降胆固醇和耐酸耐胆盐益生菌的筛选研究
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摘要
本研究通过含有胆固醇胶束的MRS-胆固醇液体培养基从五株益生菌中筛选出降胆固醇能力较强的益生菌,接着对筛选出的益生菌进行耐酸、耐胆盐试验。结果表明,五株益生菌都有降胆固醇的功能,其中干酪乳杆菌LCA-1、鼠李糖乳杆菌LR-D和植物乳杆菌LP-C的胆固醇去除率较高,分别为25.26%、22.18%和20.03%;LR-D能够短时耐受低p H值环境,在p H 2.5的发酵液中培养2h的存活率能够达到14.39%,但是耐胆盐能力较差;LP-C能够长时间耐受低p H值的环境,具有较强的胆盐耐受能力,在高胆盐环境(0.5%)中培养2 h的存活率高达133.75%,培养24 h的活菌数仍保持在1.13×105 cfu/m L,此时其余两株菌的活菌未检出,说明LP-C能够耐受高胆盐环境。植物乳杆菌LP-C具有较高的胆固醇去除率,能够耐受低p H值和高胆盐环境。因此,可用于进一步研究与开发。
In this study, probiotics with strong cholesterol-lowering ability were screened from five probiotics by using MRS-cholesterol liquid medium containing cholesterol micelles, and the selected probiotics were tested for their acid and bile tolerance. The results demonstrated that all the five strains exhibited cholesterol-lowering function, with the Lactobacillus casei(LCA-1), Lactobacillus rhamnosus(LR-D) and Lactobacillus plantarum(LP-C) having higher cholesterol degradation rates(as 25.26%, 22.18% and 20.03% respectively). LR-D could tolerate a low pH environment for a short time period but exhibited poor bile salt tolerance, with a survival rate of 14.39% after a 2 h culture in the fermentation broth at pH 2.5. LP-C could tolerate a low acid environment for a long time period and exhibited strong bile salt tolerance, with a survival rate of 133.75% and the viable cell number of 1.13×10~5 cfu/m L after being cultured in high bile salt medium(0.5%) for 2 h and 24 h, respectively. In these cases, the other two strains remained undetected. These results indicated that LP-C could tolerate the high bile salt environment. LP-C possessed a high cholesterol-removing rate and could withstand a low pH and high bile salt environment. Therefore, this probiotic can be used for further research and development.
In this study, probiotics with strong cholesterol-lowering ability were screened from five probiotics by using MRS-cholesterol liquid medium containing cholesterol micelles, and the selected probiotics were tested for their acid and bile tolerance. The results demonstrated that all the five strains exhibited cholesterol-lowering function, with the Lactobacillus casei(LCA-1), Lactobacillus rhamnosus(LR-D) and Lactobacillus plantarum(LP-C) having higher cholesterol degradation rates(as 25.26%, 22.18% and 20.03% respectively). LR-D could tolerate a low pH environment for a short time period but exhibited poor bile salt tolerance, with a survival rate of 14.39% after a 2 h culture in the fermentation broth at pH 2.5. LP-C could tolerate a low acid environment for a long time period and exhibited strong bile salt tolerance, with a survival rate of 133.75% and the viable cell number of 1.13×10~5 cfu/m L after being cultured in high bile salt medium(0.5%) for 2 h and 24 h, respectively. In these cases, the other two strains remained undetected. These results indicated that LP-C could tolerate the high bile salt environment. LP-C possessed a high cholesterol-removing rate and could withstand a low pH and high bile salt environment. Therefore, this probiotic can be used for further research and development.
