Predicting Wine Ageability Using Iron Redox Reactions

In the initial reactions of the wine oxidation pathway, oxygen consumption and phenol oxidation are coupled through the redox cycling of iron between two oxidation states: oxygen oxidizes iron(II) to iron(III) while phenols reduce iron(III) back to iron(II). Wine has been observed to consume oxygen more slowly the more oxygen it has consumed. Given the dependence of oxygen uptake on the availability of iron(II), it was hypothesized this decline is due to exhaustion of wine’s capacity to reduce iron(III). Experiments have been done to better understand iron reduction and its relationship to oxygen consumption, as well as how it may be used to gauge wine ageability.

The reduction of iron was monitored in model wines to determine how reaction rates vary with composition. 4-methylcatechol and pyrogallol, models for o-dihydroxy phenols (e.g. catechin) and 1,2,3-trihydroxy phenols (e.g. epigallocatechin) respectively, differed in their ability to reduce iron, indicating the phenolic compounds in wine are not equally oxidizable. Furthermore, the inability of 4-methylcatechol to reduce iron in the absence of a nucleophile suggests wine oxidation may depend on the availability of nucleophiles (e.g. SO2, glutathione), the depletion of which could hinder further reactions.

Oxygen consumption was found to occur more quickly as pH increased from 3.0 to 4.0, though unexpectedly the opposite was observed for iron reduction. However, rates of oxygen consumption never exceeded those of iron reduction, indicating wine phenols, in the presence of sufficient nucleophiles, maintain a supply of iron(II) that does not limit oxygen consumption, i.e. wine is constantly “primed” to receive more oxygen. This suggests wine ages at a rate limited not by the reactions therein, but by oxygen ingress, thus ageability may be conceptualized not in terms of a rate, but rather the capacity for oxidation.

The ferric reducing antioxidant power (FRAP) assay was modified to quantify the “lifespan” of wine in terms of its capacity to reduce iron(III) and supply the iron(II) required for oxygen consumption. A 2016 UC Davis Petite Syrah was stirred continuously under constant air exposure for several days to test whether its iron-reducing capacity would decrease with oxygen exposure, though this treatment ultimately did not produce any significant changes to FRAP measurements. It is possible FRAP is not a suitable metric for wine ageability as previously hypothesized, though additional trials are necessary to confirm this, before work can be done towards an alternative definition and measurement of ageability. However, it is also possible continuous stirring does not give rise to the same chemical changes as would years of aging, thus FRAP analysis of vertical series of wines aged “naturally” may reveal iron-reducing capacity to be a function of wine age.

The unexpected slowing of iron reduction/phenol oxidation with increasing pH may be explained by increased complexation of iron by wine acids (e.g. tartaric), though this remains an active area of investigation. It is additionally worth studying the effects of iron complexation on other reactions in wine, such as the Fenton reaction and subsequent oxidation of ethanol into acetaldehyde.

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.

Grape smoke exposure effects: Determining the compounds that cause smoke impacts in wine

The overall goal of this work is to identify those compounds from smoke that cause negative impacts to wine quality. We are taking a different approach than other research on this topic. In short, we are creating label smoke compounds by growing barley (our fuel source) in a 13CO2 environment. We are then able to track the labeled carbon in the smoke, grapes and wine using NMR, which is very sensitive to 13C.

The accomplishments for the project for the 6 months has been to design the chambers needed to grow barley in elevated CO2 environments (Objective 1A). Our first barley lots (0-4) were/are being run using regular CO2 due to the expense of isotopic 13CO2. Once we have determined the optimal CO2 levels in the chambers we will switch to 13CO2. Development of the chambers included not only how to increase carbon fixation of the 13C in the plant, but determined how to measure the isotopic CO2 and other necessary plant measurements. We are on track to have 13C labeled barley to burn and produce 13C smoke for the 2020 vintage.

