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Controlling secondary fermentation with new preservativesAthanassiadis, Constantine Menelaos 11 May 1955 (has links)
Graduation date: 1955
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Isolation and characterisation of the antimicrobial peptides produced by acetic acid bacteriaOelofse, Adriaan 03 1900 (has links)
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.
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Characterisation, evaluation and use of non-Saccharomyces yeast strains isolated from vineyards and mustJolly, N. P. (Neil Paul) 03 1900 (has links)
Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Wine is the product of a complex biological and biochemical interaction between
grapes and different microorganisms (fungi, yeasts, lactic acid bacteria and acetic
acid bacteria, as well as the mycoviruses and bacteriophages affecting them) in
which yeasts play the most important role regarding the alcoholic (primary)
fermentation. These wine-associated yeasts can be divided into Saccharomyces and
non-Saccharomyces yeasts. During fermentation, there is a sequence of dominance
by the various non-Saccharomyces yeasts, followed by Saccharomyces cerevisiae,
which then completes the fermentation. This is especially evident in spontaneously
fermenting must, which has a low initial S. cerevisiae concentration. Some non-
Saccharomyces yeasts can also be found throughout the fermentation. The non-
Saccharomyces presence in the fermentation can affect wine quality, either positively
or negatively. A positive contribution could be especially useful to improve wines
produced from grape varieties with a neutral flavour profile due to non-optimal
climatic conditions and/or soil types. As part of a comprehensive South African
research programme, the specific objectives of this study were: the isolation of
indigenous non-Saccharomyces yeasts from vineyards and musts; the identification
of these isolates; the characterisation and evaluation of predominant species under
winemaking conditions; and the development of a protocol for their use in enhancing
wine quality.
Initially, 720 isolates representing 24 different species, were isolated from grape
(vineyard) and must samples taken over three vintages from four distinctly different
wine producing regions. The isolates were characterised and grouped utilising
biochemical profiles and DNA karyotyping, whereupon representative isolates were
identified. The yeast species that had the highest incidence of predominance in the
vineyard was Kloeckera apiculafa. However, some vineyard samples were
characterised by low numbers or absence of this yeast, which is not according to
generally accepted norms. Other species that also predominated in a few of the
vineyard samples were Candida pulcherrima, Kluyveromyces thermofolerans,
Rhodotorula sp. and Zygosaccharomyces bailii. Generally, there was a greater
diversity of yeasts in the processed must than from the vineyard samples. Furthermore, while each sample showed a different yeast population, no pattern
linking species to climatic zone was observed.
Four species i.e. Candida collieulosa, Candida pulcherrima, Candida stel/ata and
Kloeckera apiculata, were found to predominate in grape must samples.
Representative strains consequently received further attention during laboratory and
small-scale winemaking trials. A protocol was developed whereby individual species
could be used in co-inoculated fermentations with S. cerevisiae in the small-scale
production of wine. An improvement in wine quality was achieved and it was found
that there was a link between specific species and grape cultivar. The ability of
C. pulcherrima to improve Chenin blanc wine quality was investigated further. Results
over three vintages showed that the wine produced by the co-inoculated fermentation
was superior to that of a reference wine (produced by S. cerevisiae only). The
improvement in wine quality was not linked to increased ester content nor were the
standard chemical analyses adversely affected. The effects of pH and wine
production parameters i.e. 802, fermentation temperature and use of di-ammonium
phosphate (DAP), on this yeast followed the same pattern as that known for
S. cerevisiae. This study was successfully completed and the developed protocol can
be used for the improvement of Chenin blanc wine where additional aroma and
quality is needed. / AFRIKAANSE OPSOMMING: Wyn is die produk van 'n komplekse biologiese en biochemiese interaksie tussen
druiwe en mikroorganismes (swamme, giste, melksuurbakterieë, asynsuurbakterieë,
asook die mikovirusse en bakteriofage wat hul beïnvloed) waar gis die belangrikste
rol speel ten opsigte van die alkoholiese (primêre) fermentasie. Die betrokke giste
kan in Saccharomyces- en nie-Saccharomyces-giste verdeel word. Tydens gisting
vind daar 'n opeenvolging van dominansie deur die verskillende nie-Saccharomyces
giste plaas, gevolg deur Saccharomyces cerevisiae, wat dan die gisting voltooi. Dit is
veral in spontaan fermenterende mos, waarin aanvanklik lae konsentrasies
S. cerevisiae-gisselle voorkom, waarneembaar. Sekere nie-Saccharomyces-giste
kan ook regdeur die verloop van fermentasie gevind word. Die teenwoordigheid van
nie-Saccharomyces-giste kan 'n bydrae maak tot wynkwaliteit, hetsy positief of
negatief. 'n Positiewe bydrae kan veral nuttig wees vir die verbetering van wyn
geproduseer van druifsoorte met neutrale geurprofiele as gevolg van nie-optimale
klimaatstoestande en/of grondtipes. As deel van 'n uitgebreide Suid-Afrikaanse
navorsingsprogram, was die doelwitte van hierdie studie soos volg: die isolasie van
inheemse nie-Saccharomyces-giste vanuit wingerde en mos; die identifikasie van
hierdie isolate; die karakterisering en evaluering van spesies wat tydens
wynbereiding oorheers; en die ontwikkeling van 'n protokol waarin geselekteerde nie-
Saccharomyces-giste gebruik kan word vir die verbetering van wynkwaliteit.
