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1 Research Paper 研究报告 Acta Microbiologica Sinica 48(2) 164~168; 4 February 2008 ISSN ; CN /Q 壳聚糖抑菌机制的初步研究 1, 2, 陈威 3, 吴清平 2*, 张菊梅 2, 吴慧清 ( 1, ) ( 2, ) ( 3, ) 摘要 :,,,,, (0.5%) ATP, ATP 2 4, ATP, 关键词 ; ; ; 中图分类号 Q935 文献标识码 A 文章编号 (2008) N- β-1, 4,, [1~3],,,, [4], ph,,, [5], 4, ATP, 1 材料和方法 1.1 材料 (Escherichia coli)atcc8099 (Staphylococcus aureus)atcc6538 (Candida albicans)atcc10231 (Pseudomonas aeruginosa)cmcc10104 (Bacillus subtilis)atcc9372 基金项目 : (2006A , 2007B ) * Tel/Fax: ; wuqp@gdas. ac. cn 作者简介 : (1983 ),,, frog18575@ yahoo. com. cn 收稿日期 : ; 修回日期 :

2 / (2008) 48(2) ,, NaOH ATP 5kDa 10kDa 20kDa 50kDa 100kDa, 90% SHG-C 1.2 不同分子量壳聚糖抑菌活性的检测 0.5%, (W/V) 1% 0.5% 0.25% 0.125% 0.063% 0.031%, 10mol/L NaOH ph 5.5 ( ), 10 5 cfu/ml, 0.5% (ph 5.5), 0.1mL ( ), 3, 37 24h [6] : B=(O A)/O 100%, O,A 1.3 金属离子对壳聚糖抑菌活性的影响 50kDa, 0.5% (W/V) 0.5% 0.25% 0.125% 0.063% 0.031% 0.016%, 0.5%, ph 5.5, 10 5 cfu/ml, 3, 0.1mL, 24h, 1.4 不同分子量壳聚糖引起金黄色葡萄球菌和白色念 [7] 珠菌钾离子渗漏的研究, 5kDa 1% 50kDa 1%, 0.5%, ph 5.5 2, OD 600 =0.9,, 0.5%,, 15min 30min 1h 2h 4h 8h,, 30, 不同分子量壳聚糖引起金黄色葡萄球菌和白色念 [7] 珠菌 ATP 渗漏的研究, 1min 2min 5min 10min 20min,, 30, 3 ATP, 4SHG-C ATP 2 结果和分析 2.1 不同分子量壳聚糖的抑菌活性 1,,,,,,,,,,, 2.2 金属离子对壳聚糖溶液抑菌效果的影响 50kDa 0.5%,, ( 2),,,,

3 166 Wei Chen et al. /Acta Microbiologica Sinica (2008) 48(2) Fig. 1 图 1 不同分子量壳聚糖的抑菌活性 Antibacterial activity of chitosan. A: 5kDa; B: 10kDa; C: 20kDa; D: 50kDa; E: 100kDa. ( ): Pseudomonas aeruginosa ; ( ) : Escherichia coli; ( ): Bacillus subtilis; ( ): Staphylococcus aureus; ( ): Candida albicans. 1% 0.5%, 50kDa 1%, 图 2 金属离子对壳聚糖抑制金黄色葡萄球菌 (A) 和白色念珠菌 (B) 效果的影响 Fig. 2 Influence of metal ion to antibacterial activity of chitosan against Staphylococcus aureus (A) and Candida albicans (B). ( ): K + ; ( ): Na + ; ( ): Mg 2+ ; ( ): Ca 壳聚糖溶液引起金黄色葡萄球菌和白色念珠菌钾离子的渗漏情况 5kDa 50kDa 1%, ( 3) 0.5% (ph 5.5),,, 0.5% 5kDa 图 3 金黄色葡萄球菌 (A) 和白色念珠菌 (B) 经过壳聚糖处理后钾离子渗漏情况 Fig. 3 Leakage of potassium of Staphylococcus aureus (A) and Candida albicans (B) treated by chitosan. ( ): 50kDa; ( ): 5kDa; ( ): 0.5% acetic acid.

