970 characteristics, 4
Methacrylated glycol chitosan (MeGC), 62 pharmacological properties, 7
Porphyra yezoensis, 204205, 208
N
National Parks and Wildlife Services S
(NPWS), 202 Seaweed aquaculture, 202
Nonstarch polysaccharides (NSPs) Single-walled carbon nanotube (SWCNT),
-galactosidase enzyme, 266, 266t 69
raffinose families, 266 Soybean meal (SBM), 273
Special areas of conservation (SAC), 201202
O Spirulina, 158
Oligosaccharides, 261, 263266 Sulfated polysaccharides (SPs), 214
cellcell recognition, 263 anticancer activity, 204205
feed products, 263 anti-HIV activity, 207
soybean meal, 273 antilipidemic activity, 208
antioxidant activity, 205207
P blood coagulation, 203204
Pharmaceuticals immunomodulating effects, 208209
alginate, 165 ionic regulation, 199
and biological application, 210t molecular weight, 199
anticancer activity, 204205, 206t Superparamagnetic iron oxide nanoparticles
antilipidemic activity, 208 (SPIONs), 9495
antioxidant activity, 205207
antiviral activity, 207 T
blood coagulation system, 203204 Terrestrial polysaccharides, 267268, 268t
immunomodulating effect, 208209
chitosan (see Chitosan) U
CSCG nanoparticles, 165 Ulva pertusa, 269
holothurin, 161
implications, 189191 W
pH-sensitive chitosan Wastewater treatment
imidazole modification, 9192 activated carbon, 110
PEI modification, 92 adsorption, 109110
Platelet-rich plasma (PRP), 50, 52 agar
Polyelectrolyte complexation (PEC), 5051 gelling properties, 117
Polyethylene glycol (PEG), 162163 marine polysaccharides, 126127
Polyglycol alginate (PGA), 150 structure, 116f
Polysaccharides, 261. See also alginate
Exopolysaccharides (EPSs); Marine barium alginate (BA) entrapment,
polysaccharides 125126
marine animals, 246247 biopolymer film/coating component,
nutritional values and functional 116
properties, 266267 brown algae, 114115
oligomers, 265f calcium alginate, 125
terrestrial polysaccharides, 267268, 268t chemical structure, 115f
Porphyran immobilization, 124
antilipedemic feature, 208 methylene blue (MB), 123
Index 295
oil and derivative, 125 crystallinity, 115f

Pb(II) ions, 125 derivatives, 121t
removal of cationic dyes, 123 dilute acidic solutions, 113
sodium alginate, 124 fouling, 122
biosorbents, 110111 graft copolymer, 120
carrageenan heteropolymer, 112113
-carrageenan, 117 removal of dyes, 122123
marine polysaccharides, 126127 chlorination, 108
structure, 117f coagulation and flocculation, 108109,
chitin 168
-chitin, 111112 composition, 105107
amino and hydroxyl groups, 112 dyes, 168
-chitin, 111112 methods and limitations,
chemical structure, 112f 109, 109t
marine polysaccharides, 118119 polysaccharides, 111
chitosan primary treatment, 107108
amine groups, 120 secondary treatment, 107108
amino groups, 113 sources, 105
biosorbent, 120 tertiary treatment, 107108
chemical structure, 113f toxic metals, removal of, 167

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ADVISORY BOARDS

MARY ELLEN CAMIRE

First edition 2014

Copyright 2014, Elsevier Inc. All Rights Reserved

For information on all Academic Press publications

Department of Chemistry, Pukyong National University, Busan, South Korea

organisms have received much attention in screening marine natural prod-

(MG-blocks), which represent the primary structure of alginate, depend on

2.4. Chitin and its derivatives

alternating parallel and antiparallel chains (Minke & Blackwell, 1978).

3. PHARMACOLOGICAL PROPERTIES OF MARINE

PMA plus A23187-stimulated HMC-1 cells ( Jeong et al., 2006). Notice-

IgE-expressing and IgE-secreting B cells from OVA-sensitized mice. On the

of Th2 responses by chitin was found due to decreases of Th2 cytokines

3.5. Chitosan nanoparticles

Antioxidant Effects of Chitin,

Department of Chemistry, Pukyong National University, Busan, South Korea

2. ANTIOXIDANTS AND OXIDATIVE STRESS

3. ANTIOXIDANT ACTIVITY OF CHITIN, CHITOSAN,

50% NaOH solution in a microwave for 10 min at 1400 W power.

OH and O2 radicals using electron spin resonance (ESR) spectrometer.

of OHT-chitin and NHT-chitosan was 30.9% and 31.9% at 5 mg/mL,

radicals in a dose- and time-dependent manner. The strong binding capacity

3.2. Antioxidant activity of chito-oligomers and its derivatives

hetero-COSs increased with a dose-dependent manner, and it depends on

effect of AE-COS was determined by 20 ,70 -dichlorofluorescein intensity and

Antidiabetic Activities of Chitosan

carbohydrate prepared by N-deacetylation of chitin in the presence of alka-

Beside oligomerization, other main derivatization for chitin and its

3. ANTIDIABETICS AND ANTIOBESITY APPLICATIONS

3.1. Indirect activity

To conclude, chitosan-based polymers show great potential for treat-

3.2. Direct activity

body weight and triglyceride levels (Hernandez-Gonzalez, Gonzalez-Ortiz,

complication rate of diabetes onset in animal models, given that chitosan-

mushrooms changes adipocytokine profile in diet-induced obese mice, a phenomenon

Role of Alginate in Bone Tissue

2. ALGINATE GENERAL PROPERTIES

G-C4 G-C4 M-C1 M-C1 G

Hydrogels are three dimensionally cross-linked networks composed of

2.2. Molecular weight and solubility

hydroxyapatite/chitosanalginate composite scaffolds were prepared

This ability to retain/regulate rhBMP-2 delivery and stimulate new bone

construct is a promising alternative approach for bone regeneration (Xia

4.2. Alginate hydrogels in bone TE

(e.g., degradation, bioactivities, or mechanical properties). In further studies,

Chitin and Chitosan Composites

2. NATURALLY OCCURRING BIOPOLYMERS

antibacterial properties, fungistatic properties, hydrophilicity, gel-forming

3. TISSUE ENGINEERING APPLICATIONS OF CHITIN

Chitosan is a promising polymer for tissue engineering for its nontoxicity,

Genipin-cross-linked Col/chitosan biodegradable porous scaffolds were

and chitosan as a scaffold for rabbit chondrocyte culture was studied by

4. APPLICATIONS OF CHITIN AND CHITOSAN FOR BONE

PLLAchitosan hybrid scaffolds could be suitable to release bioactive com-

In a laboratory experimental study, microdrilled bone defects were cre-

of the HA nanoparticles in the chitosan matrix and showed that the

phase transition process, and rheological property made the CS/HA/Col

microscopy. Cell proliferation in composite scaffolds was relatively higher

A novel biodegradable composite scaffold was made using

CSKGM polymer, showed potential for controlled drug delivery vehicle

Chemical Modification of Chitosan

School of Pharmacy, China Pharmaceutical University, Nanjing, PR China