002 微生物学通报 2009, Vol.36, No.7 against indicat bacterias still; the inhibitory activity was totally lost after treatment with protease; The bacteriocin

Size: px

Start display at page:

Download "002 微生物学通报 2009, Vol.36, No.7 against indicat bacterias still; the inhibitory activity was totally lost after treatment with protease; The bacteriocin"

悟迭养
5 years ago
Views:

1 微生物学通报 JUL 20, 2009, 36(7): 00~007 Microbiology 2009 by Institute of Microbiology, CAS 研究报告动物乳杆菌的分离鉴定及其抑菌蛋白的特性分析李明雄李妮李征向文良杨志荣,2* 罗璠 ( ) ( ) 摘要 : 通过滤纸片法从健康肉猪猪大肠小肠中分离得到 22 株抗生物质产生菌, 以杯碟法复筛, 得到株对溶壁微球菌 (Micrococcus lysodeikticus) 金黄色葡萄球菌(Staphylococcus aureus) 枯草芽孢杆菌 (Bacillus subtilis) 蜡状芽孢杆菌(Bacillus cereus) 等革兰氏阳性菌和大肠杆菌 (Escherichia coli) 鼠伤寒沙门氏菌(Salmonella typhimurium) 等革兰氏阴性菌以及部分真菌如禾谷镰刀霉 (Fusarium graminearum) 均有强烈抑制作用的乳酸菌经形态学生理生化特征及 6S rdna 序列同源性分析等手段鉴定该菌株为动物乳杆菌 (Lactobacillus animalis) 排除酸和过氧化氢的干扰后, 该菌株的发酵上清液对指示菌仍有明显抑菌活性 ; 用蛋白酶处理该菌株的发酵上清液后, 抑菌活性丧失 ; 发酵液粗提物具有较好的热稳定性 ( 经 2 C 处理 20 min 仍有较强抑菌活性 ) 以及较宽的抑菌活性 ph 值范围 (3.5~5.5), 因此初步认为该菌株产生一类具有广谱抑菌活性的类细菌素物质关键词 : 猪肠道, 动物乳杆菌, 类细菌素, 鉴定, 抑菌特性 Screening and Identification of Lactobacillus Animalis Strain and Its Inhibitory Protein Characteristics LI Ming-Xiong LI Ni LI Zheng XIANG Wen-Liang YANG Zhi-Rong LUO Fan,2* (. Key Laboratory of Microbial-resource and Microbial-technique, College of Life Sciences, Sichuan University, Chengdu, Sichuan 60064, China) (2. College of Life Sciences and Technoligy, Southwest University for Nationalitise, Chengdu, Sichuan 60064, China) Abstract: A bacteriocin-like substance producing strain T2 was screened from 22 lactic acid bacteria isolated from intestine of healthy pigs by paper disk test and was identified as Lactobacillus. The supernatant of T2 exhibited strong inhibitory activity against the gram-positive bacteria, including Micrococcus lysodeikticus, Staphylococcus aureus, Bacillus subtilis and Bacillus cereus. A strong activity was also observed against the gram negative bacteria and some eumycete, such as Escherichia coli, Salmonella typhimurium and Fusarium graminearum. Through detection of its appearance, physiological and biochemical characteristics and 6S rdna gene sequence homology analysis, it was identified as Lactobacillus animalis. Eliminated the organic acids and hydrogen peroxide the fermented production exhibited strong inhibitory activity 基金项目 :(No. 07SG0832) * 通讯作者 :Tel: ; : luofan77@sina.com 收稿日期 : ; 接受日期 :

2 002 微生物学通报 2009, Vol.36, No.7 against indicat bacterias still; the inhibitory activity was totally lost after treatment with protease; The bacteriocin-like substance was heat-stable even at the autoclaving temperature (2 C for 20 min) and was active over a wide ph range (3.5~5.5). The protein nature and restricted spectrum of the substance points to its being bacteriocin-like substance. Keywords: Pig intestinal tract, Lactobacillus animalis, Bacteriocin-like substance, Identification, Characterization,,,,,,, [,2], [3,4],,,,,, ph, [5,6],,,, [7],,,, 6S rdna,,, 材料与方法. 材料.2 菌种.2. 革兰氏阴性指示菌 : (E. coli), (S. typhimurium).2.2 革兰氏阳性指示菌 : (), (M. lysodeikticus), (B. subtilis), (B. cereus).2.3 真菌 : (Aspergillus flavus), (F. graminearum), (Gibberella fujikuroi), (Aspergillus oryzae), (Saccharomyces cerevisiae).3 试剂和培养基.3. 试剂 : (Amersco ) (Sigma ) K(Sigma ); (TIANprep MiNi Plasmid Kit); E. coli DH5 (Tiangen );.3.2 培养基 : MRS [8] ; [8] ; LB [9].4 分析方法.4. 分离培养 :, 0 0, 0 7 ~ ml MRS, 30 C 48 h, MRS,, 4 C.4.2 细菌数测定 : (CFU/mL).4.3 抑菌试验 :,, 0 8 CFU/mL, 00 µl 200 µl, 3 h, 36 C 6 h,.4.4 排除干扰因素 : ; (0 mg/ml), 30 C h,

