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 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.

Functional genomics of O-glucosyltransferases from Concord grape (Vitis labrusca)

Grape (Vitis spp.) is a culturally and economically important crop plant that has been cultivated for thousands of years, primarily for the production of wine. Grape berries accumulate a myriad of phenylpropanoid secondary metabolites, many of which are glucosylated in plantae More than 90 O-glucosyltransferases have been cloned and biochemically characterized from plants, only two of which have been isolated from Vitis spp. The world-wide economic importance of grapes as a crop plant, the human health benefits associated with increased consumption of grape-derived metabolites, the biological relevance of glucosylation, and the lack of information about Vitis glucosyltransferases has inspired the identification, cloning and biochemical characterization of five novel "family 1" O-glucosyltransferases from Concord grape (Vitis labrusca cv. Concord). Protein purification and associated protein sequencIng led to the molecular cloning of UDP-glucose: resveratrollhydroxycinnamic acid O-glucosyltransferase (VLRSGT) from Vitis labrusca berry mesocarp tissue. In addition to being the first glucosyltransferase which accepts trans-resveratrol as a substrate to be characterized in vitro, the recombinant VLRSGT preferentially produces the glucose esters of hydroxycinnamic acids at pH 6.0, and the glucosides of trans-resveratrol and flavonols at 'pH 9.0; the first demonstration of pH-dependent bifunctional glucosylation for this class of enzymes. Gene expression and metabolite profiling support a role for this enzyme in the bifuncitonal glucosylation ofstilbenes and hydroxycinnamic acids in plantae A homology-based approach to cloning was used to identify three enzymes from the Vitis vinifera TIGR grape gene index which had high levels of protein sequence iii identity to previously characterized UDP-glucose: anthocyanin 5-0-glucosyltransferases. Molecular cloning and biochemical characterization demonstrated that these enzymes (rVLOGTl, rVLOGT2, rVLOGT3) glucosylate the 7-0-position of flavonols and the xenobiotic 2,4,5-trichlorophenol (TCP), but not anthocyanins. Variable gene expression throughout grape berry development and enzyme assays with native grape berry protein are consistent with a role for these enzymes in the glucosylation of flavonols; while the broad substrate specificity, the ability of these enzymes to glucosylate TCP and expression of these genes in tissues which are subject to pathogen attack (berry, flower, bud) is consistent with a role for these genes in the plant defense response. Additionally, the Vitis labrusca UDP-glucose: flavonoid 3-0-glucosyltransferase (VL3GT) was identified, cloned and characterized. VL3GT has 96 % protein sequence identity to the previously characterized Vitis vinifera flavonoid 3-0-glucosyltransferase (VV3GT); and glucosylates the 3-0-position of anthocyanidins and flavonols in vitro. Despite high levels of protein sequence identity, VL3GT has distinct biochemical characteristics (as compared to VV3GT), including a preference for B-ring methylated flavonoids and the inability to use UDP-galactose as a donor substrate. RT-PCR analysis of VL3GT gene expression and enzyme assays with native grape protein is consistent with an in planta role for this enzyme in the glucosylation of anthocyanidins,but not flavonols. These studies reveal the power of combining several biochemistry- and molecular biology-based tools to identify, clone, biochemically characterize and elucidate the in planta function of several biologically relevant O-glucosyltransferases from Vitis spp.

The effect of pH and ethanol on the astringent sub-qualities of red wine, and the implications for optimum grape maturity /

