关键氨基酸调控啤酒酵母适应高浓酿造环境胁迫机制的研究

Nitrogen limitation, particularly prevailing in the case of high gravity beer brewing, restricts the tolerance of yeast cells and results in poor yeast viability and even stuck or sluggish fermentations. Therefore, firstly, the key amino acids promoting yeast cells adapt to the high osmotic pressure and high ethanol toxicity were revealed, respectively. Based on the optimum ratio and addition of key amino acids with the goal of improving physiological characteristics of yeast, fermentation performance and beer harmonious characteristic, transcriptomics and proteomics analysis was employed to identify the genes and proteins with significantly different expression, and then the correlation between the different expressed genes and proteins were evaluated. The bioinformatics (NCBI, KEGG, UniProt) were used to study gene sequence blast, protein sequence blast, metabolic pathway analysis, protein domain analysis, and analysis of enzyme substrate. The physiological functions of genes and proteins with different expression were unraveled from the gene level, protein level and metabolic levels. Finally, the roles of key amino acids in the response of yeast to high gravity environmental stress mechanism were revealed. Thus, results of this project will play an important role in both the development of scientific theories and the industrial applications of high gravity beer brewing.

氮限制是啤酒高浓酿造中普遍存在的技术难题，很大程度上损害了酵母细胞的耐受性，导致酵母活力降低、发酵滞缓甚至停滞。因此，本项目基于分别研究能够促进酵母适应高渗透压和高乙醇毒性两大环境胁迫条件的关键氨基酸；以改善酵母生理特征、发酵性能和啤酒口感协调性为目标,在确定关键氨基酸最佳配比及添加量的基础上，对酵母细胞进行转录组和蛋白组分析，从而鉴定出具有显著表达差异的基因和蛋白，对两者之间进行相关性评价；运用生物信息学工具进行基因序列比对、蛋白序列比对、代谢通路分析、蛋白结构域分析、酶底物分析，从基因水平、蛋白水平和代谢水平对差异表达基因和蛋白的生理功能进行深入剖析，最终揭示关键氨基酸调控酵母适应高浓酿造环境胁迫的分子机制。因此，本研究对啤酒高浓酿造的学科理论发展和工业化应用均具有至关重要的作用。

本项目研究了在高浓（18°P）和超高浓（24°P）发酵过程中，添加葡萄糖、蔗糖和麦芽糖对Lager酵母（Saccharomyces pastorianus）氨基酸代谢、发酵性能、胁迫耐受性的影响。结果表明，在18°P发酵过程中，添加葡萄糖或蔗糖显著改善了Lager酵母的发酵性能。而在24°P麦汁发酵过程中，与添加蔗糖相比，麦芽糖则显著改善了酵母的发酵性能。此外，添加麦芽糖后，随着麦芽汁浓度从18°P增加到24°P，酵母细胞中HSP12表达水平升高，胞内海藻糖积累增多，说明酵母细胞应激反应增强。添加麦芽糖可以促进Gly和Ala的分泌以及促进Pro在发酵后期的吸收；通过高渗透压环境胁迫条件下酵母对游离氨基酸的同化量与发酵性能各指标之间的相关性分析说明： Ser、Met、Phe、Trp、His可改善酵母对高渗透压环境胁迫的适应性。通过乙醇环境胁迫条件下酵母对游离氨基酸的同化量与发酵性能各指标之间的相关性分析说明： Asp、Ser、Gly、Arg、Tyr、Val、Met、Lys、Ile、Leu可改善酵母对高乙醇环境胁迫的适应性。研究关键氨基酸不同添加倍数（0.5倍、1倍、2倍）对啤酒高浓酿造（24 °P）中酵母发酵性能的影响。结果表明，十种关键氨基酸的补充可显著提高啤酒高浓酿造的发酵度、乙醇产量，显著促进酵母细胞数的生长并提高酵母活细胞率，改善啤酒色值。以脱脂核桃粕为原料，分别用中性蛋白酶、复合蛋白酶和风味蛋白酶酶解，制得蛋白水解物。研究了啤酒高浓酿造中应用蛋白水解物提高酵母发酵性能的可行性。结果表明，超声辅助酶解同步处理和超声预处理后酶解的水解度分别至少是传统酶解的1.43倍和0.71倍，复合蛋白酶的DH最高。与对照相比较，超声辅助酶解得到的蛋白水解物显著提高啤酒酵母的发酵性能，此外，添加蛋白水解物显著增加了酵母的生长和细胞活力，促进了酵母细胞中糖原和海藻糖的积累，促进了应激标记基因HSP12和SSA3的表达，最终促进了高级醇和酯的形成，提高了啤酒的醇酯比。