Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Monoterpene alcohols (monoterpenols) play an important role in the flavour and aroma of
grapes and wine. This is especially applicable to wines of a muscat variety, but these
flavour compounds are also present in other non-muscat grape varieties, where they
supplement other varietal flavours and aromas. These monoterpenols can be found in grapes and wine as free, volatile and odorous molecules, as well as in flavourless, nonvolatile
glycosidic complexes. These complexes most often occur as
6-0-a-L-arabinofuranosyl-p-D-glucopyranosides (vicianosides), 6-0-P-D-xylopyranosyl-
P-D-gluco-pyranosides (primverosides), 6-0-P-D-glucopyranosyl-p-D-glucopyranosides
(gentio-biosides ), 6-0-a-L -rhamnopyra nosyl-p-D-g lucopyra nos ides (rutinos ides), or
6-0-p-D-apiofuranosyl-p-D-glucopyranosides of mainly linalool, geraniol, nerol, a-terpineol
and hotrienol. These precursors are, however, hydrolyzed only to a limited extent by
endogenous glycosidases during the fermentation process, as they exhibit very low activity
in wine conditions.
The monoterpenols can be released from their sugar moieties by one of two
methods: either an acid or an enzymatic hydrolysis. The enzymatic hydrolysis mechanism
is fully understood, and the process functions in two successive steps: firstly, depending on
the precursor, the glycosidic linkage is cleaved by an a-L-arabinofuranosidase, an
a-L-rhamnosidase, a p-D-xylosidase, or a p-D-apiosidase. The second step involves the
liberation of the monoterpene alcohol by a p-glucosidase. This enzymatic hydrolysis does
not influence the intrinsic aromatic characteristics of the wine, as opposed to acid
hydrolysis.
As the endogenous grape glycosides of Vitis vinifera and the yeast Saccharomyces
cerevisiae show very low activity towards these aromatic precursors during the handling of
the juice and winemaking processes, the focus has increasingly fallen on introducing
exogenous p-glucosidases to wines and juices. Genes encoding p-glucosidases and
a-L-arabinofuranosidases have been cloned from various organisms, including bacteria,
fungi and yeasts. However, the activities and properties of these enzymes are not always
suitable for exploitation under winemaking conditions, where a low pH, low temperatures,
and high ethanol and glucose concentrations prevail. A genetically engineered wine yeast
strain of S. cerevisiae that expresses glycosidases that are active in these conditions would
be useful in improving the flavour and aroma of wines, thereby adding to the complexity
and value of the wine.
Two p-glucosidase genes, BGL 1 and BGL2 from Saccharomycopsis fibufigera, were
subcloned into two Escherichia coli-yeast shuttle vectors. A dominant selectable marker
gene (SMR1) was also inserted onto these plasmids. These plasmids were designated pBGL 1 (containing the BGL 1 gene) and pBGL2 (containing the BGL2 gene) respectively.
Introduction of the two plasmids into two strains of S. cerevisiae then followed. A laboratory
strain, L1278, was transformed to confirm the effective secretion of the expressed protein.
An industrial yeast strain, VIN13, was subsequently transformed by making use of the
selectable marker (resistance against sulfometuron). Enzyme assays with the synthetic
substrate p-nitrophenol-j3-D-glucopyranoside (pNPG) were performed to determine the
activity of the j3-glucosidases over a period of days, as well as at certain temperatures and
pH values. The stability of the enzymes was also investigated.
These recombinant yeasts were able to degrade the pNPG efficiently. They showed
promising results concerning pH optima, with a substantial amount of activity found at the
pH levels as found in the wine environment. There was also a slight increase in specific
activity at lower temperatures. The recombinant yeast strains were also tested in smallscale
fermentations. Three wines were made, of which two were from white cultivars
(Chenin blanc and GewOrtztraminer) and one from red (Pinotage). Results obtained from
micro-extraction from the finished wines showed that the terpenol content did increase,
although this was not the only wine component influenced. Other flavour compounds also
showed increases, especially the esters. This also played a role in the flavour increase in
the wine.
Future work would include optimizing the available results. This would entail the
addition of another glycosidic enzyme, such as a-L-arabinofuranosidase, to the genome of
the wine yeast to aid the further breakdown of glycosidic bonds. The cloning or engineering
of a j3-glucosidase enzyme that is more active at low temperatures would also yield better
results and release even more of the aroma of the wine. / AFRIKAANSE OPSOMMING: Monoterpeenalkohole (monoterpenole) speel 'n belangrike rol in die geur en aroma van
druiwe en wyn. Dit is veral van toepassing op wyne van Muskaat-varieteite, maar hierdie
geurkomponente is ook teenwoordig in ander nie-Muskaat druifsoorte, waar dit bydra tot
die varieteitsqeur en aroma. Hierdie monoterpenole kom voor in druiwe as vry, vlugtige en
aromatiese molekules, of as geurlose, nie-vlugtige glikosidies-gebonde komplekse. Hierdie
komplekse is meestal in die vorm van 6-0-a-L-arabinofuranosiel-~-D-glukopiranosiede, 6-
O-~-D-xilopiranosiel-~-D-glukopiranosiede (primverosiede), 6-0-~-D-glukopiranosiel-~-Dglukopiranosiede
(gentiobiosiede), 6-0-a-L-ramno-pyranosiel-~-D-glukopiranosiede
(rutinosiede), of 6-0-~-D-apiofuranosiel-~-D-glukopirano-siede van hoofsaaklik linalool,
geraniol, nerol, a-terpineol en hotrienol. Hierdie geurvoorlopers word egter slegs tot In
beperkte mate tydens die proses van fermentasie deur die endogene glikosidase ensieme
gehidroliseer, aangesien hulle baie min aktiwiteit toon onder wynbereidingstoestande.
