The effects of yeast inoculation rate and acclimatization to juice on icewine fermentation /

Icewine is an intensely sweet, unique dessert wine fennented from the juice of grapes that have frozen naturally on the vine. The juice pressed from the frozen grapes is highly concentrated, ranging from a minimum of 35° Brix to approximately 42° Brix. Often Icewine fennentations are sluggish, taking months to reach the desired ethanol level, and sometimes become stuck. In 6 addition, Icewines have high levels of volatile acidity. At present, there is no routine method of yeast inoculation for fennenting Icewine. This project investigated two yeast inoculum levels, 0.2 gIL and 0.5 gIL. The fennentation kinetics of inoculating these yeast levels directly into the sterile Icewine juice or conditioning the cells to the high sugar levels using a step wise acclimatization procedure were also compared. The effect of adding GO-FERM, a yeast nutrient, was also assessed. In the sterile fennentations, yeast inoculated at 0.2 gIL stopped fennenting before the required ethanol level was achieved, producing only 7.8% (v/v) and 8.1 % (v/v) ethanol for the direct and conditioned inoculations, respectively. At 0.5 gIL, the stepwise conditioned cells fennented the most sugar, producing 12.2% (v/v) ethanol, whereas the direct inoculum produced 10.5% (v/v) ethanol. The addition of the yeast nutrient GO-FERM increased the rate of biomass accumulation, but reduced the ethanol concentration in wines fennented at 0.5 gIL. There was no significant difference in acetic acid concentration in the final wines across all treatments. Fennentations using unfiltered Icewine juice at the 0.5 gIL inoculum level were also compared to see if the effects of yeast acclimatization and micronutrient addition had the same impact on fennentation kinetics and yeast metabolite production as observed in the sterile-filtered juice fennentations. In addition, a full descriptive analysis of the finished wines was carried out to further assess the impact of yeast inoculation method on Icewine sensory quality. At 0.5 gIL, the stepwise conditioned cells fennented the most sugar, producing 11.5% (v/v) ethanol, whereas the direct inoculum produced 10.0% (v/v) ethanol. The addition of the yeast nutrient GO-FERM increased the peak viable cell numbers, but reduced the ethanol concentration in wines fennented at 0.5 gIL. There was a significant difference 7 in acetic acid concentration in the final wines across all treatments and all treatments affected the sensory profiles of the final wines. Wines produced by direct inoculation were described by grape and raisin aromas and butter flavour. The addition of GO-FERM to the direct inoculation treatment shifted the aroma/flavour profiles to more orange flavour and aroma, and a sweet taste profile. StepWise acclimatizing the cells resulted in wines described more by peach and terpene aroma. The addition of GO-FERM shifted the profile to pineapple and alcohol aromas as well as alcohol flavour. Overall, these results indicate that the addition of GO-FERM and yeast acclimatization shortened the length of fermentation and impacted the sensory profiles of the resultant wines.

Methoxypyrazine removal in grape juice: Development of a removal system using odorant and pheromone binding proteins coupled with bentonite fining

