酿酒酵母乳酸脱氢酶发酵和酶学性质及其基因序列分析
|
摘要
在酿酒酵母的正常发酵过程中,乳酸脱氢酶催化丙酮酸还原为乳酸。高级醇是啤酒发酵中最重要的风味物质之一,大多数高级醇和酒精发酵同步形成,主要是由合成途径产生的。理论上分析高级醇的生成与乳酸的生成是反向的关系,因此可以通过增加乳酸的生成量来降低啤酒发酵中高级醇的生成量。然而在啤酒酿造中乳酸脱氢酶对高级醇的调控机制尚不清楚。本文拟研究酿酒酵母乳酸脱氢酶的发酵性质,在发酵过程中通过添加镁锌离子,调控乳酸脱氢酶的活力控制高级醇的含量。建立酿酒酵母乳酸脱氢酶分离纯化的方法,并分析不同菌株乳酸脱氢酶编码基因的序列差异,阐明不同酵母乳酸脱氢酶活力差异,为调节控制啤酒高级醇含量提供理论依据。
本研究测定5株不同酿酒酵母菌株乳酸脱氢酶的发酵性质,结果表明不同酵母菌株之间的乳酸脱氢酶酶活力存在差异。在啤酒发酵过程中随着LDH活性增加,啤酒中的高级醇含量则下降,显示了LDH活性与高级醇生成量呈负相关性。
通过向发酵液中添加金属离子Mg~(2+)(500mg/L)和Zn~(2+)(0.17mg/L),测定发酵过程中发酵液的乳酸脱氢酶活力和高级醇生成量,结果显示Mg~(2+)和Zn~(2+)的添加,显著提高了酵母的乳酸脱氢酶活力,生成产物中高级醇的含量比对照降低,并且Mg~(2+)比Zn~(2+)的促进作用更为显著。
酵母乳酸脱氢酶的分离纯化条件进行优化。结果表明:发酵液经细胞破碎,透析, 40%的(NH_4)_2SO_4盐析, DEAE-Sepharos(eDE-52)离子交换层析,Sephadex-G25凝胶过滤等纯化步骤,分离的乳酸脱氢酶经SDS-聚丙烯酰胺凝胶电泳检测为单一蛋白条带,纯化倍数为14.3,回收率为24%。凝胶法测定该酶的分子量约为32.2kD。对酵母乳酸脱氢酶的性质测定结果表明:该酶的最适反应温度为45℃,在35℃温度下比较稳定,最适反应pH值为9.4。金属离子对该酶活性具有显著影响,在0.5 mmol/L和1.0 mmol/L的金属离子浓度下:Mg~(2+),Zn~(2+)对酶活有明显的促进作用,其中1.0mmol/L Ca~(2+)离子作用最显著,Ni~+和K~+对它有明显的抑制作用,Fe~(2+)的作用不是太明显。
以酿酒酵母的基因组DNA为模板,根据Genbank发表的全序列,设计3对引物,扩增LDH基因的不同片断,对扩增片断测序。对本研究中5株酵母菌株和酿酒酵母(FLH201473.01X)的LDH基因(GenBank,accession No.DQ332540)之间的序列差异进行比对分析。结果表明: YZU03与其中的ST01和SH01的差异在LDH基因的第609-725碱基之间。而它们和FLH201473.01X的LDH基因的差异在该基因的第213、216、217、228、314和315碱基。
In the fermentation process, LDH catalytic pyruvate into lactic acid.Higher alcohols are important component of beer favor , Most of which formed simultaneously with alcohol during fermentation of beer brewing. Theorically, Higher alcohols producs mainly by the Harris pathway and the relation of the formation of the higher alcohols and the lactic acid is the reverse. Therefore we can increasing the production of lactic acid to reduce higher alcohols.However in brewing the regulation mechanism of LDH to control the higher alcohols is not clear. In this paper ,we studied the fermentation performance of different yeast lactic dehydrogenase enzyme.Adding Mg~(2+) and Zn~(2+) ions in the fermentation process to control vitality of LDH and control the production of higher alcohols.Morever , we are also studied in this paper the separation and purification methods of LDH of the Saccharomyces cerevisiae ,which laid the base for researches on the LDH enzyme nature. and analized lactic dehydrogenase coding sequence of the five strains of yeasts at the molecular level.we clarified the difference of different yeast LDH dynamic, which for the conditioning control high alcohol content of beer and provide a theoretical basis.
