Thesis (PhD (Chemistry and Polymer Science))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: The present study was initiated by the wine industry of South Africa to overcome the lack of available information on the flavor and aroma of South African wines. The aim was to develop new analytical methods and improve existing ones for the analysis of volatile compounds in the South African wines. Initially a new analytical method based on stir bar sorptive extraction (SBSE) in the headspace mode for the analysis of 37 pre-selected volatile compounds was developed and validated. Consequently, the method was improved by making important modifications and increasing the number of compounds analyzed to 39. This method was successfully applied to a large number of Pinotage wines of vintages 2005 and 2006. The quantitative data of these wines were subjected to chemometric analysis in order to investigate possible co- /variances. A clear distinction was observed between the two vintages, where the 2005 wines were more characterized by wood-related compounds and the 2006 wines by the fermentation compounds. The developed method was further applied to other cultivars of vintage 2005, including two white (Sauvignon Blanc and Chardonnay) and three red (Shiraz, Cabernet Sauvignon and Merlot) cultivars. In a similar fashion, the quantitative data of the six cultivars of vintage of 2005 were analysed by chemometric methods. Significant differences were observed between the two white cultivars and among the four red cultivars. It was shown that among these cultivars, the major role-players were the wood and fermentation related volatiles. A striking observation was the confirmation of the unique character of the Pinotage wines compared to the other red cultivars, mainly influenced by the high level of isoamyl acetate and low level of isoamyl alcohol, the former being categorized as a varietal compound for Pinotage expressed by a fruity (banana) odor. In addition, advanced chromatographic technology in the form of comprehensive twodimensional gas chromatography (GC × GC) coupled to time-of-flight mass spectrometry (TOFMS) was investigated for the detailed analysis of volatile compounds in young South African wines. This work focused primarily on Pinotage wines. In the first instance, solid phase micro extraction (SPME) in the headspace mode in combination with GC × GC-TOFMS was used. Due to the high resolution and large peak capacity of GC × GC, more than 200 compounds previously reported as wine components were identified. These compounds were dominated by the highly volatile and less polar compounds, mainly due to the characteristics of SPME. In an attempt to further extend these results, another selective extraction method, solid phase extraction (SPE) was used in combination with GC × GC-TOFMS analysis. Using this technique, more than 275 compounds, most of them unidentified using the previous method, were detected. These groups of compounds include volatile phenols, lactones as well as mostly aromatic esters and norisoprenoids, which can potentially influence the aroma and flavor of wine. The techniques developed as part of this study have extended our knowledge of the volatile composition of South African wines. / AFRIKAANSE OPSOMMING: Hierdie studie is geïnisieer deur die wyn industrie van Suid-Afrika om die tekort aan beskikbare inligting aangaande wyn aroma van Suid-Afrikaanse wyne te oorkom. Die doel was om nuwe analitiese metodes te ontwikkel en die huidige metodes te verbeter vir die analise van vlugtige verbindings in Suid-Afrikaanse wyne. Oorspronklik is ʼn nuwe analitiese metode ontwikkel en gevalideer gebaseer is op ‘stir bar sorptive extraction’ (SBSE) in die gas fase vir die analise van 37 vooraf geselekteerde vlugtige verbindings. Die metode is verbeter deur belangrike modifikasies aan te bring en die hoeveelheid verbindings wat analiseer word te vermeerder na 39. Hierdie metode is suksesvol aangewend op ʼn groot hoeveelheid Pinotage wyne van oesjare 2005 en 2006. Die kwantitatiewe data van hierdie wyne is onderwerp aan verskillende chemometriese analises om moontlike ko-/variasies te ondersoek. ʼn Duidelike onderskeid is opgemerk tussen die twee oesjare, waar die 2005 wyne gekarakteriseer is deur hout-verwante verbindings en die 2006 wyne weer meer deur fermentasie verbindings. Die verbeterde metode is verder aangewend vir analiese van ander kultivars van oesjare 2005, wat twee wit (Sauvignon Blanc en Chardonnay) en drie rooies (Shiraz, Cabernet Sauvignon en Merlot) ingesluit het. Die kwantitatiewe data van die ses kultivars van oesjaar 2005 is op ʼn soortgelyke wyse geanaliseer deur verskillende chemometriese metodes te