降胆固醇乳酸菌的筛选和分析比较
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摘要
目的比较7株乳酸菌各项生理特性,筛选高效、安全降解胆固醇的益生菌株,为后期开发应用奠定基础。方法对选定菌株依次进行体外降胆固醇、胆盐水解酶(BSH)活力、人工胃肠液及胆盐耐受性、细胞粘附性能的测定和比较,并分析抗生素耐药性和抑制病原菌活性,最后利用16S rRNA基因测序确定细菌种属。结果不同菌株各项生理指标存在差异。其中菌株ZL-2、JQI-7和PC-26的BSH酶活分别为0.010、0.015和0.030 U,可高效清除体外胆固醇,3株菌体外降解胆固醇率在58%以上;并且耐酸、耐胆盐,具备较好的HT--29细胞粘附性,不存在抗生素耐药性,可有效抑制大肠埃希菌、金黄色葡萄球菌、铜绿假单胞菌和阴沟肠杆菌生长。结论菌株ZL-2、JQI-7和PC-26满足降胆固醇益生菌株的生物学特性,可用于进一步研究和开发。
Objective To analyze the biological characteristics of seven Lactobacillus strains,screen cholesterollowering strains with safety and high-efficiency,and lay foundation for industrial production.Methods The capabilities of lowering cholesterol in vitro,activity of bile salt hydrolase(BSH),tolerance to artificial gastric intestinal juice and bile salt,cell adhesion capacity,tolerance to antibiotics and antimicrobial activity of the selected strains were detected and compared.16 S rRNA sequencing was conducted to identify the Lactobacillus species.Results Among the seven strains,the BSH activities of ZL-2,JQI-7 and PC-26 were 0.010 U,0.015 U and 0.030 U,respectively.The degradation rates of cholesterol by the three strains were over 58%.They showed good tolerance to acid and bile salt,were sensitive to 13 antibiotics,and demonstrated strong adhesion to HT-29 cells and inhibitory effect to Escherichia coli,Staphylococcus aureus,Pseudomonas aeruginosa and Enterobacter cloacae.Conclusion The strains ZL-2,JQI-7 and PC-26 can be further applied in the industrial production since they have good biological characteristics and significant cholesterol-lowering effect.
Objective To analyze the biological characteristics of seven Lactobacillus strains,screen cholesterollowering strains with safety and high-efficiency,and lay foundation for industrial production.Methods The capabilities of lowering cholesterol in vitro,activity of bile salt hydrolase(BSH),tolerance to artificial gastric intestinal juice and bile salt,cell adhesion capacity,tolerance to antibiotics and antimicrobial activity of the selected strains were detected and compared.16 S rRNA sequencing was conducted to identify the Lactobacillus species.Results Among the seven strains,the BSH activities of ZL-2,JQI-7 and PC-26 were 0.010 U,0.015 U and 0.030 U,respectively.The degradation rates of cholesterol by the three strains were over 58%.They showed good tolerance to acid and bile salt,were sensitive to 13 antibiotics,and demonstrated strong adhesion to HT-29 cells and inhibitory effect to Escherichia coli,Staphylococcus aureus,Pseudomonas aeruginosa and Enterobacter cloacae.Conclusion The strains ZL-2,JQI-7 and PC-26 can be further applied in the industrial production since they have good biological characteristics and significant cholesterol-lowering effect.
引文
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[8]Gagnon M,Berner AZ,Chervet N,et al.Comparison of the Caco-2,HT-29 and the mucus-secreting HT29-MTX intestinal cell models to investigate Salmonella adhesion and invasion[J].J Microbiolog Methods,2013,94(3):274-279.
[9]YU Zhihui.Screening,fermentation properties and in vivo efficacy of cholesterol-lowering probiotic Lactobacillus Plantarum strains[D].Changchun:Jilin University,2013.(in Chinese)于志会.益生性降胆固醇植物乳杆菌的筛选,发酵特性及体内功效研究[D].长春:吉林大学,2013.
[10]Gismondo MR,Drago L,Lombardi A.Review of probiotics available to modify gastrointestinal flora[J],Int J Antimicrob Agents,1999,12(4):287-292.
[11]ZHAO Jiarui,YANG Hong.Progress in the effect of probiotics on cholesterol and its mechanism[J],Acta Microbiologica Sinica,2005,45(2):315-319.(in Chinese)赵佳锐,杨虹.益生菌降解胆固醇的作用及机理研究进展[J].微生物学报,2005,45(2):315-319
[12]Begley M,Hill C,Gahan CGM Bile salt hydrolase activity in probiotics[J].Appl Environ Microbiol,2006,72(3):1729-1738.
[13]Gilliland SE,Speck ML.Deconjugation of bile acids by intestinal lactobacilli[J].Appl Environ Microbiol,1977,33(1):15-18.
[14]Jones ML,MartoniCJ,Parent M,etal.Cholesterol-lowering efficacy of a microencapsulated bile salt hydrolase-active Lactobacillus reuteri NCIMB 30242 yoghurt formulation in hypercholesterolaemic adults[J].Br J Nutr,2012,107(10):1505-1513.
