Microsoft Word tb 李文静t-研究报告

Transcription

1 微生物学通报 Microbiology China May 20, 2016, 43(5): DOI: /j.microbiol.china 研究报告 3 株罗伊氏乳杆菌生物学特性的分析比较 * 李文静梁运祥赵述淼 ( ) 摘要 : 目的 对 3 株罗伊氏乳杆菌的生物学特性进行分析比较, 为后期生产应用提供一定的参考 方法 对实验室保藏的 3 株罗伊氏乳杆菌的生长曲线 ph 曲线 耐受人工胃液能力 耐受猪胆盐能力 黏附能力 抑菌能力和对抗生素的耐药性等特性进行了分析比较 结果 3 株菌生长趋势大致相同 ;3 株菌对人工胃液均具有良好的耐受性, 且可以有效地抑制大肠杆菌和金黄色葡萄球菌的生长 ; 菌株 L0 和 L2 对高胆盐的环境耐受性较差, 菌株 L1 则对高胆盐环境具有极强的耐受性 ; 菌株 L1 和 L2 具有很强的黏附能力 ;3 株菌对 20 种抗生素表现出不同的耐受性 结论 菌株 L1 的生物学特性明显优于其他两株菌株, 有利于后期的生产应用 关键词 : 罗伊氏乳杆菌, 耐受性, 黏附, 抑菌 Comparative analysis of the biological characteristics of three strains of Lactobacillus reuteri LI Wen-Jing LIANG Yun-Xiang ZHAO Shu-Miao * (State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei , China) Abstract: [Objective] The study researched the biological characteristics of three Lactobacillus reuteri strains, providing guidance in industrial production. [Methods] We analyzed and compared diverse characteristics and capabilities of three Lactobacillus reuteri strains preserved in the lab, such as growth curve, ph curve, tolerance to artificial gastric intestinal juice, tolerance to porcine bile salt, adhesive capacity, antimicrobial activity, and tolerance to antibiotics. [Results] The growth trend of these 3 strains was similar. All of the 3 strains had a good tolerance to artificial gastric intestinal juice, and could inhibited the growth of Escherichia coli and Staphylococcus aureus effectively. L0 and L2 showed poor tolerance in high porcine bile salt environment, while the performance of L1 was satisfactory. In addition, L1 and L2 had strong adhesive ability and the tolerances of 3 strains to 20 antibiotics were different. [Conclusion] L1 can be further applied in the industrial production since its biological characteristics are significantly better than the other two strains. Keywords: Lactobacillus reuteri, Tolerance, Adhesion, Antibacterial activity Foundation item: Key Technologies R & D Program of China (No. 2013BAD10B02) *Corresponding author: Tel: ; shumiaozhao@mail.hzau.edu.cn Received: October 17, 2015; Accepted: January 05, 2016; Published online ( January 11, 2016 基金项目 : (No. 2013BAD10B02) * 通讯作者 :Tel shumiaozhao@mail.hzau.edu.cn 收稿日期 : 接受日期 : 优先数字出版日期 (

2 1036 微生物学通报 Microbiol. China 2016, Vol.43, No.5 [1-3] [4] 10 6 CFU/mL [5] Hill [6] ph 3 3 ph HT-29 1 材料与方法 1.1 材料 实验菌株与细胞 :Lactobacillus reuteri ( ) L0 Lactobacillus reuteri ( CICC6118) L1 Lactobacillus reuteri ( CICC6132) L2 Escherichia coli ( CICC10389) Staphylococcus aureus ( CICC10384) HT 培养基 :MRS LB [7] 主要试剂 : ( 1:10 000) ( 250 NFu/mg) 主要仪器 :DHG-9070A I B HQL150BLC HH s 1.2 方法 菌株鉴定 : L0 L1 L2 MRS 3 16S rrna PCR NCBI MEGA 生长曲线和 ph 曲线的比较 : 3 1% MRS 37 C h OD 600 ph 3 ph 耐受人工胃液能力的比较 : [8] 3 1 ml 9 ml 37 C min 耐受猪胆盐能力的比较 : 0 0.1% 0.2% 0.3% 0.4% 0.5% MRS 3 1 ml 9 ml MRS 37 C 12 h μl 30 ( CFU/mL) 5 30

