高产乳糖酶酵母菌的筛选、培养基优化及生长模型的研究
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摘要
本文对分离自牧民家庭酸马奶酒样品中的18株酵母菌进行高产乳糖分解酶的酵母菌菌株筛选,在同容量牛乳中接入相同比例酵母菌(1%),培养48h,在接种前测量原乳中的乳糖含量及pH值,在接种后12h,24h,48h测量接入酵母菌牛乳中的乳糖含量及pH值,由实验数据比较接入不同菌株的牛乳中乳糖变化量及PH值,从中筛选出1株产乳糖酶量高的菌株(J20-3)。测得接入酵母菌J20-3前牛乳中乳糖含量为59.69mg/mL,接入后12后变为17.64 mg/mL,为接种前29.55%;24h变为1.87 mg/mL,为接种前3.13%,24h后乳糖量基本不再变化。在此基础上,对酵母菌(J20-3)培养基组成成分乳糖、酵母汁、蛋白胨比例依据二次相应曲面法(Box-Behnken)优化,设计了三因素三水平的响应面分析(RSA)试验,共有15个试验点。优化结果为1000mL酵母菌培养基中乳糖为18.31g,酵母汁为5.23g,蛋白胨为10.43g。利用优化过的培养基培养酵母菌J20-3,参照中性乳糖酶的测定方法—吉斯特·布洛卡兹法测定粗酶液中酶活力。定在37℃,pH为7.3的条件下测定其单位菌体干重量所含乳糖酶,作为判别其乳糖酶活力的一个客观指标。测得单位重量酵母菌的乳糖酶含量:23.11IU/g。连续培养酵母菌J20-324h,每隔2h测量其菌体干重,以logistic模型为基础,建立该酵母菌在30℃下,菌体干重关于时间的生长模型。由试验数据确定模型参数:Cm=1.04kg/m3,Co=0.32kg/m3。将试验数据代入LN(Cm/C0-1)+LN[Cx/(Cm-Cx)]并对时间t作图,得到:斜率为k=0.194,截距intercept=0.04.最后建立方程: Cx=0.32*EXP(0.04+0.194*t)/(1-(0.32/1.04)*(1-EXP(0.04+0.194*t)))。实验数据与理论值相对误差在5%左右,模型拟和较好。
The paper studied the screening of the yeast with high vitalisticβ-Glycosidase, 18 strains of yeasts isolated from herdsman-made koumiss were used. The same proportion of yeast (1%) was inoculated into the milk with the same volume, and cultivated for 48h. The measurements of lactose content and pH value were made respectively before cultivation and in 12h, 24 hand 48h after cultivation. By comparison of the changes in lactose content and pH values, one strain with high vitalisticβ-Glycosidase was screened out, named as J20-3. The lactose content of the milk was 59.69mg/mL before inoculation, and it changed to 17.64mg/mL in 12h after inoculation, accounted for 29.55% of that before inoculation; it changed to 1.87mg/mL in 24h after inoculation, accounted for 3.13% of that before inoculation. After 24h, the lactose content did not change nearly. Based on the tests, the constitution of moiety medium, lactose, yeast juice and peptone were optimized by Box-Behnken method, three-factor and three levels Response Surface Methodology was designed and 15 experiment points were gotten. The optimization results were 18.31g for lactose, 5.23g for yeast juice and 10.43g for peptone in the 1000ml moiety medium. The enzyme activity of crude enzyme was determined by Gist & Brocades method. The activity ofβ-Galactosidase were decided by content ofβ-Galactosidase at 37℃and pH of 7.3 per thalline dry weight,and the content was 23.11IU/g. The thalline dry weight was detected every 2 hours after yeasts J20-3 cultivation within 24 hours at 30℃. The thalline dry weight vs. time referred to Logistic model was used. The parameter Cm=1.04kg/m3, Co=0.32kg/m3 were got. Experimental data were plot LN (Cm/C0-1) +LN [Cx/ (Cm-Cx)] vs. time. The slope was K=0.194, the intercept=0.04. The model
Cx=0.32*EXP (0.04+0.194*t)/ {1-[0.32/1.04-EXP (0.04+0.194*t)]} was definited. As the proportional error between experimental data and theoretical value was about 5%, the model was fit.