Druif- en mosmonsters is oor drie oestye vanuit vier duidelik onderskeibare
wynproduserende gebiede geneem en 720 isolate, verteenwoordigend van 24
verskillende spesies, is hieruit geïsoleer. Hierdie isolate is volgens biochemiese
profiele en DNA-kariotipering gekarakteriseer en gegroepeer waarna
verteenwoordigende isolate geïdentifiseer is. Die gisspesie wat die meeste in
wingerde voorgekom het, was Kloeckera apiculata. Sommige wingerde is egter deur
lae getalle of afwesigheid van dié gis gekenmerk, In feit wat afwyk van die algemeen
aanvaarde norm. Ander spesies, nl. Candida pulcherrima, Kluyveromyces
thermotolerans, Rhodotorula sp. en Zygosaccharomyces bailii, het ook in enkele
gevalle in die wingerdmonsters oorheers. Oor die algemeen was daar 'n groter
diversiteit van giste in die geprosesseerde mos as in die wingerdmonsters. Verder is elke monster gekenmerk deur verskillende gispopulasies, maar geen verband tussen
gisspesie en klimaatsone is waargeneem nie.
Vier spesies, nl. Candida collieulosa, Candida pulcherrima, Candida stel/ata en
Kloeckera apiculata, het in hoë getalle in die druiwemosmonsters oorheers en
verteenwoordigende rasse het verdere aandag tydens laboratorium- en kleinskaalse
wynmaakproewe geniet. 'n Protokol, waar hierdie rasse individueel gebruik is in
gesamentlike geïnokuleerde fermentasies met S. cerevisiae vir die kleinskaalse
produksie van wyn, is ontwikkel. 'n Verbetering in wynkwaliteit is verkry en daar is 'n
verband tussen spesifieke gisspesies en druifvariëteit gevind. Gevolglik is die vermoë
van C. pulcherrima om die gehalte van Chenin blanc wyn te verbeter, verder
ondersoek.
Resultate oor drie oesjare het gewys dat die wyn wat met die C. pulcherrima /
S. cerevisiae kombinasie geproduseer is, beter was as 'n verwysingswyn (deur slegs
S. cerevisiae geproduseer). Die waargenome verbetering in wynkwaliteit was egter
nie aan 'n verhoging in esterinhoud te danke nie en die standaard chemiese analises
het geen negatiewe afwyking uitgewys nie. Verder is gevind dat die effek van pH en
wynproduksieparameters, nl. die gebruik van S02, fermentasietemperatuur en die
gebruik van di-ammoniumfosfaat (DAP), dieselfde patroon as die bekend vir
S. cerevisiae gevolg het. Die ontwikkelde protokol kan nou aangewend word waar
verhoogde Chen in blanc wynaroma en kwaliteit verlang word.
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Formation of mousy off-flavour in wine by lactic acid bacteria / by Peter James Costello.Costello, Peter James January 1998 (has links)
Bibliography: leaves 200-214. / xi, 214 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Three structurally related compounds, 2-acetyltetrahydropyridine (ACTPY), 2-ethyltetrahydropyridine (ETPY) and N-heterocycle, 2-acetyl-1-pyrroline (ACPY), were quantified and found to be unique components of mousy wines. 35 lactic acid bacteria (LAB) were screened for the ability to produce mousy off-flavour. In addition to Lactobacillus brevis and L. cellobiosus, a diversity of LAB species, particularly heterofermentative Lactobacillus spp. and Oenococcus oeni exhibited this ability in a range of ethanolic and wine-based media. The substrates and metabolism of mousy compound formation by LAB were also investigated. A pathway for the formation of ACPY and ACTPY by heterofermentative LAB was proposed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Horticulture, Viticulture & Oenology, 1999
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Formation of mousy off-flavour in wine by lactic acid bacteriaCostello, Peter James. January 1998 (has links) (PDF)
Bibliography: leaves 200-214. Three structurally related compounds, 2-acetyltetrahydropyridine (ACTPY), 2-ethyltetrahydropyridine (ETPY) and N-heterocycle, 2-acetyl-1-pyrroline (ACPY), were quantified and found to be unique components of mousy wines. 35 lactic acid bacteria (LAB) were screened for the ability to produce mousy off-flavour. In addition to Lactobacillus brevis and L. cellobiosus, a diversity of LAB species, particularly heterofermentative Lactobacillus spp. and Oenococcus oeni exhibited this ability in a range of ethanolic and wine-based media. The substrates and metabolism of mousy compound formation by LAB were also investigated. A pathway for the formation of ACPY and ACTPY by heterofermentative LAB was proposed.
