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.

Instant puddings were prepared using ultra-high-temperature (UHT) processed and conventionally (HTST) processed milk at 6°C and 23°C in six replicates of a 2X2 factorial design to determine the effect of milk type and temperature on apparent viscosity and gel strength of pudding. Apparent viscosity was estimated from linespread readings on separate 25 ml samples of pudding measured at 0, 5, 10, 15, and 30 minutes after preparation. Gel strength was determined from penetrometer readings on separate warm and refrigerated 100 ml samples of pudding measured at 15, 30, 60, and 90 minutes after preparation. Consumer evaluations of flavor and texture of the puddings were obtained as well as word descriptors of UHT milk by those consumers who had tried the product. A five-point hedonic scale of 1=dislike extremely to 5=like extremely was used by 200 consumers to register their perceptions of flavor and texture of the puddings. Apparent viscosity was greater with HTST milk, warm milk, and longer elapsed time. The combination of cold milk and shorter time was least viscous. Gel strength of refrigerated pudding was greater for HTST milk, cold HTST, and longer time. Nonrefrigerated pudding was firmer for HTST milk and cold milk. Shortest time resulted in softest gel strength, with no difference between other time periods. Although values from objective measures differed between puddings made with UHT and with HTST milk, consumer responses to the texture and the flavor of the puddings were similar for the four milk type by temperature variations. / M.S.

Fish and crab processing wastes were ground and ensiled with corn stover or peanut hulls alone and with 5% dry molasses or 1% formic acid in 3.8 liter cardboard containers double lined with polyethylene. The wastes and roughages were ensiled in proportion to give dry matter levels of 40, 50 and 60%. The seafood wastes were also ensiled with wilted Johnsongrass with and without molasses. After ensiling, average pH for mixtures with fish waste was 6.5, compared to 8.0 for mixtures with crab waste. Addition of dry molasses resulted in a decrease (P <.01) of pH to 5.6 for the ensiled fish mixture but had no effect on the crab waste mixtures. Lactic acid was higher (P< .01) for ensiled mixtures containing fish waste than for those containing crab waste. Substantial levels of acetic acid were present in the silages. Butyric acid levels were higher in silages containing crab waste and decreased linearly (P< .01) with increased dry matter levels. Desirable ensiling was observed for the mixture of fish waste and Johnsongrass. Coliforms and fecal coliforms were decreased or elIminated by ensiling. In a large silo study, mixtures of finfish and crab processing wastes were mixed with wheat straw and ensiled in 210 liter metal drums, double lined with polyethylene bags. Proportions of the fish and straw were 70:30 and 51:49, wet basis, while that of the crab was 60:40 and 40:60. Acetic acid was added to the crab waste mixtures to lower the initial pH to 4.5. After ensiling all mixtures containing fish and straw showed a decrease in pH. Addition of acetic acid to mixtures containing crab waste inhibited fermentation, but resulted in a very stable product. In a sheep digestion trial, dry matter digestibility was higher (P <.01) for the 70:30 diet than for the 51:49 fish diet. There was no difference in dry matter digestibility between the crab silages. Crude protein digestibility was higher (P <.01) for diets containing ensiled fish, compared to diets containing ensiled crab. Nitrogen retention was positive for sheep receiving all diets. Nitrogen retention was higher (P <.01) for animals fed the crab silage diets, compared to those receiving diets containing fish silage. There was a trend for P absorption to be higher in animals fed crab silage. In the sheep palatability trial, intake of dry matter was higher (P < .01) for sheep consuming the crab silage diet and lowest (P <.01) for sheep fed the 70:30 fish silage diet. / Ph. D.