In the beer fermentation process, the fermentation performance of different yeast lactic dehydrogenase enzyme had been measured. The data revealed the interrelationship between LDH activity and higher alcohols content. With LDH activity increasing , the higher alcohols content decreased during the beer fermentation ,which suggested competitive and inhabit relationship between LDH activity and higher alcohols.
The metal ions Mg~(2+) and Zn~(2+) were added to the medium, and the lactic dehydrogenase activity and production of higher alcohols were determined in the fermentation process, the results showed that adding a metal ions Mg~(2+), Zn~(2+) in the beer fermentation increased the lactic dehydrogenase activity of yeast (P<0.05) significantly, higher alcohols in control were lower than formented products significantly. Also,Mg~(2+) plays more significant promotion role than Zn~(2+).
The separation and purification methods of LDH from Saccharomyces cerevisiae had been established and optimized.The optimized purification process includes the steps that removing cells by the centrifugation,35%saturation ammonium sulfate precipitation , dialysis,ion exchange chromatography with DE52 and gel filtration chromatography with Superdex-25.Sedium dodecyl-sulfate-polyacrylamide gel electrophovesis(SDS-PAGE)was used to examine the purification effect ,and the results indicated that the final product obitained was homogeneous and had a molocular weight around 32.2KD.the purification rate and activity recovery of LDH was 14.3 and 24.2% respectively.The optimal reaction temperature and pH of LDH was 45℃and 9.4. The stability temperature of LDH enzyme was 35℃. Metalions such as Ca~(2+)、Mg~(2+) and Zn~(2+) can activate LDH and 1.0mmol/L Ca~(2+) can activate LDH signicantly . Influence of Fe~(2+) was not apparent .However,K+ was found to deactive LDH signicantly as well as Ni+.
Three pairs of primers were synthesized according to the sequence of Saccharomyces cerevisiae LDH published on the GenBank to amplify LDH genes of five srains of Saccharomyces cerevisiae with genomic DNA as the template. Amplified fragments had been sequenced . In this study analysis of the gene sequence differences among the laboratory five strains and the published Saccharomyces cerevisiae strain FLH201473.01X . It show that the difference among YZU03 , ST01 and SH01 mainly existed in 609-725th base of the whole sequence. These sequences were different from published at the site of the 213th,216th,217th,228th,314th and 315th bases.
本研究测定5株不同酿酒酵母菌株乳酸脱氢酶的发酵性质,结果表明不同酵母菌株之间的乳酸脱氢酶酶活力存在差异。在啤酒发酵过程中随着LDH活性增加,啤酒中的高级醇含量则下降,显示了LDH活性与高级醇生成量呈负相关性。
通过向发酵液中添加金属离子Mg~(2+)(500mg/L)和Zn~(2+)(0.17mg/L),测定发酵过程中发酵液的乳酸脱氢酶活力和高级醇生成量,结果显示Mg~(2+)和Zn~(2+)的添加,显著提高了酵母的乳酸脱氢酶活力,生成产物中高级醇的含量比对照降低,并且Mg~(2+)比Zn~(2+)的促进作用更为显著。
酵母乳酸脱氢酶的分离纯化条件进行优化。结果表明:发酵液经细胞破碎,透析, 40%的(NH_4)_2SO_4盐析, DEAE-Sepharos(eDE-52)离子交换层析,Sephadex-G25凝胶过滤等纯化步骤,分离的乳酸脱氢酶经SDS-聚丙烯酰胺凝胶电泳检测为单一蛋白条带,纯化倍数为14.3,回收率为24%。凝胶法测定该酶的分子量约为32.2kD。对酵母乳酸脱氢酶的性质测定结果表明:该酶的最适反应温度为45℃,在35℃温度下比较稳定,最适反应pH值为9.4。金属离子对该酶活性具有显著影响,在0.5 mmol/L和1.0 mmol/L的金属离子浓度下:Mg~(2+),Zn~(2+)对酶活有明显的促进作用,其中1.0mmol/L Ca~(2+)离子作用最显著,Ni~+和K~+对它有明显的抑制作用,Fe~(2+)的作用不是太明显。
以酿酒酵母的基因组DNA为模板,根据Genbank发表的全序列,设计3对引物,扩增LDH基因的不同片断,对扩增片断测序。对本研究中5株酵母菌株和酿酒酵母(FLH201473.01X)的LDH基因(GenBank,accession No.DQ332540)之间的序列差异进行比对分析。结果表明: YZU03与其中的ST01和SH01的差异在LDH基因的第609-725碱基之间。而它们和FLH201473.01X的LDH基因的差异在该基因的第213、216、217、228、314和315碱基。
In the fermentation process, LDH catalytic pyruvate into lactic acid.Higher alcohols are important component of beer favor , Most of which formed simultaneously with alcohol during fermentation of beer brewing. Theorically, Higher alcohols producs mainly by the Harris pathway and the relation of the formation of the higher alcohols and the lactic acid is the reverse. Therefore we can increasing the production of lactic acid to reduce higher alcohols.However in brewing the regulation mechanism of LDH to control the higher alcohols is not clear. In this paper ,we studied the fermentation performance of different yeast lactic dehydrogenase enzyme.Adding Mg~(2+) and Zn~(2+) ions in the fermentation process to control vitality of LDH and control the production of higher alcohols.Morever , we are also studied in this paper the separation and purification methods of LDH of the Saccharomyces cerevisiae ,which laid the base for researches on the LDH enzyme nature. and analized lactic dehydrogenase coding sequence of the five strains of yeasts at the molecular level.we clarified the difference of different yeast LDH dynamic, which for the conditioning control high alcohol content of beer and provide a theoretical basis.