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Optimization of fermentation processes for the production of indigenous fruit wines (Marula)Fundira, Margaret 03 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: The importance of indigenous fruit wines is not well researched and documented. There
is a need to develop and exploit these valuable food resources through improved
production practices, storage, preservation and utilization technologies. The maruia fruit
is beneficial in many ways, it can be used for making juice, jam, beer or can be eaten as
a whole fruit. The highly nutritive nature of the fruit, its distinctive tropical flavor, its wild
occurrence and demand by the local and international communities for the by-products
of the fruit necessitated efforts to optimize the technological processes for the
production of the possible by-products. This study focuses on the fermentation
technology of the maruia fruit.
The effect of enzymes prior to the fermentation process and post-fermentation
was evaluated. For pre-fermentation processes we focused on the ability of commercial
enzymes to increase juice yield, improve the clarification and filterability. For pre- and
post-fermentation applications, aroma release was considered. The results indicated a
significant increase in the yield depending on the enzyme used. An increase of at least
2% was recorded and a maximum of 12% yield increase was observed. The enzymes
also had a phenomenal effect on the release of bound monoterpenes and hence
enhancing the flavor of the juice. The panel of judges confirmed the results from the gas
chromatography analyses by noting an increase in flavor intensity in the enzyme treated
juice.
The possibility of selecting a yeast strain that performs best during the
fermentation of maruia pulp was also looked at. This study aimed at selecting a strain
that produces wine and distillate with the typical maruia flavor complex. We showed the
effect of the different yeast strains, in the wines and distillates, on the principal volatile
compounds. We then correlated the performance of the different strains as perceived by
the panel to the various volatile compounds. The effect of fermentation temperature on
the performance of the different yeast strains was also considered. Fermenting the
maruia pulp at different temperatures resulted in the production of wines and distillates
with different volatile profiles for the different yeast strains. The wines and distillates
fermented at a low temperature of 15°C were preferred to the wines and distillates
fermented at 30°C. However, not all strains performed well at 15°C, strains like NT116
performed better at 30°C. The different commercial strains produced wines and
distillates with significantly different flavor profiles. These differences in the flavor profiles
were reflected in the sensory evaluation where, depending on the interaction of the
volatile compounds some wines and distillates were preferred to others. The effect of the
different commercial enzymes and yeast strains should thereof be further evaluated and
optimized on a larger scale. This would greatly help prevent variation in quality of the
fermented by-products of the maruia fruit. / AFRIKAANSE OPSOMMING: Die belang van inheemse vrugtewyne is nie goed nagevors en gedokumenteer
nie. Daar is 'n behoefte om hierdie waardevolle voedselbronne te ontwikkel en te benut,
deur verbeterde produksiepraktyke, storing, preservering en benuttingstegnologieë. Die
maroelavrug is veelsydig op baie wyses, deurdat dit gebruik word vir die maak van sap,
konfyt, bier, of as heel vrug geëet kan word. Die vrug is hoog in voedingswaarde, het In
kenmerkende tropiese geur, kom wild voor, en is in aanvraag by plaaslike en
internasionale gemeenskappe vir die by-produkte van die vrug. Dit maak dit essensieel
om die tegnologiese prosesse vir die produksie van hierdie moontlike by-produkte te
optimiseer. Hierdie studie fokus op die fermentasie-tegnologie van die maroelavrug.
Die effek van ensieme voor en na die fermentasie-proses is geëvalueer. Vir
prosesse wat voor fermentasie plaasvind, het ons gefokus op die vermoë van
kommersiële ensieme om sapopbrengs te verhoog, asook om verheldering en filtrering
te verbeter. Vir beide voor- en na-fermentasie toepassings is die vrystelling van aroma
gemonitor. Die resultate dui op 'n betekenisvolle verhoging in die sapopbrengs,
afhangende van die ensiem wat gebruik is. 'n Verhoging van ten minste 2% is
opgeteken, en 'n maksimum van 12% opbrengsverhoging is waargeneem. Die ensieme
het ook 'n geweldige effek op die vrystelling van gebonde monoterpene gehad, en dus
die verhoging in die geur van die sap. Die proepaneel het die resultate bevestig van die
gaschromatografie-analises, deur 'n verhoging in die geurintensiteit in die ensiembehandelde
sap te bemerk.
