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Evaluation of parameters to determine optimum ripeness in Cabernet Sauvignon grapes in relation to wine quality /Botes, Matthys Petrus. January 2009 (has links)
Thesis (MScAgric)--University of Stellenbosch, 2009. / Bibliography. Also available via the Internet.
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A study of y̲a̲y̲i̲n̲Louie, Wallace. January 1983 (has links)
Thesis (Th. M.)--Grace Theological Seminary, 1983. / Abstract. Includes bibliographical references (leaves 104-112).
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'n Vertalerswoordeboekmodel vir die Suid-Afrikaanse wynbedryf /Venter, Rudi. January 2005 (has links)
Thesis (MPhil)--University of Stellenbosch, 2005 / Bibliography. Also available via the Internet.
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Monitoring the quality control chain from vineyard to wine : an industrial case study /Swanepoel, Marinda. January 2006 (has links)
Thesis (MSc)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.
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A study of y̲a̲y̲i̲n̲Louie, Wallace. January 1983 (has links)
Thesis (Th. M.)--Grace Theological Seminary, 1983. / Abstract. Includes bibliographical references (leaves 104-112).
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Chemical, sensory and consumer profiling of a selection of South African Chenin blanc wines produced from bush vinesHanekom, Evette 12 1900 (has links)
Thesis (MScFoodSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Twenty five commercial Chenin blanc wines produced solely from bush vine vineyards and including three vintages, three styles and five production areas, were sourced for this study. Descriptive sensory analysis (DSA) and chemical analyses including GC-FID (gas chromatography fitted with a flame ionisation detector) and FTMIR (Fourier transform mid-infrared) spectroscopy were employed to establish the sensory and chemical characteristics, whereas consumer tests were conducted to determine consumer perception and liking of bush vine Chenin blanc wines. DSA (a profiling technique) was also compared to the sorting task (a classification technique) with a description assignment to evaluate the sorting task’s ability to profile wines.
According to the results of DSA, the wines separated into two groups. One group associated with sensory attributes which can be considered indicative of the Fresh and Fruity Chenin blanc style. The other group associated with sensory attributes which can be considered indicative of the Rich and Ripe style of Chenin blanc. No separation between the wooded and unwooded Rich and Ripe styles was apparent.
According to the results of the chemical analyses, the wines also separated into two groups. This separation seemed to be caused by vintage and the chemical changes associated with ageing as the wines from the youngest vintage (2010) was strongly associated with high levels of esters and malic acid. The older wines were situated farthest away from these attributes indicating low concentrations. When comparing the results from the sorting task and DSA, it could be seen that similar wine style groupings formed, indicating that DSA can also be regarded as an effective tool when categorising wines. The differences in the positioning of some of the wines and attributes on the DSA multivariate plots and the sorting task plots could be attributed to the difference in panels used. The sorting task was conducted using an expert panel with persons illustrating significant technical knowledge of Chenin blanc wines. Experience, exposure and technical knowledge tend to establish a common language amongst wine experts which could have caused the expert panel to perceive some wines differently when comparing the results of the latter panel to that of the trained panel. DSA was found to remain the most effective method for establishing a comprehensive sensory profile.
Consumer analyses showed that regular white wine drinkers prefer the unwooded styles (Fresh and Fruity and Rich and Ripe unwooded) of Chenin blanc more than the wooded style. It was also found that consumers with a higher level of objective wine knowledge tend to associate the terms ‘bush vine’ and ‘old bush vine’ with the Rich and Ripe style of Chenin blanc, whereas consumers with a lower level of objective wine knowledge associated ‘old bush vine’ with the Fresh and Fruity style. Since all the wines used in the consumer analysis were produced from old bush vines, it is evident that consumer education on the impact of bush vine training system and vine age on wine quality is needed. Better understanding of these principles could lead to elevated product appraisals and consumer satisfaction. / AFRIKAANSE OPSOMMING: Vyf en twintig kommersiële Chenin blanc wyne, uitsluitlik van bosstok wingerde geproduseer, is bekom vir hierdie studie. Die wyne het drie style, drie oesjare en vyf produksiestreke ingesluit. Beskrywende sensoriese analise (BSA) en chemiese analises, wat GC-FID (gas chromatografie gekoppel met vlam-ioniserende deteksie) en FTMIR (Fourier-transformering mid-infrarooi) spektroskopie insluit, is uitgevoer om onderskeidelik die sensoriese en chemiese eienskappe van die wyne te bepaal. Verbruikerstoetse is ook uitgevoer om verbruikerspersepsie en -voorkeure vir bosstok Chenin blanc wyne te bepaal. BSA (‘n profilerings tegniek) was ook vergelyk met ‘n sorterings taak (‘n klassifikasie tegniek) met ‘n beskrywings opdrag, primêr om die sorterings taak se vermoë om wyne te profileer te ondersoek.