Multicoloured Asian Lady Beetles (MALB) and 7-spot Lady Beetles that infect vineyards can secrete alkyl-methoxypyrazines when they are processed with the grapes, resulting in wines containing a taint. The main methoxypyrazine associated with this taint is 3-isopropyl-2-methoxypyrazine (IPMP). The wines are described as having aroma and flavours of peanut butter, peanut shells, asparagus and earthy which collectively, have become known as “ladybug taint”. To date, there are no known fining agents used commercially added to juice or wine that are effective in removing this taint. The goal of this project was to use previously identified proteins with an ability to bind to methoxypyrazines at low pH, and subsequently develop a binding assay to test the ability of these proteins to bind to and remove methoxypyrazines from grape juice. The piglet odorant binding protein (plOBP) and mouse major urinary protein (mMUP) were identified, cloned and expressed in the Pichia pastoris expression system. Protein expression was induced using methanol and the proteins were subsequently purified from the induction media using anion exchange chromatography. The purified proteins were freeze-dried and rehydrated prior to use in the methoxypyrazine removal assay. The expression and purification system resulted in yields of approximately 78% of purified plOBP and 62% of purified mMUP from expression to rehydration. Purified protein values were 87 mg of purified plOPB per litre of induction media and 19 mg of purified mMUP per litre of induction medium. In order to test the ability of the protein to bind to the MPs, an MP removal assay was developed. In the assay, the purified protein is incubated with either IPMP or 3-isobutyl-2-methoxypyrazine (IBMP) for two hours in either buffer or grape juice. Bentonite is then used to capture the protein-MP complex and the bentonite-protein-MP complex is then removed from solution by filtration. Residual MP is measured in solution following the MP removal assay and compared to that in the starting solution by Gas Chromatography Mass Spectrometry (GC/MS). GC/MS results indicated that the mMUP was capable of removing IBMP and IPMP from 300 ng/L in buffer pH 4.0, buffer pH 3.5 and Riesling Juice pH 3.5 down to the limit of quantification of the instrument, which is 6ng/L and 2ng/L for IBMP and IPMP, respectively. The results for the plOBP showed that although it could remove some IBMP, it was only approximately 50-70 ng/L more than bentonite treatment followed by filtration, resulting in approximately 100 ng/L of the MPs being left in solution. pIOBP was not able to remove IPMP in buffer pH 3.5 using this system above that removed by bentonite alone. As well, the pIOBP was not able to remove any additional MPs from Chardonnay juice pH 3.5 above that already removed by the bentonite and filtration alone. The mouse MUP was shown to be a better candidate protein for removal of MPs from juice using this system.

The osmoadaptive response of the wine yeast Saccharomyces cerevisiae K1-V1116 during icewine fermentation

The adapted metabolic response of commercial wine yeast under prolonged exposure to concentrated solutes present in Icewine juice is not fully understood. Presently, there is no information regarding the transcriptomic changes in gene expression associated with the adaptive stress response ofwine yeast during Icewine fermentation compared to table wine fermentation. To understand how and why wine yeast respond differently at the genomic level and ultimately at the metabolic level during Icewine fermentation, the focus ofthis project was to identify and compare these differences in the wine yeast Saccharomyces cerevisiae KI-Vll16 using cDNA microarray technology during the first five days of fermentation. Significant differences in yeast gene expression patterns between fermentation conditions were correlated to differences in nutrient utilization and metabolite production. Sugar consumption, nitrogen usage and metabolite levels were measured using enzyme assays and HPLC. Also, a small subset of differentially expressed genes was verified using Northern analysis. The high osmotic stress experienced by wine yeast throughout Icewine fermentation elicited changes in cell growth and metabolism correlating to several fermentation difficulties, including reduced biomass accumulation and fermentation rate. Genes associated with carbohydrate and nitrogen transport and metabolism were expressed at lower levels in Icewine juice fermenting cells compared to dilute juice fermenting cells. Osmotic stress, not nutrient availability during Icewine fermentation appears to impede sugar and nitrogen utilization. Previous studies have established that glycerol and acetic acid production are increased in yeast during Icewine fermentation. A gene encoding for a glycerollW symporter (STL1) was found to be highly expressed up to 25-fold in the i Icewine juice condition using microarray and Northern analysis. Active glycerol transport by yeast under hyperosmotic conditions to increase cytosolic glycerol concentration may contribute to reduced cell growth observed in the Icewine juice condition. Additionally, genes encoding for two acetyl CoA synthetase isoforms (ACSl and ACS2) were found to be highly expressed, 19- and II-fold respectively, in dilute juice fermenting cells relative to the Icewine juice condition. Therefore, decreased conversion of acetate to acetyl-CoA may contribute to increased acetic acid production during Icewine fermentation. These results further help to explain the response of wine yeast as they adapt to Icewine juice fermentation. ii

Metabolite changes during Riesling icewine fermentation when yeast micronutrients are used /

