Study On Detection Method Of Killer Activity And Isolation Of Killer Factor From Saccharomyces Cerevisiae

Fruit wine is low-alcohol beverage fermented by using fresh fruits as raw material and yeast as starter culture.In recent years,wine is popular with consumers for its high nutrition and health function.Mixed fermentation between Saccharomyces cerevisiae and non-Saccharomyces of wine is being used to produce wine with improved flavor and mouthfeel.However studies showed that,there may be a killer activity of S.cerevisiae on non-Saccharomyces in mixed fermentation system.Killer activity of S.cerevisiae on non-Saccharomyces will affect the use of non-Saccharomyces in wine fermentation,and lower the contribution of non-Saccharomyces to wine flavor.Therefore,it has significant value to carry out the research of S.cerevisiae killer activity.In view of this,one S.cerevisiae and ten non-Saccharomyces kept in our lab will be used as strains to develop the detection method for killer activity of S.cerevisiae.Then the killer activity of S.cerevisiae on the ten non-Saccharomyces will be analyzed,the killing mechnism of S.cerevisiae on the ten non-Saccharomyces will be preliminary studied.Finally,the killer factor from mixed fermentation supernatant between S.cerevisiae and non-Saccharomyces will be concentrated,isolated and identified.The main results are the following:1 Development of detection method for killer activity of S.cerevisiaeThe feasibility of Oxford cup method,seeding method and filter paper method in studying killer activity of S.cerevisiae were compared according to size and clarity of inhibition zone and difficulty in operation,while detection medium of killer activity containing methylene blue was used.The results indicated that filter paper method was the best method to analyze killer activity of S.cerevisiae.Furtherly,the detection conditions of filter paper method were optimized.The optimum detection methods were the following:200μL 10~7 cells/m L sensitive yeast cells solution was spread onto the killer-sensitive detection medium in advance,after cells solution was totally absorbed,filter paper was put gently onto the killer-sensitive detection medium with sterile tweezers.Then,total 20μL S.cerevisiae fermentation broth was dropped twice onto each filter paper.Finally,the plate was cultured at 28℃for 6 d.2 Analysis of killer acticity of S.cerevisiae on ten non-Saccharomyces strainskiller acticity of S.cerevisiae on ten non-Saccharomyces strains were analyzed by filter paper detection method.The results showed that Barnettozyma californica,Hanseniaspora uvarum,Hanseniaspora occidentalis,Hanseniaspora opuntiae,Pichia kudriavzevii,Candida humilis,Clavispora lusitaniae,Torulaspora delbrueckii were sensitive to S.cerevisiae.H.uvarum,H.occidentalis,H.opuntiae were the most sensitive to S.cerevisiae.3 Preliminary study on producing mechanism of S.cerevisiae killer activitySingle fermentation supernatant of S.cerevisiae at different time had no killer activity,and all the mixed fermentation supernatant at different time had strong killer activity.Therefore,killer activity of S.cerevisiae was not caused by its cells aging or cells density increasing,but caused by cell-cell contact between S.cerevisiae and non-Saccharomyces.4 Isolation and identification of killer factor from S.cerevisiaeIn order to isolate and identify the killer factor from S.cerevisiae,mixed fermentation supernatant between S.cerevisiae and H.uvarum at 5 d was concentrated and ultrafiltrated.Each fraction of ultrafiltrated liquid was analyzed through killer activity and Tricine-SDS-PAGE.The results indicated that killer factor was a low molucular peptide and its molecular weight was located 3～10 k Da.