gebruik. Beduidende verskille is opgemerk tussen die twee wit kultivars en tussen die vier rooi kultivars. Die hoof rolspelers tussen die ses kultivars was weereens die verbindings wat ʼn hout en fermentasie aard het. Die unieke karakter van die Pinotage wyne in vergelyking met die ander rooi kultivars was opvallend. Hierdie wyn word gekarakteriseer deur hoë vlakke van isoamiel asetaat en lae vlakke van isoamiel alkohol, waar eersgenoemde gekatogiseer word as ʼn verbinding wat ʼn vrugte (piesang) geur in Pinotage uitdruk. Verder is gevorderde chromatografiese tegnologie in die vorm van ‘comprehensive two-dimentional gas chromatography’ (GC x GC) gekoppel met ‘time-of-flight mass spectroscopy’ (TOFMS) ondersoek vir die analiese van vlugtige verbindings in jong Suid-Afrikaanse wyne. Hierdie werk het hoofsaaklik op Pinotage wyne gefokus. Eerstens is ‘solid phase micro extraction’ (SPME) in die gas fase gekombineer met GC x GC-TOFMS. As gevolg van die hoë resolusie en groot piek kapasiteit van GC x GC is meer as 200 verbindings wat voorheen gerapporteer is as wyn komponente geïdentifiseer. Hierdie verbindings is gedomineer deur hoë vlugtige polêre verbindings, hoofsaaklik as gevolg van die karaktersitieke van SPME..In ʼn poging om die metode verder te verbeter is ʼn selektiewe ekstraksie metode naamlik ‘solid phase extraction’ (SPE) in kombinasie met GC x GC-TOFMS gebruik. Met hierdie tegniek is meer as 275 verbindings geïdentifiseer, waarvan die meeste nie met die vorige metode waargeneem is nie. Hierdie verbindings sluit vlugtige fenole, laktone en meestal aromatiese esters en norisoprenoïdes in, wat moontlik die reuk en smaak van wyn kan beïnvloed. Die metodes ontwikkel gedurende die studie het nuwe informasie verskaf aangaande die vlugtige komponente teenwoordig in Suid Afrikaanse wyne.

Cheddar Cheese samples (good cheese, weak cheese, cheese made with pasteurized milk, cheese made with heat-shocked milk, cheese from production plant A, cheese from production plant B, cheese made with adjunct culture, and cheese made without adjunct culture), were evaluated during the ripening stage. Proteolysis was studied by a fractionation scheme, resulting in an insoluble fraction analyzed by urea polyacrylamide gel electrophoresis (Urea-PAGE), and a soluble fraction which was further investigated through water soluble nitrogen (WSN), trichloroacetic acid soluble nitrogen (TCA-SN) and phosphotungstic acid soluble nitrogen (PTA-SN) analyzed by total Kjeldahl nitrogen content (TKN). Reversed phase high performance liquid chromatography (RP-HPLC) was used to study the peptide profile of the water soluble fraction. Lipolyisis was studied by levels of individual free fatty acids determined through gas chromatography-flame ionization detection (GC-FID) after isolation employing solid phase extraction (SPE). Volatile sulfur compounds were studied using head space solid phase micro-extraction (SPME) coupled with gas chromatography-pulsed flame photometric detection (PFPD). It was found that Urea-PAGE is capable to differentiate samples according their age, but cannot discriminate samples regarding the treatment assessed, quality or origin of the samples. However, measurements of total Kjeldahl Nitrogen (TKN) of the WSN, TCA-SN, and PTA-SN fractions, and the principal component analysis of the RP-HPLC peptide profile of the WSN fraction, revealed differences in the rate and pattern of proteolysis for each one of the manufacturing cases. Good cheese, cheese produce in plant TCCA, cheese made in plant CRP with adjunct culture isolated from plant TCCA cheese, and cheese made with heat-shocked milk developed higher level of total nitrogen for the WSN, TCA-SN and PTA-SN fractions, indicating that primary and secondary proteolysis were faster for these samples. This is supported by a PCA model with three principal components that account for the 80-83% of the variability of the data from the RP-HPLC peptide profile analysis, which discriminates the samples according to age and manufacturing practice. In addition, FFA profiles demonstrated higher levels of low and medium chain free fatty acids for good cheese, cheese produce in plant TCCA, cheese made in plant CRP with adjunct culture, and cheese made with heat-shocked milk samples, which suggest faster lipolysis during ripening. The Volatile Sulfur Compounds (VSC) analysis showed higher levels of DMS and MeSH and lower levels of H2S, suggesting faster catabolism of sulfur containing amino acids in good cheese, cheese produce in plant TCCA, cheese made in plant CRP with adjunct culture, and cheese made with heat-shocked milk. / Graduation date: 2013