Icewine is an intensely s\veet dessert \vine fermented from the juice of naturally frozen grapes. Icewine fermentation poses many challenges such as failure to reach desired ethanol levels and production of high levels of volatile acidity in the fonn of acetic acid. This study investigated the impact of micronutrient addition (GO-FERM® and NATSTEP®) during the rehydration stage of the commercial \vine yeast Saccharomyces cerevisiae KI-VIII6 during Ice\vine fermentation. Sterile-filtered and unfiltered Riesling Ice\vine juice was inoculated \vith yeast rehydrated under four different conditions: in water only; with GO-FERM®; with NATSTEP®; or the combination of both micronutrient products in the rehydration water. Using sterile-filtered Icewine juice, yeast rehydration had a positive impact of reducing the rate of acetic acid produced as a function of sugar consumed, reducing the ratio of acetic acid/ethanol and reducing the ratio of acetic acid/glycerol. In the sterile-filtered fermentation, yeast rehydrated with micronutrients generated 9-times less acetic acid per gram of sugar in the first 48 hours compared to yeast rehydrated only \vith water and resulted in a 17% reduction in acetic acid in the final \vine \vhen normalized to sugar consumed. However, the sterile-filtered fermentations likely became stuck due to the overc1arification of the juice as evidenced from the low sugar consumption (117 gIL) that could not be completely overcome by the micronutrient treatments (144 gIL sugar consumed) to reach a target ethanol of IO%v/v. Contrary to \vhat \vas observed in the sterile-filtered treatements, using unfiltered Ice\vine juice, yeast micronutrient addition had no significant impact of reducing the rate of acetic acid produced as a function of sugar consumed, reducing the ratio of acetic acid/ethanol and reducing the ratio of acetic acid/glycerol. However, in the unfiltered fermentation, micronutrient addition during yeast rehydration caused a reduction in the acetic acid produced as a function of sugar consumed up to 150 giL sugar consumed.. In contrast to the sterile-filtered fermentations, the unfiltered fermentations did not become stuck as evidenced from the higher sugar consumption (l47-174g1L). The largest effects of micronutrient addition are evident in the first two days of both sterile and unfiltered fermentations.

The role of Stl1p in glycerol accumulation in osmotically stressed icewine yeast Saccharomyces cerevisiae K1 V1116

The high sugar concentration in Icewine juice exerts hyperosmotic stress in the wine yeast causing water loss and cell shrinkage. To counteract the dehydration, yeast synthesize and accumulate glycerol as an internal osmolyte. In a laboratory strain of S. cerevisiae, STLl encodes for Stllp, an H+ /glycerol symporter that is glucose inactivated, but induced upon hyperosmotic stress. STLl, was found to be a highly upregulated gene in Icewine fermenting cells and its expression was 25-fold greater than in yeast cells fermenting diluted Icewine juice, making it one of the most differentially expressed genes between the two fermentation conditions. In addition, Icewine fermenting cells showed a two-fold higher glycerol production in the wine compared to yeast fermenting diluted Icewine juice. We proposed that Stllp is (1) active during Icewine fermentation and is not glucose inactivated and (2) its activity contributes to the limited cell growth observed during Icewine fermentation as a result of the dissipation of the plasma membrane proton gradient. To measure the contribution ofStl1p in active glycerol transport (energy dependent) during Icewine fermentation, we first developed an Stllp-dependent (14C]glycerol uptake assay using a laboratory strain of S. cerevisiae (BY 4742 and LiSTLl) that was dependent on the plasma membrane proton gradient and therefore energy-dependent. Wine yeast K1-Vll16 was also shown to have this energy dependent glycerol uptake induced under salt stress. The expression of STLl and Stllp activity were compared between yeast cells harvested from Icewine and diluted Icewine fermentations. Northern blot analysis revealed that STLl was expressed in cells fermenting Icewine juice but not expressed under the diluted juice conditions. Glycerol uptake by cells fermenting Icewine juice was not significantly different than cells fermenting diluted Icewine juice on day 4 and day 7 of Vidal and Riesling fermentations respectively, despite encountering greater hyperosmotic stress. Furthermore, energy- dependent glycerol uptake was not detected under either fermentation conditions. Because our findings show that active glycerol uptake was not detected in yeast cells harvested from Icewine fermentation, it is likely that Stllp was glucose inactivated despite the hyperosmotic stress induced by the Icewine juice and therefore did not play a role in active glycerol uptake during Icewine fermentation.