Volgens die resultate van BSA, het die wyne in twee groepe verdeel. Een groep het met die sensoriese eienskappe wat op ‘n Vars-en-Vrugtige-styl dui, geassosieër. Die ander groep het met sensoriese eienskappe geassosieër wat met die Volrond-styl verband hou. Geen verdeling tussen die gehoute en ongehoute wyne binne die Volrond-styl was sigbaar nie.
Volgens die resultate van die chemiese analises, het die wyne ook in twee groepe verdeel. Die verdeling blyk asof dit veroorsaak is deur oesjaar en die chemiese veranderinge wat met wynveroudering gepaard gaan. Wyne van die jongste oesjaar (2010) het ‘n sterk verband met hoë vlakke van esters en appelsuur getoon. Die ouer wyne was verder weg van hierdie eienskappe geleë, wat op laer ester en appelsuur konsentrasies dui. Wanneer die meerveranderlike resultate van die sorterings taak (met en sonder die aanduiding van sensoriese eienskappe) en dit van BSA vergelyk word, kon soortgelyke groeperings gesien word. Dit is ‘n aanduiding dat BSA ook wyne effektief kan kategoriseer. Die verskil in posisionering van sommige wyne tussen die BSA en sorterings taak resultate, kan toegeskryf word aan die verskillende panele wat gebruik is om die tegnieke uit te voer. ‘n Deskundige paneel (wynkenners) is gebruik om die sortingstaak uit te voer. Ervaring, blootstelling en tegniese kennis is geneig om te lei tot die vestiging van ‘n gemeenskaplike taal onder wynkenners. Hierdie gemeenskaplike taal kan as rede aangevoer word vir die uiteenlopende analise van sommige wyne wanneer die resultate van die deskundige paneel met dié van die opgeleide paneel (in BSA gebruik) vergelyk word. Dit is gevind dat BSA, wanneer ‘n omvattende sensoriese profiel bepaal moet word, die mees effektiefste metode bly.
Verbruikerstoetse het getoon dat gereelde witwyn-verbruikers die ongehoute Chenin blanc style (Vars-en-Vrugtig en ongehoute Volrond) bo die gehoute styl verkies. Dit is ook bepaal dat verbruikers met ‘n hoër vlak van objektiewe wynkennis neig om die terme ‘bosstok’ en ‘ou bosstok’ met die Volrond-styl van Chenin blanc te assosieer, terwyl verbruikers met ‘n laer vlak van objektiewe wynkennis die term ‘ou bosstok’ met die Vars-en-Vrugtige Chenin blanc styl assosieër. Aangesien al die wyne wat in die verbruikerstoetse ingesluit is van ou bosstok wingerde geproduseer is, is dit duidelik dat verbruikeropvoeding insake die effek van die gebruik van bosstokke en ou wingerdstokke op wynkwaliteit noodsaaklik is. ‘n Beter begrip van hierdie beginsels sal lei tot verhoogde produkwaardasie, asook ‘n toename in verbruikertevredenheid.
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Anaerobic bioconversion of liquid and solid wastes from the winemaking processde Kock, Michelle 18 February 2015 (has links)
Thesis (MSc Food Sc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: South Africa is a developing country that relies on its agricultural sector as a main source of overall
economic welfare. Development does not only give rise to new technology and new products but
also results in increased amounts of liquid and solid waste.
Generally, the production of wine is considered an environmentally friendly process, but
significant amounts of natural resources and organic amendments are necessary, while generating
large amounts of liquid and solid wastes. Anaerobic digestion (AD) is an attractive and proven
treatment option for both liquid and solid wastes as valuable products and depollution can be
obtained. AD of liquid waste results in an effluent and biogas, while anaerobic composting of solid
waste results in an organic amendment, leachate and biogas.
The overall objective of this study was to investigate the operational feasibility of the cotreatment
of leachate produced during the anaerobic composting (AnC) of grape skins in an upflow
anaerobic sludge blanket (UASB) reactor while treating winery wastewater. This first aim of this
study was to investigate the efficiency of the anaerobic composting of grape skins. Laboratoryscaled
digesters (1L) were utilised as anaerobic composting units. The most important operational
parameters were identified (pH, moisture content and inoculum (size, ratio, composition)) in order
to produce a pH stable, odour free compost in 21 days.
