牦牛酸奶酪中高产乳糖酶菌株分离鉴定及其酶学性质
|
摘要
为获得藏区牦牛酸奶酪中的产乳糖酶活力高的菌株,采用含X-gal的培养基进行初筛,从购买的10份样品中筛选到10株产乳糖酶细菌;发酵复筛后,发现其中RTM-111菌株的产酶活力最高。综合形态学特征、生理生化实验及16S rDNA序列分析,确定该菌株为枯草芽孢杆菌(Bacillus subtilis)。对该菌株所产乳糖酶的酶学性质研究表明,该酶的最适反应温度为50℃,最适pH为7.5。稳定性实验表明,该酶在温度40～50℃、pH6.5～8.0时具有较高稳定性。金属离子Na~+、Mg~(2+)和Mn~(2+)对酶活性具有较强的激活作用,而Cu~(2+)、Zn~(2+)和EDTA对酶活有显著的抑制作用。枯草芽孢杆菌RTM-111产乳糖酶活力高、酶的性质稳定,具有潜在应用价值。
Using the screening medium containing X-gal,10 strains with high producing lactase activity were isolated from 10 yak yogurt cheese samples purchased from Xizang.An efficient lactase producing strain RTM-111 rescreened out from the 10 strains was identified as Bacillus subtilis by the means of morphological feature,physiological and biochemical characteristics measurement and 16S rDNA sequence analysis.Lactase enzyme properties research showed that the optimal reaction temperature and pH were 50 ℃ and 7.5.The enzyme was stable at 40 ～50 ℃ and pH6.5 ～8.0. The lactase activity was promoted by Na~+,Mg~(2+)and Mn~(2+),and strongly inhibited by Cu~(2+),Zn~(2+)and EDTA.The high producing activity of B. subtilis RTM-111 and the stability of the lactase made it potentially useful.
Using the screening medium containing X-gal,10 strains with high producing lactase activity were isolated from 10 yak yogurt cheese samples purchased from Xizang.An efficient lactase producing strain RTM-111 rescreened out from the 10 strains was identified as Bacillus subtilis by the means of morphological feature,physiological and biochemical characteristics measurement and 16S rDNA sequence analysis.Lactase enzyme properties research showed that the optimal reaction temperature and pH were 50 ℃ and 7.5.The enzyme was stable at 40 ～50 ℃ and pH6.5 ～8.0. The lactase activity was promoted by Na~+,Mg~(2+)and Mn~(2+),and strongly inhibited by Cu~(2+),Zn~(2+)and EDTA.The high producing activity of B. subtilis RTM-111 and the stability of the lactase made it potentially useful.
引文
[1]刘芳宁,梁琪,张炎,等.微生物β-半乳糖苷酶的研究进展[J].中国酿造杂志,2012,31(12):5-9.
[2]贺璐,龙承星,刘又嘉,等.微生物乳糖酶研究进展[J].食品与发酵工业,2017,43(6):268-273.
[3]Berni C R,Pezzella V,Amoroso A,et al.Diagnosing and treating intolerance to carbohydrates in children[J].Nutrients,2016,8(3):157.
[4]Park AR,Oh D K.Galacto-oligosaccharide production using microbialβ-galactosidase:Current state and perspectives[J].Applied Microbiology and Biotechnology,2010,85(5):1279-1286.
[5]Ana I R M,Marta C M,Antonia M,et al.Presence of mono-,di-and galactooligosaccharides in commercial lactose-free UHTdairy products[J].Journal of Food Composition and Analysis,2012,28(2):164-169.
[6]于嗣祥,杨林华,徐爱玲,等.乳清的营养价值及开发利用现状[J].农产品加工,2009(8):36-38.
[7]朱培,莫蓓红,刘振民.乳清生物技术处理研究进展[J].食品工业科技,2014,35(9):355-359.
[8]姜旭.乳糖酶在乳品工业中的应用[J].商品与质量·学术观察,2014(1):119.
[9]赵晓慧,苏佳,史玉东,等.牛乳工业中乳糖酶的应用研究进展[J].食品科技,2016,41(4):228-231.
