Anthocyanin-Cell Wall Interactions Effect on Tannin Extraction

The objectives of this proposal have been to do the following:

1) Perform extractions on white grape skins at 5 time points (13,17,20,22, and 25 °Brix) throughout maturity with varying levels of added anthocyanins.

• Extracted tannin will be analyzed for concentration, average molecular size, subunit composition, activity, and fraction extracted.
• Measure the levels of pectin methylation in the white grape skins

2) Conduct red-styled fermentations on Sauvignon blanc and Cabernet Sauvignon at 300 pounds per fermentation, with and without an anthocyanin addition. Measurements are the same as Objective 1.

The overall purpose of this proposal is to determine anthocyanins role on the extraction of procyanidin material due to interactions with cell wall material of grape skins. Activity to date has been the isolation and purification of color along with the sampling and extraction of skins with varying concentration of anthocyanins added. Furthermore, 300lb fermentations were conducted, in triplicate, under standard red wine making conditions with both Sauvignon Blanc and Cabernet Sauvignon with a 1.4% (v/v) addition of color concentrate.

Investigating Fruitiness Perception in Red and White Wines

This report details activities that occurred from February 2018 – January 2019. The final date of this project is August 2019 and the next 6 months will include completing the last of the sensory panels and combining all data analysis. A final report will be submitted in January 2020. We are still slightly behind on the timeline due to issues detailed in last year’s report and we also had to renew our IRB (human ethics approval) in June 2018. Once the renewal is submitted it is illegal to run sensory tests on the project until approval is given, which was obtained in September 2018. In January 2019 we completed the last of the Pinot noir sensory panels, although we have not yet done data analysis on the January 2019 panel. Also as stated previously, we have not been able to complete any predictive modeling, some initial reviewer comments said that this objective might have been too ambitious in the timeline and after the 1st year we have to agree and have since removed this objective. We plan on working on predictive modeling in the future but this would be after the current grant is completed.

To date we have investigated 80 different compound combinations and their impact to fruit aroma in Pinot noir wine. We have also completed a panel that shows the influences of phenolic content on fruity aromas in Pinot noir and one panel that shows the impact of ethanol content on fruity aroma in Pinot noir. We have 2 potential marker compounds for red fruit aroma in Pinot noir and 4 red fruit solution sets using fsQCA that show the cause of red fruit aroma in Pinot noir. We have also found 5 solution sets for dark fruit aroma in Pinot noir using fsQCA.

We have also investigated 49 compound combinations for fruitiness in white wine. We are still working on using fsQCA to analyze this data. Preliminary results suggest a combination of low thiols and high esters are responsible for tropical fruit aromas, low to no esters are needed for citrus aromas, and esters and terpenes cause pear, peach and apricot aromas.

We will be running the last 4 Pinot gris sensory panels from February2019-June 2019 and completing the final data analysis. We are in the process of writing the first paper for publication and have done 2 presentations at domestic conferences on the analytical data analysis. Spring /Summer 2019 we will be presenting at 4 different international conferences in Europe and have plans for at least 3 more peer-reviewed publications.

Effect of grapevine red blotch disease (GRBD) on flavor and flavor precursor formation in the grape and on wine quality

