Biological activities of chitosan and chitooligosaccharides

Food Hydrocolloids 25 (2011) 170e179

Contents lists available at ScienceDirect

Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd

Biological activities of chitosan and chitooligosaccharides Wenshui Xia a, b, *, Ping Liu c, Jiali Zhang a, d, Jie Chen a a

State key laboratory of food science and technology, Jiangnan University, Lihu Road 1800, Wuxi 214122, Jiangsu, PR China School of food science and technology, Jiangnan University, Lihu Road 1800,Wuxi 214122, Jiangsu, PR China c Jiangsu Animal Husbandry and Veterinary College, Fenghuang East-Road 8th, Taizhou 225300, Jiangsu, PR China d School of medicine and pharmaceutics, Jiangnan University, Lihu Road 1800, Wuxi 214122, Jiangsu, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 March 2009 Accepted 4 March 2010

Chitosan and its oligosaccharides, which are known to possess multiple functional properties, have attracted considerable interest due to their biological activities and potential applications in the food, pharmaceutical, agricultural and environmental industries. Many researchers have focused on chitosan as a potential source of bioactive materials in the past few decades. This review focuses on the biological activities of chitosan and chitooligosaccharides based on our and others’ latest research results, including hypocholesterolemic, antimicrobial, immunostimulating, antitumor and anticancer effects, accelerating calcium and iron absorption, anti-inflammatory, antioxidant and Angiotensin-I-converting enzyme (ACE) inhibitory activities and so on, which are all correlated with their structures and physicochemical properties. The bioactivities summarized here may provide novel insights into the functions of chitosan, its derivatives or oligosaccharides and potentially enable their use as functional-food components and additives. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Chitosan Chitooligosaccharides Biological activities Hypocholesterolemic Antimicrobial Antitumor

1. Introduction Chitosan is a natural nontoxic biopolymer produced by the deacetylation of chitin, a major component of the shells of crustaceans such as crab, shrimp, and crawfish. Currently, chitosan has received considerable attention for its commercial applications in the biomedical, food, and chemical industries (Knorr, 1984; Kurita, 1998; Muzzarelli, 1996; Razdan & Pettersson, 1994). Chitosan contains three types of reactive functional groups, an amino/acetamido group as well as both primary and secondary hydroxyl groups at the C-2, C-3 and C-6 positions, respectively. The amino contents are the main reason for the differences between their structures and physicochemical properties as well as are correlated with their chelation, flocculation and biological functions (Xia, 2003). Chitooligosaccharides (COS) are the degraded products of chitosan or chitin, which have recently been produced by several methods such as enzymatic and acidic hydrolysis. Enzymatic preparation methods have received great interest due to their safety and ease of control. Many nonspecific enzymes, such as cellulases, lipases and proteases as well as chitosanases, have been used to prepare COS (Lee, Xia, & Zhang, 2008; Lin, Lin, & Chen, 2009). Generally, the molecular weights of chitosan oligosaccharides (COS) are 10 kDa or less, and during the preparation of * Corresponding author. Tel/fax: þ86 510 85919121. E-mail address: [email protected] (W. Xia). 0268-005X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2010.03.003

chitosans with different molecular weights viscosity is used as a parameter for determining the molecular weight. Chitosan and chitooligosaccharides have attracted considerable interest due to their biological activities, namely, antimicrobial (Allan & Hadwiger, 1979; Kendra & Hadwiger, 1984; No, Park, Lee, & Meyers, 2002; Sekiguchi et al., 1994; Sudarshan, Hoover, & Knorr, 1992; Uchida, Lzume, & Ohtakara, 1989; Wei & Xia, 2003; Zhao & Xia, 2006), hypocholesterolemic (Kim & Rajapakse, 2005; Liao, Shieh, Chang, & Chien, 2007; Maezaki et al., 1996; Sugano et al., 1980; Sugano, Watanabe, Kishi, Izume, & Ohtakara, 1988; Zhou, Xia, Zhang, & Yu, 2006), immunity-enhancing and antitumor effects (Suzuki et al., 1986; Tokoro et al., 1988; Xia, 2003), drug delivery (Agnihotri, Mallikarjuna, & Aminabhavi, 2004; BravoOsuna, Millotti, Vauthier, & Ponchel, 2007; Liao et al., 2007; Park, Saravanakumar, Kim, & Kwon, 2010; Sinswat & Tengamnuay, 2003; Thanou, Verhoef, & Junginger, 2001) and accelerating Calcium and Ferrum absorption (Bravo-Osuna et al., 2007; Deuchi, Kanauchi, Shizukuish, & Kobayashi, 1995; Jeon, Shahidi, & Kim, 2000; Jung, Moon, & Kim, 2006; Liao et al., 2007; Sinswat & Tengamnuay, 2003; Xia, 2003) and so on. Studies on the biological activities of chitosan and its oligomers have been increasing, as no single type of chitosan or its oligomers exerts all of the above bioactivities. Moreover, different chitosan derivatives and enzymatic products have different structures and physicochemical properties, which may result in novel bioactivities or novel findings in known bioactive compounds.

W. Xia et al. / Food Hydrocolloids 25 (2011) 170e179

Based on our recent findings and the results of other studies, this review focuses on the biological activities of chitosan and its derivatives, which are correlated with their structures and physicochemical properties, including the hypocholesterolemic effects of different chitosan samples in vitro and in vivo due to adsorption, electrostatic interactions and entrapment, antimicrobial effects, immunity-enhancing, antitumor and anticancer effects, the acceleration of calcium and iron absorption, and other biological activities such as anti-inflammatory and antioxidant activities. This overview provides novel insights into these functions and for developing chitosan, its derivatives or oligosaccharides with special biological activity. 2. Hypocholesterolemic effects Growing evidence indicates that chitosan can lower plasma and liver triacylglycerol (TG) as well as total cholesterol (TC) levels (Cho, No, & Meyers, 1998; Fukada, Kimura, & Ayaki, 1991; Ikeda et al., 1993; Maezaki et al.,1996; Sugano et al, 1980), exhibiting hypocholesterolemic and hypolipidemic effects. It has been reported that chitosan can reduce the risk of cardiovascular diseases (Maezaki et al.,1996) and had potent fat-binding capacity in vitro (Zhou et al., 2006). In addition, it was shown to increase fecalneutral-steroid and bile-acid excretion in rats (Cho et al., 1998; Fukada et al., 1991; Sugano et al., 1980) and lower the postprandial plasma TG level in broiler chickens (Razdan & Pettersson, 1996). Among these, Maezaki et al. (1996) reported the hypocholesterolemic effect of chitosan in humans for the first time and found that chitosan effectively decreased plasma lipid levels without side effects. However, the mechanisms of the hypocholesterolemic and hypolipidemic effects of different chitosans remained unclear. Recently, our group systematically studied the mechanism of the hypocholesterolemic and hypolipidemic effects of different chitosans in vitro and in vivo. 2.1. Fat-binding, cholesterol- and bile-salt-binding capacities of different chitosan samples in vitro Chitosans with different physicochemical properties can be prepared under different reaction conditions. The degree of deacetylation and the molecular weight of a chitosan are two important characteristics which greatly affect its chemical and physiological properties. Our recent work studied the effects of the degree of deacetylation (DD) and the viscosity-average molecular weight (Mv) of chitosan samples on their fat-binding, cholesteroland bile-salt-binding capacities in vitro (Liu, Xia, & Zhang, 2008; Liu, Zhang, & Xia, 2008; Zhou et al., 2006). The results indicated that the fat-binding capacity of chitosan was significantly higher than that of cellulose, and it increased with the increase of both DD and Mv, while the cholesterol-binding capacity did not show a regular variation with changes of DD and Mv but was affected by the particle size. The bile-salt-binding capacity was greatly affected by the viscosity-average molecular weight, and the chitosan sample with highest molecular weight showed the best binding capacity for bile salts, while the degree of deacetylation seemed to have no effect on the bile-salt-binding capacity. These results suggested that the physicochemical properties of chitosan affected its binding capacities and hypocholesterolemic and hypolipidemic activities in vitro. 2.2. Hypocholesterolemic effects of different chitosan samples in vivo Chitosans with higher degrees of deacetylation (DD) have more free amino groups and a more positive charge in solution. The