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2009. / The PGU1 gene encodes an endo-polygalacturonase, an enzyme that degrades pectin. Although the presence and function of this gene is well characterized in Saccharomyces cerevisiae, its regulation is very complex and not yet fully understood. Yeast producing a highly active polygalacturonase (PG) during alcoholic fermentation could potentially improve filtration and turbidity and also enhance extraction of certain aroma compounds. This could replace the addition of expensive commercial enzyme preparations that often contain unwanted enzymes. The first objective of this study was to evaluate PGU1 expression in recombinant strains of S. cerevisiae that originally lacked the PGU1 gene. A functional PGU1 gene and its promoter were successfully re-introduced into their native position in the genomes of five wine strains. Three of these strains recovered PG activity while two did not transcribe the gene and subsequently lacked activity. The three strains that recovered activity were used in microvinification experiments to determine the effect of PG-producing yeast on the aroma profile of the wine. No significant differences were observed in the volatile compounds production between the recombinants and their respective wild types, but some tendencies arose, especially for the monoterpene geraniol. The second objective of this study was to analyze the PGU1 gene and promoter from Saccharomyces paradoxus RO88 (a strain that exhibits high PG activity) and to compare it to those of S. cerevisiae S288C in order to identify differences that could potentially be responsible for the difference in their PG activities. Comparison of the gene sequences revealed several amino acid differences, one of which was in the peptide secretion signal. Analyses of the promoters also indicated some potentially important differences. Furthermore, S. cerevisiae strain VIN13, RO88 as well as two interspecies hybrids (all displaying varying PG activities) were compared under winemaking conditions. Clear differences were observed for the production of certain compounds. RO88 and the hybrids produced higher concentrations of certain volatile compounds, although they were not strong fermenters. Two recombinants, each containing a PGU1-overexpressing plasmid (one with the PGU1 gene from S. paradoxus and the other from S. cerevisiae), were also used in vinification to determine the effects of the different PGU1 gene on the aroma profile of the wine. Unfortunately, the plasmids were unstable and lost during the fermentation. Nevertheless, some tendencies were observed that indicated possible higher production of certain compounds by the recombinants compared to their wild types. This study identified that regulation of the PGU1 gene differs between strains with different genetic backgrounds. Certain differences were observed in the PGU1 gene and promoter sequences between S. cerevisiae and S. paradoxus that could potentially be the reason for the difference in their PG activities. From an oenological point of view, the presence of PGU1 in the genome of a fermenting strain tends to increase the aromatic potential of wine. These results provide a good platform for further studies on the PGU1 gene.

Thesis (MScAgric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The distinctive varietal flavour of wines is a combination of absolute and relative concentrations of chemical compounds. Volatile compounds are responsible for the odour of wine and non-volatiles cause the sensation of flavour. Accompanying these senses, a third, tactile, sense of ‘mouth-feel’ is recognizable. This forms the complete organoleptic quality of wine. Several hundred different compounds are simultaneously responsible for the odour release in wine, and since there is no real character impact compound, the aroma of wine can be described as a delicate balance of all these compounds. One of the most important groups of volatiles is the monoterpenes, which play a role in both aroma and flavour. This is especially significant for the Muscat varieties, but these flavour compounds are also present in other non-muscat grape varieties, where they supplement the varietal aroma. Monoterpenes occur in wine as free, volatile and odorous molecules, as well as flavourless non-volatile glycosidic complexes. The latter