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The production of volatile phenols by wine microorganismsNelson, Lisha 12 1900 (has links)
Thesis (MScAgric (Viticulture and Oenology))--Stellenbosch University, 2008. / The production of good quality wine is essential to ensure competitiveness on an international level. Wine quality is usually evaluated for the visual, olfactory and taste characteristics of that specific wine. The winemaking process starts with the grapes in the vineyard followed by oenological practises in the winery until the final wine is bottled. Factors that could influence wine quality include the grape quality from which the wine is made and different techniques used during wine production. Other factors include the presence as well as the interaction between microorganisms found in the grape juice and wine, and the biochemical effect these microorganisms have on certain chemical compounds in the wine. The different microorganisms found in grape juice and wine can either have a negative or positive contribution to the final quality of the wine. During certain stages of the winemaking process the growth and metabolic activity of certain microorganisms is a necessity to produce good wine. During other stages the presence of certain microorganisms can lead to the development of compounds that is regarded as off-flavours and therefore lead to unpalatable wines of low quality.
Yeast strains that naturally present on the grapes and in the winery can also contribute to the final quality of the wine. Brettanomyces yeasts are part of the natural flora of winemaking and can drastically influence the aroma characters of a wine through the production of volatile phenols. The general aroma descriptions of volatile phenols include "smoky", "spicy", "barnyard", "animal" and "medicinal". Although some wine drinkers believe that these characters can add to the complexity of a wine, high levels of volatile phenols is mostly regarded as off-flavours and mask the natural fruity flavours of a wine.
With this study we wanted to generate a better understanding of the effect of different winemaking practises on the production of volatile phenols by B. bruxellensis. We evaluated the difference in volatile phenol production when B. bruxellensis was introduced before or after alcoholic fermentation. We have shown that B. bruxellensis could grow and produce volatile phenols during alcoholic fermentation. Results obtained also showed that commercial wine yeast strains could produce the vinyl derivatives that serve as precursors for Brettanomyces yeast to produce the ethyl derivatives. The commercial yeast strains differed in their ability to produce vinyl derivatives.
Different malolactic fermentation scenarios were evaluated, namely spontaneous versus inoculated, and with or without yeast lees. Results showed that spontaneous malolactic fermentation had higher volatile phenol levels in the wine than inoculated malolactic fermentation. The treatment with lees reduced the level of volatile phenols, probably due to absorption by yeast cells.
The presence of the phenyl acrylic decarboxylase (PAD1) gene and the production of volatile phenols by S. cerevisiae commercial yeast strains were evaluated in Shiraz grape juice and in synthetic grape juice. The results indicated that the yeast strains differ in their ability to produce 4-vinylphenol and 4-vinylguaiacol. All the yeast strains tested had the PAD1 gene. We also evaluated the presence of the phenolic acid decarboxylase (padA) gene and the ability of different lactic acid bacteria strains to produce volatile phenols in synthetic wine media. Although some of these strains tested positive for the phenolic acid decarboxylase gene most of them only produced very low levels of volatile phenols.
This study made a valuable contribution on the knowledge about the effect of Brettanomyces yeast on the volatile phenol content of red wines during different stages of the winemaking process and when applying different winemaking practices. It also showed the effect between Brettanomyces yeast and other wine microorganisms and the possible influence it could have on the final quality of wine. Research such as this can therefore aid the winemaker in making certain decisions when trying to manage Brettanomyces yeast spoilage of wines.
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Extracellular acid proteases of wine microorganisms : gene identification, activity characterization and impact on wineReid, Vernita Jennilee 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Non-Saccharomyces yeasts of oenological origin have previously been associated with spoilage or
regarded as undesired yeasts in wine. However, these yeasts have recently come under investigation for
their positive contribution towards wine aroma especially when used in sequential or co-inoculated
fermentations with Saccharomyces cerevisiae. These yeasts are also known to secrete a number of
enzymes that could be applicable in wine biotechnology. Amongst these enzymes are aspartic proteases.
The secreted proteases from some non-Saccharomyces yeast may play a role in protein haze reduction,
as demonstrated by some authors, while simultaneously increasing the assimilable nitrogen content of
the wine for the utilization and growth of fermentative microorganisms. Moreover, the proteases may have
an indirect effect on wine aroma by liberating amino acids that serve as aroma precursors. Although
many screenings have been performed detecting protease activity in non-Saccharomyces yeasts, no
attempts have been made to characterize these enzymes. This study set out to isolate and characterize
genes encoding extracellular aspartic proteases from non-Saccharomyces yeasts.
An enzymatic activity screening of a collection of 308 Saccharomyces and non-Saccharomyces yeasts,
isolated from grape must, was performed. The aspartic protease-encoding genes of two non-
Saccharomyces yeasts, which showed strong extracellular proteolytic activity on plate assays, were
isolated and characterized by in silico analysis. The genes were isolated by employing degenerate and
inverse PCR. One gene was isolated from Metschnikowia pulcherrima IWBT Y1123 and named MpAPr1.
