Revisiting Tartrate Stability

Despite the claim in the initial proposal that there were no known barriers to completing this project in a timely fashion, the world conspired to make that a lie. Between March of 2020 and the end of October, on campus research at UC Davis was restricted to time sensitive research activities (“Phase 2 activities”) and was expanded to include some research that needed on site access on 30th October (“Phase 2x”). Restrictions continue regarding number of personnel, as do guidelines for high-risk employees. This was again changed when the state of California entered a second lockdown between Dec 22 to Jan 25 of 2021. It is anticipated that there is the potential for additional restrictions in on-campus access as the virus status changes.

In April, when it became clear that these restrictions would be a serious barrier, the project investigators purchased, and installed, equipment required for this project in an off-site location to allow the work to take place outside of these restrictions. This process was slowed by all the now familiar issues of operating in a COVID world. This offsite location now can run all the tests described in the original project proposal Table 3 (below) and will continue to be the research location until COVID restrictions are removed completely (UC Davis “Phase 4”)

As of December 2020, the equipment was installed and functional. The researchers are in the process of validating the equipment and proceeding on the original project’s Goal 1:

Goal 1: Standard precision tests will be done by repeated analysis with specific variations in sample, subsample, day, equipment and/or analyst to provide information on method repeatability and replicability. If adequate collaborators can be found, a reproducibility value will also be generated; if not, standard multiples of repeatability can be used as a working estimate of reproducibility.

The adjustment to the original timeline in response to this situation shows a delay of about eight months. We do not foresee additional delays due to COVID related restrictions other than any normal disruptions in deliveries or access.

One potential advantage of the new set up is the ability to increase the output of experimentation over the next several months and make up for loss of time. If that proves the case, the researchers will move onto the proposed future goals proposed in the original proposal.

Goal 4: Correlate the analytical responses of selected non-redundant tests to specific “risk” storage or transportation conditions (such as short time-extreme cold or long time- mild cold storage, with or without other extenuating conditions) which could impact the “potassium bitartrate stability” of the wines. This will determine the analytical method’s predictive abilities.

Measuring Grape Smoke Taint Protection

Smoke taint has become a recurring issue since 2008, and one that has led to crop losses as well as legal disputes between growers and processors. With the expectation that the intensity and frequency of wildfires will continue to increase in California and impact the winegrape producing regions, it is important to investigate potential ways to mitigate the impact of grape smoke exposure. There are now a number of products and processes available in the market to treat the wine, but also materials to prevent or limit the impact of smoke exposure by applying a protective material to the grapes. As these are new problems and new remedies, it is important to have a means to test the effectiveness of such treatments. We created a simple standardized evaluation of grape protection treatments in order to compare their relative effectiveness

Tropical Fruit Aroma in Wine

The overall goal of this project is to produce Chardonnay wines with increased tropical
fruit aroma perception. In a previous study developed by Dr. Elizabeth Tomasino research group,
we found that wines with higher concentrations of fermentation esters and volatile thiols imparted
more intense tropical fruit aroma nuances. Therefore, in this project specific winemaking processes
(skin contact, β-lyase addition, and two fermentation gradient temperature regimes) were
performed with the intent to either increase or decrease these aromas in the wine. The first two
processes (skin contact and β-lyase addition,) are known for increasing volatile thiol
concentrations in wine. The latter (fermentation temperature) is expected to increase thiol
concentrations and preserve fermentation esters.

The accomplishments for the project for the 6 months have been to design and perform the
winemaking experiment for objective 1, measure the basic wine quality parameters (pH, titratable
acidity, malic acid, acetic acid, and ethanol content), and collect wine samples for the analytical
chemistry analysis. We have also performed a preliminary sensory descriptive analysis panel in
December 2020 (Projective Mapping combined with Ultra-Flash Profiling), but the results have
not been analyzed. Despite the Covid-19 lockdowns we are on track with this project and we are
currently developing a method to measure fermentation esters in the wines. To keep on track with
the project timetable we are working with a colleague at the University of Adelaide to have thiols
analyzed, as the access to the OSU MS facilities is limited due to COVID.

