基于PCR-DGGE的咖啡湿法发酵酵母菌的分离鉴定

Separation and Determination of the Yeasts Based on PCR-DGGE Technologies in Coffee Wet Fermentation

为了更全面系统的反映出咖啡发酵体系中的原始状态,分别对发酵堆的上部、中部、下部以及混合样品(上、中、下的混合)进行采集,并对发酵堆这3部分的温度进行了测定,同时采用了7种培养基,分别对上、中、下以及混合样进行酵母菌的培养、分离、纯化及鉴定。之后通过PCR-DGGE方法确定了酵母的优势菌种。结果发现发酵堆的上部、中部和下部的温差很小,随着发酵的进行,整体变化不大,基本保持在20℃左右,且出现了大量产果胶酶的酵母菌,包括异常毕赤酵母Pichia anomala,毕赤克鲁维酵母P.kluyveri,葡萄汁有孢汉生酵母Hanseniaspora uvarum等。其中,毕赤克鲁维酵母P.kluyveri,葡萄汁有孢汉生酵母H.uvarum为优势菌种,并对其在发酵过程中所起的作用进行了深入剖析,这些酵母菌在咖啡湿法发酵中起到了关键的作用。

In order to reflect a more comprehensively original state about the coffee fermentation processing,the samples were collected from the upper,middle,lower and the mixed of the fermentation pile,respectively.The temperatures of upper,middle and lower of the fermentation pile were measured.All the yeasts were cultivated firstly by seven different culture mediums,then separated,purified and determined.Later the dominant strains of yeasts were determined by PCR-DGGE technologies.Results showed that the changes of the temperatures from the upper,middle and lower of the fermentation pile were not obvious,and kept about at 20℃.A large number of yeasts which could produce pectinase were found in this research,including Pichia anomala,P.kluyveri,Hanseniaspora uvarum etc.Among them,P.kluyveri and H.uvarum were detected as the dominant strains.In the end of this study,the important roles of these yeasts played in the coffee wet fermentation process were analyzed in deeply.