slowly releases monoterpenes by acidic hydrolysis, but the impact on varietal aroma is considered insufficient for wines that are consumed young. It is therefore important to supplement the release mechanism, in order to enhance the varietal aroma of the wine. The enzymatic hydrolysis mechanism functions in two successive steps: firstly, depending on the precursor, the glycosidic linkage is cleaved by α-L-arabinofuranosidase, α-L-rhamnosidase, β-D-xylosidase or β-D-apiosidase. The second step involves the liberation of the monoterpene alcohol by a β-glucosidase. This enzymatic hydrolysis does not influence the intrinsic aromatic characteristics of the wine, as opposed to acid hydrolysis. Pectolytic enzymes play an important role in cell elongation, softening of tissue and decomposition of plant material. These enzymes are used to improve juice yields, release colour and flavour compounds from grape skins, as well as improve clarification and filterability. Pectolytic enzymes work synergistically to break down pectins in wine. Protopectinase produce water-soluble and highly polymerised pectin substances from protopectin, it acts on non-methylated galacturonic acid units. Pectin methylesterase split methyl ester groups from the polygalacturonic chain. Polygalacturonase break down the glycosidic links between galacturonic acid units. Pectin and pectate lyases have a β-eliminative attack on the chain and it results in the formation of a double bond between C4 and C5 in the terminal residues. From the above it can be seen that enzymes play a pivotal role in the winemaking process. Unfortunately, in winemaking a lot of factors can influence the effects of enzymes. One possible factor in the wine medium is the presence of acidprotease, from yeast and/or fungal origin. This type of enzyme utilizes other enzymes as substrates and renders them useless. Pure enzyme preparations were used to study the interactions of a yeast acid-protease and a report activity (β-glucosidase) in vitro. A bottled wine and a buffer were used as in vitro conditions. Enzyme assays were performed to determine the relative activity over a number of days. The results indicated that even though both enzymes showed activity in both the media, the yeast protease did not have any significantly affect on the report activity. Subsequently wine was made from Sauvignon blanc grapes, with varying enzyme preparation additions. Enzyme assays were performed during the fermentation; and chemical, as well as sensory analysis were done on the stabilized wine. The results confirmed that the yeast protease did not have any significant affect on the report activity in these conditions. The protease’s inability to affect the report activity seems unlikely due to the fact that it is active at a low pH range and has been suggested as the only protease to survive the fermentation process. It seems possible that a winerelated factor, possibly ethanol, is responsible. Thus it seems that yeast protease does not threaten the use of commercial enzymes in the winemaking process in any significant way. Future work would entail more detailed enzyme studies of interactions between protease, both from yeast and fungal origin, and other report activities in specified conditions. The degradation capability could be directed towards unwanted enzyme activities that cause oxidation and browning of the must. The characterization of interactions between protease and β-glucosidase activities may hold key to producing wines with enhanced aroma and colour potential, as well as the elimination of unwanted enzyme activities. / AFRIKAANSE OPSOMMING: Die herkenbare kultivar karakter van wyn is ‘n kombinasie van absolute en relatiewe konsentrasies van verskeie chemiese komponente. Vlugtige komponente is verantwoordelik vir die geur, of aroma, van wyn en die nie-vlugtige komponente veroorsaak die sensasie van smaak. ‘n Derde, fisiese sensasie, die ‘mondgevoel’, is ook herkenbaar. Dit vorm die omvattende organoleptiese kwaliteit van die wyn. ‘n Paar honderd verskillende komponente is gelyktydig verantwoordelik vir die aroma vrystelling in wyn en omdat daar geen werklike karakter ‘impak’ komponent is nie, kan die aroma van wyn beskryf