In the beer fermentation process, the fermentation performance of different yeast lactic dehydrogenase enzyme had been measured. The data revealed the interrelationship between LDH activity and higher alcohols content. With LDH activity increasing , the higher alcohols content decreased during the beer fermentation ,which suggested competitive and inhabit relationship between LDH activity and higher alcohols.
The metal ions Mg~(2+) and Zn~(2+) were added to the medium, and the lactic dehydrogenase activity and production of higher alcohols were determined in the fermentation process, the results showed that adding a metal ions Mg~(2+), Zn~(2+) in the beer fermentation increased the lactic dehydrogenase activity of yeast (P<0.05) significantly, higher alcohols in control were lower than formented products significantly. Also,Mg~(2+) plays more significant promotion role than Zn~(2+).
The separation and purification methods of LDH from Saccharomyces cerevisiae had been established and optimized.The optimized purification process includes the steps that removing cells by the centrifugation,35%saturation ammonium sulfate precipitation , dialysis,ion exchange chromatography with DE52 and gel filtration chromatography with Superdex-25.Sedium dodecyl-sulfate-polyacrylamide gel electrophovesis(SDS-PAGE)was used to examine the purification effect ,and the results indicated that the final product obitained was homogeneous and had a molocular weight around 32.2KD.the purification rate and activity recovery of LDH was 14.3 and 24.2% respectively.The optimal reaction temperature and pH of LDH was 45℃and 9.4. The stability temperature of LDH enzyme was 35℃. Metalions such as Ca~(2+)、Mg~(2+) and Zn~(2+) can activate LDH and 1.0mmol/L Ca~(2+) can activate LDH signicantly . Influence of Fe~(2+) was not apparent .However,K+ was found to deactive LDH signicantly as well as Ni+.
Three pairs of primers were synthesized according to the sequence of Saccharomyces cerevisiae LDH published on the GenBank to amplify LDH genes of five srains of Saccharomyces cerevisiae with genomic DNA as the template. Amplified fragments had been sequenced . In this study analysis of the gene sequence differences among the laboratory five strains and the published Saccharomyces cerevisiae strain FLH201473.01X . It show that the difference among YZU03 , ST01 and SH01 mainly existed in 609-725th base of the whole sequence. These sequences were different from published at the site of the 213th,216th,217th,228th,314th and 315th bases.
引文
[1] Stryer L.生物化学[M].第2版.唐有棋,张惠珠,译.北京大学出版社,1990:270.
[2] Al-Jassabi.S. Purification and kinetic properties of skeletal muscle lactate dehydrogenase from the Lizard Agama stellio stellio[M].Biochemistry (Moscow),2002,67(7):786～789.
[3] Laidler K J,Bunting P S.The chemical kinetic of enzyme action[M].2nd ed.London:Oxford University Press,1973:342.
[4]郑国爱.细菌乳酸脱氢酶的纯化及其性质研究[J].生物技术,1999,9(1):11～15.
[5]顾国贤.啤酒风味物质代谢控制[J].中国啤酒,2002,(11): 3～5.
[6]金应世.啤酒中一些物质的风味特征[J].四川食品与发酵,2000, (4) :21～25.
[7]黄亚东,印伯星.关于啤酒酵母产苯乙醇的研究[J].酿酒,2003,30(5):62～63.
[8]林志国.啤酒中高级醇的控制[J].酿酒科技,2001,(2):55～56.
[9]高庆.降低啤酒中高级醇含量的研究[D].扬州大学硕士论文,2005:15～16.