Two field experiments were established to investigate the effects of grapevine red blotch disease (GRBD) on flavor and flavor precursor formation in the grape berry and on resulting wine quality. The two objectives of the overall study were to 1) investigate the effect of GRBD on grape berry development with a specific focus on flavor and flavor precursor formation; and to 2) investigate the effect of GRBD on wine quality. Both experiments were located in the same vineyard located near the town of Jacksonville, OR. In both experiments, data vines were identified by visual disease symptoms (or lack thereof), and disease status was confirmed using PCR-based assays in Dr. Achala KC’s laboratory at SOREC.
To evaluate the response of flavor and flavor precursor compounds to GRBV infection during berry development (objective 1), clusters from GRBV+ and GRBV- vines were sampled weekly beginning from just before veraison through to commercial harvest. Vine water status, berry growth, and development were also monitored in those plots subjected to different irrigation treatments. Vine water status was monitored by measurements of midday stem water potential (Ψstem). Results showed that there was no significant interaction between irrigation treatment and disease status on Ψstem. However, there were significant effects of irrigation treatment and disease status on Ψstem independently. Berry size (fresh weight; FW) was consistently higher in GRBV+ vines, significant differences in TSS between GRBV+ and GRBV- vines were observed. There were no significant differences in berry pH between vines of different disease status over the entire course of berry development. Berry titratable acidity (TA; g L-1) were lower in GRBV+ fruit. These responses were only observed after veraison, but they are not as consistent. Flavor and flavor precursor analysis in the grapes is underway.
To evaluate the response of wine quality to GRBV infection (Objective 2), replicate wines were produced from field plots under the supervision of Dr. James Osborne using a standard protocol.
Wines were analyzed for volatile aroma compounds using different techniques including headspace-GC-FID, solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS) and stir bar sorptive extraction-gas chromatography-mass spectrometry method (SBSE-GC-MS), stale-isotope compounds were used as internal standards for accurate analysis. Monomeric anthocyanin and total phenolic contents in wine were also analyzed. Results showed that the RB negative wines under irrigation condition have the highest level of monomeric anthocyanin than other three groups. Total phenolic content varies in wines with different irrigation conditions. Fermentation derived aroma compounds did not show any difference between RB+ and RB-, nor free form grape-derived aroma compounds. Since the free form of grape derived aroma compounds only exist in a small portion, and the majority of these compounds exist in the bound form, analysis is underway for the bound form of volatile flavor compounds in the wine.

Utilizing malolactic fermentation as a tool to prevent Brettanomyces bruxellensis wine spoilage

Brettanomyces bruxellensis is considered one of the most problematic wine spoilage yeasts due to the difficulty of controlling it, the potential significant financial losses due to loss of wine quality, and the cost of prevention and remediation measures. Wine is particularly vulnerable to B. bruxellensis infection during and shortly after the malolactic fermentation (MLF) as SO2 cannot be added until this process is complete. It has been suggested that conducting a rapid MLF initiated by inoculation of Oenococcus oeni is a useful strategy to prevent B. bruxellensis spoilage as this minimizes the length of time the wine is not protected by SO2. This project investigates an additional benefit of conducting a rapid MLF, the prevention of B. bruxellensis growth due to inhibitory interactions with O. oeni. Pinot noir wine (no SO2 additions, no MLF) was produced and used to test the ability of a number of commercial O. oeni strains to inhibit B. bruxellensis growth at the end of MLF. Sterile filtered wine was inoculated with various O. oeni strains and growth and malic acid monitored. When MLF is complete, wines will be inoculated with a select strain of B. bruxellensis and growth and volatile phenol production monitored.

The sensitivity of a number of B. bruxellensis strains to O. oeni is also being determined. B. bruxellensis strains have been sourced representing B. bruxellensis isolates from a wide range of winemaking regions including Oregon. A model wine system was identified for use to improve the rate that B. bruxellensis strains can be tested for inhibition by O. oeni. Results from the model wine system will be used to select which strains will be used in wine experiments that take significantly longer to complete.

Investigation of different amelioration techniques to remove smoke taint character from wine

Research regarding smoke taint has mostly been undertaken in Australia with a focus on vine susceptibility, potential mitigation actions during winemaking to limit smoke taint expression and potential ways to remove smoke taint in the final wines. Thorough review of published smoke taint research indicated large gaps in knowledge and inconsistent results. The objective of the proposed research is to compare all the suggested amelioration techniques using the same wine and follow the changes in free and bound smoke taint compounds before and after treatment as well as with wine aging up to one year. Results from this study will enable us to better advice the wine industry during future smoke events. SPME-GC-MS and UPLC-Q-TOF-MS methods employing stable isotope dilution methodology (SID) have been implemented. Smoke-exposed Cabernet Sauvignon wine was made from Oakville Experimental Station fruit. Wines were treated for one to six weeks with a range of different enzymes (Lafazym AROM, Lyvarome A5, Sumizyme BGA and Zimarom) at two different addition levels (2 and 4 g/hL). Control and enzyme-treated wines (those showing elevated volatile phenols) will be treated with activated charcoal fining, reverse osmosis, Conetech smoke removal technology and molecular imprinted polymers. Those treatments showing a significant decrease in free and/or bound volatile phenols will be evaluated by descriptive analysis.