word as ‘n delikate balans van al die betrokke komponente. Een van die mees belangrike groepe vlugtige komponente is die monoterpene wat ‘n rol speel in beide aroma en smaak. Dit is veral belangrik by Muskaat kultivars, maar hierdie aroma komponente is ook teenwoordig in niemuskaat druif kultivars, waar hulle bydra tot die kultivar karakter en aroma. Monoterpene kom in wyn voor as vry, vlugtige en aromatiese molekules en in geurlose, nie-vlugtige glikosidies-gebonde komplekse. Die gebonde vorm word stadig vrygestel deur ‘n suurhidrolise, maar dit word as onvoldoende beskou vir wyne wat vroeg gedrink word. Dit is dus belangrik dat die vrystelling van geurstowwe verhoog word om die kultivar karakter van die wyn te versterk. Die ensiematiese hidrolise proses behels twee opeenvolgende stappe: eerstens, afhangende van die aard van die voorloper, word die glikosidiese verbinding deur α-L-arabinofuranosidase, α-Lramnosidase, β-D-xilosidase, of β-D-apiosidase gebreek. In die tweede stap word die monoterpeen-alkohol deur β-glukosidase vrygestel. Hierdie ensiematiese afbraak proses verander nie die intrinsieke aromatiese kenmerke van die wyn, soos met suurhidrolise die geval is nie. Pektolitiese ensieme speel ‘n fundamentele rol in selverlenging, sagwording en afbraak van plant materiaal. Hierdie ensieme word gebruik om sap opbrengs te verhoog, aroma en smaak komponente vry te stel uit die doppe, asook om sapverheldering en filtrasie te verbeter. Die pektolitiese ensieme werk op ‘n sinergistiese wyse om pektien in wyn af te breek. Protopektinase produseer wateroplosbare en hoogs gepolimeriseerde pektien uit protopektien, slegs uit niegemetileerde galakturoonsuur eenhede. Pektien metielesterase verwyder metielester groepe van die poligalakturoonsuurketting. Die glikosidiese bindings tussen galakturoonsuur eenhede word deur poligalakturonase afgebreek. Pektien- en pektaat-liase het ‘n β-eliminasie aanslag op die ketting en as gevolg daarvan word dubbelbindings tussen C4 en C5 in die terminale residue gevorm. Vanuit bogenoemde is dit dus duidelik dat ensieme ‘n kardinale rol speel in die wynbereidingsproses. Ongelukkig is daar ‘n verskeidenhied van faktore wat die werking van ensieme in die wynbereidingsproses kan beïnvloed. Een moontlike faktor is die teenwoordigheid van ‘n suur-protease, van fungisidiese en/of gis oorsprong, in die wynmedium, omdat dit ander ensieme as substraat kan benut en degradeer. Suiwer ensiem preparate is gebruik om die ensiem interaksie tussen ‘n gis suur-protease en ‘n verslag aktiwiteit (β-glukosidase) in vitro te ondersoek. ‘n Gebotteleerde wyn en ‘n buffer is gebruik om die in vitro kondisies na te boots. Relatiewe ensiem aktiwiteit is ontleed oor ‘n aantal dae. Beide die ensieme het aktiwiteit getoon in die media, maar gis protease het geen statisties beduidende invloed gehad op die aktiwiteit van die verslag ensiem nie. Daaropvolgend is wyn berei van Sauvignon blanc druiwe, met verskillende ensiempreparaat toevoegings. Die ensiemaktiwiteit is deurlopend tydens fermentasie gemeet. Na afloop van stabilisasie is chemiese, sowel as sensoriese ontledings op die wyn gedoen. Die resultate het bevestig dat gis protease, onder hierdie kondisies, geen beduidende invloed op die verslag aktiwiteit gehad het nie. Die protease se onvermoë om die verslag aktiwiteit beduidend te beinvloed blyk onwaarskynlik aangesien die suurprotease aktief is by lae pH vlakke en dit as die enigste protease voorgestel is wat die fermentasie proses kan oorleef. Dit blyk asof ‘n wyn-verwante faktor, moontlik etanol, hiervoor verantwoordelik kan wees. Dus hou protease geen gevaar in vir die gebruik van kommersiële ensieme in wynbereiding nie. Navorsing kan in die toekoms fokus op meer gedetailleerde ensiem interaksie studies tussen protease en ander ensiem aktiwiteite, in gespesifiseerde kondisies. Die degradasie kapasiteit kan moontlik aangewend word om ongewenste ensiem aktiwiteite, wat byvoorbeeld oksidasie en verbruining veroorsaak, te verminder. Die karakterisering van die interaksies tussen protease en β-glukosidase kan dus die sleutel wees tot die produksie van wyne met verhoogde aroma potensiaal, asook die eliminasie van ongewenste ensiematiese aktiwiteite.