171

hypocholesterolemic effects of chitosans with different degrees of deacetylation were investigated in vivo, and the effects tended to strengthen as the DD of chitosan increased. Rats fed diets containing the highest-DD chitosan showed significantly lowered plasma triglyceride, total cholesterol and low-density-lipoprotein cholesterol (LDL-C) levels as well as elevated high-density-lipoprotein cholesterol (HDL-C) levels, although not all differences were significant. Moreover, the food-efficiency ratio also decreased with increasing DD (Li, Zhang, Liu, & Xia, 2007; Liu, Zhang, et al., 2008). This is consistent with the results observed by Deuchi et al. (1995). Chitosans with higher molecular weights limited the bodyweight gain of adult rats significantly, reduced the food-efficiency ratio and lowered plasma lipids (Li et al., 2007; Liu, Zhang, et al., 2008). These results verified the effect of viscosity on hypocholesterolemic activity although the effect of molecular weight on the lipid-lowering activity of chitosan was not obvious. They also indicated that the viscosity was not the major factor influencing the hypocholesterolemic effects of chitosan in the upper gastrointestinal tract. Above a certain viscosity, the effect was small with increasing molecular weight. These results confirmed that the viscosity of chitosan was required for its hypocholesterolemic effect, although a glucosamine oligomer was not effective (Sugano et al., 1988). The particle size of chitosan also affected its hypocholesterolemic effect. Chitosan with a fine particle size effectively lowered plasma and liver lipid levels in rats (Sugano et al., 1980). In addition, the powdered form of chitosan exhibited a greater rate of adsorption of oil than the flake type (Ahmad, Sumathi, & Hameed, 2005). We also found that the particle size of chitosan was the main property affecting its hypocholesterolemic effect. This is consistent with the report that powdered chitosans exhibited better cholesterol-binding capacity than cellulose, while chitosan in the flake form bound less cholesterol than cellulose. From the above, we can conclude that the physicochemical properties of chitosans affect their hypocholesterolemic activities. The effects are more pronounced when the particles are finer as well as the degree of deacetylation and the molecular weight are both relatively high. 2.3. Chitosan exerting hypocholesterolemic activity via adsorption, electrostatic interaction and entrapment 2.3.1. Fat-, cholesterol- and bile-salt-binding mechanism of chitosan The hypocholesterolemic activity of chitosan was higher when its DD was higher (90% deacetylated) at the same molecular weight, which might be due to the electrostatic attraction between chitosan and anionic substances such as fatty acids and bile acids (Deuchi et al., 1995; Vahouny, Satchithanandam, Cassidy, Lightfoot, & Furda, 1983). This hypothesis was confirmed by the FT-IR and SEM analysis of chitosan and the chitosan-fat complex in vitro (Figs.1 and 2). As seen in Fig. 1, the FT-IR spectrum of the chitosanfat sample showed the presence of absorption peaks at 2,924.69, 2,854.81 cm1, and 1,744.38 cm1, corresponding to the saturated alkyl and the carbonyl groups, respectively, of a fatty acid, which suggested that chitosan had absorbed fatty acids by electrostatic interaction; the process was deduced to likely be the following: Chitosan  NH2 þ HOOC-R / chitosan  N  Hþ 3 ..OOC  R The SEM analysis of a chitosan-oil complex (Fig 2) also showed that after binding, instead of the sandlot shape of chitosan alone, the shape of the chitosan-oil complex became a white flock of

172

W. Xia et al. / Food Hydrocolloids 25 (2011) 170e179

65

CI S

70

60

1376. 21

3415. 99

5 0

2924. 69

10

1091. 32 1152. 21

15

2854. 81

20

1376. 66

25

1591. 99

1744. 36

30

1082. 24

2882. 44 3408. 12

%T

35

CI S-oi l

45 40

1630. 49

50

1153. 90

55

-5 -10 3500

3000

2500

2000

1500

1000

500

W avenumbers (cm-1)

Fig 1. The IR spectrum of chitosan and chitosan-fat compound. Among these, the absorption peak at 2924.69 m and 2854.81 cm1 were resulted from saturated hydrocarbon radical. Absorption peak at1744.38 cm1 indicated the existence of carboxyl.

blocky particles, which was probably solidified bound oil. In addition, when fat and chitosan are eaten together, the viscous chitosan will entrap the fat droplets in the stomach. When the complex arrives at the small intestine, chitosan precipitates together with the entrapped fat at neutral pH to prevent the digestion of fat; this has been proven in vitro (Zhou et al., 2006). When the DD and the particle size are comparable, the fatbinding capability of chitosan is enhanced with increasing viscosity-average molecular weight, suggesting that during the fatbinding process, the fat molecules are embedded in the long chain of chitosan; hence, a larger molecular weight means a longer chain and thus more fats will be embedded. Therefore, the electrostatic interactions with and entrapment of fat by the viscous polysaccharide chitosan, which would reduce the absorption of fat in the diet, was regarded as one factor in the fat-binding mechanism (Kanauchi, Deuchi, Imasato, Shizukuishi, & Kobayashi, 1995; Liu, Zhang, et al., 2008). This factor also resulted in a better fatbinding capability of chitosan than for cellulose. Moreover, at the same molecular weight, compared to flake chitosan, powdered chitosan has a smaller particle size, a higher total surface area and a more open pore structure, facilitating adsorption. This suggests that the interaction between chitosan and bile salts as well as cholesterol is adsorption, which also contributes

to its hypocholesterolemic effect. However, this adsorption function is likely weakened in vivo as chitosan can be dissolved in the acidic conditions of the stomach. 2.3.2. Distribution and metabolism of fluorescein-isothiocyanatelabeled chitosan (FITC-CIS) in vivo From the above, we deduced that the combined effects of electrostatic attraction, embedding, adsorption and entrapment were the probable mechanisms of the hypocholesterolemic effects of chitosan. To verify it, the distribution and metabolism of chitosan was further studied by measuring the content of FITC-labeled chitosan (FITC-CIS) in plasma and tissues following oral administration of FITC-CIS in mice. The results showed that FITC-CIS was distributed mainly in the liver, kidney and muscle; the concentration in the liver was highest after 1 h and then highest in the kidney later (Fig 3). As seen in Fig. 3, the concentration of chitosan in the stomach decreased with time and dietary fat was adsorbed by chitosan; the highest concentrations were at 1 h and 2 h in the small and large intestine, respectively. The appearance of the highest concentrations in the feces or urine corresponded to those in the large intestine and kidney, respectively; in the feces, chitosan was mostly excreted after 2.5e5 h in its original form, while in the urine chitosan was excreted mostly after 6e12 h and partially

Fig 2. The SEM profile of chitosan and chitosan-fat compound. The left is chitosan while the right one is chitosan-fat compound.

