Accepted Manuscript

Title: Antimicrobial and antitumor activities of chitosan from

shiitake stipes, compared to commercial chitosan from crab
shells

Author: Rao-Chi Chien Ming-Tsung Yen Jeng-Leun Mau

PII: S0144-8617(15)01151-0
DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2015.11.061
Reference: CARP 10580

To appear in:

Received date: 11-8-2015

Revised date: 18-11-2015
Accepted date: 23-11-2015

Please cite this article as: Chien, R.-C., Yen, M.-T., and Mau, J.-L.,Antimicrobial
and antitumor activities of chitosan from shiitake stipes, compared to
commercial chitosan from crab shells, Carbohydrate Polymers (2015),
http://dx.doi.org/10.1016/j.carbpol.2015.11.061

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 1 Antimicrobial and antitumor activities of chitosan from shiitake stipes, compared to

2 commercial chitosan from crab shells

3 Rao-Chi Chiena,b,c, Ming-Tsung Yend, Jeng-Leun Maua,b,c*

a
4 Department of Food Science and Biotechnology, National Chung Hsing University

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5 (NCHU), Taichung, Taiwan, R.O.C.;
b
6 NCHU/University of California at Davis, Plant and Food Biotechnology Center, NCHU,

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7 Taichung, Taiwan, R.O.C.;

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c
8 Agricultural Biotechnology Center, NCHU, Taiwan, R.O.C.;
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9 Department of Applied Life Science and Health, Chia Nan University of Pharmacy and Science, Jenteh

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10 71710, Tainan, Taiwan, R.O.C.

11
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12 Running Title Header: Antimicrobial and antitumor activities of chitosan

13
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14 * Corresponding author at: Department of Food Science and Biotechnology, National

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15 Chung Hsing University (NCHU), 250 Kuokuang Road, Taichung 40227, Taiwan, ROC.
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16 Tel: +886–4–2285–4313; Fax: +886–4–2287–6211.

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17 Email address: jlmau@dragon.nchu.edu.tw (J-L Mau).

18
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19 ABSTRACT

20 Chitosan was prepared by alkaline N-deacetylation of chitin obtained from shiitake

21 stipes and crab shells and its antimicrobial and antitumor activities were studied. Chitosan

22 from shiitake stipes and carb shells exhibited excellent antimicrobial activities against eight

23 species of Gram positive and negative pathogenic bacteria with inhibition zones of 11.4-

24 26.8 mm at 0.5 mg/ml. Among chitosan samples, shiitake chitosan C120 was the most

25 effective with inhibition zones of 16.4-26.8 mm at 0.5 mg/ml. In addition, shiitake and crab

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26 chitosan showed a moderate anti-proliferative effect on IMR 32 and Hep G2 cells. At 5

27 mg/ml, the viability of IMR 32 cells incubated with chitosan was 68.8-85.0% whereas that

28 of Hep G2 cells with chitosan was 60.4-82.9%. Overall, shiitake chitosan showed slightly

29 better antimicrobial and antitumor activities than crab chitosan. Based on the results

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30 obtained, shiitake and crab chitosan were strong antimicrobial agents and moderate

31 antitumor agents.

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32

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33 Key words: shiitake chitosan; crab chitosan; antimicrobial; antitumor; Lentinula edodes

34

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35

36 Highlights

37

38 ► Chitosan was prepared by alkaline N-deacetylation of chitin from shiitake and crab.

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39 ► Chitosan exhibited excellent antimicrobial activities against pathogenic bacteria.

40 ► Shiitake chitosan C120 showed inhibition zones of 16.4-26.8 mm at 0.5 mg/ml.

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41 ► Chitosan showed a moderate anti-proliferative effect on IMR 32 and Hep G2 cells.

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42 ► Chitosan from shiitake stipes showed better antimicrobial and antitumor activities.

43

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44

45 1. Introduction
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46 Chitin exists widely in the exoskeletons of invertebrates, insect cuticles and fungal

47 cell walls and is the second abundant biopolymer on earth after cellulose (Knorr, 1984).
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48 Chitin is a linear nitrogenous polysaccharide which is composed of N-acetylglucosamine

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49 linked via β-1,4-glycosidic bond and classified into α-, β- and γ-chitin (Cabib, 1981). The
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50 color of chitin is light yellow to brown and the appearance of chitin is flocculence or
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51 filiform solid, and chitin is insoluble in water (Tanigawa, Tanaka, Sashiwa, Saimoto, &

52 Shigemasa, 1992). With hot concentrated sodium hydroxide treatment, chitin can be
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53 deacetylated to produce chitosan (No & Mayers, 1997).

