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.