Aude Vernhet

Aggregation of a Proline-Rich Protein Induced by Epigallocatechin Gallate and Condensed Tannins: Effect of Protein Glycosylation

Astringency is one of the most important organoleptic qualities of numerous beverages, including red wines. It is generally thought to originate from interactions between tannins and salivary proline-rich proteins (PRPs). In this work interactions between a glycosylated PRP, called II-1, and flavan-3-ols were studied in aqueous solutions and at a colloidal level, by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). The flavan-3-ols were a monomer, epigallocatechin gallate (EGCG), and polymerized flavan-3-ol fractions extracted from grape seeds. In aqueous solutions containing EGCG and protein II-1, protein aggregation took place when protein concentration and the EGCG/protein ratio exceeded a threshold. The aggregates had a small size, comparable with the dimensions of protein monomers, and formed stable dispersions (no phase separation). Most proteins remained free in solution. This behavior is in sharp contrast with the phase separation observed for nonglycoslated PRP in the same conditions. Moreover, this slight aggregation of II-I in the presence of EGCG was disrupted by the addition of 12% ethanol. Increasing the flavan-3-ol molecular weight strongly enhanced II-I/tannin aggregation: the threshold was at a lower protein concentration (0.2 mg/mL) and a lower tannin/protein ratio. Still, in most cases, and in contrast with that observed with a nonglycosylated PRP, the aggregates remained of discrete size and stable. Only at low ethanol content (2%) did the addition of tannin polymers finally lead to phase separation, which occurred when the molar ratio of tannins to proteins exceeded 12. This systematic effect of ethanol confirmed the strong effect of cosolvents on protein/tannin interactions.

Stability of White Wine Proteins: Combined Effect of pH, Ionic Strength, and Temperature on Their Aggregation

Protein haze development in white wines is an unacceptable visual defect attributed to slow protein unfolding and aggregation. It is favored by wine exposure to excessive temperatures but can also develop in properly stored wines. In this study, the combined impact of pH (2.5-4.0), ionic strength (0.02-0.15 M), and temperature (25, 40, and 70 °C) on wine protein stability was investigated. The results showed three classes of proteins with low conformational stability involved in aggregation at room temperature: β-glucanases, chitinases, and some thaumatin-like protein isoforms (22-24 kDa). Unexpectedly, at 25 °C, maximum instability was observed at the lower pH, far from the protein isoelectric point. Increasing temperatures led to a shift of the maximum haze at higher pH. These different behaviors could be explained by the opposite impact of pH on intramolecular (conformational stability) and intermolecular (colloidal stability) electrostatic interactions. The present results highlight that wine pH and ionic strength play a determinant part in aggregation mechanisms, aggregate characteristics, and final haze.

Protein Aggregation in White Wines: Influence of the Temperature on Aggregation Kinetics and Mechanisms

High temperatures (typically 80 °C) are widely used to assess wine stability with regard to protein haze or to study mechanisms involved in their formation. Dynamic light scattering experiments were performed to follow aggregation kinetics and aggregate characteristics in white wines at different temperatures (30-70 °C). Aggregation was followed during heating and cooling to 25 °C. Results were coupled with the study of the time-temperature dependence of heat-induced protein aggregation. At low temperature (40 °C), aggregation developed during heating. Colloidal equilibria were such that attractive interactions between species led to the rapid formation of micrometer-sized aggregates. At higher temperatures (60 and 70 °C), enhanced protein precipitation was expected and observed. However, high temperatures prevented aggregation, which mainly developed during cooling. Depending on the wine, cooling induced the formation of sub-micronic metastable aggregates stabilized by electrostatic repulsions, or the rapid formation of micrometer-sized aggregates, prone to sedimentation.

Sorption of grape proanthocyanidins and wine polyphenols by yeasts, inactivated yeasts, and yeast cell walls.

Inactivated yeast fractions (IYFs) can be used in enology to improve the stability and mouthfeel of red wines. However, information concerning the mechanisms involved and the impact of the IYF characteristics is scarce. Adsorption isotherms were used to investigate interactions between grape proanthocyanidin fractions (PAs) or wine polyphenols (WP) and a commercial yeast strain (Y), the inactivated yeast (IY), the yeast submitted to autolyzis and inactivation (A-IY), and the cell walls obtained by mechanical disruption (CW). High affinity isotherms and high adsorption capacities were observed for grape PAs and whole cells (Y, IY, and A-IY). Affinity and adsorbed amount were lower with wine PAs, due to chemical changes occurring during winemaking. By contrast to whole cells, grape PAs and WP adsorption on CW remained very low. This raises the issue of the part played by cell walls in the interactions between yeast and proanthocyanidins and suggests the passage of the latter through the wall pores and their interaction with the plasma membrane.

White Wine Proteins: How Does the pH Affect Their Conformation at Room Temperature?