54 Chitosan, a hydrophilic polyelectronic polymer, contains positive charges in acidic

55 condition, and the properties of fluid behavior of chitosan can be influenced by the

56 molecular configuration, the number of hydrogen bonds and the electrostatic repulsion of

57 neighboring molecules (Launry, Doublier, & Cuverlier, 1986). The different sources and

58 the various preparation methods of chitin can affect the content of acetyl group, the

59 molecular weight and physicochemical properties of chitin and chitosan (Yen & Mau,

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60 2007a). The biological properties of chitin and chitosan have been listed, such as

61 biodegradability, biocompatibility and haemostatic, analgesic, anticholesterolemic,

62 antitumor, antimicrobial, and antioxidative activities (Aranaz et al., 2009). Besides, many

63 literatures have marked that chitin and chitosan exhibit positive functions and benefits; and

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64 therefore, chitin and chitosan have widely used on various industries, such as cosmetics,

65 water engineering, textile industry, food processing, health food, agriculture, photography,

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66 tissue engineering, wound healing, and drug delivery systems (Dutta, Dutta, & Tripathi,

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67 2004; Aranaz et al., 2009).

68 In mushroom and fermentation industries, tremendous amounts of fungal materials are

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69 discarded. However, these fungal wastes can be a good source of chitin and chitosan.

70 Using these fungal by-products to prepare functional value-added products is eco-friendly.

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71 Moreover, chitin and chitosan from the fungal source are more available than those from

72 crustacean shells, which contain high levels of inorganic materials, need demineralization
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73 and are unacceptable for vegetarians (Teng, Khor, Tan, Lim, & Tan, 2001).
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74 Shiitake mushroom (Lentinula edodes [Berkeley] Pegler) is a generally edible and

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75 medical mushroom and is one of the most cultivated mushrooms worldwide. Because of
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76 the tough texture, shiitake stipes are discarded normally. However, shiitake stipes are a

77 good source of fungal chitin and chitosan. The different preparations for chitin and chitosan
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78 from shiitake have been studied (Yen & Mau, 2007a,b), and the general physicochemical

79 characterization and antioxidant properties of chitin and chitosan from shiitake stipes have

80 also been evaluated (Yen & Mau, 2007b; Yen, Tseng, Li, & Mau, 2007). The objective of

81 this study was to evaluate the antimicrobial and antitumor activities of chitosan from

82 shiitake stipes, which were prepared by different N-deacetylation treatments. Commercial

83 chitin and chitosan derived from crab shells were also used for comparison.

84

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 85 2. Materials and methods

86 2.1. Materials and chemicals

87 Air-dried shiitake stipes (strain 271) were purchased at a local mushroom farm in

88 Taichung County, Taiwan. Fresh shiitake stipes normally with 90% moisture content have

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89 been dried at 40-60 ºC hot air for 22 h. Three air-dried samples (~200 g each) were

90 randomly selected and ground into a coarse powder (0.4 mm). Sodium hydroxide was

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91 obtained from Wako Pure Chemical Co. (Osaka, Japan). Potassium permanganate, L-

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92 glutamine, non-essential amino acids, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium

93 bromide (MTT), crude crab chitin and crab chitosan were the products of Sigma Chemical

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94 Co. (St. Louis, MO, USA). Commercial crab α-chitosan from the snow crab (Chionoecetes

95 opilio) was obtained from Dalian City, Liaonin, China. Ethanol (95% pure) was supplied
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96 by Taiwan Tobacco & Wine Monopoly Bureau (Taipei, Taiwan). Dulbecco’s modified

97 eagle medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin-glutamine

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98 (PSG), trypan blue stain 0.4%, trypsin-EDTA, plate count agar (PCA) and tryptic soy broth
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99 (TSB) were purchased from Difco Laboratories (Detroit, MI, USA). Other reagents were of
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100 analytical grade.

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101

102 2.2 Preparation of chitin and chitosan

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103 The preparation of crude chitin and chitosan from shiitake stipes was according to the

104 methods of Yen and Mau (2007a,b), and the purified crab chitin and crab chitosan were

105 produced from crude crab chitin following the methods of Yen, Yang and Mau (2008) and

106 Yen, Yang and Mau (2009). Shiitake stipe powder was treated with 1 N sodium hydroxide

107 solution at the ratio of 1:10 (w/v) at 100 ºC for 3 h to remove protein whereas the crude

108 chitin from crab shells was treated with 1 N hydrochloric acid solution at room temperature

109 for 6 h to remove minerals and then conducted with similar sodium hydroxide treatment.

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110 The mixtures were separately filtered and washed with deionized water to neutral. Crude

111 shiitake chitin was treated further with ethanol (treatment B) or with potassium

112 permanganate (treatment C) to decolorize whereas crab shell chitin was treated with

113 treatment C. Following decolorization, two kinds of fungal chitin (chitin B and chitin C)

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114 and purified crab shell chitin (purified chitin) were washed with deionized water to neutral

115 and freeze-dried.

