Spelling suggestions: "subject:"line anda wide making - microbiology"" "subject:"line anda wide making - microbiologyc""
11 |
Applied and basic aspects of sulfite metabolism in Saccharomyces cerevisiaePark, Hoon 16 December 1999 (has links)
In an effort to understand the basis for sulfite detoxification in S. cerevisiae,
the functions of two genes were analyzed. SSU1, which encodes a plasma membrane
protein, was found to be required for efficient sulfite efflux. FZFl-4, a dominant
allele of a transcriptional activator of SSUl, was also found to be involved in efficient
sulfite efflux. Analysis of an SSUl promoter-lacZ fusion showed that FZFl-4
conferred sulfite resistance through hyperactivation of SSUl. Efflux assays in cells
expressing multicopy SSUl or FZFl-4 suggested that Ssulp specifically mediates
efflux of the free form of sulfite. Sulfite resistance, mediated by either FZFl-4 or
multicopy SSUl, was found to be a useful marker for selecting transformants of
industrial and laboratory strains of S. cerevisiae. FZFl-4 was found to be more
efficient than multicopy SSUl, and in the case of the laboratory strains, was found to
be about half as efficient a selectable marker as URA3.
Sulfite transport was studied to clarify the mechanism of sulfite uptake in S.
cerevisiae. The kinetics of uptake were saturable, indicating a carrier-mediated
process. Uptake was significantly reduced in cells pretreated with carbonyl cyanide
m-chlorophenylhydrazone (CCCP) or 2,4-dinitrophenol (DNP), both of which
dissipate proton gradients. Extracellular alkalization was observed during sulfite
uptake. These findings suggest that an anionic form of sulfite, HSO₃, is taken up by
carrier-mediated proton symport.
As an alternative to costly disposal of spent cherry brine, a sulfite-containing
waste stream generated during maraschino cherry processing, brine was tested as a
substrate for ethanol production by S. cerevisiae. Initially, the toxic level of sulfite in
brine was reduced by raising brine pH to 8.5 with Ca(OH)₂ to precipitate calcium
sulfite. Because the alkalization was found to result in a 10-fold reduction of
phosphorus, brine was subsequently titrated with phosphoric acid to pH 6.0 prior to
inoculation with S. cerevisiae. All strains of S. cerevisiae tested were able to
efficiently ferment all lots of Ca(OH)₂-treated and phosphorus-enriched brine. / Graduation date: 2000
|
12 |
Growth and metabolism of `Leuconostoc oenos` and `Lactobacillus plantarum` in wine / by Thomas Henick-KlingHenick-Kling, Thomas January 1986 (has links)
Bibliography: leaves 172-187 / xxiv, 187 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1986
|
13 |
Engineering yeast for the production of optimal levels of volatile phenols in wineSmit, Annel 12 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Phenolic acids (principally p-coumaric and ferulic acids), which are generally esterified with
tartaric acid, are natural constituents of grape must and wine, and can be released as free
acids during the winemaking process by certain cinnamoyl esterase activities. Free phenolic
acids can be metabolised into 4-vinyl and 4-ethyl derivatives by several microorganisms
present in wine. These volatile phenols contribute to the aroma of the wine.
The Bretfanomyces yeasts are well known for their ability to form volatile phenols in
wine. However, these species are associated with the more unpleasant and odorous
formation of the ethylphenols and the formation of high concentrations of volatile phenols.
Other organisms, including some bacterial species, are responsible for the formation of
volatile phenols at low concentrations, especially the 4-vinylphenols, and this enhances the
organoleptic properties of the wine.
The enzymes responsible for the decarboxylation of phenolic acids are called phenolic
acid decarboxylases; and several bacteria and fungi have been found to contain the genes
encoding these enzymes. The following genes have been characterised: PAD1 from
Saccharomyces cerevisiae, fdc from Bacillus pumilus, pdc from Lactobacillus plantarum and
padc from Bacillus subtilis. PadA from Pediococcus pentosaceus was also identified.
S. cerevisiae contains the PAD1 (phenyl acrylic acid decarboxylase) gene, which is
steadily transcribed in yeast. The activity of the PAD1-encoded enzyme is low. Phenolic
acid decarboxylase from B. subtilis, as well as p-coumaric acid decarboxylase from
L. plantarum displays substrate inducible decarboxylating activity with phenolic acids. Both
the p-coumaric acid decarboxylase (pdc) and phenolic acid decarboxylase (padc) genes
were cloned into PGK1 PT expression cassette. The PGK1 PT expression cassette consisted
of the promoter (PGK1 p) and terminator (PGK1 T) sequence of the yeast
phosphoglyceratekinase I gene (PGK1). Episomal and yeast integration plasmids were
constructed for the PAD1 gene under the control of the PGK1 PT for overexpresion in yeast.
Industrial strains with the PAD1 gene disrupted were also made. Overexpression of pcoumaric
acid decarboxylase (pdc) and phenolic acid decarboxylase (padc) in S. cerevisiae
showed high enzyme activity in laboratory strains. The overexpressed PAD1 gene did not
show any higher enzyme activity than the control strain. Both bacterial genes, under the
control of the PGK1 PT cassette, were also cloned into a yeast-integrating plasmid, with the
SMR1 gene as selective marker. The cloning and transformation of pdc and padc into
industrial wine yeast strains can therefore be used to detect the effect of phenolic acid
decarboxylase genes in the winemaking process for the possible improvement of wine
aroma. Wine was made with all three strains (the bacterial genes overexpressed and PAD1
disrupted). The effect of these genes in wine was determined through GC analysis. The results showed that the bacterial genes could effectively produce higher levels of volatile
phenols in the wine. The manipulated strains also produced enzymes capable of producing
large amounts of favourable monoterpenes in the wine.