3 : 黏附 HT-29 细胞能力的比较 : HT-29 10% DMEM 37 C 5% CO 2 5 d d HT MRS 37 C r/min 5 min DMEM CFU/mL 24 2 h PBS 5 MRS 体外抑菌能力的比较 : LB MRS 37 C 15 h MRS ph ph 100 μl LB 6.00±0.10 mm 4 C 6 h 37 C 18 h [9] 对抗生素耐药性的比较 : 3 1% MRS 37 C 15 h CFU/mL 100 μl 37 C 18 h 数据处理 : 3 x ±s SPSS 结果与分析 2.1 菌株鉴定 3 MRS GenBank 3 16S rrna PCR L0 L. reuteri CICC S rrna 99% L1 L. reuteri ZJ615 16S rrna 100% L2 L. reuteri CICC S rrna 99% 3 (Lactobacillus reuteri) 生长曲线和 ph 曲线的比较 h 3 9 h OD h 3 L0>L2>L1 3 ph 耐受人工胃液能力的比较 ph ph 3.0 图 1 基于 16S rrna 基因序列的系统发育树 Figure 1 Phylogenetic tree based on 16S rrna gene sequences 图 2 罗伊氏乳杆菌的生长曲线和 ph 曲线 Figure 2 Growth curve and ph curve of Lactobacillus reuteri

4 1038 微生物学通报 Microbiol. China 2016, Vol.43, No h [10] 1 3 ph min 10 9 CFU/mL 80% 3 ph 2.4 耐受猪胆盐能力的比较 ph 0.03% 0.30% [11] 2 L0 L2 0.3 g/100 ml L1 0.4 g/100 ml 2.5 黏附 HT-29 细胞能力的比较 表 1 罗伊氏乳杆菌对 ph 2.5 人工胃液的耐受性 Table 1 Tolerance of Lactobacillus reuteri to artificial gastric intestinal juice at ph 2.5 (10 8 CFU/mL) Time (min) L ± ±1.12 ** 20.52±1.51 ** L ± ±0.52 ** 16.00±0.85 ** L ± ± ±1.00 ** 0 min * (P<0.05) ** (P<0.01). Note: In a row, compared with 0 min, * : P<0.05, ** : P<0.01. 表 2 3 株罗伊氏乳杆菌在不同浓度猪胆盐中的存活情况 Table 2 Survival of the three Lactobacillus reuteri at high porcine bile salt concentrations Bile salt concentration (g/100 ml) L0 + (+) L (+) L (+). Note: +: Growth well; (+): Weak growth; : No growth. 3 L0 HT-29 L1 L2 HT 体外抑菌能力的比较 3 3 ph 对抗生素耐药性的比较 3 5 S I R [12] 呋 表 3 罗伊氏乳杆菌对 HT-29 细胞的黏附性 Table 3 Adhesion of Lactobacillus reuteri to HT-29 cells /100 Adhesion bacterias/100 cells (±s) L ±2.46 L ±17.66 L ±49.01 表 4 3 株罗伊氏乳杆菌的抑菌活性 Table 4 Antibacterial activity of the three Lactobacillus reuteri Escherichia coli Staphylococcus Tested strains (mm) aureus (mm) CK 11.58± ±0.10 L ±1.08 ** 15.19±0.91 ** L ±1.24 ** 15.08±0.66 ** L ±1.12 ** 15.23±0.58 ** ** (P<0.01). Note: In a column, compared with CK, ** : P<0.01.

5 : Table 5 Name of antibiotics 表 5 3 株罗伊氏乳杆菌对 20 种抗生素的耐药性比较 Antimicrobial activity of the three strains of Lactobacillus reuteri to twenty antibiotics Dosage of drug (μg) The standard of bacteriostatic circle (mm) (R) (I) (S) Resistant Intermediate Susceptible L0 L1 L2 Penicillin 10 IU R R R Oxacillin R R R Ampicillin I S S Carbenicillin R I S Piperacillin S I S Cephalexin S R I Cefamezin R S S Cefradine R R R Cefuroxim S S S Ceftazidime S R I Ceftriaxone S I I Cefoperazone S R S Aboren S S S Norfloxacin R R R Ofloxacin R R R Ciprofloxacin R R R Vancomycin R R R B Polymyxin B 300 IU R R R Compound sulfamethoxazole I R R Furazolidone R R R 咗 B 3 讨论 3 3 h 9 h 3 3 L0>L2>L1 3 ph ph ph ph ph 3.0 [13]