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116 For the purpose of N-deacetylation, 1 g of chitin B or C or purified chitin was treated

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117 with 30 ml of 40% sodium hydroxide solution at 105 ºC for 90 and 120 min, respectively.

118 After filtration, washing to neutral with deionized water and then freeze drying, the fungal

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119 chitosan obtained from different treatment times was designated as chitosan B90, B120,

120 C90 and C120 whereas the crab shell chitosan obtained were designated as chitosan CC90
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121 and CC120, respectively. For further uses, these freeze-dried chitosan products were

122 dissolved in 0.2 M acetic acid solution (pH = 6). Two different sources of commercial
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123 chitosan, crab chitosan (CCS) and crab α-chitosan (CCC), were used for comparison.
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124
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125 2.3. Preparation of Pathogenic Microorganisms

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126 Eight species of pathogenic bacteria, including three Gram-positive species, Bacillus

127 cereus (BCRC 10603), Listeria monocytogenes (BCRC 14848) and Staphylococcus aureus
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128 (BCRC 15211), and five Gram-negative species, Escherichia coli (BCRC 10675),

129 Flavobacterium sp. (BCRC 10098), Pseudomonas aeruginosa (BCRC 10261), Salmonella

130 typhimurium (BCRC 10747) and Vibrio parahaemolyticus (BCRC 10806), were obtained

131 from the Bioresource Collection and Research Center, Food Industry Research and

132 Development Institute.

133 Each bacterium was initially cultured in PCA at 30 ºC for 48 h to activate them and

134 then stored at 4 ºC for further uses. To measure the growth rate of each bacterium, a loopful

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135 of bacteria in PCA was transferred to 5 ml of TSB and incubated at 30 ºC for 12 h. Then,

136 the absorbance of medium containing bacteria was measured at 660 nm. The saline solution

137 (85%, w/v) was used to dilute the concentration of each bacterium to an applicable range for

138 further uses.

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139

140 2.4. Evaluation of Antimicrobial Activity

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141 The antimicrobial activity of different kinds of chitin and chitosan was measured

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142 according to the previous studies with some modification (Barry & Thornsberry, 1985;

143 Kim, Marshall, & Wei, 1995). Each culture (100 µl) was transferred by a sterilized microtip

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144 to a dish, and then 15 ml PCA was added to the dish and gently mixed. After 20 min, the

145 sterilized stainless steel ring (8 mm inner diameter) was put on the solidified PCA, and each
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146 concentration of different samples (50 µl) was put into the ring. After 20 min, the PCA

147 plates were put into the incubator at 30 ºC for 16 h. Antimicrobial activity was evaluated by
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148 measuring with a caliper the diameter of clear zone (growth inhibition zone) around the
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149 ring.
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150
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151 2.5. Cell Culture

152 The human neuroblastoma cell line (IMR 32, BCRC 60014) and the human
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153 hepatocellular liver carcinoma cell line (Hep G2, BCRC 60025) were obtained from the

154 Institute of Molecular Biology, National Chung Cheng University, Chiayi, Taiwan. Both

155 cells were maintained in DMEM medium, which contained 10% (v/v) FBS, 2 mM L-

156 glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM

157 sodium pyruvate, at 37°C in a humidified 5% CO2 incubator.

158

159 2.6. Cell Cytotoxicity Assay

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160 The cytotoxic effect of different samples was estimated by MTT reagent. The density

161 of IMR 32 and Hep G2 cell lines was adjusted to 8 × 103 and 6 × 103 cells/100 µl/well,

162 respectively, and seeded separately into 96 well plates and incubated at 37°C for 24 h.

163 Then, each well was filled with various concentrations of different samples and incubated

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164 with cells for 72 h. After 72 h, the medium was removed and fresh medium containing 0.5

165 mg/ml MTT was added into each well and incubated for 4 h. After 4 h, the medium was

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166 gently removed, and 100 µl of MTT lysis buffer was added in each well to react for 14-16 h.

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167 The absorbance of each well was measured at 540 nm by a VMax Kinetic ELISA

168 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).

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169

170 2.7. Statistical analysis

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171 Each preparation and measurement was conducted in triplicate, and the variance of

172 experimental data was analyzed by a Statistical Analysis System (SAS Institute, Inc., Cary,
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173 NC, USA). Duncan's multiple range test was used to estimate the variance of each group,
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174 and P value < 0.05 was considered statistically significant.