This study paves the way for the development of wine yeast starter culture strains for
the production of optimal levels of volatile phenols, thereby improving the sensorial quality of
wine. / AFRIKAANSE OPSOMMING: Die fenoliese sure (p-kumaarsuur en ferolsuur), wat as natuurlike komponente in mos en wyn
voorkom, word gewoonlik as esterverbindings in wynsteensuur gevind. Seker
esterase-aktiwiteite kan die fenoliese sure as vrye sure vrystel gedurende die
wynmaakproses. Hierdie vrye fenoliese sure kan dan weer deur verskillende mikroorganismes
na 4-viniel en 4-etiel derivate omgesit word. Hierdie derivate staan as vlugtige
fenole bekend en kan tot die aroma van wyn bydra.
Die Brettanomyces giste is baie bekend vir hulle vermoeë om vlugtige fenole in wyn te
vorm, maar dit is gewoonlik die formasie van hoë konsentrasies van vlugtige fenole, veral die
4-etiel derivate, wat met af geure geassosieer word. Ander organismes besit egter die
vermoeë om vlugtige fenole teen lae konsentrasies te vorm, veral die 4-viniel derivate, wat 'n
aanvullende effek op die wyn aroma kan hê. .
Die ensieme wat verantwoordelik is vir die dekarboksilasie van fenoliese sure staan as
fenolsuurdekarboksilases bekend. Verskeie bakterieë en fungi bevat gene wat vir hiedie
ensieme kodeer. Die volgende gene is reeds gekarakteriseer: PAD1 van
Saccharomyces cerevisiae, fdc van Bacillus pumilus, pdc van Lactobacillus plantarum en
padc van Bacillus subtilis. PadA van Pediococcus pentosaceus is ook reeds geïdentifiseer.
S. cerevisiae bevat die PAD1- (fenielakrielsuurdekarboksilase) geen, wat teen 'n vaste
tempo in gis getranskribeer word. Die aktiwiteit van hierdie ensiem is egter laag.
Fenolsuurdekarboksilase van B. subtilis, sowel as p-kumaarsuurdekarboksilase van
L. plantarum, vertoon "n substraat-induseerbare dekarboksilerende aktiwiteit met fenoliese
sure. Beide die p-kumaarsuur dekarboksilase en die fenolsuurdekarboksilase gene is in die
PGK1PT ekspressie kasset gekloneer. Episomale en gisintegreringsplasmiede is vir die
PAD1-geen onder beheer van die PGK1 PT ekspressiekasset gekonstrueer vir die
ooruitdrukking van hierdie geen in gis. Die PGK1 PT ekspressiekasset het bestaan uit die
promotor- (PGK1 p) en termineerdersekwense (PGK1 T) van die gisfosfogliseraatkinasegeen
(PGK1). Industriële gisrasse is ontwikkel waarin die PAD1-geen onderbreek is.
Ooruitdrukking van p-kumaarsuurdekarboksilase (Pdc) en fenolsuurdekarboksilase (pade) in
S. cerevisiae toon hoë ensiemaktiwiteit in laboratoriumgisrasse. Die ooruitdrukking van die
PAD1-geen het nie hoër aktiwiteit as die kontroleras gewys nie. Albei die bakteriële gene,
onder die beheer van die PGK1 PT ekspressiekasset, is ook in "n gisintegreringsplasmied met
die SMR1-geen as selektiewe merker geplaas. Die klonering en transformasie van pdc en
padc in industriële wyngiste kan dus gebruik word vir die bepaling van die effek van fenolsuur
dekarboksilases in die wynmaakproses en die moontlike verbetering van wynaroma. Wyn is
met al drie die industriële rasse (die ooruitgedrukte bakteriële gene en die ontwrigte PAD1- geen) gemaak. Die effek van die teenwoordigheid van hierdie gene in die wynmaakproses is
deur gaschromatografie bepaal. Die resultate het aangedui dat die bakteriële gene op In
effektiewe wyse vlugtige fenole in die wyn kan produseer. Sekere monoterpene is ook in In
verhoogde mate gedurende hierdie proses gevorm.
Hierdie studie baan die weg vir die ontwikkeling van reingisinentingskulture vir die
produksie van optimale vlakke van vlugtige fenole om sodoende die sensoriese gehalte van
die wyn te verbeter.
|
14 |
The evaluation of β-glucosidase activity produced by wine-isolated yeastsPotgieter, Nydia, 1977- 03 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: ~-Glucosidases constitute a major group of biologically important enzymes that
catalyze the hydrolysis of glycosidic linkages in ~-glucosides, as well as in glycosides
that contain only carbohydrate residues, e.g. cellobiose. These enzymes occur in all
living kingdoms and perform a variety of functions in organisms ranging from bacteria
to highly evolved mammals. Three different types of ~-glucosidases are found in
humans, each with its own function: glucocerebrosidase (a deficiency causes
Gaucher disease), lactase-phlorizin hydrolase (a deficiency results in lactose
intolerance) and cytosolic ~-glucosidase (responsible for the hydrolysis of ~-
glucosides ingested with foods of plant and animal origin). In plants, the functions of
~-glucosidases include pigment metabolism, biomass conversion and cyanogenesis,
a function it shares with insect ~-glucosidases. Microbial ~-glucosidases, as part of
the cellulase enzyme system that is responsible for the hydrolysis of cellobiose and
short-chain oligosaccharides into glucose, playa role in the conversion of cellulosic
biomass to liquid fuel. These microbial ~-glucosidases also playa very important role
in the enhancement of fruit and wine aromas through the liberation of monoterpenols.
Monoterpenols play an invaluable role in the flavor and aroma of grapes and
wine, and are present as free, volatile and odorous molecules, as well as flavorless,
non-volatile glycosidic complexes. These complexes most often occur as 6-0-~-Dxvlopyranosyl-
B-Dcqlucopyranosides, 6-0-~-D-glucopyranosyl-~-D-glucopyranosides,
6-0-a-L-arabinofuranosyl-~-D-glucopyranosides, 6-0-a-L-rhamnopyranosyl-~-Dglucopyranosides,
or 6-0-~-D-apiofuranosyl-~-D-glucopyranosides of mainly linalool,
geraniol, nerol, a-terpineol and hotrienol. Two mechanisms exist for the release of
monoterpenes from glycosidically bound non-volatile precursors: acid hydrolysis and
enzymatic hydrolysis. As high temperature acid hydrolysis causes a rearrangement
of the monoterpene aglycones, the focus has shifted towards the more efficient
enzymatic hydrolysis that does not result in modifications of the intrinsic aromatic
character of the wine.