W. Xia et al. / Food Hydrocolloids 25 (2011) 170e179

173

1h 2h 4h 6h 9h 14 h 24 h

35

Concentration (µg/g)

30 25 20 15 10 5

t ic

ru

es T

te U

--

le

s

e cl

M

us

ra

in

y B

ne id

K

Lu

ee pl

ng

n

r ve S

Li

ea H

P

la

sm

rt

a

0

Sample Fig 3. The concentration in plasma and tissues at different time following oral administration of FITC-CIS.

degraded. These results indicated that chitosan could directly bind dietary fat in the in the digestive tract and then be excreted with the feces, and a fraction of the chitosan was degraded into COS to regulate lipid metabolism. 2.3.3. Effects of different chitosans on lipid metabolism in rats This study examined the effects of chitosan in preventing the lipid levels in plasma and liver from rising during the feeding of high-fat diets and in improving the symptoms of hypercholesterolemia in rats. The results showed that chitosan exhibited remarkable hypolipidemic effects. In animals, chitosan was reported to reduce body-weight gain in rats by 5% (wt/wt) compared to the control group (Kanauchi, Deuchi, Imasato, & Kobayashi, 1994). In our study, the rats in the chitosan groups showed similar responses. As seen in Table 1, rats in the HF and CIS groups fed the same diets except for cellulose in the HF group being replaced with chitosan in the CIS group and had similar food intakes, but the body-weight gains of rats in the CIS groups were significantly lower than those in the HF group. Moreover, the retardation of body-weight gain in rats was obvious for a long time with chitosan treatment. This might indicate that chitosan could be used as weight-loss agent for both healthy and obese humans because of its binding of lipids in the gastrointestinal tract to reduce fat absorption (Shields, Smock, McQueen, & Bryant, 2003). Interestingly, as chitosan is known to form a highly viscous solution in the stomach and precipitate in the small intestine, some researchers have found that the small intestine and the cecum of rats fed diets supplemented with 8% chitosan (wt/wt) were

noticeably bloated and their weights significantly increased (Trautwein, Jurgensen, & Erbersdobler, 1997). However, in our study the weights of the small and large intestines of the rats showed no significant differences among the five groups. Similarly, ileal digesta viscosity was not significantly influenced by feeding chitosan at 3% (wt/wt) in the diet of broiler chickens (Razdan & Pettersson, 1996). Thus, chitosan exhibited a different hypocholesterolemic mechanism from dietary fiber that increased the viscosity in the intestine. Additionally, chitosan significantly lowered serum total triglyceride (TG), total cholesterol (TC) and low-density-lipoprotein cholesterol (LDL-C) concentrations and elevated the high-densitylipoprotein cholesterol (HDL-C) level; liver TC and TG concentrations were also lowered significantly: the TC and the TG levels of the plasma and liver in the CIS2 group were a little higher than those in the CIS1 group (Table 2). This result suggested that chitosan could effectively prevent hypercholesterolemia with a highfat diet, and a longer treatment time brought a greater hypocholesterolemic effect. Rats fed chitosan showed more excretion of fecal fat and cholesterol than those fed a high-fat control diet, and the fatty acid composition in the fecal fat was the same as that in the diet (as shown in Fig. 4 and Table 3), which was consistent with the previous reports (Kanauchi et al., 1995; Sugano et al., 1980, 1988). The liver hepatic and lipoprotein lipase activities were also reduced by chitosan (Table 4), which indicated that chitosan could regulate lipase activity; chitosan could not only prevent the hyperlipemia induced by long-term administration of a high-fat diet but also reduce serum lipid levels and liver-fat accumulation in

Table 1 Tissue index of rats in five groups.

Liver index Heart index Small intestine index Cecum index Epididymal fat index

NF

HF

CR

CIS1

CIS2

2.99  0.30a 0.32  0.02a 1.26  0.29a 0.60  0.14a 1.24  0.15a

4.62  0.35b 0.34  0.03a 1.70  0.21a 0.56  0.08a 1.66  0.17b

2.70  0.36a 0.33  0.02a 1.61  0.24a 0.63  0.17a 1.43  0.23ab

3.43  0.32ac 0.32  0.01a 1.52  0.17a 0.57  0.09a 1.37  0.16ab

3.57  0.27ac 0.33  0.01a 1.42  0.14a 0.48  0.11a 1.40  0.16ab

Values are expressed as means  SE (n ¼ 8). Values with different superscript letters (a,b,c) within the same line are significantly different at P < 0.05 as determined by Student-Newman-Keuls multiple range test. The test lasted 6 weeks. NF indicates normal-fat-administered group; HF, high-fat þ cellulose (5%)-supplemented group; CR, high-fat diet þ cholestyramine (5%)-supplemented group; CIS1 is the group in which rats are fed high-fat diet þ chitosan (5%) at the beginning; CIS2 is the group in which rats are fed high-fat diet þ chitosan (5%) after two weeks. Cited from Zhang, Liu, Li, and Xia (2008).

174

W. Xia et al. / Food Hydrocolloids 25 (2011) 170e179

Table 2 The change of plasma lipid concentrations of rats during the experimental period (6 weeks). HF

CR

CIS1

CIS2

TC (mmol/L) 0wk 2.23  0.40a 2wk 2.02  0.30a 4wk 2.11  0.45a 6wk 1.96  0.32a

NF

2.20  0.30a 4.03  0.60b 4.96  0.34b 5.43  0.52b

1.97  0.31a 3.86  0.62b 2.13  0.29a 1.76  0.33a

2.33  0.45a 3.96  0.39b 2.64  0.65a 2.67  0.51c

1.99  0.21a 3.99  0.64b 2.77  0.60a 2.78  0.63c

TG (mmol/L) 0wk 1.31  0.33a 2wk 1.10  0.15a 4wk 1.09  0.28a 6wk 0.85  0.20a

1.14  0.27a 1.21  0.14a 1.38  0.29a 1.45  0.21b

1.18  0.18a 1.23  0.28a 1.27  0.27a 1.28  0.10b

1.23  0.26a 1.13  0.35a 0.79  0.15ab 0.63  0.06a

0.97  0.28a 1.07  0.30a 0.92  0.25a 0.65  0.13a

HDL-C (mmol/L) 0wk 0.86  0.14a 2wk 0.80  0.18a 4wk 0.81  0.13a 6wk 0.82  0.20a

0.83  0.18a 0.78  0.16a 0.60  0.16a 0.51  0.11b

0.77  0.16a 0.72  0.16a 0.75  0.15a 0.80  0.18a

0.82  0.15a 0.85  0.19a 0.87  0.10a 0.91  0.21a

0.78  0.19a 0.73  0.26a 0.75  0.19a 0.83  0.17a

LDL-C 0wk 2wk 4wk 6wk

0.80  0.15a 2.75  0.30 b 3.70  0.23b 4.16  0.52b

0.71  0.14a 2.59  0.18b 0.89  0.21a 0.50  0.20a

0.91  0.16a 2.60  0.20b 1.45  0.21c 1.40  0.19c

0.78  0.15a 2.72  0.19 b 1.65  0.16c 1.56  0.20c

(mmol/L) 0.77  0.12 a 0.76  0.15a 0.80  0.22 a 0.78  0.16a

Values are expressed as means  SE (n ¼ 8). Values with different superscript letters (a,b,c) within the same line are significantly different at P < 0 .05. Cited from Zhang et al. (2008).

hyperlipemic rats. Compared with cholestyramine, a hypocholesterolemic medicine, chitosan added at the same rate had less effect, but it was safe and had no side effects. 2.3.4. Molecular regulation by chitosan of several proteins correlated with lipid metabolism in the liver of rats The molecular regulation by chitosan of several proteins correlated with lipid metabolism in rat liver was studied using semiquantitative RT-PCR (see Figs. 5 and 6). The results indicated that chitosan could significantly upregulate LDL-R and PPARa mRNA levels, moderately upregulate LCAT and CYP7A1 mRNA levels as well as downregulate the HMG-CoA reductase mRNA level, which indicated that chitosan could regulate the dynamic balance of cholesterol metabolism at the molecular level and thus exert a hypolipidemic effect. The antioxidant activity of chitosan was also studied in vitro and in vivo. The results showed that chitosan at an addition of 0.02% had antioxidant effects in lard and crude rapeseed oil, but the

