The yeast Saccharomyces cerevisiae is used in many industrial applications including beer brewing, bread making, and winemaking. Winemaking yeast strains have the ability to convert grape sugars into alcohol and other metabolites consistent with good wine. An exploratory comparative approach was undertaken to identify the genes and corresponding proteins that give wine yeast strains of S. cerevisiae their distinctive phenotype, with a focus on studying genes that provide tolerance to ethanol. / Thesis (Ph.D.)--School of Agriculture, Food and Wine, 2006.

The grape flavonoids include anthocyanins, tannins and flavonols, all of which contribute to grape and wine quality by influencing the colour and mouthfeel of red wine. These compounds are synthesized in different parts of the berry and during different stages of berry development. In addition, environmental and viticultural factors such as light exposure can also alter the flavonoid composition of grapes. An understanding of how synthesis of these compounds is coordinated, their relationship to wine quality and the influence of bunch light exposure on the flavonoid composition of grapes, could be used to improve fruit quality by enhanced viticultural management. The first part of this study sought to investigate the relationship between the different products of the flavonoid biosynthetic pathway (anthocyanins, flavonols and tannins), from two climatic regions (warm and cool) and determine their role in grape and wine quality. In collaboration with a major winery, whole Shiraz grapes were sampled at the weighbridge from a range of different vineyards from two climatic regions; warm (Riverland) and cool (McLaren Vale) in 2003 and 2004. A total of 80 grape samples were collected in each season and processed (i.e. 100 berries, separated into skin, seeds and juice, weighed and frozen). Anthocyanins and flavonols were measured, in triplicate, in skins by HPLC. Tannins were determined in the skins and seeds by two methods; phloroglucinol hydrolysis (HPLC) and protein precipitation (UV-VIS spectrophotometer). A comprehensive comparison of the two methods is discussed. In both years, the grapes from warm and cool climates formed two distinct data sets based on flavonoid composition. There was a correlation between anthocyanins and flavonols for both the warm and cool climate samples in both years, however those from the warm region had lower anthocyanin for a given level of flavonol. As expected, the level of tannin in the seeds was greater than in skin for all samples. In both years, there was a weak correlation between anthocyanin levels in the skin and skin tannins, but no relationship with seed tannins. These results suggest there is some co-ordination in the synthesis of anthocyanins, flavonols and skin tannins. Also, the two regions clearly separated based on yield and despite the weak correlations in both regions, the levels of total anthocyanins were inversely related to yield. In addition, there was no relationship with any of the flavonoids and grape quality, indicating the need for improvement in streaming fruit for quality using these flavonoid compounds. The second part of the study was to investigate the effect of bunch light exposure on flavonol synthesis and accumulation in Shiraz and Chardonnay grapes during development. Light-excluding boxes were applied to bunches at budburst. Boxes were removed at four sampling times; flowering, pre-veraison, veraison and harvest. At each sampling time, berry skins were sampled when the boxes were removed and then every second day (light induced), along with exposed controls for one week. Flavonol accumulation and flavonol synthase (VvFLS1) gene expression was determined by HPLC and Real Time-PCR (RT-PCR) respectively. As expected, for all four sampling times, flavonol accumulation and VvFLS1 expression in the boxed fruit was significantly less than bunches exposed to light. On removal of boxes at flowering, pre-veraison and veraison, flavonols accumulated to levels similar to that of the exposed control fruit over a period of 4-6 days. There was a significant increase in VvFLS1 expression 2 days after exposure to light in parallel with the accumulation of flavonols. At harvest, in Chardonnay, VvFLS1 expression peaked by day 4, while in Shiraz VvFLS1 expression increased linearly and was highest at day 6. In contrast to the results for the earlier sampling times, the total amount of flavonols accumulated at harvest was less than 50% of exposed controls in Chardonnay and Shiraz grapes. These results show that flavonols are able to be induced by bunch light exposure at different times during berry development, including times when flavonols are not normally being synthesised. This suggests bunch light exposure can override the developmental control of flavonol accumulation. To further investigate the light induced expression of VvFLS1 in grapevines the molecular mechanism of transcriptional control was explored. Using genomic walking PCR techniques, two Shiraz VvFLS1 promoter sequences were cloned and their sequences were analysed. These promoter sequences were ~800bp in length and were 99% identical. A putative MYB responsive element (MRE) and several light responsive elements (LRE) were identified in the promoter region of these genes. To functionally test the VvFLS1 promoter(s), a transient assay was developed in Chardonnay suspension cells. Cells were bombarded with constructs containing potential transcription factors and the VvFLS1 promoter(s), fused to a luciferase reporter vector. After 48hrs incubation in the dark, cells were harvested and luciferase activity measured as an indicator of VvFLS1 promoter activity. Of the different transcription factors tested with the VvFLS1 promoter(s) the highest luciferase activity was observed using AtMYB12 (a flavonol-specific regulator of AtFLS1 in Arabidopsis (Mehrtens et al. 2005). While this result shows activation of the VvFLS1 promoters by AtMYB12 and the development of a transient reporter assay for testing the VvFLS1 promoter(s) a grapevine transcription factor specific for VvFLS1 was sought. Two techniques were employed to identify potential transcription factor regulators of the VvFLS1 promoter(s). The first involved BLAST sequence search analysis in a grapevine expression (EST) database with AtMYB12 and the second involved using DNA microarray technology to identify candidate transcription factors that were up-regulated in light exposed Chardonnay cell suspension cultures. Thirteen potential transcription factors were identified and after correlative RT-PCR analysis (with VvFLS1 expression patterns) two candidates were selected for further isolation and characterisation. These results have made significant progress in unravelling the molecular mechanisms of regulation of the flavonol biosynthetic, however additional experiments are required to unravel the transcriptional control of flavonol biosynthesis. This investigation contributes to our knowledge of flavonoid synthesis in grapes; how it is coordinated, the relationship with wine quality, and the influence of light particularly on synthesis of flavonols. It also explores the molecular mechanisms of VvFLS1 control, through isolation of the VvFLS1 promoter and identification of potential transcription factors, which may regulate it. An understanding of the synthesis of flavonoids and how they may be coordinated, particularly in response to light, could be used to improve fruit quality by enhanced viticultural management. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1326767 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Bibliography: leaves 138-158. This study identified and investigated the properties, functions and patterns of accumulation of prominent berry proteins associated with white wine haze. Detailed analysis was conducted on two PR-like proteins of V. vinifera, VVPR-4a and VVTL1. In vitro fungal growth inhibition assays suggested that berry PR-like proteins may play an important role in plant defence, particularly against fungal attack. Results of this study also have future implications for controlling the ripening process of grapes.