The endogenous ~-glucosidases of grapes (Vitis vinifera), as well as of the wine
yeast Saccharomyces cerevisiae, exhibit very low activity towards the glycoside
precursors, and thus the focus has increasingly fallen on the addition of exogenous
~-glucosidases to enhance wine flavor. Fungal, bacterial and some yeast ~-
glucosidases have been indicated as effective aroma liberators, but these enzymes
are not always suitable for use under the harsh conditions that prevail during
winemaking (i.e. low pH, low temperatures, and high ethanol and glucose
concentrations). The limited enzyme activities of the abovementioned
microorganisms have resulted in a search among non-Saccharomyces yeasts for ~-
glucosidases that can withstand these conditions.
The ~-glucosidase activities of 20 wine-associated non-Saccharomyces yeasts
were quantified, characterized and assessed to determine the efficiency with which
they could liberate monoterpenols from their terpenyl-glycosides. The Debaryomyces pseudopolymorphus l3-glucosidase from intracellular crude cell extracts exhibited the
most suitable combination of properties in terms of functionality at wine pH,
resistance to wine-associated inhibitory compounds (glucose, ethanol and sulfur
dioxide), high substrate affinity and large aglycone-substrate recognition. This yeast
strain was also used, in conjunction with S. cerevisiae VIN13, for the small-scale
fermentation of Chardonnay juice. The results indicated that the l3-glucosidase of D.
pseudopolymorphus had definite potential as a wine aroma-enhancing enzyme, as
the concentrations of free terpenols (nerol, geraniol and citronellol) were significantly
increased during fermentation.
Future experimental work would include an in-depth study of the kinetic
characteristics of the l3-glucosidases (both cytosolic and cell-associated) exhibiting
the highest terpenol-liberating activity under winemaking conditions. The next step
would then be the cloning and expression of the most efficient l3-glucosidase gene in
a commercial wine yeast. Such a recombinant wine yeast would release grapederived
aroma compounds from their non-volatile precursors during single culture
fermentations, thereby increasing the sensorial quality of wine. / AFRIKAANSE OPSOMMING: I3-Glukosidases vorm deel van 'n groot groep biologies belangrike ensieme wat die
hidrolise van glikosidiese bindings binne l3-glukosiede,sowel as binne glikosiede wat
slegs uit koolhidraatresidue bestaan, soos bv. sellobiose, kataliseer. Hierdie ensieme
kom in alle koningkryke van lewende organismes voor en verrig 'n wye
verskeidenheid funksies binne organismes wat wissel van bakterieë tot hoogs
ontwikkelde soogdiere. Drie verskillende tipes l3-glukosidases,elk met sy eie funksie,
kom in mense voor: glukoserebrosidase ('n gebrek hieraan lei tot Gaucher-siekte),
laktaseflorizinhidrolase ('n gebrek hieraan gee aanleiding tot laktose-intoleransie) en
sitosol l3-glukosidase (verantwoordelik vir die hidrolise van l3-glukosiede wat saam
met voedsel van plant en dier oorsprong ingeneem word). Die funksies van 13-
glukosidase binne plante sluit in pigmentmetabolisme, biomassa-omsetting en
sianogenese, wat ook 'n funksie van insek l3-glukosidases is. Mikrobiese 13-
glukosidases, as deel van die sellulase-ensiemsisteem wat verantwoordelik vir die
hidrolise van sellobiose en kortketting-oligosakkariede na glukose is, speel 'n rol in
die omsetting van sellulosebiomassa na brandstof. Hierdie mikrobiese 13-
glukosidases speelook 'n baie belangrike rol in die verbetering van vrugte- en
wynaroma deur die vrystelling van monoterpenole.
Monoterpenole speel 'n belangrike rol in die geur en aroma van druiwe en wyn,
en kom voor as vry, vlugtige en aromatiese molekules, asook geurlose, nie-vlugtige
glikosidies-gebonde komplekse. Hierdie komplekse is meestal in die vorm van 6-0-
I3-D-xilopiranosiel-I3-D-glukopiranosiede, 6-0-a-L-arabinofuranosiel-I3-D-glukopiranosiede,
6-0-I3-D-glukopiranosiel-I3-D-glukopiranosiede, 6-0-a-L-ramnopiranosiel-
I3-D-glukopiranosiede,of 6-0-I3-D-apiofuranosiel-I3-D-glukopiranosiedevan
hoofsaaklik linalool, geraniol, nerol, a-terpineol en hotrienol. Monoterpenole kan op
een van twee maniere van hul suikermolekules vrygestel word: suurhidrolise of
ensimatiese hidrolise. Die hoë temperature waarby suurhidrolise plaasvind
veroorsaak 'n herrangskikking van die monoterpeen aglikone, en die fokus het
gevolglik verskuif na die meer effektiewe ensimatiese hidrolise wat nie verandering
van die intrinsieke aromatiese karakter van die wyn tot gevolg het nie.
Die endogene l3-glukosidases van druiwe (Vitis vinifera) en die wyngis
Saccharomyces cere visiae , toon baie lae aktiwiteit ten opsigte van die aromatiese
voorlopers, en dus word daar toenemend op die toevoeging van eksogene 13-
glukosidases tot die wyn gefokus om meer geur vry te stel. Daar is bevind dat 13-
glukosidases van fungiese, bakteriële en gis oorsprong effektiewe aromavrystelIers
is, maar hierdie ensieme is nie altyd gepas vir gebruik in wyn nie, aangesien dit 'n
omgewing is met 'n lae pH, lae temperatuur, en hoë etanol- en glukosekonsentrasies.