O sabor da cerveja é determinado pela matéria-prima, pelo tipo de processo e pela levedura utilizada, além dos compostos produzidos durante a fermentação e maturação, que exercem maior impacto. A produção de amidos modificados é uma alternativa praticada com o objetivo de superar limitações dos amidos nativos e aumentar a utilidade deste polímero em aplicações industriais. O objetivo da pesquisa foi avaliar a aplicação de amidos de milho e mandioca modificados (fosfatação e oxidação) no processo de clarificação de cerveja artesanal, visando a obtenção de uma cerveja “limpa” e translúcida. Foram elaboradas oito cervejas testando os amidos aniônicos: amido de milho oxidado 1% (MiO 1%); amido de milho oxidado 2% (MiO 2%); amido de mandioca oxidado 1% (MaO 1%); amido de mandioca oxidado 2% (MaO 2%); amido de milho fosfatado baixo (MiFB); amido de milho fosfatado alto (MiFA); amido de mandioca fosfatado baixo (MaFB); amido de mandioca fosfatado alto (MaFA). Essas amostras foram comparadas com uma cerveja submetida ao mesmo processo utilizando o clarificante goma carragena (GCar) e com uma cerveja sem adição de clarificante (SC). As amostras foram submetidas às análises de compostos fenólicos, flavonóides, biureto, espectrofotometria, turbidez e viscosidade relativa para verificar a possível ação clarificante. Foi realizada a caracterização físico-química das amostras de cerveja artesanal (pH, extrato real, acidez, teor alcóolico, extrato primitivo e grau real de fermentação). A aplicação de amidos modificados reduziu o teor de compostos fenólicos, flavonóides, proteínas, e a viscosidade relativa no decorrer do processo. Além disso, a clarificação foi notada nas análises de turbidez e espectrofotometria. Pode-se concluir que alguns amidos destacaram-se, superando os resultados apresentados pela goma carragena na etapa final do processo. No entanto, notou-se que a eficiência destes amidos ocorre a partir da maturação, diferente da goma carragena que clarifica principalmente na etapa da fervura. Em um aspecto global, sugere-se que na fosfatação, a fonte botânica não influenciou, e as amostras destaques na clarificação foram o amido de milho fosfatado baixo (2,8%) (amostra 7), o amido e milho fosfatado alto (8,6%) (amostra 8) e o amido de mandioca fosfatado alto (amostra 10). Para a oxidação a amostra que apresentou melhor desempenho foi o amido de mandioca oxidado 1% (amostra 4). / The flavor of beer is determined by the feedstock, the type of process and the yeast used, in addition to the compounds produced during fermentation and maturation, that ecxercises greater impact. The production of modified starch is an alternative practiced with the aim of overcoming the limitations of the native starches and increase the usefulness of this polymer in industrial applications. The objective of the research was evaluate the application of corn and cassava modified starches (phosphatation and oxidation) in the craft beer clarification process to getting a beer "clean" and translucent. Were elaborated eight beers testing the anionic starches: oxidized corn starch 1% (MiO 1%); oxidized corn starch 2% (myo 2%); oxidized cassava starch 1% (MaO 1%); oxidized cassava starch 2% (MaO 2%); phosphate corn starch low (MiFB); phosphated corn starch high (MIFA); phosphated cassava starch low (MAFB); phosphate cassava starch high (MAFA). These samples were compared with a beer submitted to the same process using the clarifier carrageenan gum (CARG) and with a beer without added clarifier (SC). The samples were submitted to analysis of phenolic compounds, flavonoids, biuret, spectrophotometry, relative viscosity and turbidity to verify possible clarifier action. Was realized physicochemical characterization of craft beer samples (pH, real extract, acidity, alcohol content, original extract and real degree of fermentation). The application of modified starches reduced the content of phenolic compounds, flavonoids, proteins, and the relative viscosity during the process. In addition, the clarification was noted in turbidity analysis and spectrophotometry. It can be concluded that some starches stood out, overcoming the results presented by carrageenan gum in the final stage of the process. However, it was noted that the efficiency of these starches occurs from the maturation, different of carrageenan gumthat clarifies mainly in the boiling step. In a global aspect, it is suggested that in the phosphatation, the botanical source did not influence, and the highlight samples in clarification were phosphated corn starch low (2.8%) (sample 7), phosphated corn starch high (8 , 6%) (sample 8) phosphated cassava starch high (sample 10). To the Oxidation. the sample that showed better performance was oxidized cassava starch 1% (sample 4).

