Thesis (MSc (Institute for Wine Biotechnology))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: The process of winemaking involves a number of microorganisms, contributing both positively
and negatively to the final product. Lactic acid bacteria (LAB) are present at all stages of
vinification and therefore play a major role in the production of wine, especially red wine. LAB
are responsible for malolactic fermentation (MLF), which can be desirable or unwanted
depending on the style of wine. LAB can also be responsible for spoilage, and production of off flavours
resulting in a decrease in the quality of the finished wine. Spoilage occurs if the wrong
species are present at the wrong time and can also occur as a result of spontaneous MLF. It is
therefore necessary to control the population of indigenous LAB present in the wine.
Plantaricins are bacteriocins produced by Lactobacillus plantarum strains and have the
potential to inhibit closely related strains that occupy the same ecological niche. This makes
them promising for the control of LAB during the winemaking process. Inhibition of the
indigenous LAB microflora could help to prevent the formation of undesirable off-flavours, as
well as allowing for control over MLF. The use of plantaricin-producing starter cultures could
also lead to a reduction in the amount of sulphur dioxide used in wine.
The purpose of this study was to investigate the potential of L. plantarum strains isolated
from wine to produce plantaricins under winemaking conditions. This potential was evaluated by
investigating the expression of plantaricin genes under winemaking conditions.
The first objective was to screen nineteen strains of L. plantarum isolated from South
African red wines, as well as a commercial strain; for various genes responsible for the
production of plantaricins, including structural, transport and regulatory genes. Results showed
that the twenty strains contained at least 16 of the 24 genes (previously reported to be
associated with the plantaricin locus for various L. plantarum strains) screened for. Only orfZ123
and orf345 genes yielded no positive results in any of the strains.
The second objective was to sequence selected plantaricin genes (plnE, plnF, plnN, plnG
and plnB) to determine the variation in nucleotide and amino acid sequences of these genes
among the different wine L. plantarum isolates. High homology was found between the
nucleotide sequences of the strains and none of the amino acid substitutions in the protein
sequences occurred in conserved regions. The nucleotide sequence of plnN was identical in all
but one of the strains and similarity of the plnB sequence ranged from 96% to 100%. Similarity
of the plnG nucleotide sequence ranged from 99% to 100%. The plnE nucleotide sequence was
identical in all but two strains and there were only two groups in terms of nucleotide sequence
for plnF, with only two changes between the groups.
The third objective was the evaluation of plantaricin production using plate assays
mimicking certain wine parameters (pH and ethanol concentration). All twenty strains showed
inhibitory activity to varying degrees against a panel of nine indicator microorganisms, including
Enterococcus faecalis, Listeria monocytogenes and potential wine spoilage organisms,
Lactobacillus spp, Pediococcus spp and Leuconostoc mesenteroides. Addition of 10% ethanol
and a low pH of 3.5 decreased both the bacteriocin production as well as the spectrum of
activity. Seven of the twenty strains, however, showed good bacteriocin activity under all
conditions.
The fourth objective was to investigate the expression of two plantaricin structural genes
(plnEF and plnJK) and the transporter gene (plnG) under winemaking conditions. Two strains
(R1122 and 113.1) were chosen, based on the results from the previous objectives, as starter
cultures for MLF in synthetic wine media and Riesling wine. Low wine pH (3.2) and high wine
pH (3.8) levels were investigated in conjunction with ethanol concentrations of 0%, 12% and
15%. All three of the genes were expressed to varying degrees depending on the fermentation
condition. High ethanol and low pH generally decreased expression of the structural plantaricin
genes. The influence on expression of the transporter gene was different, with low pH and
presence of ethanol resulting in an increase in gene expression. The genes were also
expressed in wine, although at a lower level relative to expression in the synthetic wine media.
The presence of sensitive bacteria in the wine seemed to increase expression of the structural
genes. Furthermore, expression of the mle gene responsible for MLF was investigated under
the same winemaking conditions. Expression was shown to be inducible by malic acid, and
negatively affected by the presence of ethanol but positively influenced by a lowering in pH from
3.8 to 3.2.
This study confirms that plantaricin genes are expressed under winemaking conditions,
which in turn indicates that the plantaricins could be produced under winemaking conditions.
This confirms the potential use of these plantaricin-producing strains as starter cultures for MLF
with the ability to inhibit indigenous LAB, however, presence of the plantaricin protein in wine
still needs to be confirmed. It will also need to be established whether the protein is biologically
active and not inhibited by wine-related factors. / AFRIKAANSE OPSOMMING: Die proses van wynmaak bevat 'n verskeidenheid mikroorganismes, wat postiewe en negatiewe
bydrae kan lewer tot die finale produk. Melksuurbakterieë is teenwoordig by alle stadiums van
wynmaak en speel 'n belangrike rol in die produksie van wyn. Melksuurbakterieë is
verantwoordelik vir appelmelksuur gisting (AMG), wat gewens of ongewens kan wees,
afhangende van die styl van die wyn. Melksuurbakterieë kan ook verantwoordelik wees vir
bederf van wyn, asook die produksie van ongewenste geure wat bydrae tot ʼn toename in die
kwaliteit van die wyn. Bederf van wyn kan gebeur as die verkeerde spesies voorkom op die
verkeerde tyd en kan ook gebeur as ʼn gevolg van spontane AMG. Dit is dus nodig om die
populasie van natuurlike melksuurbakterieë in wyn te beheer.
