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.