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16S rDNA PCR-RFLP Analysis of Different Oenococcus Oeni Strains

Author: YuDongLiang
Tutor: LiuShuWen
School: Northwest University of Science and Technology
Course: Grapes and wine ,
Keywords: malolactic fermentation Oenococcus oeni specific primers RFLP analysis
CLC: TS261.1
Type: Master's thesis
Year: 2010
Downloads: 124
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Abstract


Recent research showed that Oenococcus oeni is the main lactic acid bacteria which can bear the harsh condition of wine and carry out malolactic fermentation. This process, basically consisting of malic acid decarboxylation, benefit to most red and some white wines because of the resulting improvement in their organoleptic properties and microbiological stability. In some wine developed countries, strains’ selection of O. oeni which have better applicability and specialty in wine-marking has become a industry. Many researchers have examined the diversity of O. oeni strains within and around wineries, an outcome of this analysis is the general view that Oenococcus is a genetically homogenous genus. Several reports demonstrated that resistance to wine conditions and influence on organoleptic quality are strain-dependent, so it is necessary to study O. oeni in China where microbe germplasm resources are rich.In this study, species-specific PCR were used for rapid and accurate identification of O. oeni Strains, isolated and screened from the main wine producing regions of Ningxia. Two primers, based on unique, highly conserved regions with the 16S rRNA gene of O. oeni, were applied to amplify a 995bp fragment which is specific for O. oeni. The factors of species-specific PCR reaction were optimized in this experiment. Then, 16S rDNA PCR-RFLP were employed in the study of 23 O. oeni Strains, saved in laboratory, with the aim of differentiating O. oeni strains.The results were as follows:1. 25 strains isolated and screened from the main wine producing regions of Ningxia in China were corried out some physiological and biochemical tests, the results compared with the evaluation form of O. oeni designed by Boulton et al. were shown they were consistent.2. The methods of genome DNA extraction were studied. The genome DNA digested with lysozyme was markedly polluted by RNA (A260/A280>2.0). The genome DNA digested with proteinase k and RNase was qualified for Polymerase Chain Reaction through detection of bands by electrophoresis.3. In this study, species-specific PCR were used for rapid and accurate identification of O. oeni Strains. The factors of species-specific PCR reaction were optimized. The optimal PCR system was as follows: 2.5UTaq polymerase, 200μmol/L dNTP, 0.2μmol/L forward and reverse primers each, 2.0mmol/L Mg2+,40ng DNA template, in 50μL reaction system. 25 strains isolated and screened from the main wine producing regions of Ningxia in China were all amplified a 995bp fragment which is specific for O. oeni.4. 23 O. oeni Strains saved in laboratory were all amplified the region of 16S rDNA, then digested with various restriction endonucleases. Results revealed that the patterns of HindⅢand MseⅠprofiled identical to each of the strains. While, the patterns of AluⅠanalyzed by NTSYSPC2.1 software indicated that all 23 strains were clustered into 2 subgroups at the similarity level of 63%. 5 strains randomly selected of 5 patterns in 23 O. oeni Strains were carried out fermentation characteristics test, the results showed the patterns of SD-2a and 31-DH can bear high alcoholic concentration; the patterns of SD-2g、SD-2a and SX-1a have steady acid-resistance ability; the patterns of HB-1a、SD-2a and SD-2g can strongly grow in the high SO2 concentration.

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