This research investigated the effects of Pediococcus spp. on Oregon Pinot noir wines. Pediococcus (P. parvulus (7), P. damnosus (1), P. inopinatus (1)) isolated from Oregon and Washington state wines demonstrated differences in their susceptibility to SO��� with some isolates growing well in model media at 0.4 mg/L molecular SO���. All isolates were all able to degrade p-coumaric acid to 4-vinyl phenol. The conversion of p-coumaric acid to 4-VP by pediococci resulted in accelerated production of 4-EP by B. bruxellensis in a model system. Growth of the pediococci isolates in Pinot noir wine resulted in a number of chemical and sensory changes occurring compared to the control. Very low concentrations of biogenic amines were measured in the wines with only wine inoculated with P. inopinatus OW-8 having greater than 5 mg/L. D-lactic acid production varied between isolates with OW-7 producing the highest concentration (264 mg/L). Diacetyl content of the wines also varied greatly. Some wines contained very low levels of diacetyl (< 0.5 mg/L) while others contained very high concentrations (> 15 mg/L) that were well above sensory threshold. Despite suggestions to the contrary in the literature, glycerol was not degraded by any of the isolates in this study. Color and polymeric pigment content of the wines also varied with wine inoculated with OW-7 containing 30% less polymeric pigment than the control. This may be related to acetaldehyde as a number of Pediococcus isolates, including OW-7, reduced the acetaldehyde content of the wine. Sensory analysis revealed differences in the aroma and mouthfeel of the wines compared to each other and to the control. In particular growth of some isolates produced wines with higher intensities of butter, plastic, and vegetal aromas while other also had lower perceived astringency. / Graduation date: 2013

Graduation date: 1994

Yeast mannoproteins released during the process of aging wine on the yeast lees have been reported to make important contributions to wine quality. However, few mannoproteins have been identified in wine and their lifespan during aging is unknown. As a first step towards better understanding the contributions of yeast mannoproteins to wine quality, a model system was used to follow changes in yeast protein release, and to identify the released proteins over a 9-month time course following completion of fermentation. Model musts were fermented in duplicate by a number of commercial yeast strains, including BM45 and RC212, and were stored on the yeast lees post-fermentation with monthly stirring at 15°C. Wine samples were taken during and after fermentation, and following removal of suspended solids, total protein and total mannoproteins were measured, and individual proteins were identified--but not quantified--by HPLC-MS of tryptic fragments. The total number of identified proteins in all samples increased from between 3-15 following inoculation, to between 70-80 after one month on the lees, and decreased to about 20 after 6 months on the lees. Over 50% of the identified proteins were shared among all yeast samples. For most strains, protein and mannoprotein concentrations increased during, but decreased by the end of fermentation. Both protein and mannoprotein concentrations were found to increase again post-fermentation, reaching values about 2- and 6-fold higher than values measured at 2 days, respectively. Consistent with the increase in mannoprotein concentration, cell wall mannoproteins were the predominant proteins identified after 6 months of aging on the lees. Most cytosolic proteins found during and soon after fermentation were not found after 6 months of aging on the lees. / Graduation date: 2009

Thesis (doctoral)--Johannes Gutenberg-Universität, 2002. / Vita. Includes bibliographical references (p. 204-209).

