食品与发酵工业 >

2021 , Vol. 47 >Issue 13: 98 - 106

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.026722

研究报告

适量产乙酸酯酿酒酵母菌株的选育

  • 魏亚楠 ,
  • 王金晓 ,
  • 林良才 ,
  • 陈博超 ,
  • 崔丹瑶 ,
  • 张翠英
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  • 1(工业发酵微生物教育部重点实验室(天津科技大学),天津,300457)
    2(天津科技大学 生物工程学院,天津,300457)
硕士研究生(张翠英教授为通讯作者,E-mail:cyzhangcy@tust.edu.cn)

收稿日期: 2021-01-15

  修回日期: 2021-02-20

  网络出版日期: 2021-08-02

基金资助

国家自然科学基金项目(31471724);国家重点研发计划重点专项(2016YFD0400505)

收起

Breeding of Saccharomyces cerevisiae strains with appropriate yield of acetate esters

  • WEI Yanan ,
  • WANG Jinxiao ,
  • LIN Liangcai ,
  • CHEN Bochao ,
  • CUI Danyao ,
  • ZHANG Cuiying
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  • 1(Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,Tianjin University of Science and Technology,Tianjin 300457,China)
    2(College of Bioengineering,Tianjin University of Science and Technology,Tianjin 300457,China)

Received date: 2021-01-15

  Revised date: 2021-02-20

  Online published: 2021-08-02

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摘要

乙酸乙酯、乙酸异戊酯等酯类物质是白酒中重要的风味物质,含量过低会导致酒体风味寡淡,而过高会破坏整体香气的平衡。实验室前期以工业酿酒酵母AY12的单倍体a45为出发菌株,构建了低产高级醇高产酯酿酒酵母菌株a45-AWT,但其存在乙酸异戊酯含量过高的问题,影响白酒品质。通过过表达编码乙酸异戊酯水解酶的基因IAH1的方法,可达到调控高产酯酵母乙酸异戊酯产量的目的。采用胞内同源重组的方法,在a45-AWT菌株的Gal80位点过表达IAH1基因,获得a45-BAI菌株。模拟白酒发酵结果表明,与高产酯酵母a45-AWT相比,a45-BAI的乙酸异戊酯的质量浓度仅为10.73 mg/L,降低了83.2%,乙酸乙酯和乙酸异戊酯的质量浓度比提高了87.3%。该研究通过调节酯合成、分解途径成功选育了具有高产乙酸乙酯、适量产乙酸异戊酯低产高级醇性状的酿酒酵母菌株,为白酒酿造用菌的选育提供了新思路。

关键词： 酿酒酵母; 乙酸异戊酯水解酶; 乙酸乙酯; 乙酸异戊酯; 浓醪发酵; 白酒

本文引用格式

魏亚楠 , 王金晓 , 林良才 , 陈博超 , 崔丹瑶 , 张翠英 . 适量产乙酸酯酿酒酵母菌株的选育[J]. 食品与发酵工业, 2021 , 47(13) : 98 -106 . DOI: 10.13995/j.cnki.11-1802/ts.026722

Abstract

Ethyl acetate and isoamyl acetate are two important volatile flavor substances in Chinese Baijiu. Low concentration of ester could not give Baijiu the characteristic taste, while the flavor of Baijiu was also damaged by hyper-production of ester. In previous work, ATF1 overexpression with BAT2 deletion in Saccharomyces cerevisiae a45 (a45-AWT) enhanced the content of acetate esters and decreased the content of higher alcohols. However, the concentration of isoamyl acetate was quite high, thereby causing negative effects on the quality of Baijiu. In this work, isoamyl acetate hydrolyzing esterase coding gene IAH1 was deleted to evaluate the effect on the content of acetate esters and higher alcohols in Baijiu. The strain a45-AWT was used as the parental strain. IAH1 was overexpressed and a45-BAI strain was obtained. Fermentation analysis showed that the isoamyl acetate production of a45-BAI was only 10.73 mg/L, which was decreased by 83.2%. The proportion of ethyl acetate and isoamyl acetate of strain a45-BAI was increased by 87.3%. In this study, The strain with high yield of ethyl acetate was constructed by regulating the of ester biosynthesis and biolysis. It could moderate production of isoamyl acetate and low production of higher alcohols. The results provided new strategies for strain breeding.

