Enhanced robustness and fermentation characteristics of lager yeast in high gravity brewing through accumulation of intracellular proline

In beer production, lager yeasts are subjected to harsh environment in high-gravity brewing (HGB, 24°P or more), thus leading to reduced fermentation performance, increased mortality and formation of off-flavors. This study aimed to improve the vitality, viability and fermentation characteristics of lager yeast during HGB through the accumulation of intracellular proline and to reveal the potential mechanism. A mutant lager yeast Y-100 with significantly increased intracellular proline fluorescence intensity of 37.37 % was obtained by Atmospheric and Room Temperature Plasma (ARTP) mutagenesis. Compared to parental YY, the mutant Y-100 had 13.94 % higher intracellular ATP content, 23.01 % lower ROS accumulation and 77.71 % lower mortality rate at the end of serial batch fermentation for 5 times. Moreover, the time for beer matureness by Y-100 strain was shorted by one day while the real degree of fermentation value was 2.76 % higher using 24°P wort. Through genome resequencing, RT-qPCR analysis and gene knockout and overexpression, the up-regulation of GNP1 and SUA7 genes in Y-100 strain might contribute to the proline accumulation in lager yeast cells, thus resulting in energy supply and stress protection for lager yeast during HGB. The results not only provided new insights into the role of proline in lager yeast towards unfavorable industrial condition, but also obtained a high-efficient Y-100 strain for potential HGB application.