山西老陈醋高产糖化酶霉菌的筛选、鉴定及产酶条件优化
|
摘要
从山西老陈醋大曲中共分离出75株霉菌,对高产糖化酶霉菌进行筛选、鉴定及产酶条件优化。结果表明,筛选出1株优良黑曲霉(Aspergillus niger)186,在麸皮固态培养基中其糖化酶、蛋白酶和纤维素酶酶活分别为3 963.79 U/g、368.80 U/g和4 476.60 U/g。糖化酶基因聚合酶链反应(PCR)测序结果显示,该酶脱氧核糖核酸(DNA)序列编码区长1 449 bp,共编码482个氨基酸,预测酶的分子质量为51.75 kDa,等电点为3.99。最佳产酶条件为:麸皮4%,硫酸铵0.5%,初始pH值为7.0,接种量8%,装液量100 m L/250 m L,转速145 r/min,培养7 d。在此优化条件下,黑曲霉186糖化酶活力可达892.98 U/m L,是优化前的1.77倍。
75 mold strains were isolated from Shanxi mature vinegar Daqu, the molds with high-yield saccharifying enzyme were screened and identified, and the enzyme production conditions were optimized. The results showed that a dominate strain Aspergillus niger 186 was screened, and the activity of saccharifying enzyme, protease and cellulase in bran solid medium was 3 963.79 U/g, 368.80 U/g, and 4 476.60 U/g, respectively. The sequencing result of polymerase chain reaction(PCR) of saccharifying enzyme gene showed that the sequences length of coding region for DNA of glycosylase gene was 1 449 bp, which encoded a total of 482 amino acids. The calculated molecular weight and isoelectric point of the enzyme were51.75 kDa and pH 3.99, respectively. The optimal enzyme production conditions for the A. niger 186 were bran 4%, ammonium sulfate 0.5%, initial pH 7.0, inoculum 8%, liquid loading volume 100 ml/250 ml, rotation speed 145 r/min and fermentation time 7 d. Under the optimal conditions, the saccharifing enzyme activity could reach 892.98 U/ml, which was 1.77 times higher than that of before optimization.
75 mold strains were isolated from Shanxi mature vinegar Daqu, the molds with high-yield saccharifying enzyme were screened and identified, and the enzyme production conditions were optimized. The results showed that a dominate strain Aspergillus niger 186 was screened, and the activity of saccharifying enzyme, protease and cellulase in bran solid medium was 3 963.79 U/g, 368.80 U/g, and 4 476.60 U/g, respectively. The sequencing result of polymerase chain reaction(PCR) of saccharifying enzyme gene showed that the sequences length of coding region for DNA of glycosylase gene was 1 449 bp, which encoded a total of 482 amino acids. The calculated molecular weight and isoelectric point of the enzyme were51.75 kDa and pH 3.99, respectively. The optimal enzyme production conditions for the A. niger 186 were bran 4%, ammonium sulfate 0.5%, initial pH 7.0, inoculum 8%, liquid loading volume 100 ml/250 ml, rotation speed 145 r/min and fermentation time 7 d. Under the optimal conditions, the saccharifing enzyme activity could reach 892.98 U/ml, which was 1.77 times higher than that of before optimization.
引文
[1]NIE Z,ZHENG Y,DU H,et al.Dynamics and diversity of microbial community succession in traditional fermentation of Shanxi aged vinegar[J].Food Microbiol,2015,47:62-68.
[2]LI S,LI P,LIU X,et al.Bacterial dynamics and metabolite changes in solid-state acetic acid fermentation of Shanxi aged vinegar[J].Appl Microbiol Biot,2016,100(10):4395-4411.
[3]LI P,FENG F,LUO L,et al.Microbiota dynamics associated with environmental conditions and potential roles of cellulolytic communities in traditional Chinese cereal starter solid-state fermentation[J].Appl Environ Microbiol,2015,81(15):5144-5156.
[4]ZHENG X W,TABRIZI M R,NOUT M J R,et al.Daqu-a traditional Chinese liquor fermentation starter[J].J I Brewing,2012,117(1):82-90.
[5]张琳,张也,王如福,等.大曲中高产糖化酶菌株的筛选及环境耐受性分析[J].山西农业大学学报(自然科学版),2016,36(10):740-744.
[6]刘茗铭,周阳子,袁乐梅,等.酒曲中高产糖化酶霉菌的筛选及其固态发酵产酶条件优化[J].食品与发酵工业,2018,44(10):118-123.
[7]唐国敏,徐雁漪,龚辉,等.黑曲霉糖化酶高产株糖化酶c DNA的合成、克隆和序列分析[J].生物工程学报,1993,9(2):117-121.
[8]BOEL E,HJORT I,SVENSSON B,et al.Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs[J].Embo J,1984,3(5):1097-1102.
[9]LI P,LIN W,LIU X,et al.Effect of bioaugmented inoculation on microbiota dynamics during solid-state fermentation of Daqu starter using autochthonous of Bacillus,Pediococcus,Wickerhamomyces and Saccharomycopsis[J].Food Microbiol,2017,61:83-92.
[10]LI P,LIANG H,LIN W T,et al.Microbiota dynamics associated with environmental conditions and potential roles of cellulolytic communities in traditional chinese cereal starter solid-state fermentation[J].Applied&Environmental Microbiology,2015,81(15):5144-56.
[11]LI P,AFLAKPUI F W K,YU H,et al.Characterization of activity and microbial diversity of typical types of Daqu for traditional Chinese vinegar[J].Ann Microbiol,2015,65(4):2019-2027.
