酸马奶源发酵乳杆菌对致病性大肠杆菌O_8的体外抑菌研究
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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 O824h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399.5μg/mL;最低抑菌浓度(MIC)和最低杀菌浓度(MBC)分别为25.0和49.9μg/mL;CFS能使致病性E.coli O8的碱性磷酸酶(AKP)含量在1h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。
In order to study the antibacterial mechanism of Lactobacillusfermentumisolated 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 16 S 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 hwas 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 onpathogenic E.coli O8 isolated from koumiss,and the best strain was Lactobacillus fermentumafter 16 S rDNA sequence and phylogenetic tree analysis.Protein was the main bacteriostatic substance of CFS,and its content was 399.5μg/mL.The minimum inhibitory concentration(MIC)and the minimum bactericidal concentration(MBC)were 25.0 and 49.9μg/mL,respectively.CFS could rapidly increase the AKP content of pathogenic E.coli O8 within 1 hand 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.
In order to study the antibacterial mechanism of Lactobacillusfermentumisolated 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 16 S 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 hwas 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 onpathogenic E.coli O8 isolated from koumiss,and the best strain was Lactobacillus fermentumafter 16 S rDNA sequence and phylogenetic tree analysis.Protein was the main bacteriostatic substance of CFS,and its content was 399.5μg/mL.The minimum inhibitory concentration(MIC)and the minimum bactericidal concentration(MBC)were 25.0 and 49.9μg/mL,respectively.CFS could rapidly increase the AKP content of pathogenic E.coli O8 within 1 hand 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.
引文
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[17] PAPADEMAS P,PARMAXI I,ASPRI M.Probiotic,antimicrobial,antioxidant and sensory properties of fermented donkey milk with Lactobacillus fermentum ME-3and 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 O8and 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 shelflife extension[J].International Dairy Journal,2006,16(9):1058-1071.
[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 bacteria[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 ofLactobacillus fermentum CECT5716enhances 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-3as 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 5716in 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 ApcMin/+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 213strains 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-3and 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 O8and 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 shelflife extension[J].International Dairy Journal,2006,16(9):1058-1071.