Daar is ook gekyk na die moontlikheid om 'n gisras te selekteer wat die beste
presteer tydens die fermentasie van maroela-pulp. Hierdie studie het die doelstelling
gehad om In gisras te selekteer wat wyn en distillaat produseer met In tipiese maroelageurkompleks.
Ons het die effek van verskillende gisrasse aangedui in die wyne en
distillate, op grond van van vlugtige komponente. Ons het dan die prestasie van die
verskillende rasse, soos waargeneem deur die paneel, gekorrelleer met die verskeie
vlugtige komponente. Die effek van fermentasie-temperatuur op die werkverrigting van
die verskillende gisrasse is ook in ag geneem. Fermentasie van die maroela-pulp by
verskillende temperature het gelei tot die produksie van wyne en distillate met
verskillende vlugtige profiele vir die verskillende gisrasse. Die wyne en distillate wat by
In laer temperatuur van 15°C gefermenteer is, is verkies bo die wyne en distillate wat by
30°C gefermenteer is. Alle rasse het egter nie baie goed presteer by 15°C nie, soos
byvoorbeeld NT116 wat beter presteer het by 30°C. Die verskillende kommersiële rasse
het wyne en distillate geproduseer met betekenisvol verskillende geurprofiele. Hierdie
verskille in geurprofiele is gereflekteer in die sensoriese evaluering waar, afhangende
van die interaksie van die vlugtige komponente, sommige wyne en distillate bo ander
verkies is. Die effek van die verskillende kommersiële ensieme en gisrasse moet verkieslik verder op groter skaal geëvalueer en geoptimiseer word. Dit sal veral help om
variasie in kwaliteit van die gefermenteerde by-produkte van die maroelavrug te
voorkom.
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Genetic characterisation and breeding of wine yeastsVan der Westhuizen, T. J. (Theunes Johannes) January 1990 (has links)
Thesis (MSc)--Stellenbosch University, 1990. / ENGLISH ABSTRACT: To remain competitive in the market place, the South African wine industry will
have to direct well-planned yeast strain-development programmes. However, the
winemaker can only benefit from the extensive biochemical and molecular
information of the yeast cell and the impressive arsenal of genetic techniques
available, if the wine industry defines its requirements in genetic terms. The
successful application of these genetic and recombinant deoxyribonucleic acid
(DNA) techniques in breeding programmes depends on the availability of rapid
and reliable techniques to differentiate between parental and hybrid strains.
Ten strains of Saccharomyces cerevisiae used for commercial production of
wine in South Africa, were characterised by electrophoretic banding patterns of
total soluble cell proteins, DNA restriction fragments and chromosomal DNA.
Variations in the protein and DNA profiles of strains N6, N21, N66, N76, N95
and N97 were apparent in the number, position and intensity of the bands.
Strains N93 and N181 originated from the same culture and, as expected,
displayed the same characteristic protein, DNA restriction fragment and
chromosomal banding patterns. Similar protein and DNA profiles were also
obtained for killer strain N96 and strain N91. Strain N91 is a derivative of strain
N96, cured of the K2 killer character. Results obtained by electrophoretic
fingerprinting and karyotyping corresponded well, indicating that these
techniques are valuable in the identification and quality control of industrial wine
yeasts.
The value of electrophoretic fingerprinting and karyotyping was also
demonstrated in a breeding programme. The aim of this breeding programme
was to obtain hybrids that combine the desired oenological characteristics of
strains N76 and N96, and of strains N96 and N181. The protein banding patterns
of hybrids USM21, USM22 and USM23 were identical and contained a
combination of prominent unique bands present in the profiles of parental
strains, N76 and N96H (N96H is a haploid derived from N96). The DNA
restriction fragment profiles of hybrids USM21, USM22 and USM23 contained
slight variations, whereas their profiles were quite different from those of their
parental strains, N76 and N96H. The contour clamped homogeneous electric
field (CHEF) karyotypes of hybrids USM21, USM22 and USM23 were identical
but differed from those of their parental strains, N76 and N96H. The protein
profiles of hybrid USM30 and its parental strains, N96H and N181, were similar,
whereas their DNA restriction fragment banding patterns and CHEF karyotypes
showed discrete differences. In conclusion, protein and DNA fingerprinting techniques were found to be valuable in selecting four hybrid killer strains after
mass spore-cell mating. These four killer hybrids contain desirable oenological
properties long sought after by the South African wine industry. Fermentation
trials and evaluation of these hybrids were conducted independently by the
Deparment of Oenology, University of Stellenbosch and by Stellenbosch Farmers'
Winery and they have now been released for commercial wine production. / AFRIKAANSE OPSOMMING: Om mededingend in die handel te bly, sal die Suid-Afrikaanse wynbedryf weloorwoe
gisras-ontwikkelingsprogramme moet loads. Die wynmaker sal egter
slegs voordeel kan trek uit die omvattende biochemiese en molekul...Lre inligting
oor die gissel en die indrukwekkende arsenaal van genetiese tegnieke wat
beskikbaar is, indien die wynbedryf sy vereistes in genetiese terme definieer. Die
suksesvolle toepassing van hierdie genetiese en rekombinante
deoksiribonuklei"ensuur (DNA) tegnieke in telingsprogramme sal afhang van die
beskikbaarheid van vinnige en betroubare tegnieke om tussen ouerlike en
hibried-rasse te onderskei.