The other putative gene was isolated from Candida apicola IWBT Y1384 and named CaAPr1. The
MpAPr1 gene is 1137 bp long, encoding a 378 amino acid putative protein with a predicted molecular
weight of 40.1 kDa. The CaAPr1 putative gene is 1101 bp long and encodes a 367 amino acid putative
protein with a predicted molecular weight of 39 kDa. These features are typical of extracellular aspartic
proteases. The deduced protein sequences showed less than 40% homology to other yeast extracellular
aspartic proteases. By heterologous expression of MpAPr1 in S. cerevisiae, it was confirmed that the
gene encodes an extracellular acid protease. The expression of MpAPr1 was shown to be induced in
media containing proteins as sole nitrogen source and repressed when a preferred nitrogen source was
available. The gene was expressed in the presence of casein, bovine serum albumin (BSA) and grape
juice proteins and repressed in the presence of ammonium sulphate. Expression was most induced in the
presence of grape juice proteins, which was expected since these proteins are present in the natural
habitat of the yeast. A genetic screening confirmed the presence of the MpAPr1 gene in 12 other
M. pulcherrima strains isolated from grape juice. The extracellular protease activity of the strains was also
visualized on plates. As far as we know, this is the first report on the genetic characterization of secreted
aspartic proteases from non-Saccharomyces yeasts isolated from grape must and provides the
groundwork for further investigations. / AFRIKAANSE OPSOMMING: Nie-Saccharomyces giste is voorheen met wynbederf geassosieer en hul teenwoordigheid in wyn is
ongewens. Hierdie giste is onlangs ondersoek vir hulle positiewe bydrae tot wyn aroma, in veral
sekwensiële en ko-inokulerings met Saccharomyces cerevisiae. Sommige van die nie-Saccahromyces
giste skei ‘n verskeidenhied ensieme af wat moontlik vir die wynmaker van nut kan wees. Een groep van
hierdie ensieme is die aspartiese suurproteases. Soos deur sommige navorsers aangetoon word, kan die
proteases die vorming van proteïenwaasverlaging, terwyl dit terselfdertyd die assimilerende
stikstofinhoud van die wyn vir die gebruik en groei van fermentasie-mikroörganismes verhoog. Die
proteases kan moontlik ook ‘n indirekte uitwerking op die aromaprofiel van die wyn hê deur die vrystelling
van aminosure wat as aromavoorlopers dien. Alhoewel baie studies gedoen is wat die ekstrasellulêre
teenwoordigheid van proteases bevestig in nie-Saccharomyces giste wat van druiwesap/wyn afkoms is,
is daar geen dokumentasie oor die genetiese karakterisering van hierdie ensieme beskikbaar nie. Die
doel van hierdie studie was om gene wat aspartiese proteases in nie-Saccharomyces giste enkodeer, te
isoleer en gedeeltelik te karakteriseer.
‘n Versameling van 308 Saccharomyces en nie-Saccharomyces giste wat uit druiwe sap geïsoleer is, is
gesif vir ensiematiese aktiwiteit deur plaattoetse uit te voer. Twee gene wat aspartiese protease
enkodeer, is geïsoleer van twee nie-Saccharomyces giste. Dit hetpositief gedurende die aktiwiteitstoetse
getoets en is deur in silico–analise gekarakteriseer. Die gene is deur die uitvoering van gedegenereerde
en inverse PKR geïdentifiseer. Een geen is vanaf Metschnikowia pulcherrima IWBT Y1123 geïsoleer en
is MpAPr1 genoem, terwyl die ander van Candida apicola IWBT Y1384 geïsoleer en CaAPr1 genoem is.
Die MpAPr1-geen is 1137 bp lank en enkodeer ‘n proteïen wat uit 378 aminosure bestaan met ‘n
voorspelde molekulêre massa van 40.1 kDa. Daar teenoor is die CaAPr1-geen 1101 bp lank en enkodeer
vir ‘n proteïen wat uit 367 aminosure met ‘n molekulêre massa van 39 kDa bestaan. Hierdie eienskappe
is kenmerkend van aspartiese protease. Die afgeleide proteïenvolgorde het minder as 40% homologie
met ander ekstrasellulêre aspartiese proteases vertoon, wat dui op die nuwigheid van hierdie ensieme.
Die MpAPr1-geen is heterologies in S. cerevisiae YHUM272 uitgedruk en dit het bevestig dat die geen
inderdaad ‘n ekstrasellulêre aspartiese protease enkodeer. Die MpAPr1-geen is uitgedruk in media wat
alleenlik proteïen as stikstofbron bevat het, terwyl dit onderdruk is in gevalle waar ‘n verkose stikstofbron
beskikbaar was. Die geen is uitgedruk in die teenwoordigheid van kaseïen, BSA en proteïene afkomstig
vanaf druiwesap en in die teenwoordigheid van ammoniumsulfaat onderdruk. Die hoogste uitdrukking
was in die teenwoordigheid van druifproteïene. Hierdie proteïene is teenwoordig in die natuurlike habitat
van die gis en is dus dalk ‘n bekende stikstofbron vir die gis. ‘n Genetiese sifting het die teenwoordigheid
van die MpAPr1-geen in 12 ander M. pulcherrima–rasse, wat ook van wynkundige oorsprong is, bevestig.
