Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Wine quality is greatly influenced by the number of microorganisms, which occur
throughout the winemaking process. Yeasts are responsible for the alcoholic
fermentation, the lactic acid bacteria (LAB) are responsible for malolactic
fermentation (MLF), while acetic acid bacteria (AAB) are responsible for converting
ethanol to acetic acid. These microorganisms are present on the grapes and in the
cellar and these consequently serve as gateways to the fermentation tanks where
they will affect the wine quality. However, these microorganisms can be seen either
as beneficial or as wine spoilage microorganisms, depending on the conditions that
prevail throughout the winemaking process. It is thus very important to prevent any
process that could lead to the lowering of the wine quality. In this regard, some of the
factors that should always be evaluated include the quality of the grapes, winemaking
techniques and quality control.
One of the measures that have been implemented during winemaking to ensure
the microbial stability is the use of chemical preservatives. Sulphur dioxide (502) has
been, and is, used widely as primary preservative in winemaking. However, an
ever-increasing consumer resistance against the use of chemical preservatives has
developed as it poses possible health risks and decreases the sensorial quality of
wine. An alternative approach to chemical preservation that has triggered numerous
new investigations, is biological preservation or biopreservation. This is the use of the
natural microbial flora and/or their antimicrobial products, such as bacteriocins, to
inhibit or destroy the other sensitive microorganisms that are unwanted in the same
environment.
Evidence in the wine industry has shown that bacterial spoilage still is a very
common problem in many wineries. This bacterial spoilage can lead to, amongst
other, two main problems, which are of great concern to winemakers. This include
high levels of volatile acidity, resulting in the wine having a vinegary off-flavour, and
sluggish/stuck fermentation, which is the result of compounds such as acetic- and
other fatty acids that causes inhibition of the yeast's growth. With acetic acid being
the common link in both cases, it became evident that investigations should be
performed on the main producer of acetic acid, namely AAB. As a result, AAB turned
out to be one of the main spoilage microorganisms associated with winemaking.
Most of the research on biopreservation in the food and beverage industry has
been performed on the Gram-positive LAB. The fact that their spectrum of inhibition
currently excludes most Gram-negative bacteria, specifically AAB, indicated that AAB
should be screened in search of possible antimicrobial compounds that could be
applied to control their cell numbers during winemaking. No evidence of antimicrobial
action amongst AAB could be found in literature, therefore this work was considered
novel.
The main objectives of this study were to screen wine isolates of AAB for the
production of antimicrobial compounds. This was followed by the isolation and preliminary characterisation of the antimicrobial substances produced. Various
attempts to optimise the production of the antimicrobial compounds and isolation
procedures, were also included. This study forms part of a larger research
programme that has been initiated at the Institute for Wine Biotechnology at
Stellenbosch University on the biopreservation in wine.
Our results indicated that possible antimicrobial compounds of proteinaceous
nature, produced by AAB isolated from wine, do exist. It was found that two different
species of AAB, namely Acetobacter aeeti and Gluconobacter frateurii, produced
antimicrobial compounds that inhibited other species of AAB. Preliminary results
indicated that these compounds are heat sensitive and stable in a wide pH range. It
was also shown that after the action of proteolytic enzymes, such as proteinase K
and a-chemotrypsin, all inhibitory activity was lost. This study also revealed the
existence of the species Gluconobacter frateurii, which have not yet been associated
with the winemaking environment.
This study made a valuable contribution to the limited amount of information and
understanding of AAB, not only in the wine environment, but also elsewhere. The
results and findings of this research would serve as platform for further projects. This
might soon lead to the development of antimicrobial substances or tailored
wine-yeasts with antimicrobial abilities, which can be applied during winemaking to
assist the winemaker in combatting high cell numbers and subsequent spoilage by
AAB. / AFRIKAANSE OPSOMMING: Wynkwaliteit word beïnvloed deur 'n verskeidenheid van mikroorganismes wat
regdeur die wynrnaakproses teenwoordig is. Die giste is vir die alkoholiese
fermentasie, die melksuurbakterieë (MSB) vir die appelmelksuurgisting, terwyl die
asynsuurbakterieë (ASB) vir die omskakeling van etanol na asynsuur verantwoordelik
is. AI hierdie mikroorganismes is teenwoordig op die druiwe en in die kelder, en dit
dien gevolglik as 'n weg waardeur hulle in die fermentasietenke kan kom om
sodoende die wynkwaliteit te beïnvloed. Hierdie mikroorganismes kan egter gesien
word as óf voordelig óf as wynbederfmikroorganismes, afhangende van die
heersende kondisies gedurende die wynrnaakproses. Dit is daarom baie belangrik
om enige proses te voorkom wat tot 'n verlaging in wynkwaliteit kan lei. Wat
laasgenoemde aanbetref, is daar sekere faktore wat altyd geëvalueer moet word,
naamlik die druifkwaliteit, wynrnaaktegnieke en kwaliteitsbeheer.