[12]谢玉球,时晓,周二干,等.洋河大曲中糖化酶高产霉菌的筛选鉴定及固态发酵条件优化[J].酿酒科技,2016(4):39-42.
[13]郭红珍.山西老陈醋大曲红曲霉分离鉴定[J].中国食品添加剂,2006(3):99-103.
[14]ZHENG X W,YAN Z,HAN B Z,et al.Complex microbiota of a Chinese"Fen"liquor fermentation starter(Fen-Daqu),revealed by culturedependent and culture-independent methods[J].Food Microbiol,2012,31(2):293-300.
[15]谭婷婷,王家林,桑戈,等.北方黄酒麦曲中真菌的筛选·鉴定及系统发育分析[J].安徽农业科学,2015,43(17):15-16,71.
[16]班世栋,王晓丹,陈孟强,等.酱香型大曲中具产酶功能霉菌的分离筛选[J].酿酒,2014,41(4):31-36.
[17]黄永光,徐岩.真菌Aspergillus hennebergii酸性蛋白酶小麦固态发酵性能研究[J].食品工业科技,2014,35(21):135-139.
[18]班世栋.酱香大曲中霉菌类群和酶系研究[D].贵阳:贵州大学,2015.
[19]王旭亮,王异静,王德良,等.白酒发酵高糖化性能霉菌的筛选及鉴定[J].酿酒科技,2012(9):22-28.
[20]黄永光.酱香型白酒酿造中Aspergillus hennebergii及其分泌酸性蛋白酶的研究[D].无锡:江南大学,2014.
[21]李兰晓.黑曲霉(Aspergillus niger sp.)固态发酵啤酒糟生产纤维素酶及其酶学性质与发酵产物的研究[D].泰安:山东农业大学,2008.
[2]LI S,LI P,LIU X,et al.Bacterial dynamics and metabolite changes in solid-state acetic acid fermentation of Shanxi aged vinegar[J].Appl Microbiol Biot,2016,100(10):4395-4411.
[3]LI P,FENG F,LUO L,et al.Microbiota dynamics associated with environmental conditions and potential roles of cellulolytic communities in traditional Chinese cereal starter solid-state fermentation[J].Appl Environ Microbiol,2015,81(15):5144-5156.
[4]ZHENG X W,TABRIZI M R,NOUT M J R,et al.Daqu-a traditional Chinese liquor fermentation starter[J].J I Brewing,2012,117(1):82-90.
[5]张琳,张也,王如福,等.大曲中高产糖化酶菌株的筛选及环境耐受性分析[J].山西农业大学学报(自然科学版),2016,36(10):740-744.
[6]刘茗铭,周阳子,袁乐梅,等.酒曲中高产糖化酶霉菌的筛选及其固态发酵产酶条件优化[J].食品与发酵工业,2018,44(10):118-123.
[7]唐国敏,徐雁漪,龚辉,等.黑曲霉糖化酶高产株糖化酶c DNA的合成、克隆和序列分析[J].生物工程学报,1993,9(2):117-121.
[8]BOEL E,HJORT I,SVENSSON B,et al.Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs[J].Embo J,1984,3(5):1097-1102.
[9]LI P,LIN W,LIU X,et al.Effect of bioaugmented inoculation on microbiota dynamics during solid-state fermentation of Daqu starter using autochthonous of Bacillus,Pediococcus,Wickerhamomyces and Saccharomycopsis[J].Food Microbiol,2017,61:83-92.
[10]LI P,LIANG H,LIN W T,et al.Microbiota dynamics associated with environmental conditions and potential roles of cellulolytic communities in traditional chinese cereal starter solid-state fermentation[J].Applied&Environmental Microbiology,2015,81(15):5144-56.
[11]LI P,AFLAKPUI F W K,YU H,et al.Characterization of activity and microbial diversity of typical types of Daqu for traditional Chinese vinegar[J].Ann Microbiol,2015,65(4):2019-2027.
[12]谢玉球,时晓,周二干,等.洋河大曲中糖化酶高产霉菌的筛选鉴定及固态发酵条件优化[J].酿酒科技,2016(4):39-42.
[13]郭红珍.山西老陈醋大曲红曲霉分离鉴定[J].中国食品添加剂,2006(3):99-103.
[14]ZHENG X W,YAN Z,HAN B Z,et al.Complex microbiota of a Chinese"Fen"liquor fermentation starter(Fen-Daqu),revealed by culturedependent and culture-independent methods[J].Food Microbiol,2012,31(2):293-300.
[15]谭婷婷,王家林,桑戈,等.北方黄酒麦曲中真菌的筛选·鉴定及系统发育分析[J].安徽农业科学,2015,43(17):15-16,71.
[16]班世栋,王晓丹,陈孟强,等.酱香型大曲中具产酶功能霉菌的分离筛选[J].酿酒,2014,41(4):31-36.
[17]黄永光,徐岩.真菌Aspergillus hennebergii酸性蛋白酶小麦固态发酵性能研究[J].食品工业科技,2014,35(21):135-139.
[18]班世栋.酱香大曲中霉菌类群和酶系研究[D].贵阳:贵州大学,2015.
[19]王旭亮,王异静,王德良,等.白酒发酵高糖化性能霉菌的筛选及鉴定[J].酿酒科技,2012(9):22-28.
[20]黄永光.酱香型白酒酿造中Aspergillus hennebergii及其分泌酸性蛋白酶的研究[D].无锡:江南大学,2014.
[21]李兰晓.黑曲霉(Aspergillus niger sp.)固态发酵啤酒糟生产纤维素酶及其酶学性质与发酵产物的研究[D].泰安:山东农业大学,2008.