16:0 18:0 18:1 18:2

Fa tty a cid c ontent (%)

35 30 25 20 15 10 5 0 NF

HF

CR Group

C IS

H F d ie t

Fig 4. Fatty acid composition of the high-fat diet and feces in the NF, HF, CR and CIS group.

activity was lower than ascorbic acid; when the addition increased, chitosan and ascorbic acid had similar activities. Chitosan could significantly reduce serum FFA and MDA concentrations as well as elevate SOD, CAT and GSH-PX activities, the latter being the major antioxidant enzymes in the body, which indicated that chitosan regulated antioxidant enzyme activities and reduced lipid peroxidation. In this study, the chitosan with best hypolipidemic activity was chosen, a novel method for the determination of fluorescently labeled chitosan in plasma and tissues was developed, the hypolipidemic mechanism of chitosan was elucidated with respect to the mRNA expression of proteins correlated with lipid metabolism, the distribution and excretion of chitosan in mice was evaluated and the reduction of lipid peroxidation and fat-binding activity demonstrated in vitro. According to the above results, we confirmed that chitosan exhibited the hypocholesterolemic effects by adsorption, electrostatic interaction and entrapment (Liu, 2008). 3. Antimicrobial effects The antimicrobial activity of chitosan and its derivatives or oligomers has been recognized and is considered to be one of the most important properties, corresponding directly to their possible biological applications (Wei & Xia, 2003; Xia, 2003; Zhao & Xia, 2006). Allan and Hadwiger (1979) first reported chitosan and its derivatives had broad-spectrum antimicrobial effects. Since then many studies have been performed on the antimicrobial activity of chitosan and its derivatives and oligosaccharides, confirming that chitosan showed antimicrobial properties with bacteria, yeasts and fungi. The antibacterial activities of six chitosans and six chitosan oligomers with different molecular weights (Mv) were examined against four gram-negative (Escherichia coli, Pseudomonas fluorescens, Salmonella typhimurium, and Vibrio parahaemolyticus) and seven gram-positive bacteria (Listeria monocytogenes, Bacillus megaterium, Bacillus cereus, Staphylococcus aureus, Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus bulgaricus) by No et al. (2002). They found that chitosans showed higher antibacterial activities than chitosan oligomers and markedly inhibited the growth of most of the tested bacteria, although the inhibitory effects differed with the Mw of chitosan and the bacterial species. Chitosan generally showed stronger bactericidal effects on grampositive bacteria than gram-negative bacteria at a concentration of 0.1%. The minimum inhibitory concentration (MIC) of chitosans ranged from 0.05% to more than 0.1% depending on the bacterial species and the Mw of the chitosan. As a chitosan solvent, 1% acetic acid was effective in inhibiting the growth of most tested bacteria except for Lactobacillus, which was more effectively suppressed with 1% lactic or formic acids. The antibacterial activity of chitosan was inversely affected by pH (over the pH 4.5e5.9 range tested) and exerted better effects at a lower pH value. Uchida et al. (1989) previously reported that the MIC of chitosan for E. coli and S. aureus were 0.025% and 0.05%, respectively. Hence, chitosan was recognized as the best candidate among natural antimicrobial preservatives, although the antimicrobial activity and MIC acquired from different researchers differs, probably due to differences in the experimental methods, type of chitosan or pH. However, Zheng and Zhu (2003) used E. coli and S. aureus to study the antimicrobial activity of chitosans with different molecular weights (Mw). They found that for chitosans with Mw below 300 kDa, the antimicrobial effect on S. aureus was strengthened as the Mw increased; in contrast, the effect on E. coli was weakened. The antibacterial activities of water-soluble N-alkylated disaccharide chitosan derivatives against E. coli and S. aureus were also investigated by Yang, Chou, and Li (2005). They found that the

W. Xia et al. / Food Hydrocolloids 25 (2011) 170e179

175

Table 3 Fecal fat and cholesterol excretion of rats in five groups.

Fecal fat (g/100 g dry wt) Fecal cholesterol (mmol/g dry wt)

NF

HF

CR

CIS1

CIS2

4.31  0.56a 1.02  0.02a

12.56  1.23b 6.87  0.96b

15.81  1.22c 7.95  0.89b

17.82  1.35c 10.98  1.22c

16.81  1.08c 10.05  1.29c

Values are expressed as means  SE (n ¼ 8). Values with different superscript letters (a,b,c) within the same line are significantly different at P < 0.05.

antibacterial activity of chitosan derivatives was affected by the degree of disaccharide substitution (DS) and the kind of disaccharide present in the molecule. Regardless of the kind of disaccharide linked to the chitosan molecule, a DS of 30e40%, in general, exhibited the most pronounced antibacterial activity against both test organisms. E. coli and S. aureus were the most susceptible to cellobiose-chitosan derivatives and maltose-chitosan derivatives, both with DS values of 30e40%, among the various examined chitosan derivatives. Although the disaccharide chitosan derivatives showed less antibacterial activity than native chitosan at pH 6.0, they exhibited higher activity at pH 7.0. The antibacterial activity of the chitosan derivatives (DS 30e40%) against E. coli increased as the pH increased above 5.0 and reached a maximum around pH 7.0e7.5. The effect of pH on the antibacterial activity of chitosan derivatives against S. aureus was not as significant as that observed with E. coli. Population reductions of E. coli or S. aureus in nutrient broth increased markedly when the concentration of chitosan derivatives was increased from 0 to 500 mg/kg, while no marked increase in population reduction was found with further increases, even up to 2,000 mg/kg. Additionally, chitosan has shown inhibitory effects on the growth of fungi and other microorganisms, especially plant pathogens. Chitosan can induce plants to produce defense enzymes (chitosanases) with antimicrobial activity. It has a higher antimicrobial activity than chitin because it carries a positive charge. Hirano and Nagao (1989) studied the relative molecular weights of chitosan on the inhibition of plant pathogens. The results indicated that chitooligosaccharides (DP2-8) and partially degraded lowmolecular-weight chitosan (LMWC) showed higher inhibitory activities on Fusarium oxysporum, Phomopsis fukushi and Alternaria alternata than high-molecular-weight chitosan. Uchida et al. (1989) found that the inhibition of fungi and bacteria by chitooligosaccharides with higher degrees of polymerization (DP) was much stronger than those by chitosan and chitooligosaccharides with lower DP; simultaneously, their inhibitory effects increased with increasing DD. This result was confirmed by later research (Jeon & Kim, 2001). Jeon and Kim (2000) produced and isolated three kinds of COS using an ultra filtration membrane bioreactor; among these, the COS with Mw of 5,000-10,000 showed strong antimicrobial activity on the tested pathogens. Later, they produced a COS with a DP of 3e6 by the same methods, which showed a higher inhibitory effect on E. coli with increasing concentration; a 0.5% COS solution completely inhibited the growth of E. coli. In our previous study, we also studied the antimicrobial activity of partially hydrolyzed chitosan oligomers against the common

Table 4 Liver and plasma lipase activities of rats in five groups. NF

HF

CR

CIS1

Liver HL (U/g prot) 98.3  10.0a 51.9  9.0b 47.3  5.2b 81.1  9.7c LPL (U/g prot) 12.0  1.0a 7.7  1.5b 10.7  1.8a 17.7  2.9c Plasma LPL (U/g prot) 73.0  6.8a

51.6  3.2b 70.2  5.4a

CIS2 79.9  8.2c 11.1  1.1a

57.6  5.3b 51.3  3.8b

Values are expressed as means  SE (n ¼ 8). Values with different superscript letters within the same line are significantly different at P < .05. The test lasted 6 weeks. HL, hepatic lipase; LPL, lipoprotein lipase.

