酸马奶源发酵乳杆菌对致病性大肠杆菌O8的体外抑菌研究

doi:10.16431/j.cnki.1671-7236.2018.10.023

摘要/Abstract

摘要：

为了探究天然发酵酸马奶中发酵乳杆菌对致病性大肠杆菌O₈（E.coli O₈）的抑菌机理，本试验从酸马奶中分离筛选出抑菌效果最佳的菌株，对其进行16S rDNA序列鉴定，经NCBI网站BLAST对比鉴定菌种；对菌株进行培养发酵，制备无细胞发酵上清液（CFS）；通过排酸、排过氧化氢（H₂O₂）、不同蛋白酶处理等方法初步确定CFS中的抑菌活性物质性质及其含量；采用牛津杯法和二倍稀释法确定CFS对致病性E.coli O₈ 24 h生长曲线的最佳抑菌浓度；试剂盒法测定CFS对致病性E.coli O₈的细胞膜和细胞壁通透性的影响。结果显示，从酸马奶中分离出22株对致病性E.coli O₈有抑制作用的菌株，对抑菌作用最好的菌株进行16S rDNA序列鉴定及系统进化树分析后确定其为发酵乳杆菌属；CFS中主要的抑菌物质为蛋白，含量为399.5 μg/mL；最低抑菌浓度（MIC）和最低杀菌浓度（MBC）分别为25.0和49.9 μg/mL；CFS能使致病性E.coli O₈的碱性磷酸酶（AKP）含量在1 h内快速升高，之后呈缓慢增长趋势，且使致病菌培养液中的蛋白含量明显升高。综上所述，发酵乳杆菌CFS的主要抑菌物质为蛋白，蛋白浓度越高抑菌能力越强；CFS通过破坏或改变致病性E.coli O₈细胞膜和细胞壁的通透性，使其释放出AKP和胞内蛋白，从而在短时间内起到抑制致病性E.coli O₈生长的作用。

关键词: 酸马奶; 发酵乳杆菌; 致病性E.coli O₈; 抑菌作用; 无细胞发酵上清液(CFS)

Abstract:

In order to study the antibacterial mechanism of Lactobacillus fermentum isolated from natural fermented koumiss on pathogenic Escherichia coli O₈ (E.coli O₈),the strains with best bacteriostasis isolated from koumiss was screened out in this study,and then 16S rDNA sequence was identified by BLAST of NCBI website.After fermentation,the cell-free fermentation supernatant (CFS) was prepared.The properties and content of antibacterial substances in CFS were preliminarily determined by acid excretion,hydrogen peroxide excretion and different protease treatments.The best inhibitory concentration of CFS on the growth curve of pathogenic E.coli O₈ within 24 h was determined using the Oxford cup method and two fold dilution method.The influence of CFS on cell membrane and cell wall permeability of pathogenic E.coli O₈ was determined by kit method.The results showed that there were 22 strains which had antibacterial effects on pathogenic E.coli O₈ isolated from koumiss,and the best strain was Lactobacillus fermentum after 16S rDNA sequence and phylogenetic tree analysis.Protein was the main bacteriostatic substance of CFS,and its content was 399.5 μg/mL.The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were 25.0 and 49.9 μg/mL,respectively.CFS could rapidly increase the AKP content of pathogenic E.coli O₈ within 1 h and then increase slowly.Moreover,it could obviously increase the protein content in the culture of the pathogen.In conclusion,the protein of Lactobacillus fermentum was the antibacterial substance on pathogenic E.coli O₈.And with the increase of protein concentration,the bacteriostasis ability was stronger.CFS could release AKP and intracellular proteins by destroying or altering the permeability of cell membrane and cell wall.Thus,it could inhibit the growth of pathogenic E.coli O₈ in a short time.

Key words: koumiss; Lactobacillus fermentum; pathogenic E.coli O₈; bacteriostasis; cell-free fermentation supernatant (CFS)

中图分类号:

S853.7

郭雪梅, 王纯洁, 斯木吉德, 吉米斯, 崔银雪, 敖日格乐. 酸马奶源发酵乳杆菌对致病性大肠杆菌O₈的体外抑菌研究[J]. 《中国畜牧兽医》, 2018, 45(10): 2856-2865.