引文
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[10] Gilliland S E, Speck M L. Deconjugation of bile acids by intestinal Lactobacilli[J]. Applied and Environmental Microbiology, 1977, 33(1):15-18
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[14] Pato U, Surono I S. Bile and acid tolerance of lactic acid bacteria isolated from tempoyak and their probiotic potential[J]. International Journal of Agricultural Technology, 2013,9(7):1849-1862
[15] Chiu Ch, Lu T Y, Tseng Y Y, et al. The effects of Lactobacillus-fermented milk on lipid metabolism in hamsters fed on high-cholesterol diet[J]. Applied Microbiology and Biotechnology, 2006, 71(2):238-245
[16]郭春锋.人源性益生菌降胆固醇机制及影响因素研究[D].哈尔滨:哈尔滨工业大学,2011GUO Chun-feng. Study on cholesterol-lowering mechanism and influencing factors of human probiotics[D]. Harbin:Institute of Technology, 2011
[17] Liong M T, Shah N P. Acid and bile tolerance and cholesterol removal ability of Lactobacilli strains[J]. Journal of Dairy Science, 2005, 88(1):55-66
[18] Sridevi N, Vishwe P, Prabhune A.hypocholesteremic effect of bile salthydrolase from Lactobacillus buchneri ATCC 4005[J]. Food Research International, 2009, 42(4):516-520
[19]毕洁.胆盐水解酶提高乳酸菌胆盐耐受能力的酶学与生理学机制研究[D].无锡:江南大学,2016BI Jie. Enzymatic and physiological mechanisms of bile salth ydrolase to improve the tolerance of lactic acid bacteria bile salts[D]. Wuxi:Jiangnan University, 2016
[20] Xu F, Guo F, hu X J, et al. Crystal structure of bile salth ydrolase from Lactobacillus salivarius[J]. Acta Crystallographica, 2016, 72(5):376-381
[21]姜华.降胆固醇功能乳酸菌的筛选及其特性的研究[D].南京:南京农业大学,2010JIANG Hua. Screening and characteristics of cholesterollowering lactic acid bacteria[D]. Nanjing:Nanjing Agricultural University, 2010
[22]李贵节.降胆固醇乳杆菌的综合评价及BSH酶活研究[D].上海:上海交通大学,2008LI Gui-jie. Comprehensive evaluation of lactobacillus cholerae and study of BSH enzyme activity[D].Shanghai:Shanghai Jiao Tong University, 2008
[23]胡梦坤,岳喜庆.植物乳杆菌LP1103的筛选及其降胆固醇作用机理的研究[J].食品科学,2008,29(6):226-229HU Meng-kun, YUE Xi-qing. Screening of Lactobacillus plantarum LP1103 and its mechanism of cholesterol reduction[J]. Food Science, 2008, 29(6):226-229
[24] Duary R K, Batish V K, Grover S. Relative gene expression of bile salth ydrolase and surface proteins in two putative indigenous Lactobacillus plantarum strains under in vitro gut conditions[J]. Molecular Biology Reports, 2012, 39(3):2541-2552
[25] Jones M L, Martoni C J, Parent M, et al. Cholesterollowering efficacy of a microencapsulated bile salth ydrolase-active Lactobacillus reuteri NCIMB 30242 yoghurt formulation in hypercholesterolaemic adults[J]. British Journal of Nutrition, 2012, 107(10):1505-1513
[26] Singh T P, Malik R K, Katkamwar S G, et al.hypocholesterolemic effects of Lactobacillus reuteri LR6 in rats fed on high-cholesterol diet[J]. International Journal of Food Sciences and Nutrition, 2015, 66(1):71-75
[27] Kumar R, Dahiya J S, Singh D, et al. Biotransformation of cholesterol using Lactobacillus bulgaricus in a glucose-controlled bioreactor[J]. Bioresource Technology,2001, 78(2):209-211
[28]彭兴兴.乳酸菌发酵南瓜浆的研究[D].广州:华南理工大学,2016PENG Xing-xing. Study on fermentation of pumpkin pulp by lactic acid bacteria[D]. Guangzhou:South China University of Technology, 2016
