Year: 2007
Type: dissertation
Abstract: Wine is a complex medium.Wine aroma, flavour and colour are important quality factors, but these can be influenced by many factors, such as grape-derived compounds that exist as free volatiles and also as glycosidically bound.The chemical composition of wine is determined by factors such as grape variety, geographic position, viticulture condition, microbial ecology of the grape and the winemaking process.The varietals aroma is determined by both the volatile and the non-volatile compounds, such as monoterpenes, norisoprenoids and benzene derivatives, which are naturally present in the wine.Monoterpenes are very important in the flavour and aroma of grapes and wine.They can be found in grapes and wine either in the free, volatile and odorous form, or in the glycosidically-bound, non-volatile and non-odorous form.The ratio of glycosidically-bound compounds to free aroma compounds is very high in the Gewürztraminer, Muscat and Riesling cultivars in particular.The glycosidic bonds can be hydrolysed either by the acid method or by using enzymes.The acid method is disadvantageous because it can modify the monoterpenes, whereas enzymatic hydrolysis has the advantage of not modifying the aroma character.The enzyme method of breaking the glycosidic bonds occurs in two successive steps: initial separation of glucose from the terminal sugar by a hydrolase (α-L-arabinofuranosidase, α-L-rhamnosidase or β-apiosidase, depending on the aglycone moiety), followed by the breaking of the bond between the aglycone and glucose by β-glucosidase.The enzyme β-glucosidase can be obtained from many plant (Vitis vinifera), bacterial, yeast or fungal sources.Most of the enzymes produced by these sources are not functional under the winemaking conditions of low pH, low temperature, high glucose and high ethanol content.However, β-glucosidases from fungal origins, particularly from Aspergillus spp., are tolerant of winemaking conditions.The idea of using the β-glucosidase gene from the fungus Aspergillus kawachii (BGLA), which is linked to the cell wall and the free β-glucosidase, was to determine if anchoring the enzyme to the cell wall will increase the activity of the enzyme compared to the free enzyme.Four plasmids, pCEL 16, pCEL 24, pDLG 97 and pDLG 98, were used in this study.BGLA that was cloned into the plasmids pCEL 24 and pDLG 97 was linked to CWP2, and in pDLG 98 it was linked to AGα1 anchor domains.All the plasmids were genome-integrated and expressed in the reference strain Saccharomyces cerevisiae 303-1A.All the transformants were grown in 2% cellobiose and showed higher biomass production compared to the reference strain.β-Glucosidase activity was also assayed and transformed strain W16 showed a fourfold increase in activity compared to the reference strain.There was no significant increase in the activity of the other transformed strains, W24, W97 and W98.Enzymatic characterisation for optimum pH and temperature was Stellenbosch University http://scholar.sun.ac.za OPSOMMING OPSOMMING OPSOMMING OPSOMMINGWyn is 'n komplekse medium.Wynaroma, -geur en -kleur is belangrike kwaliteitsfaktore, hoewel hierdie kwaliteite deur verskeie faktore beïnvloed kan word, soos druifafgeleide verbindings wat as vry vlugtige stowwe teenwoordig kan wees of glikosidies gebind is.Die chemiese samestelling van wyn word bepaal deur faktore soos druifvariëteit, geografiese ligging, wingerdkundige toestande, mikrobiese ekologie van die druif en die wynbereidingsproses.Die variëteitsaroma word bepaal deur vlugtige en nie-vlugtige verbindings, soos monoterpene, norisoprenoïede en benseenderivate, wat natuurlik in die wyn voorkom.Monoterpene is baie belangrik vir die geur en aroma van druiwe en wyn.Monoterpene is teenwoordig in die druiwe en wyn in vry, vlugtige en geurige, of in glikosidiesgebinde, nie-vlugtige en nie-geurige vorms.Die verhouding van glikosidiesgebonde verbindings tot vry aromaverbindings is baie hoog, veral in die Gewürztraminer-, Muscat-en Riesling-kultivars.Glikosidiese verbindings kan deur óf die suurmetode óf die ensiemmetode gehidroliseer word.Die nadeel van die suurmetode is dat dit monoterpene kan modifiseer, terwyl die ensiemmetode die voordeel het dat dit nie die aromakarakter modifiseer nie.Die ensiemmetode waarmee die glikosidiese verbinding afgebreek word, vind in twee opeenvolgende stappe plaas: aanvanklike skeiding van glukose van die terminale suiker deur 'n hidrolase (α-L-arabinofuranosidase, α-Lramnosidase of β-apiosidase, afhangende van die aglikoongedeelte), gevolg deur die verbreking van die verbinding tussen die aglikoon en glukose deur βglukosidase.Die β-glukosidase-ensiem kan vanaf 'n verskeidenheid plant-(Vitis vinifera), bakterie-, gis-en swambronne verkry word.Die meerderheid van die ensieme wat deur hierdie bronne geproduseer word, is nie onder die wynbereidingstoestande van lae pH, hoë temperatuur, hoë glukose en hoë etanol funksioneel nie.β-Glukosidase vanaf 'n swamoorsprong, veral vanaf Aspergillus-spesies, kan egter wynbereidingstoestande verdra.Die idee agter die gebruik van die β-glukosidasegeen afkomstig van die swam Aspergillus kawachii (BGLA), wat aan die selwand en die vry β-glukosidase gekoppel is, was om te bepaal of die aktiwiteit van die ensiem in vergelyking met dié van die vry ensiem verhoog sou word indien die ensiem aan die selwand geanker is.Vier plasmiede, pCEL 16, pCEL 24, pDLG 97 en pDLG 98, is in hierdie studie gebruik.BGLA, wat in die plasmiede pCEL 24 en pDLG 97 gekloneer is, is gekoppel aan CWP2, en in pDLG 98 is dit aan AGα1-ankergebiede gekoppel.Al die plasmiede is in verwysingsras Saccharomyces cerevisiae 303-1A genoomgeïntegreer en uitgedruk.Al die transformante is in 2% sellobiose gegroei en het hoër biomassaproduksie as die verwysingsras getoon.β-Glukosidaseaktiwiteit is ook geëssaieer en die getransformeerde ras W16 het 'n viervoudige
Author: Ntanganedzeni Ranwedzi
Language: English
Subfield: Food Science
Domain: Life Sciences
Open Access status: green
Funder: National Research Foundation
OpenAlex ID: W2105816879