GUO Xuemei, WANG Chunjie, SIMUJIDE, JIMISI, CUI Yinxue, AORIGELE. Study on Antibacterial Mechanisim of Lactobacillus fermentum Isolated from Koumiss on Pathogenic Escherichia coli O₈[J]. , 2018, 45(10): 2856-2865.

参考文献

[1] 芒来,布仁马虎雅尔,杨永平.策格[M].呼和浩特:内蒙古人民出版社,2013.MANG L,BURENMAHUERYA,YANG Y P.Chigo[M].Hohhot:Inner Mongolia Peoples Publishing House,2013.(in Chinese)
[2] 额尔登孟克,潘琳,阿木尔图布兴,等.传统发酵乳制品酸马奶的研究进展[J].内蒙古农业大学学报(自然科学版),2016,37(2):134-140.EERDENGMENGKE,PAN L,AMUERTUXING et al.Research progress on traditional fermented dairy products koumiss[J].Journal of Inner Mongolia Agricultural University (Natural Science Edition),2016,37(2):134-140.(in Chinese)
[3] JI X,LI X,MA Y,et al.Differences in proteomic profiles of milk fat globule membrane in yak and cow milk[J].Food Chemistry,2017,221:1822-1827.
[4] DIMOV S,IVANOVA P,HARIZANOVA N.Genetics of bacteriocins biosynthesis by lactic acid bac-teria[J].Biotechnology & Biotechnological Equipment,2005,19(2):4-10.
[5] 史瑞芳.复合乳酸菌胶囊联合蒙脱石散治疗小儿轮状病毒性肠炎疗效分析[J].甘肃科技纵横,2018,47(1):80-82.SHI R F.Analysis of the effect of Lactobacillus complex capsules combined montmorillonite powder on infantile rotavirus enteritis[J].Scienfic & Technical Information of Gansu,2018,47(1):80-82.(in Chinese)
[6] 张梅梅,索化夷,赵欣,等.传统发酵牦牛酸乳中细菌素产生菌的筛选与鉴定[J].食品与机械,2017,33(3):21-26.ZHANG M M,SUO H Y,ZHAO X,et al.Screening and identification of lactobacillin-producing strain in traditional yak yogurt[J].Food & Machinery,2017,33(3):21-26.(in Chinese)
[7] GESUDU Q,ZHENG Y,XI X,et al.Investigating bacterial population structure and dynamics in traditional koumiss from Inner Mongolia using single molecule real-time sequencing[J].Journal of Dairy Science,2016,99(10):7852-7863.
[8] OLIVARES M,DIAZ-ROPERO M P,SIERRA S,et al.Oral intake of Lactobacillus fermentum CECT5716 enhances the effects of influenza vaccination[J].Nutrition,2007,23(3):254-260.
[9] MIKERLSAAR M,ZILMER M,KULLISAAAR T,et al.Strain of microorganism Lactobacillus fermentum ME-3 as novel anti-microbiol and anti-oxidative probiotic.EP:US7244424[P].2007.
[10] VERA-PINGTORE E,JIMENEZ M E,DALLAGNOL A,et al.Screening and characterization of potential probiotic and starter bacteria for plant fermentations[J].LWT-Food Science and Technology,2016,71:288-294.
[11] 张燕.酸马奶源乳酸乳球菌代谢物和大黄联合作用对小鼠盲肠微生物以及营养代谢的影响[D].呼和浩特:内蒙古农业大学,2016.ZHANG Y.The combined effects of lactic acid galactococcus metabolites and rhubarb on nutrient metabolism and caecum microorganism in mice[D].Hohhot:Inner Mongolia Agricultural University,2016.(in Chinese)
[12] 徐瑾鼎,王悦.母乳中发酵乳杆菌CECT 5716对人体免疫调节作用的研究进展[J].浙江医学,2017,39(6):493-495.XU J D,WANG Y.Research progress of Lactobacillus fermentum CECT 5716 in breast milk on human immune regulation[J].Zhejiang Medical Journal,2017,39(6):493-495.(in Chinese)
[13] KAHOULI I,MALHOTRA M,WESTFALL S,et al.Design and validation of an orally administrated active L.fermentum-L.acidophilus,probiotic formulation using colorectal cancer Apc^Min/+ mouse model[J].Applied Microbiology & Biotechnology,2017,101(5):1999-2019.
[14] SUN Z,HARRIS HM,MCCANN A,et al.Expanding the biotechnology potential of Lactobacilli through comparative genomics of 213 strains and associated genera[J].Nature Communications,2015,6:8322.