Grape Smoke Exposure Effects: Determining the Compounds that Cause Smoke Impacts in Wine

The goal of this project is to determine the volatile chemicals in smoke that affect the flavor and aroma of wine and wine grapes for Pinot noir and Chardonnay. To differentiate chemicals derived from smoke from ambient grape or wine compounds, a fuel source, barley, was chemically “tagged” using 13CO2. As 13CO2 is assimilated by the barley, it is distributed throughout the plant and its chemical components, including the precursor smoke material, such as lignin. By identifying the major components from smoke, future studies may be able to better prevent off-flavors caused by smoke from nearby wildfires. More immediate conclusions may even begin to identify thresholds when crops are exposed to smoke to the point of sensory perception prior to harvest or fermentation.

To achieve these goals, in this year we have:

  • Implemented 13CO2 incubation cages for growing isotopically labelled barley (Figure 1)
  • Tested and concluded ideal practical conditions to maximize growth of 13C-labelled barley
  • Grew over 2 kg (dry weight!) 13C-labelled barley from January through September
  • Adjusted protocols to account for Covid-19 lockdowns and personnel distancing as outlined by state and university policies, continuing the project with minimal interruption
  • Begun processing and milling barley samples to determine lignin, carbohydrate, and dissolved composition through chemical methods, and 13C assimilation via isotope ratio mass spectrometry
  • Designed and constructed smokers and smoke tents for smoking wine grapes using chemically labelled barley as a fuel source (Figure 2)
  • Designed methodology to recreate high density smoke conditions while balancing cost[1]effectiveness of administering labelled smoke while ensuring the viability of the wine
  • Exposed over 20 kg Chardonnay and 20 kg Pinot noir grape clusters to labelled smoke, consistently holding smoke labelled smoke density >20 mg/m3 over the course of 3 days in October (Figure 3)
  • Made wine from grapes exposed to 13C-labelled smoke (Figures 4 and 5)

Preliminary aroma and flavor of smoke-exposed wine show a strong incorporation of chemicals associated heavily smoked grapes, with a marked difference between the grapes exposed to smoke in the tents and the control grapes that were exposed to smoke during the wildfire events in Oregon in September. Moreover, we expect to be able to differentiate the chemicals’ origins based on the NMR experiments and confirmed by GC- or LC-MS in 2021.

Effect of Polysaccharides on Wine Aroma Binding and Perception

The organoleptic properties of red wine are among the essential wine quality parameters. Besides volatile aroma compounds, the nonvolatile has seen a growing interest in the last decades, comprising taste molecules as well as substances evoking a broader range of mouthfeel sensations. The influence of polysaccharides on wine organoleptic qualities is associated with their ability to interact and aggregate with tannins and to decrease perceived tannin astringency in wine. Also, wine polysaccharides have been shown to interact with aroma compounds. The interaction of polysaccharides with tannin and aroma compounds depends on the polysaccharides’ chemical structure and composition. This work aims to isolate and characterize polysaccharides from Pinot noir wine and study their interaction with wine aroma compounds.

From yeast, polysaccharides were sequentially extracted with hot water (20% yeast cells aqueous solution, 121°C, 3.5 h) and alkali (3% sodium hydroxide solution, 80°C, 6 h). Grape polysaccharides were isolated from Pinot noir grapes with hot water (90℃, 2 h) and alkali solution. Polysaccharides in wine were obtained via membrane ultrafiltration with molecular weight cut-off of 2-100 kDa and over 100 kDa. All the polysaccharides were precipitated with ethanol and dried under a vacuum oven.