bacteria, molds and yeasts in found food and found it to be much higher than that of chitosan, with an MIC of 1e10 g/L, and the inhibitory effects were enhanced with increasing chitooligosaccharide concentration. Moreover, the relations between their structures and antimicrobial effects were also examined. It was found that the antimicrobial activity of chitooligosaccharides was correlated with the content of protonated amino groups and relative molecular weights. Additionally, when COS was applied as a preservative in apple juice, the storage period of juice at 37  C was prolonged from nine days to 70 days with a COS concentration of 4 g/L, showing good preservative effects (Wei & Xia, 2003). Chitin, chitosan, and their oligomers have been reported to exhibit elicitor activities in several plants and have been widely used as elicitors for the induction of secondary products in plantcell cultures. When attacked by pathogens such as fungi, bacteria, and viruses, higher plants have various defense reactions including the production of phytoalexins, enzymes such as chitinase and bglucanase, proteinase inhibitors, hydroxyproline-rich glycoproteins, proteinase, active oxygen species as well as lignification. Chitin oligomers (DP3-6) were active as elicitors of defense reactions in higher plants, whereas chitosan oligomers (DP3-6) had almost no eliciting activity. However, higher chitosan oligomers, e.g., octamers, were efficient elicitors for inducing pisatin accumulation and inhibiting fungal growth. These results suggest that elicitor activities of chitin and chitosan oligomers are highly dependent on their polymerization and the presence of N-acetylglucosamine. From the above, we can conclude that although there are many reports discussing chitosan’s antimicrobial activity in different conditions with conflicting results, they all confirmed that chitosan and its oligosaccharides have strong antimicrobial effects and are safe for human use. Hence, the antimicrobial characteristics of chitosan and its oligosaccharides present a profitable potential for developing natural food preservatives for food-processing applications and functional-food additives. 4. Immunity-enhancing, antitumor, and anticancer effects The immunostimulating activity of chitosan and COS has been reported for several decades. Nishimura et al. (1984) first reported that chitosan, especially 70%-DD chitosan, could stimulate rats to produce a nonspecific host repellence when infected with E. coli and Sendai virus. They concluded that 70%-DD chitosan was an immune regulator that can activate macrophages (Mcp) and natural killer cells (NK) and improve the delayed-type hypersensitive reaction, increase cytotoxicity and induce mitosis in cells producing interleukins, breeding factors and interferon. Later, Suzuki et al. (1986) reported enhanced immune regulation with the increased water-solubility of chitosan. They proved that chitooligosaccharides inhibited tumor growth through an increase in immune effects. The chitooligosaccharides with DP4-7 showed strong inhibition of ascites cancer in BALB/c mice, while (GlcNAC)6 and (GlcN)6 showed very strong inhibiting effects for S-180 and MM156 solid tumor growth in syngenic mice, which was also reported by Tokoro et al. (1988). Additionally, (GlcNAC)6 was also reported to show an antitransfer effect on Lewis lung cancer in mice (Tsukada).

176

W. Xia et al. / Food Hydrocolloids 25 (2011) 170e179

Fig 5. The mRNA expression of each proteins in rat liver; I.LDL-R; II. LCAT; III.HMG-CoA reductase; IV.CYP7A1; V. PPARa;a1,a2,a3:normal fat (NF) groups;b1,b2,b3:High-fat groups; c1,c2,c3:chitosan groups.

Chitosan also showed an immunity-enhancing effect by enhancement of antibody response. The effect of chitosan as a novel adjuvant to an inactivated influenza vaccine was studied (Chang, Chen, Fang, & Chen, 2004). Here, BALB/c mice were abdominally inoculated with vaccine and chitosan together twice every three weeks. Blood serum was prepared and testing for levels of antibodies IgG, IgG1, and IgG2a as well as IgA antibody in nasal secretions. One week after the immunization regimen, the mice were challenged with the deadly flu virus A/PR/8/34(H1N1) and the weights of the mice and levels of antibody protection were measured. The results indicated that using chitosan as an adjuvant increased the antibody content in serum remarkably and increased the antiviral defense in the mice, enhancing the immune reaction to the vaccine. The antitumor mechanism of these chitooligosaccharides was probably related to their induction of lymphocyte factor, increasing T-cell proliferation to produce the tumor inhibitory effects. Through analysis of the splenic cell changes in cancerous mice, Suzuki et al. (1986) proved that the antitumor mechanism of chitooligosaccharides is to enhance acquired immunity by accelerating T-cell differentiation to increase cytotoxicity and maintain T-cell activity. While several studies have reported the importance of chitosan derivatives for their anticancer activity, no clear information is

available describing the relationship between their charge properties and their observed activities. Huang, Mendis, Rajapakse, and Kim (2006) studied the anticancer activities of differently charged chitooligosaccharide (COS) derivatives using three cancer-cell lines: HeLa, Hep3B and SW480. Neutral red and MTT cell-viability studies revealed that highly charged COS derivatives could significantly reduce cancer-cell viability, regardless of their positive or negative charge. Furthermore, fluorescence microscopic observations and DNA-fragmentation studies confirmed that the anticancer effect of these highly charged COS derivatives were due to necrosis. However, the exact molecular mechanism for the anticancer activity of strongly charged COS compared to their poorly charged counterparts is not clear. Additionally, chitosan is also known as a drug carrier which can improve drug absorption, stabilize drug components to increase drug targeting and enhance drug release. As a gene carrier, chitosan can protect DNA and increase the expression period of genes. Hence, chitosan has broad prospects for applications as drug and gene carriers. For example, chitosan could also used as a drug carrier to provide anticancer and antitumor chemotherapy. It has been reported that the conjugates of some kinds of anticancer agents with chitin and chitosan derivatives display good anticancer effects with a decrease in side effects over the original

W. Xia et al. / Food Hydrocolloids 25 (2011) 170e179

LDL-R HMG-CoA PPARa

relative expression level of mRNA

1.6

LCAT CYP7A1

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 NF

HF

CIS

group Fig 6. The relative expression of each mRNA level in rat liver.

form due to a predominant distribution in the cancer tissue and a gradual release of free drug from the conjugates. For instance, doxifluridine and 1-b-D-arabinofuranosylcytosine (Ara-C) could be conjugated with chitosan via a glutaric acid spacer. The conjugates of Ara-C with chitosan, in particular, showed improved antitumor effects against a P388-bearing leukemia model in mice. Glycol-chitosan (G-Chi) was distributed mainly in the systemic circulation and the kidney after i.v. administration in normal mice and retained long-term in the kidney. The therapeutic effects of the conjugates of mitomycin C (MMC) with G-Chi were not necessarily improved over that of the free drug, but the toxic side effects were significantly decreased. Conjugates of MMC with 6-O-carboxymethyl-chitin showed an almost complete suppression of tumor growth at 10 mg eq. MMC/kg, while a lethal adverse effect was also observed. The conjugates of MMC with N-succinylchitosan showed good antitumor activities against various tumor models due to their predominant distribution into the tumor tissue and sustained-release characteristics with both waterinsoluble and -soluble formulations (Kato, Onishi, & Machida, 2005). From the above, it is believed that chitin and chitosan derivatives are good candidates for polymeric drug carriers in cancer chemotherapy. 5. Acceleration of calcium and iron absorption in vivo The ability of chitosan and its derivatives to bind cations is well known. Chitosan can accelerate the absorption of calcium and iron, and the chelation of metal ions may also be related to its drugdelivery characteristics (Liao et al., 2007; Park et al., 2010), but there is some disagreement on this point. Some researchers found that with oral administration of 1e5% chitosan in mice instead of cellulose, the whole-body retention of radioactive 47Ca decreased greatly, indicating that dietary chitosan affected the calcium metabolism in the animals (Jeon et al., 2000). Other studies found a decrease in the absorption of calcium and the bone content of calcium after oral administration of chitosan for two weeks, so oral administration of native chitosan apparently reduced the absorption of calcium and iron ions (Deuchi et al., 1995). However, several patents have claimed that chitosan-Fe and chitosan-hemochrome complexes could enhance iron absorption in the intestines as well