[29]贺珊珊,鲍志宁,林伟锋,夏枫耿.一种含有胆固醇胶束的培养基溶液的制备方法:中国,CN108795767A[P]. 2018-11-13HE Shan-shan, BAO Zhi-ning, LIN Wei-feng, XIA Feng-geng. Preparation of a medium containing cholesterol micelles:China, CN108795767A[P], 2018-11-13
[30]鲍志宁,贺珊珊,林伟锋.一种胆固醇浓度的测定方法:中国,2018112126193[P],2018-10-18BAO Zhi-ning, HE Shan-shan, LIN Wei-feng. A method for determination of cholesterol concentration:China, 201811212619.3[P], 2018-10-18
[31]凌代文.乳酸细菌分类鉴定及实验方法[M].北京:中国轻工业出版社,1999LING Dai-wen. Classification and Identification of Lactic Acid Bacteria and Experimental Methods[M]. Beijing:China Light Industry Press, 1999
[32]张水华.食品分析[M].北京:中国轻工业出版社,2012ZHANG Shui-hua. Food Analysis[M]. Beijing:China Light Industry Press, 2012
[33]王永华.食品分析.第2版[M].北京:中国轻工业出版社,2010WANG Yong-hua. Food Analysis. 2nd ed[M]. Beijing:China Light Industry Press, 2010
[34] Dora I A P, Glenn R G. Cholesterol assimilation by lactic acid bacteria and bifido bacteria isolated from the human gut[J].Applied and Environmental Microbiology, 2002, 68(9):4689-4693
[35] Kimotoh, Ohmomo S, Okamoto T. Cholesterol removal from media by lactococci[J]. Journal of Dairy Science, 2002,85(12):3182-3188
[36]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):43-47WANG Yi-ming, FAN Xiao-bing, HANG Xiao-min, et al.Screening and mechanism of cholesterol removal in vitro[J].Microbiology China, 2006, 33(6):43-47
[37]纪旭,侯红漫,王秀玲,等.干酪乳杆菌GL-B体外降胆固醇的作用机理[J].中国乳品工业,2010,38(4):10-12,25JI Xu, HOU hong-man, WANG Xiu-ling, et al. Mechanism of cholesterol lowering in Lactobacillus casei GL-B in vitro[J]. China Dairy Industry, 2010, 38(4):10-12, 25
[38]李婷婷,张洋,林超,等.降胆固醇优良菌株的筛选及其胆盐水解酶特性研究[J].食品工业科技,2012,33(24):191-194LI Ting-ting, ZHANG Yang, LIN Chao, et al. Screening of excellent cholesterol-lowering strains and characteristics of bile salth ydrolase[J]. Science and Technology of Food Industry, 2012, 33(24):191-194
[39]陈大卫,郭飞翔,顾瑞霞,等.具有降胆固醇能力的人源乳酸菌筛选[J].现代食品科技,2014,(3):114-120CHEN Da-wei, GUO Fei-xiang, GU Rui-xia, et al. Screening of human lactic acid bacteria with cholesterol-lowering ability[J]. Modern Food Science and Technology, 2014,(3):114-120
[40]郭峰,樊磊,廖宇欣,等.植物乳杆菌M1-UVs29体外降解胆固醇的作用[J].中国生物制品学杂志,2014,27(6):803-806.GUO Feng, FAN Lei, LIAO Yu-xin, et al. Degradation of cholesterol by Lactobacillus plantarum M1-UVs29 in vitro[J]. Chinese Journal of Biological Products, 2014, 27(6):803-806
[2] Martoni C, Bhathena J, Urbanska A M, et al.Microencapsulated bile salthydrolase producing Lactobacillus reuteri for oral targeted delivery in the gastrointestinal tract[J]. Applied Microbiology and Biotechnology, 2008, 81(2):225-233
[3] Ridker P M. Evaluating novel cardiovascular risk factors:can we better predictheart attacks?[J]. Annals of Internal Medicine, 1999, 130(11):933-937
[4]林晓斐.中国居民营养与慢性病状况报告(2015年)发布[J].中医药管理杂志,2015,13:89-89Lin Xiao-fei. The Report on the Status of Nutrition and chronic diseases of chinese residents(2015)[J]. Journal of Traditional Chinese Medicine Management, 2015, 13:89-89
[5] Manson J E, Tostesonh, Ridker P M, et al. The primary prevention of myocardial infarction[J]. The New England Journal of Medicine, 1992, 326(21):1406-1416
[6] Moran A, Gu D, Zhao D, et al. Future cardiovascular disease in china:Markov model and risk factor scenario projections from the coronary heart disease policy model-china[J]. Circ Cardiovasc Qual Outcomes, 2010, 3(3):243-252