[10]甘水洋.啤酒中高级醇的产生及控制办法[J].福建轻纺,1998,(1):1～4.
[11]陈思纭,萧熙佩.酵母生物化学[M].山东科学技术出版社,1990: 250～320.
[12]魏运平.低产高级醇猕猴桃酒酵母菌株的筛选[D].江南大学,2004.
[13]彭智辉,林炜铁,冯杰龙.酶共催化降低啤酒中双乙酰含量的研究设想[J].酿酒科技,2001,(1):66～67.
[14]石海英,徐伟.谈啤酒中上头物质-高级醇的形成与降低措施[J].食品科技,2003,113(3):49～50.
[15]周爱国,姚永春,孙文斌,等.使用酵母营养盐对啤酒高级醇含量的探讨[J].酿酒,2000,141(6):76～77.
[16] Verachtert, H .Kumara, S, Dawond, E. Yeast in mixed cultures with special emphasis on lambic beer brewingYeast Biotechnology and Catalysis,1990,90:429～449.
[17] Priest ,F.G,Campbell, I.Brewing Microbiology(second ED)[M].Chapman and Hall,1995.
[18]周爱国,姚永春,孙文斌,等.使用酵母营养盐对啤酒高级醇含量的探讨[J].酿酒,2000,141(6):76～77.
[19] Priest ,F.G,Campbell, I.Brewing Microbiology(second ED)[M].Chapman and Hall,1995
[20] Stewart, G. G, Russell, I. One hundred years of yeast research and development in the brewing industry .Journal of the institute of Brewing ,1986,92,537-558
[21]管敦仪.啤酒工业手册(修订版)[M].北京:中国轻工业出版社,1998.
[22]祝忠付.锌离子在啤酒酿造中的作用与控制[J].酿酒科技,2003(6):65～66.
[23]张惟广,吾永娴.Zn2+对酵母凝集性的影响研究[J].西南农业大学学报,1998,20(2):170～174.
[24]李俊林,刘东亚.麦汁中的锌离子的控制与测定[J].酿酒,2002,29(4):43～44.
[25]李云芝,康红雨,姜经宁,等.啤酒酵母富铁、富锌的生物学特性研究[J].生物技术,1997,7(3):43～44.
[26]张和笙,梁卫红.浅谈镁离子对啤酒发酵的影响[J].酿酒科技,1991,1(91):53～54.
[27]董亮,赵长新,李峰,等.大麦发芽过程中添加金属离子对纤维素酶和蛋白酶活力影响研究[J].食品科技,2006,2:18～21.
[28]汪志君,余晓红,方维明.富铁、富锌麦汁对啤酒酵母代谢副产物的影响[J].酿酒科技,2003,5(119):73～76.
[29]王志坚.酵母发酵副产物与啤酒风味[J].酿酒科技,2001,107(5):69～70.
[30]王刚.试论啤酒中的高级醇与上头[J].酿酒,1999,26(3):78.
[31]戴仁泽.啤酒发酵进展[M].北京:轻工业出版社,1985: 190～208.
[32]姜涌明,戴祝英等.分子酶学导论[M].中国农业大学出版社,2000: 56～83.
[33]郑国爱.细菌乳酸脱氢酶的纯化及其性质研究[J].生物技术,1999,9(1):11～15.
[34]朱文淼,肖敏,刘复今,等.枯草芽孢杆菌BS12乳酸脱氢酶的分离纯化与部分性质的研究[J].生物技术,1996,6(2):23～25.
[35]苏虹,汪建军,杨海麟,王武.植物乳杆菌产乳酸脱氢酶的发酵条件优化及酶的纯化[J].吉林大学学报,2006,11,44(6):1015～1018.
[36]潘丽军,余赟,郑志,等.米根霉乳酸脱氢酶的特性研究[J].食品科学,2003,24(11),23～26.
[37]王立艳,汪建军,杨海麟,等.猪心肌乳酸脱氢酶的提纯工艺及酶反应最适条件[J].南京师大学报,2004,27(2):70～73.
[38]周静,郑玉才,金素钰,等.牛蛙乳酸脱氢酶同工酶的分离纯化及其性质研究[J].四川动物,2006,25(2):244～246.
[39]金黎明,张乐,蒋本国,等.羊心肌乳酸脱氢酶的纯化及性质研究[J].大连民族学院,2006,30(1):20～23.
[40]司晓辉,赵兴波,郑玉才,等.藏系绵羊乳酸脱氢酶A的分离纯化和酶学性质[J].农业生物技术学报,2006,14(2):178～182.
