酿酒酵母拮抗作用的蛋白组分析

Proteomic analysis on the antagonism of Saccharomyces Cerevisiae

为研究混菌发酵过程中酿酒酵母胞外蛋白对克鲁维酵母的拮抗作用并初步分离毒蛋白,采用超滤法提取酿酒酵母纯培养及透析培养的胞外蛋白,分别加入克鲁维酵母纯培养液中,通过平板菌落计数法,动态观测酿酒酵母胞外蛋白对克鲁维酵母生长的影响;双向电泳结合PDQuest软件分离解析酿酒酵母两种培养条件下的胞外蛋白图谱。实验结果表明,纯培养下的酿酒酵母胞外蛋白对克鲁维酵母无拮抗作用,而透析培养得到的酿酒酵母胞外蛋白可以导致克鲁维酵母提前死亡;纯培养图谱共79个蛋白点;透析培养图谱共109个蛋白点,比纯培养的新增30个蛋白点。由此推断:酿酒酵母可以分泌导致克鲁维酵母提前死亡的分子量大于10ku的蛋白类毒素,并且这种毒素的分泌不是自发的,而是在克鲁维酵母分子量小于10ku分泌物的诱导作用下才会产生。这30个新增蛋白点包括对克鲁维酵母的拮抗蛋白。

To study the antagonism of Saccharomyces Cerevisiae＇s extracellular proteins against Kluyveromyces Thermotolerans in the mixed culture,and to preliminarily separate the toxic proteins,extracellular proteins of Saccharomyces Cerevisiae from single culture and dialysis tube fermentation were extracted by ultrafiltration,added into the single culture of Kluyveromyces Thermotolerans respectively,for further population dynamic analysis of Kluyveromyces Thermotolerans under the influence of Saccharomyces Cerevisiae extracellular proteins by plate count method.The extracellular proteins of Saccharomyces Cerevisiae from the two cultures were seperated and analyzed using two-dimensional electrophoresis combined with PDQuest software.Statistics showed that extracellular proteins of Saccharomyces Cerevisiae from single culture displayed no antagonistic action on Kluyveromyces Thermotolerans,while those extracted from the dialysis tube fermentation induced the early death Kluyveromyces Thermotolerans.79 spots were detected on the single culture profile while 109 spots were found on the dialysis tube fermentation one.By comparison,30 new spots appeared on the dialysis tube fermentation image.In conclusion Saccharomyces Cerevisiae could secrete high molecular weight（10ku） proteins that could be toxic to Kluyveromyces Thermotolerans.Moreover,the secretion of toxic proteins was not spontaneous,but by the induction of secretion smaller than 10ku from non-Saccharomyces Cerevisiae.The antagonistic proteins were included within the 30 new spots.