Tien rasse van Saccharomyces cerevisiae wat vir kommersiele
wynproduksie in Suid-Afrika gebruik word, is met behulp van elektroforetiese
bandpatrone van totale oplosbare selprotei"ene, DNA-restriksiefragmente en
chromosomale DNA gekarakteriseer. Variasies in die protei"en- en DNA-profiele
van rasse N6, N21, N66, N76, N95 en N97 het geblyk uit die aantal, posisie en
intensiteit van die bande. Rasse N93 en N181 het uit dieselfde kultuur ontstaan
en het, soos verwag, dieselfde karakteristieke protei"en-, DNA-restriksiefragmenten
chromosomale bandpatrone getoon. Soortgelyke protei"en en DNA profiele is
ook vir killerras N96 en ras N91 verkry. Ras N91 is 'n variant van ras N96 wat die
K2 killerkenmerk verloor het. Resultate wat met behulp van elektroforetiese
vingermerking en kariotipering verkry is, het goed ooreengestem en dui daarop
dat hierdie tegnieke waardevol is vir die identifisering en beheer van industriele
giste.
Die waarde van elektroforetiese vingermerking en kariotipering in
telingsprogramme is ook gedemonstreer. Die doel van hierdie telingsprogram
was om hibriede te kry waarin die gewenste kenmerke van rasse N76 en N96, en
van rasse N96 en N181, gekombineer is. Die protei"en-bandpatrone van hibriede
USM21, USM22 en USM23 was identies en het 'n kombinasie van prominente
unieke bande, teenwoordig in die profiele van hul ourlike rasse, N76 en N96H
(N96H is 'n haploi"de afstammeling van N96), bevat. Die DNArestriksiefragment-
profiele van hibriede USM21, USM22 en USM23 toon geringe
onderlinge verskille, maar hul profiele het wesenlik van die van hul ouerlike rasse,
N76 en N96H, verskil. Die kontoergeklampde-homogene-elektriese-veld
(CHEF) elektroforetiese kariotipes van hibriede USM21, USM22 en USM23 was
identies, maar het verskil van die van hul ouerlike rasse, N76 en N96H. Die
protei"enprofiele van hibried USM30 en sy ouerlike rasse, N96H en N181, was
soortgelyk, terwyl hul DNA-restriksiefragment-bandpatrone en CHEF-kariotipes diskrete verskille getoon het. Ten slotte is gevind dat prote'ien- en DNAvingermerkingstegnieke
waardevol was in die seleksie van vier hibried-killerrasse
na massa spoor-sel paring. Hierdie vier killerhibriede beskik oor gewenste
wynkundige eienskappe waarna die Suid-Afrikaanse wynbedryf reeds lank soek.
Fermentasie-proewe en evaluering is onafhanklik deur die Departement
Wynkunde, Universitiet van Stellenbosch en deur Stellenbosch-Boerewynmakery
gedoen en hulle is nou vir kommersiele wynproduksie vrygestel.
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Genetic investigation and characterization of killer toxins secreted by non-Saccharomyces yeastsMehlomakulu, Ngwekazi Nwabisa 04 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: In the current study, two isolates showing killer activity against several wine yeast species in a
previous study were identified to strain level and found to belong to the yeast species Candida
pyralidae. The identified yeast strains and a Kluyveromyces wickerhamii yeast strain used as a
control exhibited killer activity against B. bruxellensis known for its spoilage characteristics in
red wine, and against several strains of the genus Brettanomyces on white and red grape juice
medium. The killer yeasts inhibited neither the growth of S. cerevisiae nor that of the lactic acid
bacteria Oenococcus oeni and Lactobacillus plantarum strains. Yeasts are reported to secrete
killer toxins, which can play a role in yeast microbial interactions under winemaking conditions.
The C. pyralidae strains were found to secrete two novel killer toxins, designated CpKT1 and
CpKT2. These killer toxins were stable and active under winemaking conditions, pH 3.5 - 4.5
and temperature ranges between 15 and 25°C. Ethanol and sugar concentrations found during
winemaking did not affect the activity and stability of these killer toxins. Although, the killer
toxins differed with regards to their biochemical and environmental stability and activity, they
were found to have a similar mode of action. The killer toxins induced a fungistatic effect on
B. bruxellensis sensitive cells in addition to binding to the cell wall of the sensitive cells, inducing
cell surface and plasma membrane damage as did the Kwkt killer toxin secreted by
K. wickerhamii. According to the author’s knowledge this is the first report on the identification of
novel killer toxins secreted by C. pyralidae strains isolated from a wine environment as well as
the identification of the mode of action of killer toxins on B. bruxellensis cells. This indeed
provides great research scope in this field.