[41]茅凌翔,孟泽,史利宁,等.人红细胞乳酸脱氢酶的提纯及其性质研究[J].临床检验杂志2001,19(1):4～7.
[42]姜玉梅,杨歌德,范业鹏,等.乳酸脱氢酶同工酶的提取及活力测定[J].哈尔滨医科大学学报,1999 (2): 71～72.
[43]周静,郑玉才,金素钰,等.牛蛙乳酸脱氢酶同工酶的分离纯化及其性质研究[J].四川动物,2006,25(2):244～246.
[44]朱文淼,肖敏,刘富今,等.枯草芽孢杆菌BS12乳酸脱氢酶的分离纯化与部分性质的研究[J].生物技术1996,6(2):23～25.
[45]石轩峰,杨海麟,王武,等.高纯度诊断用乳酸脱氢酶的制备和酶促反应[J].食品与生物技术学报,2005,11,24(6):38～42.
[46]高庆.降低啤酒中高级醇含量的研究[D]扬州大学硕士论文,2005:15～16.
[47]金黎明,张乐,蒋本国,等.羊心肌乳酸脱氢酶的纯化及性质研究[J].大连民族学院学报,2006,1:20～23.
[48]石轩峰,谢慧,杨海麟,等.植物乳杆菌乳酸脱氢酶的分离纯化工艺研究[J].河南工业大学学报,2005,6,26(3):49～53.
[49]司晓辉,赵兴波,郑玉才,等.藏系绵羊乳酸脱氢酶A的分离纯化和酶学性质[J].农业生物技术学报,2006,14(2):178～182.
[50]郑国爱.细菌乳酸脱氢酶的纯化及其性质研究[J].生物技术,1999,9(1):11～15.
[51]朱文淼,肖敏,刘复今,等.枯草芽孢杆菌BS12乳酸脱氢酶的分离纯化与部分性质的研究[J].生物技术,1996,6(2):23～25.
[52]王立艳,汪建军,杨海麟,等.猪心肌乳酸脱氢酶的提纯工艺及酶反应最适条件[J].南京师大学报,2004,27(2):70～73.
[53]周静,郑玉才,金素钰,等.牛蛙乳酸脱氢酶同工酶的分离纯化及其性质研究[J].四川动物,2006,25(2):244～246.
[54]石轩峰,谢慧,杨海麟,等.植物乳杆菌乳酸脱氢酶的分离纯化工艺研究[J].河南工业大学学报,2005,6,26(3):49～53.
[55]苏虹,汪建军,杨海麟,等.植物乳杆菌产乳酸脱氢酶的发酵条件优化及酶的纯化[J].吉林大学学报,2006,11,44(6):1015～1018.
[56]金黎明,张乐,蒋本国,等.羊心肌乳酸脱氢酶的纯化及性质研究[J].大连民族学院,2006,30(1):20～23.
[57]司晓辉,赵兴波,郑玉才,等.藏系绵羊乳酸脱氢酶A的分离纯化和酶学性质[J].农业生物技术学报,2006,14(2):178～182.
[58]茅凌翔,孟泽,史利宁,等.人红细胞乳酸脱氢酶的提纯及其性质研究[J].临床检验杂志2001,19(1):4～7.
[59] Skory CD.Lactic acid production by Saccharomyces cerevisiae expressing aRhizopus oryzae lactate dehydrogenase gene.J Ind Microbiol Biotechnol 2003,30(1):2-7.
[60] Ishida NS.Saitoh K,Tokuhiro eatl Efficient production of L-lactic acid by metabolically engineered Saccharomyces cerevisiae with a genome–integratedL-lactate dehydrogenase gene.Appi Environ.Microbiol 2005,71:1964-1970.
[61] Saitoh S,Ishida N,Onishi T,et al genetically Engineered Wine Yeast Produces High Concentration of L-Lactic Acid of Extremely High Optical Purity. Applied and Environmental Microbiology,2005,71,(5):2789-2792.
[62]管敦仪主编.啤酒工业手册(中册)[M]轻工业出版社.1985,7,108～109.
[63]杨智.酿酒酵母ST01发酵特性与SCAR标记研究[D].扬州大学硕士论文,2007:22.
[64]张和笙,梁卫红.浅论镁离子对啤酒发酵的影响[J].酿酒科技,1991,91(1):53～54.
[65]汪志君,余晓红,方维明.富铁、富锌麦汁对啤酒酵母代谢副产物的影响[J].酿酒科技,2003,119(5):73～76.
