微生物学通报 Microbiology China 研究报告 NOV 20, 2010, 37(11): by Institute of Microbiology, CAS 茶多酚对金黄色葡萄球菌和铜绿假单胞菌的抑菌机理 * 钱丽红陶妍

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1 微生物学通报 Microbiology China 研究报告 NOV 20, 2010, 37(11): by Institute of Microbiology, CAS 茶多酚对金黄色葡萄球菌和铜绿假单胞菌的抑菌机理 * 钱丽红陶妍谢晶 ( ) 摘要 : 以革兰氏阳性的金黄色葡萄球菌和革兰氏阴性的铜绿假单胞菌为试验菌, 通过测定茶多酚与两种菌作用前后细菌培养液的电导率和可溶性总糖的变化, 以及菌体在磷代谢和蛋白质表达方面的变化, 初步阐明了茶多酚对这两种菌的抑菌机理研究结果表明, 茶多酚对金黄色葡萄球菌和铜绿假单胞菌均有抑菌活性, 但对金黄色葡萄球菌的抑菌活性更强经茶多酚处理后, 细菌培养液的电导率和总糖浓度均增大, 表明了茶多酚可破坏细胞膜的结构导致细胞通透性增加, 进而使细胞内容物外泄另一方面, 经茶多酚处理后的两种菌对磷的消耗量降低, 以致严重影响了核酸磷脂等细胞重要成分的合成以及能量代谢 ; 通过 SDS-PAGE 分析, 证实茶多酚可以阻碍细菌蛋白质的正常表达, 以致影响其细胞的结构组成以及酶的催化活性, 最终导致细菌正常生理功能的丧失关键词 : 茶多酚, 金黄色葡萄球菌, 铜绿假单胞菌, 抑菌机理 Antimicrobial Mechanisms of Tea Polyphenol Against Staphyloccocus aureus and Pseudomonas aeruginosa QIAN Li-Hong TAO Yan * XIE Jing (College of Food Science and Technology, Shanghai Ocean University, Shanghai , China) Abstract: The antimicrobial mechanisms of tea polyphenol against Gram-positive S. aureus and Gram-negative P. aeruginosa were investigated by determining the changes in electric conductivity and total sugar concentration of broth for bacteria, as well as the changes in phosphorous metabolism and protein expression of bacteria. As the results showed, tea polyphenol had antimicrobial activity against S. aureus and P. aeruginosa, but stronger against the former. The facts that the electric conductivity and total sugar concentration of microbial broth increased indicated that tea polyphenol could damage the structure of cell membrane, which resulted in the increase of permeability of cell membrane and release of cell components. Besides, the consumption of phosphorous decreased in the tea-polyphenol-treated bacteria, which seriously influenced the synthesis of important cell components such as nucleic acid and phospholipid and energy metabolism. SDS-PAGE assay 基金项目 : 2008 (No ); 2009 (No. 09XD ) * 通讯作者 :Tel: ; : ytao@shou.edu.cn 收稿日期 : ; 接受日期 :

2 : 1629 demonstrated that tea polyphenol could block the protein expression in bacteria, which influenced the cell structure composition and catalyzing activity of enzyme, and finally leading to the lost of normal physiological function of bacterium. Keywords: Tea polyphenol, S. aureus, P. aeruginosa, Antimicrobial mechanism,,, 60% 80% [1],, [2 3] ; [4 6], [7],, [8] ; María [9], Weiduo [10],,,,,, 1 材料与方法 1.1 材料与试剂 (Staphyloccocus aureus) (Pseudomonas aeruginosa) ; ( ) ; ;, 1.2 实验方法茶多酚的抑菌活性测定 :, 37 C 12 h (5000 r/min, 10 min),,, 10 4 CFU/mL, 2 g/l, min, 50 μl, 37 C 24 h, ; 3 = ( ) / 100% 菌液电导率的测定 : [11] 0.1 mol/l (ph 7.4) 3, 10 8 CFU/mL, 5 ml, 1 g/l 2 g/l, 10 min ; 菌体可溶性总糖的测定 : [12] 5 ml (1 g/l 2 g/l), h 1 ml (11000 r/min, 2 min), 5, 50 μl, 200 μl, 5 min, 10 min, 10 min, 620 nm ; ; 菌体磷代谢的测定 : [13],, 37 C 12 h (5000 r/min, 10 min),, 0.1 mol/l (ph 7.4), 10 6 CFU/mL ; 0.5 ml 0.5 ml (1 g/l), 200 μl 200 μl (2 g/l), h 0.1 ml, -