The utility of single-bounce attenuated total reflectance (SB-ATR) and heterospectral two-dimensional correlation spectroscopy (H2D-CS) in quantitative analysis by Fourier transform infrared (FTIR) spectroscopy was investigated by exploring several potential applications of these techniques. Enzymatic hydrolysis of lactose in milk was monitored by SB-ATR/FTIR spectroscopy, and changes in the concentrations of glucose, galactose and lactose during the process were successfully measured quantitatively. SB-ATR/FTIR spectroscopy was shown also to perform comparably to Fourier transform near-infrared (FT-NIR) spectroscopy for the determination of the alcohol content of distilled liquors and better than FT-NIR spectroscopy and comparably to transmission FTIR spectroscopy for the analysis of alcohol, total reducing sugar, total acidity and pH in wines. In addition, a set of 149 pre-analyzed wine samples was employed to develop and validate an SB-ATR/FTIR calibration for 11 different parameters and constituents in wines with the use of partial-least-squares (PLS) regression, demonstrating the potential utility of this method in the routine analysis of wines. The application of SB-ATR/FTIR spectroscopy and H2D-CS in the selection of wavelengths for multiple linear regression (MLR) calibration for FT-NIR analysis of ternary aqueous solutions of fructose, glucose and galactose was also investigated. NIR wavelengths were identified for the three sugars by H2D-CS of the SB-ATR/FTIR spectra of binary sugar solutions in relation to their FT-NIR spectra. An MLR calibration developed based on these wavelengths gave better results than PLS calibrations and comparable results to those obtained by MLR using wavelengths selected by examination of 1st and 2nd derivative spectra. H2D-CS was extended to include 2D correlations between high-pressure liquid chromatography (HPLC) and SB-ATR/FTIR data for the purpose of identifying HPLC peaks without the need to isolate the eluted compounds. The potential utility of this approach, termed spectroscopic/chromatographic 2D correlation (SC2D-C), was investigated by generating FTIR slice spectra corresponding to the HPLC peaks of wines spiked with sucrose, glucose and fructose and comparing them to 404 reference spectra in an IR spectral library. It was found that these constituents were correctly identified provided there was sufficient random variability of their concentrations in the samples analyzed.

No description available.

Two enzymatic systems from the lactic acid bacterium Oenococcus oeni, isolated from wine, have been studied. The first one is the H+-ATPase for which the activity was characterized under various conditions of growth. The activity gradually increased by l.6 to 1.9-fold upon inoculation at pH 3.5. The H+-ATPase activity did not vary significantly in function of the growth rate or with and without malic acid. However, acidification of the medium in the absence of malic acid induced the activity by 1.5 to 2.2-fold depending on the initial pH. The partially cloned H+-ATPase genes shared high homologies with those from other bacterial F0F1-ATPases. A mRNA of about 7 kb was detected by Northern blot and its size suggests that the genetic organization of O. oeni atp operon is similar to most F0F 1-ATPases. Furthermore, the amount of atp mRNA was shown to increase in acidic conditions. O. oeni H +-ATPase activity was pH-inducible and regulation of the expression seems to occur at the level of mRNA synthesis. Thus, the results confirmed the proposed role of the H+-ATPase in acid tolerance in O. oeni. / The second system studied was a chromosome-encoded P-type ATPase (CopB) and its putative transcriptional regulator (CopR). The copB gene encodes a protein showing great similarities with other Cu2+-ATPases of the CPx-type family of heavy-metal ATPases like Enterococcus hirae copB. Another gene (copR) was found 250 bp upstream of copB and displays great similarities with proteins of the MecI/BlaI family of transcriptional regulators, including En. hirae CopY repressor. O. oeni was shown to be highly resistant to copper and growth occurred in up to 30 mM CuSO4. Northern blot analyses indicated that the amount of copB mRNA increased upon a 0.2 to 4.0 mM copper stress suggesting that expression of the enzyme might be regulated at the level of mRNA synthesis. Whether CopR is involved in this regulation remains to be determined, but the results suggest that copRB genes might be involved in copper resistance in O. oeni.