Die beperkte ensiemaktiwitiet van bogenoemde mikroorganismes het gelei tot 'n
soeke onder nie-Saccharomyces giste na l3-glukosidases wat in die wynomgewing
kan funksioneer. Die ~-glukosidase-aktiwiteit van twintig wyn geassosieerde nie-Saccharomyces
giste is gekwantifiseer en gekarakteriseer om te bepaal tot watter mate dit
monoterpenole van hul terpeniel glikosiede kan vrystel. Die intrasellulêre ~-
glukosidase teenwoordig in the selekstrak van Debaryomyces pseudopolymorphus,
het die belowendste resultate getoon ten opsigte van funksionaliteit by wyn se pH,
weerstand teen wyn geassosieerde inhibeerders (glukose, etanol en
swaweidioksied), hoë substraataffiniteit en breë aglikoon-substraat herkenning.
Hierdie gisras is ook in kombinasie met S. cerevisiae VIN13 gebruik vir die
kleinskaalse fermentasie van Chardonnay sap. Die resultate het getoon dat die ~-
glukosidase van D. pseudopolymorphus wel potensiaal het om wynaroma te
verhoog, aangesien die konsentrasie van ongebonde terpenole (nerol, geraniol en
citronellol) aansienlik tydens fermentasie toegeneem het.
Toekomstige eksperimentele werk sluit in, onder meer, In in-diepte studie van die
kinetiese eienskappe van die ~-glukosidases (beide sitesolies en sel-geassosieerd)
wat die meeste terpenole onder wynrnaakkondisies vry stel, asook die klonering en
uitdrukking van die enkele ~-glukosidasegeen met die hoogste aktiwiteit, in In
kommersiële wyngis. Só In rekombinante wyngis sal die vrystelling van druifgebaseerde
aromakomponente van hul nie-vlugtige, geurlose voorlopers tydens
enkel-kultuur fermentasies teweeg bring.
|
15 |
The taxonomy and physiology of the lactic acid bacteria in South African dry winesDu Plessis, L. de W. (Ludwig de Wet) 12 1900 (has links)
Thesis (DSc)--Stellenbosch University, 1961. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming
|
16 |
Deletion analysis of the Ure2p in Saccharomyces cerevisiae and effect of NCR on the production of ethyl carbamate during wine fermentationsErasmus, Daniel J. 12 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The wine yeast Saccharomyces cerevisiae has the ability to utilize several different
nitrogenous compounds to fulfill its metabolic requirements. Based upon different
growth rates of the yeast in a particular nitrogen source, nitrogen compounds have
been classified as either good or poor nitrogen sources. In an environment which
contains different quality nitrogen sources, such as grape must, the yeast first utilizes
good and then the poor nitrogen sources. This discrimination between good and
poor nitrogen sources is referred to as nitrogen catabolite repression (NCR).
Examples of good nitrogen sources are ammonia, glutamine and asparagine.
Nitrogen sources such as allantoin, y-aminobutyrate (GABA), arginine and proline
are poor quality nitrogen sources.
Several regulatory proteins, Ure2p, Gln3p, Da180p,Gat1pand Deh1p, mediate NCR
in S. cerevisiae. These trans-acting factors regulate transcription of NCR sensitive
genes. All these proteins, except Ure2p, bind cis-acting elements in the promoters
of genes that are responsible for degradation of poor nitrogen sources. Gln3p is an
activator of NCR sensitive genes in the absence of good nitrogen sources. The
predominant mechanism by which NCR functions is by using Ure2p to inactivate the
activator Gln3p in the presence of a good nitrogen source.
Several research groups have studied the Ure2p, mainly due to its prion-like
characteristics. The Ure2p has two domains: a prion inducing domain located in the
N-terminal region and a NCR regulatory domain located in the C-terminal domain.
The aims of this study were (i) to determine the part of the C-terminal domain which
is responsible for NCR, (ii) to establish if ure2 deletion mutants produce less ethyl
carbamate during wine fermentations and (iii) if NCR functions in industrial yeast
strains. Nested deletions of the URE2 gene revealed that the NCR regulatory
domain resides in the last ten amino acids of the Ure2p. This was established by
Northern blot analysis on the NCR sensitive genes DAL5, CAN1, and GAP1 genes.
Ethyl carbamate in wine is produced by spontaneous chemical reaction between
urea and ethanol in wine. Urea is produced by S. cerevisiae during the metabolism of arginine. Arginine is degraded to ornithine and urea by arginase, the product of
the CAR1 gene. Degradation of urea by S. cerevisiae is accomplished by urea
amidolyase, a bi-functional enzyme and product of the DUR1,2 gene which is subject
to NCR. This study investigated if a ure2 mutant strain produced less ethyl
carbamate during wine fermentations.
Wine fermentations were conducted with diploid laboratory strains: a ure2 mutant
strain and its isogenic wild type strain. GC/MS analysis of the wine revealed that the
ure2 mutant produced less ethyl carbamate but more ethanol than the wild type
strain when arginine, di-ammoniumphosphate, asparagine or glutamine were added
as nitrogen sources, in combinations and separately. There was no significant
difference between the wild type fermentation and the ure2 mutant fermentation
when no nitrogen was added. It was found that a combination between the deletion
of URE2 and the addition of a good nitrogen source resulted in lower levels of ethyl
carbamate.
High density micro array analysis done on an industrial strain wine yeast in
Chardonnay grape must revealed that the GAP1, CAN1, CAR1 and DUR1,2 genes,
responsible for transport and metabolism of arginine and degradation of urea, are
NCR sensitive. These data strongly suggest that NCR functions in industrial yeast
strains. / AFRIKAANSE OPSOMMING: Die wyngis Saccharomyces cerevisiae kan verskillende stikstofbronne gebruik om in
sy stikstofbehoeftes te voldoen. Stikstofbronne word as goeie of swak stikstofbronne
geklassifiseer op grond van die groeitempo van die gis op die betrokke stikstofbron.