: 003.4.5 蛋白酶处理 : mg/ml, ph ph ( K ph 7.6, ph 7.0), 37 C h ph ph 4.5, 22, 60,, T2 T2 (,.5 菌株的鉴定.5. 形态学和生理生化特征 : ),, T2 [8].5.2 基因组 DNA 提取 : 2.2 菌株 T2 所产抑菌物质的确定 [0] DNA 2.2. 酸抑制作用的排除 : T2.5.3 6S rdna 的 PCR 扩增 : PCR(50 µl):,, 0 PCR, 5.

3 : 蛋白酶处理 : mg/ml, ph ph ( K ph 7.6, ph 7.0), 37 C h ph ph 4.5, 22, 60,, T2 T2 (,.5 菌株的鉴定.5. 形态学和生理生化特征 : ),, T2 [8].5.2 基因组 DNA 提取 : 2.2 菌株 T2 所产抑菌物质的确定 [0] DNA 2.2. 酸抑制作用的排除 : T S rdna 的 PCR 扩增 : PCR(50 µl):,, 0 PCR, 5.0 µl; Mg 2+ (5 mmol/l), 3.0 µl; dntp( 2.5 mmol/l), 4.0 µl; (0 µmol/l), 24 h 2.0 µl; Taq DNA 0.5 µl; DNA 2.0 µl;, ph 4.5, 33.5 µl : 94 C 4 min; 94 C min,,, ph 45 C.5 min, 72 C 2 min, 26 ; 72 C 0 min; 4.5, ph 4 CPCR % 4.5,.5.4 产物克隆与序列测定 : PCR Tiangen 过氧化氢作用的排除 : pmd8-t, E. coli DH5,,,, (Invitrogen) (M. lysodeikticus) (S..5.5 序列分析和系统树的构建 : NCBI aureus) (E. coli), Blast, GenBank T2,,, Clustal X.83 2,,, MEGA 3., N-J [,2] 2 结果与分析酶解检测 :, 2. 菌株筛选 T2 K MRS, 2, 图 T2 菌株对 3 种指示菌的抑制作用 Fig. Results of antibacterial from supernatant of T2 with three indicators : : ; 2: ; 3: ; a: ; b: MRS. Note: : E. coli; 2: ; 3: M. lysodeikticus; a: Untreated supernatant; b: MRS liquid medium control.

004 微生物学通报 2009, Vol.36, No.7 表排除酸作用的抑菌试验结果 Table Results of antibacterial by supernatant without acid Sample The supernatant fluid femented by T2 ph 4.5 ph 4.5 of the supernatant ph 4.5 ph 4.5 of the lactic acid ph 4.

4 004 微生物学通报 2009, Vol.36, No.7 表排除酸作用的抑菌试验结果 Table Results of antibacterial by supernatant without acid Sample The supernatant fluid femented by T2 ph 4.5 ph 4.5 of the supernatant ph 4.5 ph 4.5 of the lactic acid ph 4.5 ph 4.5 of the hydrochloric acid : :. Note: : No inhibition. (mm) M. lysodeikticus E. coli 表 2 过氧化氢酶处理后的抑菌试验结果 Table 2 Results of antibacterial after treatment with hydrogen peroxidase Sample (mm) M. lysodeikticus E. coli Control Supernatant treated with catalase (%) Percentage of activity decrease Fig. 2 Results of antibacterial from supernatant after treatment with protease : A: ; B: ; : ; 2: ; 3: K; 4: T2. Note: A: E. coli; B: ; : Trypsase; 2: Caroid; 3: Protease K; 4: Supernatant of T2. K, T2, T2 2.3 菌株 T2 抑菌物质特性分析 2.3. 菌株 T2 粗提液热稳定性 : T2 20 min,, () (E. coli), 3,, T2, 2 C 20 min, T 不同 ph 值对 T2 粗提液抑菌活性的影响 : T2 mol/l NaOH mol/l HCl ph , ph, 3 4, T2 Table 3 3 Effects of various temperatures on inhibition activity of fermented production ( C) Temperature ( C) (mm) (%) Percentage of activity decrease (%) E. coli (mm) (%) Percentage of activity decrease (%) Control