Key words： Saccharomyces cerevisiae; isoamyl acetate hydrolysing esterase; ethyl acetate; isoamyl acetate; concentrated fermentation; Chinese Baijiu

参考文献

[1] 范光森, 吴秋华,刘朋肖,等.脂肪酶在白酒酯类化合物合成中的作用研究进展[J].中国食品学报,2021,21(1):351-360.
FAN G S,WU Q H,LIU P X,et al.Research progress on the role of lipase in the synthesis of esters in Baijiu[J].Journal of Chinese Institute of Food Science and Technology,2020(12):1-8.
[2] ZHU M X,FAN W L,XU Y,et al.1,1-Diethoxymethane and methanethiol as age markers in Chinese roasted-sesame-like aroma and flavour type liquor[J].European Food Research & Technology,2016,242(11):1 985-1 992.
[3] ZHANG R,WU Q,XU Y.Aroma characteristics of Moutai-flavour liquor produced with Bacillus licheniformis by solid-state fermentation[J].Letters in Applied Microbiology,2013,57(1):11-18.
[4] JIN G Y,ZHU Y,XU Y.Mystery behind Chinese liquor fermentation[J].Trends in Food Science & Technology,2017,63:18-28.
[5] 孙丽萍, 樊金梁,王德良.啤酒生产过程中酯类的形成以及影响因素[J].酿酒,2002,29(4):58-59.
SUN L P,FAN J L,WANG D L.The formation and influencing factors of esters in beer production[J].Liquor Making,2002,29(4):58-59.
[6] 张翠英, 崔丹瑶,李维,等.高产乙酸酯酿酒酵母菌种的选育与提高白酒酯香物质含量的研究[J].酿酒科技,2018(7):92-97.
ZHANG C Y,CUI D Y,LI W,et al.Breeding of S.cerevisiae strains with high-yield of acetate[J].Liquor-Making Science & Technology,2018(7):92-97.
[7] 肖冬光, 赵树欣,陈叶福.白酒生产技术[M].北京:化学工业出版社,2011.
XIAO D G,ZHAO S X,CHEN Y F,et al.Baijiu production technology[M].Beijing:Chemical Industry Press,2011.
[8] MA Y H,QIAO H,WANG W,et al.Variations in physicochemical properties of Chinese Fenjiu during storage and high-gravity technology of liquor aging[J].International Journal of Food Properties,2014,17(4):923-936.
[9] 王旭亮, 王异静,王德良,等.清香型白酒优质产酯酵母菌筛选[J].酿酒,2012,39(4):28-33.
WANG X L,WANG Y J,WANG D L,et al.Ccreening of high ethyl acetate producing yeasts for Fen-flavor Chinese spirits fermentation[J].Liquor Making,2012,39(4):28-33.
[10] 马金鸣. 酵母乙酸异戊酯水解酶和V型ATP酶F亚基的结构生物学研究[D].合肥:中国科学技术大学,2010.
MA J M.The structural biology research of IAH1 and VMA7 from Saccharomyces cerevisiae[D].Hefei:University of Science and Technology of China,2010.
[11] 曾伟, 朱力红,付毅华.白酒溶液各主要溶质功能作用及调味启示[J].酿酒,2005(5):28-29.
ZENG W,ZHU L H,FU Y H.The function and seasoning enlightenment of main solutes in Baijiu[J].Liquor Making,2005(5):28-29.
[12] PENEDA J,BAPTISTA A,LOPES J M.Interaction of the constituents of alcoholic beverages in the promotion of liver damage[J].Acta Medica Portuguesa,1994,7:S51-S55.
[13] 范光森, 王允中,刘朋肖,等.醇酰基转移酶催化合成白酒中的乙酸乙酯研究进展[J].中国食品学报,2020,20(11):294-304.
FAN G S,WANG Y Z,LIU P X,et al.Research progress in catalytic synthesis of ethyl acetate in Baijiu by alcohol acyltransferase[J].Journal of Chinese Institute of Food Science and Technology,2020,20(11):294-304.
[14] VERSTREPEN K J,VAN LAERE S D M,VANDERHAEGEN B M P,et al.Expression levels of the yeast alcohol acetyltransferase genes ATF1,Lg-ATF1,and ATF2 control the formation of a broad range of volatile esters[J].Applied & Environmental Microbiology,2003,69(9):5 228-5 237.
[15] LILLY M,LAMBRECHTS M G,PRETORIUS I S.Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates[J].Applied and Environmental Microbiology,2000,66(2):744-753.
[16] FUKUDA K,KUWAHATA O,KIYOKAWA Y,et al.Molecular cloning and nucleotide sequence of the isoamyl acetate-hydrolyzing esterase gene(EST2) from Saccharomyces cerevisiae[J].Journal of Fermentation and Bioengineering,1996,82(1):8-15.