Investigating Fruitiness Perception in Red and White wines

This provides results and conclusions from the entire project, although we may refer to previous reports. We have had a very exciting accomplishment with the adaptation of a chemometric method that can calculate chemical interactions resulting in specific sensory perceptions. This method, fuzzy set qualitative comparative analysis (fsQCA), overcomes the issues with traditional correlation analysis that made determining aroma chemical interactions very difficult. To date we have investigated 87 different compound combinations and their impact to fruit aroma in Pinot noir wine. By applying fsQCA we have found 5 compound sets that result in red fruit aroma in Pinot noir wine and 2 compound sets that result in dark fruit aroma in Pinot noir wine. The necessary and sufficient conditions found in these sets are supported by other work, but our results are the first to show the multiple combinations of compounds that can result in specific fruity aromas. We have also investigated 57 compound combinations for fruitiness in white wine. Since we were less sure about the calibrations used for fsQCA we ended up creating unique set variables to determine the necessary and sufficient conditions for different fruity aromas in white wine. Overall we found 5 compound sets that cause tropical fruit aroma, 2 compounds sets that cause red apple aroma, 1 compound set for pineapple aroma, 2 compounds sets for pear aroma, 4 compound sets for peach aroma, 1 compound set for orange aroma and 1 compound set for lychee aroma. For the compounds we investigated we did not get any compounds sets for citrus aroma and green apple aroma. We also incorporated nonvolatile factors into the analysis. In red wine we are investigating the effect of phenolic composition on fruitiness perception and in white wine we changed the residual sugar and ethanol concentration to determine its impact on fruity perception in white wines. Phenolic content in red wine was found to alter fruitiness perception, but the change in ethanol and sugar in white wine did not have an impact to aromas perception. This work has shown huge strides in understanding compound interactions that cause specific aromas in wines. To the point that we will be able to start building predictive models in the future that have much better success than any previous ones. With these models it will be possible to determine the impacts of different viticulture and winemaking practices to these wines without having to go through lengthy sensory studies and make real time decisions during the season for the desired wine quality parameter.

Identification of smoke odorants by gas chromatography/olfactometry and assessment of smoke odorants in grapes and wine

Smoke taint has become a significant concern for the wine industry, particularly in Southern Oregon and California, partly due to climate change. Smoke taint is an off-aroma describing the wine with smoky, medicinal, and ashy characters, and this unpleasant taint is caused by grapes or grapevine exposed to bushfire smoke before. Wine made from smoke-tainted grapes is often characterized by smoky, burnt, burnt rubber, ashy, smoked salmon, smoked meats, salami, leather, disinfectant/hospital, medicinal, dusty, and earthy aromas. Guaiacol, 4-methylguaiacol, and syringol have smoky odors with low sensory thresholds, and these compounds are likely to contribute to the overall smoke flavor.

When guaiacol and other smoke-related compounds are absorbed by the grapevine, the grapevine will convert them to the corresponding glycosides or other bound forms. These glycosides as well as the bound form precursors do not exhibit aroma themselves, but can be converted back to the odorants during winemaking and wine aging process. Grape maturity, grape varieties, and bottle aging can all influence the intensity of smoke taint in wines.

Smoke taint precursors, including glycosides, can persist in the wine and directly affect flavor perception during consumption. Guaiacol β-D-glucoside and m-cresol β-D-glucoside in model wine were found to give rise to a smoky or ashy flavor in-mouth, due to the release of respective free volatiles in-mouth. It has been confirmed that the enzymes present in human saliva can release the volatile smoke compounds from their glycoconjugates even under low pH and elevated ethanol conditions. Smoke taints in grapes and their conversion during winemaking, and wine aging are very complex, the mechanisms of transformation need to be thoroughly investigated to mitigate the issue.