The first objective of this study was to identify appropriate sensory descriptors to assess the astringent sub-qualities of red wine. The influence of pH and ethanol on the sensation of astringency in red wine was evaluated, using a de-alcoholized red wine. A portion of the wine was adjusted to the pH values of 3.2, 3.4, 3.6 and 3.8, and another portion was adjusted to ethanol concentrations of 0%, 6%, 12%, and 15%. In addition, the pH 3.4 and 3.6 treatments were adjusted to an ethanol concentration of 12% and 15% all wines were then assessed sensorially and seventeen terms were identified, through panel discussion, to describe the mouth-feel and taste qualities: velvet, aggressive, silk/satin, dry, fleshy, unripe, pucker viscosity, abrasive, heat, chewy, acidity, grippy/adhesive, bitter, balance, overall astringency, and mouth-coat. Descriptive analysis profiling techniques were used to train the panel and measure the intensity of these attributes. It was found that decreasing pH values (averaged across all ethanol concentrations) showed an increase in the overall astringency of the wine. The combined treatments of ethanol and pH, real wine parameters (pH 3.4 and 3.6; 12% and 15% ethanol) did not have an effect on the perception of the astringent sub-qualities of the wine. A time intensity study was also included using the pH and ethanol adjusted wines, which showed that as the ethanol level of the wines increased so did the time to maximum intensity. The second objective was to identify appropriate sensory descriptors to evaluate the influence of grape maturity and maceration technique (grape skin contact) on the astringency sub-qualities of red vinifera wines from Niagara. The grapes were harvested across two dates, representing an early harvest and a late harvest. A portion of the Cabernet Sauvignon grapes wine was divided into three maceration treatments of oneweek maceration, standard two-week maceration, three-week maceration, and MCM. Another portion of both the early and late harvest Cabernet Sauvignon grapes were chaptalized to yield a final ethanol concentration of 14.5%. The wines were assessed sensorially and thirteen terms were identified, through panel discussion, to describe the mouth-feel and taste qualities: carbon dioxide, pucker, acidity, silk/chamois, dusty/chalky/powdery, sandpaper, numbing, grippy/adhesive, dry, mouthcoat, bitter, balance and, overall astringency. Descriptive analysis techniques were used to train the panel and measure the intensity of these attributes. The data revealed few significant differences in the mouth-feel of the wines with respect to maturity; which included differences in overall astringency and balance. There were varietal differences between Cabernet Sauvignon, Cabernet Franc, and Pinot Noir and differences for Cabernet Sauvignon wines due to the length and manner of maceration and as a result of chaptalization. Statistical analysis revealed a more complex mouth-feel for the Pinot Noir wines; and an increase in the intensity of the astringent sub-qualities as a result of the addition of sugar to the wines. These findings have implications for how processing decisions, such as optimum grape maturity and vinification methods may affect red wine quality.

Niagara Grape and Wine Festival Records 1955, 1964-1991, n.d.

Ontario Editorial Bureau (O.E.B.)

Ontario Grape Growers Marketing Board Records 1947, 1968-1993, n.d.

Ontario Editorial Bureau (O.E.B.)

A case study investigation of the learning needs of the Niagara grape and wine community

The Niagara Grape and Wine Community (NGWC) is an industry that has undergone rapid change and expansion as a result of changes in governmental regulations and consumer preferences. As a result of these changes, the demands of the wine industry workforce have changed to reflect the need to implement new strategies and practices to remain viable and competitive. The influx of people into the community with little or no prior practical experience in grape growing (viticulture) or winemaking (oenology) has created a need for additional training and learning opportunities to meet workforce needs. This case study investigated the learning needs of the members of this community and how these needs are currently being met. The barriers to, and the opportunities for, members acquiring new knowledge and developing skills were also explored. Participants were those involved in all levels of the industry and sectors (viticulture, processing, and retail), and their views on needs and suggestions for programs of study were collected. Through cross analyses of sectors, areas of common and unique interest were identified as well as formats for delivery. A common fundamental component was identified by all sectors - any program must have a significant applied component or demonstration of proficiency and should utilize members as peer instructors, mentors, and collaborators to generate a larger shared collective of knowledge. Through the review of learning organizations, learning communities, communities of practices, and learning networks, the principles for the development of a Grape and Wine Learning Network to meet the learning needs of the NGWC outside of formal institutional or academic programs were developed. The roles and actions of members to make such a network successful are suggested.

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.

Impact of different irrigation strategies on grapes and wine quality of four grapevine cultivars (Vitis sp.) in cool climate conditions. An investigation into the relationships among ABA, water status, grape cultivar and wine quality