Experimental studies highlighted the importance of shredding waste as well as the addition
of calcium oxide and green waste to increase the initial pH of the composting mixture. After
optimising a 50% (m.m-1) cow manure inoculum, lower inoculum concentrations (10, 15 and 25%
(m.m-1)) were investigated to make the process more economically viable. A 10% (m.m-1)
anaerobic compost (AC) inoculum was found to produce the most favourable results in terms of pH
stabilisation and leachate generation. A 50% (m.m-1) moisture level performed the best by
attaining a pH > 6.5 on day 6 and having the highest end pH (7.65) on day 21, while white and red
grape skins in an equal ratio were found to generate a higher end pH. With all these optimum
parameters in place (shredded waste, green waste, CaO, inoculum, moisture, grape skins), a
compost with a final pH (7.09), moisture (58%), nitrogen (2.25%), phosphorous (0.22%) and
potassium content (1.7%) was obtained. The optimised parameters were scaled-up (1:10) by
using polyvinyl chloride anaerobic digesters (20 L) to suit the operational requirements of the AnC
process and also produced a stable compost within 21 days. The second aim of this study was to investigate the combined anaerobic digestion of winery
wastewater (WWW) and leachate obtained from the anaerobic composting of grape skins in an
upflow anaerobic sludge blanket (UASB). This involved the operation of a 2.3 L laboratory-scale
UASB reactor for 205 days. The reactor successfully co-treated WWW and leachate at
ca. 8.5 kgCOD.m-3d-1 with a final chemical oxygen demand (COD) reduction of over 90%, a stable
reactor effluent pH (7.61) and alkalinity (3 281 CaCO3 mg.L-1). This study showed the feasibility
for the combined treatment of liquid and solid waste from the winemaking process. Although the legal limits for reactor effluent disposal onto land was not met, significant reduction in COD
concentrations were achieved, whilst producing a soil amendment that could potentially result in
cost savings for chemical fertilisers. The benefits related to using anaerobic bioconversion as a
treatment option for liquid and solid waste could possibly be advantageous to the wine industry as
an environmental control technology, by converting liquid and solid waste into valuable resources. / AFRIKAANSE OPSOMMING: Suid-Afrika is 'n ontwikkelende land wat staatmaak op sy landbousektor as 'n hoofbron van
algehele ekonomiese welstand. Ontwikkeling gee nie net aanleiding tot nuwe tegnologie en nuwe
produkte nie, maar lei ook tot die verhoogde bydrae van vloeistof sowel as vaste afval.
Oor die algemeen, word die produksie van wyn beskou as 'n omgewingsvriendelike proses,
maar aansienlike hoeveelhede natuurlike hulpbronne en organiese kunsbemesting word benodig,
terwyl groot hoeveelhede vloeistof en vaste afval gegenereer word. Anaërobiese vertering (AV) is
'n aantreklike en bewese behandelingsopsie vir beide vloeistof en vaste afval aangesien
waardevolle produkte en suiwering verkry kan word. AV van vloeistowwe lewer uitvloeisel sowel
as biogas, terwyl anaërobiese kompostering van vaste afval 'n organiese kunsbemesting, loog en
biogas lewer.
Die oorhoofse doel van hierdie studie was om die operasionele doeltreffendheid van die
mede-behandeling van loog wat gegenereer word tydens die anaërobiese kompostering (AnK) van
druiwe doppe in 'n opvloei-anaërobiese-slykkombers (OAS) reaktor terwyl kelderafvalwater
behandel word, te ondersoek. Die eerste mikpunt van hierdie studie was om die doeltreffendheid
van die anaërobiese komposteringsproses van druiwe doppe te ondersoek. Laboratorium-skaal
verteerders (1L) is gebruik as anaërobiese komposteringseenhede. Die belangrikste operasionele
parameters is geïdentifiseer (pH, voginhoud en inokulum (grootte, verhouding, samestelling)) om ‘n
'n pH-stabiele, reukvrye kompos te produseer in 21 dae. Die belangrikheid van gesnipperde afval asook die byvoeging van kalsiumoksied en groen
afval om die aanvanklike pH van die komposmengsel te verhoog, is deur eksperimentele studies
beklemtoom. Na die optimering van 'n 50% (m.m-1) koeimis inokulum, is laer inokulum
konsentrasies (10, 15 en 25% (m.m-1)) geondersoek om die proses meer ekonomies uitvoerbaar te
maak. Daar is gevind dat ‘n 10% (m.m-1) anaërobiese kompos (AK) inokulum die mees gunstige
resultate lewer in terme van pH stabilisering en loog generering. ‘n 50% (m.m-1) vloeistof vlak het
die beste presteer deur 'n pH> 6.5 te bereik teen Dag 6 asook die hoogste eind pH (7.65) teen Dag
21, terwyl wit en rooi druiwe doppe in dieselfde verhouding gevind is om ‘n hoër eind pH te
genereer. Met al hierdie optimum parameters in plek (gesnipperde afval, groen afval,
kalsiumoksied, inokulum, vog, druiwe doppe) is 'n kompos met 'n finale pH (7.09), vog (58%),
stikstof (2.25%), fosfor (0.22%) en kalium inhoud (1.7%) verkry. Die optimale parameters is
opgeskaal (1:10) deur gebruik te maak van polivinielchloried anaërobiese verteerders (20 L) om
aan die operasionele vereistes van die AnK proses te voldoen en ook om 'n stabiele kompos binne
21 dae te produseer.