[7] Mann G V. Studies of a surfactant and cholesteremia in the Maasai[J]. The American Journal of Clinical Nutrition, 1974,27(5):464-473
[8] Mann G V. A factor in yogurt which lowers cholesteremia in man[J]. Atherosclerosis, 1977, 26(3):335-340
[9] Hepner G, Fried R, St J S, et al. hypocholesterolemic effect of yogurt and milk[J]. American Journal of Clinical Nutrition,1979, 32(1):19-24
[10] Gilliland S E, Speck M L. Deconjugation of bile acids by intestinal Lactobacilli[J]. Applied and Environmental Microbiology, 1977, 33(1):15-18
[11] Gilliland S E, Nelson C R, Maxwell C. Assimilation of cholesterol by Lactobacillus acidophilus[J]. Appl Environ Microbiol, 1985, 49(2):377-381
[12] Klaver F A, Van D M R. The assumed assimilation of cholesterol by Lactobacilli and Bifidobacterium bifidum is due to their bile salt-deconjugating activity[J]. Applied and Environmental Microbiology, 1993, 59(4):1120-1124
[13]张佳程,骆承庠.乳酸菌对食品中胆固醇脱除作用的研究-乳酸菌菌种(株)的筛选[J].食品科学,1998,19(3):20-22ZHANG Jia-cheng, LUO Cheng-xiang. Study on the removal of cholesterol from food by lactic acid bacteria-screening of lactic acid bacteria[J]. Food Science, 1998, 19(3):20-22
[14] Pato U, Surono I S. Bile and acid tolerance of lactic acid bacteria isolated from tempoyak and their probiotic potential[J]. International Journal of Agricultural Technology, 2013,9(7):1849-1862
[15] Chiu Ch, Lu T Y, Tseng Y Y, et al. The effects of Lactobacillus-fermented milk on lipid metabolism in hamsters fed on high-cholesterol diet[J]. Applied Microbiology and Biotechnology, 2006, 71(2):238-245
[16]郭春锋.人源性益生菌降胆固醇机制及影响因素研究[D].哈尔滨:哈尔滨工业大学,2011GUO Chun-feng. Study on cholesterol-lowering mechanism and influencing factors of human probiotics[D]. Harbin:Institute of Technology, 2011
[17] Liong M T, Shah N P. Acid and bile tolerance and cholesterol removal ability of Lactobacilli strains[J]. Journal of Dairy Science, 2005, 88(1):55-66
[18] Sridevi N, Vishwe P, Prabhune A.hypocholesteremic effect of bile salthydrolase from Lactobacillus buchneri ATCC 4005[J]. Food Research International, 2009, 42(4):516-520
[19]毕洁.胆盐水解酶提高乳酸菌胆盐耐受能力的酶学与生理学机制研究[D].无锡:江南大学,2016BI Jie. Enzymatic and physiological mechanisms of bile salth ydrolase to improve the tolerance of lactic acid bacteria bile salts[D]. Wuxi:Jiangnan University, 2016
[20] Xu F, Guo F, hu X J, et al. Crystal structure of bile salth ydrolase from Lactobacillus salivarius[J]. Acta Crystallographica, 2016, 72(5):376-381
[21]姜华.降胆固醇功能乳酸菌的筛选及其特性的研究[D].南京:南京农业大学,2010JIANG Hua. Screening and characteristics of cholesterollowering lactic acid bacteria[D]. Nanjing:Nanjing Agricultural University, 2010
[22]李贵节.降胆固醇乳杆菌的综合评价及BSH酶活研究[D].上海:上海交通大学,2008LI Gui-jie. Comprehensive evaluation of lactobacillus cholerae and study of BSH enzyme activity[D].Shanghai:Shanghai Jiao Tong University, 2008
[23]胡梦坤,岳喜庆.植物乳杆菌LP1103的筛选及其降胆固醇作用机理的研究[J].食品科学,2008,29(6):226-229HU Meng-kun, YUE Xi-qing. Screening of Lactobacillus plantarum LP1103 and its mechanism of cholesterol reduction[J]. Food Science, 2008, 29(6):226-229
[24] Duary R K, Batish V K, Grover S. Relative gene expression of bile salth ydrolase and surface proteins in two putative indigenous Lactobacillus plantarum strains under in vitro gut conditions[J]. Molecular Biology Reports, 2012, 39(3):2541-2552