Die monoterpenole kan op een van twee wyses van hul suikermolekules vrygestel
word: 'n suurhidrolise, of ensiematiese hidrolise. Die ensiematiese hidroliseproses word
baie goed begryp en behels twee opeenvolgende stappe: eerstens, afhangende van die
aard van die voorloper, word die glikosidiese verbinding deur In a-L-arabinofuranosidase, In
a-L-ramnosidase, In ~-D-xilosidase, of 'n ~-D-apiosidase gebreek. In die tweede stap word
die monoterpeenalkohol deur In ~-glukosidase vrygestel. Hierdie ensiematiese
afbraakproses verander nie die intrinsieke aromatiese kenmerke van die wyn, soos wat met
suurhidrolise die geval is nie.
Omdat die endogene glikosidases van Vitis vinifera en die van die gis
Saccharomyces cerevisiae baie lae aktiwiteit ten opsigte van die aromatiese voorlopers
gedurende die hantering van die druiwesap en wynmaakprosesse toon, val die fokus al hoe
meer op die inkorporering van eksogene ~-glukosidases in wyn en sappe. Gene wat vir ~-
glukosidases en a-L-arabinofuranosidases kodeer, is al vanuit verskeie organismes
gekloneer, insluitende bakteriee, fungi en giste. Die aktiwiteite en kenmerke van hierdie
ensieme is egter nie altyd wenslik vir hul gebruik in wyn nie, aangesien dit In omgewing is
met 'n lae pH, lae temperatuur, en hoe etanolvlakke en glukose-konsentrasies. In geneties
veranderde wyngis van S. cerevisiae wat in staat is om glikosidases uit te druk wat onder
hierdie kondisies aktief is, sal baie handig te pas kom in die verbetering van die geur en
aroma van wyne, om daardeur die kompleksiteit en die waarde van die wyn te verhoog.
Twee ~-glukosidasegene, BGL 1 en BGL2 vanaf die gis Saccharomycopsis
fibuligera , is in twee afsonderlike Esccherichia coli-gis-pendelplasmiede gesubkloneer. In
Dominante selekteerbare merkergeen (SMR1) is ook in hierdie plasmiede gekloneer.
Hierdie plasmiede word onderskeidelik pBGL 1 (met die BGL 1-geen) en pBGL2 (bevattende die BGL2-geen) genoem. Hierdie twee plasmiede is hierna apart na twee rasse van
S. cerevisiae getransformeer. Eerstens is 'n laboratoriumras, L1278, getransformeer om te
bevestig dat effektiewe sekresie en uitdrukking van die proteTen plaasvind. Hierna is 'n
industriele gisras, VIN13, getransformeer deur gebruik te maak van die selektiewe merker
(bestandheid teen sulfometuron). Ensiem-bepalings met behulp van die sintetiese
substraat p-nitrofeniel-p-O-glukopiranosied (pNPG) is gedoen om die aktiwiteit van die
p-glukosidqses oor 'n aantal dae te bepaal, asook om die aktiwiteit by sekere temperature
en pH-vlakke te meet. Die stabiliteit van die ensieme is ook bepaal.
Hierdie rekombinante giste was in staat om pNPG effektief af te breek. Hulle het
belowende resultate betreffende die pH-optima getoon, met 'n aansienlike hoeveelheid
aktiwiteit by die pH-vlakke soos dit in die wynomgewing voorkom. Daar was ook 'n effense
verhoging in die ensieme se aktiwiteite by laer temperature. Die rekombinante gisrasse is
ook in kleinskaalse wynfermentasies gebruik. Drie verskillende wyne is gemaak, waarvan
twee wit kultivars was (Chenin blanc en GewOrtztraminer) en een 'n rooi kultivar (Pinotage).
Resultate wat deur die mikro-ekstraksie van die voltooide wyne verkry is, het getoon dat die
terpenolinhoud wei verhoog het, alhoewel dit nie die enigste wynkomponente was wat
beinvloed is nie. Ander geurkomponente het ook 'n verhoging in konsentrasie getoon, veral
die esters. Hierdie verbindings het ook 'n rol in die verhoging van geur in die wyne gespeel.
Toekomstige werk sal die beskikbare resultate verder optimaliseer. Dit sal insluit die
byvoeging van nog 'n glikosidiese ensiem, soos a.-L-arabinofuranosidase, tot die genoom
van die wyngis, om verdere afbraak van glikosidiese verbindings teweeg te bring. Die
klonering of verandering van 'n p-glukosidase-ensiem met verhoogde aktiwiteit by laer
temperature sal ook beter resultate toon en meer geur in die wyn kan vrystel.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52586 |
| Date | 12 1900 |
| Creators | Stidwell, Tanya Gwendryth |
| Contributors | Pretorius, I. S., Van Rensburg, P., Lambrechts, M. G., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 96 p. : ill. |
| Rights | Stellenbosch University |
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