The paper studied the screening of the yeast with high vitalisticβ-Glycosidase, 18 strains of yeasts isolated from herdsman-made koumiss were used. The same proportion of yeast (1%) was inoculated into the milk with the same volume, and cultivated for 48h. The measurements of lactose content and pH value were made respectively before cultivation and in 12h, 24 hand 48h after cultivation. By comparison of the changes in lactose content and pH values, one strain with high vitalisticβ-Glycosidase was screened out, named as J20-3. The lactose content of the milk was 59.69mg/mL before inoculation, and it changed to 17.64mg/mL in 12h after inoculation, accounted for 29.55% of that before inoculation; it changed to 1.87mg/mL in 24h after inoculation, accounted for 3.13% of that before inoculation. After 24h, the lactose content did not change nearly. Based on the tests, the constitution of moiety medium, lactose, yeast juice and peptone were optimized by Box-Behnken method, three-factor and three levels Response Surface Methodology was designed and 15 experiment points were gotten. The optimization results were 18.31g for lactose, 5.23g for yeast juice and 10.43g for peptone in the 1000ml moiety medium. The enzyme activity of crude enzyme was determined by Gist & Brocades method. The activity ofβ-Galactosidase were decided by content ofβ-Galactosidase at 37℃and pH of 7.3 per thalline dry weight,and the content was 23.11IU/g. The thalline dry weight was detected every 2 hours after yeasts J20-3 cultivation within 24 hours at 30℃. The thalline dry weight vs. time referred to Logistic model was used. The parameter Cm=1.04kg/m3, Co=0.32kg/m3 were got. Experimental data were plot LN (Cm/C0-1) +LN [Cx/ (Cm-Cx)] vs. time. The slope was K=0.194, the intercept=0.04. The model
Cx=0.32*EXP (0.04+0.194*t)/ {1-[0.32/1.04-EXP (0.04+0.194*t)]} was definited. As the proportional error between experimental data and theoretical value was about 5%, the model was fit.
引文
1 郭勇,郑穗平.酶在食品工业中的应用[M].北京:中国轻工业出版社,1996.67-69
2 王福潭.现代食品发酵技术[M].北京:中国轻工业出版社,2002.
3 马春丽.乳糖酶及其在乳品工业中的应用[[J].食品研究与开发,2003,24(5):62-64
4 Wallenfels K, R Weil. The Enzymes [M]. New York: Academic Press, 1972.
5 李玉强,王昌禄,顾小波,等.β—半乳糖昔酶的研究与应用[[J].中国食品添加剂,2001,2:30-34
6 秦燕,宁正祥.β—半乳糖昔酶的应用研究进展[[J].广州食品工业科技,2001,16(3):38-42
7 陆淳,檀志芬,邱泉若,等.乳糖酶及其在乳品工业中的应用[[J].中国乳品工业,2005,33(8):41-42
8 Ana C Adam,Marta Rubio-Texeira,Julio Polaina.Lactose:The milk sugar from a biotechnological perspective[J]. Critical Review in Food Science and Nutrition, 2004, 44(7-8):553-557
9 高焕明.乳糖酶的特性及其在乳品工业中的应用[J].中国乳品工业,1996,24(3):19-21
10 王晓路,邵靖宇.消化道酶类药物剂型研究一乳糖酶复合微囊[J].中国医学科学院学报,1991,13(6):435-438
11 归莉琼,魏东芝.生物活性物质一低聚半乳糖[J].中国乳品工业,1998,26(4),9-12
12 骆承痒编.乳与乳品工业.北京:中国农业出版社,2001.
13 母智深.乳糖酶高活力酵母菌株的筛选及酶的特性研究[D]内蒙古农业大学,2002.
14 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
15 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
16 VRESE M,STEGELMANN A,RICHTER B,et al.Probiotic-compensation for lactase nsufficiency [J].Am J Clin Nutr, 2001,73:421-429
17 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):1131-1135
18 李强军,全文海.乳清中乳糖的酶法水解[[J].无锡轻工业学报,1991.10(3):15-23
19 黄北阳.开发低乳制品促进我国乳品产业发展[J].当代畜牧,2001,3:1-2
20 Dimitris G Hatzinikolaou, Efstathios Katisfas, Diomi Mamma, et al. Modeling of the simultaneous hydrolysis-ultrafiltration of whey permeate by a thermostable β —galactosidase from Aspergillusniger[J]. Biochemical Engineering Journal, 2005, 24(2):161-172
21 郑建仙,李漩.低聚半乳糖的制备[J].食品与机械,1999,1:20～21
22 归莉琼,魏东芝.生物活性物质一低聚半乳糖[J].中国乳品工业,1998,26(4):9～12
23 王筱兰,魏东芝,史凌洋,等.半乳糖寡糖的生物活性[[J].华东理工大学学报,1999,25(4):374～376.