Thesis (Msc (Viticulture and Oenology. Institute for Wine Biotechnology))--University of Stellenbosch, 2009. / Monoterpenes are important aroma compounds in certain grape varieties such as Muscat, Gewürztraminer and Riesling and are present as either odourless, glycosidically bound complexes or as free aromatic monoterpenes. These complexes occur as monoglucosides or, when present as diglycosides, most commonly as 6-O-α-L-arabinofuranosyl-β-D-glucopyranosides of mainly linalool, geraniol, nerol and citronellol. The release of monoterpenes from non-volatile glycosidically bound precursors occurs either by acid hydrolysis or enzymatic hydrolysis. High temperature acid hydrolysis causes a rearrangement of the monoterpene aglycones and a decrease in the aroma and changes in the aromatic characteristics of monoterpenes and is therefore not suitable. Enzymatic hydrolysis does not modify the monoterpene aglycones and can be an efficent method to release potentially volatile monoterpenes. α-L-arabinofuranosidase and β-glucosidase are important enzymes responsible for the liberation of monoterpene alcohols from their glycosides. Glycosidases from Vitis vinifera and Saccharomyces cerevisiae are severely inhibited by winemaking conditions and this leads to unutilized aroma potential, while commercial preparations of aroma liberating enzymes are crude extracts that often have unwanted and unpredictable side effects. It is therefore of interest to investigate alternative measures to release glycosidically bound monoterpenes to increase the floral aroma of wine without side activities that impact negatively on wine. Heterologous α-L-arabinofuranosidases and β-glucosidases have previously been expressed in S. cerevisiae and these studies have evaluated and found increased glycosidic activities against both natural and synthetic substrates. In this study, we expressed the Aspergillus awamori α-L-arabinofuranosidase (AwAbfB) in combination with either the β-glucosidases Bgl2 from Saccharomycopsis fibuligera or the BglA from Aspergillus kawachii in the industrial yeast strain S. cerevisiae VIN13 to facilitate the sequential enzymatic hydrolysis of monoterpene diglycosides. Enzyme assays and GC-FID (Gas Chromatography with a Flame Ionization Detector) results show a significant increase in the amount of free monoterpene concentrations under winemaking conditions in the strain co-expressing the AwAbfB and the Bgl2. The increases in free monoterpene levels obtained were similar to those obtained with a commercial enzyme preparation, LAFAZYM AROM. Sensorial evaluation confirmed the improvement in the wine aroma profile, particularly the floral character. This yeast strain permits a single culture fermentation which improves the sensorial quality and complexity of wine. Further investigations on the factors influencing the stability and reactivity of monoterpenes during alcoholic fermentation are needed.

Thesis (Msc (Viticulture and Oenology))--University of Stellenbosch, 2007. / Among the factors contributing to wine complexity and quality, wine aroma is one of the most important factors. Wine aroma is the outcome of interaction among different compounds produced from the grapes, during fermentation as well as during the ageing process. Apart from its origin from grapes, fungi and yeasts, wine aroma can also be derived from the metabolic activity of wine lactic acid bacteria (LAB). These microorganisms are usually associated with malolactic fermentation (MLF) which normally occurs after alcoholic fermentation. MLF is beneficial to wine due to its contribution to deacidification, microbiological stabilisation and wine aroma formation, with the latter being the most important area of interest in our study. The production of volatile aromatic components in wine can, in part, be achieved through the hydrolytic action of enzymes produced by LAB associated with wine. These enzymes include β-glucosidase, protease, esterase, lipase and glucanase. Most of the work done on bacterial enzymes has been on LAB from food sources other than wine, in which these enzymes contribute to the flavour development of some cheeses, yoghurt and other fermented foods. The activity of these enzymes during wine fermentation has mostly been concerned with β-glucosidase from Oenococcus oeni. Only in recent years has there been a renewed interest in evaluating the activity of β-glucosidase in other genera of wine LAB. The overriding goal of this study was to screen and characterise wine-related enzymes produced by LAB associated with wine. All the LAB isolates tested in this study were obtained from IWBT culture collection and were previously isolated from five different wineries situated in the Western Cape region, South Africa. We first screened isolates using classical methods. The isolates were grown on agar medium supplemented with appropriate substrate analogues in order to evaluate the activity of enzymes (i.e. β- glucosidase, glucanase, lipase and esterase). The colonies exhibiting enzymatic activity ...