[10]张萃荟,杨瑞金,张文斌,等.适合乳果糖生产的β-半乳糖苷酶高产株的筛选及培养基优化[J].食品工业科技,2008,29(4):68-74.
[11]杜海英,于宏伟,韩军,等.原生质体诱变选育乳糖酶高产菌株[J].微生物学通报,2006,33(6):48-51.
[12]高兆建,侯进慧,孙会刚,等.耐高温β-半乳糖苷酶的分离纯化与酶学性质分析[J].食品科学,2010,23(31):151-156.
[13]章沙沙.β-半乳糖苷酶基因的克隆、表达及应用[D].长沙:湖南农业大学,2010.
[14]兆娟,徐颖,石磊,等.凝结芽孢杆菌中β-半乳糖苷酶基因的克隆及表达[J].南京林业大学学报:自然科学版,2015,39(6):35-39.
[15]Zhang H,Chen X,Dan T,et al.Traditional Chinese fermented dairy foods[M].Zhang H,Cai Y.Lactic acid bacteria fundamentalsand practice.New York:Springer,2014:493-535.
[16]贾宏信,吴正钧,刘振工,等.干酪内微生物及其功能的研究进展[J].食品与发酵工业,2012,38(2):35-140.
[17]甘伯中,李晓鹏,张卫兵,等.天祝牧区牦牛乳酸奶中优良乳酸菌的分离机筛选[J].食品科学,2010,31(5):184-189.
[18]罗毅皓,孙万成.牦牛酸奶和酥油中乳酸菌的分离和鉴定[J].中国乳品工业,2017,45(11):11-13.
[19]蒋厚阳,陈芝兰,赵国华,等.PCR-DGGE法分析西藏传统发酵乳制品中乳酸菌的多样性[J].食品科学,2014,35(1):167-173.
[20]张兴吉,葛武鹏,刘阳,等.西部传统发酵乳品中乳酸菌筛分及其亚硝酸盐降解能力[J].食品科学,2018,39(14):199-205.
[21]刘珊春,赵欣,骞宇,等.西藏羊八井地区牦牛酸乳中乳酸菌菌种鉴定[J].食品工业科技,2016,37(10):208-216.
[22]Yu J,Sun Z,Liu W,et al.Rapid identification of lactic acid bacteria isolated from home-made fermented milk in Tibet[J].Journal of General and Applied Microbiology,2009,55(3):181-190.
[23]巨蕾,梁琪,乔海军,等.甘南牧区牦牛乳酸奶中产乳糖酶菌株的筛选、鉴定及产酶条件研究[J].食品工业科技,2012,33(12):239-254.
[24]东秀珠,蔡妙英,等编著.常见细菌系统鉴定手册[M].北京:科学出版社,2001:62-63.
[25]张卫兵,张炎,文鹏程,等.Enterobacter SYA2乳糖酶酶学性质研究[J].中国酿造,2012,31(11):93-98.
[26]李文婷,边斐,王翠萍,等.凝结芽孢杆菌RY237β-半乳糖苷酶酶学性质研究[J].食品工业科技,2017,38(14),111-115.
[27]潘琳琳,赵新淮.嗜热脂肪芽孢杆菌产β-半乳糖苷酶发酵条件优化[J].食品工业科技,2009,30(7):187-193.
[28]沈莲莲,杨瑞金,华霄,等.产低温乳糖酶菌株的筛选、鉴定及酶学性质的研究[J].工业微生物,2013,43(3):12-17.
[29]Venkateswarulu TC,Prabhakar KV,Kumar RB,et al.Optimization of variables for lactase production from isolated Bacillus subtilis strain VUVD001 through submerged fermentation[J].Current Trends in Biotechnology&Pharmacy,2017,11(4):370-375.
[30]王景会,李达,姜媛媛,等.枯草芽孢杆菌JAASB4的安全性评价[J].吉林农业科学,2015,40(5):102-103.
[31]Simon M.Bacillus probiotics[J].Food Microbiology,2011,28(1):214-220.