Die aspartiese protease-aktiwiteit van die 12 rasse is ook op agarplate waargeneem. Na ons wete, is dit
die eerste verslag oor die genetiese karakterisering van afgeskeide aspartiese proteases van nie-
Saccharomyces giste van wynkundige oorsprong en verskaf die grondslag vir verdere ondersoek.
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Optimization and evaluation of heterologous lysozyme production in saccharomyces cerevisiaeWilcox, Dale Adrian 03 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Hen egg white lysozyme (HEWL; muramidase; EC 3:2:1:17) is an enzyme
present in high concentrations in chicken (Gallus gallus) egg whites. It hydrolyses
the link between N-acetylmuramic acid and N-acetylglucosamine in Gram positive
bacterial cell walls, resulting in cell death. It is thus active against lactic acid
bacteria (LAB), which may be present in grape juices and musts. These bacteria
are responsible for malolactic fermentation of wines although many species, particularly
of the genera Lactobacillus and Pediococcus, are considered spoilage organisms.
The growth of LAB is therefore closely monitored and controlled during
winemaking. The most common means of control is growth inhibition by chemical
treatment (usually with SO2). Lysozyme is a commonly used wine processing aid,
complementing the antimicrobial activity of SO2 . It allows for lower doses of SO2
to be used, thus improving the wholesomeness of wine. The OIV (Organisation
Internationale de la Vigne et du Vin) approved its use in quantities up to 500 mg per
liter of wine in 1997.
This study evaluated the effect of different secretion signals on the secretion of
lysozyme by the haploid auxotroph Saccharomyces cerevisiae strain FY23. Secretion
by an industrial strain (VIN13) transformed with a single copy of the HEWL gene with the MF-a secretion signal under the control of the PGK1 (phosphoglycerate
kinase 1) prompter and terminator was also evaluated. In the case of FY23 four
secretion signals were used, namely the native lysozyme signal and the S. cerevisiae
mating factor-a signal as well as mutants of these signals. These mutants incorporated
two additional arginines at the N-terminus of the signals immediately downstream
of the terminal methionine. The effect of these mutations was to increase the
positive charge of the secretion signal N-terminals. The secretion signal-lysozyme
fusions were placed under the regulation of the S. cerevisae PGK1 gene’s promoter
and terminator. The resulting expression cassettes were cloned into integrating vectors
YIpLac211 and pDMPOF1b and episomal vector pHVX2. These were used to
transform FY23 and VIN13.
FY23 as well as VIN13 transformants were evaluated in an artificial medium
designed to reflect the nutrient content of grape juice, with particular attention being
paid to assiminable nitrogen. Three hexose concentrations were tested in order to
determine the effect thereof on lysozyme secretion titer.
Lysozyme secreted under all tested growth conditions was found to be too low
for detection by either enzymatic assay or HPLC-FLD. For this reason secreted
lysozyme was isolated and concentrated 10x by means of cation-exchange. Subsequently,
lysozyme concentrations in the concentrates was determined by means of
the aforementioned techniques. SDS-PAGE analysis of lysozyme concentrates was
also performed.
No significant differences were found between native or MF-a secretion signals
and their mutated counterparts in terms of secretion titer or proteolytic maturation.
Lysozyme secreted with the MF-a signal was found to be misprocessed in all cases,
with both an authentically processed and a larger form, in which the secretion signal
was not completely removed, being present. Lysozyme secreted with the native
signal appeared to be correctly processed in all cases. Secretion titer from high
copy number episomal FY23 tranformants was similar to that of integrants containing
a single copy of the gene. Sugar concentration affected lysozyme production,
with higher quantities of the enzyme being secreted when higher initial sugar concentrations
were used. Lysozyme titers were extremely low (< 0:25 mg/L) with
all expression cassettes under all the tested conditions with both FY23 and VIN13.
In the case of the VIN13’s a lower final biomass was found for the secretor strain
tested in comparrison to the VIN13 wild-type. / AFRIKAANSE OPSOMMING: Hoendereierwitlisosiem (HEWL; muramidase, EG 3:2:1:17) is ´n ensiem teenwoordig
in hoë konsentrasies in hoender (Gallus gallus) eierwitte. Dit hidroliseer
die binding tussen N-asetielmuramiensuur en N-asetielglukosamien in Gram positiewe
bakteriese selwande, wat tot seldood lei. Dit is dus aktief teen melksuurbakterieë
(MSB), wat in druiwesap en mos teenwoordig kan wees. Hierdie bakterieë
is verantwoordelik vir appelmelksuurgisting van wyne, hoewel baie spesies, veral
van die genera Lactobacillus en Pediococcus, ook as bederforganismes beskou
word. Die groei van MSB word dus noukeurig tydens wynbereiding gemoniteer en
beheer. Die algemeenste wyse van beheer is groei-inhibisie deur chemiese behandeling
(gewoonlik SO2). Lisosiem is ´n algemeen gebruikte wyntoevoegingsmiddel
en vul die antimikrobiese aktiwiteit van SO2 aan. Met lisosiem kan ´n laer dosis
van SO2 gebruik word, wat lei tot ´n verbetering van die heilsaamheid van die wyn.