[15] SONG Y,SUN Z,GUO C,et al.Genetic diversity and population structure of Lactobacillus delbrueckii subspecies bulgaricus isolated from naturally fermented dairy foods[J].Scientific Reports,2016,6:22704.
[16] 杨斯琴,敖日格乐,王纯洁,等.内蒙古呼和浩特地区牛源致病性大肠杆菌的血清型、毒力基因检测及耐药性分析[J].中国预防兽医学报,2015,37(10):761-764.YANG S Q,AORIGELE,WANG C J,et al.Prevalence of serotypes virulence genes and antimicrobial susceptibility of bovine pathogenic Escherichia coli isolates in Inner Mongolia[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(10):761-764.(in Chinese)
[17] PAPADEMAS P,PARMAXI I,ASPRI M.Probiotic,antimicrobial,antioxidant and sensory properties of fermented donkey milk with Lactobacillus fermentum ME-3 and Lactobacillus acidophilus (ATCC 4356)[J].BAOJ Microbiology,2015,1(4):2-8.
[18] PEI J,LI X,HAN H,et al.Purification and characterization of plantaricin SLG1,a novel bacteriocin produced by Lb.plantarum,isolated from yak cheese[J].Food Control,2017,84:111-117.
[19] IRANMANESH M,EZZATPANAH H,MOJGANI N.Antibacterial activity and cholesterol assimilation of lactic acid bacteria isolated from traditional Iranian dairy products[J].LWT-Food Science and Technology,2014,58(2):355-359.
[20] JAY J M.Modern Food Microbiology[M].7th Edition.Germany:Springer,2005.
[21] CHEN Y J,CHEN A,WANG C J,et al.Screening and extracting mycocin secreted by yeast isolated from koumiss and their antibacterial effect[J].Journal of Food & Nutrition Research,2015,3(1):52-56.
[22] LEVY J,BOYER R R,NEILSON A P,et al.Evaluation of peanut skin and grape seed extracts to inhibit growth of foodborne pathogens[J].Food Science & Nutrition,2017,5(6):1130-1138.
[23] CHEN Y J,WANG C J,HOU W Q,et al.Effects of antibacterial compounds produced by Saccharomyces cerevisiae in koumiss on pathogenic Escherichia coli O₈ and its cell surface characteristics[J].Journal of Integrative Agriculture,2017,16(3):742-748.
[24] 张燕,敖日格乐,王纯洁,等.nisin和EDTA对牛源致病性大肠杆菌体外抑菌效果的研究[J].中国农业大学学报,2016,21(5):98-103.ZHANG Y,AORIGELE,WANG C J,et al.Antibacterial effects of nisin and EDTA on bovine pathogenic Escherchia coli in vitro[J].Journal of China Agricultural University,2016,21(5):98-103.(in Chinese)
[25] 彭齐,谢丽玲,谢俊.中药抑菌机制的研究方法[J].生物技术进展,2015,5(1):66-69.PENG Q,XIE L L,XIE J.Research methods for antibacterial mechanism of traditional Chinese medicine[J].Current Biotechnology,2015,5(1):66-69.(in Chinese)
[26] 阿琪玛,敖日格乐,王纯洁,等.五谷虫蛋白粗提液对牛源致病性Escherichia coli O₁和E.coli O₇₈体外抑菌作用[J].中国农业大学学报,2017,22(3):102-108.AQIMA,AORIGELE,WANG C J,et al.Antibacterial effects of crude protein extract from maggot on bovine pathogenic Escherichia coli O₁ and E.coli O₇₈[J].Journal of China Agricultural University,2017,22(3):102-108.(in Chinese)
[27] 张燕,敖日格乐,王纯洁,等.乳酸链球菌素与大黄联合作用对牛源致病性Escherichia coli O₇₈的抑菌作用[J].中国农业大学学报,2016,21(7):92-97.ZHANG Y,AORIGELE,WANG C J,et al.Study on the antibacterial mechanism of nisin combined with rhubarb on bovine pathogenic Escherichia coli O₇₈[J].Journal of China Agricultural University,2016,21(7):92-97.(in Chinese)
[28] DEEGAN L H,COTTER P D,HILL C,et al.Bacteriocins:Biological tools for bio-preservation and shelf-life extension[J].International Dairy Journal,2006,16(9):1058-1071.