Niagara Peninsula of Ontario is the largest viticultural area in Canada. Although it is considered to be a cool and wet region, in the last decade many water stress events occurred during the growing seasons with negative effects on grape and wine quality. This study was initiated to understand and develop the best strategies for water management in vineyards and those that might contribute to grape maturity advancement. The irrigation trials investigated the impact of time of initiation (fruit set, lag phase and veraison), water replacement level based on theoretical loss through crop evapotranspiration (ETc; 100,50 and 25%) and different irrigation strategies [partial root zone drying (PRD) versus regulated deficit irrigation (RD!)] on grape composition and wine sensory profiles. The irrigation experiments were conducted in a commercial vineyard (Lambert Vineyards Inc.) located in Niagara-on-the-Lake, Ontario, from 2005 through 2009. The two experiments that tested the combination of different water regimes and irrigation time initiation were set up in a randomized block design as follows: Baco noir - three replicates x 10 treatments [(25%, 50% and 100% of ETc) x (initiation at fruit set, lag phase and veraison) + control]; Chardonnay - three replicates x seven treatments [(25%, 50% and 100% of ETc) x (initiation at fruit set and veraison) + control]. The experiments that tested different irrigation strategies were set up on two cultivars as follows: Sauvignon blanc - four replicates x four treatments [control, fully irrigated (100% ETc), PRD (100% ETc) and RDI (25% ETc)]; Cabemet Sauvignon - four replicates x five treatments [control, fully irrigated (100% ETc), PRD (100% ETc), RDI (50% ETc) and RDI (25% ETc)]. The controls in each experiment were nonirrigated. The irrigation treatments were compared for many variables related to soil water status, vine physiology, berry composition, wine sensory profile, and hormone composition [(abscisic acid (ABA) and its catabolites]. Soil moisture profile was mostly affected by irrigation treatments between 20 and 60 em depth depending on the grapevine cultivar and the regime of water applied. Overall soil moisture was consistently higher throughout the season in 100 and 50% ETc compare to the control. Transpiration rates and leaf temperature as well as shoot growth rate were the most sensitive variables to soil water status. Drip irrigation associated with RDI treatments (50% ETc and 25% ETc) had the most beneficial effects on vine physiology, fruit composition and wine varietal typicity, mainly by maintaining a balance between vegetative and reproductive parts of the vine. Neither the control nor the 100 ETc had overall a positive effect on grape composition and wine sensory typicity. The time of irrigation initiation affected the vine physiology and grape quality, the most positive effect was found in treatments initiated at lag phase and veraison. RDI treatments were overall more consistent in their positive effect on grape composition and wine varietal typicity comparing to PRD treatment. The greatest difference between non-irrigated and irrigated vines in most of the variables studied was found in 2007, the driest and hottest season of the experimental period. Soil water status had a greater and more consistent effect on red grapevine cultivars rather than on white winegrape cultivars. To understand the relationships among soil and plant water status, plant physiology and the hormonal profiles associated with it, abscisic acid (ABA) and its catabolites [phaseic acid (PA), dihydrophaseic acid (DPA), 7-hydroxy-ABA (TOH-ABA), 8' -hydroxy-ABA, neophaseic acid and abscisic acid glucose ester (ABA-GE)] were analyzed in leaves and berries from the Baco noir and Chardonnay irrigation trials over two growing seasons. ABA and some of its catabolites accurately described the water status in the vines. Endogenous ABA and some of its catabolites were strongly affected in Baco noir and Chardonnay by both the water regime (i.e. ET level) and timing of irrigation initiation. Chardonnay grapevines produced less ABA in both leaves and berries compared to Baco noir, which indicated that ABA synthesis is also cultivar dependant. ABA-GE was the main catabolite in treatments with high water deficits, while PA and DPA were higher in treatments with high water status, suggesting that the vine produced more ABA-GE under water deficits to maintain rapid control of the stomata. These differences between irrigation treatments with respect to ABA and catabolites were particularly noticeable in the dry 2007 season. Two trials using exogenous ABA investigated the effect of different concentrations of ABA and organs targeted for spraying, on grape maturation and berry composition of Cabemet Sauvignon grapevines, in two cool and wet seasons (2008-2009). The fIrst experiment consisted of three replicates x three treatments [(150 and 300 mg/L, both applications only on clusters) + untreated control] while the second experiment consisted in three replicates x four treatments [(full canopy, only clusters, and only leaves sprayed with 300 ppm ABA) + untreated control]. Exogenous ABA was effective in hastening veraison, and improving the composition of Cabemet Sauvignon. Ability of ABA to control the timing of grape berry maturation was dependant on both solution concentration and the target organ. ABA affected not only fruit composition but also yield components. Berries treated with ABA had lower weight and higher skin dry mass, which constitutes qualitative aspects desired in the wine grapes. Temporal advancement of ripening through hormonal control can lead to earlier fruit maturation, which is a distinct advantage in cooler areas or areas with a high risk of early frost occurrence. Exogenous ABA could provide considerable benefits to wine industry in terms of grape composition, wine style and schedule activities in the winery, particularly in wet and cool years. These trials provide the ftrst comprehensive data in eastern North America on the response of important hybrid and Vitis vinifera winegrape cultivars to irrigation management. Results from this study additionally might be a forward step in understanding the ABA metabolism, and its relationship with water status. Future research should be focused on ftnding the ABA threshold required to trigger the ripening process, and how this process could be controlled in cool climates.