Die OIV (Organisasie Internationale de la Vigne et du Vin) het die gebruik daarvan
goedgekeur tot en met 500 mg per liter wyn vanaf 1997. Hierdie studie het
die effek van verskillende sekresieseine op die uitskeiding van lisosiem deur die haploïede ouksotrofe Saccharomyces cerevisiae stam, FY23, geëvalueer. Uitskeiding
deur ´n industriële stam (VIN13), wat getransformeer is met ´n enkelkopie van die
HEWL-gene met die MF-a sekresiesein onder die beheer van die PGK1 (Fosfogliseraat
kinase 1) promotor en termineerder, is ook geëvalueer. In die geval van FY23
is vier sekresieseine gebruik, naamlik die inheemse lisosiemsein, S. cerevisiae MF-
a sein, asook mutante van hierdie seine. Hierdie mutante het twee bykomende
arginienresidu’s by die N-terminus van die seine direk stroom-af van die terminale
metionien. Die effek van hierdie mutasies was om die positiewe lading van die
uitskeidingsein N-terminale te verhoog. Die gevolglike uitdrukkingskassette is in
die integrasievektor YIpLac211 en pDMPOF1b, en die episomale vektor pHVX2,
gekloneer. Dit is gebruik om VIN13 en FY23 te transformeer. FY23, sowel as
VIN13-transformante, is geëvalueer in ´n kunsmatige medium wat ontwerp is om
die voedingsinhoud van druiwesap te weerspieël, met besondere aandag aan assimileerbare
stikstof. Drie heksose konsentrasies is getoets om te bepaal wat die
uitwerking daarvan op die lisosiemsekresietiter is. Onder alle groeitoestande was
die isosiem wat uitgeskei is, te laag om deur ensimatiese toetse of HPLC-FLD
bepaal te word. Om hierdie rede is uitgeskeide lisosiem geïsoleer en 10x gekonsentreer
deur middel van katioon-uitruiling. Daarna is lisosiemkonsentrasies bepaal
deur middel van bogenoemde tegnieke. SDS-PAGE-ontleding van lisosiemkonsentraat
is ook uitgevoer. In terme van sekresietiter of proteolitiese maturasie, is geen
beduidende verskille gevind tussen inheemse of MF-a sekresieseine en hul gemuteerde
eweknieë nie. Lisosiem wat deur die MF-a sein uitgeskei is, is in alle gevalle
foutief geprosesseer, met ´n teenwoordigheid van beide die regte produk en ´n groter
produk, waarin die uitskeidingsein nie heeltemal verwyder word nie. Lisosiem wat
met die inheemse sein uitgeskei is, blyk in alle gevalle korrek verwerk te wees.
Sekresietiter van ´n aantal hoë-kopie episomale FY23-transformante was soortgelyk
aan dié van integrante met ´n enkelkopie van die geen. Suikerkonsentrasie beïnvloed
lisosiemproduksie, met ´n hoër hoeveelheid van die ensiem wat uitgeskei word wanneer
die aanvanklike suiker in hoër konsentrasies gebruik is. Lisosiemtiters was
baie laag (< 0:25 mg/L), met al die kassette onder al die getoetste toestande vir
beide FY23 en VIN13. In die geval van die VIN13’s, is ´n laer finale biomassa vir
die uitskeidingstam in vergelyking met die VIN13 wilde-tipe gevind.
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Investigating the role of Brettanomyces and Dekkera during winemakingOelofse, Adriaan 12 1900 (has links)
Thesis (PhD (Genetics. Plant Biotechnology))--Stellenbosch University, 2008. / Wine quality is greatly influenced by the number of microorganisms, which occur
throughout the winemaking process. These microorganisms are naturally present on
the grapes and in the cellar from where they can be introduced to the winemaking
process at any given time and consequently impart specific contributions to the wine
quality. However, these microorganisms can be seen either as beneficial or as wine
spoilage microorganisms, depending on the conditions under which they can
proliferate during the winemaking process. Wine yeasts (Saccharomyces spp.) are
typically responsible for the alcoholic fermentation; lactic acid bacteria (LAB) are
responsible for malolactic fermentation (MLF), while acetic acid bacteria (AAB) and
other wild yeasts (non-Saccharomyces spp.) are typically associated with the
formation of off-flavours under poorly controlled winemaking conditions.
In recent years, evidence from the wine industry has highlighted a specific group
of non-Saccharomyces yeast species as a serious cause for wine spoilage that
required more research investigations. Yeast of the genus Brettanomyces or its
teleomorph Dekkera has been identified as one of the most controversial spoilage
microorganisms during winemaking as they can produce several compounds that are
detrimental to the organoleptic quality of wine. This has triggered the research
initiative behind this doctoral study on the significance of Brettanomyces and Dekkera
yeasts during winemaking.
In this dissertation, various aspects of the detection, isolation and identification
methods of Brettanomyces yeast from the winemaking environment were
investigated. As a first objective, a culture collection of Brettanomyces bruxellensis
wine isolates had to be established. This followed after the isolation of
Brettanomyces yeasts from various red wine cultivars from South African wineries
from different stages of the winemaking process. Different conventional
microbiological methods such as plating on selective agar media and microscopy
were investigated along with molecular identification techniques such as the
polymerase chain reaction (PCR) in this regard.
