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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52823 |
| Date | 12 1900 |
| Creators | Smit, Annel |
| Contributors | Van Rensburg, Pierre, Cordero Otero, R. R., Lambrechts, M. G., Pretorius, I. S., 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 | 65 p. : ill. |
| Rights | Stellenbosch University |
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