| Oenococcus oeni is the main lactic acid bacteria in malolactic fermentation(MLF)which is a secondary fermentation.O.oeni can survive in the wine environment with low p H and high ethanol concentration.However,the spontaneous MLF fermentation process often is hard to begin.Previous research found that glutathione(GSH)can enhance the survival ability of O.oeni under acid and ethanol stress.However,the related mechanism remains unknown.Therefore,O.oeni SD-2a was selected as a research object in this study,and the mechanisms of glutathione affecting the O.oeni response to acid and ethanol stress at the cellular and molecular level were investigated.The results showed that the optimal addition amount of GSH was 1500 mg/L.GSH could increase the growth rate and biomass of O.oeni SD-2a under acid stress(p H 3.5,4)and ethanol stress(6%,10% and12%).The mechanism maybe responsible for the reducing the leakage of extracellular ?-galactosidase in acid or ethanol stress environment for O.oeni SD-2a.Thus,the permeability of cell membrane was reduced.The activity of intracellular lactate dehydrogenase was increased by GSH supplement,and that could help to keep the integrity of cell membrane.GSH addition could increase the content of intracellular ATPase and avoid the great change in cell environment so as to relieve the damage on O.oeni by stress.However,GSH had no significant effect on the protein and nucleic acid spillover of O.oeni SD-2a under acid and ethanol stress.This study investigated the expression of cfa,hsp18,cit E,mle A,clp P,clp X,cts R,rml B,rdx1,rdx2,rdx3 and trx A3 genes in O.oeni SD-2a under acid or ethanol stress and control conditions.Under different p H(3.2 and 3.5)and different ethanol(8% and 10%)stress conditions,the expression patterns of these genes were different.GSH could improve the O.oeni tolerance to acid or ethanol.The mechanism maybe responsible for protect O.oeni from acid or ethanol stress by increasing the expression of stress response gene hsp18.The expression level of the cfa and rml B genes were increased by GSH supplement that could help O.oeni synthesized more cyclopropane cell membrane fatty acids,reduce the fluidity of cell membrane,promote the formation of cell wall and maintain the integrity of cell.The mle A and cit E genes expression levels were increased by GSH addition that could rapidly consume malic acid and arginine to cope with the changes of environment.The expression levels of clp P,clp X and the regulatory gene cts R were increased.Therefore,GSH maybe involved in the degrade and clear irreversible damage protein which were produced in the stress response.Moreover,the expression of rdx1,rdx2,rdx3 and trx A3 genes were increased by GSH supplement that could contribute to maintain the cell redox balance.The tolerance of strain to fermentation environment could be improve by pre-adaptation cultivation.Therefore,in this study,O.oeni SD-2a was cultured in acid or ethanol pre-adaptation medium with GSH,and the bacterial cultures were inoculated to the Cabernet Sauvignon wine for MLF.The results showed that the best pre-adaptation medium was FMATB medium with 5 mmol/L GSH and p H was 3.5.Compared with the control,the contents of total phenols,total flavanols,total anthocyanins and the abilities of scavenging DPPH free radicals in the final Cabernet Sauvignon wine which inoculated the pre-adaptation strains were increased.However,there was no significant effect on the procyanidins content,wine color and chroma. |
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