[15]Bordoni A,Amaretti A,Leonardi A,et al.Cholesterol-lowering probiotics:In vitro selection and in vivo testing of Bifidobacteria[J].Appl Microbiol&Biotechnol,2013,97(18):8273-8281.
[16]LI Wenjing.LIANG Yunxiang.ZHAO Shumiao.Comparative analysis of the biological characteristics of three strains of Lactobacillus reuteri[J].Microbiol China,2016,43(5):1035-1041.(in Chinese)李文静,梁运祥,赵述淼.3株罗伊氏乳杆菌生物学特性的分析比较[J].微生物学通报,2016,43(5):1035-1041.
[17]Hechard Y,Dherbomez M,Cenatiempo Y,et al.Antagonism of lactic acid bacteria from goats'milk against pathogenic strains assessed by the"sandwich method"[J].Lett Appl Microbiol,1990,11(4):185-188.
[18]Maragkoudakis PA,Zoumpopoulou G,Miaris C,et al.Probiotic potential of Lactobacillus strains isolated from dairy products[J].Int Dairy J,2006,16(3):189-199.
[19]LI Qing,WANG Ying,LIU Xiaoli,et al.Isolation of a broadspectrum antibacterial lactic acid bacterium and evaluation of probiotic properties[J].Microbiol China,2015,42(2):332-339.(in Chinese)李清,王英,刘小莉,等.一株广谱抑菌活性乳酸菌的筛选及特性研究[J].微生物学通报,2015,42(2):332-339.
[20]Hamon E,Horvatovich P,Izquierdo E,et al.Comparative proteomic analysis of Lactobacillus plantarum for the identification of key proteins in bile tolerance[J].BMC Microbiol,2011,11(63):1471-1481.
[21]WEI Yinping,WANG Bin,WEI Hong.Study of adhesion capability of three probiotics to HT-29 cells and adhesion inhibition of bacterial pathogens[J].China Pharmac,2006,15(8):7-8.(in Chinese)魏银萍,王斌,魏泓.益生菌株对HT-29细胞的粘附及对致病菌的粘附抑制性能研究[J].中国药业,2006,15(8):7-8.
[22]LI Pinglan,YANG Hua,ZHANG Chi.Study on adhesion ability of lactic acid bacteria to the colonic adenocarcinoma cell line HT-29[J].J China Agric Univ,2002,7(1):19-22.(in Chinese)李平兰,杨华,张篪.乳酸菌体外粘附人结肠腺癌细胞系HT-29细胞的研究[J].中国农业大学学报,2002,7(1):19-22.
[23]LIU Hanlin.Study on the cholesterol-reducing characteristic of Lactobacillus isolated from traditional dairy products in pasture region of Inner Mongolia[D].Hohhot:Inner Mongolia Agricultural University,2006.(in Chinese)柳翰凌.内蒙古牧区传统乳制品中乳杆菌降胆固醇特性的研究[D].呼和浩特:内蒙古农业大学,2006.
[2]JIN Zhiqiang,WANG Yanxiang,LI Pinglan,et al.Acid and bile tolerance of Lactobacillus plantarum in vitro and its cholesterol-lowering effect[J].J Chin Institute Food Sci&Technol,2009,9(5):24-28.(in Chinese)靳志强,王延祥,李平兰,等.植物乳杆菌耐酸耐胆盐的体外试验及其降胆固醇作用[J].中国食品学报,2009,9(5):24-28.
[3]Martoni C,Bhathena J,Urbanska AM,et al.Microencapsulated bile salt hydrolase producing Lactobacillus reuteri for oral targeted delivery in the gastrointestinal tract[J].Appl Microbiol&Biotechnol,2008,81(2):225-233.
[4]LI Ni.Screening and mechanism of cholesterol-lowering Lactobacillus[D].Xiamen:Jimei University,2012.(in Chinese)李昵.降胆固醇乳酸菌的筛选及其降解机理研究[D].厦门:集美大学,2012.
[5]WANG Yiming,FAN Xiaobing,HANG Xiaomin,et al.In vitro selection of probiotic Lactobacillus from the human feces and their mechanism on cholesterol-lowering[J].Microbiol China,2006,33(6):43-47.(in Chinese)王一鸣,范小兵,杭晓敏,等.体外去除胆固醇菌株的筛选及其作用机理研究[J].微生物学通报,2006,33(6):43-47.
[6]CHEN Chen.Properties of the adhesion of Lactobacillus plantarum ST-Ⅲon intestinal epithelial cells and its mechanism study[D].Wuxi:Jiangnan University,2008.(in Chinese)陈臣.植物乳杆菌ST-Ⅲ对肠上皮细胞的粘附性质及机理的研究[D].无锡:江南大学,2008.