3 1630 微生物学通报 2010, Vol.37, No.11, 630 nm 菌体总蛋白的聚丙烯酰胺凝胶电泳 (SDS-PAGE): Ke [14], 0.1 mol/l (ph 7.4), OD 600 = 0.5, 5 ml (2 g/l), h 5 ml (10000 r/min, 5 min), 50 ml 200 μl, 5 min, 40 μl SDS-PAGE 4% 10%; 0.1% R-250, 25% 7% 2.2 茶多酚对菌体细胞膜通透性的影响经茶多酚处理后细菌培养液电导率的变化 :,,,, [16] g/l,,,, 2 结果与分析 2.1 茶多酚对两种菌的抑菌活性 1,,, 2 g/l 20 min 45.2%, 18.0%, 40 min (P < 0.05), Negi [15] 图 2 茶多酚处理后金黄色葡萄球菌菌液的电导率变化 Fig. 2 Changes in electric conductivity of broth for S. aureus 图 1 茶多酚对两种菌的抑菌活性 Fig. 1 Antimicrobial activity of tea polyphenol against two types of bacteria 图 3 茶多酚处理后铜绿假单胞菌菌液的电导率变化 Fig. 3 Changes in electric conductivity of broth for P. aeruginosa

4 : g/l 60 min 80 min,,,,, [17] ;,,,, 经茶多酚处理后细菌培养液中总糖浓度的变化 :,,,, [18] 4 5, 2 g/l,,, ;,,, 1 g/l 1 h,, 图 5 茶多酚处理后铜绿假单胞菌菌液中总糖浓度的变化 Fig. 5 Changes in total sugar concentration of broth for P. aeruginosa 2.3 茶多酚对细菌磷代谢的影响, [19],, [13] 6 7,,,,, 图 6 茶多酚处理后金黄色葡萄球菌的磷代谢变化 Fig. 6 Changes in phosphorous metabolism for S. aureus 图 4 茶多酚处理后金黄色葡萄球菌菌液中总糖浓度的变化 Fig. 4 Changes in total sugar concentration of broth for S. aureus 2.4 茶多酚对细菌蛋白质表达的影响 8 1 4, 1 3 h,,,

5 1632 微生物学通报 2010, Vol.37, No.11, 6 h,, ( ) 图 7 茶多酚处理后铜绿假单胞菌的磷代谢变化 Fig. 7 Changes in phosphorous metabolism for P. aeruginosa 图 8 茶多酚处理后两种菌菌体总蛋白的 SDS-PAGE 电泳图谱 Fig. 8 SDS-PAGE patterns of total proteins for two types of bacteria : 1 4: 0 3 h; 5 11: 0 6 h; M:. Note: 1 4: 0 3 h of S. aureus; 5 11: 0 6 h of P. aeruginosa; M: Molecular mass markers. 3 讨论,,,, ;,,,, [18],, [17],,, SDS-PAGE, HepG2 Bcl-2 [20],,, ;,,, [21],, 参考文献 [1],,,.. 1. :, 1998: [2],,,.., 1997(5): 3 5. [3] Almajano MP, Carbó R, Jiménez JAL, et al. Antioxidant and antimicrobial activities of tea infusions. Food Chemistry, 2008, 108(1): [4],,,.. :, 2008, 34(6):

6 : 1633 [5],.., 1996, 14(2): [6],,,.., 2009, 36(3): 3 5. [7],,.., 2008, 30(3): [8],,,. (Nisin)., 2008, 186(9): [9] Tumay Y, Belma A, Sahlan O. Antimicrobial and antioxidant activities of Russula delica Fr. Food and Chemical Toxicology, 2009, 47(8): [10] Weiduo S, Joshus G, Rong T, et al. Bioassay-guided purification and identification of antimicrobial components in Chinese green tea extract. Journal of Chromatography A, 2006, 1125(2): [11],,.., 2008, 17(3): [12].. :, 1995: [13],,,.., 2006, 26(11): [14] Ke X, Xi GC, Ming K, et al. Effect of oleoyl-chitosan nanoparticles as a novel antibacterial dispersion system on viability, membrane permeability and cell morphology of Escherichia coli and Staphylococcus aureus. Carbohydrate Polymers, 2009, 76(1): [15] Negi PS, Jayaprakasha GK, Jena BS. Antioxidant and antimutagenic activities of pomegranate peel extracts. Food Chemistry, 2003(3): [16] MT, JM.. 11.,. :, 2009: [17].., 2008(2): [18],,,.., 2009, 30(5): [19].. 2. :, 2008: [20],,. HepG2., 2005, 34(1): [21],,.. :, 2009: 稿件书写规范专论与综述论文的撰写要点,,,,,,,,,,,,, : 5,

Microsoft Word - 陈威_new_.doc

Microsoft Word - 陈威_new_.doc Research Paper 研究报告 Acta Microbiologica Sinica 48(2) 164~168; 4 February 2008 ISSN 0001-6209; CN11-1995/Q http://journals.im.ac.cn 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, 430071) ( 2, 510070)