The exoproteomes consisting of the killer toxins Kwkt, CpKT1 and CpKT2 revealed the
presence of exo-glucanases and glucosidases, respectively. The enzymes KwExg1 (exoglucanase)
and KwSun4 (glucosidase) retrieved from K. wickerhamii’s exoproteome were
identified as the potential toxins, but their killer activity could not be confirmed. These findings
suggest that hydrolytic enzymes possess killer activity, as previously reported in literature.
However, further investigation is needed to identify the killer toxins characterized in this study. / AFRIKAANSE OPSOMMING: In die huidige studie is twee isolate wat in ’n vorige studie “killer” aktiwiteit teenoor verskeie
wyngisspesies vertoon het, tot op rasvlak geïdentifiseer en daar is gevind dat hulle aan die
gisspesie Candida pyralidae behoort. Die geïdentifiseerde gisrasse en ’n Kluyveromyces
wickerhamii gisras wat as kontrole gebruik is, het “killer” aktiwiteit getoon teenoor B.
bruxellensis, wat bekend is vir sy bederfkarakter in rooi wyn, en ook teenoor verskeie rasse van
die genus Brettanomyces in wit en rooi druiwesapmedium. Die “killer” giste het nie die groei van
óf S. cerevisiae óf van die melksuurbakteria Oenococcus oeni en Lactobacillus plantarum-rasse
geïnhibeer nie. Giste word berig om “killer” gifstowwe uit te skei, wat ’n rol kan speel in gis
mikrobiese interaksies onder wynbereidingstoestande.
Die C. pyralidae-rasse is gevind om twee nuwe “killer” gifstowwe af te skei, wat CpKT1 en
CpKT2 genoem is. Hierdie “killer” gifstowwe was stabiel en aktief onder
wynbereidingstoestande, pH 3.5 - 4.5 en temperatuur tussen 15 en 25°C. Die etanol- en
suikerkonsentrasies wat onder wynbereiding voorkom, het nie die aktiwiteit en stabiliteit van
hierdie “killer” gifstowwe beïnvloed nie. Hoewel die “killer” gifstowwe met betrekking tot hulle
biochemiese en omgewingstabiliteit en aktiwiteit verskil het, is daar gevind dat hulle ’n eenderse
modus van aksie het. Die “killer” gifstowwe het ’n fungistatiese effek op B. bruxellensis
sensitiewe selle geïnduseer, buiten dat dit aan die selwand van die sensitiewe selle gebind het,
en het seloppervlak- en plasma-membraanskade geïnduseer, net soos die Kwkt “killer” gifstof
wat deur K. wickerhamii afgeskei is. So ver die skrywer weet, is hierdie die eerste verslag van
die identifisering van nuwe “killer” gifstowwe wat deur C. pyralidae rasse afgeskei word wat uit
’n wynomgewing geïsoleer is, asook van die identifikasie van die modus van aksie van “killer”
gifstof op B. bruxellensis selle. Dit verbreed dus beslis die navorsingsomvang van hierdie
gebied.
Die eksoproteome, bestaande uit die “killer” gifstowwe Kwkt, CpKT1 en CpKT2, het die
teenwoordigheid van ekso-glukanases en glukosidases onderskeidelik onthul. Die ensieme
KwExg1 (eksoglukanase) en KwSun4 (glukosidase) wat vanuit K. wickerhamii se eksoproteoom
herwin is, is as die potensiële gifstowwe geïdentifiseer, maar hulle “killer” aktiwiteit kon nie
bevestig word nie. Hierdie bevindings suggereer dat hidrolitiese ensieme “killer” aktiwiteit besit,
soos voorheen in die literatuur berig is. Verdere ondersoeke word egter benodig om die “killer”
gifstowwe wat in hierdie studie gekarakteriseer is, te identifiseer.
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Characterisation of L-malic acid metabolism in strains of Saccharomyces and the development of a commercial wine yeast strain with an efficient malo-ethanolic pathwayVolschenk, Heinrich 12 1900 (has links)
Dissertation (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: L-Malic and tartaric acid are the most prominent organic acids in wine and playa crucial role in
winemaking processes and wine quality, including the organoleptic quality and the physical,
biochemical and microbial stability of wine. The production of premium wines depends on the
oenologist's skill to accurately adjust wine acidity to obtain the optimum balance with other wine
components to produce wine with optimum colour and flavour.
