The Stress Adaptive Response Of Oenococcus OeniSD-2a And Its Mechanism

During the process of winemaking,malolactic fermentation (MLF) follows alcoholicfermentation.Different bacteria genera have been reported to carry out MLF in wine.Amongthem,Oenococcus oeni is recognized as the most advantageous and tolerant bacterium.MLFdeacidifies wine and results in a softer feeling in the mouth.MLF also improvesmicrobiological stability and organoleptic characteristics.However,due to very harshenvironmental conditions in the wine for bacterial survival and growth,induction of MLF hasshown failures.There are two reasons for such failures.On one hand,strains lose their naturaladaptation to survive and growth in wine,which results in low inoculation viability;On theother hand,lyophilization offers stress conditions such as freezing,drying,and concentrationstress,which diminish cell viability.Therefore,expanding interest in freeze-driedready-to-use malolactic starter cultures has placed more emphasis on developing starterproduction and preservation methods that promote high cell viability and activity.O.oeniSD-2a isolated by our lab from spontaneous MLF wine of YanTai area,is a highlystress adaptive strain.It is of theory and practical importanc e to study on O.oeniSD-2a stressadaptive response mechanism and develop ready-to-use malolactic starter cultures with ourown property right.Using O.oeniSD-2a as experimental strain,we investigated the effect ofmedium composition,acid stress treatment,alcohol stress treatment on MLF activity afterdirectly inoculated into wine-like medium and freeze-drying viability,in order to evaluate thefeasibility of application of stress induced cross protection into preparation of starter cultures,and supply basic experimental dates.In addition,from the point of intracellular MLE activity,H~+-ATPase activity and changes in membrane fatty acid composition,the paper clarifiedO.oeniSD-2a acid and alcohol stress adaptive response and cross protective responsemechanism.Main results were displayed as follows:1.The effect of 3 kinds of culture media was investigated on the direct inoculationviability,freeze-drying viability of O.oeniSD-2a.ATB medium without supplementation ofDL-malate had weak pH buffering capability.Compared with FMATB and MATB,O.oenicells cultured in ATB increased inoculation viability and freeze-drying viability.Though ATBmedium decreased intracellular MLE activity,H+-ATPase activity was the highest among 3kinds of culture media.Concerning the membrane fatty acid composition,it was observed that ATB medium increased distinctly the relative concentration of lactobacillic acid (C19cyc11)and U/S ratio in cell membrane lipid composition of O.oeniSD-2a.The increased resistance towine stressor and freeze-drying is probably a result of the cross protection conferred by selfacid stress response induced in ATB medium,which might be related with changes inmembrane fatty acid composition of O.oeniSD-2a.2.FMATB medium is mixed with glucose,fructose and DL-malate,which makes forbacterial growth and MLF activity.Compared with ATB and MATB medium,the growth rateincreased by 12.1% and 94.5% in FMATB medium,intracellular MLE activity increased by33.9% and 7.1%.3.Acid stress treatment decreased the growth rate of O.oeniSD-2a,but took a certaineffect on cell biomass in FMATB medium.Acid stress treatment increased inoculationviability,and pH3.2 treatment increased by 9 folds compared with control treatment.Thistreatment could successfully initiate and complete MLF.Acid stress treatment all increasedfreeze-drying viability,and the freeze-drying viability of pH3.5 treatment was 103%,whichwas highest survival rate.Therefore,acid stress treatment was an effective technique forpreparation of ready-to-use O.oeniSD-2a starter cultures.pH stress conditions should be setbetween 3.5-3.2.4.Acid stress adaptive response mechanisms of O.oeniSD-2a were studied;The resultsshowed that acid stress treatment increased bacterial intracellular MLE activity andH~+-ATPase activity.Membrane fatty acid composition analysis showed acid stress treatmentdistinctly increased CFAs relative concentration,and pH3.5 and pH3.2 treatment significantlyincreased membrane U/S ratio,which should closely related with enhancement of inoculationviability and freeze-drying viability.Accumulation of CFAs in membrane lips might be oneof basic substance of acid induced cross protection response mechanism.5.Alcohol stress treatment distinctly decreased bacterial growth rate and cell biomass inFMATB medium.Compared with acid stress treatment,alcohol stress treatment had littleimpact on inoculation viability.5% alcohol stress treatment decreased freeze-drying viability,and 10% alcohol stress treatment increased freeze-drying viability.However,as a whole,alcohol stress treatment was not an effective technique for preparation of O.oeniSD-2a startercultures.6.Alcohol stress adaptive response mechanisms of O.oeniSD-2a were studied;Theresults showed that alcohol stress treatment increased bacterial intracellular MLE activity,butgreatly inhibited H~+-ATPase activity,which might be related with high medium pH.Membrane fatty acid composition analysis showed 5% alcohol stress treatment distinctlyincreased SFAs relative concentration,decreased membrane U/S ratio,which might be related with lower freeze-drying viability.But 10% alcohol stress treatment distinctly decreasedSFAs relative concentration,increased UFAs relative concentration,increased membrane U/Sratio,which might be related with enhancement of inoculation viability and freeze-dryingviability.7.In the presence of 10% alcohol stress,medium composition plays an important role ingrowth rate.The results showed malic acid in medium notably increased intracellular MLEactivity,and distinctly increased membrane U/S ratio.It was assumed that MLF pathwaycould be involved in changes in membrane fatty acid profiles,which increased bacterialalcohol tolerance.8.O.oeniSD-2a cells in the early stationary phase survived better after freeze-drying thanthose in the mid-exponential phase.Acid stress conditions and medium compositionsignificantly affect bacterial freeze-drying viability,and interaction between those two factorswas also significant.The freeze-drying viability of O.oeniSD-2a cultured in pH3.5 FMATBmedium was highest.9.The correlation matrix calculated by DPS software showed the freeze-drying viabilityexhibited a significant positive correlations with the levels of C 19cyc11,and had a significantnegative correlations with the levels of C16:0,which indicated C19cyc11,rather than C16:0,could offer more tolerance ability of O.oeniSD-2a cells to freeze-drying.However,fatty acidcomposition and freeze-drying viability of O.oeniSD-2a cells were determined by the mediumpH when cells harvested.There were significant negative correlations between the mediumpH and the levels of C19cyc11,and between the medium pH and freeze-drying viability,suggesting that low growth pH stimulated the synthesis of C19cyc11,which might play animportant role in the resistance of O.oeniSD-2a cells to freeze-drying.10.The innovation of the paper was as follows:(1) it is the first time to study O.oenistress adaptive response mechanism by using a native O.oeni strain.(2) a new idea was putforward that O.oeni acid stress adaptive response could be applied into preparation ofready-to-use malolactic starter cultures,and good experimental results were obtained.(3) Thefreeze-drying tolerance mechanisms of O.oeni were studied for the first time.