[66]高庆.降低啤酒中高级醇的含量的研究[D].扬州大学硕士学位论文,2004.
[67]钟玲,汪天虹.氯化苄法提取染色体DNA[J]微生物学杂志,1997,173:62～63.
[68]王关林,方宏筠.植物基因工程.第二版.北京:科学出版社,1998,755.
[69]李建武.生物化学实验原理和方法[M]北京大学出版社.1994,9,174～176.
[70]姜玉梅,杨歌德,等.乳酸脱氢酶同工酶的提取及活力测定[J].哈尔滨医科大学学报,1999 (2): 71～72.
[71]黄伟坤.食品检验与分析[M].轻工业出版社,1989:660～661.
[72]黄伟坤.食品检验与分析[M].轻工业出版社,1989:641～642.
[73]高庆.降低啤酒中高级醇的含量的研究[D].扬州大学硕士学位论文,2004.
[74]戴仁泽.啤酒发酵进展[M].北京:轻工业出版社,1985: 176～208.
[75]李俊林,刘东亚.麦汁中的锌离子的控制与测定[J].酿酒,2002,29(4):43～44.
[76]甘水洋.啤酒中高级醇的产生及控制办法[J].福建轻纺,1998,(1):1～4.
[77]石轩峰,谢慧,杨海麟,等.植物乳杆菌乳酸脱氢酶的分离纯化工艺研究[J].河南工业大学学报,2005,6,26(3):49～53.
[78]金黎明,张乐,蒋本国,等.羊心肌乳酸脱氢酶的纯化及性质研究[J].大连民族学院,2006,30(1):20～23.
[79]董亮,赵长新,李峰,等.大麦发芽过程中添加金属离子对纤维素酶和蛋白酶活力影响研究[J].食品科技,2006,2:18～21.
[2] Al-Jassabi.S. Purification and kinetic properties of skeletal muscle lactate dehydrogenase from the Lizard Agama stellio stellio[M].Biochemistry (Moscow),2002,67(7):786～789.
[3] Laidler K J,Bunting P S.The chemical kinetic of enzyme action[M].2nd ed.London:Oxford University Press,1973:342.
[4]郑国爱.细菌乳酸脱氢酶的纯化及其性质研究[J].生物技术,1999,9(1):11～15.
[5]顾国贤.啤酒风味物质代谢控制[J].中国啤酒,2002,(11): 3～5.
[6]金应世.啤酒中一些物质的风味特征[J].四川食品与发酵,2000, (4) :21～25.
[7]黄亚东,印伯星.关于啤酒酵母产苯乙醇的研究[J].酿酒,2003,30(5):62～63.
[8]林志国.啤酒中高级醇的控制[J].酿酒科技,2001,(2):55～56.
[9]高庆.降低啤酒中高级醇含量的研究[D].扬州大学硕士论文,2005:15～16.
[10]甘水洋.啤酒中高级醇的产生及控制办法[J].福建轻纺,1998,(1):1～4.
[11]陈思纭,萧熙佩.酵母生物化学[M].山东科学技术出版社,1990: 250～320.
[12]魏运平.低产高级醇猕猴桃酒酵母菌株的筛选[D].江南大学,2004.
[13]彭智辉,林炜铁,冯杰龙.酶共催化降低啤酒中双乙酰含量的研究设想[J].酿酒科技,2001,(1):66～67.
[14]石海英,徐伟.谈啤酒中上头物质-高级醇的形成与降低措施[J].食品科技,2003,113(3):49～50.
[15]周爱国,姚永春,孙文斌,等.使用酵母营养盐对啤酒高级醇含量的探讨[J].酿酒,2000,141(6):76～77.
[16] Verachtert, H .Kumara, S, Dawond, E. Yeast in mixed cultures with special emphasis on lambic beer brewingYeast Biotechnology and Catalysis,1990,90:429～449.
[17] Priest ,F.G,Campbell, I.Brewing Microbiology(second ED)[M].Chapman and Hall,1995.
[18]周爱国,姚永春,孙文斌,等.使用酵母营养盐对啤酒高级醇含量的探讨[J].酿酒,2000,141(6):76～77.
[19] Priest ,F.G,Campbell, I.Brewing Microbiology(second ED)[M].Chapman and Hall,1995
[20] Stewart, G. G, Russell, I. One hundred years of yeast research and development in the brewing industry .Journal of the institute of Brewing ,1986,92,537-558
[21]管敦仪.啤酒工业手册(修订版)[M].北京:中国轻工业出版社,1998.
[22]祝忠付.锌离子在啤酒酿造中的作用与控制[J].酿酒科技,2003(6):65～66.
