The Primary Exploration Of Inhibitory Mechanism Of Saccharomyces Cerevisiae On Growth Of Non-saccharomyces

Non-Saccharomces such as Hanseniaspora uvarum and Hanseniaspora guilliermondii can be used to ferment wine with Saccharomyces cerevisiae to enhance wine flavor.However,the growth of non-Saccharomces are strongly inhibited by S.cerevisiae during mixed fermentation which will limit the application of non-Saccharomces in wine fermentation.Therefore,it is very important to study the inhibition molecular mechanism of non-Saccharomces from S.cerevisiae.More and more researches show that the inhibition factors of non-Saccharomces from S.cerevisiae are antimicrobial peptides(AMPs)from GAPDH in S.cerevisiae,and the producing of AMPs may be related to the changing of transcription,expression and aggregation of GAPDH.Moreover,the interaction between two yeasts can enhance stress reaction and oxidation pressure which cause the changing of transcription and expression level and aggregation of GAPDH S.cerevisiae.In view of these,firstly,a fermentation device was developed which can separate two yeasts and was used to study the interaction between two yeasts.Then,the dynamic changing of ROS in S.cerevisiae was studied by fluorescent probe method and the changing of transcription and expression level and aggregation of GAPDH in S.cerevisiae was analyzed by RTFQ PCR,SDS-PAGE and Western-blot during mixed fermentation with H.uvarum and H.guilliermondii.All the result will indicated the inhibition molecular mechanism of non-Saccharomces from S.cerevisiae.Mainly research findings as follows.1 Development of separated fermentation deviceIn order to analyze inhibition mechanism of non-Saccharomces from S.cerevisiae.Fistly,the transmission on antimicrobial peptides and yeast cells were analyzed,and a fermentation device fitting for studying the interaction between two yeasts has been deveploped.Results showed that the growth of H.uvarum and H.guilliermondii was inhibited during separated fermentation with S.cerevisiae,but its inhibitory effect of S.cerevisiae to H.uvarum and H.guilliermondii was weaker than that of mixed fermentation.Moreover,the inhibition effect of S.cerevisiae was enhanced with the increasing cut-off molecular weight of Dialysis tube.This separation fermentation device can be used in learning the interaction between S.cerevisiae and non-Saccharomces.2 Analysis of oxidative stress in S.cerevisiae in separated fermentationIn order to study whether the stress reaction will occurre or not in S.cerevisiae during separation fermentation with H.uvaruma and H.guilliermondii.Firstly the detection condition of fluorescence probe was optimized in ROS analysis.The changing of ROS in S.cerevisiae was analyzed with the changing of fermentation time and the cut-off molecular weight of dialysis tube during separated fermentation.Additionally,activity of SOD,POD and CAT in S.cerevisiae was analyzed during separated fermentation.Results showed that 30 min incubation and 20?M DCFH₂-DA was the optimum detected condition.The ROS increasing in S.cerevisiae happened and reached maximum on 3^th d during separation fermentation.The activity of SOD,POD and CAT of S.cerevisiae in separated fermentation developed by 300 k dialysis tube is higher than that in single fermentation.During single fermentation,activity of SOD,POD and CAT of S.cerevisiae were 94.23?109.41 U/10⁸CFU×m L^-1,34.65?46.80U/10⁸CFU×m L^-1,11.28?23.71 U/10⁸CFU×m L^-1,respectively.During separated fermentation with H.uvarum,activity of SOD,POD and CAT of S.cerevisiae were121.46?134.20 U/10⁸CFU×m L^-1,36.63?48.72 U/10⁸CFU×m L^-1,36.06?49.60U/10⁸CFU×m L^-1,respectively.During separated fermentation with H.guilliermondii,activity of SOD,POD and CAT of S.cerevisiae were 93.08?128.15 U/10⁸CFU×m L^-1,36.06?49.60 U/10⁸CFU×m L^-1,17.25?26.34 U/10⁸CFU×m L^-1,respectively.During S.cerevisiae inhibits the growth of non-Saccharomyces,oxidative stress of S.cerevisiae is increased,and activity of enzyme related to ROS eliminate is increased,too.3 Analysis of GAPDH in S.cerevisiae in separated fermentationIn order to study the changing of GAPDH in S.cerevisiae during separated fermentation with H.uvaruma and H.guilliermondii,the changing of transcription,expression and aggregation of GAPDH in S.cerevisiae was analyzed by RTFQ-PCR,SDS-PAGE and Western-blot,respectively.Results showed that the transcription level of GAPDH coding gene was remarkablely increased in S.cerevisiae separating fermentation with H.uvarum and H.guilliermondii and reached maximum value on 3^thd and 2^th d,respectively,which was 8 times and 7 times higher than that in S.cerevisiae during single fermentation.At the same time,the expression of was not changed,but12 k Da protein was produced,70 k Da and 80 k Da protein was disappeared.Additionally,the expression level of GAPDH was increased on the 3^th d and GAPDH was polymerized.However,the expression level of GAPDH obtained from SDS-PAGE and Western-Blot existed difference which should be studied furtherly.