Plantarisiene, geproduseer deur Lactobacillus plantarum wyn-isolate, het die potensiaal om
naby verwante stamme se groei te inhibeer wat in dieselfde nis voorkom. Hierdie eienskap
maak hul belowend vir die beheer van melksuurbakterieë se groei gedurende die
wynmaakproses. Inhibering van die natuurlike mikroflora kan help om die vorming van
ongewenste geure te verhoed, sowel as om AMG te beheer. Die gebruik van aanvangskulture,
wat plantarisiene kan produseer, kan lei tot ’n vermindering in die gebruik van swaweldioksied
in die wynindustrie.
Die doel van hierdie studie was om die potensiaal van L. plantarum stamme, geïsoleer
vanuit wyn, te ondersoek vir hul vermoë om plantaricins te produseer in toestande wat die
wynmaakproses naboots. Die potensiaal was ondersoek deur te kyk na die uitdrukking van
plantarisien-produserende gene onder wynmaak toestande.
Die eerste objektief was om die 19 L. plantarum stamme, geïsoleer vanuit Suid-Afrikaanse
rooi wyne, asook n kommersiele stam, te ondersoek vir die teenwoordigheid van verskeie gene
wat verantwoordelik is vir die produksie van plantarisiene, sowel as strukturele, transporter en
regulerende gene. Al twintig van hierdie stamme het ten minste 16 uit die 24 gene bevat
waarvoor ondersoek was. OrfZ123 en orf345 het egter geen positiewe resultate opgelewer in
enige van die stamme nie.
Die tweede objektief was om die DNA-volgorde te bepaal van spesifieke gene (plnE, plnF,
plnN, plnG, sowel as plnB) en sodoende die variasie in nukleotied en aminosuur volgorde van
hierdie gene in die verskillende L. plantarum wyn-isolate te bepaal. Hoë vlakke van homologie
was gevind en geen van die aminosuur veranderings het in behoue gebiede plaasgevind nie.
Die nukleotied volgorde van plnN was identies in al die stamme, behalwe vir een, en die
ooreenkomste tussen die plnB volgorde het varieër van 96% tot 100%. Die ooreenkomste
tussen die plnG nukleotied volgorde het varieër van 99% to 100%. Die plnE nukleotied volgorde
was identies in al die stamme, behalwe vir twee, en daar was net twee groepe in terme van
nukleotied volgorde vir plnF, met net twee veranderinge tussen die groepe.
Die derde objektief was om die vermoë van die stamme om plantaricins the produseer,
deur gebruik te maak van plaat assays, onder verskillende wyntoestande te ondersoek. Die
twinting stamme het verskillende vlakke van inhibering teenoor die nege toets-organismes
getoon, wat Enterococcus faecalis, Listeria monocytogenes sowel as potensiele wyn bederf
organismes, Lactobacillus spp, Pediococcus spp and Leuconostoc mesenteroides insluit. Die
byvoeging van 10% etanol en ’n lae pH van 3.5, het beide bakteriosien produksie inhibeer,
sowel as die spektrum van aktiwiteit verminder. Sewe van die stamme het egter steeds goeie
aktiwiteit getoon onder al die kondisies wat getoets was.
Die vierde objektief was om die uitdrukking van twee plantaricin strukturele gene (plnEF en
plnJK), sowel as die transporter geen (plnG) onder wynmaak omstandighede te ondersoek.
Twee stamme (R1122 en 113.1) was gekies as aanvangskulture vir AMG in sintesiese wyn
media, sowel as Riesling wyn. Hierdie twee stamme was gekies op grond van die resultate wat
van die vorige objektiewe verkry was. Lae wyn pH (3.2) en hoë wyn pH (3.8) was ondersoek in
samewerking met verskillende etanol konsentrasies wat 0%, 12% en 15% etanol insluit. Al drie
hierdie gene was uitgedruk teen verskillende vlakke, afhangende van die verskeie fermentasie
kondisies. Hoë etanol en lae pH lei oor die algemeen tot ʼn toename in uitdrukking van die
strukturele plantarisien gene. Die invloed op uitdrukking van die transporter geen was
verskillend, want lae pH en die teenwoordigheid van etanol het gelei tot ʼn verhoging in geen
uitdrukking. Die gene was uitegdruk in wyn, maar was teen laer vlakke relatief tot uitdrukking in
die sintetiese wyn media. Dit blyk dat die teenwoordigheid van sensitiewe bakterieë in die wyn
tot ‘n hoër uitdrukking van die strukturele gene lei. Die uitdrukking van die mle geen,
verantwoordelik vir AMG, was ook onder dieselfde wynmaak kondisies ondersoek. Die
uitdrukking was geïnduseer deur appelsuur, negatief beïnvloed deur die teenwoordigheid van
etanol, maar positief beïnvloed deur ’n verlaging in pH van 3.8 tot 3.2.
Hierdie studie toon dat plantaricin gene uitegedruk word onder wynmaak toestande en dat
plantaricins moontlik onder hierdie toestande geproduseer kan word. Die potensiaal van hierdie
stamme word getoon om as aanvangskulture gebruik te word vir AMG, om sodoende die groei
van natuurlike melksuur bakterieë te inhibeer. Die teenwoordigheid van die plantarisien peptied
in die wyn moet egter nog bewys word. Daar sal ook vasgestel moet word of die peptied
biologies aktief is en nie deur wynverwante faktore geïnhibeer word nie.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/5463 |
| Date | 12 1900 |
| Creators | Miller, Bronwen Jayne |
| Contributors | Du Toit, Maret, Franz, Charles, Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | iii, 86 p. : ill. |
| Rights | Stellenbosch University |
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