6 1040 微生物学通报 Microbiol. China 2016, Vol.43, No.5 ph h 10 6 CFU/mL [14] ph h 10 8 CFU/mL 31.62% 3 ph min 10 9 CFU/mL 80% 3 ph [15] L0 L2 0.3 g/100 ml L1 0.4 g/100 ml [16] [17] 3 L0 HT-29 L1 L2 HT-29 [18] 24 HT ±1.8 / L ±0.18 / 3 ph ph 3 L1 L1 参考文献 [1] Thacker PA. Alternatives to antibiotics as growth promoters for use in swine production: areview[j]. Journal of Animal Science and Biotechnology, 2013, 4(1): 35 [2] Shen ZY, Zhao SM, Liang YX. Screening of lactic acid bacteria strains for pig additives and the study of its probiotic properties in vitro[j]. Journal of Huazhong Agricultural University, 2007, 26(3): (in Chinese),,. [J]., 2007, 26(3): [3] Wang Z, Zeng XM, Mo YM, et al. Identification and characterization of a bile salt hydrolase from Lactobacillus salivarius for development of novel alternatives to antibiotic growth promoters[j]. Applied and Environmental Microbiology, 2012, 78(24): [4] Pang J, Zhou N, Liu P, et al. Beneficial effects of Lactobacillus reuteri to human and animals[j]. China Biotechnology, 2011, 31(5): (in Chinese),,,. [J]., 2011, 31(5): [5] Gismondo MR, Drago L, Lombardi A. Review of probiotics available to modify gastrointestinal flora[j]. International Journal of Antimicrobial Agents, 1999, 12(4): [6] Hill C, Guarner F, Reid G, et al. Expert consensus document: The international scientific association for probiotics and prebiotics consensus statement on the scope and appropriate use of the term probiotic[j]. Nature Reviews Gastroenterology & Hepatology, 2014, 11(8): [7] He SJ, Chen WL. The Experiment of Microbiology[M]. Beijing: China Agriculture Press, 2007: (in Chinese),. [M]. :, 2007: [8] Pedersen C, Jonsson H, Lindberg JE, et al. Microbiological characterization of wet wheat distillers grain, withfocus on isolation of lactobacilli with potential as probiotics[j]. Applied and Environmental Microbiology, 2004, 70(3): [9] Wu HF, Mao SY, Yao W, et al. Lactic acid production and antagonistic effect of lactic acid bacteria from piglet intestine[j]. Microbiology China, 2005, 32(1): (in Chinese),,,. [J]., 2005, 32(1): [10] Xiong T, Song SH, Huang JQ, et al. Tolerance of Lactobacillus plantarum NCU116 in stimulated digestive environments[j]. Food Science, 2011, 32(11): (in Chinese),,,. NCU116 [J]., 2011, 32(11): [11] Wang JC, Guo Z, Yan LY, et al. Comparison of transit tolerance and fermentation properties of probiotic Lactobacillus casei Zhang with commercial probiotic strains[j]. Journal of Chinese Institute of Food Science and Technology, 2009, 9(5): (in Chinese),,,. Lactobacillus casei Zhang [J]., 2009, 9(5): [12] Ministry of Health of the People s Republic of China. WS/T Standard of antidbiotics susceptibility test(kirby-bauer method)[s]. Beijing: China Standard Press, 2000 (in Chinese). WS/T

7 : [S]. :, 2000 [13] Shao JH, Yang YH, Li L, et al. Phenotype investigation on a strain of Lactobacillus reuteri[j]. China Dairy Industry, 2012, 40(3): (in Chinese),,,. [J]., 2012, 40(3): [14] Li Q, Wang Y, Liu XL, et al. Isolation of a broad-spectrum antibacterial lactic acid bacterium and evaluation of probiotic properties[j]. Microbiology China, 2015, 42(2): (in Chinese),,,. [J]., 2015, 42(2): [15] Pan XD, Chen FQ, Wu TX, et al. The acid, bile tolerance and antimicrobial property of Lactobacillus acidophilus NIT[J]. Food Control, 2009, 20(6): [16] Bengmark S. Ecological control of the gastrointestinal tract. The role of probiotic flora[j]. Gut, 1998, 42(1): 2-7 [17] Bjorkman PJ. Colonization of the Human Gastrointestinal Tract by Two Formulations of Lactobacillus reuteri Dissertation[M]. Helsinki, Finland: University of Helsinki, 1999: [18] Li PL, Yang H, Zhang C. Study on adhesion ability of lactic acid bacteria to the colonic adenocarcinoma cell line HT-29[J]. Journal of China Agricultural University, 2002, 7(1): (in Chinese),,. HT-29 [J]., 2002, 7(1): 年中国微生物学会及各专业委员会学术活动计划表 (2-1) / lvxz@cma.org.cn