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175
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176 3. Results and discussion

177 3.1. Antimicrobial activity of chitin

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178 Crude chitin from crab shells at 10.0 mg/ml did not showed any antimicrobial activity,

179 as evidenced by finding no inhibition zone (Table 1). After purification, purified chitin only

180 showed an inhibitory effect on the growth of E. coli. At 10.0 mg/ml, both chitins B and C

181 from shiitake stipes could inhibit the growth of B. cereus and E. coli. However, chitin C

182 was more effective than chitin B. In addition, chitin C also showed antimicrobial activities

183 against Flavobacterium sp., S. typhimurium and V. parahaemolyticus. Chen (1998) had

184 proposed that the inhibition zones of 6 and 12 mm are regarded as good and better

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185 inhibition, respectively. In Table 1, all inhibition zones were around 11.3-15.1 mm and

186 could be considered as nearly better inhibition at 10.0 mg/ml.

187 It seems that the purification of crude chitin from crab shells, which removed minerals

188 and proteins and then decolorized, yielded purified chitin with an antimicrobial activity only

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189 against E. coli. Besides, the difference between chitin B and C was the decolorization

190 method in the preparation process. It reveals that chitin C treated with potassium

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191 permanganate (treatment C) was more effective in antimicrobial activity than chitin B

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192 treated with ethanol (treatment B). Furthermore, although both purified chitin and chitin C

193 were decolorized with treatment C, the chitin source was the factor responding for the

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194 antimicrobial activity. In other words, chitin from shiitake stipes exhibited more effective

195 antimicrobial activity than that from crab shells.

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196

197 3.2. Antimicrobial activity of chitosan

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198 Unlike chitin, all chitosan samples from shiitake stipes showed a dose-dependently
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199 inhibitory effect on eight species of Gram positive and Gram negative pathogenic bacteria
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200 at the concentration as low as 0.5 mg/ml (Table 2). At 0.5 mg/ml, all chitosan samples
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201 exhibited a higher inhibition zone than better inhibition of 12 mm except for chitosan B90

202 (11.4 mm). It indicates that these chitosan samples made from shiitake stipes were effective
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203 antimicrobial agents. At three concentrations tested, all chitosan samples inhibited most

204 effectively the growth of L. monocytogenes but least effectively the growth of B. cereus, as

205 evidenced by the inhibition zone of 22.7-28.8 mm and 11.4-19.7 mm, respectively.

206 Besides, chitosan C were more effective than chitosan B whereas chitosan 120 were more

207 effective than chitosan 90. This is consistent with the finding in Table 1 that chitin C was

208 more effective than chitin B. Overall, antimicrobial activities were in the descending order

209 of chitosan C120 > chitosan B120 > chitosan C90 > chitosan B90.

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210 Several mechanisms of antimicrobial activity of chitosan have been proposed. The

211 interaction between polycationic chitosan and anionic groups on the microbial cell

212 membranes causes the leakage of intracellular constitutes (Papineau, Hoover, Knorr, &

213 Farkas, 1991; Sudharshan, Hoover, & Knorr, 1992). Besides, chitosan can be a chelating

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214 agent to bind metals to inhibit the microbial growth and toxin production (Cuero, Osuji, &

215 Washington, 1991). Small molecular weight of chitosan can permeate into cell nucleus to

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216 inhibit the synthesis of RNA and protein and thereby, suppressing the growth of bacteria

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217 (Liu, Yun, Dong, Zhi, & Kang, 2001). However, better antimicrobial activity of chitosan

218 120 over chitosan 90 might be due to the fact that the prolonged reaction time yielded

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219 chitosan with higher degrees of N-deacetylation and more cationic groups.

220 Like chitosan from shiitake stipes, chitosan from crab shells showed a similarly dose-
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221 dependently inhibitory effect on all pathogenic bacteria at 0.5 mg/ml (Table 3). At 0.5

222 mg/ml, all chitosan samples exhibited a higher inhibition zone than better inhibition of 12
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223 mm except for chitosan CCS (10.8 mm). Obviously, all chitosan samples from crab shells
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224 were also effective antimicrobial agents but slightly less effective than chitosan from
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225 shiitake stipes. Similarly, at three concentrations tested, all chitosan samples inhibited most
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226 effectively the growth of L. monocytogenes but least effectively the growth of B. cereus, as

227 evidenced by the inhibition zone of 18.8-25.6 mm and 10.8-18.6 mm, respectively. Overall,
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228 antimicrobial activities were in the descending order of chitosan CC120 > chitosan CCC >

229 chitosan CC90 > chitosan CCS. However, based on the results in Tables 1 and 2,

230 antimicrobial activity of chitosan C120 was higher than that of chitosan CC120.

