Highly Selective Enzymatic Degradation of Undesirable Phenols in Brettanomyces and Smoke Taint Contaminated Wines.

This is the first-year progress report on a multi-year project divided into two-phases with the first (current) phase focusing on discovery and design of enzymes with exclusive degradation of 4-ethylguaiacol (4EG) and 4-ethylphenol (4EP), the main sensory-active components in wine contaminated with Brettanomyces and responsible for a portion of the ‘taint’ phenols found in smoke-tainted wine (Licker 1998, Mirabelli-Montan 2021).  A second phase of this project—beyond the scope of the current proposal—will use the base of both engineered laccases and the specific knowledge of their active site modifications to create additional enzymes capable of fully, and exclusively, degrading the remaining smoke taint marker compounds including guaiacol, 4-methylguaiacol, o-, p-, m-cresol, and syringol.

Summary of Major Research Accomplishments and Results by Objective

Objective 1 – Library screening:  Library screening resulted in the discovery of 13 laccases with almost 100% 4EG degradation in a buffered pH 7 environment, with three also showing 90% 4EP degradation. When the environment is made more acidic, at pH 4, only laccases #33 and #36 degrade 80-90% 4EG with minimal 4EP activity. When testing in model wine with 4g/L tartaric acid, 13% ethanol, at pH 3.5, only laccases #15 and #16 completely degrade 4EG with small amounts of 4EP activity; with laccases #11 and #34 also degrading 60% 4EG. When the phenols are isolated, then #11, #15, and #36 degrade 80-90% 4EG with #36 degrading almost 30% 4EP after one week. A sequence similarity network that clustered laccases by 70% homology showed multiple clusters had activity on 4EG and 4EP at pH 7. Activity in both pH environments were limited to one cluster. High activity on 4EG in model wine was slightly spread out over three clusters, with additional low to mild activity on 4EP in two of them.

Objective 2 – Computational analysis & design:  Library screening resulted in two pairs of homologous laccases (#15 & #16; #33 & #36) representing two different paths for design towards an exclusively 4EG and 4EP degrading enzyme. However, structural analysis of the active site with the docked phenol revealed a wide-open site on all active laccases. These open active sites greatly increase the risk of off-target degradation of beneficial flavor aromatic compounds. This slowed down the next design steps with first needing to map out the active site landscape for residues required for desired activity by building and testing point mutations to alanine. Current plans include testing these point mutants for altered activities. Then, computational design with newly developed open-sourced AI-tools will be utilized to close the active site with increased specificity and exclusivity on 4EG and 4EP.

Raman for Smoke Exposure

During the 2022-2023 funding cycle, we have finished the volatile phenol analysis of 82 wine samples having various degrees of smoke exposure and about 60 pooled samples from different wineries. Those samples will be used for Raman spectra collection once the nanoparticle and Raman signal enhancement protocol is finalized. We have collected grape samples throughout the 2022 season from eight vineyards in California and Oregon and an enology lab. Out of the 214 independent samples, 180 are pinot noir. The samples were collected between September 9 and October 24. About half of the samples were collected from Salem, OR, where the wildfire created a lot of worry during harvest. The lab offered us samples and enology test results in an anonymous way. More grape samples will be collected in 2023. All the grape samples will be analyzed in the 2023-2024 funding cycle simultaneously. Those samples will be used for Raman spectra collection once the nanoparticle and Raman enhancement protocols are finalized. Different sizes of silver nanoparticles have been synthesized, and the Raman enhancements were observed. However, the enhancement depends on the size, shape, and concentration of the nanoparticles. More nanoparticles with different sizes will be synthesized and evaluated in the 2023-2024 funding cycle in conjunction with a linker agent such as benzenethiol. An automated sampling system for the Raman Spectrometer was developed by Dr. Feng Ye’s team from Spectra Scientific company to replace manual spectra collection. The system is based on an alumina 3D printed 117 well plate to eliminate Raman interference from plastics. Because alumina is not stable at acidic pH, we will obtain another 3D printing plate and coat it with gold so it can be used for wine samples. 18 We have established benchmark results for the Raman fingerprints of wine samples, especially using a combination of steady-state electronic and vibrational spectroscopies in a table-top optical setup. We have implemented FSRS with a tunable Raman pump and probe pulses in a femtosecond laser amplifier system for assessing the smoke-exposed wine. We have combined FSRS with AgNPs to optimize conditions for SE-FSRS and achieve higher sensitivity than the resonance Raman enhancement alone. We are investigating nanoparticle receptors for smoke compounds to enhance the Raman signal. Raman spectrum of 82 smoke-exposed wines and 214 smoke-exposed grape samples (grape juice) were collected on the 1064nm FT-Raman system. We have developed an algorithm for machine learning. First, we applied a background subtraction algorithm to remove the Rayleigh scattering and background fluorescence. After backgrounds were removed, the intensities of each peak were captured through a regression algorithm. After the base models were built using average spectra, each sample was measured using juice or wine base models to report the intensity of each peak. Intensities of extracted peaks were subsequently analyzed using a correlation matrix and principal component analysis. The above metrics were fed into Orange Data Mining for data processing and ML development. The model showed an excellent correlation of Raman to titratable acidity. The undergoing research is to use nanoparticles for surface enhancement Raman Spectroscopy and smoke compound receptor to enhance the signal so that the GC-MS data can be correlated with the smoke compounds.