Investigation of the efficacy of winery cleaning and sanitization chemistries

This research focused on optimizing cleaner and sanitizer concentration and contact time for several different chemicals and spoilage microorganisms relevant to the wine industry. Minimum inhibitory concentration (MIC) and minimum biocidal concentration (MBC) assays were performed, which expose the microbes to dilution series of antimicrobial agents to determine at which concentration different species are either inhibited (MIC) or inactivated (MBC) by exposure to the antimicrobial. As the MIC/MBC assay involves contact times for the microorganisms that are greater than would be reasonable for the wine industry (24 hrs), fluorescence spectroscopy was employed to provide complementary kinetic inactivation data. Peracetic acid was used at several different concentrations to determine the minimum contact time for inactivating S. cerevisiae cells in suspension. In a similar experimental design as the MIC/MBC assay, a minimum biofilm eradicating concentration (MBEC) assay was employed to assess whether sessile communities would require elevated concentrations in order to inactivate or remove the biofilm populations from the microtiter plates. While many of the chemicals did require higher concentrations to inactivate sessile communities, cleaners that contained surfactants and other detergents were effective at lower concentrations, possibly due to the fact that they physically removed the biofilm from the well plate regardless of whether the cells were inactivated. In combination with previous research efforts (Final Report 2017_2123) these results were used to develop an optimized cleaning and sanitation framework for assessment in the winery at the pilot scale (2000 L), which were assessed using ATP swabbing and traditional plate counts. Results from those trials indicate that cleaning and sanitizing contact times are less important beyond 5-minute exposure than proper attention to critical control points in the shadow of spray balls or mechanical agitation. Worker diligence in manually addressing and cleaning these sensitive areas may have a greater impact in cleaning and sanitizing success than increasing contact time several fold.

Winery cleaning and sanitization, monitoring methodology and efficacy of cleaning and sanitization chemistries

This project analyzed the ability of cleaners and sanitizers frequently used in the wine industry to inactivate microbial populations in solution (planktonic) and stationary (biofilm) physiologies. A screening of 20 different cleaner and sanitizer chemistries was conducted using 96-well plates and the crystal violet method. Next, biofilms were grown on 304 stainless steel coupons by incubating the coupons in an inoculated grape juice medium. These coupons were treated with chemicals at varying concentrations and contact times and then swabbed with ATP luminescence swabs and traditional plate count swabs to determine the microbial load and soil after treatment. Further trials were then conducted in the UC Davis teaching winery facility at the 200 L and 2000 L scale. For the 200 L fermentations, a custom device was constructed to prove 110 replicate soiled coupons that could be used for further treatment in the laboratory setting. These trials allowed for the development of an optimized protocol that could be tested against other similar treatments at the 2000 L-scale. For these larger scale trials, a five-step cleaning and sanitizing framework was employed, and again ATP and traditional plate count data were collected. The results of these experiments show that the vulnerable areas of tanks (gaskets and areas in the shadow of spray arms) have consistent microbial contamination, regardless of the cleaning protocol or contact time. These need to be areas of focus in any cleaning and sanitation protocol, and winemakers must be prudent to develop a system that exceeds the typical visual inspection protocol often employed in the winery environment.

Characterization of Bitter and Astringent Proanthocyanidins during Winemaking

Polyphenols, including proanthocyanidins (i.e., tannins), are widely distributed in foods and
beverages, including grapes and wines and they are key constituents impacting bitter and
astringent perception. Due, at least in part, to their chemical complexity, the changes in
proanthocyanidin concentration and chemical structure that occur during winemaking and that
impact sensory properties have not been fully evaluated.

We have completed development and validation of an ultra-high performance liquid
chromatography quadrupole time-of-flight mass spectrometry (UHPLC-qTOF MS) approach to
characterize the subunit composition and molecular weight/average degree of polymerization of
wine proanthocyanidins. Wines with different maceration treatments were analyzed and we were
able to demonstrate differences in proanthocyanidin composition as a function of maceration
treatment. This work provides important insight into the impact of maceration treatments on
proanthocyanidin composition of wines.

