Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: During winemaking, a number of biochemical changes occur as a result of the metabolic
activity of wine lactic acid bacteria (LAB) associated with malolactic fermentation (MLF). The
latter process, which occurs mostly after alcoholic fermentation by wine yeasts, involves the
conversion of L-malate to L-lactate and CO2, thus resulting to wine acidity reduction,
microbiological stabilization and alterations of wine organoleptic quality.
Although Oenococcus oeni is predominantly the most preferred species suitable for
carrying out MLF in wine owing to its desirable oenological properties, Lactobacillus plantarum
has also been considered as a potential candidate for MLF induction. Other species in the
genera of Lactobacillus and Pediococcus are often associated with wine spoilage. These
microorganisms induce wine spoilage by producing off-flavours derived from their metabolic
activity. It is therefore of paramount importance to understand the mechanism by which wine
microbiota cause spoilage.
The purpose of this study was to investigate the presence of genes encoding enzymes of
oenological relevance in wine-associated LAB strains. In order to achieve this, different sets of
specific primers were designed and employed for a wide-scale genetic screening of wine LAB
isolates for the presence of genes encoding enzymes involved in various metabolic pathways,
such as citrate metabolism, amino acid metabolism, hydrolysis of glycosides, degradation of
phenolic acids as well as proteolysis and peptidolysis. PCR detection results showed that the
majority of the tested strains possessed most of the genes tested for. It was also noted that,
among the O. oeni strains tested for the presence of the pad gene encoding a phenolic acid
decarboxylase, only two strains possessed this gene. None of the O. oeni strains has
previously been shown to possess the pad gene, and this study was the first to report on the
presence of this gene in O. oeni strains. In an attempt to genetically characterize this putative
gene, DNA fragments from the two positive O. oeni strains were sequenced. The newly
determined sequences were compared to other closely related species. Surprisingly, no match
was found when these sequences were compared to the published genomes of three O. oeni
strains (PSU-1, ATCC BAA-1163 and AWRI B429). This reinforced a speculation that the pad
gene in these two strains might have been acquired via the horizontal gene transfer. In
addition, it remains to be further determined if the presence of this gene translates to volatile
phenol production in wine.
In this study, a novel strain isolated from South African grape and wine samples was also
identified and characterized. The identification of this strain was performed through the 16S
rDNA sequence analysis, which indicated that this strain belongs to Lactobacillus florum
(99.9% sequence identity). A novel PCR assay using a species-specific primer for the rapid
detection and identification of Lb. florum strains was also established. For further
characterization, this strain was also investigated for the presence of genes encoding
enzymes of oenological relevance. PCR detection results indicated that the Lb. florum strain
also possess some of the genes tested for.
In addition to genetic screening of wine LAB isolates for the presence of different genes,
this study was also aimed at evaluating the regulation of the mleA gene encoding malate
decarboxylase in three oenological strains of O. oeni. The regulation of this gene was tested in
a synthetic wine medium under various conditions of pH and ethanol. From the expression
analysis, it was observed that the mleA gene expression was negatively affected by high
ethanol content in the medium. On the other hand, low pH of the medium seemed to favour
the expression of this gene as the mleA gene expression was more pronounced at pH 3.2 than
at pH 3.8.
The findings from this study have shed more light on the distribution of a wide array of
enzyme-encoding genes in LAB strains associated with winemaking. However, it remains
unknown if the enzymes encoded by these genes are functional under oenological conditions,
given that wine is such a hostile environment encompassing a multitude of unfavourable
conditions for the enzymes to work on. Evaluating the expression of these genes will also help
give more insights on the regulation of the genes under winemaking conditions. / AFRIKAANSE OPSOMMING: Gedurende wynmaak, sal 'n aantal biochemiese veranderinge plaasvind as gevolg van die
metaboliese aktiwiteit van wyn melksuurbakterieë (MSB) wat betrokke is by appelmelksuurgisting
(AMG). Die laasgenoemde proses, wat meestal na alkoholiese fermentasie deur wyngiste
plaasvind, behels die omskepping van L-malaat na L-laktaat en CO2, om sodoende die wyn se suur
te verminder, mikrobiologiese stabiliteit en verandering van wyn organoleptiese kwaliteit.