[23]张惟广,吾永娴.Zn2+对酵母凝集性的影响研究[J].西南农业大学学报,1998,20(2):170～174.
[24]李俊林,刘东亚.麦汁中的锌离子的控制与测定[J].酿酒,2002,29(4):43～44.
[25]李云芝,康红雨,姜经宁,等.啤酒酵母富铁、富锌的生物学特性研究[J].生物技术,1997,7(3):43～44.
[26]张和笙,梁卫红.浅谈镁离子对啤酒发酵的影响[J].酿酒科技,1991,1(91):53～54.
[27]董亮,赵长新,李峰,等.大麦发芽过程中添加金属离子对纤维素酶和蛋白酶活力影响研究[J].食品科技,2006,2:18～21.
[28]汪志君,余晓红,方维明.富铁、富锌麦汁对啤酒酵母代谢副产物的影响[J].酿酒科技,2003,5(119):73～76.
[29]王志坚.酵母发酵副产物与啤酒风味[J].酿酒科技,2001,107(5):69～70.
[30]王刚.试论啤酒中的高级醇与上头[J].酿酒,1999,26(3):78.
[31]戴仁泽.啤酒发酵进展[M].北京:轻工业出版社,1985: 190～208.
[32]姜涌明,戴祝英等.分子酶学导论[M].中国农业大学出版社,2000: 56～83.
[33]郑国爱.细菌乳酸脱氢酶的纯化及其性质研究[J].生物技术,1999,9(1):11～15.
[34]朱文淼,肖敏,刘复今,等.枯草芽孢杆菌BS12乳酸脱氢酶的分离纯化与部分性质的研究[J].生物技术,1996,6(2):23～25.
[35]苏虹,汪建军,杨海麟,王武.植物乳杆菌产乳酸脱氢酶的发酵条件优化及酶的纯化[J].吉林大学学报,2006,11,44(6):1015～1018.
[36]潘丽军,余赟,郑志,等.米根霉乳酸脱氢酶的特性研究[J].食品科学,2003,24(11),23～26.
[37]王立艳,汪建军,杨海麟,等.猪心肌乳酸脱氢酶的提纯工艺及酶反应最适条件[J].南京师大学报,2004,27(2):70～73.
[38]周静,郑玉才,金素钰,等.牛蛙乳酸脱氢酶同工酶的分离纯化及其性质研究[J].四川动物,2006,25(2):244～246.
[39]金黎明,张乐,蒋本国,等.羊心肌乳酸脱氢酶的纯化及性质研究[J].大连民族学院,2006,30(1):20～23.
[40]司晓辉,赵兴波,郑玉才,等.藏系绵羊乳酸脱氢酶A的分离纯化和酶学性质[J].农业生物技术学报,2006,14(2):178～182.
[41]茅凌翔,孟泽,史利宁,等.人红细胞乳酸脱氢酶的提纯及其性质研究[J].临床检验杂志2001,19(1):4～7.
[42]姜玉梅,杨歌德,范业鹏,等.乳酸脱氢酶同工酶的提取及活力测定[J].哈尔滨医科大学学报,1999 (2): 71～72.
[43]周静,郑玉才,金素钰,等.牛蛙乳酸脱氢酶同工酶的分离纯化及其性质研究[J].四川动物,2006,25(2):244～246.
[44]朱文淼,肖敏,刘富今,等.枯草芽孢杆菌BS12乳酸脱氢酶的分离纯化与部分性质的研究[J].生物技术1996,6(2):23～25.
[45]石轩峰,杨海麟,王武,等.高纯度诊断用乳酸脱氢酶的制备和酶促反应[J].食品与生物技术学报,2005,11,24(6):38～42.
[46]高庆.降低啤酒中高级醇含量的研究[D]扬州大学硕士论文,2005:15～16.
[47]金黎明,张乐,蒋本国,等.羊心肌乳酸脱氢酶的纯化及性质研究[J].大连民族学院学报,2006,1:20～23.
[48]石轩峰,谢慧,杨海麟,等.植物乳杆菌乳酸脱氢酶的分离纯化工艺研究[J].河南工业大学学报,2005,6,26(3):49～53.
[49]司晓辉,赵兴波,郑玉才,等.藏系绵羊乳酸脱氢酶A的分离纯化和酶学性质[J].农业生物技术学报,2006,14(2):178～182.
[50]郑国爱.细菌乳酸脱氢酶的纯化及其性质研究[J].生物技术,1999,9(1):11～15.