Ultra high temperature (UHT) processing can extend shelf life of milk to several months without refrigeration, which is more convenient and energy saving than pasteurized milk. However, the poor acceptance caused by "cooked" flavors limits its marketing growth, especially in United States. Ohmic heating, which has a more uniform and rapid heating than conventional UHT process, may minimized the flavor change during the thermal treatment. Flavor composition between Ohmic heated UHT milk and other traditionally processed UHT milk (direct steam injection and indirect plate heating) during 36 weeks storage were investigated in this study. A total of 20 volatile compounds were analyzed based on their importance to UHT milk as well as their representation to different chemical classes including sulfur-containing compounds, ketones, lactones, aldehydes and others. Dimethyl sulfide (DMS) and methyl ketones were significant different among three types of UHT heated milk. δ-lactones showed higher amount in Ohmic heating after stored for four weeks, which might generate creamy, fruity intermediate aroma. Other compounds showed no significant difference among three heating processes. Aroma recombination test revealed that the overall aroma of the ultra pasteurized (UP) milk could be mimicked by recombining 15 important reference odorants in the same concentrations as they occurred in the UHT milk using commercial pasteurized milk as the matrix. / Graduation date: 2012

Thesis (MSc Food Sc (Food Science))--University of Stellenbosch, 2009. / This study focused on a serious quality-related problem in the global wine industry, including the South African Wine Industry, namely cork taint in wine. Annually, large financial losses are incurred by cork suppliers and wine producers, as a result of cork-tainted wine. Although contaminated new unused corks are frequently implicated as the origin of this taint, contaminated cellar equipment and water can also be the source of the problem. An explorative investigation into the incidence of cork taint in South African wines showed that 3.8% of the 133 wines tested, contained 2,4,6-trichloroanisole (TCA) concentrations of 3.5 ng/L and higher, as determined by gas chromatography coupled with electron capture detection (GC-ECD). TCA concentrations higher than 1 ng/L were found in 18% of the wines tested. All affected wines were sealed with solid or agglomerate cork stoppers. These wines were sourced from various wineries in the Western Cape region, South Africa and were of different cultivars. None of the wines sealed with synthetic closures had any detectable TCA, 2,4,6-tribromoanisole (TBA) or pentachloroanisole (PCA) levels and only very low 2,3,4,6- tetrachloroanisole (TeCA) levels (1 ng/L or less). Another group of 28 wines that were rejected by the official South African wine regulatory body on the basis of the presence of mouldy taint during wine certification, was also included in this study. GC-ECD analysis showed that 30% of the wines in this group contained TCA at concentrations of 3.5 ng/L and higher. These results pointed to a relative high incidence of TCA in the wines investigated, especially those sealed with cork stoppers. Although no general conclusions should be made on the incidence of cork taint in the wider wine industry based on the results found within this explorative investigation, these findings confirmed the presence of cork taint in South African wines. Detection threshold values were determined for TCA, TeCA, TBA and PCA in three wine cultivars using the standard ASTM method. Results indicate that factors relating to the wine cultivar seemed to affect threshold values considerably. Our research proposes a detection range rather than an average detection threshold. Detection ranges established for TCA, TeCA, TBA and PCA in Chenin blanc, Pinotage and Shiraz coincide with reported values in literature. This result can be regarded as a valuable expansion of the existing knowledge of detection threshold values. Descriptive sensory analysis indicated significant (P 0.05) changes in the aroma profile of Chenin blanc, Pinotage and Shiraz after TCA, TeCA, TBA or PCA was added to the respective base wines that contained no detectable levels of the haloanisoles. The mouldy taint induced by these haloanisoles were described as mouldy, mouldy-chemical, mouldychlorine, as well as mouldy-acidic. In Chenin blanc, additions of TCA, in the concentration range 1 to 17 ng/L, resulted in a marked increase in the mouldy aroma and was accompanied by an immediate decrease in fruitiness. This change was already evident at added TCA concentrations of 1 ng/L. Similar trends were observed in Pinotage, while the addition of low levels of TCA to Shiraz (2 ng/L) resulted in a significant (P 0.05) decrease in the herbaceous character of the wine. The aroma changes observed were prominent enough to render the wine totally unacceptable in comparison to its original character. Consumers’ degree of liking did not seem to be affected by very low concentration levels of TCA in Chenin blanc, Pinotage or Shiraz, but rejection increased as the concentration increased beyond detection threshold level. A slight gender effect was also noticed. Female consumers appeared to be more sensitive to increasing levels of TCA, whereas male consumers did not respond as negatively to higher concentration levels of TCA. This study makes an important contribution towards understanding the sensory impact of especially TCA contamination in wine, through the establishment of concentration ranges at which these compounds exert a noticeable detrimental effect on the aroma profile of wine. Additional insight into cork taint in wine is provided by the consumer preference studies, where the effects of the taint on the product acceptance by consumers are demonstrated. The development of a modus operandi to ensure that sensory panels provide reliable data, can be regarded as an important contribution to wine-related research. This study is one of the first where advanced sensometric techniques were applied in sensory studies on cork tainted wines.