Alhoewel Oenococcus oeni hoofsaaklik die mees gewenste spesies is wat geskik is vir die
uitvoering van AMG in wyn weens sy geskikte wynkundige eienskappe, Lactobacillus plantarum
word ook beskou as 'n potensiële kandidaat vir AMG induksie. Ander spesies in die genera
Lactobacillus en Pediococcus word dikwels geassosieer met wynbederf. Hierdie mikro-organismes
veroorsaak wynbederf deur die produksie van wangeure as gevolg van hul metaboliese aktiwiteite.
Dit is dus van kardinale belang dat die meganisme van die wynbederf verstaan word.
Die doel van hierdie studie was om die teenwoordigheid van koderend ensieme gene van
wynkundige belang in wynverwante MSB stamme te ondersoek. Ten einde dit te bereik, was
verskillende stelle van spesifieke peilers ontwerp en toegepas vir 'n groot skaal se genetiese
toetsing van wyn MSB isolate vir die teenwoordigheid van ensiemkoderende gene betrokke by
verskeie metaboliese paaie, soos sitraat metabolisme, aminosuur metabolisme, hidrolise van
glikosiede, agteruitgang van fenoliese sure sowel as proteolise en peptidolise. PKR opsporings
resultate het getoon dat die meerderheid van die stamme getoets, die meeste van die gene
getoets voor besit. Dit is ook opgemerk dat, onder die O. oeni stamme getoets vir die
teenwoordigheid van die pad geen, slegs twee stamme hierdie geen besit. Geen O. oeni stamme
het voorheen gewys dat hul die pad geen besit, en hierdie studie was die eerste bewys oor die
teenwoordigheid van hierdie geen in O. oeni stamme. In 'n poging om die geen geneties te
karakteriseer, is DNA-fragmente van die twee positiewe O. oeni stamme se sekwens volgorde
bepaal. Die DNA volgorde is vergelyk met ander nouverwante spesies. Verrassend, was geen
passende DNA volgorde gevind met die gepubliseerde genome van drie O. oeni stamme (PSU-1,
ATCC BAA-1163 en AWRI B429) nie. Dit versterk die spekulasie dat die pad geen in hierdie twee
stamme via die horisontale geen-oordrag verkry is. Verder moet dit nog bepaal word of die
teenwoordigheid van hierdie geen lei na vlugtige fenol produksie in wyn.
In hierdie studie, is ongeïdentifiseerde stam geïsoleerd van Suid-Afrikaanse druiwe en wyn
monsters ook geïdentifiseer en karakteriseer. Die identifisering van hierdie stam is uitgevoer deur
middel van die 16S rDNA volgorde analise, wat aangedui het dat hierdie stam behoort aan
Lactobacillus florum (99.9% volgorde identiteit). PKR toetse met behulp van die spesie-spesifieke
peiler vir die vinnige opsporing en identifikasie van Lb. florum stamme is ook ontwikkel. Vir verdere
karakterisering, was hierdie stam ook ondersoek vir die teenwoordigheid van koderende ensiem
gene van wynkundige belang. PKR opsporings resultate het aangedui dat die Lb. florum stam ook
oor 'n paar van die gene getoets voor besit.
Bykomend tot genetiese toetsing van wyn MSB isolate vir die teenwoordigheid van
verskillende gene, het die studie ook die evaluering van die regulering van die mleA geen,
kodering malaatdekarboksilase in drie wyn stamme van O. oeni. Die regulering van hierdie geen
was getoets in die sintetiese wynmedium onder verskillende pH en etanol kondisies. Van die
uitdrukkingsresultate, is daar waargeneem dat die mleA geenuitdrukking is negatief geraak deur
hoë etanol-inhoud in die medium. Aan die ander kant, in die lae pH medium was die uitdrukking
van hierdie geen bevoordeel by pH 3.2 as by pH 3.8.
Die bevindinge van hierdie studie het meer lig gewerp op die verspreiding van die wye
verskeidenheid van ensiem-koderende gene in MSB stamme wat verband hou met wynmaak. Dit
bly egter steeds onbekend of die ensieme gekodeer deur hierdie gene funksioneel is onder
wynkondisies, gegewe dat wyn so 'n vyandige omgewing is menigte ongunstige toestande vir die
werking van ensieme. Evaluering van die uitdrukking van hierdie gene sal ook help om meer
insigte gee oor die regulering van die gene onder wynmaak toestande.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/6496 |
| Date | 03 1900 |
| Creators | Mtshali, Phillip Senzo |
| Contributors | Du Toit, Maret, Divol, Benoit, University of Stellenbosch. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 139 p. : ill. |
| Rights | University of Stellenbosch |
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