'n Goeie stikstofbron laat die gis vinniger groei as wat dit op 'n swak stikstofbron sou
groei. In omgewings soos druiwemos waar daar 'n verskeidenheid van
stikstofbronne teenwoordig is, sal die gis eers die goeie bronne en daarna die swak
bronne benut. Stikstofbronne soos ammonium, asparagien en glutamien word
geklassifiseer as goeie bronne. Allantoïen, y-amino-butaraat (GABA), prolien en
arginien word as swak stikstofbronne geklassifiseer. Die meganisme waarmee S.
cerevisiae tussen die stikstofbronne onderskei, staan as stikstof kataboliet
onderdrukking (NCR) bekend.
Die proteïene wat vir verantwoordelik is NCR naamlik Ure2p, Gln3p, Gat1 p, Dal80p
en Deh1 p, bind met die uitsondering van Ure2p, almal aan cis-werkende elemente in
die promoters van NCR-sensitiewe gene. Die trans-werkende faktore reguleer die
transkripsie van NCR-sensitiewe gene. NCR werk hoofsaaklik deur die inhibering
van Gln3p deur Ure2p in die teenwoordigheid van 'n goeie stikstofbron. Die oorgrote
meerderheid NCR-sensitiewe gene word deur Gln3p in die afwesigheid van 'n goeie
stikstofbron geaktiveer.
Heelwat navorsing is op die prionvormings vermoë van Ure2p gedoen. Ure2p het
twee domeine: 'n N-terminale domein wat vir prionvorming verantwoordelik is en die
C-terminale domein waar die NCR funksie van Ure2p gesetel is. Die doel van die
studie was (i) om te bepaal waar in die C-terminale domein van Ure2p die NCR
regulering geleë is, (ii) of ure2 delesie mutante minder etielkarbamaat tydens
wynfermentasies produseer en (iii) of NCR in industriële gisrasse funksioneel is.
Delesie analises van URE2 het getoon dat die NCR regulerings domein in die laaste
tien aminosure gesetel is. Dit is vas gestel m.b.v. noordlike klad tegniek analises op
die OALS, CAN1 en GAP1 gene.Etielkarbamaat in wyn word deur die spontane chemiese reaksie tussen ureum en
alkohol geproduseer. Ureum word gedurende die metabolisme van arginien in S.
cerevisiae geproduseer. Arginien word deur arginase, produk van die CAR1 geen,
na ornitien en ureum afgebreek. Die bi-funksionele ureum amidoliase, gekodeer
deur die DUR1,2 geen, breek ureum na CO2 en NH/ af. As gevolg van die NCRsensitiwiteit
van dié gene is ondersoek ingestel na In ure2 mutant se vermoë om
minder etielkarbamaat tydens wynfermentasies te produseer. Chardonnay
druiwemos is met In diploiede laboratorium ras en die isogeniese ure2 mutant
gefermenteer. GC/MS analise op die wyn het getoon dat die ure2 mutant minder
etielkarbamaat, maar meer alkohol in vergelyking met die wilde tipe gis produseer,
as arginien, di-ammoniumfosfaat, asparagien en glutamien, afsonderlik of
gesamentlik byvoeg is. Daar was egter nie In merkwaardige verskil tussen die
fermentasies waar geen stikstof bygevoeg is nie. Dit dui daarop dat In kombinasie
van In URE2 delesie en die byvoeging van stikstof etielkarbamaat vlakke verlaag.
Mikro-skyfie analise van In industriële gis in Chardonnay mos het getoon dat die
GAP1, CAN1, CAR1 en DUR1,2 gene wat verantwoordelik is vir die transport en
metabolisme van arginien en degradasie van ureum, wel NCR-sensitief is. Dit dui
daarop dat NCRwel in industriële gisrasse funksioneel is.
|
17 |
Fermentation optimization of pediocin PD-1 production and a comparative study of the effect of pediocin PD-1, plantaricin 423 and nisin on biofilms of Oenococcus oeniNel, Hannes Augustinus 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Lactic acid bacteria are present in many foods and beverages and are used as starter cultures in
the production of a variety of fermented products. Many of these bacteria produce ribosomally
synthesized antimicrobial peptides (bacteriocins), which inhibit the growth of bacteria genetically
closely related to the producer cell. Since many of these target bacteria include foodbome
pathogens such as Bacillus spp., Clostridium spp., Listeria spp., and Staphylococcus spp., the
practical importance of these peptides as food preservatives has been well documented and, in the
case of nisin and pediocin PA-I, commercially explored.
The increased demand from health conscious consumers for foods with no chemical
preservatives is putting renewed pressure on the producer to supply a "clean and green" product,
but with the same or even an extended shelf life. Various research groups are screening lactic
acid bacteria for production of novel broad-spectrum antimicrobial peptides or are exploring the
possibilities of altering known bacteriocins to inhibit Gram-negative bacteria, yeasts and molds.
Pediocin PD-I, produced by Pediococcus damnosus NCFB 1832, belongs to the class Ila
bacteriocins, i.e. heat-stable Listeria-active peptides, containing the YGNGV -consensus sequence
in the N-terminal region. Little is known about the production and mode of activity of pediocin
PD-I.
In this study, production of pediocin PD-I was significantly increased by optimizing the
growth medium, De Man Rogosa and Sharpe (MRS) broth. Addition of bacteriological peptone
(1.7%, w/v), manganous sulphate (0.014%, w/v) and Tween 80 (3%, v/v), and lowering of the pH
during fermentation stimulated pediocin PD-I production and the level of organic acids
produced. Maximum levels of bacteriocin activity were recorded at an initial pH of 6.7 in the
latter medium. Under these conditions the specific bacteriocin activity increased by a factor of
approximately six after 55 h of fermentation.
The effect of pediocin PD-I, plantaricin 423, produced by Lactobacillus plantarum 423, and
commercial grade nisin (Aplin and Barrett Ltd., Trowbrige, Wilts, England) was tested against
planktonic cells of Oenococcus oeni and a biofilm of the cells established on stainless steel
surfaces identical to those used in wineries. After 5 h of treatment with 3000 AU (arbitrary
units )/ml of each bacteriocin, all planktonic cells of 0. oeni in a modified Chardonnay must
medium were killed. All viable cells in the biofilm were killed after only 1 h in the presence of 3000 AU/ml of anyone of the bacteriocins. In addition, pediocin PD-I, plantaricin 423 and nisin
removed the biofilms from the surfaces and reduced the biomass either completely, as in the case
of pediocin PD-I, or by 58% and 50% as in the case of plantaricin 423 and nisin, respectively.
