Exploring the influence of S. cerevisiae mannoproteins on wine astringency and color: Impact of their polysaccharide part.

The impact of the polysaccharide moiety of mannoproteins (MPs) on the color and astringency of red wines was studied respectively through spectrophotometry and their impact on tannin interactions with BSA. To this end, MPs with conserved native structures from four different Saccharomyces cerevisiae strains were used: a Wild-Type strain (BY4742, WT) taken as reference, mutants ΔMnn4 (with no mannosyl-phosphorylation) and ΔMnn2 (linear N-glycosylation backbone), and a commercial enological strain. MPs affected tannin-BSA interactions by delaying aggregation kinetics. To achieve it, a well-balanced density/compactness of the polysaccharide moiety of MPs was a key factor. MP-WT and MP-Mnn2 acted as weak copigments and induced a slight increase in the absorbance of Malvidin-3-O-Glucoside. The same MPs also promoted a synergistic effect during the copigmentation of Quercetin-3-O-Glucoside with Malvidin-3-O-Glucoside. The intensity of these hyperchromic effects was related to the accessibility of anthocyanins to negatively charged mannosyl-phosphate groups within the polysaccharide moiety.

Diffusion of phenolic compounds during a model maceration in winemaking: role of flesh and seeds.

BACKGROUND: During red winemaking, diffusion of phenolic compounds from the grape berry cells into the liquid phase occurs simultaneously with the adsorption of the same compounds onto the pulp. In previous studies, we quantified the proportions of polyphenols diffusing from the skins and then assessed the amounts that can be fixed by the pulp. In this work, we added the impact of seeds, also present during vinification, by carrying out macerations in a model medium with the following berry compartments: skins, seeds, skins + seeds, skins + seeds + pulp. RESULTS: Interestingly, the seeds alone released a rather high amount of polyphenols. As soon as they were in the presence of cell walls of skin/flesh, and/or anthocyanins, the concentration of seed tannins in the solution dropped dramatically, due to a combined effect of adsorption and/or precipitation and/or chemical reactions. The pulp certainly adsorbed tannins, but they also tended to shift the extraction equilibria, and it seems that more tannins could be extracted from skins and seeds when pulp was present. Polyphenol amounts extracted in model systems with skins + seeds + pulp were close to what was extracted in microvinification. CONCLUSION: These model experiments reflect relatively well extraction during microvinification experiments and highlight the respective impact of the grape berry's different compartments in the wine's final phenolic composition as well as some of the mechanisms involved. © 2022 Society of Chemical Industry.

Focus on the relationships between the cell wall composition in the extraction of anthocyanins and tannins from grape berries.

Concentrations of anthocyanins and tannins after extraction from berries in wines and from skin macerations in model solutions have been studied for two grape varieties, two maturation levels and two vintages berries. Characterization of the cell wall polysaccharides has also been performed, the classical method based on the analysis of the neutral sugars after depolymerization being completed by a comprehensive microarray polymer profiling (CoMPP). Extraction was lower in model solutions than in wines, with the same ranking: non acylated anthocyanins> tannins > p-coumaroylated anthocyanins. The polysaccharidic composition suggested a role of homogalacturonans, rhamnogalacturonans and extensins in the extraction process. A global explanation of the interactions between anthocyanins, tannins and polysaccharides is proposed.

Multi-method study of the impact of fermentation on the polyphenol composition and color of Grenache, Cinsault, and Syrah rosé wines.

Rosé wines show large color diversity, due to different phenolic pigment compositions. However, the mechanisms responsible for such diversity are poorly understood. The present work aimed at investigating the impact of fermentation on the color and composition of rosé wines made from Grenache, Cinsault, and Syrah grapes. Targeted MS analysis showed large varietal differences in must and wine compositions, with higher concentrations of anthocyanins and flavanols in Syrah. UV-visible spectrophotometry and size exclusion chromatography data indicated that Grenache and Cinsault musts contained oligomeric pigments derived from hydroxycinnamic acids and flavanols which were mostly lost during fermentation due to adsorption on lees. Syrah must color was mainly due to anthocyanins which were partly converted to derived pigments through reactions with yeast metabolites with limited color drop during fermentation. This work highlighted the impact of must composition, reflecting varietal characteristics, on changes occurring during fermentation and consequently wine color.