Een van die maatreëls wat geïmplementeer is om mikrobiologiese stabiliteit
tydens die wynrnaakproses te handhaaf, is die gebruik van chemiese
preserveermiddels. Swaweidioksied (S02) word algemeen gebruik as primêre
preserveermiddel tydens wynrnaak. Daar is egter 'n toenemende
verbruikersweerstand teen die gebruik van chemiese preserveermiddels, aangesien
dit moontlike gesondheidsrisiko's kan inhou, asook tot 'n verlaging in sensoriese
kwaliteit van die wyn kan lei. 'n Alternatiewe benadering vir chemiese preservering,
wat reeds tot verskeie nuwe ondersoeke gelei het, is biologiese preservering of
biopreservering. Dit is die gebruik van die natuurlike mikroflora en/of hulle
antimikrobiese produkte, soos bv. bakteriosiene, om die sensitiewe mikroorganismes
wat in dieselfe omgewing voorkom, se groei te inhibeer óf om hulle dood te maak.
Aanduidings vanuit die wynbedryf dui daarop dat bakteriese bederf steeds 'n
algemene probleem is wat in baie kelders ondervind word. Hierdie bakteriese bederf
kan onder andere twee hoofprobleme veroorsaak, wat 'n groot bekommernis vir
verskeie wynmakers is. Dié probleme sluit in hoë vlakke van vlugtige suurheid, wat
gevolglik die wyn 'n asyn-afgeur gee, en slepende/gestaakte fermentasies, wat die
gevolg is van komponente soos asynsuur en ander vetsure, wat die gis se groei
inhibeer. Die feit dat asynsuur die gemeenskaplike faktor in beide gevalle was, het
daarop gedui dat 'n ondersoek rakende die hoofproduseerder van asynsuur, naamlik
ASB, benodig word. ASB word gevolglik as een van die hoofbederforganismes wat
met die wynrnaakproses geassosieer word, beskou.
Die meeste navorsing oor biopreservering in die voedsel -en drank bedryf is op
die Gram-positiewe MSB gedoen. Die spektrum van inhibisie van die bakteriosiene
van MSB sluit egter die meeste Gram-negatiewe bakterieë uit, veral ASB, en dit dui
daarop dat ASB gesif moet word in 'n soektog na antimikrobiese substanse wat
moontlik gebruik kan word om hul getalle tydens die wynrnaakproses te beheer.
Geen bewyse kon tot dusver uit die literatuur gekry word met betrekking tot antimikrobiese aktiwiteit teen ASB nie, daarom word hierdie navorsing dus as nuut
beskou.
Hierdie studie se hoofdoelwittewas om die wyn-isolate van ASB vir die produksie
van antimikrobiese peptiede te sif. Dit is gevolg deur die isolasie en voorlopige
karakterisering van die geproduseerde antimikrobiese komponente. Daar is ook
verskeie pogings aangewend om die produksie van die antimikrobiese substanse,
asook die isolasieprosedures, te optimiseer. Hierdie studie vorm deel van 'n groter
navorsingsprogram oor biopreservering van wyn wat deur die Instituut vir
Wynbiotegnologie by die Universiteit van Stellenbosch geïnisieer is.
Die resultate het daarop gedui dat antimikrobiese substanse van proteïenagtige
aard, afkomstig vanaf wyn-isolate van ASB, wel bestaan. Daar is gevind dat twee
veskillende spesies, naamlik Aeefobaefer aeefi en Glueonobaefer frafeurii,
antimikrobiese peptiede produseer, wat ander spesies van ASB kan inhibeer.
Voorlopige resultate het getoon dat hierdie substanse hitte-sensitief is en ook stabiel
is oor 'n wye pH-reeks. Daar was ook aanduidings dat, ná die aksie van proteolitiese
ensieme, soos bv. proteïnase K en a-chemotripsien, al die inhibitoriese aktiwiteit
verlore gegaan het. Hierdie studie het ook die voorkoms van die spesies
Glueonobaefer frafeurii aangedui, wat nog nie tot dusver met die wynrnaakomgewing
geassosieer is nie.
Hierdie studie maak 'n waardevolle bydrae tot die beperkte hoeveelheid inligting
oor en begrip van ASB, nie net in die wynomgewing nie, maar ook in die algemeen in
die natuur. Die bevindinge en resultate van hierdie navorsing sal as basis dien vir
verdere projekte wat sal volg. Dit kan moontlik binnekort lei tot die ontwikkeling van
antimikrobiese substanse, en ook pasgemaakte wyngiste met antimikrobiese
vermoëns, wat tydens die wynrnaakproses gebruik kan word om sodoende die
wynmaker in staat te stelom die hoë bakteriese getalle en die gevolglike bederf deur
ASB, te beheer.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53479 |
| Date | 03 1900 |
| Creators | Oelofse, Adriaan |
| Contributors | Du Toit, M., Pretorius, I. S., Lambrechts, M. G., 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 | 98 pages : illustrations |
| Rights | Stellenbosch University |
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