5 : 005 Table 4 表 4 不同 ph 值对菌株发酵产物抑菌活性的影响 Effects of various ph on inhibitory activity of fermented production ph ph value Control (mm) E. coli (%) Percentage of activity decrease (%) (mm) M. lysodeikticus (%) Percentage of activity decrease (%) ph ph ph ph ph ph ph ph : :. Note: : No inhibition. ph : ph 4.5, ph,, ; ph 5.0,, 57.95%; ph 5.5, 6.59%; ph 6.0 T2, ph 3.5~5.5, T2 ph, ph 酶的敏感性 : 3 T2, ph ph, mg/ml K, 37 C h ph ph 4.5,, 3,, 3, 菌株 T2 粗提液的抑菌谱 :, T2, ph, 3 5, T2, 表 5 T2 菌株发酵产物的抑菌谱试验结果 Table 5 Inhibitory spectrum of the products from T2 fermented production Test organism (mm) Average diameter of inhibition Control M. lysodeikticus B. subtilis E. coli S. typhimurium B. cereus F. graminearum A. flavus G. fujikuroi A. oryzae S. cerevisiae : :. Note: : No inhibition. 2.4 菌株鉴定 2.4. 形态学特征 :,,,, mm~2 mm 生理生化特征 :,

6 006 微生物学通报 2009, Vol.36, No.7,, H 2 S, ph C, (Lactobacillus) MR, V-P, S rdna 序列分析 : T2 DNA, 6S rdna PCR 6 Table 6 Results of Saccharide fermentation test / Saccharide or alcohol Results Glucose + Fructose + Sucrose + D- D-Galactose + Lactose + Maltose + Sorbose Rhamnose Arabinose Ribose Xylose Trehalose Sorbitol Inositol Mannitol : +: ; :. Note: +: Positive; : Negative.,.5 kb T2 6S rdna (FJ67349) GenBank 6S rdna, T2 6S rdna (98%~ 00%), Lactobacillus animalis ATCC S rrna 99% 6S rdna T2 3 T2 Lactobacillus animalis ATCC 35046, 6S rdna, T2 Lactobacillus animalis 6S rdna 99%, [3], T2 (L. animalis) 3 讨论, [5] T2,, ph 4.5 (M. lysodeikticus) (S. aureus) (E. coli) ; ph 5.5 (M. lysodeikticus), 图 3 菌株 T2 基于 6S rrna 基因序列的 N-J 法系统发育树图 Fig. 3 Neighbour-joining tree showing the phylogenetic position of strain T2 and representatives of some other related taxa bsed on 6S rrna sequences Note: Bar: 0.02 substitutions per nucleotide position.

7 : 007, T2 Reid, (Lactobacillus casei subsp. rhamnosus GR-) 80 C 2 h, [6] 6 00 C 5min [7] 2 C 20 min, () (E. coli) 92%, T2, [8] K, () (E. coli), T2 T2 Virginia (Lactobacillus salivarus), [9], T2, K ph 3.5~5.5 T2,, 参考文献 [],,,. SB900., 995, 5(6): [2] Kyriakis SC, Tsiloyianni VK, V lemmas J, et al. Res Vet Sci, 999, 67: [3].., 999, (): 58. [4],.., 2000, 5 (): 820. [5],,.. :, 996, pp [6].., 998, 26 (2): 420. [7] Simon S, Jadamus A, Vahjen W. J Anim Feed Sci, 200, 0: 567. [8],.. :, 999, pp.727. [9] Joseph S, David WR. Molecular Clone: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory, 200, p [0].. :, 2007, pp [] Thompson JD, Gibson TJ, Plewniak F, et al. The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res, 997, 24: [2] Kumar SK, Tamura I, Nei M, et al. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform, 2004, 5: [3] RE, NE... :, 984. [4],,,.., 2005, 32(): [5],,.., 998, 25(5): [6] Reid G, Mcgroarty JA, Angotti R, et al. Can J Microbiol, 988, 34: [7],,,., 200, 37: 80. [8] Daba H, Pandian S, Gosselin JF, et al. Appl Environ Microbiol, 99, 57: [9] Virginia S. Characterization of a bacteriocin-like substance produced by a vaginal Lactobacillus salivarius strain. Appl Environ Microbiol, 999, 65:

untitled

untitled 理類 CN9626 行 96 年 1 1 96 年 12 31 參行 97 年 2 28 1 行離 Lactic acid bacteria 金 Staphylococcus aureus 行離 159 API 50 CHL 行離 159 不度了行 H 2 O 2 1 Lactobacillus plantarum 了識益益來不利來益切了流 Joerger 2003