Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Lactic acid bacteria (LAB) play a vital role in reducing wine acidity and also contributing to its
aroma and flavour. However, they can also be responsible for many wine spoilage problems that
compromise the quality and value of wine. While Oenococcus oeni contributes positive characteristics to
the sensory properties of wine, certain species of the genera, Lactobacillus and Pediococcus can affect the
wholesomeness of wine by producing undesirable compounds, such as biogenic amines and ethyl
carbamate.
Chemical preservatives like sulphur dioxide (SO2) are used to prevent the growth of spoilage
micro-organisms during the winemaking process. SO2 also acts as a reducing agent and maintains the
benefits of antioxidant properties of the polyphenols of wine. However, there is a worldwide demand to
reduce SO2 levels due to the increasing health related risks and other factors.
All these considerations have increased the interest in research to look for new preservation
strategies, and LAB-produced bacteriocins seem to be a potential alternative that has been explored in the
last decade. Various types of bacteriocins have been identified and characterized. However, there are few
reports on bacteriocins produced by LAB of oenological origin or on bacteriocins present in the finished
wine.
The present study screened 155 LAB isolates from the IWBT culture collection for bacteriocin
production. The isolates originated from South African red wines undergoing spontenous malolactic
fermentation (MLF). Eight strains (5%) were identified to be producers, as evidenced by strong inhibition
zones formed against sensitive organisms on agar plates. The producers demonstrated a broad spectrum of
antimicrobial activity by inhibiting Lactobacillus spp., Leuconostoc mesenteroides, Listeria
monocytogenes and Pediococcus pentosaceus strains. Some of these bacterial genera are important in
winemaking since they are potential wine spoilage bacteria. Hence these strains and/or the bacteriocins
they produce could possibly find application in the food fermentation industry. The physiological results, biochemical tests and sugar fermentation profiles all gave the same
results for the seven isolates, which were indicative of enterococci. The identification through 16S rRNA
gene sequencing revealed that the seven tested isolates were all Enterococccus faecium. RAPD-PCR
fingerprinting gave the same profile for the seven strains confirming that they were all identical on
genetic level. Determining the molecular weight using SDS-PAGE showed the peptides to be below
4.6 kDa in size. PCR amplification of the enterocin P gene, sequencing and BLAST search results
confirmed that all eight strains contained the enterocin P gene from Ent. faecium.
The enterocin tested in this study was heat stable at 100°C (30 min), but lost 50% of its activity at
121°C (15 min). Factors such as bacteriocin production and heat resistance are among many that enable
enterococci to be dominant in fermented products such as dairy foods or meat. Therefore, enterococci
producing bacteriocins have potential applications in various foods and fermented products. The pH tests
showed enterocin to be active over a broad pH range (2-10). Enterocin activity over a wide pH range make them potentially more suitable as natural preservatives of foods and fermented products where
products are acidified or pH decreases due to natural LAB present. They also have potential applications
in oenological process where pH levels are as low as 3 and 4.
Proteolytic enzyme treatments with lysozyme, lipase, lyticase and catalase could not inhibit
enterocin activity. This indicated that their antimicrobial activity was independent of lipid or carbohydrate
moieties or hydrogen peroxide. α-Chymotrypsin and proteinase K inactivated enterocin, which indicated
that the compound was proteinaceous in nature.
Bacteriocin production tested in two of the isolates, #16.3 and 128.1, coincided with the
exponential growth phase which occurred after 6 hours of incubation at 30°C, which was an indication of
primary metabolite kinetics. The highest production of 400 AU/ml was observed after eight hours and
was maintained for several hours (46 hours) in the stationery phase. The bactericidal effect of the cell free
supernatants from #16.3 and 128.1 against the sensitive culture of Lactobacillus pentosus DSM 20314
was clearly demonstrated by complete inhibition of growth for most of the experimental period, while the
control increased exponentially throughout the experiment.
In conclusion, this study has confirmed the isolation and identification of Ent. faecium strains
from wine, a genus that is rarely found in the wine environment. Although one can speculate on the origin
of this bacterium in the wine e.g. human handling and contaminated water, these bacterial isolates
produced enterocin P which have antimicrobial action against wine-related LAB genera and therefore
have a potential role in wine spoilage control. / AFRIKAANSE OPSOMMING: Melksuurbakterieë (MSB) speel ‘n belangrike rol in die redusering van die suurgehalte van wyn
en dra ook by tot die aroma en smaak daarvan. Hulle kan egter ook verantwoordelik wees vir vele
wynbederfprobleme wat die gehalte en waarde van wyn negatief beïnvloed. Hoewel Oenococcus oeni
positiewe karaktertrekke aan die sensoriese eienskappe van wyn verleen, kan sekere spesies van die
genus, Lactobacillus en Pediococcus, die heilsaamheid van wyn beïnvloed deur ongewenste verbindings,
soos biogeniese amienes en etielkarbamaat, te produseer.
Chemiese preserveermiddels, soos swaweldioksied (SO₂), word gebruik om die groei van
bederfmikro-organismes tydens die wynbereidingsproses te voorkom. SO₂ fungeer ook as ‘n
reduseermiddel en onderhou die voordele van die antioksidant eienskappe van die poli-fenole van wyn.
Daar is egter ‘n wêreldwye vraag na die redusering van SO₂-vlakke as gevolg van die toename in
gesondheidsverwante risiko’s en ander faktore.