Our studies focused on the determination of aggregation mechanisms of proteins occurring in wine at room temperature. Even if the wine pH range is narrow (2.8 to 3.7), some proteins are affected by this parameter. At low pH, the formation of aggregates and the development of a haze due to proteins sometimes occur. The objective of this work was to determine if the pH impacted the conformational stability of wine proteins. Different techniques were used: circular dichroism and fluorescence spectroscopy to investigate the modification of their secondary and tertiary structure and also SAXS to determine their global shape. Four pure proteins were used, two considered to be stable (invertase and thaumatin-like proteins) and two considered to be unstable (two chitinase isoforms). Two pH values were tested to emphasize their behavior (pH 2.5 and 4.0). The present work highlighted the fact that the conformational stability of some wine proteins (chitinases) was impacted by partial modifications, related to the exposure of some hydrophobic sites. These modifications were enough to destabilize the native state of the protein. These modifications were not observed on wine proteins determined to be stable (invertase and thaumatin-like proteins).

p-Hydroxyphenyl-pyranoanthocyanins: An Experimental and Theoretical Investigation of Their Acid-Base Properties and Molecular Interactions.

The physicochemical properties of the wine pigments catechyl-pyranomalvidin-3-O-glucoside (PA1) and guaiacyl-pyranomalvidin-3-O-glucoside (PA2) are extensively revisited using ultraviolet (UV)-visible spectroscopy, dynamic light scattering (DLS) and quantum chemistry density functional theory (DFT) calculations. In mildly acidic aqueous solution, each cationic pigment undergoes regioselective deprotonation to form a single neutral quinonoid base and water addition appears negligible. Above pH = 4, both PA1 and PA2 become prone to aggregation, which is manifested by the slow build-up of broad absorption bands at longer wavelengths (λ ≥ 600 nm), followed in the case of PA2 by precipitation. Some phenolic copigments are able to inhibit aggregation of pyranoanthocyanins (PAs), although at large copigment/PA molar ratios. Thus, chlorogenic acid can dissociate PA1 aggregates while catechin is inactive. With PA2, both chlorogenic acid and catechin are able to prevent precipitation but not self-association. Calculations confirmed that the noncovalent dimerization of PAs is stronger with the neutral base than with the cation and also stronger than π–π stacking of PAs to chlorogenic acid (copigmentation). For each type of complex, the most stable conformation could be obtained. Finally, PA1 can also bind hard metal ions such as Al3+ and Fe3+ and the corresponding chelates are less prone to self-association.

Protein/Polysaccharide Interactions and Their Impact on Haze Formation in White Wines

Proteins in white wines may aggregate and form hazes at room temperature. This was previously shown to be related to pH-induced conformational changes and to occur for pH <3.5. The aim of the present work was to study the impact of wine polysaccharides on pH-induced haze formation by proteins but also the consequences of their interactions with these proteins on the colloidal stability of white wines. To this end, model systems and purified global pools of wine proteins and polysaccharides were used first. Kinetics of aggregation, proteins involved, and turbidities related to final hazes were monitored. To further identify the impact of each polysaccharide, fractions purified to homogeneity were used in a second phase. These included two neutral (mannoprotein and arabinogalactan) and two negatively charged (rhamnogalacturonan II dimer (RG-II) and arabinogalactan) polysaccharides. The impact of major wine polysaccharides on wine protein aggregation at room temperature was clearly less marked than those of the pH and the ionic strength. Polysaccharides modulated the aggregation kinetics and final haziness, indicating that they interfere with the aggregation process, but could not prevent it.

Review: Characterization and Role of Grape Solids during Alcoholic Fermentation under Enological Conditions

During wine production, grape solids have a large impact on the fermentation characteristics and organoleptic qualities of the resulting wine. Here we review the research on grape solids. We begin by focusing on the origin, physical characteristics, and composition of these solids and on the changes in these factors that occur during fermentation. We then consider the impact of solids on fermentation, the role of sterols, the control of solids, and interactions between solids and other nutrients. Solids exert their effects on alcoholic fermentation mainly by modulating lipid supply. The balance between solids content and nitrogen is a key factor in fermentation control. The study of grape solids is in its infancy and requires further development. Knowledge of the composition of these solids and of sterol uptake mechanisms by yeast should facilitate improvements in fermentation control.

Condensed Tannin Changes Induced by Autoxidation: Effect of the Initial Degree of Polymerization and Concentration

Condensed tannins are a major class of polyphenols and play an important part in organoleptic properties of beverages. Because of their structure, they are chemically reactive. During food processing, reactions take place, leading to structural changes of the native structures to give modified tannins and pigments. Average degrees of polymerization (DPs) determined by standard depolymerization methods become irrelevant, because bonds created from oxidation are uncleavable. Small-angle X-ray scattering was used to determine the conformation of native and autoxidized tannins and assess the impact of tannins initial DP and concentration on changes induced by autoxidation. Different behaviors were observed: (i) slight increase of the DP when tannins were oxidized in dilute solutions; (ii) increase of the DP with tannins in concentrated solutions, leading to the formation of longer linear chains or branched macromolecules depending on the initial DP.