These same results were recorded after 5 h of treatment with 3000 AU/ml in a modified
Chardonnay must medium.
To our knowledge this is the first report of controlling biofilm formation of malolactic bacteria
on stainless steel surfaces with natural antimicrobial peptides. This implies that, apart from being
very effective in controlling the cell numbers of free-living cells of 0. oeni, the three
bacteriocins, especially pediocin PD-I, could also be used as natural sanitizers. The fact that the
production and activity levels ofpediocin PD-I could be increased without genetically modifying
the producer strain is an added advantage. / AFRIKAANSE OPSOMMING: Melksuurbakterieë is teenwoordig in verskeie soorte voedsel- en drankprodukte en word as
suurselkulture in die produksie van 'n verskeidenheid gefermenteerde produkte gebruik. Baie van
hierdie bakterieë produseer ribosomaal-vervaardigde antimikrobiese peptiede (bakteriosiene) wat
die groei van ander bakterieë, geneties naverwant aan die produserende organisme, inhibeer.
Omdat baie van hierdie bakterieë voedselpatogene soos Bacillus spp., Clostridium spp., Listeria
spp. en Staphylococcus spp. insluit, is die praktiese belang van hierdie peptiede reeds deeglik
ondersoek en word, soos in die geval van nisien en pediosien PA-I, kommersieel gebruik.
Die toenemende behoefte van die verbruiker na voedselprodukte met geen chemiese
preserveermiddels plaas nuwe druk op die vervaardiger om veilige voedselprodukte te produseer,
maar met dieselfde of selfs langer rakleeftyd. Verskeie navorsingsgroepe bestudeer
melksuurbakterieë vir die produksie van unieke antimikrobiese peptiede met 'n wye spektrum van
inhibisie en ondersoek ook die moontlikhede om hierdie bakteriosiene geneties te manipuleer ten
einde Gram-negatiewe bakterieë, giste en swamme te inhibeer.
Pediosien PD-l, geproduseer deur Pediococcus damnosus NCFB 1832, word as 'n klass na
bakteriosien geklassifiseer. Hierdie groep sluit in die hitte-stabiele Listeria-aktiewe peptiede, met
'n YGNGV-konsensus volgorde in die N-terminale deel van die peptied. Min is egter bekend oor
die meganisme van werking van hierdie bakteriosiene.
In hierdie studie is die produksie van pediosien PD-l betekenisvol verhoog met die
optimalisering van die vloeibare groeimedium De Man Rogosa en Sharpe (MRS). Die
toevoeging van bakteriologiese peptone (1.7%, miv), mangaan sulfaat (0.014%, miv) en Tween
80 (3.0%, v/v) en 'n afname in die pH gedurende groei het pediosien PD-l-poduksie gestimuleer
en sodoende ook die vlak van organiese sure wat geproduseer is. Maksimum vlakke van
bakteriosien-aktiwiteit is in hierdie medium met 'n aanvangs-pH van 6.7 waargeneem. Onder
hierdie omstandighede, en na 55 uur van fermentasie, het die spesifieke aktiwiteit van die
bakteriosien met 'n faktor van ongeveer ses verhoog.
Die effek van pediosien PD-l, plantarisien 423, geproduseer deur Lactobacillus plantarum
423, en 'n kommersiële graad nisien (Aplin and Barrett Ltd., Trowbride, Wilts, Engeland) is teen
die planktoniese selle van Oenococcus oeni en 'n biofilm van hierdie selle, gevestig op 'n vlekvrye
staaloppervlak identies aan wat in wynkelders gebruik word, getoets. Na 5 ure van behandeling met 3000 AB (arbitrêre eenhede)/ml van elke bakteriosien, is al die planktoniese selle van O. oeni
in 'n gemodifiseerde Chardonnay mos-medium vernietig. Alle lewensvatbare selle in die biofilm
is ook na slegs 1 uur in die teenwoordigheid van 3000 AE/ml van enige een van hierdie
bakteriosiene vernietig. Verdermeer het pediosien PD-I, plantarisien 423 en nisien ook die
biofilm op die vlekvrye staal-oppervlak verwyder. In die geval van pediosien PD-I is 'n totale
afname van die biomassa-oppervlak waargeneem, terwyl plantarisien 423 en nisien 58% en 50%
van die totale biomassa verwyder het. Hierdie resultate is na 5 ure van behandeling (3000
AE/ml) in 'n gemodifiseerde Chardonnay mos-medium waargeneem.
Sover ons kennis strek is hierdie die eerste verslag rakende die gebruik van natuurlike
antimikrobiese peptiede om biofilm-vorming deur appel-melksuurbakterieë op vlekvrye staal
oppervlaktes te beheer. Dit impliseer dat bakteriosiene, spesifiek pediosien PD-I, benewens die
beheer van planktoniese selle van appel-melksuurbakterieë, ook as natuurlike oppervlak-reinigers
gebruik kan word. Die feit dat die produksie en aktiwiteitsvlakke van pediosien PD-I verhoog
kon word sonder om die organisme geneties te modifiseer is 'n verdere voordeel.
|
18 |
Characterisation of biogenic amine genes in lactic acid bacteria isolated from wineDowning, Lynn,1978- 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The winemaking process involves a complex microbial flora where the interaction of
yeasts, lactic acid bacteria and acetic acid bacteria play an important role in the
quality and wholesomeness of the final product. Yeasts are primarily responsible for
alcoholic fermentation. Malolactic fermentation follows alcoholic fermentation and is
conducted by lactic acid bacteria. These bacteria are important in winemaking and
can have a positive or negative effect on the wine quality. Biogenic amines are one
of the compounds produced by lactic acid bacteria, which affect the hygienic quality
and wholesomeness of the wine negatively and directly pose a health risk to the
consumer. The demand of consumers for higher quality and healthier foods has led
to renewed interest in studies on biogenic amines. Biogenic amines occur in a wide
variety of food products, such as cheese, dried sausage, sauerkraut, fishery
products, chocolates, wine and beer. This thesis focussed on the presence of
biogenic amines in wine.