24 George Tzortzis, Athanasios K Goulas, Jennifer M Gee, et al. A Novel Galacto-oligosaccharide Mixture Increases the Bifidobacterial Population Number in a Continuous In Vitro Fermentation System and in the Proximal Colonic Contents of Pigs In Vivo[J]. The American Society for Nutritional Sciencese, 2005, 135: 1726-1731.
25 R Sharp,S Fishbain,G T Macfarlane.Effect of short-chain carbohydrates on human intestinal Bifidobacteria and Escherichia coli in vitro[J].J Med Microbiol, 2001, 50:152-160.
26 M V W Wijnands,H C Schoterman,J P Bruijntjes,et al.Effect of dietary galacto-oligosaccharides on azoxymethane-induced aberrant crypt foci and colorectal cancer in Fischer 344 rats[J].Carcinogenesis, 2001,22(1):127-132.
27 Tuge Goktug,Mustafa Kemal Sezginturk,Erhan Dinckaya.Glucose oxidase-, β-galactosidase hybrid biosensor based on glassy carbon electrode modified with mercury for lactose determination[J].Analytica Chimica Acta,2005,551(1-2):51 -56
28 Stahl B,Thrul S,Zeng J,et al.Oligosaccharides from human milk as revealed by matrix-assisted laser desorption/ionization mass spectrometry [J].Analytical Biochemistry,1994,223:218-226
29 Roy I,Gupta M N. Lactose hydrolysis by Lactase immobilized on cellulose beads in batch and fluidized bed modes[J].Process Biochemistry,2003,39:325-332
30 秦燕.乳糖酶及其合成低聚半乳糖的研究[D].广州:华南理工大学,2001.
31 M nor Zairossani, I famer melih, Mehrva mehrab, et al. Improvement of intracellular β -galactosidase production in fed-batch culture of Kluyveromyces fragilis [J].Biotechnology Letters, 2001,23:845 一 849
32 Becerra M, Diaz prado S, Takatoshi itoh, et al. New secretory strategies for Kluyveromyces lactis β-galactosidase [J].Protein Engineering, 2001,14(5):379～386
33 Michael T Flood,Mitsuru Kondo.Toxicity evaluation of a β-galactosidase preparation produced by Penicillium multicolor[J]. Regulatory Toxicology and Pharmacology,2004,40 (3):281～292.
34 (日)相 孝亮.酶应用手册.黄文涛,胡学智译.上海:上海科学技术出版社,1989. 87～90
35 贺银凤,李少英,母智深等.酸马奶酒中微生物的分离鉴定及抗菌特性的研究.农业工程学报.2002.18(2):91-95
36 Zwietering,M.H.Jongenburs,I.,Rombouts,F.M.,&Van Rist,K.Modeling of the bacterialgrowth ezrve.Appl.Environ.Microbiol.1990,56(6):1875-1881
37 Moser A.In:Brauer H ed.Biotechnoligy[J].1985,(2):243-246
38 URS LENDENMANN,MARIO SNOZZI,THOMAS EGLI. Growth kinetics cf escherichia coli with galactose and several other sugars in carbon limited chemostat culture[J].Can JMicrobiol,2000,46(1):72-80
39 任善强,雷鸣.数学模型(M).重庆:重庆大学出版社,1996.6-12,17-19
40 刘振民.乳酸菌分批培养的菌体增殖动力学研究. 徐州工程学院学报,2005,01:84-86
41 汤琳,曾光明,孙伟等.Logistic 方程在微生物分批培养动力学中的应用. 湖南大学学报.(自然科学版), 2004:03:23-28
42 郝士海等著.现代细菌学培养基和生化试验手册.北京:中国科学技术出版社,1995.
43 陈天寿等著.微生物培养基的织造与应用.北京:中国农业出版社,1995.
44 黄伟坤等编著.食品检验与分析.北京:轻工业出版社,1989.
