PCR-based DGGE identification of bacteria and yeasts present in South African grape must and wine

• Main Author: Siebrits, Leoni
• Other Authors: Witthuhn, R.C.
• Format: Others
• Language: en_ZA
• Published: Stellenbosch : Stellenbosch University 2012
• Subjects: Bacteria > Identification; Yeast > Identification; Must > Microbiology; Wine and wine making > Microbiology > South Africa; Theses > Food science; Dissertations > Food science
• Online Access: http://hdl.handle.net/10019.1/19865

Thesis (MSc)--University of Stellenbosch, 2007. === ENGLISH ABSTRACT: Wine production involves complex interactions between a variety of yeasts and bacteria. Conventional microbiological methods can be used to identify the different microorganisms present in wine, but prove to be time-consuming and certain microbial species may not grow on synthetic isolation media. The aim of this study was to evaluate the microbial population present in two South African red wines, Pinotage and Merlot, as well as five spoilt commercial South African wines by using a non-culturable approach, polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE). The results from the non-culturable approach were compared to conventional platings. Unique PCR-based DGGE fingerprints were obtained for the Bacteria and yeasts present in the South African Pinotage and Merlot wines. Using yeast specific primers the Pinotage wine showed the presence of non-Saccharomyces yeasts at the beginning of the alcoholic fermentation, while Saccharomyces cerevisiae was present until the completion of the malo-lactic fermentation (MLF). This yeast was also identified during both the alcoholic fermentation and MLF of the Merlot wine using PCR-based DGGE and conventional plating. Using Bacteria specific primers, Lactobacillus plantarum and Lactobacillus sp. was identified in the Pinotage wine using PCR-based DGGE, while Lactobacillus brevis were isolated from Merlot wine using conventional platings. Although the presence of S. cerevisiae is expected during wine fermentation, the presence of this microbe in bottled wine could lead to spoilage. Four of the spoilt commercial wine samples (RW1, RW2, RoW1 and WW1) were found to be spoilt by S. cerevisiae, while a fifth wine sample (RW3) was found to be spoilt by an Acetobacter sp. using PCR-based DGGE. Members of the family Enterobacteriaceae were identified from all the wines using PCR-based DGGE, while Enterobacter sakazakii was identified from RW1 using PCR-based DGGE and conventional plating. The members of the family Enterobacteriaceae could possibly have contributed to the spoilage of the wine by producing undesirable secondary metabolites. PCR-based DGGE proved to be an alternative to conventional microbiological methods for the identification of the microbial species in South African red grape must and wine. This method also proved to be useful in the identification of spoilage microbes in spoilt commercial South African wines. === AFRIKAANSE OPSOMMING: Die produksie van rooi wyn behels komplekse interaksies tussen ‘n verskeidenheid van giste en bakterieë. Konvensionele mikrobiologiese metodes kan gebruik word om die verskillende mikro-organismes wat in rooi wyn teenwoordig is te identifiseer, maar dit blyk tydrowend te wees, terwyl sekere mikro-organismes nie groei op sintetiese media nie. Die doel van hierdie studie was om die mikrobiologiese populasie wat in twee Suid-Afrikaanse rooi wyne, Pinotage en Merlot, en vyf bederfde kommersiële wyne teenwoordig is, te evalueer met die gebruik van ‘n kultuur-onafhanklike benadering, polimerase ketting-reaksie (PKR)-gebaseerde denaturerende gradiënt jel elektroforese (DGJE). Die resultaat van die kultuur-onhafhanklike benadering was vergelyk met konvensionele uitplating tegnieke. Unieke, ongeëwenaarde PKR-gebaseerde DGGE vingerafdrukke was verkry van die Bakterieë en giste aanwesig in die Pinotage en Merlot wyne. Deur gebruik te maak van gis-spesifieke inleiers het die Pinotage wyn die teenwoordigheid van nie-Saccharomyces giste getoon, terwyl Saccharomyces cerevisiae teenwoordig was tot en met die afhandeling van die appel-melksuur gisting (AMG). Hierdie gis is ook geïsoleer gedurende beide die alkoholiese gisting en AMG van die Merlot wyn deur gebruik te maak van PKR-gebaseerde DGGE en konvensionele uitplating tegnieke. Met Bakterieë-spesifieke inleiers, was Lactobacillus plantarum en Lactobacillus sp. geïdentifiseer in die Pinotage wyn deur gebruik te maak van PKR-gebaseerde DGGE, terwyl Lactobacillus brevis geïsoleer is uit Merlot wyn deur gebruik te maak van konvensionele uitplatings. Alhoewel die teenwoordigheid van S. cerevisiae verwag word gedurende wynfermentasie, kan die teenwoordigheid van hierdie mikrobe in gebottelde wyn tot bederwing lei. Vier van die bedorwe kommersiële wynmonsters (RW1, RW2, RoW1 en WW1) was bederf deur S. cerevisiae, terwyl ‘n vyfde wynmonster (RW3) bederf was deur ‘n Acetobacter sp. deur die gebruik van PKR-gebaseerde DGGE. Van al die wyne is lede van die Enterobacteriaceae familie geïdentifiseer deur gebruik gemaak te maak van PKR-gebaseerde DGGE, terwyl Enterobacter sakazakii geïsoleer is van RW1 met konvensionele uitplating. Die lede van die familie Enterobacteriaceae kon moontlik bygedra het tot die bederwing van die wyn deur ongewenste sekondêre metaboliete te produseer. PKR-gebaseerde DGGE bewys ‘n alternatief tot die konvensionele mikrobiologiese metodes vir die identifikasie van die mikrobiese spesies in Suid-Afrikaanse rooi druif mos en wyn te wees. Hierdie metode het ook die bruikbaarheid in die identifikasie van mikrobes wat kommersiële Suid-Afrikaanse wyne bederf, bewys.