The isolated polysaccharides were analyzed for purity, and all isolated polysaccharides have high purity. The polysaccharides isolated from yeast have the highest purity. Size exclusion liquid chromatography was used to analyze the isolated polysaccharides’ molecular distribution. Matrix-assisted laser desorption/ionization and time-of-flight mass spectrometry (MALDI-TOF) was used for partial sugar sequencing.

The sugar composition of the isolated polysaccharides was analyzed by gas chromatography and gas chromatography-mass spectrometry after derivatization. The results showed that sugar composition differed greatly depending on the polysaccharides’ source and the methods used for polysaccharide isolation. Water extracted polysaccharides from yeast mainly consist of mannose, glucose, xylose, and alkali extracted yeast polysaccharides mainly consisting of mannose and glucose. In comparison, water extracted grape polysaccharides are mostly glucose, arabinose, rhamnose, galactose, mannose, galacturonic acid, and xylose. Alkali extracted grape polysaccharides are composed primarily of arabinose, galactose, glucose, rhamnose, galacturonic acid, xylose, and mannose. The polysaccharides isolated from the wine with molecular weight 2- 100 kDa are composed mainly of galacturonic acid, mannose, rhamnose, glucose, galactose, and arabinose. Polysaccharides with a molecular weight of over 100 kDa are composed mainly of arabinose, mannose, galactose, rhamnose, galacturonic acid, and glucose.

Our next step is to understand how the sugar composition, structure, molecular weight will interact with wine aroma compounds and alter aroma perception.

Identification of Smoke Odorants by Gas Chromatography/Olfactometry and Assessment of Smoke Odorants in Grapes and Wine

There are two primary objectives for this funding cycle. One is to build reliable analytic methods for smoke compounds quantification, and another one is to build a database for smoke assessment. We developed a headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method and a stir bar sorptive extraction-gas chromatography-mass spectrometry (SBSE-GC-MS) method to analyze 13 smoke-related compounds. When stable-isotope internal standards are used, the HS-SPME-GC-MS is fast and reliable and can analyze smoke-related compounds in different wines. However, the stable[1]isotope internal standards are not commercially available for all 13 smoke-related compounds. In comparison, the SBSE-GC-MS method is more sensitive and less dependent on the wine matrix.

To respond industry’s smoke exposure concern, we developed a rapid method based on the SPME-GC-MS technique with isotope compounds as the internal standards. We can analyze guaiacol, 4-methylquaiacol, 4-ethylguaiacol, o-cresol, m-cresol, and p-cresol, 4-ethylphenol in 30 min. Our analytical results were verified with a certified commercial lab.

Using this rapid analytical method, we analyzed over 370 smoked exposed red wines and 90 smoke-exposed white wines (including rose wine). A standard was run every ten samples to verify recovery and instrument performance, and a duplicate sample was analyzed every 20 samples. Both free phenols and total phenols (after acid hydrolysis, pH 1, 100C/4h) were analyzed for all the samples. Out of the 370 smoke-exposed red wine, 84 samples had guaiacol concentration in the range of 0-5 μg/L, 159 samples had guaiacol concentration in the range of 5- 10 μg/L. Most of the samples had 4-methylguiacol less than 3 μg/L. In red smoke exposure wine, the average ratio of free guiacol to 4-methylguaiacol was 4.5. But the average ratio of total guaiacol to 4-methylguaiacol was 6.5, higher than the free forms.

A total of 86 control wines were randomly selected from 2013-2016 vintages with about 20 samples from each year. The samples were obtained from industry fermentation without barrel aging. Both free and total volatile phenols were analyzed. Multiple statistical methods were used to analyze the data. Univariate data analysis was used to identify differences between the control and smoke-exposed red wine. The results showed that except for free 4-methylguaiacol and bounded m-cresol, all compounds were significantly higher in smoke-exposed wine. Statistical analysis suggests that the total p-cresol is the best biomarker of smoked 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 large number of commercial O. oeni strains to inhibit B. bruxellensis growth at the end of MLF. Sterile filtered wine was inoculated with one of eleven commercial O. oeni strains and growth and malic acid monitored. When MLF was complete, wines were inoculated with a select strain of B. bruxellensis and growth and volatile phenol production monitored.