Al hierdie oorwegings het belangstelling in die navorsing van nuwe preserveringstrategieë laat
toeneem en MSB-geproduseerde bakteriosiene lyk na ‘n potensiële alternatief wat in die laaste dekade
ondersoek word. Verskeie tipes bakteriosiene is geïdentifiseer en getipeer. Daar is egter nog weinig
gerapporteer oor bakteriosiene wat deur MSB van wynkundige oorsprong geproduseer is of oor
bakteriosiene wat in afgeronde wyn teenwoordig is.
Die huidige studie het 155 MSB isolate van die Instituut vir Wynbiotegnologie se
kultuurversameling vir bakteriosien-produsering gegradeer. Agt stamme (5%) is as produseerders
geïdentifiseer, soos gestaaf is deur sterk inhibisiesones wat teen sensitiewe organismes op agarplate
gevorm het. Die produseerders het ‘n breë spektrum van antimikrobiese aktiwiteit by inhiberende
Lactobacillus spp., Leuconostoc mesenteroides, Listeria monocytogenes en Pediococcus pentosaceus
stamme gedemonstreer. Sommige van hierdie bakteriese genera is belangrik in wynbereiding, omdat dit
potensiële wynbederfbakterieë is. Hierdie isolate en/of die bakteriosiene wat dit produseer, kan dus moontlik toepassing in die voedselfermentasiebedryf vind.
Die fisiologiese resultate, biochemiese toetse en suikerfermentasieprofiele het almal dieselfde
resultate vir die sewe isolate, wat indikatief van enterococci was, gelewer. Die identifisering deur 16S
rRNA-basispaaropeenvolging het onthul dat die sewe getoetste isolate almal Enterococccus faecium was.
RAPD-PKR-vingerafdrukke het dieselfde profiel vir die sewe rasse gelewer, wat bevestig dat die
rasse almal identies op genetiese vlak was. Deur die molekulêre gewig vas te stel deur middel van SDSPAGE,
het dit getoon dat die peptiede kleiner as 4.6 kDa in grootte is. PKR-amplifikasie van die
enterosien-P geen, die bepaling van basispaaropeenvolging en BLAST-soekresultate het bevestig dat al
agt rasse die enterosien-Pgeen van Ent. faecium bevat.
Die enterosien wat in hierdie studie getoets is, was hitte-stabiel teen 100°C (30 min), maar het
50% van sy aktiwiteit teen 121°C (15 min) verloor. Faktore soos bakteriosienproduksie en
hittebestandheid, is van die vele faktore wat enterococci in staat stel om dominant in gefermenteerde produkte, soos suiwelprodukte of vleis te wees. Enterococci wat bakteriosiene produseer het dus
potensiële toepassings in verskeie kossoorte en gefermenteerde produkte. Die pH-toetse het getoon dat
enterosien-P oor ‘n breë pH spektrum (2-10) aktief was. Enterosienaktiwiteit oor ‘n wye pH spektrum
maak dit potensieel meer geskik as natuurlike preserveermiddels vir kossoorte en gefermenteerde
produkte waar produkte versuur word of die pH afneem as gevolg van natuurlike MSB wat teenwoordig
is. Dit het ook potensiële toepassings in enologiese prosessering waar pH-vlakke so laag as 3 en 4 is.
Proteolitiese ensiembehandelings met lisosiem, lipase, litikase en katalase kon nie
enterosienaktiwiteit inhibeer nie. Daar is getoon dat hul antimikrobiese aktiwiteit onafhanklik was van
lipiede, koolhidraatdele óf waterstofperoksied. α-Chymotripsien en proteïenase-K het enterosien onaktief
gemaak, wat getoon het dat die samestelling proteïenagtig van nature is.
Bakteriosienproduksie wat in twee van die stamme #16.3 en 128.1 getoets is, het ooreengestem
met die eksponensiële groeifase wat na 6 ure van inkubasie teen 30°C plaasgevind het, en wat ‘n
aanduiding is van primêre metabolitiese kinetika. Die hoogste produksie van 400 AU/ml is na agt ure
waargeneem en is vir etlike ure (46 uur) in die stasionêre fase gehandhaaf. Die bakterie-dodende effek
van die selvrye supernatant van #16.3 en 128.1 teenoor die sensitiewe kultuur van Lactobacillus pentosus
DSM 20314 is duidelik gedemonstreer deur totale inhibisie van groei vir die grootste deel van die
eksperimentele periode, terwyl die kontrole eksponensieel deur die hele eksperiment toegeneem het.
Hierdie studie het dus die isolering en identifisering van Ent. faecium-stamme, ‘n genus wat baie
selde gevind word in ‘n wynomgewing, vanuit wyn bevestig. Alhoewel daar gespekuleer kan word oor
die oorsprong van hierdie bakterie in wyn bv. menslike hantering en besmette water, het hierdie rasse wel
enterosien geproduseer en daarom die potensiaal om ‘n rol te speel in beheer teen verskeie bederf-MSB-genera. / TIA, NRF and THRIP
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/80322 |
| Date | 03 1900 |
| Creators | Ndlovu, Joseph Buyani |
| Contributors | Du Toit, Maret, Hamman, Heidi, 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 | 96 p. : ill. |
| Rights | Stellenbosch University |
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