高效发酵啤酒酵母原生质体融合实验条件的优化

Optimizing process of protoplast fusion in efficient fermentation beer yeast

探索原生质体融合技术应用于高效啤酒酵母育种中关键步骤的最优条件。本实验以啤酒酵母b与啤酒酵母c为实验菌株，研究菌体酶解浓度、酶解时间、灭活条件以及融合时间对原生质体融合的影响。当酶解温度为28℃时，酶解时间为30min，酶解浓度为2％，原生质体制备率和生成率最佳。啤酒酵母b采用15W紫外灯下30cm照射2min，100％灭活。啤酒酵母C采用60℃水浴4min，100％灭活。聚乙二醇4000（PEG-4000）作为融合剂，PEG-4000浓度选取30％，处理30min为最佳融合时间。结论：通过最佳条件的确定，后续融合实验的效率得到大幅提升，最终筛选出了高效发酵的啤酒酵母菌株，发酵速度较融合亲本提高了108％，双乙酰含量降低了60.7％。

Exploring the optimum conditions of the protoplast fusion which applied to the key steps ofSacchararomyces cerevisiae. With Sacchararomyces cerevisiae b and c as the tested material, we explored the effects of several factors including enzyme concentration, hydrolysis time, inactivation conditions, fusion time on protoplast fusion. The results show that the ratios of preparation and generation arrives the best when the temperature of enzymolys is 25℃, enzymatic hydrolysis time is 60 min, concentration of enzyme solution is 2%. Sacchararomyces cerevisiae b is inactivated completely under the condition that 15W UV lamp irradiated for 2min, 30cm. Sacchararomyces cerevisiae c is inactivated completely under the condition that 60℃ wafer bath works for 4 min. Polyethylene glycol 4000 （PEG - 4000） is fusion agents, 30% PEG - 4000 was selected, 40 min is the best fusion processing time. Conclusion： through the determination of optimum condition, experiment got a lot of improvement in the efficiency of the subsequent fusion. In the end, the efficient fermentation Sacchararomyces cerevisiae strain is selected： fermentation speed increased by 108% than fusion parents, content of diacetyl reduced by 60.7%.