Die tweede mikpunt van hierdie studie was om die gekombineerde anaërobiese vertering
van kelderafvalwater en loog, verkry vanaf die anaërobiese kompos van druiwe doppe in 'n OAS
reaktor, te ondersoek. Dit het die bedryf van 'n 2.3 L laboratorium-skaal OAS reaktor vir 205 dae
ingesluit. Die reaktor het kelderafwater en loog suksesvol behandel by ongeveer 8.5 kgCSV.m-3d-1
met 'n finale chemiese suurstof vereiste (CSV) vermindering van meer as 90%, 'n stabiele reaktor
uitvloeisel pH (7.61) en alkaliniteit (3 281 CaCO3mg.L-1). Hierdie studie het die uitvoerbaarheid
van die gekombineerde behandeling van vloeistof en vaste afval van die wynmaakproses getoon.
Alhoewel die wetlike vereistes van die reaktor uitvloeisel vir storting op grond nie bereik is nie, is ‘n
beduidende vermindering in CSV konsentrasies bereik, asook die vervaardiging van
kunsbemesting wat die potensiële aankoopkoste van chemiese kunsmis kan verminder. Die
voordele verbonde aan die gebruik van anaërobiese bio-omskakeling as 'n behandelingsopsie vir
vloeistof en vaste afval kan moontlik voordelig wees vir die wynbedryf as 'n omgewingsbeheerende
tegnologie deur om vloeistof en vaste afval om te skakel na waardevolle bronne.
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The production of resveratrol by wine yeastArmstrong, Gareth Owen 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Grapevine is constantly under attack from a wide variety of pathogens including viruses,
bacteria and fungi. In order to ensure survival, the grapevine has developed a vast array of
defense mechanisms to combat invading organisms. A key element of this disease
resistance is the production of phytoalexins, of which resveratrol is the primary component.
The synthesis of resveratrol, together with other structural and biochemical defense
mechanisms equips the plant to combat a number of pathogens resulting in the production
of healthy grapes for the vinification of top quality wine. As part of the active disease
response resveratrol is synthesised de novo in the berry skin at the site of infection, on
recognition of the pathogen. Here it is able to limit the damage caused by the pathogen as
well as preventing it from spreading. This gives the plant the opportunity to initiate its
systemic acquired resistance thereby protecting the rest of the plant and preventing
secondary infections.
The fermentation of red wine on the grape skins allows for the extraction of resveratrol
from the skin into the wine. Red wines therefore have a significantly higher concentration
of resveratrol than white varieties, which contain little or no resveratrol at all. It is for this
reason that the moderate consumption of wine, in particular red wine, is synonymous with
a healthy lifestyle. The antioxidant and anti-inflammatory activities of resveratrol are
important contributors to the cardiovascular benefits derived from the consumption of red
wine. It now seems, however, that significant cardiovascular protection is derived from the
synergistic action of resveratrol, the polyphenols and the alcohol in wine.
With the wholesomeness of any food or beverage being of extreme importance, the
aim of this project was to manipulate wine yeast to produce resveratrol during
fermentation. This required the introduction of an entire metabolic pathway, by integrating
plant genes into the yeast. Resveratrol synthase utilises three malonyl-CoA and one pcoumaroyl-
CoA molecules to produce one molecule of resveratrol, Saccharomyces
cerevisiae produces malonyl-CoA but no p-coumaroyl-CoA. Therefore, the following genes
were obtained to enable yeast to produce p-coumaroyl-CoA: PAL, encoding phenylalanine
ammonia-lyase to convert phenylalanine into cinnamic acid; C4H, encoding cinnamate-4-
hydroxlyase to convert cinnamic acid into p-coumaric acid; and 4CL9 or 4CL216 encoding
CoA-ligases to convert the p-coumaric acid into p-coumaroyl-CoA. To attain high-level
expression, the genes were subcloned under the control of the phosphoglycerate kinase
gene (PGK1) promoter and terminator. Due to integration problems with these expression
cassettes and the fact that the yeast was able to consume p-coumaric acid, the 4CL9,
4CL216 and Vst1 (encoding resveratrol synthase) genes were subcloned under the control
of the alcohol dehydrogenase (ADH2) and PGK1 promoters into episomal plasmids,
respectively. A laboratory yeast strain containing both the Vst1 and 4CL9, or the Vst1 and
4CL216 genes was evaluated for its ability to utilise p-coumaric acid and produce
resveratrol. Northem analysis confirmed that the Vst1, 4CL9 and 4CL216 genes were transcribed and over-expressed compared to the control strain. The transformants
expressing the CoA-ligase genes utilised the p-coumaric acid faster than the control,
although it was not possible to determine whether p-coumaroyl-CoA was produced. No
resveratrol was produced under the assay conditions used. The results indicated that the
yeast is unable to produce active resveratrol synthase, which is required to catalyse the
final reaction in the production of resveratrol. Posttranslational modification, such as overglycosylation
and disulphide formation, of the heterologous protein in yeast has been
indicated as the possible reason for the lack of enzyme activity. This introduces an exciting
area of research for the development of biotechnological tools with the ability to increase
the production of active heterologous proteins in yeast. / AFRIKAANSE OPSOMMING: Wingerde word voortdurend deur 'n groot verskeidenheid patogene, insluitende virusse,
bakteriee en swamme, aangeval. Ten einde oorlewing te verseker, het die wingerdstok In
wye reeks verdedigingsmeganismes ontwikkel om weerstand te bied teen indringerorganismes.