Although guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-ethylphenol, eugenol, and furfural are related to bushfire smoke, but not all of them are directly generated from smoke, some of them can naturally exist in grapes or extracted from the wine barrel. These compounds are essential contributors to wine flavor at low concentrations.
Our initial experiments were designed to extract the smoky chemicals from wine. Complementary analytical methods have been evaluated. The results showed that smoky odorants can be obtained from wine using different methods. Further investigations were under progress to identify the smoky odorants.

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 sensitivity of a range of B. bruxellensis strains to O. oeni was also determined in 2018 Pinot noir using O. oeni strain Alpha. While B. bruxellensis UCD2049 populations declined rapidly when inoculated into 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 is sensitive to O. oeni while the other B. bruxellensis strain were not is unknown at this point but is being investigated in ongoing experiments. Additional experiments are 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. The mechanism of inhibition is also being investigated to determine if inhibition occurs via cell to cell contact, nutrient depletion, and/or production of an inhibitory compound by O. oeni. While wineries must continue to use sound winemaking practices to prevent the growth of Brettanomyces, results from this study may provide winemakers with an additional strategy/tool to help prevent wine spoilage by Brettanomyces.

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.

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.

Using a genome-scale metabolic model for Saccharomyces cerevisiae for 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. Predictions for nutrient utilization and production of metabolites such as ethanol, glycerol, and organic acids are quite good. Biomass is somewhat underpredicted using conditions that we would expect. However, predicted biomass increases if we vary amino acid utilization and oxygen utilization at the beginning of fermentation. We have also found that the biomass composition and amino acid composition of proteins are important parameters in predicting maximum biomass concentration. Experimentally, however,
we have found that composition changes between strains and over time. We are now generating a more complete set of this data. In past work, no measurements of this biomass composition were conducted—researchers just assumed composition from old data sets—thus limiting the utility of their predictions. We have also made progress on curating the model for aromatic compounds derived from yeast metabolism and have begun our experiments to quantify the aromatic compounds as a function of time and yeast 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 and phenolic extraction. We have developed a reactor-engineering model for red wine fermentations that includes the fundamentals of fermentation kinetics, heat transfer, diffusion, compressible fluid flow, and extraction of phenolics (anthocyanins, skin tannins, and seed tannins).

COMSOL was used to solve all components of the model simultaneously utilizing a Finite Elements Analysis (FEA) approach. Prediction of phenolic concentration gradients and temperature gradients from this model were validated against measurements in 2000 L pilot fermentations. Model prediction and experimental data showed excellent agreement for anthocyanin and tannin concentrations and distributions over the course of fermentation.

After validation, this model was applied to examine how fermentor design (e.g. scale and aspect ratio) and operational decisions (temperature set point, pump over frequency) would affect phenolic extraction rates, relative concentrations of skin to seed tannins, and distribution of phenolics throughout the fermentor in the absence of cap management. These results were a follow up to 2018-2019’s work, where the model was used to explore fermentation dynamics and temperature control in red wine cylindrical fermentors and white wine concrete egg fermentors. Example findings include optimization of skin tannin extraction via cap management, with 1x/day pump overs being found superior to both no cap management and 8x/day pump overs, a finding made possible via the combined spatial fermentation-extraction model.

Our results have opened up two exciting avenues of further investigation. The first is applying our reactor engineering models to isothermal fermentation process acceleration, where wine fermentation process cycle time could be greatly decreased by the judicious application of yeast nutrients throughout the fermentation, maximizing yeast biomass. This would greatly improve productivity in existing wine fermentors and lower the capital cost of new winery equipment. The second is the application of COMSOL extraction models to external grape pomace extraction columns, allowing for the fine-tuning of phenolic profiles in the end wine, potentially in a much more rapid fashion than in-tank extraction.

In the nineteen months since this grant began, we have been highly productive having published six primary research papers, along with a review of wine fermentation process modeling. We have also published three papers in cooperative works stemming from this grant. We have presented this work at various extension venues, as well as technical conferences throughout the nation.