Ontario Tender Fruit Grower’s Marketing Board Fonds, 1960, 1967, 1969-1977, 1980-1981, 1987

The Ontario Tender Fruit Marketing Board operates under the Farm Producers Marketing Act. It covers all tender fruit farmers who produce either fresh or canned products. Today the board has over 500 grower-members. Tender fruit in the Niagara region includes: peaches, pears, plums, grapes and cherries. The fruits are used in a number of different ways, from jams and jellies to desserts, sauces and wine. Peaches were first harvested along the Niagara river in 1779. Peter Secord (Laura Secord’s uncle) is thought to be the first farmer to plant fruit trees when he took a land grant near Niagara in the mid 1780s. Since the beginnings of Secord’s farm, peaches, pears and plums have been grown in the Niagara region ever since. However, none of the original varities of peach trees remain today. Peaches were often used for more than eating by early settlers. The leaves and bark of the tree was used to make teas for conditions such as chronic bronchitis, coughs and gastritis. Cherries have been known to have anti-inflammatory and pain relieving properties. Like peaches and cherries, pears had many uses for the early pioneers. The wood was used to make furniture. The juice made excellent ciders and the leaves provided yellow dyes. Plums have been around for centuries, not only in the Niagara region, but throughout the world. They have appeared in pre-historic writings and were present for the first Thanksgiving in 1621. The grape industry in Ontario has also been around for centuries. It began in 1798 when land was granted to Major David Secord (brother-in-law to Laura Secord) slightly east of St. David’s, on what is Highway No. 8 today. Major Secord’s son James was given a part of the land in 1818 and in 1857 passed it onto Porter Adams. Adams is known to be the first person to plant grapes in Ontario1. Tender fruits are best grown in warm temperate climates. The Niagara fruit belt, stretching 65km from Hamilton to Niagara on the Lake, provides the climate necessary for this fruit production. This belt produces 90% of Ontario’s annual tender fruit crop. It is one of the largest fruit producing regions in all of Canada.

Study of new yeast strains as novel starter cultures for Riesling icewine production

Icewine is a sweet dessert wine fermented from the juice of grapes naturally frozen on the vine. The production of Icewine faces many challenges such as sluggish fermentation, which often yields wines with low ethanol, and an accumulation of high concentration of volatile acidity, mainly in the form of acetic acid. This project investigated three new yeast strains as novel starter cultures for Icewine fermentation with particular emphasis on reducing acetic acid production: a naturally occurring strain of S. bayanus/S. pastorianus isolated from Icewine grapes, and two hybrids between S. cerevisiae and S. bayanus, AWRI 1571 and AWRI 1572. These strains were evaluated for sugar consumption patterns and metabolic production of ethanol, glycerol and acetic acid, and were compared to the performance of a standard commercial wine yeast KI-VI116. The ITS rONA region of the two A WRI crosses was also analyzed during fermentations to assess their genomic stability. Icewine fermentations were performed in sterile filtered juice, in the absence of indigenous microflora, and also in unfiltered juice in order to mirror commercial wine making practices. The hybrid A WRI 1572 was found to be a promising candidate as a novel starter culture for Icewine production. I t produced 10.3 % v/v of ethanol in sterile Riesling Icewine fermentations and 11.2 % v/v in the unfiltered ones within a reasonable fermentation time (39 days). Its acetic acid production per gram sugar consumed was approximately 30% lower in comparison with commercial wine yeast K I -V 1116 under both sterile filtered and unfiltered fermentations. The natural isolate S. bayanus/S. pastorianus and AWRI 1571 did not appear to be suitable for commercial Icewine production. They reached the target ethanol concentration of approximately 10 % v/v in 39 day fermentations and also produced less acetic acid as a function of both time and sugar consumed in sterile fermentations compared to KI-V1116. However, in unfiltered fermentations, both of them failed to produce the target concentration of ethanol and accumulated high concentration of acetic acid. Both A WRI crosses displayed higher loss of or reduced copies in ITS rDNA region from the S. bayanus parent compared to the S. cerevisiae parent; however, these genomic losses could not be related to the metabolic profile.

Icewine fermentation by Saccharomyces cervisiae : fundamental stress responses and comparative fermentation dynamics

Please consult the paper edition of this thesis to read. It is available on the 5th Floor of the Library at Call Number: Z 9999.5 B63 P54 2007