231 Tsai, Su and Chen (1999) evaluated the antimicrobial effect of three kinds of chitosan

232 with different degrees of N-deacetylation, 50%, 75% and 95%, on the growth inhibition in

233 general pathogens, B. cereus, L. monocytogenes, Shigella dysenteriae, S. aureus, V.

234 cholerae, V. parahaemolyticus and Candida albicans, and demonstrated that as the degree

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235 of N-deacetylation was higher, the antimicrobial activities were more effective. The

236 physico-chemical characterization of chitosan from shiitake stipes and crab shells used in

237 this study had been measured in previous studies (Yen & Mau, 2006; Yen et al., 2008,

238 2009; Yen & Mau, 2007a,b). The degrees of N-deacetylation of chitosan B90, B120, C90

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239 and C120 were 85.5%, 90.3%, 86.3% and 90.2%, respectively, and those of chitosan CC90,

240 CC120, CCC and CCS were 88.4%, 93.3%, 87.8% and 85.2%, respectively. Apparently,

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241 chitosan B120, C120, CC120 and CCC with high degrees of N-deacetylation exhibited

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242 better antimicrobial activities, regardless of their sources. This is consistent with the

243 finding of previous studies (Tsai et al., 1999).

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244 Chitosan is derived by partially N-deacetylation of chitin, which is classified into α-,

245 β- and γ-chitin (Cabib, 1981). α-Chitin, the most abundant in nature, has a structure of
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246 antiparallel chains and is found in the crabs, shrimps and lobsters whereas β-chitin found in

247 squid has intrasheet hydrogen bondings by parallel chains (Jang, Kong, Jeong, Lee, & Nah,
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248 2004). Chitin in cell walls of fungi such as mushrooms is classified as γ-chitin, which has a
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249 mixture of parallel and antiparallel chains, which is a combination of α- and β-chitin (Jang
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250 et al., 2004). Because of weak intermolecular hydrogen bonding, β-chitin is easier to be
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251 deacetylated than α-chitin (Chandumpaia, Singhpibulpornb, Faroongsarngc, & Sornprasit,

252 2004), which would produce chitosan with different physicochemical properties. After
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253 partially N-deacetylation, chitosan might conserve its original structure. Therefore, chitosan

254 from shiitake stipes could be γ-chitosan whereas chitosan from crab shells was α-chitosan.

255 However, with respect to antimicrobial activity in this research, γ-chitosan was better than

256 α-chitosan.

257 Chitin and chitosan generated from various sources had exhibited different

258 physicochemical, antimicrobial and antioxidant activities (Abdou, Nagy, & Elsabee, 2007;

259 Limam, Selmi, Sadok, & El-abed, 2011). Moreover, different decolorization treatments had

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260 affected the yields and proximate compositions of crude shiitake chitin (Yen & Mau, 2006).

261 However, Yen et al. (2007) showed that no obvious relationship was established between

262 decolorization method and antioxidant activity of chitosan from shiitake stipes. In this

263 research, it seems that different decolorization treatments also affected the antimicrobial

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264 activity.

265 The growth of S. aureus was inhibited as the molecular weights of chitosan increased

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266 whereas the growth of E. coli was suppressed as the molecular weight decreased (Zheng &

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267 Zhu, 2003). The molecular weights of shiitake chitosan B90, B120, C90 and C120 were

268 423.43, 394.69, 402.45 and 382.73 kDa, respectively whereas those of crab chitosan CC90,

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269 CC120, CCC and CCS were 512.53, 483.22, 212.93 and 548.75 kDa, respectively (Yen &

270 Mau, 2007a; Yen et al., 2009). Among fungal chitosan, B120 and C120 with low molecular
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271 weights and high degrees of N-deacetylation had a better inhibition on bacterial growth

272 whereas among crab chitosan, CC120 and CCC exhibited a notable inhibition. Errington,
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273 Harding, Varum and Illum (1993) proposed that the antimicrobial model of chitosan is to
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274 attach on the surface of bacteria, and different kinds of bacteria, sources of bacteria, degrees
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275 of N-deacetylation, molecular weights and working concentrations would affect the
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276 antimicrobial activity.

277
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278 3.3. Antitumor activity of chitosan

279 Shiitake and crab chitosan exhibited a dose-dependently anti-proliferative effect on

280 IMR 32 cells (Fig. 1). Obviously, shiitake chitosan was more effective than crab chitosan.

281 At 5 mg/ml, the viabilities of IMR 32 cells incubated with chitosan B90, B120, C90 and

282 C120 were 78.0%, 70.3%, 71.2% and 68.8%, respectively whereas those with chitosan

283 CC90, CC120, CCS and CCC were 79.3%, 76.8%, 85.0% and 82.3%, respectively. The

284 anti-proliferative effect of shiitake chitosan on Hep G2 cells was more noticeable than that

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285 of crab chitosan (Fig. 2). At 5 mg/ml, the viability of Hep G2 cells incubated with chitosan

286 B90, B120, C90 and C120 were 65.0%, 60.4%, 62.3% and 61.3%, respectively whereas

287 those with chitosan CC90, CC120, CCS and CCC were 73.5%, 70.7%, 82.9% and 80.6%,

288 respectively. Chitosan B120, C120 and CC120 with high degrees of N-deacetylation

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289 exhibited more effectively inhibitory effect on the growth of IMR32 and Hep G2 cells.

