Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: No single chemical constituent can be accredited with giving wine and brandy their
overall aroma and flavour. The aroma and flavour of wine and brandy are rather
attributed to a number of chemical constituents reacting together and it is these
reactions that give the beverage its character. Certain chemicals within wine and
brandy do, however, make larger contributions to the flavour. These include the
esters, terpenes and volatile acids, although others also exist.
Esters are a large group of volatile compounds with variable aroma and flavour
characteristics, including banana-like (isoamyl acetate), apple-like (ethyl caproate)
and chemical/solvent-like (ethyl acetate). Esters are produced as secondary
metabolites during the conversion of sugar to ethanol and are formed when an
alcohol binds with a fatty acid. Chemically, ester metabolism is well documented and
understood; however, much work still needs to be done on a genetic level. The yeast
strain used during fermentation is one of the most important factors contributing to
the type and quantity of esters produced. This is due to differences in genetic
makeup. The metabolism of esters is controlled largely on a genetic level, with
numerous genes being involved. The alcohol acetyltransferase genes are involved in
ester anabolism, whilst esterase genes are involved in ester catabolism. Esterases
have a negative effect on the overall level of esters within an alcoholic beverage, as
they are capable of reducing the number of esters and are thus capable of altering
the beverage's aroma and flavour profile. The IAH1 and the TIP1 gene products are
believed to encode for two such esterases.
The objective of this study was to investigate the contribution of the IAH1 and
TIP1 genes to the level of esters in both wine and brandy. This was accomplished by
using two approaches. Firstly, the above genes were disrupted using a polymerise
chain reaction (PCR)-generated disruption cassette homologous to either the IAH1 or
the TIP1 gene. These cassettes were integrated into the industrial wine yeast,
Saccharomyces cerevisiae strain VIN13. The integrations were verified by Southern
blot analysis to produce yeasts VIN13-~IAH1 and VIN13-~TIP1; however, only a
single copy of each was disrupted. Secondly, the IAH1 and the TIP1 genes were
cloned from S. cerevisiae using PCR into plasmid pj between the phosphoglycerate
kinase gene (PGK1) promoter and terminator, producing plasmids pJ-IOE1 and
pJ-TOE1. The PGK1 promoter has previously been shown to constitutively express
genes at high levels. These new constructs were then used as template for PCR to
produce two overexpression cassettes, one for IAH1 and the other for TlP1. These
cassettes were integrated into S. cerevisiae VIN13 and verified by Southern blot
analysis to produce strains VIN13-IOE1 and VIN13-TOE1.
The above yeast strains including VIN13 were used for the production of wines
and base wines from Colombard must. Reverse-transcriptase (RT-PCR) confirmed
that the VIN13-IOE1 and VIN13-TOE1 strains overexpressed the appropriate gene at a higher level than the control VIN13 strain. The VIN13-AIAH1 disrupted strain
showed no difference in expression level to that of the control strain, whilst
VIN13-ATIP1 showed lower levels of expression than that of the control strain.
VIN13-IOE1 behaved as expected, with a decrease of between 30% and 60% in the
total ester level in the wine and base wine respectively, a 30% decrease in the total
acid level and no change in the higher alcohol level. The VIN13-AIAH1 strain showed
no difference to the control wine, most likely as this strain still expressed the IAH1
gene at levels consistent with the control strain. VIN13-TOE1 behaved in an
unexpected manner - instead of hydrolysing esters, it appeared to produce them.
This increase in the total ester level was most noticeable during distillation, when a
20% increase took place. Another unexpected occurrence was a large decline in the
total acid level, with acetic acid being the most significant contributor, decreasing by
up to 78%. This is a very favourable finding, as acetic acid is a known spoilage
molecule and is a cause of sluggish/stuck fermentations. VIN13-ATIP1 behaved in an
opposite manner to VIN13-TOE1, with higher total acid levels and slightly decreased
total ester levels, especially during distillation. Neither affected the total higher
alcohol levels. Sensorially, the only significant difference in the wine samples was for
the fruity flavour. A panel of judges distinguished that VIN13-TOE1 was fruitier than
the other wines, with VIN13-ATIP1 being the least fruity.
This study again proves the significant impact that a single gene can have on the
chemical makeup of wine and brandy. The relatively simple genetic alteration of an
organism can drastically change and improve not only the organoleptic properties of
the organism, but its viability as well. These alterations can produce more favourable
organisms with more desirable characteristics for the fermenting beverage industry to
produce products of higher quality and better suitability. / AFRIKAANSE OPSOMMING: Geen chemiese komponent kan uitgesonderword as die produseerder van aroma en
geur in wyn of brandewyn nie. Die aroma en geur van wyn en brandewyn word
eerder toegeskryf aan die interaksie tussen 'n groot aantal chemiese komponente
om aan die drank sy karakter te gee. Enkele van hierdie chemiese komponente sluit
in esters, terpene en vlugtige sure, om maar 'n paar te noem.
