摘要
乙酸乙酯和高级醇是啤酒中的重要风味物质,为了探究BAT2基因和Lg-ATF1基因对啤酒酵母产醇酯能力的影响,进而解决啤酒中存在的醇高酯低的问题。本研究通过酶切连接法构建重组质粒pUC-PLABBK,采用醋酸锂转化法和胞内同源重组技术,以多倍体啤酒酵母菌株S5为出发菌株,KanMX基因作为筛选标记,最终获得过量表达Lg-ATF1基因同时敲除BAT2基因的重组菌株S5-Lg。通过啤酒发酵实验和数字PCR探究重组酵母菌株S5-Lg与出发菌株S5醇酯含量与相关基因表达量的变化。结果显示,与出发菌株相比,S5-Lg的总高级醇生成量降低9.12%,其中异丁醇和异戊醇的生成量分别降低了10.63%和9.55%,乙酸乙酯生成量提升了26.81%,BAT2基因表达量降低45.72%,Lg-ATF1基因表达量大幅提升。BAT2基因和Lg-ATF1基因可以影响啤酒酵母产生高级醇和乙酸乙酯的含量,对改善啤酒风味有重要参考意义。
Ethyl acetate and higher alcohols are important flavor substances in beer. The effect of BAT2 gene and Lg-ATF1 gene in beer yeast on producing alcohol esters was explored to solve the problem of more higher alcohols and less ethyl acetate in beer. The expression vector pUC-PLABBK was constructed by using the method of enzyme digestion and connection. Saccharomyces cerevisiae multiploid S5 was used as the original strain and the KanMX gene was used as a selection marker in this study. Lithium acetate transformation and homologous recombination were employed to screen for a mutant strain S5-Lg that overexpressing Lg-ATF1 and at the same time deleting BAT2. The variation between the content of higher alcohols, ethyl acetate and expression of related genes in the engineered yeast strain S5-Lg was investigated by fermentation experiments and digital PCR. The results showed that compared with the original strain S5, the ethyl acetate production of the S5-Lg strain was increased by 26.81%, total higher alcohol production decreased by 45.72%, of which the contents of isobutanol and isoamyl alcohol were reduced by 10.63% and 9.55%, respectively. The expression of Lg-ATF1 gene was greatly increased, and the expression level of BAT2 gene was decreased by 45.72%. It effectively changed the ability of brewer's yeast to produce ethyl acetate and higher alcohols and has important significance for improving beer flavor.
作者
葛峻伶
郭莹
刘小二
张翠英
肖冬光
GE Jun-ling;GUO Ying;LIU Xiao-er;ZHANG Cui-ying;XIAO Dong-guang(College of Biotechnology,Tianjin University of Science & Technology,Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,Tianjin 300457,China)
出处
《现代食品科技》
EI
CAS
北大核心
2019年第6期171-176,272,共7页
Modern Food Science and Technology
基金
国家自然科学基金资助项目(31471724)