'n Belangrike faktor in hierdie weerstand teen siektes is die produksie van
fitoaleksiene, waarvan resveratrol die hoofkomponent is. Oeur die sintese van resveratrol,
asook ander strukturele en biochemiese verdedigingsmeganismes, word die plant
toegerus om weerstand te kan bied teen In hele aantal patogene ten einde gesonde
druiwe te produseer wat gebruik kan word vir die vinifikasie van topgehalte wyn. As deel
van die aktiewe reaksie teen siektes, word resveratrol de novo in die dop van die korrel by
die plek van infeksie gesintetiseer sodra 'n patogeen herken word. Hier kan dit die skade
deur die patogeen veroorsaak, beperk en verhoed dat dit versprei. Oit gee aan die plant
die geleentheid om sy sistemies-verworwe weerstand te inisieer, en daardeur die res van
die plant te beskerm, sowel as sekondere infeksies te verhoed.
Die fermentasie van rooiwyn op die druifdoppe maak voorsiening vir die ekstraksie van
resveratrol uit die dop na die wyn. Die konsentrasie van resveratrol in rooiwyn is dus
beduidend hoer as in die wit varietelte, wat geen of baie min resveratrol bevat. Oit is dan
juis die rede waarom die matige inname van wyn, veral rooi wyn, gesien word as In
integrale deel van 'n gesonde leefwyse. Resveratrol se aktiwiteit as antioksidant en antiinflammatoriese
middel lewer In belangrike bydrae tot die kardiovaskulere voordele wat
verkry word uit die inname van rooiwyn. Oit blyk egter nou dat die beduidende
kardiovaskulere beskerming gesetel is in die sinergistiese werking van resve ratro I, die
polifenole en die alkohol in wyn.
Aangesien die heilsaamheid van enige voedsel of drank van die uiterste belang is,
was dit die doel van hierdie projek om wyngis te manipuleer ten einde tydens die
fermentasieproses resveratrol te produseer. Hiervoor moes 'n volledige metaboliese pad
daargestel word deur plantgene in die gis te inkorporeer. Resveratrol-sintase maak
gebruik van drie maloniel-KoA-molekules en een p-kumarotel-Kos-molekule om een
molekule resveratrol te produseer. Saccharomyces cerevisiae produseer maloniel-KoA,
maar nie p-kumaroiel-Kcs, nie. Oie volgende gene is dus aangewend om die gis in staat
te stel om p-kumarolel-Koe, te produseer: PAL, wat fenielalanien-ammoniak-liase
enkodeer om fenielalanien om te sit na kaneelsuur; C4H, wat sinnamaat-4-hidroksliase
enkodeer om kaneelsuur om te sit na p-kumaarsuur; en 4CL9 of 4CL216 wat KoA-ligases
enkodeer om p-kumaarsuur om te sit na p-kumarolel-Kos, Om hoevlak-uitdrukking te
verkry, is die gene gesubkloneer onder beheer van die fosfogliseraat-kinase-geen(PGK1)-
promotor en -terminator. As gevolg van integrasieprobleme met hierdie
uitdrukkingskassette en die feit dat die gis die p-kumaarsuur kon verteer, is die 4CL9-,
4CL216- en Vst1- (wat resveratrol-sintase enkodeer) gene na episomale plasmiede
gesubkloneer onder beheer van die alkohol-dehidrogenase(ADH2)- en PGK1-promotors onderskeidelik. 'n Laboratorium-gisstam wat 6f beide die Vst1-geen en die 4CL9-geen, 6f
die Vst1-geen en die 4CL216-geen bevat het, is geevalueer vir die verrnoe om pkumaarsuur
te benut en resveratrol te produseer. Noordelike klad analises het bevestig
dat die Vst1-, 4CL9- en 4CL216-gene getranskribeer en ooruitgedruk was in vergelyking
met die kontrole-stam. Die transformante wat die KoA-ligases uitgedruk het, het die pkumaarsuur
vinniger benut as wat die kontrole dit gedoen het, alhoewel dit nie moontlik
was om vas te stel of o-kurnarotel-Kos, geproduseer is nie. Met die essai-kondisies wat
gebruik is, is geen resveratroI geproduseer nie. Die resultate het daarop gedui dat die gis
nie daartoe in staat is om aktiewe resveratrol-sintase, wat nodig is vir die katalise van die
finale reaksie in die produksie van resveratrol, te produseer nie. Naomsettingsmodifikasies
van die heteroloe protelen in die gis, soos oor-glikosilasie en
disulfiedvorming, is aangewys as die moontlike rede vir die gebrek aan ensiemaktiwiteit.