45 靳敏,夏玉宇.食品检验技术[M].北京:化学工业出版社许学与应用化学出版中心.2003:36-60
46 张意静.食品分析[M].北京:中国轻工业出版社,1999.
47 Thompson D R. Response surface experimentation[J]. J Food Proc Pres.1982,(6):155-159
48 洒荣波,石贵阳,王正祥. 基因工程菌 Pichia pastoris 高密度培养条件的摇瓶研究. 食品研究与开发.2005,26(2):52-57
49 谭树华,高向东,吴梧桐.药用乳糖酶酵母菌发酵条件研究.中国药科大学学报.1999,30(5):392-395
50 B.施特尔马赫!德].酶的测定方法. 钱嘉渊[译].北京:中国轻工业出版社,1992.
51 韩冲.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
52 李平作,章克昌.灵芝胞外多糖分批发酵非结构动力学模型[J].生物技术,1999,9(3):24-26
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54 李信,许雷,蔡昭铃.蛹虫草菌胞外多糖发酵及其发酵动力学[J].生物工程学报,1999, 15 (4):507～511
55 郭本恒.乳品化学.北京:中国轻工业出版社,2001.
56 郭杰炎 蔡武城.微生物酶.北京:科学出版社,1986.
57 袁勤生,赵健,王维育主编.应用酶学.上海:华东理工大学出版社,1994.
2 王福潭.现代食品发酵技术[M].北京:中国轻工业出版社,2002.
3 马春丽.乳糖酶及其在乳品工业中的应用[[J].食品研究与开发,2003,24(5):62-64
4 Wallenfels K, R Weil. The Enzymes [M]. New York: Academic Press, 1972.
5 李玉强,王昌禄,顾小波,等.β—半乳糖昔酶的研究与应用[[J].中国食品添加剂,2001,2:30-34
6 秦燕,宁正祥.β—半乳糖昔酶的应用研究进展[[J].广州食品工业科技,2001,16(3):38-42
7 陆淳,檀志芬,邱泉若,等.乳糖酶及其在乳品工业中的应用[[J].中国乳品工业,2005,33(8):41-42
8 Ana C Adam,Marta Rubio-Texeira,Julio Polaina.Lactose:The milk sugar from a biotechnological perspective[J]. Critical Review in Food Science and Nutrition, 2004, 44(7-8):553-557
9 高焕明.乳糖酶的特性及其在乳品工业中的应用[J].中国乳品工业,1996,24(3):19-21
10 王晓路,邵靖宇.消化道酶类药物剂型研究一乳糖酶复合微囊[J].中国医学科学院学报,1991,13(6):435-438
11 归莉琼,魏东芝.生物活性物质一低聚半乳糖[J].中国乳品工业,1998,26(4),9-12
12 骆承痒编.乳与乳品工业.北京:中国农业出版社,2001.
13 母智深.乳糖酶高活力酵母菌株的筛选及酶的特性研究[D]内蒙古农业大学,2002.
14 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
15 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
16 VRESE M,STEGELMANN A,RICHTER B,et al.Probiotic-compensation for lactase nsufficiency [J].Am J Clin Nutr, 2001,73:421-429
17 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):1131-1135
18 李强军,全文海.乳清中乳糖的酶法水解[[J].无锡轻工业学报,1991.10(3):15-23
19 黄北阳.开发低乳制品促进我国乳品产业发展[J].当代畜牧,2001,3:1-2
20 Dimitris G Hatzinikolaou, Efstathios Katisfas, Diomi Mamma, et al. Modeling of the simultaneous hydrolysis-ultrafiltration of whey permeate by a thermostable β —galactosidase from Aspergillusniger[J]. Biochemical Engineering Journal, 2005, 24(2):161-172
21 郑建仙,李漩.低聚半乳糖的制备[J].食品与机械,1999,1:20～21
22 归莉琼,魏东芝.生物活性物质一低聚半乳糖[J].中国乳品工业,1998,26(4):9～12
23 王筱兰,魏东芝,史凌洋,等.半乳糖寡糖的生物活性[[J].华东理工大学学报,1999,25(4):374～376.
24 George Tzortzis, Athanasios K Goulas, Jennifer M Gee, et al. A Novel Galacto-oligosaccharide Mixture Increases the Bifidobacterial Population Number in a Continuous In Vitro Fermentation System and in the Proximal Colonic Contents of Pigs In Vivo[J]. The American Society for Nutritional Sciencese, 2005, 135: 1726-1731.