[51]朱文淼,肖敏,刘复今,等.枯草芽孢杆菌BS12乳酸脱氢酶的分离纯化与部分性质的研究[J].生物技术,1996,6(2):23～25.
[52]王立艳,汪建军,杨海麟,等.猪心肌乳酸脱氢酶的提纯工艺及酶反应最适条件[J].南京师大学报,2004,27(2):70～73.
[53]周静,郑玉才,金素钰,等.牛蛙乳酸脱氢酶同工酶的分离纯化及其性质研究[J].四川动物,2006,25(2):244～246.
[54]石轩峰,谢慧,杨海麟,等.植物乳杆菌乳酸脱氢酶的分离纯化工艺研究[J].河南工业大学学报,2005,6,26(3):49～53.
[55]苏虹,汪建军,杨海麟,等.植物乳杆菌产乳酸脱氢酶的发酵条件优化及酶的纯化[J].吉林大学学报,2006,11,44(6):1015～1018.
[56]金黎明,张乐,蒋本国,等.羊心肌乳酸脱氢酶的纯化及性质研究[J].大连民族学院,2006,30(1):20～23.
[57]司晓辉,赵兴波,郑玉才,等.藏系绵羊乳酸脱氢酶A的分离纯化和酶学性质[J].农业生物技术学报,2006,14(2):178～182.
[58]茅凌翔,孟泽,史利宁,等.人红细胞乳酸脱氢酶的提纯及其性质研究[J].临床检验杂志2001,19(1):4～7.
[59] Skory CD.Lactic acid production by Saccharomyces cerevisiae expressing aRhizopus oryzae lactate dehydrogenase gene.J Ind Microbiol Biotechnol 2003,30(1):2-7.
[60] Ishida NS.Saitoh K,Tokuhiro eatl Efficient production of L-lactic acid by metabolically engineered Saccharomyces cerevisiae with a genome–integratedL-lactate dehydrogenase gene.Appi Environ.Microbiol 2005,71:1964-1970.
[61] Saitoh S,Ishida N,Onishi T,et al genetically Engineered Wine Yeast Produces High Concentration of L-Lactic Acid of Extremely High Optical Purity. Applied and Environmental Microbiology,2005,71,(5):2789-2792.
[62]管敦仪主编.啤酒工业手册(中册)[M]轻工业出版社.1985,7,108～109.
[63]杨智.酿酒酵母ST01发酵特性与SCAR标记研究[D].扬州大学硕士论文,2007:22.
[64]张和笙,梁卫红.浅论镁离子对啤酒发酵的影响[J].酿酒科技,1991,91(1):53～54.
[65]汪志君,余晓红,方维明.富铁、富锌麦汁对啤酒酵母代谢副产物的影响[J].酿酒科技,2003,119(5):73～76.
[66]高庆.降低啤酒中高级醇的含量的研究[D].扬州大学硕士学位论文,2004.
[67]钟玲,汪天虹.氯化苄法提取染色体DNA[J]微生物学杂志,1997,173:62～63.
[68]王关林,方宏筠.植物基因工程.第二版.北京:科学出版社,1998,755.
[69]李建武.生物化学实验原理和方法[M]北京大学出版社.1994,9,174～176.
[70]姜玉梅,杨歌德,等.乳酸脱氢酶同工酶的提取及活力测定[J].哈尔滨医科大学学报,1999 (2): 71～72.
[71]黄伟坤.食品检验与分析[M].轻工业出版社,1989:660～661.
[72]黄伟坤.食品检验与分析[M].轻工业出版社,1989:641～642.
[73]高庆.降低啤酒中高级醇的含量的研究[D].扬州大学硕士学位论文,2004.
[74]戴仁泽.啤酒发酵进展[M].北京:轻工业出版社,1985: 176～208.
[75]李俊林,刘东亚.麦汁中的锌离子的控制与测定[J].酿酒,2002,29(4):43～44.
[76]甘水洋.啤酒中高级醇的产生及控制办法[J].福建轻纺,1998,(1):1～4.
[77]石轩峰,谢慧,杨海麟,等.植物乳杆菌乳酸脱氢酶的分离纯化工艺研究[J].河南工业大学学报,2005,6,26(3):49～53.
[78]金黎明,张乐,蒋本国,等.羊心肌乳酸脱氢酶的纯化及性质研究[J].大连民族学院,2006,30(1):20～23.
[79]董亮,赵长新,李峰,等.大麦发芽过程中添加金属离子对纤维素酶和蛋白酶活力影响研究[J].食品科技,2006,2:18～21.