乳糖酶高活力酵母菌株的筛选及酶的特性研究
摘要
本研究主要以内蒙古中西部地区牧区采集的19个发酵乳样品（10份酸马奶、3份发酵山羊奶、4份酸牛奶和2份发酵驼奶）为研究对象，分离获得30株酵母菌，从中筛选乳糖酶高活力酵母菌株。根据乳糖代谢率和乳糖酶比活力的测定结果，选出菌株STK-1-1为优胜菌株。经鉴定，该菌株属克鲁维酵母属（Kluyveromyces）的马克斯克鲁维酵母（Kluyveromyces marxianus）。本文还就其发酵特性、乳糖酶的生物学特性和低乳糖乳生产工艺优化等作了进一步研究。结果表明：菌株STK-1-1的对数生长期为18～30h，适宜生长温度范围为25～30℃，最佳接种量为6%，在此条件下，可获得数量和活力较高的酵母细胞；STK-1-1所产的乳糖酶为中性乳糖酶，最适pH=6.5，酶的最适作用温度范围为35～45℃；在实验所研究的离子中，CU2+、Fe2+、Ca2+对酶的活力有抑制作用，K+、Na+、Mg2+对酶有激活作用，其中Mg2+可使酶的活力提高18.4%；利用STK-1-1所产的乳糖酶生产低乳糖乳，其优化工艺条件为加酶量1.5%，保温温度37℃，保温时间3h，在此条件下，乳中的乳糖可由原来的4.64%降解到2.2%。感官质量指标评价结果显示：除色泽指标外，低乳糖乳的风味、甜度、口感等指标均优于普通乳。
In This paper ,thirty strains of yeast were isolated from nineteen samples of fermented milk(ten koumiss samples,three fermented goat milk samples,four fermented milk samples,two shubat samples) which were collected from the west-centre region of Inner Mongolia from which we aimed to select the yeast with high vitalistic β—Galactosidase .According to the results of dedermination that were the metabolism rate and ratio vitality of β—Galactosidase,STK-1-1 has been selected .The result of identification showed that STK-1-1 belonged to Kluyveromyces marxianus of Kluyveromyces.
In the research,fermenting properties of STK-1-1, biological properties ofβ—Galactosidase and the optimization of producting technology of low-lactose-milk have also been studied.The results showed that the logarithmic growth phase of STK-1-1 is 18～30h,suitable temperature range 25～30℃,reasonable inoculating content 6%.Under this condition,a large number of higher vitality of yeast cells could been achieved; β—Galactosidase of STK-1-1 is neutral,suitable pH=6.5, suitable temperature range 35～45℃.Within six ions, CU2+、Fe2+、Ca2+ can inhibit the vitality ofβ—Galactosidase while K+、Na+、Mg2+ can promote the vitality of β—Galactosidase. Therein，vitality of β—Galactosidase can be increased 18.4% by Mg+2; Using theβ—Galactosidase producting low-lactose-milk, the optimum producting technology are the density of β—Galactosidase 1.5%,the temperature of heat preservation 37℃,the time of heat preservation 3h.Under this condition,the lactose of milk have been decreased from 4.64% to 2.2%.
The result of the qulity index of sensory orgar showed that except the colour index,flavour,sweet and taste of low-lactose-milk are all superior to general milk.
In This paper ,thirty strains of yeast were isolated from nineteen samples of fermented milk(ten koumiss samples,three fermented goat milk samples,four fermented milk samples,two shubat samples) which were collected from the west-centre region of Inner Mongolia from which we aimed to select the yeast with high vitalistic β—Galactosidase .According to the results of dedermination that were the metabolism rate and ratio vitality of β—Galactosidase,STK-1-1 has been selected .The result of identification showed that STK-1-1 belonged to Kluyveromyces marxianus of Kluyveromyces.