290 However, the concentration at 50% inhibition (IC50) was not established for both cells,

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291 indicating that chitosan showed a moderate anti-proliferative effect on both cells.

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292 Chitinous materials produced by chitinase could inhibit the survival of CT 26 cell line

293 (Liang, Chen, Yen, & Wang, 2007) and the hydrolyzates derived from chitinous materials

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294 by bromelain also exhibited antitumor and antimicrobial activities (Wang et al., 2008). If

295 shiitake and crab chitosan in this research could be hydrolyzed to chitinous materials with
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296 smaller molecular weights and higher degrees of N-deacetylation and their anti-proliferation

297 on cancer cells would be greater enhanced.

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298 Chitosan C120 showed the best antimicrobial and antitumor activities among
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299 chitosans from shiitake stipes whereas CC120 was the best among those from crab shells.
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300 However, C120 was more effective than CC120. The degrees of N-deacetylation of C120
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301 and CC120 were 90.2% and 93.3% (Yen & Mau, 2007a; Yen et al., 2009). The degree of

302 N-deacetylation usually affects the antimicrobial activity of chitosan. Unfortunately, the
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303 degree of N-deacetylation was not the major factor accounting for their difference in

304 antimicrobial and antitumor activities.

305 The molecular weights of C120 and CC120 were 382.73 and 483.22 kDa, respectively

306 (Yen & Mau, 2007a; Yen et al., 2009). The molecular weight and degree of N-

307 deacetylation are two crucial factors for inhibiting the growth of cancer cells. Besides, the

308 high molecular weight chitosan was less effective than the low molecular weight chitosan

309 oligosaccharide. (Azuma, Osaki, Minami, & Okamoto, 2015). Moreover, chitosan

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310 oligosaccharides (COS) obtained from fungi had been used to study their effect on

311 antitumor and antimetastatic in Hep G2 cells. COS could inhibit cell proliferation by

312 retarding cell cycle at S phase (Shen, Chen, Chan, Jeng, & Wang, 2009). Therefore, the

313 better antitumor activity of C120 might be due to its low molecular weight. Similarly, the

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314 low molecular weight of C120 might also be responsible for its better antimicrobial activity.

315 The structure of chitin and chitosan from shiitake stipes and crab shells used in this

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316 study had been examined previously (Yen & Mau, 2007a; Yen et al., 2009). Under electron

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317 microscopic examination, C120 showed the aggregated flakes with dense and firm structure

318 and without porosity whereas CC120 exhibited distinctly ordered porosity and arranged

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319 microfibrillar crystalline. The wide-angle X-ray diffraction (WAXD) pattern of C120

320 exhibited characteristic crystalline peaks at 2θ = 9.65° and 19.87° whereas CC 120 showed
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321 two crystalline reflections at 8.8-9.0° and 18.9-19.1°. The two characteristic crystalline

322 peaks of C120 and CC120 with slightly fluctuated diffraction angles found in the WAXD
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323 patterns indicated that two types of α- and γ-chitosan exhibited comparable degree of
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324 crystallinity. However, fungal chitosan contained high amount of polysaccharides in the
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325 complex (Yen & Mau, 2006) and could not exhibit microfibrillar or filamentous structure as
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326 crab chitosan did. The difference in the structure between chitosans from shiitake stipes

327 and crab shells might also be accounting for the slight efficiency difference in antimicrobial
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328 and antitumor activities.

329

330 4. Conclusion

331 Crude chitin from crab shells did not show any antimicrobial activity but purified

332 chitin showed an antimicrobial activity against E. coli. Chitin from shiitake stipes exhibited

333 a better inhibitory effect on bacterial growth than chitin from crab shells. After purification,

334 chitosan from shiitake stipes and carb shells exhibited excellent antimicrobial activities

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335 against eight species of pathogenic bacteria. However, chitosan from shiitake stipes was

336 slightly more effective than that from crab shells. Among chitosan samples, shiitake

337 chitosan C120 was the most effective. In addition, shiitake and crab chitosan showed a

338 moderate anti-proliferative effect on IMR 32 and Hep G2 cells. However, shiitake chitosan

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339 showed a better anti-proliferative effect than carb chitosan. Based on the results obtained,

340 shiitake and crab chitosan were strong antimicrobial agents and moderate antitumor agents.

