| Malolactic acid fermentation(MLF) takes place after alcoholic fermentation(AF)and consists of the decarboxylation of malic acid into lactic acid, producing a reduction in total acidity of the wine. As a consequence, the organoleptic quality and the microbiological stability of wine are improved. Oenococcus oeni is the most widespread used lactic acid bacterium in triggering and completing MLF. However, it is hard to start the MLF because of nutrient deprivation, high ethanol concentration,and low pH after AF. The high acid environment is mainly caused by malic acid,tartaric acid, citric acid, etc. in raw material. Whereas, winemakers always solved by adding calcium carbonate or potassium bicarbonate to accomplish dropping acid.However, such modusoperandi directly reduce the resistance to disease, lower the quality of the wine. As a result, a better screening acid stress strain, and an understanding of the acid resistance gene possess of O. oeni to survive in low-pH environment is thus of great importance.In this study, three O. oeni screened from Shaanxi province was used as parental strains in ion implantation mutagenesis, then the artificial mutagenesis strains were direct transfer to the stress environment(ATB pH 3.0 / pH 9.0). The acid-resistant ability of all mutant strains and corresponding parental strains were determined in pH3.0 ATB medium, growth curves were draw according the OD600 values. Winemaking characteristics(beta-glycosidase activity and L-malic acid consumption) were also detected. Six mutans with discrepant tolerance phenotypic were selected for de novo,and the sequencing data were analysis by genome-wide-association method in order to identify targets of acid tolerance genes in O. oeni. The main results are as follows:(1) O. oeni SX-1a, SX-1b and CS-1b were selected from 40 O. oeni strains,conserved in our College, as parental strains for ion implantation mutagenesis. After ion implantation mutagenesis, stress environment training, purification and again stress environment training, we final have obtained 10 mutant strains in total. Mutants screened from pH 3.0 acid stress environment including: one SX-1a mutant, twoSX-1b mutants, two CS-1b mutants; mutants screened from pH 9.0 alkaline stress conditions: two SX-1a mutants, one SX-1b mutant, two CS-1b mutants. In addition,each continuous cultivation were more than 12 generations(N≥12), to ensure stable property of each obtained mutant. Besides, the original strain SX-1a, SX-1b, CS-1b have lost their growth abilities after 3-5 continuous cultuvation under pH 3.0 and pH9.0 stress environment.(2) Growth profiles(OD600) of all mutants and parental strains under pH 3.0showed that, the activities of all acid-resistant mutants(mutants screening from pH3.0 ATB medium) were much higher than the starting strains; and the activities of all acid-sensitive mutants(mutants screened from pH 9.0 ATB medium) were lower than the original strains.(3) Measurement of β-glucosidase activities of all mutants and parental strains also showed that, the enzyme activities of acid-tolerance mutants were much higher than the original strains, and enzyme activities of acid-sensitive mutants were lower than the original strains.(4) Results of the L-malic acid consumptiom test shown that, all mutations preserved MLF ability. Besides, the L-malic acid consumption abilities of acid-tolerance mtants were stronger than that of acid-sensitive mutants.(5) GWAS statistics show that a total of 3916 SNP significantly correlated with acid-resistance property. Besides, those SNPs are located within 181 genes in O.oeni.GO and KEGG annotations results showed that, most of these genes paly a fundamental or most important function in microorganisms. |
PDF Full Text Request