All O. oeni strains tested inhibited the growth of B. bruxellensis UCD2049 in Pinot noir wine with O. oeni strain variation observed. O. oeni strains Alpha, 350, VP41, MBR31 and PN4 most strongly inhibited growth of B. bruxellensis UCD2049, while strains CH11, Omega, Beta, and VFO 2.0 inhibited B. bruxellensis to a lesser extent. The potential mechanism of this inhibition was investigated by using a dialysis membrane to physically separate O. oeni and B. bruxellensis cells but allow free movement of nutrients and other potential inhibitory compounds. The physical separation of O. oeni from B. bruxellensis relieved the inhibition of B. bruxellensis by O. oeni that occurred when the two microorganism were in present together. These results indicate that inhibition is not due to nutrient depletion by O. oeni as nutrients could flow freely across the dialysis membrane. It is also unlikely that B. bruxellensis inhibition was due to the production of an inhibitory compound by O. oeni as any potential inhibitory compound would also have passed through the dialysis membrane. Instead, these results provide strong evidence that the inhibition of B. bruxellensis by O. oeni is due to cell-cell contact.

The sensitivity of additional B. bruxellensis strains to O. oeni was also determined. While B. bruxellensis UCD2049 populations declined rapidly when inoculated into Pinot noir wine that had just completed MLF with O. oeni Alpha, growth of the other B. bruxellensis strains tested was not impacted. Why B. bruxellensis strain UCD2049 was inhibited by O. oeni while the other B. bruxellensis strains were not was subsequently investigated. Initial experiments considered whether ethanol tolerance between B. bruxellensis strains impacted inhibition by O. oeni. Given that earlier experiments had been conducted in 13% (v/v) wine, wines were instead adjusted to 12.5% or 14% (v/v) ethanol. In low (12.5%) ethanol wine that had undergone MLF, B. bruxellensis UCD2049 grew well, in contrast to what was observed in 13% wine where growth was inhibited. B. bruxellensis strains AWRI-1499 and Copper Mountain also grew well in low ethanol wine with no difference between treatments. In higher ethanol wine, B. bruxellensis UCD2049 struggled to grow whether the wine had undergone MLF or not. In contrast, B. bruxellensis strains AWRI-1499 and Cooper Mountain grew well in the higher ethanol wine. B. bruxellensis strains AWRI-1499 populations recovered slower in wine that had undergone MLF while the opposite occurred for strain Copper Mountain. These results demonstrate that ethanol tolerance differences between B. bruxellensis strains impact their inhibition by O. oeni. For example, strain UCD2049 was not inhibited by O. oeni in wine at 12.5% ethanol but was inhibited in 13% and 14% ethanol wine. Additional experiments will be conducted where pH will also be considered as tolerance to this factor is known to differ between B. bruxellensis strains. Experiments are also underway exploring how long MLF induced B. bruxellensis inhibition last as well as whether B. bruxellensis inhibition occurs if infection happens at the beginning or mid-point of MLF.

Investigation of the Impact of Grapevine Red Blotch Virus (GRBV) on Phenolic Extractability and Winemaking Strategies to Mitigate

Since its identification in 2012, grapevine red blotch (RB) disease has been found to be widespread in the United States1 . This disease is caused by grapevine red blotch virus infection of grapevines2 . Previous research in the Oberholster lab indicates mostly a substantial impact on berry ripening in all varieties studied, along with variable impacts on primary and secondary metabolites depending on site and season, which had a larger impact than variety3–5 . RB diseased grapes show transcriptional suppression of primary and secondary metabolic pathways, specifically restricting the biosynthesis and accumulation of phenylpropanoids and derivatives6 . Our research indicates a clear trend of decreasing anthocyanin content in red grapes where the impact on sugar accumulation was severe (16 to 20% reduction). There were also significant differences in the volatile composition of RB(+) versus RB(-) grapes, with mostly a suppression in aroma compounds due to RB disease.

