| Malolactic fermentation(MLF)involved in the deacidification process is a recommended secondary fermentation of wines,especially red wines,because of its positive impact on wine stability and sensorial quality.Oenococcus oeni(O.oeni)is commonly used for the production of MLF starter because of its high resistance to acid and ethanol stresses.As a moderate drying technique,freeze-drying is widely applied in the preparation of MLF starters.Nevertheless,freeze-dried O.oeni starter culture during the manufacture is under the influences of freezing,drying,concentration stress and oxidative stress,which affect the physiological status and bacterial survival.So far,studies of O.oeni starter mainly focused on the preparation of starter culture and changes of physiological and biochemical status during the preparation process,but the mechanisms involved in the resistance of O.oeni to freeze-drying were rarely reported.Studies have shown that stress pretreatments could produce the cross-protective effects to enhance the activity of freeze-dried O.oeni.However,the mechanism involved is still unclear.Therefore,it is important to explore the mechanism of acid and ethanol acclimation on the resistance of O.oeni to freeze-drying.In this study,O.oeni SD-2a was used to explore the effects of acid and ethanol acclimation on the resistance of O.oeni to freeze-drying.It has been found that acid and ethanol acclimation could improve the viability of freeze-dried O.oeni.Based on this result,cell morphology,proteomics,metabolomics and gene expression analysis were applied to investigate and discuss the involved mechanisms.The main contents and results of this research were as follows:(1)The adaptive response of O.oeni SD-2a to acid and ethanol stressThe effects of acid and ethanol stress on the damage and membrane potential of O.oeni SD-2a were determined using fluorescence dyes.And the changes of intracellular ATP content and glutathione content of O.oeni SD-2a with acid or ethanol stresses were detected using kit.The results showed that acid(p H 3.2)and ethanol(8%,12%)stresses both caused the damage of cell membrane and decreased cellular ATP concentration.At the same time,acid stress increased the uptake of glutathione,while ethanol stress led to cell depolarization.Moreover,transcriptional analysis was applied to discover the key genes involved in stress tolerance.The RT-PCR results showed that the expression of stress relevant genes(hsp20,clp P,trx A,cts R,Rec O,Usp A)increased highly with ethanol and acid stress treatments.(2)Proteomic analysis insights on the response of O.oeni SD-2a to freeze-drying stressIn this work,the two-dimensional gel electrophoresis maps of O.oeni SD-2a without freeze-drying(No-FD),with freeze-drying(FD)and freeze-drying with monosodium glutamate(MSG)(FD-MSG)were established.In the basis of spot-to-spot comparison and statistical analysis,101 spots were differentially expressed among No-FD,FD and FD-MSG cells.And a total of 93 proteins were successfully identified by MALDI-TOF/TOF MS among 101 differential abundance proteins(DAPs)to analyze the relationship between DEPs and freeze-drying process.Bioinformatics analysis revealed that varying proteins were mainly involved in carbon,lipid and nucleic acids metabolism,stress response,oxidoreductase activity and signal sensing.Among the identified proteins,the highlighted proteins were those involved in polysaccharides production and quorum sensing for freeze-drying resistance and cyclopropane fatty acid metabolism for MSG addition.(3)Analysis of proteomic responses of freeze-dried O.oeni to access the molecular mechanism of acid acclimation on cell freeze-drying resistanceIn order to study the relationship between freeze-dried O.oeni SD-2a proteome and acid acclimation pretreatments,two-dimensional gel electrophoresis maps of acid acclimated freeze-dried O.oeni SD-2a were established.Totally 83 protein spots were analyzed with statistically significant(P-value < 0.05)and 82 protein spots were successfully identified using MALDI-TOF/TOF-MS-based analysis.Bioinformatics analysis indicated that DAPs were involved in carbohydrate metabolism process,amino acid metabolism and transport,quorum sensing and bacterial secretion system and stress response.KEGG analysis showed that different abundance proteins were notably enriched in carbohydrate metabolism process,especially amino sugar and nucleotide sugar metabolism.The key linkers in protein-protein networks were related to carbohydrate metabolism.(4)The regulation mechanisms of ethanol acclimation for freeze-drying resistance of O.oeni SD-2a as revealed by comparative proteomic analysisTwo-dimensional gel electrophoresis(2-DE)maps of ethanol acclimated freeze-dried O.oeni SD-2a were established.Based on the 2-DE map of freeze-dried cells without ethanol acclimation,84 proteins with statistical significance were selected and 82 protein spots were successfully identified using MS/MS analysis.Comparative proteomics combined with bioinformatics revealed the important role of central carbohydrate metabolism,polysaccharides production,cell envelope biosynthesis and stress responses related proteins with ethanol acclimations in freeze-dried O.oeni SD-2a.Moreover,higher content ethanol acclimation(12%)promoted the over-production of cell wall composition related proteins,whereas inhibited the expression of cell membrane biosynthesis and Clp protease associated proteins.(5)Changes of cell membrane and the expression of key genes in freeze-dried O.oeni SD-2a with acid and ethanol acclimationThe surface characteristics of O.oeni SD-2a with different treatments were observed using scanning electron microscopy(SEM).The results showed that the extracellular polymeric substance(EPS)produced by O.oeni SD-2a during freeze-drying process would contribute to the accumulation of cells,and moderate acid and ethanol stress contributed to the production of EPS.Cell damage of freeze-dried O.oeni SD-2a was detected using flow cytometry combined with fluorescence dye.It was shown that acid and ethanol acclimation could improve the cell membrane integrity of lyophilized cells.Analysis of key genes in freeze-dried O.oeni SD-2a with acid and ethanol acclimation showed that the expression of small heat shock protein gene(hsp20)and citric acid lyase gene(cit F)was significantly up-regulated.(6)Metabolomics analysis insights on the response of freeze-dried O.oeni SD-2a with acid and ethanol acclimationBased on UHPLC-Q-TOF/MS,the untargeted metabolomics approach was established for freeze-dried O.oeni SD-2a.The metabolites changes of lyophilized O.oeni without stress treatment(CK)and with acid(A)or ethanol(E)acclimations were evaluated using this approach.The results of PCA and OPLS-DA analysis on O.oeni SD-2a metabolome showed that there was a trend of separation among O.oeni with CK,A,E treatments.Based on multivariate and univariate analysis,acid treatment significantly altered the contents of 10 metabolites in freeze-dried cells and ethanol treatment significantly altered the contents of 22 metabolites in freeze-dried cells.In addition,metabolites of palmitic acid,L-histidine and Phe-Asp may be potential biomarkers for acid acclimation treatment,metabolites of oleic acid,linoleic acid,adenine,cytosin,erucamide,AMP,myristic acid and 2-Oxoadipic acid may be potential biomarkers for ethanol acclimation treatment.All in all,acid and ethanol stress induced the expression of genes related to stress response,which is useful for the stress adaptability of O.oeni SD-2a.On the basis of stress adaptation,O.oeni SD-2a regulated the expression of proteins involved in carbohydrate metabolism,amino acid metabolism,polysaccharide synthesis,quorum sensing,cell membrane biosynthesis,stress response and other pathways during freeze-drying process.Then the content of substances involved in the metabolic pathways of fatty acids,amino acids and nucleotides would be changed.These changes would promote the production of EPS and be helpful for maintaining the integrity of bacterial structure to resist freeze-drying stresses.These results provide theoretical support for the production of active dry powder of O.oeni and would be contribute to reveal the mechanism of O.oeni resistance to environmental stresses. |
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