Detection and identification of wine spoilage microbes using PCR-based DGGE analysis
Date
2009-03
Authors
Bester, Linka
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : University of Stellenbosch
Abstract
Grape juice is transformed into wine through the complex processes of alcoholic and
malolactic fermentation that is performed by yeasts, lactic acid bacteria and acetic acid
bacteria. However, the microbes involved in these processes do not only take part in
ensuring the successful production of wine, but also cause spoilage of the wine if their
growth is not controlled.
Conventional, culture-dependent methods of microbiology have been used as the
main technique in detecting and identifying these spoilage microbes. Cultureindependent
techniques of molecular biology have recently become more popular in
detecting possible spoilage microbes present in must and wine, since it allows the
detection and identification of viable, but non-culturable microbes and are not as timeconsuming
as conventional microbiological methods.
The aim of this study was to investigate the sustainability of polymerase chain
reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis in
detecting 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 part
of mixed microbial populations. Three methods of DNA isolation from SSS, sterile white
wine and sterile red wine inoculated with reference microbial strains were compared in
terms of DNA concentration and purity, as well as simplicity of the technique. These
three DNA isolation methods were the TZ-method, the proteinase K-method and the
phenol extraction method. DNA could not successfully be isolated from red wine using
any of the three DNA isolation methods. The TZ-method was the method of choice for
the isolation of DNA from inoculated SSS and sterile white wine as this technique gave
the 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 Acetobacter
pasteurianus, Lactobacillus plantarum, Pediococcus pentosaceus, Oenococcus oeni,
Brettanomyces bruxellensis and Saccharomyces cerevisiae were amplified with the
appropriate primers and successfully resolved with DGGE analysis. PCR and DGGE
detection limits were successfully determined when 106 cfu.ml-1 of the reference
microbes, A. pasteurianus, Lb. plantarum, Pd. pentosaceus and B. bruxellensis were
separately inoculated into SSS and sterile white wine. It was possible to detect low
concentrations (101 cfu.ml-1) with PCR for A. pasteurianus, Lb. plantarum, Grape juice is transformed into wine through the complex processes of alcoholic and
malolactic fermentation that is performed by yeasts, lactic acid bacteria and acetic acid
bacteria. However, the microbes involved in these processes do not only take part in
ensuring the successful production of wine, but also cause spoilage of the wine if their
growth is not controlled.
Conventional, culture-dependent methods of microbiology have been used as the
main technique in detecting and identifying these spoilage microbes. Cultureindependent
techniques of molecular biology have recently become more popular in
detecting possible spoilage microbes present in must and wine, since it allows the
detection and identification of viable, but non-culturable microbes and are not as timeconsuming
as conventional microbiological methods.
The aim of this study was to investigate the sustainability of polymerase chain
reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis in
detecting 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 part
of mixed microbial populations. Three methods of DNA isolation from SSS, sterile white
wine and sterile red wine inoculated with reference microbial strains were compared in
terms of DNA concentration and purity, as well as simplicity of the technique. These
three DNA isolation methods were the TZ-method, the proteinase K-method and the
phenol extraction method. DNA could not successfully be isolated from red wine using
any of the three DNA isolation methods. The TZ-method was the method of choice for
the isolation of DNA from inoculated SSS and sterile white wine as this technique gave
the 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 Acetobacter
pasteurianus, Lactobacillus plantarum, Pediococcus pentosaceus, Oenococcus oeni,
Brettanomyces bruxellensis and Saccharomyces cerevisiae were amplified with the
appropriate primers and successfully resolved with DGGE analysis. PCR and DGGE
detection limits were successfully determined when 106 cfu.ml-1 of the reference
microbes, A. pasteurianus, Lb. plantarum, Pd. pentosaceus and B. bruxellensis were
separately inoculated into SSS and sterile white wine. It was possible to detect low
concentrations (101 cfu.ml-1) with PCR for A. pasteurianus, Lb. plantarum,
iv
Pd. pentosaceus, and B. bruxellensis in SSS when amplified with the HDA1-GC and
HDA2 primer pair. A PCR detection limit of 102 cfu.ml-1 was determined in sterile white
wine for Pd. pentosaceus and 103 cfu.ml-1 for B. bruxellensis using this primer pair. The
results obtained from the PCR amplification with the WBAC1-GC and WBAC2 primer
pair 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 to
detect lower concentrations (101 – 102 cfu.ml-1) of A. pasteurianus, Lb. plantarum and
Pd. pentosaceus with the HDA1-GC and HDA2 primer pair than the WBAC-GC and
WBAC2 primer pair (102 – 104 cfu.ml-1). Lower detection limits were also determined for
B. 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 mixed
populations in SSS and sterile white wine compared well with the results obtained from
the reference microbes inoculated as single microbial species. PCR detection limits of
101 cfu.ml-1 were determined for all three reference microbes inoculated as part of
mixed populations when amplified with the HDA1-GC and HDA2 and the WBAC1-GC
and WBAC2 primer pairs. It was observed that similar or higher DGGE detection limits
were 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 used
successfully with the universal, wine-bacteria and yeast specific primer pairs for the
detection of A. pasteurianus, Lb. plantarum, Pd. pentosaceus and B. bruxellensis. The
culture-independent technique makes the early detection of possible spoilage microbes
at low concentrations in wine possible.
Description
Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2009.
Keywords
PCR-based DGGE, Wine spoilage microbes, Brettanomyces, Dissertations -- Food science, Theses -- Food science