酸马奶源发酵乳杆菌对致病性大肠杆菌O₈的体外抑菌研究

Study on Antibacterial Mechanisim of Lactobacillus fermentum Isolated from Koumiss on Pathogenic Escherichia coli O₈

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 10

编辑推荐

Metrics

本文评价

[1]	邵明珠, 赵允清, 李松励, 侯绍华, 张艳英. 河北省奶牛源大肠杆菌的分离鉴定、致病性及中草药体外抑菌效果研究[J]. 中国畜牧兽医, 2022, 49(9): 3683-3691.
[2]	朱怡萱, 刘诗芃, 张伟, 李金荣, 彭菲, 林娜, 王语嫣, 刘丹丹. 抗马链球菌马亚种组方加味优选及其体内抑菌作用研究[J]. 中国畜牧兽医, 2022, 49(10): 4085-4096.
[3]	薛文慧, 赵千惠, 梁天, 王明迪, 孙鹏, 霍书英, 李永. 禽源烟曲霉菌和黄曲霉菌的分离鉴定及几种中药抑菌作用研究[J]. 中国畜牧兽医, 2022, 49(1): 375-385.
[4]	尹珺伊, 刘秋瑾, 田秋丰, 王岩, 张军, 陈楠楠, 秦平伟, 白长胜, 孟维珊, 史同瑞, 高圣玥, 宋岩, 王佳辉, 唐玲玲, 黄宣凯, 李梦姝. 一株干酪乳杆菌的分离鉴定及生物学特性研究[J]. 中国畜牧兽医, 2021, 48(2): 516-524.
[5]	夏亿, 张元可, 徐晶云, 郭双双, 丁斌鹰. 发酵乳杆菌和凝结芽孢杆菌对产气荚膜梭菌感染肉鸡生长性能和肠道健康的影响[J]. 中国畜牧兽医, 2019, 46(10): 2927-2936.
[6]	邱园园, 韩金潭, 孙翠翠, 王赛, 刘群. "香连"溶液干膏剂体外抑菌试验研究[J]. 《中国畜牧兽医》---唯一指定的官方网站, 2015, 42(3): 708-713.
[7]	朱芝秀, 蒋新华, 邓舜洲, 张文波, 王倍, 陈龙. 解淀粉芽孢杆菌的分离鉴定及其对嗜水气单胞菌抑制作用研究[J]. 《中国畜牧兽医》---唯一指定的官方网站, 2015, 42(3): 734-740.
[8]	刘益丽, 邓霄禹, 江明锋. 溶菌酶抑菌活性及检测方法研究进展[J]. 中国畜牧兽医, 2013, 40(8): 189-194.
[9]	张国祖, 贾艳华, 刘梅, 张帅帅, 郭振环, 李荣誉, 陈献忠. 穿心莲水提物与10种临床常用抗菌药联用的体外抑菌试验[J]. , 2012, 39(12): 186-189.
[10]	林碧芬;黄志坚. 南美蟛蜞菊挥发油的抑菌活性及成分分析[J]. , 2011, 38(11): 69-72.