O sabor da cerveja é determinado pela matéria-prima, pelo tipo de processo e pela levedura utilizada, além dos compostos produzidos durante a fermentação e maturação, que exercem maior impacto. A produção de amidos modificados é uma alternativa praticada com o objetivo de superar limitações dos amidos nativos e aumentar a utilidade deste polímero em aplicações industriais. O objetivo da pesquisa foi avaliar a aplicação de amidos de milho e mandioca modificados (fosfatação e oxidação) no processo de clarificação de cerveja artesanal, visando a obtenção de uma cerveja “limpa” e translúcida. Foram elaboradas oito cervejas testando os amidos aniônicos: amido de milho oxidado 1% (MiO 1%); amido de milho oxidado 2% (MiO 2%); amido de mandioca oxidado 1% (MaO 1%); amido de mandioca oxidado 2% (MaO 2%); amido de milho fosfatado baixo (MiFB); amido de milho fosfatado alto (MiFA); amido de mandioca fosfatado baixo (MaFB); amido de mandioca fosfatado alto (MaFA). Essas amostras foram comparadas com uma cerveja submetida ao mesmo processo utilizando o clarificante goma carragena (GCar) e com uma cerveja sem adição de clarificante (SC). As amostras foram submetidas às análises de compostos fenólicos, flavonóides, biureto, espectrofotometria, turbidez e viscosidade relativa para verificar a possível ação clarificante. Foi realizada a caracterização físico-química das amostras de cerveja artesanal (pH, extrato real, acidez, teor alcóolico, extrato primitivo e grau real de fermentação). A aplicação de amidos modificados reduziu o teor de compostos fenólicos, flavonóides, proteínas, e a viscosidade relativa no decorrer do processo. Além disso, a clarificação foi notada nas análises de turbidez e espectrofotometria. Pode-se concluir que alguns amidos destacaram-se, superando os resultados apresentados pela goma carragena na etapa final do processo. No entanto, notou-se que a eficiência destes amidos ocorre a partir da maturação, diferente da goma carragena que clarifica principalmente na etapa da fervura. Em um aspecto global, sugere-se que na fosfatação, a fonte botânica não influenciou, e as amostras destaques na clarificação foram o amido de milho fosfatado baixo (2,8%) (amostra 7), o amido e milho fosfatado alto (8,6%) (amostra 8) e o amido de mandioca fosfatado alto (amostra 10). Para a oxidação a amostra que apresentou melhor desempenho foi o amido de mandioca oxidado 1% (amostra 4). / The flavor of beer is determined by the feedstock, the type of process and the yeast used, in addition to the compounds produced during fermentation and maturation, that ecxercises greater impact. The production of modified starch is an alternative practiced with the aim of overcoming the limitations of the native starches and increase the usefulness of this polymer in industrial applications. The objective of the research was evaluate the application of corn and cassava modified starches (phosphatation and oxidation) in the craft beer clarification process to getting a beer "clean" and translucent. Were elaborated eight beers testing the anionic starches: oxidized corn starch 1% (MiO 1%); oxidized corn starch 2% (myo 2%); oxidized cassava starch 1% (MaO 1%); oxidized cassava starch 2% (MaO 2%); phosphate corn starch low (MiFB); phosphated corn starch high (MIFA); phosphated cassava starch low (MAFB); phosphate cassava starch high (MAFA). These samples were compared with a beer submitted to the same process using the clarifier carrageenan gum (CARG) and with a beer without added clarifier (SC). The samples were submitted to analysis of phenolic compounds, flavonoids, biuret, spectrophotometry, relative viscosity and turbidity to verify possible clarifier action. Was realized physicochemical characterization of craft beer samples (pH, real extract, acidity, alcohol content, original extract and real degree of fermentation). The application of modified starches reduced the content of phenolic compounds, flavonoids, proteins, and the relative viscosity during the process. In addition, the clarification was noted in turbidity analysis and spectrophotometry. It can be concluded that some starches stood out, overcoming the results presented by carrageenan gum in the final stage of the process. However, it was noted that the efficiency of these starches occurs from the maturation, different of carrageenan gumthat clarifies mainly in the boiling step. In a global aspect, it is suggested that in the phosphatation, the botanical source did not influence, and the highlight samples in clarification were phosphated corn starch low (2.8%) (sample 7), phosphated corn starch high (8 , 6%) (sample 8) phosphated cassava starch high (sample 10). To the Oxidation. the sample that showed better performance was oxidized cassava starch 1% (sample 4).