Other focus areas of this study aimed at performing genetic characterisation and
differentiation studies of B. bruxellensis wine isolates. For this purpose, different
intraspecific identification methods were investigated on several strains, including
strains of European origin. The application of molecular techniques allowing strain
identification aided in the selection of specific strains that were evaluated for volatile
phenol production in synthetic media and wine. The results obtained from this work
indicated that a large degree of genetic diversity exists among B. bruxellensis strains
and that the volatile phenol production differed between the strains after evaluation in
synthetic media and wine.
In addition to the molecular intraspecific strain identification techniques that were
investigated, a feasibility study was also performed that focused on evaluating Fourier transform infrared (FTIR) spectroscopy combined with chemometrics as an
alternative approach for differentiating between B. bruxellensis strains.
The two approaches of FTIR spectroscopy that were investigated involved the
use of firstly, Fourier transform mid-infrared (FTMIR) spectroscopy to obtain spectral
fingerprints of spoiled wines by different B. bruxellensis strains; and secondly,
Attenuated total reflectance (FTIR-ATR) to obtain spectral fingerprints from whole
cells of B. bruxellensis on microbiological agar media. The results of this study
illustrated the potential of FTIR spectroscopy to become a reliable alternative to
molecular based methods for differentiating between B. bruxellensis strains and for
characterisation studies.
The formation of volatile phenols in wine by species of the genera Brettanomyces
and Dekkera is one of the primary reasons for their classification as wine spoilage
yeasts. The enzymatic activities of this reaction have been identified and involve a
phenyl acrylic (phenolic) acid decarboxylase (PAD) and a vinyl phenol reductase
(VPR). However, only a limited amount of information is available about these
enzymes from Brettanomyces/Dekkera yeasts and no genetic data have been
described. It was therefore imperative that this dissertation should include a genetic
investigation into the phenylacrylic (hydroxycinnamic) acid decarboxylase from the
species B. bruxellensis involved in the formation of volatile phenols. Strategies that
were investigated included various molecular DNA techniques and protein purification
procedures to obtain either genetic or protein sequence data. The decarboxylase
activity of this yeast species towards p-coumaric acid was demonstrated and
substantial genetic sequence data was obtained.
The results from this dissertation made a substantial contribution to the current
available knowledge about Brettanomyces/Dekkera spp. and led to a better
understanding of this wine spoilage yeast. This research developed a platform from
which further investigations could follow and the knowledge gained will be invaluable
for future Brettanomyces research projects at the Institute for Wine Biotechnology at
Stellenbosch University.
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Investigating the impact of sulphur dioxide on Brettanomyces bruxellensis at a molecular and cellular levelDuckitt, Edward 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The yeast Brettanomyces was isolated from beer in 1904 and associated with wine thereafter. A
sporulating form, Dekkera, was discovered later. Brettanomyces bruxellensis produces high
levels of volatile phenol off-flavours in wine. Sulphur dioxide (SO2) is the most widely used
chemical preservative in wine. Yeasts have several mechanisms to cope with the SO2, namely
Ssu1p, a membrane bound SO2 transporter; sulphite reduction, sulphite oxidation and
acetaldehyde production. In unfavourable environmental conditions, certain yeasts can enter a
viable-but-non-culturable (VBNC) state which is characterised by reduced metabolic rate,
inability to reproduce on solid media and a reduction of cell size. VBNC can be triggered by
chemical stress such as high SO2 levels. The objectives of this study were to examine the SO2
tolerance of B. bruxellensis and Saccharomyces cerevisiae, to quantify their rates of SO2
accumulation and efflux, determine the effect of SO2 on their energy metabolism and investigate
if B. bruxellensis possesses an orthologue to S. cerevisiae SSU1.
In this study, the identity of a number of Brettanomyces/Dekkera strains was confirmed using
5.8S rDNA-ITS RFLP analysis and DNA sequencing. Sporulation assays were used to confirm
whether these strains belonged to the Dekkera or Brettanomyces genus. A method to
accurately quantify SO2 in laboratory conditions was optimised. Molecular SO2 tolerance was
tested by spotting fresh yeast cultures on media with SO2 and/or ethanol. Tolerance to SO2
and/or ethanol showed highly strain dependent results with S. cerevisiae showing the highest
tolerance levels while B. bruxellensis tolerated SO2 and ethanol poorly but certain strains grew
well with only SO2. The SO2 accumulation and efflux rates of 3 S. cerevisiae strains and 3 B.
bruxellensis strains were determined. It was shown that the S. cerevisiae strains followed the
same trends as previously found in literature whereas B. bruxellensis strains showed similar
trends but displayed highly variable strain-dependent results. B. bruxellensis CB63 and S. cerevisiae VIN13 were investigated for their response to SO2 in two
different media, TA and SWM, over a 48-hour and 32-day period respectively. Acetic acid,
acetaldehyde, D-glucose, D-fructose (only in SWM) and ethanol (only in TA) were regularly
monitored over the time course of each experiment. SO2 had the greatest impact on
B. bruxellensis with decreased rates of glucose consumption and ethanol production as well as
increased acetic acid. Acetaldehyde peaked shortly after SO2 addition with the subsequent
restarting of sugar consumption for certain samples. This suggests that sufficient acetaldehyde
was produced to bind free SO2 to reduce SO2 stress. Volatile phenols were quantified for day 32
of the SWM experiment. An increase of 4-ethyl guaiacol was correlated to higher molecular SO2
levels. SO2 negatively affected both yeasts energy metabolism, forcing the yeasts metabolism
to adapt to ensure survival.