Esters is "n groot groep van vlugtige verbindings wat beskik oor 'n
verskeidenheid van aroma- en geurkenmerke, soos piesangagtig (isoamielasetaat),
appelagtig (etielkaproaat) en chemies/oplosmiddelagtig (etielasetaat). Esters word as
sekondêre metaboliete geproduseer wanneer suikers na etanolomgeskakel word en
word gevorm wanneer "n alkohol met "n vetsuur verbind. Estermetabolisme is
chemies goed beskryf en verstaan, maar op "n genetiese vlak is daar nog heelwat
aspekte wat nagevors moet word. Die gisras betrokke gedurende fermentasie word
beskou as een van die grootse bydraes tot die tipe en die hoeveelheid esters wat
geproduseer word. Dit word toegeskryf aan verskille in die genetiese saamestelling
van die gisras. Ester metabolisme word grootliks deur genetiese faktore beheer en
verskeie gene is betrokke. Dit is hoofsaaklik die alkoholasetieltransferasegene wat vir
esterkatabolisme verantwoordelik is, terwyl die esterasegene vir esteranabolisme
verantwoordelik is. Esterases het 'n negatiewe effek op die totale estervlak binne
alkoholiese dranke deurdat hulle in staat is om die aantal esters drasties te verminder
en sodoende die drank se aroma- en geurprofiel te verander. Daar is voorgestel dat
die IAH1- en die TlP1-geen produkte is wat vir twee sulke esterases kodeer.
Die doel van hierdie studie was om die IAH1- en die TIP1-gene se bydrae tot die
totale estervlak in wyn en brandewyn te ondersoek. Dit is deur twee benaderings
uitgevoer. Eerstens is die bogenoemde gene d.m.V. disrupsiekassette wat homoloog
aan die IAH1- of die TlP1-gene was, uitgeslaan. Die disrupsiekassette is deur die
polimerasekettingreaksie (PKR) geproduseer. Hierdie kassette is in die industriële
wyngis, Saccharomyces cerevisiae VIN13, geïntegreer. Die integrasies is deur
Southernkladanalise bevestig en het die giste VIN13-~IAH1 en VIN13-~TIP1
gelewer. Net 'n enkele kopie van elke geen is egter uitgeslaan. Tweedens is die
IAH1- en TIP1-gene d.m.V. PKR vanaf S. cerevisiae binne in plasmied pJ gekloneer,
tussen die fosfogliseraatkinasegeen (PGK1) se promotor en termineerder, om
plasmiede pJ-IOE1 en pJ-TOE1 te produseer. Die PGK1-promotor is al tevore
geïdentifiseer as "n hoë-vlak konstitutiewe uitdrukker van gene. Hierdie twee nuwe
konstrukte het vervolgens gedien as templaat vir PKR om twee
ooruitdrukkingskassette, een vir IAH1 en die ander vir TIP1, te produseer. Hierdie
kassette is in S. cerevisiae VIN13 geïntegreer en bevestig deur Southernkladanalise.
Hierdie integrasies het die giste VIN13-IOE1 en VIN13-TOE1 geproduseer.
All die nuwe gisrasse, tesame met VIN13, is gebruik vir die produksie van wyne
sowel as rebatwyne vanaf Colombard-mos. Omgekeerde-transkriptase polimerasekettingreaksie (OT-PKR) het bewys dat die VIN13-IOE1 en VIN13-TOE1
rasse die geskikte geen ooruitgedruk het, met hoêr vlakke as van die kontrole
VIN13-ras. Dit het ook aangedui dat die VIN13-i\IAH1-ras, waarvan die geen
uitgeslaan was, geen verskil in uitdrukking gehad het in vergelyking met die
kontroleras nie, terwyl VIN13-i\TIP1 'n lae uitdrukkingsvlak getoon het. VIN13-IOE1
het teen verwagting opgetree, met 'n afname van tussen 30% en 60% in die totale
estervlak in beide die wyne en rebatwyne. 'n Afname van 30% in die totale suurvlak,
asook geen waarneembare verskil in die hoêr alkoholvlak, in vergelyking met die
kontroleras, is ook opgemerk. Die VIN13-i\IAH1-ras het glad nie van die kontroleras
verskil nie, heel waarskynlik omdat hierdie ras die IAH1-geen teen dieselfde vlak as
die kontroleras kon uitdruk. Die VIN13-TOE1-ras het teen verwagting opgetree
deurdat dit esters geproduseer het i.p.v. om esters te hidroliseer. Hierdie toename in
die totale estervlak is die meeste waarneembaar tydens distillasie, met tot 'n 20%
toename. Nog 'n onverwagte effek was die groot afname in die totale suurvlak. met
asynsuur wat die betekenisvolste bydrae gelewer het deurdat dit 'n afname van tot
78% getoon het. Hierdie bevinding is baie voordelig, aangesien asynsuur, 'n bekende
bederfmolekuul, veral vir slepende/gestaakte fermentasies verantwoordelik is.
VIN13-i\TIP1 het op die teenoorgestelde wyse opgetree as VIN13-TOE1, met 'n hoêr
totale suurvlak en 'n klein afname in die totale estervlak. Weereens is dit meer
gedurende distillasie waargeneem. Beide rasse het egter geen effek op die hoêr
alkoholvlak gehad nie. Die proepaneel het, met betrekking tot die vrugtige geur, een
betekenisvolle geurverskil tussen die wyne gevind. VIN13-TOE1 was meer vrugtig as
al die ander wyne en VIN13-i\TIP1 was die minste vrugtig.
Die studie het weereens bewys dat 'n enkele geen 'n betekenisvolle effek op die
chemiese samestelling van wyn en brandewyn kan hê. Die relatief eenvoudige
genetiese verandering van 'n organisme kan die organoleptiese eienskappe asook
die lewensvatbaarheid van "n organisme, drasties verander en verbeter.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53094 |
| Date | 12 1900 |
| Creators | Hignett, Jason Satch |
| Contributors | Du Toit, M., Pretorius, I. S., Lambrechts, M. G., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology . |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 91 p. : ill. |
| Rights | Stellenbosch University |
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