This research investigated yeast populations and diversity during pre-fermentation cold maceration and alcoholic fermentation of Vitis vinifera L. cv. Pinot noir grapes from a commercial vineyard (Dayton, OR). Fermentations were conducted at the Oregon State University research winery in 100 L tanks while grapes from the same vineyard lot were fermented at a commercial winery. Samples were taken daily during pre-fermentation maceration (9°C) and alcoholic fermentation (27°C) and plated on WL and lysine media to determine Saccharomyces and non-Saccharomyces populations and diversity. Total non-Saccharomyces populations increased from 1 x 10³ cfu/mL to 1 x 10⁵ cfu/mL during pre-fermentation cold maceration and reached a maximum of 1 x 10⁷ cfu/mL during alcoholic fermentation. Thirteen distinct yeast species were tentatively identified based on appearance on WL media and were initially screened for β-glucosidase activity using 4-methyllumbelliferyl-β-D-gluconopyranoside (4-MUG) plates. The identity of the isolates screening positive for β-glucosidase activity was determined by sequence analysis of the D1/D2 domain of the 26S rDNA gene. The five isolates identified were Metschnikowia pulcherrima, Hanseniaspora uvarum, Kluveromyces thermotolerans, and two Saccharomyces cerevisiae isolates. β-glucosidase activity was further characterized and quantified using a liquid media representing grape must conditions (pH 3.5, 20° Brix) at two temperatures (25°C and 8°C). While increasing sugar concentration suppressed the β-glucosidase activity of H. uvarum (-99%), β-glucosidase activity still remained relatively high for M. pulcherrima, S. cerevisiae isolate 1, and S. cerevisiae isolate 2. At 8°C, β-glucosidase activity was reduced for M. pulcherrima compared to activity at 25°C, but activity increased for K. thermotolerans, S. cerevisiae isolate 1, and S. cerevisiae isolate 2. The yeast isolates possessing β-glucosidase activity were used in fermentations of Vitis vinifera L. cv. Pinot Noir grapes. The grapes were treated with high hydrostatic pressure (HHP) to inactivate naturally occurring yeast and bacteria. All yeast isolates grew during pre-fermentation cold maceration (7 days at 9°C) and populations increased 3 to 4 logs. Following pre-fermentation cold maceration, all ferments were warmed to 27°C and inoculated with S. cerevisiae RC212. Alcoholic fermentations were all complete within eight days and after pressing wines were analyzed for volatile aroma compounds by SPME-GC-MS. The presence of different yeast isolates during pre-fermentation cold maceration resulted in wines with unique aroma profiles. Ethyl ester concentrations were highest in the wine that did not undergo a pre-fermentation cold maceration, while concentrations of branch-chained esters were higher in the treatments with yeast present during pre-fermentation cold maceration. Pre-fermentation cold maceration with yeast isolates demonstrating β-glucosidase did not affect the concentration of β-damascenone or β-ionone. Wines that had undergone pre-fermentation cold maceration with S. cerevisiae isolate 1, S. cerevisiae isolate 2, and a combination of all isolates resulted in over twice the concentration of β-citronellol over wines that did not undergo a pre-fermentation cold maceration. / Graduation date: 2013

Department of Food Science and Technology / MSCPNT