177

as chitosan-Ca. A CaCO3-chitosan oral liquid was taken by 15 elderly osteoporosis sufferers; after 55 days, their bone-mineral density (BMD) significantly improved. It is generally considered that when it arrives in the intestines, Ca2þ or Fe3þ will be precipitated as hydrates to reduce their absorption. While chitosan could chelate Ca2þ and Fe3þ at low pH, making these metal ions soluble in the intestines thus increasing their absorption. The precondition is the solubility of chitosan in intestines, as natural chitosan is soluble in acidic conditions. To retain chitosan’s solubility and strong chelation in alkaline conditions, it could be chemically modified, e.g., by the addition of carboxymethyl groups. Chitosan derivatives have been evaluated to overcome chitosan’s limited solubility and effectiveness as an absorption enhancer at neutral pH values such as those found in the intestinal tract; they do not cause damage to the cell membrane or alter the viability of intestinal epithelial cells (Thanou et al., 2001). This kind of derivative chelated with Ca2þ and Fe3þ could remain soluble in alkaline conditions to increase absorption of these ions in the intestines, similarly to the hydrolysis product of milk casein (casein phosphate peptides, CPP) (BravoOsuna et al., 2007; Xia, 2003). Chitosan oligosaccharides also accelerated the absorption of calcium and other minerals in vivo. Jeon et al. (2000) reported that COS (DP3-7) decreased the excretion of fecal calcium and improved the antifracture capacity of rat thighbone. Moreover, the in vivo effects of COS on Ca bioavailability were further studied in a rat osteoporosis model induced by ovariectomy and concurrent low calcium intake. It was found that after the low-Ca diet the COS diet, including both normal levels of calcium and vitamin D, significantly decreased calcium loss in the feces and increased calcium retention compared to the control diet. The levels of femoral total calcium, bone-mineral density (BMD), and femoral strength were also significantly increased by the COS diet at a similar level to those of the CPP group. In the present study, the results proved the beneficial effects of low-molecular-weight chitooligosaccharides (COS) in a preventing negative mineral balance (Jung et al., 2006). 6. Anti-inflammatory effects and repair of arthritic tissue Chitosan is alkaline, having free amino groups; it can neutralize gastric acids and form a protective membrane in the stomach, so chitosan could be used to cure acid indigestion and peptic ulcer. The anti-inflammatory mechanism of chitosan is due to the acid hydrolysis of chitosan, to glucosamine hydrochloride or its sulfate, phosphate and other salt preparation by salt conversion. These monosaccharides are structural units of the proteoglycans contained in connective tissue and cartilage. These tissues can be repaired and regenerated by absorbing these monosaccharides directly when they are damaged or inflamed (Olivier et al., 2004). Therefore, these monosaccharides are an effective treatment for preventing and curing rheumatoid arthritis as well as bone hyperplasia and are used as antiarthritic drugs in clinical practice. Moreover, unlike some common antiarthritic steroidal antiinflammatory drugs or analgesic and anti-inflammatory drugs, these monosaccharides have no side effects or toxicity and can be taken for a long period. The clinic experiments indicated that taking glucosamine for two weeks can eliminate arthritic pain and improve movement in patients suffering from severe arthritis (Olivier & Jean-Yves, 2007). 7. Antioxidant activity Both chitosan and its oligosaccharides showed antioxidant effects. In our recent study, the antioxidant activity of chitosan was studied in vitro and in vivo (Liu, 2008). The results showed that

178

W. Xia et al. / Food Hydrocolloids 25 (2011) 170e179

chitosan at an addition of 0.02% had antioxidant effects 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 FFA and MDA concentrations and elevate SOD, CAT and GSH-PX activities, the latter being the major antioxidant enzymes in the body, indicating that chitosan regulated the antioxidant enzyme activities and reduced lipid peroxidation. The cellular antioxidant effects of chitosan oligosaccharides (NA-COS; Mw 229.21e593.12 Da) produced by acidic hydrolysis of crab chitin were also identified by Ngo, Kim, and Kim (2008). Their study showed that NA-COS have free-radical scavenging effects in a cellular system. They can inhibit myeloperoxidase activity and decrease free-radical oxidation of DNA and membrane proteins. Furthermore, they also stimulate an increase in intracellular GSH levels. Based on the results, they concluded that NA-COS have freeradical scavenging effects, acting in both indirect and direct ways to inhibit and prevent biological molecular damage by free radicals in living cells. This finding is similar to that of Park, Je, and Kim (2004). Hence, chitosan and COS can be used as a scavenger to control radical-induced damage to cellular systems and promises further applications in the future.

Table 5 Applications of chitosan, and their oligomers. Some of which was cited from Jeon et al (2000). Fields

Chitosan

Chitosan oligosaccharides

Food

Antimicrobial agent; Preservative agent; Edible film; Accelerating the calcium and ferrum absorption; Dietary fiber;

Antimicrobial agent; Preservative agent;

Hypocholesterolemic agent; Immuno-enhancing agent; Pharmaceutical and medical

Antitumor agent; Carrier for drug and DNA delivery system; Accelerator for wound healing; Bandage; Artificial skin; Biotechnology

9. Other biological activities Additionally, chitosan and its oligosaccharides have other biological functions such as excluding toxins from the intestines, reducing heavy-metal poisoning in humans, radio-protective properties, preventing tooth decay and tooth diseases (Xia, 2003), as a bifidus factor (BF) to regulate microbial metabolism in intestines (Lee, Park, Jung, & Shin, 2002), anti-mutagenic effects (Nam, Choi, & Shon, 2001) and so on. 10. Prospects and limitations Chitosan and COS possess various biological activities and have considerable current as well as potential applications summarized in Table 5. This review detailed the hypocholesterolemic, antimicrobial effects and their mechanisms as well as the research advances in other bioactivities. In the food industry, chitosan (edible chitosan, more than 83% DD) and COS have been used as Dietary Food Additives and functional factors for their antimicrobial, hypocholesterolemic and immune-stimulating effects as well as drug carriers. Many commercial functional products have been available, such as chitosan capsules (e.g., the 6th Element) and COS capsules in China and Norway. Edible chitosan biofilm has also been prepared for food storage utilizing its antimicrobial activity. In agriculture, chitosan and its derivatives have been utilized in plant protection and as feed additives. The drug-delivery

Protective effect on bacterial infection; Immuno-enhancing agent; Antitumor agent; Anti-inflammatory effect and repairing the arthritis tissue;

Carrier for immobilized enzymes and Cells; Porous beads for bioreactors; Resin for chromatography; Membrane material;

e

Environment

wastewater treatment;

anti-mutagenic effects

Agriculture

Seed coating preparation ; Activator of plant cells; Activator of plant cells;

8. Angiotensin-I-converting enzyme (ACE) inhibition Chitosan has also been reported to prevent increases in blood pressure. A high-salt diet can raise blood pressure because Cl activates angiotensin-converting enzyme (ACE), while chitosan can bind Cl and remove it, preventing the blood pressure from rising (Xia, 2003). Moreover, other researchers found that chitosan oligomers also had ACE-inhibitory activity. They reported that the ACEinhibitory activity of hetero-COS was dependent on the degree of deacetylation and that COS with the relatively lowest DD exhibited the highest ACE-inhibitory activity (Park, Je, & Kim, 2003); substitution of the hydrogen atom at the C-6 position of the pyranose residue with the aminoethyl group promoted the ACE-inhibitory effects of COS (800e3000 Da and 90%DD) (Ngo, Qian, Je, Kim, & Kim, 2008).