In general, SO2 was shown to have a negative impact on all aspects of a yeasts growth and
metabolism and that SO2 tolerance is highly strain dependent and a far more complicated
characteristic than currently understood. / AFRIKAANSE OPSOMMING: Die gis Brettanomyces is in 1904 uit bier geïsoleer en daarna met wyn geassosieer. 'n
sporulerende vorm, Dekkera, is later ontdek. Brettanomyces bruxellensis produseer hoë vlakke
van vlugtige fenol afgeure in wyn. Swaweldioksied (SO2) is die mees gebruikte chemiese
preserveermiddel in wyn. Giste het verskeie meganismes om SO2 te hanteer, naamlik Ssu1p, 'n
membraan-gebonde SO2 transporter, sulfietvermindering, sulfiet-oksidasie en
asetaldehiedproduksie. In ongunstige omgewingstoestande kan sekere giste 'n lewensvatbare,
maar nie-kultiveerbare (LMNK)-toestand aanneem wat gekenmerk word deur verlaagde
metaboliese tempo, onvermoë om voort te plant op soliede media en 'n vermindering van die
selgrootte. LMNK kan veroorsaak word deur chemiese stres, soos hoë SO2-vlak. Die doelwitte
van hierdie studie was om die SO2 -bestandheid van B. bruxellensis en Saccharomyces
cerevisiae te ondersoek, hul spoed van SO2 -opneming/akkumulasie en -uitskeiding te
kwantifiseer, die invloed van SO2 op energiemetabolisme te bepaal en te ondersoek of B.
bruxellensis oor ‘n soortgelyke geen as die S. cerevisiae SSU1 beskik.
In hierdie studie is die identiteit van 'n aantal Brettanomyces/Dekkera-stamme bevestig deur
5.8S rDNA-ITS RFLP-analise en DNA-opeenvolging te gebruik. Sporulasietoetse is gebruik om
te bevestig of hierdie stamme aan die genus Dekkera of Brettanomyces behoort. 'n Metode om
SO2 onder laboratoriumtoestande akkuraat te kwantifiseer, is geoptimiseer. Molekulêre SO2-
bestandheid is getoets deur vars giskulture op media met SO2 en/of etanol te groei.
Bestandheid teen SO2 en/of etanol het stam-afhanklike resultate getoon, S. cerevisiae wat die
hoogste toleransievlakke getoon het, terwyl B. bruxellensis SO2 en etanol swak tolereer, maar
sekere stamme het goed gegroei met slegs SO2. Die SO2-akkumulasie en -uitskeidingtempo
van 3 S. cerevisiae-rasse en 3 B. bruxellensis-stamme is bepaal. Daar is gevind dat die S.
cerevisiae-rasse dieselfde tendens soos voorheen in die literatuur beskryf, gevolg het, terwyl B.
bruxellensis-stamme soortgelyke tendense getoon het,maar hoogs veranderlike stamafhanklike
resultate vertoon. B. bruxellensis CB63 en S. cerevisiae VIN13 is ondersoek vir hul reaksie tot SO2 in twee
verskillende media, TA en SWM, oor 'n tydperk van 48-uur en 32-dae onderskeidelik. Asynsuur,
asetaldehied, D-glukose, D-fruktose (slegs in SWM) en etanol (slegs in TA) is gereeld
gemoniteer oor die verloop van elke eksperiment. SO2 het die grootste impak op B. bruxellensis
met ‘n verlaagde tempo van glukoseverbruik en etanolproduksie, sowel as verhoogde asynsuur.
‘n Asetaldehiedhoogtepunt is bereik kort na die SO2-byvoeging met die daaropvolgende
hervatting van suiker wat vir sekere monsters gebruik is. Dit dui daarop dat voldoende
asetaldehied geproduseer is om vry SO2 te bind om SO2-stres te verminder. Vlugtige fenole is
op dag 32 van die SWM-eksperiment gekwantifiseer. 'n Toename van 4-etiel-guajakol korreleer
met hoër molekulêre SO2-vlakke. SO2 het beide giste se energiemetabolisme negatief
beïnvloed, wat die gis dwing om sy metabolisme aan te pas om oorlewing te verseker. Oor die algemeen het SO2 'n negatiewe impak op alle aspekte van giste se groei en
metabolisme, en SO2-bestandheid is hoogs stam–afhanklik. Dit is ook 'n baie meer
ingewikkelde kenmerk as wat tans verstaan word.
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The selection and characterisation of lactic acid bacteria to be used as a mixed starter culture for malolactic fermentationLerm, Elda 03 1900 (has links)
Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The quality of wine is influenced and determined by various factors, one of which includes the
process of malolactic fermentation (MLF). MLF plays an integral role in the flavour and sensory
profile of most red wines as well as some white wines like Chardonnay. This process is conducted
by lactic acid bacteria (LAB), specifically of the genera Oenococcus, Lactobacillus, Pediococcus
and Leuconostoc. Of these, Oenococcus oeni is best adapted to survive in the harsh wine
environment.