O mercado cervejeiro passa por uma revolução voltada à produção em escala artesanal ao invés de escala industrial, devido às expectativas dos consumidores em busca de alta qualidade e novo sabor do produto final. Visando produzir uma cerveja leve com relação ao teor de extrato e álcool, com sabor diferenciado, foram realizados vários ensaios de adição de gengibre no processo de produção. O mais viável resultado foi obtido com adição de 2g L-1 de lascas de gengibre in natura na maturação. A cerveja artesanal adicionada de gengibre foi produzida em escala laboratorial, para permitir sua análise físico-química e análise sensorial. Paralelamente, foram analisadas outras vinte e oito amostras de cerveja Pilsen para efeitos comparativos com relação aos aspectos físico-químicos. De acordo com o resultado do teste sensorial, o índice de aceitabilidade global foi de 92%, estando também acima de 70% o índice dos atributos individuais avaliados. A cerveja artesanal adicionada de gengibre apresentou características mais próximas às amostras de “Cerveja” ao invés das amostras “Puro Malte”, segundo classificação com relação ao teor de malte, por meio de análises quimiométricas (PCA e HCA). Essa discriminação entre os grupos foi devida aos teores de álcool, grau real de fermentação, grau aparente de fermentação, potássio, calorias e magnésio. O produto final apresentou como características principais um baixo teor alcoólico (3,40o GL), baixo valor energético (115,44 KJ 100 mL-1) e extrato reduzido (7,81º Plato). Apesar do amargor mais acentuado (21,55 B. U.), o índice de aceitabilidade para o amargor permaneceu acima de 70%, com flavor picante e aromático. / The brewing market undergoes a revolution directing to production on a homemade scale rather than an industrial scale, due to the expectations of consumers searching high quality and new flavor of the final product. In order to produce a light beer with respect to the extract and alcohol content and different flavor, several ginger addition tests were carried out in the production process. The most viable result was obtained with addition of 2 g L-1 of ginger flakes in natura at maturation. The artisanal brewed beer of ginger was produced in laboratory scale, to allow its physical-chemical analysis and sensorial analysis. In parallel, others twenty-eight samples of Pilsen beer were analyzed for comparative purposes in relation to physico-chemical aspects. According to the result of the sensorial test, the overall acceptability index was 92% as also as individual attributes evaluated were also above 70%. The artisanal beer added with ginger showed characteristics closer to the "Beer" samples than the "Pure malt" samples, according to classification in relation to the malt content, by means of chemometric analyzes (PCA and HCA). This discrimination between groups was due to alcohol, real degree of fermentation (RDF), apparent degree of fermentation (ADF), potassium, calories and magnesium. The final product had a low alcohol content (3.40 oGL), low energy (115.44 KJ 100 mL-1) and reduced extract (7.81 oPlato). Despite the more pronounced bitterness (21.55 B. U.), the acceptability index forbitterness remained above 70%, with spicy and aromatic flavor.