Preliminary Study On Quorum Sensing Signaling Molecule 2-Phenylethanol In Non-Saccharomyces Cerevisiae And Sequential Fermentation

Quorum Quorum sensing is a widely cell-cell communication mode in the microbial community.Many fungi communicate through quorum sensing molecules（QSM）to communicate and coordinate their collective behavior.Saccharomyces cerevisiae uses quorum sensing molecules such as 2-phenylethanol to perceive changes in nitrogen sources and other metabolites in the fermentation broth,and regulates the expression of corresponding genes through transduction mechanisms,which lead to the changes in metabolic pathways of yeast,ultimately affecting the sensory quality of wine.Studies on Quorum Sensing and its Mechanism in Saccharomyces cerevisiae is clear.Non-Saccharomyces cerevisiae is evolutionarily homologous with Saccharomyces cerevisiae and in the same ecological niche.whether non-Saccharomyces cerevisiae has quorum sensing phenomenon,can it synthesize 2-phenylethanol and use 2-phenylethanol to mediate the interaction between Saccharomyces cerevisiae and non-Saccharomyces cerevisiae is still unclear.It has been found that some non-Saccharomyces cerevisiae can synthesize 2-phenylethanol.There are also non-Saccharomyces cerevisiae that do not detect any known quorum sensing molecules.Different strains of non-Saccharomyces cerevisiae have different types and synthetic quantities of quorum sensing signaling molecules,which showed species-specific characteristics.Therefore,to screen strains that can synthesize 2-phenylethanol from local non-Saccharomyces cerevisiae and Further study on their synthesis pattern and effect of 2-phenylethyl and its influence on the interaction between Saccharomyces cerevisiae and non-Saccharomyces cerevisiae will provide theoretical basis for controlling and optimizing mixed bacterial fermentation by regulating quorum sensing molecules in the future1、In this study,yeast was isolated from grape surface and natural fermented juice by plate marking method.Twenty-nine typical yeasts were isolated and purified by morphological characteristics,and 26 strains were identified as non-Saccharomyces cerevisiae by WL medium.By 26S r DNA sequencing and homologous sequence alignment analysis,the obtained non-Saccharomyces cerevisiae strains were found belonging to 4 genera.namely,Saccharomyces Hansoniana,Saccharomyces Pichia,Saccharomyces cerevisiae,and Saccharomyces Meiqi;The strains related to quorum sensing were further screened by using restricted nitrogen source plate to determine and evaluate the brewing factor tolerance of the strains.The results showed that Mp-57 and H1 were tolerant to glucose and SO₂;Mp-57 had poor tolerance to alcohol;Mp-57 and H1 grow well at 20-30;The two strains had certain acid resistance.2、The quorum sensing phenomenon of the two target strains was verified by dilution inoculation effect experiment,condition culture and study on the effect of exogenous addition of 2-phenylethanol on pseudohyphae formation of the target strains;The gas chromatography analysis showed that the target strain Mp-57 and H1 could synthesize 2-phenylethanol;Further study on the synthesis of 2-phenylethanol in the target strain revealed that the initial synthesis of 2-phenylethanol was cell density dependent,and the synthesis rate of 2-phenylethanol increased with the extension of culture time,reached the maximum value at the end of logarithmic stage,and then decreased rapidly.The change curve of 2-phenylethanol synthesis rate showed a bell shape.We also investigated the effects of exogenous addition of 2-phenylethanol on fermentation-related traits of Mp-57 and Ds strains,and found that 2-phenylethanol was similar to signal molecules,which affected the growth,biofilm formation and ethanol tolerance in a dose-dependent manner.3、The target strain Mp-57 was applied to sequential inoculation fermentation.In order to clarify the interaction between Mp-57 and Saccharomyces cerevisiae,the changes of ethanol,glycerol synthesis and reducing sugar consumption over time were monitored.It was found that the synthesis trend of 2-phenylethanol was the same in Ds pure fermentation and Mp-57/Ds sequential fermentation broth.The total amount of 2-phenylethanol synthesis in sequential fermentation broth was significantly higher than that of Ds pure fermentation;The results of fluorescence quantitative PCR showed that the expression level of ARO9 and ARO10 invovled in the synthesis of 2-phenylethy of Saccharomyces cerevisiae was significantly up-regulated at 72 hours.and the expression trends of ARO9 and ARO10 genes in Saccharomyces cerevisiae were the same;The monitoring results of yeast growth dynamics and sugar consumption showed that the presence of the target strain Mp-57 affects the growth of Ds cells,The Ds pure fermentation rate was greater than that of the sequential inoculation fermentation;The results of metabolite analysis showed that the glycerol yield was higher in the Mp-57/Ds mixedculture fermentation,but the ethanol content was significantly lower than that of Ds pure fermentation.