MLF is defined as the conversion of L-malic acid to L-lactic acid and carbon dioxide. The
conversion of the dicarboxylic malic acid to the monocarboxylic lactic acid results in a decrease in
acidity and an increase in pH, to give a softer mouthfeel and more favourable flavour profile. A
further reason for conducting MLF in wine includes the improvement of microbial stability due to
the removal of malic acid as a possible substrate for microorganisms. Recently, research focus has
shifted to the ability of MLF and LAB to alter the aroma profile of wine via the production and/or
modification of certain aroma compounds.
In order for wine LAB to conduct MLF, they need to be able to survive the harsh and
challenging wine environment. Conditions in South African wines are particularly challenging due
to the long, hot ripening seasons resulting in high sugar concentrations which give high ethanol
concentrations. Some LAB also struggle to adapt to an environment with high pH and low malic
acid concentrations. These factors, combined with the use of sulphur dioxide, cause LAB to
struggle in conducting and completing successful MLF. Many of the commercial starter cultures
that are currently available contain LAB that have not been isolated from South African wine and
are therefore not optimal for use under these challenging wine conditions. Oenococcus oeni is also
the single LAB culture present in all commercially available starter cultures.
The overriding goal of this study was to create a MLF starter culture containing a mixture of
LAB cultures, namely O. oeni and Lactobacillus plantarum, which can successfully convert malic
acid to lactic acid, ensure microbial stability, but also make a positive contribution to the wine
aroma profile. Lactobacillus plantarum has previously been considered for possible use in a
commercial starter culture. The LAB isolates used in this study were selected from the Institute for
Wine Biotechnology culture collection as well as isolated from spontaneous MLF.
The first objective was to characterise these LAB strains for important traits and for possible
use as a MLF starter culture. A total of 23 strains were identified as O. oeni and 19 strains as
Lb. plantarum. The identified strains were screened in a synthetic wine medium for their ability to
convert malic acid to lactic acid. Based on the LAB strain performance in the synthetic wine
medium, seven strains of both O. oeni and Lb. plantarum were selected. These 14 strains were
screened for the presence of genes encoding for enzymes responsible for biogenic amine
production and were found to contain none of the genes associated with the formation of
histamine, tyramine or putrescine. The LAB strains were genetically screened for enzymes associated with aroma modification by LAB during MLF. The enzymes of interest that were
screened for included β-glucosidase, esterase, protease and phenolic acid decarboxylase (PAD).
The Lb. plantarum strains were found to possess more diverse enzymatic profiles related to aroma
than O. oeni. The biggest differences were observed for the presence of β-glucosidase and PAD.
The second objective was to perform small-scale fermentations with the individual LAB
isolates. The individual isolates were evaluated in Pinotage and based on these results; three
strains of each O. oeni and Lb. plantarum were selected for evaluation in mixed culture
fermentations. The mixed cultures were evaluated in Pinotage, Shiraz and Cabernet Sauvignon in
the 2008 vintage. As a third objective, the wines were also analytically and sensorially evaluated to
investigate the changes in the aroma profile that could be attributed to the presence of the mixed
LAB isolates. Based on the fermentation data as well as data pertaining to the aroma modification,
three mixed cultures were selected for evaluation in the 2009 vintage in Pinotage, Cabernet
Sauvignon and Chardonnay. The mixed cultures were able to successfully complete MLF in
fermentation periods comparable to that of a commercial culture used as control. The different LAB
cultures had distinct and diverse effects on the wine aroma profile. The O. oeni strain played a
larger role in the ester concentration present after MLF, while the Lb. plantarum strain had a larger
effect on the higher alcohol and volatile fatty acid concentration upon completion of MLF.
The results generated by this novel study clearly indicate the potential of a mixed LAB starter
culture for conducting MLF. The mixed cultures successfully completed MLF and made a positive
contribution to the wine aroma profile. / AFRIKAANSE OPSOMMING: Die kwaliteit van wyn word beïnvloed en bepaal deur verskeie faktore en wynbereidings prosesse,
wat die proses van appelmelksuurgisting (AMG) insluit. AMG speel ’n integrale rol in die
sensoriese profiel van meeste rooiwyne, sowel as sommige witwyne soos Chardonnay.
AMG word gedefinieër as die omskakeling van L-appelsuur na L-melksuur en koolstofdioksied.
Hierdie omskakeling kan toegeskryf word aan die teenwoordigheid van melksuurbakterieë (MSB),
spesifiek spesies van die genera Oenococcus, Lactobacillus, Pediococcus en Leuconostoc. Vanuit
hierdie wyn MSB, is Oenococcus oeni die spesies wat die beste aanpas en oorleef onder
stresvolle wyn kondisies. Die omskakeling van appelsuur, ’n dikarboksielsuur, na melksuur, ’n
monokarboksielsuur, lei tot ‘n vermindering in suurheid en ’n verhoging in pH. Hierdie vermindering
in suurheid gee ’n sagter en meer geronde mondgevoel aan die wyn en dra by tot ‘n meer
aangename geurprofiel. ’n Verdere rede vir AMG in wyn is om mikrobiese stabiliteit te verseker
deurdat appelsuur verwyder word as ’n moontlike koolstof substraat vir mikroörganismes. Onlangs
het navorsing begin fokus op AMG en die vermoë van MSB om die aroma profiel van wyn te
beïnvloed deur die produksie/modifisering van sekere aroma komponente.