The first objective of the study was to determine the ability of lactic acid bacteria
isolated from South African wine to produce biogenic amines, using a decarboxylase
screening plate method. The potential to produce the biogenic amines histamine,
tyramine, putrescine and cadaverine was investigated. The results obtained showed
that Lactobacillus species (Lactobacillus brevis and Lactobacillus hilgardil) might be
the lactic acid bacteria responsible for tyramine and putrescine production and that it
can contribute significantly to the overall biogenic amine content in wines. The
results also suggest that amine production is strain dependent and not species
specific. None of the lactic acid bacteria tested had the ability to produce histamine
or cadaverine. It is important to remember that the ability of the lactic acid bacteria to
produce biogenic amines has only been investigated in synthetic media and that it
does not necessarily imply similar behaviour in wine. Wine represents a complex
environment with a wide number of factors influencing microbial growth and
decarboxylase activity and, thus, further investigation is necessary to determine if
these amine-producing bacteria behave similarly in wine conditions.
In addition, the polymerase chain reaction (PCR) amplification method was
used for the identification of the tyrosine decarboxylase (TOe) gene in some of the
tyramine-producing lactic acid bacteria. This was followed by the sequencing of the
amplified products, which are partial TOe gene sequences, of two L. brevis strains
and of a L. hilgardii strain. Only one tdc gene sequence has been described for
bacteria (Enterococcus faecalis), while a partial TOC gene sequence from L. brevis
lOEB 9809 was described. An amino acid sequence alignment of the three TOe
gene fragments, obtained in this study, with the known TOe gene fragment of
L. brevis lOEB 9809 and the tdc gene of E. faecalis showed a high degree of
relatedness and conserved regions.
To meet consumer demands, procedures are necessary to prevent the
formation of amines in food products. One way of preventing the formation of biogenic amines is to relate amine production with certain lactic acid bacteria species
involved in the winemaking process. Another possible way would be to develop a
rapid detection method for bacteria carrying amino acid decarboxylase genes. The
results of this study provide knowledge about which lactic acid bacteria in the
winemaking process could contribute to the production of biogenic amines and the
sequencing of additional partial TOe genes could possibly assist in the development
of a rapid detection method for tyramine-producing lactic acid bacteria in food
products. / AFRIKAANSE OPSOMMING: Die wynmaakproses behels 'n komplekse mikrobiese flora waar die interaksie van
giste, melksuurbakterieë en asynsuurbakterieë 'n belangrike rol speel in die kwaliteit
en heilsaamheid van die finale produk. Giste is primêr verantwoordelik vir
alkoholiese fermentasie. Appelmelksuurgisting volg op alkoholiese fermentasie en
word deur melksuurbakterieë uitgevoer. Hierdie bakterieë is belangrik in die maak
van wyn en kan 'n positiewe of negatiewe uitwerking op die kwaliteit van wyn hê.
Biogeniese amiene is een van die komponente wat deur melksuurbakterieë
geproduseer kan word en wat die higiëniese kwaliteit en heilsaamheid van die wyn
benadeel. Dit hou ook 'n gesondheidsrisiko vir die verbruiker in. Die vereiste van
verbruikers vir hoër kwaliteit en gesonder voedselprodukte het nuwe belangstelling in
studies op biogeniese amiene ontlok. Biogeniese amiene kom in 'n wye
verskeidenheid voedselprodukte voor, soos kaas, droëwors, suurkool, vis, sjokolade,
wyn en bier. Hierdie tesis fokus op die teenwoordigheid van biogeniese amiene in
wyn.
Die eerste doelwit van die studie was om melksuurbakterieë, wat uit Suid-
Afrikaanse wyn geïsoleer is, se vermoë te bepaal om biogeniese amiene op
dekarboksilase-agarplate te produseer. Die potensiaal om die biogeniese amiene
histamien, tiramien, putresien en kadawerien te produseer, is bestudeer. Die
resultate wat verkry is, toon dat Lactobacillus-spesies (Lactobacillus brevis en
Lactobacillus hilgardit) vir tiramien- en putresienproduksie verantwoordelik is en dat
hulle 'n belangrike bydrae kan lewer tot die totale biogeniese amienkonsentrasie in
wyn. Die resultate dui ook daarop dat die produksie van amiene afhanklik is van die
ras, en nié 'n spesifieke spesie nie. Geen melksuurbakterieë wat getoets is, het die
vermoë getoon om histamien of kadawerien te produseer nie. Dit is belangrik om in
ag te neem dat die vermoë van die melksuurbakterieë om amiene te produseer slegs
in sintetiese media bestudeer is en dat dit nie noodwendig dieselfde gedrag in wyn
sal toon nie. Wyn is 'n komplekse omgewing met 'n wye verskeidenheid faktore wat
die mikrobiese groei en dekarboksilase-aktiwiteit kan beïnvloed, daarom is verdere
studie nodig om vas te stelof hierdie amien-produserende bakterieë dieselfde
gedrag in wyn sal toon.
