Screening Identification And Characteristics Research On Superior MLF Lactic Acid Bacteria In Wine

Malolactic fermentation (MLF)，which usually refers to secondary fermentation，isa very important fermentation process of wine-brewing，especially for high-quality redwine．Whether MLF can be successfully started and accomplished by wine is directlyaffected by the fermentation bacteria and its environment in which alcohol content，SO2concentration，pH and temperature can directly affect the growth of lactic acid bacteria sothey can affect the malolactic fermentation of wine.In this article，fourteen strains MLF lactic acid bacteria with stronger ability toproduce lactic acid were isolated from natural fermented grape wine by using strain L450as reference strain，and excellent MLF lactic acid bacteria which with four superiortolerance characters for high alcohol concentration，high sulfur dioxide concentration，low acid concentration and low temperature．On the basis of fermentation performance，We identified these excellent strains from the features in physiological and biochemicalcharacteristics and16S rDNA molecular sequencing，and then systematically studied theoptimum growth temperature，the optimum pH and growth curve of them．This studyaims to provide new strains for wine brewing and fermentation of the lactic acidfermentation fruit juice，and to provide the theoretical basis and technical support for thecommercialization of lactic acid bacteria．The details are as follows:In the laboratory，we simulated the environment of wine natural fermentation，usingpaper chromatography technology to determine the start time of wine MLF and took thewine samples that already started MLF to isolate and culture．By comparing theseparation effect of different culture medium，we find that the separation effect of ATBmedium added sorbic acid is better than that of MRS medium added Calcium carbonateor MRS medium added nystatin．The addition of sorbic acid in the ATB medium is1.5grams per liter．It can effectively inhibit the growth of yeast，mold and part of the aerobicbacteria．Compared to conventional separation medium of lactic acid bacteria culture，theATB medium with sorbic acid significantly inhibits interference in fungal and bacterialand adopts anaerobic culture at the same time，making the separation efficiency is moreefficient and targeted.Lactic acid bacteria that can grow normally in good condition under the condition of13%alcohol content，130milligrams per liter sulfur dioxide concentration，pH3.0and 17℃w ere isolatedby using natural fermented wine as Bacteria source and using livingbacteria number as major index and using strain L450as Reference strain，and finally weidentified excellent strains G23andL42with comprehensive strongest tolerance asmalolactic fermentation (MLF) Lactic acid bacteria．We identified these excellent strainsfrom the features physiological and biochemical characteristics and16S rDNA molecularsequencing，the results showed that both of them were identified as Lactobacillusplantarum，respectively named Lactobacillus G23and L42．Breeding superior tolerancecharacters strains G23and L42can effectively breakthrough the technical bottleneck toregular tolerance of alcohol content，sulfur dioxide concentration，acid concentration andtemperature．So the technical problems of conducting MLF at lower sulfur dioxideconcentration and high temperatures cause the wine browning and bacteria multiply canbe solved.Muscat grapes and merlot grapes were used to ferment，by using the naturalfermented wine without adding strains as the reference and using MLF startup time，acidity，the wine chromaticity，fermentation aroma，taste and evaluation score as theresearch index．We can get the following conclusion：superior strains G23and L42canshorten the start-up time of MLF，reduce the acidity of the wine，reduce the winechromaticity values with a small degree，and enhance fruity fermentation，and withoutsour taste．G23and L42with the high score of wine comprehensive evaluation indicatedthat the strain has superior fermentation characteristics and ensure the quality of winefermentation strains and create the preconditions for putting the plant lactobacillus G23and L42into production.The optimum growing temperature and pH of G23and L42were identified bymeasuring the number of living bacterium at different temperatures and pH．At the sametime，we measured the change of OD and pH of G23and L42within0~36h L42anddrew a growth curve．The results showed that the most suitable growing temperature is37℃，the pH was6.0，and the best time to harvest their cells is during15~18h ofculturing G23and L42under suitable conditions.