Vir MSB om AMG te kan deurvoer, moet hulle kan oorleef in die stresvolle wynomgewing.
Wyntoestande in Suid-Afrika is veral uitdagend vir die oorlewing van mikroörganismes as gevolg
van lang, warm somers wat lei tot ’n matriks met ’n hoë suikerkonsentrasie en wyn met ’n hoë
etanolkonsentrasie. ‘n Omgewing met ‘n hoë pH en lae appelsuur konsentrasie, kan ook bydrae tot
stresvolle kondisies vir MSB. Hierdie parameters, tesame met die gebruik van swaweldioksied,
maak dit moeilik vir MSB om AMG te inisieer en te voltooi. Sommige van die kommersiële
aanvangskulture wat tans beskikbaar is, bevat nie MSB wat onder Suid-Afrikaanse wyntoestande
geïsoleer is nie en daarom is dit nie altyd optimaal vir gebruik nie. Oenococcus oeni is ook die
enkele MSB kultuur wat in alle kommersiële kulture gebruik word.
Die hoofdoelwit van hierdie studie was om ’n potensiële kommersiële aanvangskultuur te
ontwikkel wat ‘n mengsel van MSB bevat. Hierdie aanvangskultuur moet AMG suksesvol kan
voltooi, mikrobiologiese stabiliteit bevorder en steeds die wynaroma positief kan beïnvloed.
Bakterierasse van O. oeni en Lb. plantarum is geselekteer vir gebruik in hierdie studie.
Lactobacillus plantarum het reeds in vorige studies potensiaal getoon as ‘n moontlike
aanvangskultuur. Die MSB isolate vir hierdie studie is geselekteer uit die Instituut vir
Wynbiotegnologie se kultuurversameling en geïsoleer uit spontane AMG fermentasies.
Die eerste doelwit was om hierdie MSB isolate te karakteriseer vir belangrike eienskappe en
die moontlike gebruik as ’n kommersiële AMG aanvangskultuur. ‘n Totaal van 23 O. oeni en 19
Lb. plantarum isolate is geïdentifiseer. Hierdie isolate is in ’n sintetiese wynmedium geëvalueer vir
hul vermoë om appelsuur na melksuur om te skakel. Op grond van hul reaksie in die sintetiese
wynmedium, is sewe isolate van elk van die O. oeni en Lb. plantarum geselekteer. Hierdie 14
isolate is ondersoek vir die teenwoordigheid van die gene wat kodeer vir biogeenamien produksie en daar is gevind dat geen van die isolate enige van die biogeenamien gene wat ondersoek is,
naamlik histamien, tiramien en putresien besit nie. Die MSB isolate is geneties ondersoek vir die
teenwoordigheid van dié gene wat kodeer vir ensieme wat die aromaprofiel tydens AMG
beïnvloed. Dié ensieme sluit β-glukosidase, esterase, protease, fenoliese suurdekarboksilase en
sitraatliase in. Daar is gevind dat die Lb. plantarum isolate meer diverse ensiemprofiele as O. oeni
besit. Die grootste verskille in die ensiemprofiele kan toegeskryf word aan die teenwoordigheid van
β-glukosidase en fenoliese suurdekarboksilase.
Die tweede doelwit was om kleinskaalse AMG fermentasies met die individuele MSB isolate
uit te voer. Die individuele isolate is in Pinotage geëvalueer. Volgens hierdie resultate is drie isolate
van elk van die O. oeni en Lb. plantarum geselekteer om in gemengde kulture getoets te word. Die
gemengde kulture is in Pinotage, Shiraz en Cabernet Sauvignon in 2008 geëvalueer. As ’n derde
doelwit is hierdie wyne ook analities en sensories geëvalueer om die veranderinge in die
aromaprofiele as gevolg van die teenwoordigheid van die MSB te ondersoek. Op grond van die
fermentasiedata, sowel as die data oor die aromaveranderinge, is drie gemengde kulture
geselekteer vir evaluering in Pinotage, Cabernet Sauvignon en Chardonnay in 2009. Die
gemengde kulture kon AMG suksesvol voltooi met fermentasietempo’s wat vergelykbaar was met
dié van ‘n kommersiële AMG kultuur wat as kontrole gebruik is. Die verskillende MSB kulture het
spesifieke en uiteenlopende uitwerkings op die wynaroma gehad. Die O. oeni isolaat in die
gemengde kultuur blyk ‘n belangriker rol te speel in die esterkonsentrasie na AMG, terwyl die
Lb. plantarum isolaat ’n groter effek het op die hoër alkohol en vlugtige vetsuurinhoud na AMG.
Die resultate wat deur hierdie unieke studie gegenereer is, gee ’n aanduiding van die
potensiaal van ’n gemengde MSB aanvangskultuur vir AMG. Die gemengde kulture kon AMG
suksesvol voltooi en ‘n positiewe bydrae tot die aromaprofiel van die wyn lewer.
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