高通量筛选诱变菌株降低黄酒发酵氨基甲酸乙酯前体积累

High-throughput screening of mutant strain to reduce the accumulation of ethyl carbamate precursor in rice wine fermentation

摘要:

[目的]氨基甲酸乙酯是发酵食品中普遍存在的一种潜在危害物。黄酒中氨基甲酸乙酯的前体主要是尿素和乙醇。本研究通过高通量筛选策略降低黄酒发酵过程中尿素的积累，从而降低氨基甲酸乙酯积累。[方法]以一株黄酒生产工业菌株酿酒酵母XZ-11为研究对象，采用ARTP诱变和高通量筛选策略，获得尿素积累量较低菌株。使用实时定量PCR技术检测氮代谢中尿素代谢和转运相关基因（DUR1，2和DUR3）的变化。[结果]筛选得到一株尿素高效稳定性利用菌株5-11C。其尿素积累量比酿酒酵母XZ-11降低了50.6%。实时定量荧光PCR结果表明，与尿素代谢和转运相关的基因（DUR1，2和DUR3）表达量分别提高了3.3和2.2倍。[结论]高通量筛选策略可以用于降低黄酒生产过程中氨基甲酸乙酯前体尿素的含量。由于未采用基因工程手段，避免了可能的法规问题，消费者易于接受，在发酵食品行业具有较好的应用前景。

Abstract:

[Objective] Ethyl carbamate is a common potential hazard in different fermented foods. Urea and ethanol are the major precursors of ethyl carbamate in rice wine. In this study, the accumulation of urea was reduced based on a high-throughput screening strategy, therefore decrease the accumulation of ethyl carbamate in rice wine.[Methods] An industrial strain Saccharomyces cerevisiae XZ-11 was used for the study. Lower urea accumulating strains were achieved by using Atmospheric and Room Temperature Plasma (ARTP) mutagenesis and high-throughput screening strategy. RT-qPCR analysis was used to detect the change of DUR1,2 and DUR3, two important urea metabolism and transport genes.[Results] A mutant strain 5-11C was obtained with the capacity of both efficiently urea using and genetic stability. The accumulation of urea was 50.6% lower than that of S. cerevisiae XZ-11. RT-qPCR results showed that the expression levels of DUR1,2 and DUR3 increased 3.3 and 2.2 folds, respectively.[Conclusion] High-throughput screening strategy can be applied to obtain mutants with reduced accumulation of ethyl carbamate precursor in rice wine.