[17] LILLY M,BAUER F F,STYGER G,et al.The effect of increased branched-chain amino acid transaminase activity in yeast on the production of higher alcohols and on the flavour profiles of wine and distillates[J].FEMS Yeast Research,2006,6(5):726-743.
[18] FUJII T,NAGASAWA N,IWAMATSU A,et al.Molecular cloning,sequence analysis,and expression of the yeast alcohol acetyltransferase gene[J].Applied and Environmental Microbiology,1994,60(8):2 786-2 792.
[19] CUI D Y,ZHANG Y,XU J,et al.PGK1 promoter library for the regulation of acetate ester production in Saccharomyces cerevisiae during Chinese Baijiu fermentation[J].Journal of Agricultural & Food Chemistry,2018,66(28):7 417-7 427.
[20] GIETZ R D,WOODS R A.Yeast transformation by the LiAc/SS carrier DNA/PEG method[J].Yeast Protocol,2006,313:107-120.
[21] 肖冬光, 许葵,李瑞青.酒精浓醪发酵生产工艺的优化[J].酿酒科技,2004(6):40-42.
XIAO D G,XU K,LI R Q.The optimization of production conditions of high-gravity alcohol fermentation[J].Liquor-Making Science & Technology,2004(6):40-42.
[22] 段钢, 许宏贤,钱莹,等.酸性蛋白酶在玉米酒精浓醪发酵上的应用[J].食品与发酵工业,2005,31(8):34-38.
DUAN G,XU H X,QIAN Y,et al.Application of acid protease for high dry solid alcohol fermentation[J].Food and Fermentation Industries,2005,31(8):34-38.
[23] 刘代武, 彭涛,邬善远,等.玉米酒精浓醪发酵工艺研究[J].酿酒科技,2005(2):87-89.
LIU D W,PENG T,WU S Y,et al.Study on high-concentration mash fermentation technology[J].Liquor-Making Science & Technology,2005(2):87-89.
[24] 崔丹瑶. 酿酒酵母中不同强度PGK启动子文库的构建与应用[D].天津:天津科技大学,2019.
CUI D Y.Construction and application of PGK promoter library in Saccharomyces cerevisiae[D].Tianjin:Tianjin University of Science & Technology,2019.
[25] 杨林娥, 彭晓光,杨庆文,等.斐林试剂法测定还原糖方法的改进[J].中国酿造,2010,29(5):160-161.
YANG L E,PENG X G,YANG Q W,et al.Improvement of the determination of reducing sugar with Fehling’s reagent method[J].China Brewing,2010,29(5):160-161.
[26] 郑楠, 郭庆焕,何亚辉,等.乙酰辅酶A含量对酿酒酵母乙酸乙酯合成的影响[J].中国酿造,2018,37(5):150-156.
ZHENG N,GUO Q H,HE Y H,et al.Effect of acetyl-CoA content on ethyl acetate synthesis in Saccharomyces cerevisiae[J].China Brewing,2018,37(5):150-156.
[27] LU J,DONG J,WU D G,et al.Construction of recombinant industrial brewer’s yeast with lower diacetyl production and proteinase A activity[J].European Food Research and Technology,2012,235(5):951-961.
[28] 张晓晓, 任剑星,刘凯毅,等.TOR1基因缺失对酿酒酵母耐受性的影响[J].食品与发酵工业,2021,47(2):1-7.
ZHANG X X,REN J X,LIU K Y,et al.Effects of TOR1 gene deletion on the tolerance of Saccharomyces cerevisiae[J].Food and Fermentation Industries,2021,47(2):1-7.
[29] FUKUDA K,YAMAMOTO N,KIYOKAWA Y,et al.Brewing properties of sake yeast whose EST2 gene encoding isoamyl acetate-hydrolyzing esterase was disrupted[J].Journal of Fermentation & Bioengineering,1998,85(1):101-106.
[30] LI W,WANG J H,ZHANG C Y,et al.Regulation of Saccharomyces cerevisiae genetic engineering on the production of acetate esters and higher alcohols during Chinese Baijiu fermentation[J].Journal of Industrial Microbiology & Biotechnology,2017,44(6):949-960.
[31] FUKUDA K,YAMAMOTO N,KIYOKAWA Y,et al.Purification and characterization of isoamyl acetate-hydrolyzing esterase encoded by the IAH1 gene of Saccharomyces cerevisiae from a recombinant Escherichia coli[J].Applied Microbiology and Biotechnology,2000,53(5):596-600.
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