In the research,fermenting properties of STK-1-1, biological properties ofβ—Galactosidase and the optimization of producting technology of low-lactose-milk have also been studied.The results showed that the logarithmic growth phase of STK-1-1 is 18～30h,suitable temperature range 25～30℃,reasonable inoculating content 6%.Under this condition,a large number of higher vitality of yeast cells could been achieved; β—Galactosidase of STK-1-1 is neutral,suitable pH=6.5, suitable temperature range 35～45℃.Within six ions, CU2+、Fe2+、Ca2+ can inhibit the vitality ofβ—Galactosidase while K+、Na+、Mg2+ can promote the vitality of β—Galactosidase. Therein，vitality of β—Galactosidase can be increased 18.4% by Mg+2; Using theβ—Galactosidase producting low-lactose-milk, the optimum producting technology are the density of β—Galactosidase 1.5%,the temperature of heat preservation 37℃,the time of heat preservation 3h.Under this condition,the lactose of milk have been decreased from 4.64% to 2.2%.
The result of the qulity index of sensory orgar showed that except the colour index,flavour,sweet and taste of low-lactose-milk are all superior to general milk.
引文
[1]孙玉梅，朱蓓薇，牟连玉.乳糖酶的应用及固定化，食品工业科技，1995，89（3）：23-25
[2]相尺孝亮[日]编，黄文涛，胡学智译.酶应用手册.上海：上海科学技术出版社，1989
[3]骆承庠编.乳与乳品工业.北京：中国农业出版社，2001
[4]王岁楼编.食品生物技术.北京：海译出版社，1998
[5]韩雅珊编.食品化学.北京：北京农业大学出版社，1992
[6]沈同，王镜岩编.生物化学.上海；高等教育出版社，1990
[7]吕晓华，刘世贵等.生物技术在乳糖不耐受防治中的应用.中国乳品工业，2002，146（1）：44-47
[8]周晓云.酶技术.北京：石油工业出版社，1995
[9]金世林.生物技术应用于乳品工业的最新进展.中国乳品工业，1998，26（1）：18-21
[10]陈天寿编.微生物培养基的制造与应用.北京：中国农业出版社，1995
[11]根井外喜男[日]编，金连缘译.微生物保存法.上海：上海科学技术出版社，1992
[12]黄伟坤.食品检验与分析.北京：中国轻工业出版社，1989
[13]B.施特尔马赫[德]，钱嘉渊[译]. 酶的测定方法.北京：中国轻工业出版社，1992.
[14]韩冲等.一株产β—半乳糖苷酶酵母菌株的选育研究,食品工业科技,2001,136(8):33-35
[15]J.A.巴尼特等编.胡瑞卿译.酵母菌的特征与鉴定手册.青岛:青岛海洋大学出版社,1991
[16]周德庆编.微生物学实验手册.上海:上海科学技术出版社,1986
[17]杜连祥等编.工业微生物学实验手册.天津：天津科技出版社，1992
[18]付秀辉等.引起甜炼乳罐头鼓胀变质的嗜高糖酵母的研究.微生物学报，1989，29（4）：235-238
[19]乌尼编.畜牧微生物学.农业出版社，1992
[20]中国农业大学食品科学系微生物教研室编.食品微生物学实验指导.北京：中国农业大学出版社，1992
[21]严自正等.分枝梨头霉α-半乳糖苷酶的纯化和性质研究.微生物学报，1989，29（4）：265-271
[22]北京农业大学植物生化教研室编.基础生物化学实验.北京：北京农业大学出版社，1987
[23]张龙祥等.生化实验方法和技术.上海：高等教育出版社，1985
[24]扬寿均等.酶制剂工业（上册）.北京：科学出版社，1984
[25]扬寿均等.嗜热栖热菌HB8耐热α-葡萄糖苷酶的提纯和性质.微生物学报，1992，32（1）：23-29
[26]《乳品工业手册》编写组编.乳品工业手册.北京：中国轻工业出版社，1987
[27]张惟杰编.复合多糖生化研究技术.上海：上海科学技术出版社，1987
[28]张丰德，樊廷玉编.现代生物学技术.天津：南开大学出版社，1996
[29]张立名，王贤舜.现代生物化学分析原理.安徽：中国科技大学出版社，1991
[30]吴东儒编.糖类的生物化学.上海：高等教育出版社，1987
[31]朱余尧.应用模糊综合评判进行食品感官评价的实施报告.食品科学，1987，（10）：11-13
[32]张连生.模糊数学在综合评价月饼卫生状况中的应用.食品科学，1999，230（2）：63-65
[33]无锡轻工学院，天津轻工学院编.食品工厂机械与设备.北京：中国轻工业出版社，1993
[34]张裕中等编.食品加工技术装备.北京：中国轻工业出版社，2000
[35]金花等.酸马奶.中国乳品工业，1998，26（4）：16-17
[36]F．M．德赖森，Z.普汉编，顾瑞霞译.发酵乳科学与技术.广州：东南大学出版社，1991
[37]秦燕，宁正祥.乳糖水解时低聚半乳糖的生成及应用.食品与发酵工业，2001，26（5）：67-72
[38]无锡轻工学院，天津轻工学院编.食品生物化学.北京：中国轻工业出版社，1981
[39]房玉国等.灭菌乳商业无菌检验方法的探讨.中国乳品工业，2002，146（1）：29-31
[40]沃夫冈.牛乳超高温（UHT）处理. 中国乳品工业，2002，146（1）：48-49
[41]金世琳.生物技术应用于乳业的最新进展[J].中国乳品工业，1998, 26(1): 18-21.