During grape ripening, multiple factors may influence phenolic extractability such as interactions with cell walls, cell integrity, individual phenolic concentrations and interactions with each other7–10. It is commonly accepted that pectolytic enzyme degradation of skin cell walls during grape ripening increases the extractability of anthocyanins. However, GRBV impact on cell wall composition has yet to be investigated. Studies have shown that grapes with increased anthocyanin and skin tannin concentrations resulted in wines with higher tannin concentration, deeper color and better ratings by wine judges11. The question is whether winemaking protocols used to increase phenolic extraction from diseased grapes such as maceration enzymes and extended maceration can compensate for extractability differences and compositional differences. Prior research focused mostly on determining the impact of RB disease on wine composition and the grapes when harvested when the healthy controls reached 25°Brix12. Subsequent, studies determined the impact of longer hangtime of RB(+) grape to reach 25°Brix and found that extractability improved greatly resulting in wine with improved phenolic content13,14. Therefore, further research is needed to understand whether the impact of GRBV decrease with increasing ripeness levels, such as 27ºBrix for healthy vines, at which most commercial harvests occur.

Thus, the aim of the current study is two-fold. The first is to investigate the impact of GRBV and ripening on phenolic extractability by determining the changes in grape cell wall composition and how this relates to the release of phenolics under winemaking conditions. The second is to determine whether winemaking protocols such as enzyme addition and extended maceration can increase the extractability of RB(+) grapes resulting in wines more similar to those made from healthy grapes. Characterization of grape cell walls is underway. Preliminary data indicate that both the use of maceration enzymes and extended maceration increased the tannin concentrations in RB(+) fermentations compared to the controls, although not for all ripeness levels. Wines were bottled in February. However, due to the coronavirus pandemic, both the wine phenolic composition and sensory characteristics have not been completed.

Investigation of Different Winemaking Protocols to Mitigate Smoke Taint Character in 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 research project was to compare all the suggested wine protocols that evolved from the current literature using one batch of smoke impacted grapes under identical winemaking conditions except for the parameter under investigation. Results from this study will enable to us to better advice the wine industry during future smoke events. 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. Cabernet Sauvignon grapes were received from three different areas with varied amounts of smoke exposure (Oakville, Alexander Valley, and Silverado Trail AVA’s) in Northern California. Gas chromatography mass spectrometry (GC-MS) and sensory analysis were performed in order to correlate wine composition to smoke taint characteristics. The winemaking variables investigated were the use of different fermentation yeasts, oak additions and fermentation temperatures. Among other attributes, smokiness and ashy aftertaste were found to be significantly different among the wines, showing a clear difference between the wines that were made from smoke impacted fruit and the control wines that were made from non-impacted fruit. One yeast showed a significant effect by highlighting the fruitiness in the wines and reducing the ashy aftertaste. Different oak additions were not successful in masking the impact of the smoke. Similarly, different fermentation temperatures did not have a significant impact on smoke expression in the resulting wines. Findings indicate that mitigation strategies during red wine fermentation have a limited impact on the extraction of smoke taint markers as well as the expression of smoke taint sensory characteristics.

Profiling 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 depended on indirect measurement of the glycosides and acid or enzymatic hydrolysis is needed to release the volatile aglycone which can result in artifact formation. We subsequently 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; the method was used to monitor monoterpene glycosides during fermentation of red and white grapes. The method was also expanded to measure 31 volatile-phenol glycosides in grapes exposed to wildfires and to measure 102 glycosidically bound precursors of monoterpenoids, norisoprenoids, volatile phenols, aliphatic alcohols, and sesquiterpenoids in grapes.