Baseline Levels of Smoke-related Volatiles and their Glycosidic Precursors in California Grapes and Wines

The increasing incidence of wildfires in grape growing regions of California and the West Coast has highlighted the need for enhanced understanding of the levels of volatile phenols and their non-volatile glycoside precursors that contribute to smoke taint off-flavors in grapes and wines. In this project, we measured ten volatile phenols in non-smoke exposed grapes to begin to understand baseline levels of these compounds in red and white grape varieties. Free and total levels of guaiacol, creosol, phenol, 4-ethylguiacol, o-, m-, p-cresol, 4-ethylphenol, 4- methylsyringol, and syringol were measured in grapes from different regions of California. Air quality data for these regions is also presented. We will obtain data over a minimum of two years in order to begin to assess year to year variability. We have submitted a proposal to continue this work in 2022-23.

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.

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.

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.

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.

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.

Rapid Analysis of Wine Phenolics by Laser-induced Fluorescence

Under the current grant, we conducted an initial evaluation of wine fluorescence properties with a time-resolved (lifetime) fluorescence spectroscopy (TRFS) device that allows for rapid, in situ measurements of fluorescence intensity, spectra and lifetime upon UV laser light excitation and visible autofluorescence light detection. Below, we provide a synopsis of the main results along with an extended report.

First, we measured the excitation-emission matrices of model wine solutions (wine analogs with individual components typically found in wines) that may contribute to the overall fluorescence of finished wine products, together with grape seed extracts and proteins. This analysis will guide the configuration of the TRFS device, which spectral distribution can be adjusted for optimal performance in each application. Moving forward with wine applications, narrower optical filters along the main fluorescence peaks found in the EEMs of wines and their respective components have the potential to increase the detection sensitivity. Only a small subset of wine components were tested in this work, namely caffeic acid, gallic acid, rutin, catechin, and malvidin-3-glucose. Expanding the library of potential contributors to the fluorescence of the final wine product is expected to further guide the optimization procedure of the final device, which could be different depending on the specific application, i.e. it might be of interest to tune the spectral bandwidths to detect a particular contaminant instead of intrinsic wine properties.

Second, we tested the performance of the current lab configuration of the TRFS device to detect spectra (intensity ratio) and fluorescence lifetime of the wine models as well as a variety of wines. Consistently with the EEMs measured in the first place, we found a red shift of red wine with respect to most of the tested wine models, except for caffeic acid, which spectral properties closely resemble those of the finished product. Fluorescence lifetime of all tested wine models was shorter in spectral band 1 than that of red wine. However, for the rest of the spectral bands, fluorescence lifetime of all models except for caffeic acid was longer, where detectable. For caffeic acid, lifetime was always found shorter than for red wine. Commercial wine bottles were then tested. Tannin levels and fluorescence properties were measured to find that both intensity ratio and lifetime in spectral band 4 (570 – 650 nm) better correlate with tannin levels than fluorescence parameters in any other spectral range. This further confirmed some preliminary data that we had acquired previous to this award. Interestingly, the selection of wines for this analysis had a narrow range of tannin levels. Combining the results from the two experiments extends the range of tannins, and initial evaluations indicate a trend: as tannin concentration increases, fluorescence intensity increases, and fluorescence lifetime decreases. With a considerable increase of tested wines and further statistical analysis, these fluorescence parameters have the potential to be used as a proxy for tannin concentration, which would be a faster and economic assay to run compared to current methods. We also applied a multivariate analysis of fluorescence parameters to explore the potential of TRFS to identify or discriminate between different wine varieties. The presented analysis is a very simplified model, but already shows discrimination power. Applying more advanced computational methods and expanding the database with different wines could result in a classifier capable of identifying different wine types in a rapid and inexpensive manner. 2

Third, we evaluated how fluorescence parameters are affected by oxygen levels in wine. This could have potential implications in determining wine quality. For example, after opening a wine bottle, fluorescence parameters could establish when the wine gets spoiled. Our modest first trial showed that wine oxygenation changes some of the fluorescence parameters, but not all. Further measurements and analysis are required to understand these changes and establish an experimental model.

In summary, the studies enabled by this award yielded very promising results and our group plans to continue working on this space, which provides a new and exciting area of research for our time-resolved fluorescence spectroscopy technology that is complementary to our current biomedical applications.