Strains of Saccharomyces, in general, rarely degrade L-malic acid completely in grape must during
alcoholic fermentation, with relatively minor modifications in total acidity during vinification. The
degree of L-malic acid degradation, however, varies from strain to strain. Some strains of
Saccharomyces are known to be able to degrade a higher percentage of L-malic acid, but the
underlying reason for this phenomenon is unknown. The underlying mechanisms of this phenomenon
have been partially revealed during preliminary transcriptional regulation research during this study.
In contrast, S. pombe cells can effectively degrade up to 29 gil L-malic acid via the malo-ethanolic
pathway that converts L-malic acid to pyruvate and CO2, and ultimately to ethanol under fermentative
conditions. A number of reasons for the weak degradation of L-malic acid in Saccharomyces
cerevisiae have been postulated. Firstly, S. cerevisiae lacks the machinery for the active transport of
L-malic acid found in S. pombe and relies on rate-limiting simple diffusion for the uptake of
extracellular L-malic acid. Secondly, the malic enzyme of S. cerevisiae has a significantly lower
substrate affinity for L-malic acid (Km = 50 mM) than that of S. pombe (Km = 3.2 mM), which
contributes to the weaker degradation of L-malic acid in S. cerevisiae. Lastly, the mitochondrial
location of the malic enzyme of S. cerevisiae, in contrast to the cytosolic S. pombe malic enzyme,
suggests that the S. cerevisiae malic enzyme is inherently subject to the regulatory effects of
fermentative metabolism.
The malate permease gene tmael) and the malic enzyme gene (mae2) of S. pombe was therefore
cloned and co-expressed in single or multi-copy under regulation of the constitutive S. cerevisiae
3-phosphoglycerate kinase (PGK1) promoter and terminator sequences in a laboratory strain of
S. cerevisiae. This introduced a strong malo-ethanolic phenotype in S. cerevisiae where L-malic acid
was rapidly and efficiently degraded in synthetic and Chardonnay grape must with the concurrent
production of higher levels of ethanol. Functional expression of the malo-ethanolic pathway genes of
S. pombe in a laboratory strain of S. cerevisiae paved the way for the genetic modification of
industrial wine yeast strains of Saccharomyces for commercial winemaking.
A prerequisite for becoming an inherited component of yeast is the stable integration of the
malo-ethanolic genes into the genome of industrial wine yeast strains. Genetic engineering of wine
yeasts strains of Saccharomyces is, however, complicated by the homothallic, multiple ploidy and prototrophic nature of industrial strains of Saccharomyces. Transformation and integration of
heterologous genes into industrial strains of Saccharomyces require the use of dominant selectable
markers, i.e. antibiotic or toxic compound resistance markers. Integration of these markers into the
yeast genome is, however, not acceptable for commercial application due to the absence of long-term
risk assessment and consumer resistance.
A unique strategy for the integration of the S. pombe mae} and mae2 expression cassettes without the
incorporation of any non-yeast derived DNA sequences was. The malo-ethanolic cassette, containing
the S. cerevisiae PGK} promoter and terminator regions together with the S. pombe mae] and mae2
open reading frames, was integrated into the VRA3 locus of an industrial strain of Saccharomyces
bayanus EC 1118 during co-transformation with a phleomycin-resistance plasmid, pUT332. After
initial screening for phleomycin resistance, S. bayanus EC1118 transformants were cured of the
phleomycin-resistance plasmid, resulting in the loss of non-yeast derived DNA sequences. After
correct integration of the mae] and mae2 expression cassettes was verified, small-scale vinification in
synthetic and Chardonnay grape must with stable transformants resulted in rapid and complete
degradation of L-malic acid during the early stages of alcoholic fermentation. Integration and
expression of the malo-ethanolic genes in S. bayanus ECll18 had no adverse effect on the
fermentation ability of the yeast, while sensory evaluation and chemical analysis of the Chardonnay
wines indicated an improvement in wine flavour compared to the control wines, without the
production of any off-flavours. / AFRIKAANSE OPSOMMING: L-Appelsuur en wynsteensuur is die mees prominente organiese sure in wyn en speel 'n kritiese rol in
die wynbereidingsproses en organoleptiese wynkwaliteit, insluitende die fisiese, biochemiese en
mikrobiese stabiliteit van wyn. Die produksie van hoë-kwaliteit wyne berus op die vermoë van 'n
wynmaker om die suurinhoud korrek aan te pas om sodoende 'n gebalanseerde produk met optimale
geur en kleur te produseer.