[42] Aronson M. Archives of Biochemistry and Biophysics, 1952, 39: 370 -378
[43] Burvall A, Asp N-G, Dahlqvist A. Food Chemistry, 1979, 4:243-250
[44]Chonan O, Matsumoto K, Watanuki M. Biotech. Biochem., 1995, 59(2) :236-239
[45]Chonan O, Watanuki M. J. Nutr. Sci. Vitaminol., 1995, 41:95-104
[46] Cupples C G, Miller J H, Huber R E. Journal of Biological Chemistry, 1990, 265(10): 5 512- 5 518
[47]DURING M J, XU R, YOUNG D, et al. Peroral gene therapy of lactose intolerance
using an adeno-associated virus vector[J]. Nat Med, 1998, 4(10): 1 131 - 1 135.
[48] Fowler A V, Zabin I.Journal of Biological Chemistry, 1978, 258: 10204- 10207
[49]GB4789.28-94
[50]http：//www.fh21.com.cn/guanggao/pop2.htm
[51]http：//www.fh21.com.cn/guanggao/pop3.htm
[52]IBRAHIM S A, O' SULLIVAN D J. Use of chemical mutagenesis for the isolation of food grade beta-galactosidase overproducing mutants of bifidobacteria, lactobacilli and Streptococcus thermophilus[J]. J Dairy Sci, 2000, 83 (5): 923 - 930.
[53]Jacobson R H.Three Dimensional Structure of β-Galactosidase. Ph.D. thesis, 1993, University of Oregon, Corvallis, OR.
[54]KARATZAS C N, TURNER J D. Toward altering milk composition by genetic
manipulation: current status and challenges [J]. J Dairy Sci, 1997, 80(9): 2 225-2232.
[55]Mahoney R R. In Advanced Dairy Chemistry, Vol. 3., (ed. Fox P F)London:
Chapman and Hall, 1996. 77-126
[56] Mahone R R, Whitaker J R. Journal Food Biochemistry, 1977, 1: 327-350
[57] Monsan P, Paul F, Remaud M et al. Food Biotechnology, 1989, 3:11-29
[58]Mozzaffar Z, Nakashini K, Matsuno R. Applied Microbiology and Biotechnology,
1989, 31: 59-60
[59]PATEL Y. T, MINOCHA A. Lactose intolerance: diagnosis and management[J].
Compr Ther, 2000, 26(4): 246 - 250
[60]PINTADO B, GUTIERREZ - ADAN A. Transgenesis in large domestic species:
future development for milk modification[J]. Reprod Nutr Dev, 1999, 3939(5 -6):
535 - 544.
[61]Pazur J H. Science, 1953,117:355-368
[62]RUBIO-TEXEIRA M, AREVALO-RODRIGUEZ M, LEQUERICA J L,et al.
Lactose utilization by saccharomyces cerevisiae strains expressing Kluyveromyces
lactis LAC genes[J]. J Biotechnol, 2001, 84(2): 97- 106.
[63] Sinnott M L. FEBS Letters, 1978, 94: 1-9
[64]VRESE M, STEGELMANN A, RICHTER B, et al. Probiotic-compensation for
lactase insufficiency[J]. Am J Clin Nutr, 2001, 73: 421 - 429.