Dit stel In opwindende veld vir verdere navorsing voor, naamlik die ontwikkeling van
biotegnologiese middele met die vermoe om die produksie van aktiewe heteroloe
protelene in gis te verhoog.
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Manipulating the levels of ethyl acetate and isoamyl acetate formation during the production of wine and brandyBayly, Jennifer Carr,1977- 12 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: The production of wine is a complex process, which involves the conversion of sugar in
grape must to ethanol, carbon dioxide and other byproducts. The principal organism in
winemaking is yeast, of which Saccharomyces cerevisiae is the most important due to
its ability to survive winemaking conditions, its GRAS (Generally Regarded As Safe)
status and the favourable flavours it imparts during the winemaking process. However,
due to the demands of the consumer and the emergence of sophisticated wine markets,
a demand is developing for specialised yeast strains with enhanced and new
oenological properties. For these reasons, research into the contribution of wine yeast
to the aroma bouquet as well the influence of wine or brandy maturation in wood on the
aroma bouquet is important for consumer demands to be met.
The fruity aroma of wine is associated with esters, which are produced during the
alcoholic fermentation by yeast. Important acetate esters in wine and brandy are ethyl
acetate, which has a fruity, solvent-like aroma, and isoamyl acetate, which has a
banana-like aroma. These esters are produced through the action of acetyltransferases
(AATases), which catalyse the reaction between a higher alcohol and acyl Coenzyme A.
Esters are mainly a product of alcoholic fermentation. However, their concentration
changes during wood maturation and it has been found that the concentration of acetate
esters can increase during the maturation period.
In this study, the aim was to investigate the influence of AATase I and AATase II,
which are encoded by the ATF1 and ATF2 genes respectively, on the aroma bouquet of
wine and brandy. Therefore, the first objective of this study was to clone the ATF2 gene
from a commercial wine yeast strain and to overexpress this gene in a commercial wine
yeast strain and in a wine yeast strain that already has the A TF1 gene overexpressed.
Disruption cassettes were also designed in order to disrupt the ATF1 and ATF2 genes in
a commercial wine yeast strain. The resultant recombinant wine yeast strains were
used for the production of wine and brandy. GC analyses and tasting trials were
conducted to determine the effect of the overexpression or disruption of these genes on
the aroma bouquet of wine.
The results obtained indicated that there are differences in the aroma bouquet of
wine and brandy when changes are made in gene expression. The results indicated
that the A TF1 gene plays a large role in the production of ethyl and isoamyl acetate.
When this gene was overexpressed, the level of ethyl acetate was 5.6-fold more than
that of the control and the level of isoamyl acetate was 3.5-fold higher than that of the
control. However, no increase in ethyl acetate or isoamyl acetate was observed when
the A TF2 gene was overexpressed. An increase in 2-phenylethyl acetate and diethyl
succinate was observed in brandy, although there was a decrease in total ester
concentration. A decrease in acetic acid was also observed in the brandy produced,
which could be an indication of ester production. Similarly, no increase in ethyl acetate or isoamyl acetate was observed in the wine or brandy produced when both the ATF1
and ATF2 genes were overexpressed in a single yeast. Once again, a marked decrease
was observed in acetic acid concentration in both the wine and brandy.
In conclusion, it is clear that changes in gene expression can change the aroma
profile of wine or brandy. However, the role of the ATF2 gene still remains unclear and
further studies are needed to clarify its role in yeast. Future studies involving the effect
of wood maturation on ester concentration will also be of importance, so that the
winemaker or distiller can make a product that suits the ever-changing market. / AFRIKAANSE OPSOMMING: Die produksie van wyn is 'n komplekse proses wat die omskakeling van die suiker in
mos tot etanol, koolstofdioksied en ander byprodukte tot gevolg het. Die hooforganisme
betrokke in die wynmaakproses is gis, waarvan Saccharomyces cerevisiae as een van
die belangrikste geag word as gevolg van die vermoë daarvan om onder die
wynfermentasietoestande te kan oorleef, die "GRAS"-status (Generally Regarded As
Safe) daarvan en die invloed wat dit op die aroma van die uiteindelike produk het weens
die werking daarvan gedurende alkoholiese fermentasie. Die behoefte aan wyn met
nuwe, verbeterde eienskappe het die vraag na meer gespesialiseerde gisrasse deur
beide die verbruiker en nuwe wynmarkte gedurende die afgelope paar jaar drasties laat
toeneem. Dit is om dié redes dat navorsing oor die bydrae van wyngis en
houtveroudering tot die aroma van beide wyn en brandewyn so belangrik geag word.
Die vrugtige aroma van wyn word geassosieer met die esters wat gedurende die
alkoholiese fermentasie deur gis gevorm word. Die belangrikste asetaatesters in wyn en
brandewyn is etielasetaat, wat vir 'n oplosmiddelagtige, vrugtige aroma bekend is, en
isoamielasetaat, wat 'n piesangaroma veroorsaak. Die esters word geproduseer deur
die werking van asetieltransferases (AATases), wat as katalis in die reaksie tussen 'n
hoër alkohol en asetiel-Ko-ensiem A optree. Alhoewel esters hoofsaaklik 'n produk van
alkoholiese fermentasie is, wissel die konsentrasie daarvan gedurende houtveroudering.