Protective effect on bacterial infection; Immuno-enhancing agent;

Accelerating the calcium and ferrum absorption; Hypocholesterolemic agent;

Activator of plant cells; Antimicrobial activity for plant pathogen;

Antimicrobial activity for plant pathogen; Cosmetic

Cosmetics material

e

characteristics of chitosan and its derivatives has made them popular both in functional food and medicine as they can deliver drugs or functional factors to the target and control release. However, it should be noted that chitosans with different structures showed different biological activities, and not all the biological activities are found in one kind of chitosan. Each special type of bioactive chitosan should be developed for its potential application. Moreover, many of the studies carried out on chitosan and COS bioactivity have not provided detailed molecular mechanisms. In fact, it is hard to explain exactly how these molecules exert their activities. Therefore, future research should be directed towards understanding their molecular-level details, which may provide insights into the unknown biochemical functions of chitosan and COS as well as help to accelerate their future application. Acknowledgements This work was financially supported by National Nature Science Foundation of China (NSFC, No.: 20576104, No.: 20571034 and No.: 20876068), also supported by SKLF-MB-200805, PCSIRT0627, 111project-B07029 and the State high-tech project (863) (No.:2007AA100401 and No.:2006AA09Z444). References Agnihotri, S. A., Mallikarjuna, N. N., & Aminabhavi, T. M. (2004). Recent advances on chitosan-based micro- and nanoparticles in drug delivery. Journal of Controlled Release, 100(1), 5e28.

W. Xia et al. / Food Hydrocolloids 25 (2011) 170e179 Ahmad, A. L., Sumathi, S., & Hameed, B. H. (2005). Adsorption of residue oil from palm oil mill effluent using powder and flake chitosan: equilibrium and kinetic studies. Water Research, 39, 2483e2494. Allan, C. R., & Hadwiger, L. A. (1979). The fungicidal effect of chitosan on fungi of varying cell wall composition. Experimental Mycology, 3, 285e287. Bravo-Osuna, I., Millotti, G., Vauthier, C., & Ponchel, G. (2007). In vitro evaluation of calcium binding capacity of chitosan and thiolated chitosan poly (isobutyl cyanoacrylate) core-shell nanoparticles. International Journal of Pharmaceutics, 338(1e2), 284e290. Chang, H. Y., Chen, J. J., Fang, F., & Chen, Z. (2004). Enhancement of antibody response by chitosan, a novel adjuvant of inactivated influenza vaccine. Chinese Journal of Biologicals, 17(6), 21e24. Cho, Y. I., No, H. K., & Meyers, S. P. (1998). Physicochemical characteristics and functional properties of various commercial chitin and chitosan products. Journal of Agricultural and Food Chemistry, 46, 3839e3843. Deuchi, K., Kanauchi, O., Shizukuish, M., & Kobayashi, E. (1995). Continuous and massive intake of chitosan effects mineral and fat-soluble vitamin status in rats fed on a high-fat diet. Bioscience Biotechnology & Biochemistry, 59, 1211e1216. Fukada, Y., Kimura, K., & Ayaki, Y. (1991). Effect of chitosan feeding on intestinal bile acid metabolism in rats. Lipids, 26, 395e939. Hirano, S., & Nagao, N. (1989). Effects of chitosan, pectic acid, lysozyme, and chitinase on the growth of several phytopathogens. Agricultural and Biological Chemistry, 53 (11), 3065e3066. Huang, R. H., Mendis, E., Rajapakse, N., & Kim, S. K. (2006). Strong electronic charge as an important factor for anticancer activity of chitooligosaccharides (COS). Life Science, 78(20), 2399e2408. Ikeda, I., Sugano, M., Yoshida, K., Sasaki, E., Iwamoto, Y., & Hatano, K. (1993). Effects of chitosan hydrolysates on lipid absorption and on serum and liver lipid concentration in rats. Journal of Agricultural and Food Chemistry, 41, 431e435. Jeon, Y. J., & Kim, S. K. (2000). Production of chitooligosaccharides using an ultra filtration membrane reactor and their antibacterial activity. Carbohydrate Polymers, 41, 133e141. Jeon, Y. J., & Kim, S. K. (2001). Effect of antimicrobial activity by chitosan oligosaccharide N-conjugated with asparagine. Journal of Microbiology and Biotechnology, 11, 281e286. Jeon, Y. J., Shahidi, F., & Kim, S. K. (2000). Preparation of chitin and chitosan logomers and their applications in physiological functional foods. Food Reviews International, 16(2), 159e176. Jung, W. K., Moon, S. H., & Kim, S. K. (2006). Effect of chitooligosaccharides on calcium bioavailability and bone strength in ovariectomized rats. Life Science, 78, 970e976. Kanauchi, O., Deuchi, K., Imasato, Y., & Kobayashi, E. (1994). Increasing effect of a chitosan and ascorbic acid mixture on fecal dietary fat excretion. Bioscience Biotechnology & Biochemistry, 58, 1617e1620. Kanauchi, O., Deuchi, K., Imasato, Y., Shizukuishi, M., & Kobayashi, E. (1995). Mechanism for the inhibition of fat digestion by chitosan and for the synergistic effect of ascorbate. Bioscience Biotechnology & Biochemistry, 59(5), 786e790. Kato, Y., Onishi, H., & Machida, Y. (2005). Contribution of chitosan and its derivatives to cancer chemotherapy. In Vivo, 19(1), 301e310. Kendra, D. F., & Hadwiger, L. A. (1984). Characterization of the smallest chitosan oligomer that is maximally antifungal to Fusarium solani and elicits pisatin formation in Pisum sativum. Experimental Mycology 8276e8281. Kim, S. K., & Rajapakse, N. (2005). Enzymatic production and biological activities of chitosan oligosaccharides (COS): a review. Carbohydrate Research, 62, 357e368. Knorr, D. (1984). Use of chitinous polymers in foodda challenge for food research and development. Food Technology, 38, 85e97. Kurita, K. (1998). Chemistry and application of chitin and chitosan. Polymer Degradation and Stability, 59(1e3), 117e120. Lee, D. X., Xia, W. S., & Zhang, J. L. (2008). Enzymatic preparation of chitooligosaccharides by commercial lipase. Food Chemistry, 111(2), 291e295. Lee, H. Y., Park, Y. S., Jung, J. S., & Shin, W. S. (2002). Chitosan oligosaccharides, dp 2e8, have prebiotic effect on the Bifidobacterium bifidium and Lactobacillus sp. Anaerobe, 8(6), 319e324. Liao, F. H., Shieh, M. J., Chang, N. C., & Chien, Y. W. (2007). Chitosan supplementation lowers serum lipids and maintains normal calcium, magnesium, and iron status in hyperlipidemic patients. Nutrition Research, 27(3), 146e151. Li, L., Zhang, J. L., Liu, J. N., & Xia, W. S. (2007). Effects of chitosan on serum lipid and fat liver. Chinese Journal of Marine Drugs, 26(2), 7e9. Lin, S. B., Lin, Y. C., & Chen, H. H. (2009). Low molecular weight chitosan prepared with the aid of cellulase, lysozyme and chitinase: characterisation and antibacterial activity. Food Chemistry, 116(1), 47e53. Liu, J. N. (2008). Study on the hypolipidemic mechanism of chitosan. Doctor dissertation. Jiangnan university. Wuxi, China. Liu, J. N., Xia, W. S., & Zhang, J. L. (2008). Effects of chitosans physico-chemical properties on binding capacities of lipid and bile salts in vitro. Chinese Food Science, 29(1), 45e49. Liu, J. N., Zhang, J. L., & Xia, W. S. (2008). Hypocholesterolemic effects of different chitosan samples in vitro and in vivo. Food Chemistry, 107, 419e425. Maezaki, Y., Tsuji, K., Nakagawa, Y., Kawai, Y., Akimoto, M., Tsugita, T., et al. (1996). Chitin enzymology. Italy: Lyon and Ancona: European Chitin Society. pp. 217e232. Muzzarelli, R. R. A (1996). Chitin enzymology (pp. 217e232). Italy, Lyon and Ancona: European Chitin Society. Nam, K. S., Choi, Y. R., & Shon, Y. H. (2001). Evaluation of the antimutagenic potential of chitosan oligosaccharide: Rec, Ames and Umu tests. Biotechnology Letters, 23, 971e975.