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341

342 Acknowledgements

343 This study was supported by the Ministry of Science and Technology, Taiwan,

344 Republic of China (MOST-104-2911-I-005-301, NSC-103-2911-I-005-301) and the

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345 Ministry of Education, Taiwan, R.O.C. under the ATU plan.

346

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347 References

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348 Abdou, E. S., Nagy, K. S. A., & Elsabee, M. Z. (2007). Extraction and characterization of

349 chitin and chitosan from local sources. Bioresource Technology, 99, 1359–1367.

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Table 1. Antimicrobial activities of chitin from shiitake stipes and crab shells
Inhibition zone diameter a (mm) at 10.0 mg/ml
Shiitake stipe Crab shell
Strains Chitin B Chitin C Crude chitin Purified chitin
Gram positive
Bacillus cereus 11.3±0.2B 12.5±0.5A –b –
Listeria monocytogenes – – – –

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Staphylococcus aureus – – – –

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Gram negative
Escherichia coli 13.6±0.2B 15.1±0.2A – 13.0±0.6B

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Flavobacterium sp. – 11.5±0.8 – –
Pseudomonas aeruginosa – – – –
Salmonella typhimurium – 11.8±0.9 – –

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Vibrio parahaemolyticus – 12.4±0.6 – –
a
Each value is expressed as mean ± SE (n = 3). Means with different letters within a row

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differ significantly (P < 0.05).
b
No inhibition.
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Table 2. Antimicrobial activities of chitosan B and C from shiitake stipes
Inhibition zone diameter (mm) a

an
Gram positive Gram negative
Conc. Bacillus cereus Listeria Staphylococcus Escherichia Flavobacterium Pseudomonas Salmonella Vibrio

M
(mg/ml) monocytogenes aureus coli sp. aeruginosa typhimurium parahaemolyticus
B90 0.5 d11.4±0.8B c22.7±0.3C b17.8±0.3C c18.5±0.3A d17.2±0.3C b18.2±0.1C c19.7±0.3B c17.3±0.2C
1.0 c12.6±0.3AB c24.3±0.4B c19.2±0.4B d18.8±0.4A d19.5±0.4B c20.2±0.3B c20.8±0.3A c18.4±0.2B

B120
10.0
0.5
d13.7±0.4A
b15.4±0.4B ed
d25.4±0.1A
b24.3±0.4C
c20.6±0.2A
a19.2±0.4B
d19.5±0.3A d20.9±0.2A
b20.2±0.4B b19.8±0.2C
c21.2±0.1A c21.4±0.3A
a21.0±0.5B a21.8±0.9A
c21.7±0.2A
b18.7±0.1C
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1.0 b16.9±0.5B b26.2±0.4B b20.9±0.3A b22.3±0.2A b21.4±0.4B b22.2±0.3A b22.2±0.8A b19.8±0.1B
10.0 b18.7±0.4A b27.6±0.3A b21.1±0.2A b22.7±0.3A b22.8±0.4A b23.1±0.5A b22.8±0.2A b21.9±0.2A
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C90 0.5 c14.4±0.5B b23.6±0.5B b18.2±0.1C c19.0±0.3A c19.3±0.2C b18.4±0.3B b20.7±0.2B b18.3±0.2B
1.0 b16.4±0.7A b25.8±0.2A c19.6±0.1B c20.2±0.7A c20.8±0.1B c20.4±0.3A b22.1±0.4A c18.2±0.2B
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10.0 c16.7±0.2A c26.8±0.3A bc20.6±0.4A c20.5±0.1A c21.9±0.2A c20.8±0.4A c21.2±0.1AB c21.6±0.2A

C120 0.5 a16.4±0.4C a26.8±0.3B a19.7±0.4C a21.8±0.2B a20.2±0.4C a20.7±0.1B a22.1±0.4B a20.9±0.2B
1.0 a18.4±0.4B a27.2±0.1B a21.6±0.2B a23.8±0.7A a22.2±0.4B a23.1±0.4A a22.8±0.2B a21.1±0.2B
10.0 a19.7±0.3A a28.8±0.2A a23.7±0.2A a25.0±0.3A a24.4±0.6A a23.9±0.2A a23.4±0.2A a24.1±0.4A

a
Each value is expressed as mean ± SE (n = 3). Means with different capital letters within a column of a specific chitosan and means with

different lowercase letters within a column of a specific concentration differ significantly (P < 0.05).