Daar is gevind dat die konsentrasie van die asetaatesters gedurende die
verouderingsproses kan verhoog.
Die studie het ten doelom die invloed van AATase I en AATase II, wat
onderskeidelik deur die ATF1- en ATF2-gene geënkodeer word, op die aroma van wyn
en brandewyn te ondersoek. Die eerste doelwit van die studie was vervolgens om die
ATF2-geen vanaf 'n kommersiële wyngisras te kloneer en dit daarna te ooruitdruk in 'n
kommersiële wyngisras, asook die geen te ooruitdruk in 'n kommersiële wyngisras wat
reeds die ATF1-geen ooruitdruk. Disrupsiekassette is ook vir die disrupsie van die
ATF1- en ATF2-gene in 'n kommersiële wyngisras ontwerp. Die rekombinante
wyngisrasse wat gedurnde die studie gemaak is, is vir die produksie van wyn en
brandewyn gebruik. Gas chromatografise-ontledings en sensoriese evaluerings is ook
op die wyn en brandewyn uitgevoer.
Die resultate van die studie het bewys dat daar wel veranderings plaasvind
wanneer 'n verandering in geenuitdrukking gemaak is. Die resultate het weereens
bevestig dat die ATF1-geen 'n belangrike rol in die produksie van etiel- en isoamielasetaat
speel. Wanneer die ATF1-geen ooruitgedruk is, is die etielasetaatproduksie 5.6
keer meer en die isoamielasetaatproduksie 3.5 keer meer as in die kontrole. Die
ooruitdrukking van die ATF2-geen het geen verhoging in etielasetaat of isoamielasetaat
of in totale esters in die wyn getoon nie, alhoewel die ras 2.7 keer meer diëtielsuksinaat
geproduseer het. In die brandewyn wat geproduseer is met die gisras waarin ATF2 ooruitgedruk is, was daar wel 'n verlaging in die asynsuur, wat 'n aanduiding van
estervorming kan wees, alhoewel die totale esters wat geproduseer is minder was as in
die kontrole. 'n Verhoging in diëtielsuksinaat en 2-fenielasetaat is ook gevind. Daar is
geen verhoging in etiel- of isoamielasetaat getoon wanneer die ATF1- en ATF2-geen
saam ooruitgedruk is nie. Die ras het minder totale sure in wyn en brandewyn
geproduseer en ook geen verhoging in totale esters getoon nie.
Uit die resultate is dit duidelik dat veranderings in geenuitdrukking 'n verandering
in die aromaprofiel van wyn en brandewyn kan veroorsaak. Die rol van dié A TF2-geen
is nog steeds onduidelik en verdere studies sal moet plaasvind om die rol van die geen
te verduidelik. Studies wat konsentreer op die invloed van houtveroudering op
esterkonsentrasie is ook belangrik vir die toekoms, want dit sal die wyn- of
brandewynmaker meer beheer oor die uiteindelike produk gee en daardeur die wyn- of
brandewynmaker help om 'n produk te vervaardig wat sy mark bevredig.
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Expression and purification of recombinant extracellular proteases originating from non-Saccharomyces yeastsTheron, Louwrens Wiid 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: During wine fermentation, yeasts release extracellular enzymes that significantly impact wine
properties. While the extracellular proteins of Saccharomyces cerevisiae have been
characterised, those of non-Saccharomyces yeasts remain largely unknown. Most of these
enzymes break down sugar polymers or catalyse the liberation of glycosidically-bound
molecules. Another category of enzymes of oenological interest is represented by acid
proteases that are able to prevent or reduce protein haze, as reported in literature, while
simultaneously increasing the assimilable nitrogen content of wine. The liberation of amino
acids from peptides and proteins that serve as aroma precursors may also have an indirect
effect on wine aroma. In a recent study performed at the Institute for Wine Biotechnology
(IWBT), the sequences of two aspartic proteases were retrieved from non-Saccharomyces
yeast species isolated from South African wines. The genes, MpAPr1 and CaAPr1, were
isolated from two non-Saccharomyces species, Metschnikowia pulcherrima IWBT Y1123 and
Candida apicola IWBT Y1384, respectively. However, no further characterization was
undertaken. This study aimed to clone these two genes into a recombinant bacterial host for
expression and purify the corresponding enzymes as a first step toward characterizing their
kinetic properties. Considering that some non-Saccharomyces species have been shown to
produce more than one acid protease, an additional aim was to identify novel acid proteases
within M. pulcherrima IWBT Y1123.
Cloning of the genes and transformation of the expression vectors into E. coli were achieved.
Optimal conditions for induced expression were established following extensive optimization.