[32]王利宾,孙利娜.枯草芽孢杆菌作用机制及其在养殖业中的应用研究进展[J].饲料博览,2015(1):35-38.
[33]刘刚,杨雪,梅雪然,等.枯草芽孢杆菌Loq18抗菌蛋白的分离纯化及其对灰葡萄孢的抑制活性[J].四川师范大学学报(自然科学版),2015,46-49.
[34]Karasov P,Spiwok V,Mal KR,et al.Beta-galactosidase activity in psychrotrophic microorganisms and their potential use in food industry[J].Czech Journal of Food Sciences,2002,20:43-47.
[35]张红艳,刘成更.乳糖酶的酶学特性及其研究进展[J].食品研究与开发,2004,25(6):34-36.
[36]张明丽,熊华,齐金峰,等.乳酸克鲁维酵母乳糖酶性质的研究[J].中国乳品工业,2008,36(1):7-9.
[37]Rahim KAA,Lee BH.Specificity,inhibitory studies,and oligosaccharide formation byβ-Galactosidase from psychrotrophic Bacillus subtilis KL88[J].Journal of dairy science,1991,74(6):1773-1778.
[38]Lara ABCC,Li Y,Paul D,et al.Characterization of aβ-galactosidase from Bacillus subtilis with ransgalactosylation activity[J].International Journal of Biological Macromolecules,2018,120:279-287.
[2]贺璐,龙承星,刘又嘉,等.微生物乳糖酶研究进展[J].食品与发酵工业,2017,43(6):268-273.
[3]Berni C R,Pezzella V,Amoroso A,et al.Diagnosing and treating intolerance to carbohydrates in children[J].Nutrients,2016,8(3):157.
[4]Park AR,Oh D K.Galacto-oligosaccharide production using microbialβ-galactosidase:Current state and perspectives[J].Applied Microbiology and Biotechnology,2010,85(5):1279-1286.
[5]Ana I R M,Marta C M,Antonia M,et al.Presence of mono-,di-and galactooligosaccharides in commercial lactose-free UHTdairy products[J].Journal of Food Composition and Analysis,2012,28(2):164-169.
[6]于嗣祥,杨林华,徐爱玲,等.乳清的营养价值及开发利用现状[J].农产品加工,2009(8):36-38.
[7]朱培,莫蓓红,刘振民.乳清生物技术处理研究进展[J].食品工业科技,2014,35(9):355-359.
[8]姜旭.乳糖酶在乳品工业中的应用[J].商品与质量·学术观察,2014(1):119.
[9]赵晓慧,苏佳,史玉东,等.牛乳工业中乳糖酶的应用研究进展[J].食品科技,2016,41(4):228-231.
[10]张萃荟,杨瑞金,张文斌,等.适合乳果糖生产的β-半乳糖苷酶高产株的筛选及培养基优化[J].食品工业科技,2008,29(4):68-74.
[11]杜海英,于宏伟,韩军,等.原生质体诱变选育乳糖酶高产菌株[J].微生物学通报,2006,33(6):48-51.
[12]高兆建,侯进慧,孙会刚,等.耐高温β-半乳糖苷酶的分离纯化与酶学性质分析[J].食品科学,2010,23(31):151-156.
[13]章沙沙.β-半乳糖苷酶基因的克隆、表达及应用[D].长沙:湖南农业大学,2010.
[14]兆娟,徐颖,石磊,等.凝结芽孢杆菌中β-半乳糖苷酶基因的克隆及表达[J].南京林业大学学报:自然科学版,2015,39(6):35-39.
[15]Zhang H,Chen X,Dan T,et al.Traditional Chinese fermented dairy foods[M].Zhang H,Cai Y.Lactic acid bacteria fundamentalsand practice.New York:Springer,2014:493-535.
[16]贾宏信,吴正钧,刘振工,等.干酪内微生物及其功能的研究进展[J].食品与发酵工业,2012,38(2):35-140.