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Table 3. Antimicrobial activities of commercial chitosan from crab shells
Inhibition zone diameter (mm) a
Gram positive Gram negative

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Conc. Bacillus cereus Listeria Staphylococcus Flavobacterium Pseudomonas Salmonella Vibrio
(mg/ml) monocytogenes aureus Escherichia coli sp. aeruginosa typhimurium parahaemolyticus
CC90 0.5

ed
b12.1±0.4A c19.3±0.5B c18.6±0.2B d17.9±0.2B d16.5±0.4B c17.2±0.4B b20.1±0.3AB b19.4±0.2B
1.0 c12.5±1.0A c21.4±0.2A c18.3±0.1B bc18.6±0.3B b16.5±0.5B c17.0±0.2B b19.5±0.2B b19.4±0.3B
10.0 c13.4±0.2A c22.2±0.1A c20.1±0.3A b21.1±0.2A c19.1±0.3A c19.2±0.4A b20.8±0.3A c21.2±0.3A
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CC120 0.5 a14.2±0.3C a22.6±0.3C a20.4±0.2B a21.8±0.2B a20.4±0.4B a21.6±0.3B a21.2±0.3B a20.2±0.3B
1.0 a16.3±0.3B a23.6±0.3B b19.4±0.2C a21.8±0.2B a19.8±0.2B a21.2±0.3B a20.8±0.2B a21.2±0.4B
ce

10.0 a18.6±0.2A a25.6±0.1A a21.6±0.2A a23.4±0.2A a21.7±0.3A a24.1±0.3A a22.0±0.2A a22.0±0.4A

CCS 0.5 c10.8±0.3B c18.8±0.2C c18.6±0.4A c19.2±0.2B c17.8±0.1B c16.8±0.3B c19.1±0.3A c18.2±0.1B
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1.0 c12.4±0.4A d19.8±0.2B c18.6±0.4A c18.2±0.2C b17.2±0.2B c17.2±0.3AB c18.1±0.2B c18.2±0.1B

10.0 c13.1±0.3A d20.8±0.2A d19.2±0.3A c20.1±0.2A c19.1±0.3A d18.1±0.2A c19.1±0.3A d18.7±0.2A
CCC 0.5 b12.4±0.3C b21.2±0.6B b19.6±0.3B b20.8±0.4AB b18.2±0.2B b20.2±0.3B b20.5±0.2A b19.5±0.4B
1.0 b13.7±0.2B b22.3±0.2B a20.1±0.2AB b19.6± 0.5B a19.2±0.6AB b20.2±0.1B c18.6±0.4B b20.1±0.5AB
10.0 b16.2±0.2A b25.2±0.1A b20.9±0.2A b21.4±0.4A b20.1±0.3A b21.5±0.2A b21.2±0.2A b21.1±0.3A

a
Each value is expressed as mean ± SE (n = 3). Means with different capital letters within a column of a specific chitosan and means with

different lowercase letters within a column of a specific concentration differ significantly (P < 0.05).
22

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ed
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 Figure caption

Fig. 1 Effect of chitosan from shiitake stipes (A) and crab shells (B) on the cell viability of

IMR 32 cells. Each bar is expressed as mean ± SE (n = 3). Means with different

letters within a specific concentration differ significantly (P < 0.05).

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Fig. 2 Effect of chitosan from shiitake stipes (A) and crab shells (B) on the cell viability of

Hep G2 cells. Each bar is expressed as mean ± SE (n = 3). Means with different

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letters within a specific concentration differ significantly (P < 0.05).

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p te
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Figure 1

120
A Chitosan B 90

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Chitosan B 120
A
A AA

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AAAA Chitosan C 90
100 A A
A A Chitosan C 120
A AB

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AB
80 B A
Cell viability (%)

BB
C

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60

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40

20
M
0
0.0 0.1 0.5 1.0 5.0
d

Concentration (mg/mL)
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120 CC 90
CC 120
B CCS
p

AA A CCC
A AA A A
100 A AA A
ce

AB A AB A
B A
AB
80 B
Cell viability (%)

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60

40

20

0
0.0 0.1 0.5 1.0 5.0
Concentration (mg/mL)

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Figure 2

120

t
A Chitosan B 90
A

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A AA A A AA AA A Chitosan B 120
100 Chitosan C 90
B
Chitosan C 120

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A AB
80 AB
Cell viability (%)

B
A
B ABAB

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60

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40

20
M
0
0.0 0.1 0.5 1.0 5.0
d
Concentration (mg/mL)

120 CC 90
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A CC 120

AB B AB AB A
B CCS
A AA A CCC
100 AB
p

B
AB B A AB
AA
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80 BB
Cell viability (%)

60
Ac

40

20

0
0.0 0.1 0.5 1.0 5.0
Concentration (mg/mL)

26
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27
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