[7]CHEN Chen,GUO Benhuan,WANG Yinyu,et al.Properties of the adhesion of Lactobacillus plantarum ST-Ⅲon enterocyte-like cell line Caco-2[J].J Dairy Sci&Technol,2008,31(2):51-55.(in Chinese)陈臣,郭本恒,王荫榆,等.植物乳杆菌ST-Ⅲ粘附肠上皮样细胞系Caco-2性质的研究[J].乳业科学与技术,2008,31(2):51-55.
[8]Gagnon M,Berner AZ,Chervet N,et al.Comparison of the Caco-2,HT-29 and the mucus-secreting HT29-MTX intestinal cell models to investigate Salmonella adhesion and invasion[J].J Microbiolog Methods,2013,94(3):274-279.
[9]YU Zhihui.Screening,fermentation properties and in vivo efficacy of cholesterol-lowering probiotic Lactobacillus Plantarum strains[D].Changchun:Jilin University,2013.(in Chinese)于志会.益生性降胆固醇植物乳杆菌的筛选,发酵特性及体内功效研究[D].长春:吉林大学,2013.
[10]Gismondo MR,Drago L,Lombardi A.Review of probiotics available to modify gastrointestinal flora[J],Int J Antimicrob Agents,1999,12(4):287-292.
[11]ZHAO Jiarui,YANG Hong.Progress in the effect of probiotics on cholesterol and its mechanism[J],Acta Microbiologica Sinica,2005,45(2):315-319.(in Chinese)赵佳锐,杨虹.益生菌降解胆固醇的作用及机理研究进展[J].微生物学报,2005,45(2):315-319
[12]Begley M,Hill C,Gahan CGM Bile salt hydrolase activity in probiotics[J].Appl Environ Microbiol,2006,72(3):1729-1738.
[13]Gilliland SE,Speck ML.Deconjugation of bile acids by intestinal lactobacilli[J].Appl Environ Microbiol,1977,33(1):15-18.
[14]Jones ML,MartoniCJ,Parent M,etal.Cholesterol-lowering efficacy of a microencapsulated bile salt hydrolase-active Lactobacillus reuteri NCIMB 30242 yoghurt formulation in hypercholesterolaemic adults[J].Br J Nutr,2012,107(10):1505-1513.
[15]Bordoni A,Amaretti A,Leonardi A,et al.Cholesterol-lowering probiotics:In vitro selection and in vivo testing of Bifidobacteria[J].Appl Microbiol&Biotechnol,2013,97(18):8273-8281.
[16]LI Wenjing.LIANG Yunxiang.ZHAO Shumiao.Comparative analysis of the biological characteristics of three strains of Lactobacillus reuteri[J].Microbiol China,2016,43(5):1035-1041.(in Chinese)李文静,梁运祥,赵述淼.3株罗伊氏乳杆菌生物学特性的分析比较[J].微生物学通报,2016,43(5):1035-1041.
[17]Hechard Y,Dherbomez M,Cenatiempo Y,et al.Antagonism of lactic acid bacteria from goats'milk against pathogenic strains assessed by the"sandwich method"[J].Lett Appl Microbiol,1990,11(4):185-188.
[18]Maragkoudakis PA,Zoumpopoulou G,Miaris C,et al.Probiotic potential of Lactobacillus strains isolated from dairy products[J].Int Dairy J,2006,16(3):189-199.
[19]LI Qing,WANG Ying,LIU Xiaoli,et al.Isolation of a broadspectrum antibacterial lactic acid bacterium and evaluation of probiotic properties[J].Microbiol China,2015,42(2):332-339.(in Chinese)李清,王英,刘小莉,等.一株广谱抑菌活性乳酸菌的筛选及特性研究[J].微生物学通报,2015,42(2):332-339.
[20]Hamon E,Horvatovich P,Izquierdo E,et al.Comparative proteomic analysis of Lactobacillus plantarum for the identification of key proteins in bile tolerance[J].BMC Microbiol,2011,11(63):1471-1481.
[21]WEI Yinping,WANG Bin,WEI Hong.Study of adhesion capability of three probiotics to HT-29 cells and adhesion inhibition of bacterial pathogens[J].China Pharmac,2006,15(8):7-8.(in Chinese)魏银萍,王斌,魏泓.益生菌株对HT-29细胞的粘附及对致病菌的粘附抑制性能研究[J].中国药业,2006,15(8):7-8.
[22]LI Pinglan,YANG Hua,ZHANG Chi.Study on adhesion ability of lactic acid bacteria to the colonic adenocarcinoma cell line HT-29[J].J China Agric Univ,2002,7(1):19-22.(in Chinese)李平兰,杨华,张篪.乳酸菌体外粘附人结肠腺癌细胞系HT-29细胞的研究[J].中国农业大学学报,2002,7(1):19-22.
[23]LIU Hanlin.Study on the cholesterol-reducing characteristic of Lactobacillus isolated from traditional dairy products in pasture region of Inner Mongolia[D].Hohhot:Inner Mongolia Agricultural University,2006.(in Chinese)柳翰凌.内蒙古牧区传统乳制品中乳杆菌降胆固醇特性的研究[D].呼和浩特:内蒙古农业大学,2006.