| In the process of wine Malolactic Fermentation(MLF),lactic acid bacteria can degrade malic acid to produce lactic acid and improve the quality of wine,among which the most important lactic acid bacteria is Oenococcus oeni,which is an important starter of maliclactic acid fermentation.With the study of O.oeni,it was found that O.oeni grows in low p H value,high ethanol and other harsh environments that are not conducive to microbial growth.Of course,this harsh environment will greatly reduce the fermentation speed and fermentation capacity of O.oeni.Therefore,it is particularly important to improve the stress resistance of Oidium.A large number of previous studies have shown that heat shock proteins(HSP)in organisms can prevent abnormal protein folding and aggregation under stress conditions,which can improve the tolerance of microorganisms to adverse conditions.However,under stress conditions,the functions of heat shock proteins from different sources are quite different,and their effects on microbial resistance are also different.Therefore,in this study,two heat shock protein genes from different sources were cloned.First,the ethanol intolerant Lactococcus lactis was used as the host for heterologous expression,and the Hsp with the greatest influence on ethanol tolerance were screened by comparing the growth of recombinant bacteria in different ethanol content media Then,the optimized expression vector was used to transform the selected heat shock protein gene into O.oeni,and the ethanol tolerance of the transformed strain and its effect on the fermentation of O.oeni were studied.Findings were as follows:1.OoHsp18 and Lp Hsp19.5 gene cloning and L.lactis MG1363/p MG36e-OoHsp18 and L.lactis MG1363/p MG36e-Lp Hsp19.5 expression strains build.Two different heat shock protein genes OoHsp18 and Lp Hsp19.5,were cloned from the genomic DNA of O.oeni and Lactobacillus plantarum by PCR,respectively.OoHsp18 and Lp Hsp19.5 were ligated with expression vector p MG36 e by homologous recombinase and transformed into Escherichia coli Fast-T1.Positive strains were selected for verification.The two successfully verified plasmids p MG36e-OoHsp18 and p MG36e-Lp Hsp19.5 were electrically transformed into L.lactis MG1363.Recombinant L.lactis MG1363/p MG36e-OoHsp18 and L.lactis MG1363/p MG36e-Lp Hsp19.5 were constructed.2.Determination of p H and ethanol tolerance of recombinant L.lactis MG1363/p MG36e-OoHsp18 and L.lactis MG1363/p MG36e-Lp Hsp19.5 strains.The wild-type strain was used as the control group,and the two recombinant strains were used as the experimental group.The growth curves of the wild-type strain and the two recombinant strains were measured under different p H and ethanol concentrations.Results the wild type strains and recombinant strains fit to grow in the p H7 neutral conditions.Lp Hsp19.5 gene and OoHsp18 gene had little effect on the growth of L.lactis under different p H conditions.Although there was no significant difference in growth between the wild-type strain and the two recombinant strains in the absence of ethanol,the growth of the Lp Hsp19.5 expressing strain was better than that of the OoHsp18 expressing strain under ethanol stress.Furthermore,when the ethanol content was 6.0%,the relative conductivity of the wild-type strain and OoHsp18 expression strain was higher than that of the Lp Hsp19.5 strain.Therefore,we speculated that expression of Lp Hsp19.5 conferred better ethanol resistance in the strain.3.Optimization of expression vector and construction of Lp Hsp19.5 overexpression of O.oeni.The promoter of the expression vector p HH60 constructed previously in our laboratory was optimized to increase the expression of downstream exogenous genes.The results showed that the green fluorescence signal of the downstream reporter gene GFP was obviously displayed in the transformed strain of O.oeni,while the corresponding fluorescence signal was almost not observed in the transgenic strain of p HH60,indicating that the p HH40 expression vector indeed improved the expression of foreign genes in O.oeni.It can increase the influence of foreign genes on the corresponding traits of O.oeni.Subsequently,the p HH40 vector was used to recombinate with Lp Hsp19.5,which was more tolerant to ethanol of the strain,and transformed into E.coli Fast-T1.The recombinant plasmid p HH40-Lp Hsp19.5 was extracted from the successfully verified positive bacteria and transformed into O.oeni by electroporation.The positive transformation strain O.oeni/p HH40-Lp Hsp19.5 was obtained after verification.4.Ethanol tolerance of the recombinant strain O.oeni/p HH40-Lp Hsp19.5 was determined.The growth curves and relative conductivity of the wild-type strain and the recombinant strain were determined under different ethanol concentrations.The results showed that the growth of the wild type strain decreased when the ethanol content was12%,but the recombinant strain still had a high growth rate.In addition,the relative conductivity of the wild-type strain was higher than that of the Lp Hsp19.5 strain when the ethanol content was 10%.These results indicated that Lp Hsp19.5 gene could significantly improve the ethanol resistance of the strain.5.The O.oeni/p HH40-Lp Hsp19.5 strain was used for dry white wine and dry red wine apple milk fermentation.O.oeni/p HH40-Lp Hsp19.5 was used as experimental group and wild type O.oeni was used as control group to produce dry white wine(9.5(%v/v)alcohol)and dry red wine(10.2(%v/v)alcohol),respectively.The results showed that the two strains had the same utilization rate and malic acid conversion rate to dry white,and had no effect on the alcohol accuracy of dry white,and had the same performance in dry red.The contents of malic acid and citric acid in dry white and dry red wines fermented by O.oeni/p HH40-Lp Hsp19.5 strain decreased more rapidly,while the content of lactic acid increased.At the same time,compared with the wild type strain,the recombinant strain had little effect on the total amount of final esters in dry white and dry red.In conclusion,Lp Hsp19.5 from L.plantarum,as compared with OoHsp18 from O.oeni,improved the ethanol tolerance of transformed L.lactis probably by protecting the plasma membrane to maintain its stability.Subsequently,Lp Hsp19.5 gene was transformed into O.oeni by the optimized expression vector p HH40,which could improve the ethanol tolerance of the transformed strain by 20%.In addition,through the apple milk dry white wine and red wine fermentation experiment,shows that the transformation strain fermentation of wine has the acceleration. |
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