[65]WALL R J, KERR D E, BONDIOLI K R. Transgenic dairy cattle: genetic
engineering on a large scale[J]. J Dairy Sci, 1997, 80(9): 2 213 -2 224.
[66]Wallenfels K, Malhotra O P, Galactosidases. Advances in Carbohydrate Chemistry.1961, 16: 239-298
[67]Yanahira S, Morita M, Aoe S et al. J. Nutr. Sci. Vitaminol., 1995, 41:83-94
[2]相尺孝亮[日]编，黄文涛，胡学智译.酶应用手册.上海：上海科学技术出版社，1989
[3]骆承庠编.乳与乳品工业.北京：中国农业出版社，2001
[4]王岁楼编.食品生物技术.北京：海译出版社，1998
[5]韩雅珊编.食品化学.北京：北京农业大学出版社，1992
[6]沈同，王镜岩编.生物化学.上海；高等教育出版社，1990
[7]吕晓华，刘世贵等.生物技术在乳糖不耐受防治中的应用.中国乳品工业，2002，146（1）：44-47
[8]周晓云.酶技术.北京：石油工业出版社，1995
[9]金世林.生物技术应用于乳品工业的最新进展.中国乳品工业，1998，26（1）：18-21
[10]陈天寿编.微生物培养基的制造与应用.北京：中国农业出版社，1995
[11]根井外喜男[日]编，金连缘译.微生物保存法.上海：上海科学技术出版社，1992
[12]黄伟坤.食品检验与分析.北京：中国轻工业出版社，1989
[13]B.施特尔马赫[德]，钱嘉渊[译]. 酶的测定方法.北京：中国轻工业出版社，1992.
[14]韩冲等.一株产β—半乳糖苷酶酵母菌株的选育研究,食品工业科技,2001,136(8):33-35
[15]J.A.巴尼特等编.胡瑞卿译.酵母菌的特征与鉴定手册.青岛:青岛海洋大学出版社,1991
[16]周德庆编.微生物学实验手册.上海:上海科学技术出版社,1986
[17]杜连祥等编.工业微生物学实验手册.天津：天津科技出版社，1992
[18]付秀辉等.引起甜炼乳罐头鼓胀变质的嗜高糖酵母的研究.微生物学报，1989，29（4）：235-238
[19]乌尼编.畜牧微生物学.农业出版社，1992
[20]中国农业大学食品科学系微生物教研室编.食品微生物学实验指导.北京：中国农业大学出版社，1992
[21]严自正等.分枝梨头霉α-半乳糖苷酶的纯化和性质研究.微生物学报，1989，29（4）：265-271
[22]北京农业大学植物生化教研室编.基础生物化学实验.北京：北京农业大学出版社，1987
[23]张龙祥等.生化实验方法和技术.上海：高等教育出版社，1985
[24]扬寿均等.酶制剂工业（上册）.北京：科学出版社，1984
[25]扬寿均等.嗜热栖热菌HB8耐热α-葡萄糖苷酶的提纯和性质.微生物学报，1992，32（1）：23-29
[26]《乳品工业手册》编写组编.乳品工业手册.北京：中国轻工业出版社，1987
[27]张惟杰编.复合多糖生化研究技术.上海：上海科学技术出版社，1987
[28]张丰德，樊廷玉编.现代生物学技术.天津：南开大学出版社，1996
[29]张立名，王贤舜.现代生物化学分析原理.安徽：中国科技大学出版社，1991
[30]吴东儒编.糖类的生物化学.上海：高等教育出版社，1987
[31]朱余尧.应用模糊综合评判进行食品感官评价的实施报告.食品科学，1987，（10）：11-13
[32]张连生.模糊数学在综合评价月饼卫生状况中的应用.食品科学，1999，230（2）：63-65
[33]无锡轻工学院，天津轻工学院编.食品工厂机械与设备.北京：中国轻工业出版社，1993
[34]张裕中等编.食品加工技术装备.北京：中国轻工业出版社，2000
[35]金花等.酸马奶.中国乳品工业，1998，26（4）：16-17
[36]F．M．德赖森，Z.普汉编，顾瑞霞译.发酵乳科学与技术.广州：东南大学出版社，1991
[37]秦燕，宁正祥.乳糖水解时低聚半乳糖的生成及应用.食品与发酵工业，2001，26（5）：67-72
[38]无锡轻工学院，天津轻工学院编.食品生物化学.北京：中国轻工业出版社，1981
[39]房玉国等.灭菌乳商业无菌检验方法的探讨.中国乳品工业，2002，146（1）：29-31
[40]沃夫冈.牛乳超高温（UHT）处理. 中国乳品工业，2002，146（1）：48-49
[41]金世琳.生物技术应用于乳业的最新进展[J].中国乳品工业，1998, 26(1): 18-21.