Furthermore, while native extraction of the recombinant proteins was unsuccessful, denaturing
conditions allowed their recovery, suggesting that the recombinant proteins are encapsulated
into inclusion bodies. Recombinant MpAPr1 was purified by using a nickel based column
system and mass fingerprinting of the purified enzyme (MpAPr1) confirmed its identity.
Purification was followed by refolding experiments, but yielded poor recovery of active enzymes.
Unfortunately, recombinant expression of CaAPr1 could not be observed for reasons yet to be
elucidated that may include the large sequence dissimilarities between CaAPr1 and MpAPr1.
Finally, Southern blot analysis on the genomes of M. pulcherrima IWBT Y1123 and C. apicola
IWBT Y1384 revealed that both possess at least one additional protease other than those
previously described. Further analysis of the extracellular proteome of M. pulcherrima IWBT
Y1123 also confirmed the presence of at least one enzyme able to hydrolyze BSA at a low pH.
Unfortunately, mass fingerprinting performed on the entire extracellular proteome and on small
groups of proteins thereof did not allow the identification of these enzymes. / AFRIKAANSE OPSOMMING: Gedurende fermentasie van druiwe sap skei gis ekstrasellulêre ensieme af wat ‘n aanmerklike
impak op wyn eienskappe het. Terwyl die ekstrasellulêre proteïene vanaf Saccharomyces
cerevisiae al gekarakteriseer is, bly die van nie-Saccharomyces spesies grootliks onbekend.
Meeste van hierdie ensieme breek suiker polimere af of kataliseer die vrystelling van
glikosiediese verbonde molekules. ‘n Ander klas van ensieme wat van belang is vir oenologie
word voorgestel deur proteases wat in staat is daartoe om proteïenewaas te verminder, soos
voorheen geraporteer is in literatuur, terwyl dit terselfde tyd die assimileerbare stikstof inhoud
kan vermeerder. Die vrystelling van aminosure vanaf peptiede en/of proteïene wat as aroma
voorlopers dien mag ook ‘n indirekte effek op die wyn se aroma profiel hê. In ‘n onlangse studie
wat uitgevoer is by die Instituut vir Wynbiotegnologie (IWBT) was die volgordes van twee
aspartiese proteases bepaal vanaf twee nie-Saccharomyces gis spesies wat geisoleer was uit
Suid-Afrikaanse wyne. Die gene MpAPr1 en CaAPr1, was afsonderlik geisoleer vanuit twee nie-
Saccharomyces giste, Metschnikowia pulcherrima IWBT Y1123 en Candida apicola IWBT
Y1384. Egter was daar geen verder karakterisering van hierdie ensieme nie. Die doel van
hierdie studie is om die bogenoemde gene in ‘n rekombinante bakteriese gasheer te kloneer vir
uitdrukking en suiwering as ‘n eerste stap tot karakterisering van hul kinetiese eienskappe. Om
in ag te neem dat sommige nie-Saccharomyces spesies meer as een protease produseer was
‘n aditionele mikpunt om vir nuwe suur proteases te soek binne M. pulcherrima IWBT Y1123.
Klonering van hierdie gene en transformasie van die uitdrukkings vektore in E. coli was
suksesvol. Optimale kondisies vir die induksie van ekspressie was bevestig na omvattende
optimalisering. Verder, terwyl inheemse ekstraksie van die rekombinante proteïene onsuksesvol
was, het denatureerende kondisies toegelaat vir suksesvolle ekstraksie, wat voorgestel het dat
die rekombinante proteïene geinkapsileer word in inklusie liggame. Rekombinante MpAPr1 was
gesuiwer deur gebruik te maak van ‘n niekel gebaseerde kolom sisteem en massa petied
fingerafdrukke van die gesuiwerde ensiem (MpAPr1) het die identiteit bevestig. Suiwering was
gevolg deur hervouing eksperimente, maar het swak opbrengste gelewer van die aktiewe
ensiem. Ongelukkig kon die rekombinante ekspressie van CaAPr1 nie gevisualiseer word nie vir
redes wat nog bevestig moet word, maar wat mag behels dat daar groot volgorde veskille
tussen MpAPr1 en CaAPr1 kan wees. Uiteindelik was Southern blot hibridiseering analises
uitgevoer op die genome van albei M. pulcherrima IWBT Y1123 en C. apicola IWBT Y1384 wat
voorgestel het dat albei ten minste een addisionele protease, anders as die wat voorheen
geidentifiseer was, bevat. Verder analiese van die ekstrasellulêre proteoom van M. pulcherrima
IWBT Y1123 het ook die teenwoordigheid van ten minste een ensiem bevestig wat die vermoë
het om BSA te hidroliseer by ‘n lae pH. Ongelukkig het massa peptied vingerafdrukbepaling wat uitgevoer was op die hele ekstrasellulêre proteoom en op klein groepe protein nie identifikasie
van hierdie ensieme bevestig nie.
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