[17]甘伯中,李晓鹏,张卫兵,等.天祝牧区牦牛乳酸奶中优良乳酸菌的分离机筛选[J].食品科学,2010,31(5):184-189.
[18]罗毅皓,孙万成.牦牛酸奶和酥油中乳酸菌的分离和鉴定[J].中国乳品工业,2017,45(11):11-13.
[19]蒋厚阳,陈芝兰,赵国华,等.PCR-DGGE法分析西藏传统发酵乳制品中乳酸菌的多样性[J].食品科学,2014,35(1):167-173.
[20]张兴吉,葛武鹏,刘阳,等.西部传统发酵乳品中乳酸菌筛分及其亚硝酸盐降解能力[J].食品科学,2018,39(14):199-205.
[21]刘珊春,赵欣,骞宇,等.西藏羊八井地区牦牛酸乳中乳酸菌菌种鉴定[J].食品工业科技,2016,37(10):208-216.
[22]Yu J,Sun Z,Liu W,et al.Rapid identification of lactic acid bacteria isolated from home-made fermented milk in Tibet[J].Journal of General and Applied Microbiology,2009,55(3):181-190.
[23]巨蕾,梁琪,乔海军,等.甘南牧区牦牛乳酸奶中产乳糖酶菌株的筛选、鉴定及产酶条件研究[J].食品工业科技,2012,33(12):239-254.
[24]东秀珠,蔡妙英,等编著.常见细菌系统鉴定手册[M].北京:科学出版社,2001:62-63.
[25]张卫兵,张炎,文鹏程,等.Enterobacter SYA2乳糖酶酶学性质研究[J].中国酿造,2012,31(11):93-98.
[26]李文婷,边斐,王翠萍,等.凝结芽孢杆菌RY237β-半乳糖苷酶酶学性质研究[J].食品工业科技,2017,38(14),111-115.
[27]潘琳琳,赵新淮.嗜热脂肪芽孢杆菌产β-半乳糖苷酶发酵条件优化[J].食品工业科技,2009,30(7):187-193.
[28]沈莲莲,杨瑞金,华霄,等.产低温乳糖酶菌株的筛选、鉴定及酶学性质的研究[J].工业微生物,2013,43(3):12-17.
[29]Venkateswarulu TC,Prabhakar KV,Kumar RB,et al.Optimization of variables for lactase production from isolated Bacillus subtilis strain VUVD001 through submerged fermentation[J].Current Trends in Biotechnology&Pharmacy,2017,11(4):370-375.
[30]王景会,李达,姜媛媛,等.枯草芽孢杆菌JAASB4的安全性评价[J].吉林农业科学,2015,40(5):102-103.
[31]Simon M.Bacillus probiotics[J].Food Microbiology,2011,28(1):214-220.
[32]王利宾,孙利娜.枯草芽孢杆菌作用机制及其在养殖业中的应用研究进展[J].饲料博览,2015(1):35-38.
[33]刘刚,杨雪,梅雪然,等.枯草芽孢杆菌Loq18抗菌蛋白的分离纯化及其对灰葡萄孢的抑制活性[J].四川师范大学学报(自然科学版),2015,46-49.
[34]Karasov P,Spiwok V,Mal KR,et al.Beta-galactosidase activity in psychrotrophic microorganisms and their potential use in food industry[J].Czech Journal of Food Sciences,2002,20:43-47.
[35]张红艳,刘成更.乳糖酶的酶学特性及其研究进展[J].食品研究与开发,2004,25(6):34-36.
[36]张明丽,熊华,齐金峰,等.乳酸克鲁维酵母乳糖酶性质的研究[J].中国乳品工业,2008,36(1):7-9.
[37]Rahim KAA,Lee BH.Specificity,inhibitory studies,and oligosaccharide formation byβ-Galactosidase from psychrotrophic Bacillus subtilis KL88[J].Journal of dairy science,1991,74(6):1773-1778.
[38]Lara ABCC,Li Y,Paul D,et al.Characterization of aβ-galactosidase from Bacillus subtilis with ransgalactosylation activity[J].International Journal of Biological Macromolecules,2018,120:279-287.