Detection and identification of wine spoilage microbes using PCR-based DGGE analysis

[摘要] Grape juice is transformed into wine through the complex processes of alcoholic andmalolactic fermentation that is performed by yeasts, lactic acid bacteria and acetic acidbacteria. However, the microbes involved in these processes do not only take part inensuring the successful production of wine, but also cause spoilage of the wine if theirgrowth is not controlled.Conventional, culture-dependent methods of microbiology have been used as themain technique in detecting and identifying these spoilage microbes. Cultureindependenttechniques of molecular biology have recently become more popular indetecting possible spoilage microbes present in must and wine, since it allows thedetection and identification of viable, but non-culturable microbes and are not as timeconsumingas conventional microbiological methods.The aim of this study was to investigate the sustainability of polymerase chainreaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis indetecting wine spoilage microbes inoculated into sterile saline solution (SSS) (0.85%(m/v) NaCl) and sterile white wine and red wine as single microbial species and as partof mixed microbial populations. Three methods of DNA isolation from SSS, sterile whitewine and sterile red wine inoculated with reference microbial strains were compared interms of DNA concentration and purity, as well as simplicity of the technique. Thesethree DNA isolation methods were the TZ-method, the proteinase K-method and thephenol extraction method. DNA could not successfully be isolated from red wine usingany of the three DNA isolation methods. The TZ-method was the method of choice forthe isolation of DNA from inoculated SSS and sterile white wine as this technique gavethe best results in terms of simplicity, DNA concentration and purity.PCR and DGGE conditions were optimised for the universal primer pair,HDA1-GC and HDA2, the wine-bacteria specific primer pair, WBAC1-GC and WBAC2,and the yeast specific primer pair, NL1-GC and LS2. DNA from Acetobacterpasteurianus, Lactobacillus plantarum, Pediococcus pentosaceus, Oenococcus oeni,Brettanomyces bruxellensis and Saccharomyces cerevisiae were amplified with theappropriate primers and successfully resolved with DGGE analysis. PCR and DGGEdetection limits were successfully determined when 106 cfu.ml-1 of the referencemicrobes, A. pasteurianus, Lb. plantarum, Pd. pentosaceus and B. bruxellensis wereseparately inoculated into SSS and sterile white wine. It was possible to detect lowconcentrations (101 cfu.ml-1) with PCR for A. pasteurianus, Lb. plantarum, Grape juice is transformed into wine through the complex processes of alcoholic andmalolactic fermentation that is performed by yeasts, lactic acid bacteria and acetic acidbacteria. However, the microbes involved in these processes do not only take part inensuring the successful production of wine, but also cause spoilage of the wine if theirgrowth is not controlled.Conventional, culture-dependent methods of microbiology have been used as themain technique in detecting and identifying these spoilage microbes. Cultureindependenttechniques of molecular biology have recently become more popular indetecting possible spoilage microbes present in must and wine, since it allows thedetection and identification of viable, but non-culturable microbes and are not as timeconsumingas conventional microbiological methods.The aim of this study was to investigate the sustainability of polymerase chainreaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis indetecting wine spoilage microbes inoculated into sterile saline solution (SSS) (0.85%(m/v) NaCl) and sterile white wine and red wine as single microbial species and as partof mixed microbial populations. Three methods of DNA isolation from SSS, sterile whitewine and sterile red wine inoculated with reference microbial strains were compared interms of DNA concentration and purity, as well as simplicity of the technique. Thesethree DNA isolation methods were the TZ-method, the proteinase K-method and thephenol extraction method. DNA could not successfully be isolated from red wine usingany of the three DNA isolation methods. The TZ-method was the method of choice forthe isolation of DNA from inoculated SSS and sterile white wine as this technique gavethe best results in terms of simplicity, DNA concentration and purity.PCR and DGGE conditions were optimised for the universal primer pair,HDA1-GC and HDA2, the wine-bacteria specific primer pair, WBAC1-GC and WBAC2,and the yeast specific primer pair, NL1-GC and LS2. DNA from Acetobacterpasteurianus, Lactobacillus plantarum, Pediococcus pentosaceus, Oenococcus oeni,Brettanomyces bruxellensis and Saccharomyces cerevisiae were amplified with theappropriate primers and successfully resolved with DGGE analysis. PCR and DGGEdetection limits were successfully determined when 106 cfu.ml-1 of the referencemicrobes, A. pasteurianus, Lb. plantarum, Pd. pentosaceus and B. bruxellensis wereseparately inoculated into SSS and sterile white wine. It was possible to detect lowconcentrations (101 cfu.ml-1) with PCR for A. pasteurianus, Lb. plantarum,ivPd. pentosaceus, and B. bruxellensis in SSS when amplified with the HDA1-GC andHDA2 primer pair. A PCR detection limit of 102 cfu.ml-1 was determined in sterile whitewine for Pd. pentosaceus and 103 cfu.ml-1 for B. bruxellensis using this primer pair. Theresults obtained from the PCR amplification with the WBAC1-GC and WBAC2 primerpair compared well with the results of the HDA1-GC and HDA2 primer pair.The results from the DGGE detection limits indicated that it was possible todetect lower concentrations (101 – 102 cfu.ml-1) of A. pasteurianus, Lb. plantarum andPd. pentosaceus with the HDA1-GC and HDA2 primer pair than the WBAC-GC andWBAC2 primer pair (102 – 104 cfu.ml-1). Lower detection limits were also determined forB. bruxellensis amplified with the HDA1-GC and HDA2 primer pair (103 – 104 cfu.ml-1)than with the NL1-GC and LS2 primer pair (105 cfu.ml-1).PCR and DGGE detection limits for the inoculation of A. pasteurianus,Lb. plantarum and B. bruxellensis at an inoculum of 108 cfu.ml-1 as part of mixedpopulations in SSS and sterile white wine compared well with the results obtained fromthe reference microbes inoculated as single microbial species. PCR detection limits of101 cfu.ml-1 were determined for all three reference microbes inoculated as part ofmixed populations when amplified with the HDA1-GC and HDA2 and the WBAC1-GCand WBAC2 primer pairs. It was observed that similar or higher DGGE detection limitswere obtained for the reference microbes inoculated in sterile white wine(101 – 107 cfu.ml-1) than when inoculated into SSS (101 – 105 cfu.ml-1).PCR-based DGGE analysis proved to be a technique that could be usedsuccessfully with the universal, wine-bacteria and yeast specific primer pairs for thedetection of A. pasteurianus, Lb. plantarum, Pd. pentosaceus and B. bruxellensis. Theculture-independent technique makes the early detection of possible spoilage microbesat low concentrations in wine possible.

[发布机构] Stellenbosch University