Saccharomyces rasse kan gewoonlik nie appelsuur volledig tydens alkoholiese gisting benut nie en
dra dus nie noemenswaardig tot 'n verlaging van die totale suurinhoud van wyn by nie. Die mate van
appelsuur afbraak deur Saccharomyces wissel egter van ras tot ras. Sekere Saccharomyces rasse kan
'n groter persentasie appelsuur afbreek, maar die onderliggende rede vir hierdie verskynsel is
onbekend. Die onderliggende meganismes vir hierdie verskynsel is gedurende hierdie studie uitgelig
na afloop van voorlopige transkripsionele regulerings studies op die malaatensiemgeen. In
teenstelling hiermee kan S. pombe tot 29 gIl appelsuur via die malo-alkoholiese padweg afbreek
waartydens appelsuur na pirodruiwesuur en CO2, en uiteindelik na alkoholonder fermentatiewe
toestande omgeskakel word. Verskeie redes vir die swak afbraak van appelsuur deur
Saccharomyces cerevisiae is voorgestel. Eerstens beskik S. cerevisiae nie oor 'n meganisme vir die
aktiewe transport van appelsuur, soos in die geval van S. pombe nie, en is aangewese op die stadige
opname van appelsuur deur eenvoudige diffusie. Tweedens het die S. cerevisiae malaatensiem 'n baie
laer substraataffiniteit vir appelsuur (Km = 50 mM) in vergelyking met die van S. pombe (Km =
3.2 mM), wat verder bydra tot die swak afbraak van appelsuur in S. cerevisiae. Laastens dra die
mitochondriale ligging van die S. cerevisiae malaatensiem in teenstelling met die sitoplasmiese
ligging van die S. pombe malaatensiem, verder by tot die swak afbraak van appelsuur, aangesien die
mitochondria onder fermentatiewe toestande negatief gereguleer word.
Die malaatpermease geen (maely en malaatensiem geen (mae2) van S. pombe is gevolglik gekloneer
en heteroloog in 'n laboratoriumras van S. cerevisiae onder die beheer van die konstitutiewe
3-fosfogliseraat kinase (PGKI) promoter- en termineerdervolgordes uitgedruk. 'n Sterk
malo-alkoholiese fenotipe was duidelik tydens fermentasies met die rekombinante gis in sintetiese en
Chardonnay druiwemos, met 'n gepaardgaande verhoging in alkoholvlakke. Funksionele uitdrukking
van die malo-alkoholiese gene van S. pombe in 'n S. cerevisiae laboratoriumras het die weg vir die
genetiese modifisering van industriële wynrasse van S. cerevisiae vir kommersiële wynfermentasie
gebaan.
Om 'n integrale deel van die gis te word, moet die malo-alkoholiese gene stabiel in die genoom van
industriële wynrasse geïntegreer word. Genetiese manipulering van industriële wynrasse word egter
bemoeilik deur die homotalliese, multi-ploïediese en prototrofiese aard van industriële Saccharomyces rasse. Transformasie en integrasie van heteroloë gene in industriële Saccharomyces
rasse vereis die gebruik van dominante merkers, bv. weerstandbiedendheid teen antibiotika of ander
gifstowwe. Integrasie van hierdie merkers in die gisgenoom is egter nie vir kommersiële toepassing
aanvaarbaar nie weens die afwesigheid van langtermyn risikobepalings en verbruikersweerstand.
Tydens hierdie studie is daar dus gepoog om industriële wynrasse met 'n unieke strategie geneties te
verbeter sodat slegs gis-DNA tydens die integrasie van die S. pombe mae1 en mae2
uitdrukkingskassette in die gisgenoom opgeneem word. Die Malo-alkoholiese integrasiekasset wat
slegs die S. pombe mae1, mae2 oopleesrame en die S. cerevisiae PGK1 promoter en
termineerdervolgordes bevat, is in die URA3 lokus van Saccharomyces bayanus ECll18 geïntegreer
tydens parallelle transformasie met 'n 'phleomycin' weerstandbiedendheidsplasmied. Na seleksie van
transformante op 'phleomycin' -bevattende media, is die S. bayanus EC 1118 transformante in nieselektiewe
kondisies opgegroei sodat verlies van die 'phleomycin' plasmied kon plaasvind. Integrasie
van die mae1 en mae2 uitdrukkingskassette is bevestig en kleinskaalse fermentasies in sintetiese en
druiwemos het 'n vinnige en doeltreffende afbraak van appelsuur in die vroeë fases van die
alkoholiese fermentasie getoon. Integrasie en uitdrukking van die malo-alkoholiese gene in
S. bayanus ECl118 het geen nadelige effek op die fermentasievermoë van die gis getoon nie, terwyl
sensoriese en chemiese ontleding van die Chardonnay wyne 'n verbetering in aroma relatief tot die
kontrole wyne getoon het, met die afwesigheid van enige afgeure.
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Putative promoter sequences for differential expression during wine fermentationsPolotnianka, Renata Martina. January 1996 (has links) (PDF)
Includes bibliographies. This thesis describes the isolation of putative promoter sequences that can produce differential expression of a gene during anaerobic wine fermentations, the use of these sequences in the development of expression vectors and the application of this work to the production of genetically engineered wine yeasts for commercial purposes.
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