Superior MLF Lactic Acid Bacteria Selective Breeding And Enzyme Production Characteristics Research And Application

A superior malolactic fementation(MLF) lactic acid bacterium was obtained byselective breeding from natural fermented loquat wine, which with high sulfur tolerance,high MLE activity and excellent MLF ability.The growth characteristics, culture mediumoptimization, productive conditions and enzymology characteristics of malolactic enzyme(MLE) of selected lactic acid bacterium were studied, the deacidification dynamics ofloquat wine, the influence on wine quality and application in loquat juice with thebacteria were also studied. This study will provide a superior malolactic fementation lacticacid bacterium for fruit wine making or lactic acid fermentation juice, provie a theoreticalbasis and technical support for the commercial application and immobilisation of MLE ofthis lactic acid bacteriym.The results of this study were shown as follows:(1) Study on suprior lactic bacteria selective breeding.A supeior malolactic fementation(MLF) lactic acid bacterium was obtained byselective breeding from natural fermented loquat wine, which with sulfur dioxide toleranceability as the main investigation index, combined with alcohol, acid, low temperaturetolerance ability, MLF ability. The selected lactic acid bacteria was excellent with highertolerance of sulfur dioxide to120mg/L and alcohol to13%(v/v), it could keep growingwell under pH3.0.The lactic acid bacterium was identificated as Lactobacillus plantarumby the China Center of Industrial Cuture Collection(CICC), and was named Lactobacillusplantarum R23. Gene sequences of Lactobacillus plantarum R23had been set up a fileupload to the U.S. National Center for Biotechnology Information(NCBI) dater bank,which GenBank number was HQ658056.It had brokenthrough the conventional lactic acid bacteria resistant to low sulfurdioxide ability of the technical bottleneck with selective breeding of Lactobacillusplantarum R23with high resistance to the sulfur dioxide, and it solved the technicaldifficult problem that it will lead to wine browning and miscellaneous bacteria growthingunder low sulfur dioxide environment to MLF biological deacidification.(2) Study on growth charicteristics and culture medium optimization ofLactobacillus plantarum R23.The suitable growth temperature, pH value, growth curve and culture mediumoptimization of Lactobacillus plantarum R23were studied, the results showed thatLactobacillus plantarum R23was gram-positive bacteria, facultative anaerobic, the most suitable growth temperature was30℃and pH value was6.0, the cells harvest time wascultured21to24hours under the suitable growth condition. Culture medium of LH16without Mn2+was selected for the suitable growth of Lactobacillus plantarum R23. Byincubating the Lactobacillus plantarum R23in medium LH16at30℃for24hours, the celldensities were up to3.40×109cfu per mL, which was twice than incubated by MRS mediumwithout Mn2+. It could provide research foundation of high density culture, high securityDVS bacterium agent research and application with the study on growth charicteristics andnon-Mn+culture medium optimization of Lactobacillus plantarum R23.(3) Study on produing MLE conditions and enzymology characteristics ofLactobacillus plantarum R23.The produing MLE charicteristics during growth and effecting factors on produingMLE of Lactobacillus plantarum R23were studied, the results indicated that thecontent and activity of malolactic enzyme increased with increasing of bacteria biomassat logarithmic phage, enzyme activity reach a high value during4-8hours of stationaryphase; It showed positive correlation at bacteria growth early phage between thecontent and activity of malolactic enzyme, but showed negative correlation at laterphage.A moderate amount of L-malic acid or NAD+or anaerobic fermentation couldincrease enzyme production and activity.Response surface methodology was used to optimize conditions of MLE productionon the basis of pre-experiments, and a second order quadratic equation was finallyobtained.Results showed that the effecting order of three factors to the total activity of MLEwas as follows: concentration of L-malic acid＞fermentation temperature＞pH value.Theoptimized conditions were fermentation temperature33.5℃, pH value6.2, L-malic acidconcentration3.8g/L.Under the optiimal conditions, MLE production of Lactobacillusplantarum R23with anaerobic fermentation28hours was1.34mg/mL, MLE activity andthe total activity was up to462.33u and619.52U respectively.The optimum conditions of enzymatic reaction for Lactobacillus plantarum R23wereunder pH6.0and temperature at24-27℃. L-latic and SO₂showed against the enzymaticreaction of MLE. However, NAD⁺and Mn2+showed positive effects on the enzymereaction, which proper added conentration were1.0mM and100μM respectively.TheMichaelis equation of enzymatic reaction of MLE was:1/v=0.0087×1/[s]+0.0023(R²=0.9936), and the Km was3.78×10^-3mM. (4)Study on application in loquat wine and lactic acid fermented juice ofLactobacillus plantarum R23.Based on quadratic orthogonal design with the deacided index (Y₁) as target,inoculation amount(X₁),alcohol degree(X₂),total sulfur dioxide concentration (X₃)and fermented temperature(X₄)as effecting factors, the mathematical model of MLF byLactobacillus plantarum R23in loquat wine was established:Y₁=83.5168+9.4207X₁-1.8707X₂-2.4874X₃+1.9457X₄。The model had a high fitting degree,It could be able to accurately reflect the relationship between the various influencing factorsand deaciding rate.Malolactic fermentation with Lactobacillus plantarum R23in loquatwine could reduce the total acid,increase the soft degree,change the volatile compositionand contents, and it could improve the loquat wine quality.The relations between growth and acid yield of Lactobacillus plantarum R23inloquat juice was studied, as well as the organic acid and sugar metabolism were alsostudied, the results showed that Lactobacillus plantarum R23take malic acid as the maincarbon source to MLF in loquat juice fermentation before24hours.It was non-growthcoupling model for fermenting to produce acid; It was abnormity lactic acid fementationwith sugar metabolism afer MLF. This study expanded the application fields of lacticacid bacteria, and it could provide research foundations for the development of lacticacid fermented fruit juice.