Characterization of Aroma Volatiles and their Glycosidic Precursors in Grapes and Wines

The complex aroma of wine is derived from many sources, with grape-derived components being
responsible for the varietal character. The ability to monitor grape aroma compounds would allow
for better understanding of how vineyard practices and winemaking processes influence the final
volatile composition of the wine. Previously we developed a procedure using GC-MS combined
with solid-phase microextraction (SPME) for profiling the free volatile compounds in grapes and
wines. We also developed a method for monitoring the ‘aroma potential’ of grapes and wines
without the need for initial isolation of the glycoside precursor fraction. However, this method still
depends on indirect measurement of the glycosides and acid or enzymatic hydrolysis is needed to
release the volatile aglycone which can result in artifact formation. In the current project we
validated a novel method using UHPLC-qTOF MS/MS for direct analysis of intact aroma
glycosides in grapes with minimal artifactual changes in composition. Using this method we
tentatively identified 27 monoterpene glycosides including two monoterpene trisaccharide
glycosides, tentatively identified for the first time in any plant. We measured the terpene
glycosides in six cultivars at three maturity time points and demonstrated differential profiles
depending on cultivar and maturity. We also modified the method so that it can be used to monitor
monoterpene glycosides in wines and during winemaking. We have analyzed the glycoside content
during fermentation for wines made in fall 2016 and 2017 with different varieties (Chardonnay,
Merlot, Cabernet Sauvignon) and winemaking/processing methods. Monoterpenyl glycoside
profiles differed between the grapes and the first alcoholic fermentation samples. In red wines,
malonylated monoterpenol glucosides and monoterpenol hexose-pentoses decreased after the
completion of alcoholic fermentation. We also measured the volatile composition of the wines
during fermentation and we have started to relate changes in terpene volatiles to changes in the
glycoside profiles. This work sheds important insight into possible biochemical changes in
glycosylation during grape berry maturation. In addition, this research will allow us to better
understand the effects of viticultural and winemaking practices on grape and wine components
that affect flavor.

Development of a genome-scale metabolic model for Saccharomyces cerevisiae for use in understanding and modifying strain performance

Two key metabolic activities of yeast relevant to wine fermentations are nutrient utilization efficiency and wine aroma development. For nutrient utilization efficiency (NUE), variability in yeast cell metabolism results from modulation of cellular processes that include changes in membrane composition along with a range of other metabolic pathways that are not fully understood. This variability often affects the completeness of a fermentation (characterized as “dry”, ”sluggish” or ”stuck”). Moreover, variability in yeast species or strains used in wine production results in different concentrations of aroma compounds, which can lead to distinct sensory characteristics. Controlling factors affecting nutrient utilization efficiency and wine aroma profile and mouthfeel characteristics related to yeast requires a detailed understanding of cellular metabolism. To develop such understanding, studies often use large-scale data approaches (e.g. genomics and metabolomics), along with multivariate statistics, to identify key metabolic fluxes or metabolites whose presence favors a specific fermentation outcome.

Although these studies are useful in exploring variation between yeasts, they are often not comprehensive enough, especially considering that they are labor intensive and costly. An alternative method is to use genome-scale metabolic models combined with dynamic FBA (flux balance analysis) to predict the flux distribution of all the metabolites within the cell over the course of an entire fermentation. As a part of this grant, our goal is to show that this computational approach can be used to predict experimental wine fermentation data, to understand differences between commercial strains, and to suggest genetic modification strategies towards increasing strain performance and control aroma characteristics. To date, we have been able to simulate anaerobic, nitrogen-limited yeast fermentations with the latest genome-scale yeast model. Behavior predicted for changing initial nitrogen concentration matches qualitatively with experiment. We simulated fermentation of three commercial yeast strains with highly varied NUE. Utilizing multivariate statistics, we have used the simulation results to identify the metabolic pathways that differ the most between these strains. On first analysis, the results are in agreement with existing experimental data. It is also clear that having an accurate biomass composition will be critical to a good quantitative fit of the data. Therefore, we are currently pursuing measurement of these key parameters as a function of fermentation time and strain.