179

Ngo, D. N., Kim, M. M., & Kim, S. K. (2008). Chitin oligosaccharides inhibit oxidative stress in live cells. Carbohydrate Polymers, 74(2), 228e234. Ngo, D. N., Qian, Z. J., Je, J. Y., Kim, M. M., & Kim, S. K. (2008). Aminoethyl chitooligosaccharides inhibit the activity of angiotensin converting enzyme. Process Biochemistry, 43(1), 119e123. Nishimura, K., Nishimura, S., Nishi, N., Saiki, I., Tokura, S., & Azuma, I. (1984). Immunological activity of chitin and its derivatives. Vaccine, 2(1), 93e99. No, H. K., Park, N. Y., Lee, S. H., & Meyers, S. P. (2002). Antibacterial activity of chitosans and chitosan oligomers with different molecular weights. International Journal of Food Microbiology, 74(1e2), 65e72. Olivier, B., & Jean-Yves, R. (2007). Glucosamine and chondroitin sulfate as therapeutic agents for knee and hip osteoarthritis. Drugs & Aging, 24(7), 573e580. Olivier, B., Karel, P., Lucio, R., Rita, D., Marta, O., Gatterova, et al.. (2004). Glucosamine sulfate reduces osteoarthritis progression in postmenopausal women with knee osteoarthritis: evidence from two 3-year studies. Menopause, 11(2), 138e143. Park, J. H., Saravanakumar, G. S., Kim, K. Y., & Kwon, C. (2010). Targeted delivery of low molecular drugs using chitosan and its derivatives. Advanced Drug Delivery Reviews, 62(1), 28e41. Park, P. J., Je, J. Y., & Kim, S. K. (2003). Angiotensin I Converting Enzyme (ACE) inhibitory activity of hetero-chitooligosaccharides prepared from partially different deacetylated chitosans. Journal of Agricultural and Food Chemistry, 51(17), 4930e4934. Park, P. J., Je, J. Y., & Kim, S. K. (2004). Free radical scavenging activities of differently deacetylated chitosans using an ESR spectrometer. Carbohydrate Polymers, 55, 17e22. Razdan, A., & Pettersson, D. (1994). Effect of chitin and chitosan on nutrient digestibility and plasma lipid concentrations in broiler chickens. British Journal of Nutrition, 72, 277e288. Razdan, A., & Pettersson, D. (1996). Hypolipidaemic, gastrointestinal and related responses of broiler chickens to chitosans of different viscosity. British Journal of Nutrition, 76, 387e397. Sekiguchi, S., Miura, Y., Kaneko, H., Nishimura, S. I., Nishi, N., Iwase, M., et al. (1994). Molecular weight dependency of antimicrobial activity by chitosan oligomers. In K. Nishinari, & E. Doi (Eds.), Food hydrocolloids: Structures, properties, and functions (pp. 71e76). New York: Plenum. Shields, K. M., Smock, N., McQueen, C. E., & Bryant, P. J. (2003). Chitosan for weight loss and cholesterol management. American Journal of Health-System Pharmacy, 60, 1310e1312, 1315e1316. Sinswat, P., & Tengamnuay, P. (2003). Enhancing effect of chitosan on nasal absorption of salmon calcitonin in rats: comparison with hydroxypropyl- and dimethyl-b-cyclodextrins. International Journal of Pharmaceutics, 257(1e2), 15e22. Sudarshan, N. R., Hoover, D. G., & Knorr, D. (1992). Antibacterial action of chitosan. Food Biotechnol 6257e6272. Sugano, M., Fujikawa, T., Hiratsuji, Y., Nakashima, K., Fukuda, N., & Hasegawa, Y. (1980). A novel use of chitosan as a hypocholesterolemic agent in rats. American Journal of Clinical Nutrition, 33, 787e793. Sugano, M., Watanabe, S., Kishi, A., Izume, M., & Ohtakara, A. (1988). Hypocholesterolemic action of chitosans with different viscosity in rats. Lipids, 23(3), 187e191. Suzuki, K., Mikami, T., Okawa, Y., Tokoro, A., Suzuki, S., & Suzuki, M. (1986). Antitumor effect of hexa-N-acetylchitohexaose and chitohexaose. Carbohydrate Research, 151, 403e408. Thanou, M., Verhoef, J. C., & Junginger, H. E. (2001). Chitosan and its derivatives as intestinal absorption enhancers. Advanced Drug Delivery Reviews, 50(S1), S91eS101. Tokoro, A., Tatewaki, N., Suzuki, K., Mikami, T., Suzuki, S., & Suzuki, M. (1988). Growth-inhibitory effect of hexa-N-acetylchitohexaose and chitohexaose against meth-A solid tumor. Chemical & Pharmaceutical Bulletin, 36, 784e790. Trautwein, E. A., Jurgensen, U., & Erbersdobler, H. F. (1997). Cholesterol-lowering and gallstone-preventing action of chitosans with different degree of deacetylation in hamsters fed cholesterol-rich diets. Nutrition Research, 17, 1053e1065. Uchida, Y., Lzume, M., & Ohtakara, A. (1989). Preparation of chitosan oligomers with purified chitosanase and its application. In G. Skjak-Brak, T. Anthonsen, & P. Sandford (Eds.), Chitin and chitosan: Sources, chemistry, biochemistry, physical properties and applications (pp. 373e382). London: Elsevier. Vahouny, G. V., Satchithanandam, S., Cassidy, M. M., Lightfoot, F. B., & Furda, I. (1983). Comparative effects of chitosan and cholestyramine on lymphatic absorption of lipids in the rats. The American Journal of Clinical Nutrition, 38, 278e284. Wei, X. L., & Xia, W. S. (2003). Research development of chitooligosaccharides physiological activities. Chinese Pharmaceutical Bulletin, 19(6), 614e617. Xia, W. S. (2003). Physiological activities of chitosan and its application in functional foods. Journal of Chinese Institute of Food Science and Technology, 3(1), 77e81. Yang, T. C., Chou, C. C., & Li, C. F. (2005). Antibacterial activity of N-alkylated disaccharide chitosan derivatives. International Journal of Food Microbiology, 97, 237e245. Zhang, J. L., Liu, J. N., Li, L., & Xia, W. S. (2008). Dietary chitosan can effectively improve hypercholesterolemia in rats fed high-fat diets. Nutrition Research, 28, 383e390. Zhao, X. R., & Xia, W. S. (2006). Antimicrobial activities of chitosan and application in food preservation. Chinese Food Research and Development, 27(2), 157e160. Zheng, L. Y., & Zhu, J. F. (2003). Study on antimicrobial activity of chitosan with different molecular weights. Carbohydrate Polymers, 54(4), 527e530, 1. Zhou, K., Xia, W., Zhang, C., & Yu, L. (2006). In vitro binding of bile acids and triglycerides by selected chitosan preparations and their physicochemical properties. LWT- Food Science and Technology, 39, 1087e1092.