4 / (2008) 48(2) 167, ( 11mg/mL, 10.5mg/mL),, 2.4 壳聚糖溶液引起金黄色葡萄球菌和白色念珠菌 ATP 渗漏的情况 1%, ATP ( 4) 50 kda ATP 5kDa, ATP,, ATP,, ATP,,, 3 讨论,, [8],,,,,,,,,,,, [9], [10, 11],,,, ATP,,, ( 5kDa) [12],, DNA 参考文献图 4 金黄色葡萄球菌 (A) 和白色念珠菌 (B) 经过壳聚糖溶液处理后 ATP 渗漏情况 Fig. 4 Leakage of ATP of Staphylococcus aureus (A) and Candida albicans (B) treated by chitosan.( ): 50kDa ; ( ) : 5kDa; ( ) : 0.5%acetic acid. [1] Kim SK, Niranjan R. Enzymatic production and biological activities of chitosan oligosaccharides (COS): A review. Carbohydrate Polymers, 2005, 62: [2] Wang XH, Du YM, Fan LH, et al. Chitosan- metal complexes as antimicrobial agent: Synthesis, characterization

5 168 Wei Chen et al. /Acta Microbiologica Sinica (2008) 48(2) and Structure-activity study. Polymer Bulletin, 2005, 55: [3] Shepherd R, Reader S, Falshaw A. Chitosan functional properties. Glycoconjugate Journal, 1997, 14: [4] Zheng LY, Zhu JF. Study on antimicrobial activity of chitosan with different molecular weights. Carbohydrate Polymers, 2003, 54: [5] Helander IM, Ahvenainen R, Rhoades J, et al. Chitosan disrupts the barrier properties of the outer membrane of Gram-negative bacteria. International Journal of Food Microbiology, 2001, 71: [6] Gerasimenko DV, Avdienko ID, Bannikova GE, et al. Antibacterial effects of water-soluble low-molecular -weight chitosans on different microorganisms. Applied Biochemistry and Microbiology, 2004, 40: [7] Fitzgerald DJ, Stratford M, Gasson MJ, et al. Mode of antimicrobial action of vanillin against Escherichia coli, Lactobacillus plantarum and Listeria innocua. Journal of Applied Microbiology, 2004, 97: [8],,,.. (Chinese Journal of Antibiotics), 2006, 31(6): [9] Carlos P, Waldo AM, Hazel P, et al. Chitosan: An Attractive Biocompatible Polymer for Microencapsulation. Macromol Biosci, 2003, 3: [10] Wang XH, Du YM, Fan LH, et al. Chitosan- metal complexes as antimicrobial agent: Synthesis, characterization and Structure-activity study. Polymer Bulletin, 2005, 55: [11],.. (Journal of Natural Science of Heilongjiang University), 2003, 20: [12] Tokura SK, Ueno S, Nishi N, et al. Molecular weight dependent antimicrobial activity of chitosan. Macromol Symp, 1997, 120: 1 9. Antibacterial mechanism of chitosan Wei Chen 1, 2, 3, Qingping Wu 2*, Jumei Zhang 2, Huiqing Wu 2 ( 1 Wuhan Institute of Virology, Chinese Academy of Science, Wuhan , China ) ( 2 Guangdong Provincial Key Laboratory of Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou , China ) ( 3 Graduate School of Chinese Academy of Science, Beijing , China ) Abstract: In this study, we found that many metal ions could affect the antibacterial activity of chitosan, such as K +, Na +, Mg 2+ and Ca 2+. High concentration (about 0.5%) of metal ions could make chitosan completely lose its antibacterial activity, except for the effect of Na + on the antibacterial activity against Staphylococcus aureus. We also found that chitosan could weaken the barrier function of the outer membrane of different microorganisms. Large amount of K + and ATP leakages were observed during exposure of Staphylococcus aureus and Candida albicans to chitosan. Both 50kDa chitosan and 5kDa chitosan had good antibacterial activity against Staphylococcus aureus and Candida albicans, but the 50kDa chitosan could make nearly two to four times amount of K + and ATP leakages than the 5kDa chitosan. There might have been different antibacterial mechanisms of high-molecular-weight and low-molecular-weight chitosan. Keywords: chitosan; metal ion; antibacterial activity; mechanism Supported by the Programs for Science and Technology Development of Guangdong Province (2006A ,2007B ) * Corresponding author. Tel/Fax: ; wuqp@gdas. ac. cn Received: 17 September 2007/Revised: 23 October 2007

activities than commercial antibacterial agents. Moreover, "Extract Cleanser was safe to organisms as the result of toxicity evaluation identified by

activities than commercial antibacterial agents. Moreover, Extract Cleanser was safe to organisms as the result of toxicity evaluation identified by Journal of China Institute of Technology Vol.32-2005.5 Assessment of Property and Function of All-Purpose Natural Anti-Bacteria Cleanser 1 2 3 * Li-chun Wu Ying-chien Chung Sheng-shih Chang 1 2 3 ( ) (APEO)