Year: 2009
Type: dissertation
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 10 6 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 (10 1 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 10 2 cfu.ml -1 was determined in sterile white wine for Pd.pentosaceus and 10 3 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 (10 1 -10 2 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 (10 2 -10 4 cfu.ml-1 ).Lower detection limits were also determined for B. bruxellensis amplified with the HDA1-GC and HDA2 primer pair (10 3 -10 4 cfu.ml-1 ) than with the NL1-GC and LS2 primer pair (10 5 cfu.ml -1 ).PCR and DGGE detection limits for the inoculation of A. pasteurianus, Lb. plantarum and B. bruxellensis at an inoculum of 10 8 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 10 1 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 (10 1 -10 7 cfu.ml-1 ) than when inoculated into SSS (10 1 -10 5 cfu.ml -1 ). PCR-based DGGE analysis proved to bea 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.v UITTREKSEL Druiwesap word omgeskakel na wyn deur die komplekse prosesse van alkoholiese-en appelmelksuurfermentasie wat uitgevoer word deur giste, melksuurbakterieë en asynsuurbakterieë.Die betrokke mikrobes speel egter nie slegs 'n rol in die versekering van die suksesvolle produksie van wyn nie, maar kan ook tot bederf van die wyn lei as die mikrobiese groei nie beheer word nie.Konvensionele, kultuur-afhanklike mikrobiologiese tegnieke word algemeen gebruik as die hoof metode vir die deteksie en identifisering van hierdie bederfmikrobes.Molekulêre kultuur-onafhanklike tegnieke, wat die deteksie en identifisering van lewensvatbare, maar nie-kweekbare mikrobes toelaat, het onlangs meer gewild geraak vir die deteksie van moontlike bederfmikrobes wat in mos en wyn voorkom.Verder is hierdie tegnieke minder tydrowend as die konvensionele mikrobiologiese tegnieke.Die doel van hierdie studie was om die suksesvolle toepassing van polimerase ketting-reaksie (PKR)-gebaseerde denaturerende gradiënt jel elektroforese (DGJE) analise vir die deteksie van wyn bederfmikrobes, wat as enkel mikrobiese spesies en as deel van gemengde mikrobiese populasie in steriele fiosiologiese soutoplossing (FSO) (0.85% (m/v) NaCl) en steriele witwyn geïnokuleer is, te evalueer.Drie metodes vir die isolasie van DNS vanuit FSO, en steriele wit-en rooiwyn wat met verwysingsmikrobes spesies geïnokuleer, is vergelyk in terme van die DNS-konsentrasie en -suiwerheid, sowel as die eenvoudigheid van die tegniek.Die drie geëvalueerde DNS isolasie metodes was die TZ-metode, die proteinase-K metode en die fenol-ekstraksie metode.DNS kon nie suksesvol vanuit rooiwyn met enige van die drie ekstraksie metodes geïsoleer word nie.Die TZ-metode was die verkose metode vir die isolasie van DNS vanuit geïnokuleerde FSO en steriele witwyn aangesien die tegniek die beste resultate gelewer het in terme van eenvoud, DNS-konsentrasie en -suiwerheid.PKR en DGJE kondisies is geoptimiseer vir die universele inleierpaar, HDA1-GC en HDA2, die wyn-bakterieë spesifieke inleierpaar, WBAC1-GC en WBAC2, en die gis spesifieke inleierpaar, NL1-GC en LS2.DNS vanaf Acetobacter pasteurianus, Lactobacillus plantarum, Pediococcus pentosaceus, Oenococcus oeni, Saccharomyces cerevisiae en Brettanomyces bruxellensis is geamplifiseer met die toepaslike inleiers en is suksesvol geanaliseer met DGJE.PKR en DGJE deteksie limiete is suksesvol bepaal vir die 10 6 kve.ml -1 inokulum van die verwysingsmikrobes, A. pasteurianus, Lb. plantarum, Pd. pentosaceus en B. bruxellensis apart in FSO en steriele witwyn.Dit was moontlik om lae konsentrasies (10 1 kve.ml-1 ) van A. pasteurianus, Lb. plantarum, vi Pd. pentosaceus en B. bruxellensis, geïsoleer vanuit FSO, met PKR te bepaal wanneer die DNS met die inleierpaar, HDA1-GC en HDA2, geamplifiseer is.PKR deteksie limiete van 10 2 kve.ml -1 vir Pd. pentosaceus en 10 3 kve.ml-1 vir B. bruxellensis, in witwyn, wanneer dieselfde inleier paar gebruik is, is bepaal.Die PKR amplifiserings resultate vir die inleierpaar, WBAC1-GC en WBAC2, het goed vergelyk met die resultate verkry vir die inleierpaar, HDA1-GC en HDA2.Die DGJE deteksie limiet resultate het getoon dat dit moontlik is om laer konsentrasies (10 1 -10 2 kve.ml -1 ) van A. pasteurianus, Lb. plantarum en Pd. pentosaceus met die inleierpaar, HDA1-GC en HDA2, te bepaal as wanneer met die inleierpaar, WBAC1-GC en WBAC2, (10 2 -10 4 kve.ml-1 ) geamplifiseer word.Laer deteksie limiete (10 3 -10 4 kve.ml-1 ) is verder bepaal vir B. bruxellensis tydens amplifisering met die inleierpaar, HDA1-GC en HDA2, as wanneer met die inleierpaar, NL1-GC en LS2, (10 5 kve.ml -1 ) geamplifiseer word.PKR en DGJE deteksie limiete wat bepaal is vir die inokulasie van A. pasteurianus, Lb. plantarum en B. bruxellensis, teen 'n inokulum van 10 8 kve.ml -1 , as deel van 'n gemengde populasie in FSO en steriele witwyn het goed vergelyk met die resulate verkry vanaf die verwysingsmikrobes wat geïnokuleer was as enkel mikrobiese spesies.PKR deteksie limiete vir al drie verwysingsmikrobes, geïnokuleer as deel van gemengde populasies, en wat met die inleierpare, HDA1-GC en HDA2, en WBAC1-GC en WBAC2 geamplifiseer is, is bepaal as 10 1 kve.ml-1 .Vergelykbare of hoër DGJE deteksie limiete is waargeneem vir die verwysingsmikrobes wat in steriele witwyn (10 1 -10 7 kve.ml-1 ) geïnokuleer is in vergelyking met die inokulasie van die onderskeie mikrobes in FSO (10 1 -10 5 kve.ml -1 ).Hierdie studie het getoon dat PKR-gebaseerde DGJE analise suksesvol met die universele, wyn-bakterieë en gis-spesifieke inleierpare gebruik kan word vir die deteksie van A. pasteurianus, Lb. plantarum, Pd. pentosaceus en B. bruxellensis.Die gebruik van 'n kultuur-onafhanklike tegniek maak die vroeë deteksie van moontlike bederfmikrobes, teen lae konsentrasies, in wyn moontlik.
Author: Linka Bester
Language: English
Subfield: Food Science
Domain: Life Sciences
SDG: Zero hunger
Open Access status: green
OpenAlex ID: W2155249479