为了筛选出具有高胆盐水解酶活性的乳酸菌,以生鲜牛奶为样品,利用传统分离技术分离乳酸菌,并通过牛津杯法选出具有高胆盐水解酶活性的乳酸菌,进而采用16S rDNA测序技术对筛选菌株进行鉴定。同时,研究pH、紫外照射时间、温度、培养基盐浓度和培养基糖浓度对所筛选菌株胆盐水解酶活性的影响。实验中分离出2株具有高胆盐水解酶活性的植物乳杆菌,为菌株N1和N12,胆盐水解酶高活性的最适pH为7、最适温度为59 ℃。菌株N1最适糖质量分数与最适盐质量分数分别为10％和7％,菌株N12最适糖质量分数与最适盐质量分数分别为5％和4％。植物乳杆菌N1和N12具有高胆盐水解酶活性,具有降解人体血清胆固醇的潜力,可应用于降胆固醇产品的开发。
In order to screen lactic acid bacteria(LAB) with high bile salt hydrolase (BSH) activity, fresh milk was used as the sample and LAB were isolated by traditional techniques. LAB with high BSH activity was screened by Oxford Cup method, then 16S rDNA sequencing was used to identify the strains. The effects of pH, UV exposure, temperature, and salt and sugar content in the medium on BSH activity of the strains were studied. The results showed that two Lactobacillus plantarum strains with high BSH activity were isolated, named N1 and N12, the optimal pH and temperature for BSH activity is 7.0 and 59 ℃, respectively. The optimum mass fraction of sugar and salt for N1 were 10% and 7%, respectively, and those for N12 were 5% and 4%, respectively. N1 and N12 have the potential to degrade serum cholesterol due to high BSH activity, and can be used in the development of cholesterol-lowering products.
[1] JAMES W, ANDERSON, STANLEY E, et al. Effect of fermented milk (Yogurt) containing Lactobacillus acidophilus L1 on serum cholesterol in hypercholesterolemic humans[J]. Journal of the American College of Nutrition, 1999,18(1):43-50.
[2] 付永岩,赵悦含,王毅超,等.降胆固醇乳酸菌的益生特性及其作用机理[J].中国乳品工业,2019,47(7):23-27.
[3] 李尧,张羽竹,张利,等.分离自传统自然发酵食品中降胆固醇乳酸菌的筛选与评价[J].中国食品学报,2019,19(6):212-222.
[4] 范颖,陈思涵,党芳芳,等.一株降胆固醇乳酸菌的筛选及其在模拟消化环境活性的研究[J].中国乳品工业,2018,46(9):4-7.
[5] 董改香,张勇刚.具有胆盐水解酶活性乳酸菌体内降胆固醇功效机理的研究[J].甘肃畜牧兽医,2018,48(4):68-71.
[6] 王继超. 胆盐水解酶的分离纯化及酶学性质的研究[D].哈尔滨:黑龙江大学,2013.
[7] KLAVER F A, VAN DER MEER R. The assumed assimilation of cholesterol by Lactobacilli and Bifidobacterium bifidum is due to their bile salt-deconjugating activity[J]. Appl. Environ. Microbiol, 1993, 59(4): 1 120-1 124.
[8] 任大勇,曲天铭,杨柳,等.东北传统发酵食品中降胆固醇乳酸菌的筛选及降解机制研究[J].食品科学:1-12[2019-12-04].http://kns.cnki.net/kcms/detail/11.2206.TS.20190329.1349.002.html.
[9] 田建军,张开屏,李权威,等.乳酸菌调控胆固醇代谢的物质基础研究进展[J].食品科学,2019,40(19):334-339.
[10] 武昌俊,戴陈伟,李舜,等.一株安徽本地泡菜中产细菌素乳酸菌的筛选与鉴定[J].中国食品添加剂,2019,30(7):112-120.
[11] 胡虹,杜迅,张秀江,等.液体发酵饲料植物乳杆菌诱变育种的研究[J].河南科学,2019,37(4):570-574.
[12] 王玉文,刘慧,李平兰,等.产胆盐水解酶乳酸菌的分离、鉴定及降解胆固醇机理的初步研究[J].食品科学,2006(10):215-218.
[13] 欧伶, 俞建瑛, 金新根. 应用生物化学[M]. 北京:化学工业出版社, 2001:74-87.
[14] LAMBERT J M, BONGERS R S, VOS W M, et al. Functional analysis of four bile salt hydrolase and penicillin acylase family members in Lactobacillus plantarum WCFS1[J]. Appl Environ Microbiol, 2008, 74(15): 4 719-4 726.
[15] 吴诗敏,雷文平,周辉,等.南山牧场牛乳中具有抑菌活性乳酸菌的筛选及鉴定[J].中国酿造,2019,38(5):108-112.
[16] 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,2016.
[17] 汤慧勤,姚晓敏,徐一涵,等.产胆盐水解酶植物乳杆菌降胆固醇作用的研究[J].上海交通大学学报(农业科学版),2018,36(4):35-40.
[18] 姜金康. 乳杆菌属胆汁盐水解酶的活性多样性、底物特异性及相关基因研究[D].上海:上海交通大学,2008.
[19] 刘静,王瑾,秦雪梅,等.具有胆固醇去除能力和胆盐水解酶活性的乳酸菌的体外筛选[J].食品科技,2011,36(5):18-22.
[20] 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):191-194.
[21] 惠明,刘勇,张红星,等.产胆盐水解酶BSH乳酸菌的鉴定及发酵条件优化[J].中国食品学报,2011,11(1):214-221.
[22] 李奕佳. 芥菜的清洁发酵工艺及产品开发研究[D].长沙:湖南农业大学,2017.
[23] 蒲博,张驰翔,王周,等.乳酸菌降胆固醇作用及其机理的研究进展[J].中国酿造,2014,33(7):5-9.
