高产蛋白酶米曲霉菌株的选育及对酱油风味生成的影响

Breeding Aspergillus oryzae with high-productive protease and its influence on soy sauce fermentation

米曲霉蛋白酶活力对酱油发酵的蛋白质利用率和酱油风味有重要影响。为提高米曲霉突变株的选育效率,以碘化丙啶和荧光素二乙酸酯为染色剂,构建了基于流式细胞术的高通量筛选方法。经过4轮常压室温等离子体诱变,筛选得到蛋白酶活力较出发菌(米曲霉沪酿3.042)提高145.6%的突变株H34。蛋白质电泳、质谱和实时荧光定量PCR分析表明,H34中增加的蛋白酶主要包括酸性蛋白酶(aspergillopepsin-1)、碱性蛋白酶(alkaline protease)和中性蛋白酶(neutral protease 2 homolog AO090001000135)。实验室酱油模拟发酵显示,突变株H34发酵生产的酱油在总氮、氨基酸态氮、有机酸、氨基酸和风味物质成分等方面均优于出发菌。研究结果在为酱油发酵提供了优良菌株的同时,也为其他丝状真菌的诱变选育提供了重要方法学参考。

In order to improve the breeding efficiency of Aspergillus oryzae,a high-throughput screening method based on flow cytometry was constructed.After four rounds of mutation using atmospheric and room temperature plasma,the strain H34 with 145.6%increase of protease activity was screened.The increased protease activities in strain H34 were mainly aspergillopepsin-1,alkaline protease,and neutral protease 2 homolog AO090001000135.The soy sauce produced by H34 strain was superior in total nitrogen,amino acid nitrogen,organic acid,amino acid,and flavor substance.The results provide an excellent strain for soy sauce fermentation and important methodological reference for mutation breeding of other filamentous fungi.