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.

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.

Development of a prediction tool for phenolic extraction in red wines as a function of winemaking practices and fermentor design

Red wine fermentations are performed in the presence of grape skins and seeds to ensure
extraction of color and other phenolics. The presence of these solids results in two distinct phases
in the fermentor, as the solids float to the top to form a “cap.” Modeling of red wine
fermentations is, therefore, complex and must consider spatial heterogeneity to predict
fermentation kinetics. We have developed a reactor-engineering model for red wine
fermentations that includes the fundamentals of fermentation kinetics, heat transfer, diffusion,
and compressible fluid flow. To develop the heat transfer component of the model, the heat
transfer properties of grapes were experimentally determined as a function of fermentation
progression. COMSOL was used to solve all components of the model simultaneously utilizing a
Finite Elements Analysis (FEA) approach. Predictions from this model were validated using
prior experimental work. Model prediction and experimental data showed excellent agreement.
The model was then used to predict spatial profiles of active yeast cell concentration and ethanol
productivity, as well as liquid velocity profiles. The model was also used to predict how these
gradients would change with differences in initial nitrogen concentration, a key parameter in
predicting fermentation outcome in nitrogen-limited wine fermentations. After validation, this
model was applied to examine how fermentor design (e.g. scale and aspect ratio) would affect
fermentation mixing, temperature control, and chemical gradients. Along these lines,
temperature control and mixing were also evaluated for concrete eggs using the same model.
Finally, a preliminary model for phenolic extraction from skins and seeds was developed and
validated using experimental data. This led to an analysis of phenolic release of tannins from
grape seeds that we are currently pursuing. We are now in the process of the next step in
modeling—combining the two models for fermentation dynamics and phenolic extraction to be
able to predict and control phenolic profiles in finished red wines. In the seven months since this
grant began, we have been highly productive having published two papers, submitted a third that
is under review, and will be submitting a fourth paper within the next month. We have also
presented this work at various extension venues around the state.

Characterization of Bitter and Astringent Proanthocyanidins During Winemaking

Project Title: Characterization of Bitter and Astringent Proanthocyanidins during

Winemaking

Principal Investigators

  • Dr. Susan E. Ebeler, Department of Viticulture & Enology, University of California, Davis,

530-752-0380

  • Dr. Hildegarde Heymann, Department of Viticulture & Enology, University of California,

Davis, 530-752-0380

Summary

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.

During the past year we have developed 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 are in the

process of completing the data analysis and comparison of the treatments. This work is

beginning to provide important insight into the impact of maceration treatments on

proanthocyanidin composition of wines.

Significance of Oak Ellagitannin Chemical Structure to Wine Oxidation

The pathway of wine oxidation, as currently understood, encompasses the cascade of reactions incited by the oxidation of phenols in the presence of oxygen, eventually coming to the conversion of ethanol into acetaldehyde. While it is evident that wines consume oxygen over time and acetaldehyde becomes increasingly apparent with age, the rate of oxidation can vary unpredictably among different wines, and it was hypothesized that different phenolic structural features, particularly those of oak ellagitannins, are the reason for such variability in oxidation. It was originally proposed that ellagitannins and other phenols with distinct functionalities be studied for their effects on oxygen consumption and acetaldehyde production. However, in light of recent studies conducted by our laboratory demonstrating that the input of oxygen does not guarantee the output of acetaldehyde, it was decided that attempting to study the pathway of oxidation in its entirety, from oxygen to acetaldehyde, would not be an effective approach.

Given the complexity of wine oxidation, a more sensible strategy would be to study the effects of phenolic structure on individual reactions rather than the pathway as a whole. In the first step of wine oxidation, the oxidation of phenols is coupled to the reduction of oxygen by iron, which acts as a shuttle for electrons between phenols and oxygen. A more specific hypothesis now is phenolic structure affects their reactivity with iron, subsequently affecting oxygen consumption and the remainder of the wine oxidation pathway. The initial reactions of wine oxidation may be characterized by the redox cycling of iron between its two oxidation states: Fe(II) and Fe(III). The addition of electrons to oxygen occurs with the oxidation of Fe(II) to Fe(III), and in the opposite direction, the loss of electrons from phenols takes place with the reduction of Fe(III) to Fe(II). The ratio of Fe(II) to Fe(III) should thus depend on the relative reaction rates of Fe(II) with oxygen and that of Fe(III) with phenols.

A quick and simple spectrophotometric method for iron speciation, employing the complexing agent Br-PADAP, is currently being optimized and validated, to be used not only to assess differential rates of iron reduction by structurally diverse phenols, but also by the industry to more generally measure the “redox status” of their wines. Our laboratory’s modified version of the Br-PADAP assay is simple and inexpensive, requiring a sample volume of only 200 μL and a reaction time of 10 min, and is done directly in a cuvette. Validation of the assay is currently underway; difficulty lies in the fact that there does not exist a standard method for iron speciation to compare, thus alternative methods of validation are being considered. This research would not only improve management of oxidation, but also furnish a more complete understanding of phenolic oxidation, with the ultimate goal being the prediction of wine aging based on phenolic content and composition.