Innovation and adaptation in the Ontario grape and wine industry: An integrated, transdisciplinary response to climate change

With scientific consensus supporting a 4oC increase in global mean temperature over the next century and increased frequency of severe weather events, adaptation to climate change is critical. Given the dynamic and complex nature of climate change, a transdisciplinary approach toward adaptation can create an environment that supports knowledge sharing and innovation, improving existing strategies and creating new ones. The Ontario wine industry provides a case study to illustrate the benefits of this approach. We describe the formation and work of the Ontario Grape and Wine Research Network within this context, and present some preliminary results to highlight the opportunities for innovation that will drive the successful adaption of the Ontario grape and wine industry.

Functional genomics of BAHD acyltransferases in different varieties of the wine grape, Vilis vinifera

The characteristic "foxy" aroma of Vilis labrusca Concord grapes is due in large part to methyl anthranilate, a volatile ester formed by the enzyme anthraniloyl- CoA:methanol anthraniloyltransferase (VIAMAT) of the superfamily of BARD acyltransferases. The publication of the genome ofthe closely related wine grape Vilis vinifera, which does not accumulate methyl anthranilate, permitted the searching for any putative VlAU4T-like genes, with the result of 5 highly homologous candidates being found, with one candidate sharing 95% identity to VlAU4T. Probing the gene expression of 18 different cultivars of V. vinifora ripe berries by RT -PCR showed that many varieties do indeed express VlAU4T-like genes. Subsequent cloning of the full-length open reading frame of one of these genes from eDNA prepared from the cultivar Sauvignon Blanc permitted preliminary biochemical characterization of the enzyme after heterologous expression in E. coli. It was determined that this alcohol acyltransferase (named VvsbAATl) catalyzes the formation of cis-3-hexenyl acetate, a "green-leaf' volatile. Although the cloned gene from Sauvignon Blanc had 95% identity at the amino acid level to VIAMAT, it displayed an altered substrate specificity and expression pattern. These results highlight the difficulty in predicting substrate specificity and function of enzymes through the basis of sequence homology, which is a common finding in the study of BARD acyltransferases. Also, the determination of function of VvsbAATl and other BARD acyltransferases in V. vinifera could be used as a genetic marker for certain aroma characteristics in grape breeding programs.

IMPACT OF CROP LEVEL AND HANG TIME ON THE COMPOSITION OF FOUR WINE GRAPE CULTIVARS FROM THE NIAGARA REGION

This study analyzed the use of two viticultural practices: “crop level” (half crop; HC, and full crop; FC) and “hang times”, and their impact on the composition of four grape cultivars; Pinot gris, Riesling, Cabernet Franc and Cabernet Sauvignon from the Niagara Region and wine volatile composition by GC-MS. It was hypothesized that keeping a full crop with a longer hang time would have a greater impact on wine quality than reducing the crop level. In all cultivars, a reduction of crop level induced reductions in yield, clusters per vine and crop load, with increases in Brix. Extended hang time also increased Brix related to desiccation. The climatic conditions at harvest had an impact on hang time effects. The GC-MS analysis detected the presence of 30 volatile components in the wine, with different odour activity values. Harvest time had a positive impact than crop reduction in almost all compounds.

Illustration - "Grape Culture - A new Source of Canadian Wealth"

An illustration from the "Canadian Illustrated News" that shows a group of individuals "gathering the fruit" and then a "scene in the vineyard".

Letters Patent Incorporating The Ontario Grape Growing and Wine Manufacturing Company

The patent incorporates the Ontario Grape Growing and Wine Manufacturing Company for the purpose "for the manufacture of wine within the province of Ontario" and is recorded as no. 85 on June 9, 1873.