Die polimerase-kettingreaksie (PKR) amplifikasie-metode is vir die identifikasie
van die tirosiendekarboksilase-geen (TDK) in sommige van die tiramienproduserende
melksuurbakterieë gebruik. Dit is gevolg deur die volgordebepaling
van die geamplifiseerde produkte, wat gedeeltelike TDK-geenvolgordes is, van twee
L. brevis- en van een L. hilgardii-ras. Slegs een tdk-geenvolgorde is al voorheen vir
bakterieë beskryf, nl. Enterococcus faecalis, asook 'n gedeeltelike TDK-geenvolgorde
vir L. brevis lOEB 9809. 'n Vergelyking van die aminosuurvolgordes van die drie
TDK-geenfragmente wat in die studie verkry is, het 'n hoë graad van ooreenkoms en
gekonserveerde areas met die bekende TDK-geenfragment van L. brevis lOEB 9809
en die tdk-geen van E. faecalis getoon. Om verbruikers se behoeftes te bevredig, is dit noodsaaklik dat die vorming van
amiene in voedselprodukte voorkom word. Een manier van voorkoming is om
amienproduksie aan sekere melksuurbakterieë wat in die wynmaakproses betrokke
is, te koppel. 'n Ander manier sal wees om 'n vinnige metode te ontwikkel vir die
opsporing van bakterieë wat aminosuurdekarboksilase-gene dra. Die resultate van
die studie verskaf kennis van watter melksuurbakterieë in die wynmaakproses tot die
produksie van biogeniese amiene kan bydra. Die volgordebepaling van addisionele
gedeeltelike TDK-gene kan moontlik tot die ontwikkeling van 'n vinnige
opsporingsmetode van tiramien-produserende melksuurbakterieë in voedselprodukte
bydra.
|
19 |
Biochemiese veranderinge in druiwemos veroorsaak deur Botrytis cinerea en Rhizopus nigricansHofmann, Gerhard 12 1900 (has links)
Thesis (MScAgric)--Stellenbosch University, 1964. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming
|
20 |
The effect of oxygen on the composition and microbiology of red wineDu Toit, Wessel Johannes 03 1900 (has links)
Thesis (PhD(Agric) (Viticulture and Oenology))--University of Stellenbosch, 2006. / The winemaking process involves different complex chemical and biochemical
reactions, which include those of oxygen (O2). Oxygen can come into contact with the
wine through various winemaking procedures and can be used by the winemaker to
enhance the quality of red wine. In wine, the main substrates for oxidation are
phenolic molecules, which form quinones. These can influence the sensory
characteristics of the wine. O2 can be used in fresh must to remove oxidisable
phenolic molecules through a process called hyper-oxidation and can also be added
to fermenting must to enhance the fermentation performance of yeast. Controlled O2
additions during ageing can lead to the wine’s colour being increased and the
astringency of the wine decreased. This is due to the formation of acetaldehyde from
the oxidation of ethanol, which induces the polymerisation of tannin and anthocyanin
molecules. The addition of too much O2 to wine can, however, lead to unwanted
over-oxidation, with certain off-odours being formed. It can also enhance the growth
of unwanted spoilage microorganisms, such as Brettanomyces and acetic acid
bacteria. Although research on O2 in wine was started many years ago, many
questions still remain. These include the general effect of O2 on the sensory and
phenolic profile of red wine especially and the microbiology of wine during ageing. An
effective way of measuring oxidation, especially in red wine must also be developed.
In the first part of this study, the effects of O2 and sulfur dioxide (SO2) additions
on a strain of Brettanomyces bruxellensis (also known as Dekkera bruxellensis) and
Acetobacter pasteurianus were investigated. Epifluorescence microscopy and plating
revealed that the A. pasteurianus strain went into a viable but non-culturable state in
the wine after prolonged storage under relative anaerobic conditions. This state,
however, could be negated with successive increases in culturability by the addition
of O2, as would happen during the transfer of wine when air is introduced. The A.
pasteurianus strain was also relatively resistant to SO2, but the B. bruxellensis strain
was more sensitive to SO2. A short exposure time to molecular SO2 drastically
decreased the culturability of the B. bruxellensis strain, but bound SO2 had no effect
on the culturability or viability of either of the two types of microorganisms. Oxygen
addition to the B. bruxellensis strain also led to a drastic increase in viability and
culturability. It is thus clear that SO2 and O2 management in the cellar is of critical
importance for the winemaker to produce wines that have not been spoiled by
Brettanomyces or acetic acid bacteria. This study should contribute to the
understanding of the factors responsible for the growth and survival of
Brettanomyces and acetic acid bacteria in wine, but it should be kept in mind that
only one strain of each microorganism was used. This should be expanded in future
to include more strains that occur in wine.
The second part of this study investigated the effect of micro-oxygenation on four
different South African red wines. It was found that the micro-oxygenation led to an
increase in the colour density and SO2 resistant pigments of the two wines in which micro-oxygenation was started just after the completion of malolactic fermentation. In
one of these wines, a tasting panel preferred the micro-oxygenation treated wines to
the control. In the other two red wines, in which the micro-oxygenation was started
seven months after the completion of malolactic fermentation, very little colour
increase was observed. One of these two wines was also matured in an oak barrel,
where the change in phenolic composition was on par with the treated wines. A
prolonged period of micro-oxygenation, however, led to this wine obtaining an
oxidised, over-aged character. Micro-oxygenation and maturation in an oak barrel
also enhanced the survival of acetic acid bacteria and Brettanomyces in this wine.
Micro-oxygenation can hence be used by the wine producer on young red wines to
enhance the quality of the wine, but should be applied with care in older red wines.
Future research into micro-oxygenation should focus on whether it can simulate an
oak barrel. More research into the effect of micro-oxygenation on the sensory profile
of the wine is needed.
As mentioned, the addition of O2 can lead to oxidative degradation of wine. The
brown colour in wine is often used as an indication of oxidation, but oxidative aromas
can be perceived before a drastic increase in the brown colour has been observed in
red wine.
The third part of this study was to assess the possible use of Fourier Transform
Infrared Spectroscopy (FTIR) to measure the progression of oxidation in Pinotage red
wines. Three wines were used in this study and clear separation between the control
and aerated wines was observed by using Principle Component Analysis (PCA).
Sensory analysis of these wines confirmed this observation, with a reduction
especially in berry fruit and coffee characters and an increase first in potato skin and
then acetaldehyde aroma characters as the oxidation progressed. PCA analysis also
revealed that in certain wines the visible spectrum of light did not indicate the
progression of oxidation as sensitively as with the use of FTIR. This also correlated
with the inability of the panel to observe a drastic colour change. FTIR should be
further investigated as a possible means of monitoring oxidation in wine and this
study should be expanded to wines made from other cultivars as well.
|
Page generated in 0.1915 seconds