25 R Sharp,S Fishbain,G T Macfarlane.Effect of short-chain carbohydrates on human intestinal Bifidobacteria and Escherichia coli in vitro[J].J Med Microbiol, 2001, 50:152-160.
26 M V W Wijnands,H C Schoterman,J P Bruijntjes,et al.Effect of dietary galacto-oligosaccharides on azoxymethane-induced aberrant crypt foci and colorectal cancer in Fischer 344 rats[J].Carcinogenesis, 2001,22(1):127-132.
27 Tuge Goktug,Mustafa Kemal Sezginturk,Erhan Dinckaya.Glucose oxidase-, β-galactosidase hybrid biosensor based on glassy carbon electrode modified with mercury for lactose determination[J].Analytica Chimica Acta,2005,551(1-2):51 -56
28 Stahl B,Thrul S,Zeng J,et al.Oligosaccharides from human milk as revealed by matrix-assisted laser desorption/ionization mass spectrometry [J].Analytical Biochemistry,1994,223:218-226
29 Roy I,Gupta M N. Lactose hydrolysis by Lactase immobilized on cellulose beads in batch and fluidized bed modes[J].Process Biochemistry,2003,39:325-332
30 秦燕.乳糖酶及其合成低聚半乳糖的研究[D].广州:华南理工大学,2001.
31 M nor Zairossani, I famer melih, Mehrva mehrab, et al. Improvement of intracellular β -galactosidase production in fed-batch culture of Kluyveromyces fragilis [J].Biotechnology Letters, 2001,23:845 一 849
32 Becerra M, Diaz prado S, Takatoshi itoh, et al. New secretory strategies for Kluyveromyces lactis β-galactosidase [J].Protein Engineering, 2001,14(5):379～386
33 Michael T Flood,Mitsuru Kondo.Toxicity evaluation of a β-galactosidase preparation produced by Penicillium multicolor[J]. Regulatory Toxicology and Pharmacology,2004,40 (3):281～292.
34 (日)相 孝亮.酶应用手册.黄文涛,胡学智译.上海:上海科学技术出版社,1989. 87～90
35 贺银凤,李少英,母智深等.酸马奶酒中微生物的分离鉴定及抗菌特性的研究.农业工程学报.2002.18(2):91-95
36 Zwietering,M.H.Jongenburs,I.,Rombouts,F.M.,&Van Rist,K.Modeling of the bacterialgrowth ezrve.Appl.Environ.Microbiol.1990,56(6):1875-1881
37 Moser A.In:Brauer H ed.Biotechnoligy[J].1985,(2):243-246
38 URS LENDENMANN,MARIO SNOZZI,THOMAS EGLI. Growth kinetics cf escherichia coli with galactose and several other sugars in carbon limited chemostat culture[J].Can JMicrobiol,2000,46(1):72-80
39 任善强,雷鸣.数学模型(M).重庆:重庆大学出版社,1996.6-12,17-19
40 刘振民.乳酸菌分批培养的菌体增殖动力学研究. 徐州工程学院学报,2005,01:84-86
41 汤琳,曾光明,孙伟等.Logistic 方程在微生物分批培养动力学中的应用. 湖南大学学报.(自然科学版), 2004:03:23-28
42 郝士海等著.现代细菌学培养基和生化试验手册.北京:中国科学技术出版社,1995.
43 陈天寿等著.微生物培养基的织造与应用.北京:中国农业出版社,1995.
44 黄伟坤等编著.食品检验与分析.北京:轻工业出版社,1989.
45 靳敏,夏玉宇.食品检验技术[M].北京:化学工业出版社许学与应用化学出版中心.2003:36-60
46 张意静.食品分析[M].北京:中国轻工业出版社,1999.
47 Thompson D R. Response surface experimentation[J]. J Food Proc Pres.1982,(6):155-159
48 洒荣波,石贵阳,王正祥. 基因工程菌 Pichia pastoris 高密度培养条件的摇瓶研究. 食品研究与开发.2005,26(2):52-57
49 谭树华,高向东,吴梧桐.药用乳糖酶酵母菌发酵条件研究.中国药科大学学报.1999,30(5):392-395
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