[25] Jones M L, Martoni C J, Parent M, et al. Cholesterollowering efficacy of a microencapsulated bile salth ydrolase-active Lactobacillus reuteri NCIMB 30242 yoghurt formulation in hypercholesterolaemic adults[J]. British Journal of Nutrition, 2012, 107(10):1505-1513
[26] Singh T P, Malik R K, Katkamwar S G, et al.hypocholesterolemic effects of Lactobacillus reuteri LR6 in rats fed on high-cholesterol diet[J]. International Journal of Food Sciences and Nutrition, 2015, 66(1):71-75
[27] Kumar R, Dahiya J S, Singh D, et al. Biotransformation of cholesterol using Lactobacillus bulgaricus in a glucose-controlled bioreactor[J]. Bioresource Technology,2001, 78(2):209-211
[28]彭兴兴.乳酸菌发酵南瓜浆的研究[D].广州:华南理工大学,2016PENG Xing-xing. Study on fermentation of pumpkin pulp by lactic acid bacteria[D]. Guangzhou:South China University of Technology, 2016
[29]贺珊珊,鲍志宁,林伟锋,夏枫耿.一种含有胆固醇胶束的培养基溶液的制备方法:中国,CN108795767A[P]. 2018-11-13HE Shan-shan, BAO Zhi-ning, LIN Wei-feng, XIA Feng-geng. Preparation of a medium containing cholesterol micelles:China, CN108795767A[P], 2018-11-13
[30]鲍志宁,贺珊珊,林伟锋.一种胆固醇浓度的测定方法:中国,2018112126193[P],2018-10-18BAO Zhi-ning, HE Shan-shan, LIN Wei-feng. A method for determination of cholesterol concentration:China, 201811212619.3[P], 2018-10-18
[31]凌代文.乳酸细菌分类鉴定及实验方法[M].北京:中国轻工业出版社,1999LING Dai-wen. Classification and Identification of Lactic Acid Bacteria and Experimental Methods[M]. Beijing:China Light Industry Press, 1999
[32]张水华.食品分析[M].北京:中国轻工业出版社,2012ZHANG Shui-hua. Food Analysis[M]. Beijing:China Light Industry Press, 2012
[33]王永华.食品分析.第2版[M].北京:中国轻工业出版社,2010WANG Yong-hua. Food Analysis. 2nd ed[M]. Beijing:China Light Industry Press, 2010
[34] Dora I A P, Glenn R G. Cholesterol assimilation by lactic acid bacteria and bifido bacteria isolated from the human gut[J].Applied and Environmental Microbiology, 2002, 68(9):4689-4693
[35] Kimotoh, Ohmomo S, Okamoto T. Cholesterol removal from media by lactococci[J]. Journal of Dairy Science, 2002,85(12):3182-3188
[36]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):43-47WANG Yi-ming, FAN Xiao-bing, HANG Xiao-min, et al.Screening and mechanism of cholesterol removal in vitro[J].Microbiology China, 2006, 33(6):43-47
[37]纪旭,侯红漫,王秀玲,等.干酪乳杆菌GL-B体外降胆固醇的作用机理[J].中国乳品工业,2010,38(4):10-12,25JI Xu, HOU hong-man, WANG Xiu-ling, et al. Mechanism of cholesterol lowering in Lactobacillus casei GL-B in vitro[J]. China Dairy Industry, 2010, 38(4):10-12, 25
[38]李婷婷,张洋,林超,等.降胆固醇优良菌株的筛选及其胆盐水解酶特性研究[J].食品工业科技,2012,33(24):191-194LI Ting-ting, ZHANG Yang, LIN Chao, et al. Screening of excellent cholesterol-lowering strains and characteristics of bile salth ydrolase[J]. Science and Technology of Food Industry, 2012, 33(24):191-194
[39]陈大卫,郭飞翔,顾瑞霞,等.具有降胆固醇能力的人源乳酸菌筛选[J].现代食品科技,2014,(3):114-120CHEN Da-wei, GUO Fei-xiang, GU Rui-xia, et al. Screening of human lactic acid bacteria with cholesterol-lowering ability[J]. Modern Food Science and Technology, 2014,(3):114-120
[40]郭峰,樊磊,廖宇欣,等.植物乳杆菌M1-UVs29体外降解胆固醇的作用[J].中国生物制品学杂志,2014,27(6):803-806.GUO Feng, FAN Lei, LIAO Yu-xin, et al. Degradation of cholesterol by Lactobacillus plantarum M1-UVs29 in vitro[J]. Chinese Journal of Biological Products, 2014, 27(6):803-806