[42] Aronson M. Archives of Biochemistry and Biophysics, 1952, 39: 370 -378
[43] Burvall A, Asp N-G, Dahlqvist A. Food Chemistry, 1979, 4:243-250
[44]Chonan O, Matsumoto K, Watanuki M. Biotech. Biochem., 1995, 59(2) :236-239
[45]Chonan O, Watanuki M. J. Nutr. Sci. Vitaminol., 1995, 41:95-104
[46] Cupples C G, Miller J H, Huber R E. Journal of Biological Chemistry, 1990, 265(10): 5 512- 5 518
[47]DURING M J, XU R, YOUNG D, et al. Peroral gene therapy of lactose intolerance
using an adeno-associated virus vector[J]. Nat Med, 1998, 4(10): 1 131 - 1 135.
[48] Fowler A V, Zabin I.Journal of Biological Chemistry, 1978, 258: 10204- 10207
[49]GB4789.28-94
[50]http：//www.fh21.com.cn/guanggao/pop2.htm
[51]http：//www.fh21.com.cn/guanggao/pop3.htm
[52]IBRAHIM S A, O' SULLIVAN D J. Use of chemical mutagenesis for the isolation of food grade beta-galactosidase overproducing mutants of bifidobacteria, lactobacilli and Streptococcus thermophilus[J]. J Dairy Sci, 2000, 83 (5): 923 - 930.
[53]Jacobson R H.Three Dimensional Structure of β-Galactosidase. Ph.D. thesis, 1993, University of Oregon, Corvallis, OR.
[54]KARATZAS C N, TURNER J D. Toward altering milk composition by genetic
manipulation: current status and challenges [J]. J Dairy Sci, 1997, 80(9): 2 225-2232.
[55]Mahoney R R. In Advanced Dairy Chemistry, Vol. 3., (ed. Fox P F)London:
Chapman and Hall, 1996. 77-126
[56] Mahone R R, Whitaker J R. Journal Food Biochemistry, 1977, 1: 327-350
[57] Monsan P, Paul F, Remaud M et al. Food Biotechnology, 1989, 3:11-29
[58]Mozzaffar Z, Nakashini K, Matsuno R. Applied Microbiology and Biotechnology,
1989, 31: 59-60
[59]PATEL Y. T, MINOCHA A. Lactose intolerance: diagnosis and management[J].
Compr Ther, 2000, 26(4): 246 - 250
[60]PINTADO B, GUTIERREZ - ADAN A. Transgenesis in large domestic species:
future development for milk modification[J]. Reprod Nutr Dev, 1999, 3939(5 -6):
535 - 544.
[61]Pazur J H. Science, 1953,117:355-368
[62]RUBIO-TEXEIRA M, AREVALO-RODRIGUEZ M, LEQUERICA J L,et al.
Lactose utilization by saccharomyces cerevisiae strains expressing Kluyveromyces
lactis LAC genes[J]. J Biotechnol, 2001, 84(2): 97- 106.
[63] Sinnott M L. FEBS Letters, 1978, 94: 1-9
[64]VRESE M, STEGELMANN A, RICHTER B, et al. Probiotic-compensation for
lactase insufficiency[J]. Am J Clin Nutr, 2001, 73: 421 - 429.
[65]WALL R J, KERR D E, BONDIOLI K R. Transgenic dairy cattle: genetic
engineering on a large scale[J]. J Dairy Sci, 1997, 80(9): 2 213 -2 224.
[66]Wallenfels K, Malhotra O P, Galactosidases. Advances in Carbohydrate Chemistry.1961, 16: 239-298
[67]Yanahira S, Morita M, Aoe S et al. J. Nutr. Sci. Vitaminol., 1995, 41:83-94