Impact of the variety on the adsorption of anthocyanins and tannins on grape flesh cell walls.

BACKGROUND: During winemaking, after extraction from the skins, anthocyanins and tannins adsorb onto the pulp flesh cell walls. The present study aimed to quantify the amounts adsorbed and their impact on wine composition, the impact of variety and ethanol on adsorption, and whether the presence of anthocyanins plays a role and impacts tannin adsorption. RESULTS: Anthocyanin and tannin fractions obtained by mimicking winemaking conditions were mixed with fresh flesh cell walls of two varieties: Carignan and Grenache. Adsorption isotherms were measured. Adsorption of tannins was higher with Carignan than with Grenache and decreased when the ethanol content increased. In comparison, anthocyanins were adsorbed in small amounts, and their mixing with tannins had no impact on their adsorption. The differences were related to differences in pulp cell wall composition, particularly in terms of extensins and arabinans. CONCLUSION: Adsorption of tannins, which can reach 50% of the initial amount, depends on the pulp cell wall composition. This needs to be investigated further. © 2021 Society of Chemical Industry.

Structural characteristics of Saccharomyces cerevisiae mannoproteins: Impact of their polysaccharide part.

While they have many properties of interest in enology, the structure-function relationships of mannoproteins and the part played by their polysaccharide moiety are not yet well understood. Mannoproteins (MP) extracted with ß-glucanase from a laboratory yeast strain (WT), two of its mutants (Mnn2 with unbranched N-glycosylated chains and Mnn4 without mannosyl-phosphorylation), and an enological strain (Com) were purified and thoroughly characterized. The protein moiety of the four MPs had the same amino acid composition. Glycosyl-linkage and net charge analyses confirmed the expected differences in mutant strain MPs. MP-Com had the highest mannose/glucose ratio followed by MP-WT/MP-Mnn4, and MP-Mnn2 (13.5 > 5.6 ≈ 5.2 > 2.2). The molar mass dependencies of Rg, Rh, and [η], determined through HPSEC-MALLS-QELS-Viscosimetry, revealed specific conformational properties of mannoproteins related to their nature of highly branched copolymers with two branching levels. It also clearly showed structural differences between MP-Com, MP-WT/Mnn4, and MP Mnn2, and differences between two populations within the four mannoproteins.

Impact of grape variety, berry maturity and size on the extractability of skin polyphenols during model wine-like maceration experiments.

BACKGROUND: Skin cell walls modulate anthocyanin and tannin extraction from grape skins. However, relationships between the composition of alcohol-insoluble cell wall solids (AIS) and extraction are still unclear. Our objectives were to characterize the impact of variety, berry size and ripeness on skin AIS composition (polysaccharides, proteins) and polyphenol extraction during maceration. RESULTS: Grape skin composition and its impact on polyphenol extraction was compared for two varieties - Carignan and Grenache - with skins of berries sorted according to their size and density. Extractions were performed under model wine-like maceration conditions. Fresh skins had similar content of polymeric tannins, but strongly differed in their anthocyanin content (higher in Carignan and in the ripest berries) and composition (higher proportions in coumaroylated anthocyanins in Carignan). Anthocyanin extraction was proportionally much higher in Grenache, which was not just related to the Carignan's higher levels in coumaroylated anthocyanins. Chemical reactions decreased anthocyanin concentrations in solution for both varieties. Tannin extraction for Grenache was slightly higher and faster than for Carignan. Skin AISs differed slightly between the two varieties in their carbohydrate composition and protein content, but not between modalities. Polyphenol analyses in the precipitates evidenced at the end of the maceration and in residual skins highlighted differences between the two varieties and between berries with different ripeness. CONCLUSION: Structural information on the cell wall network and on its changes during maceration, along with a better understanding of the chemical reactions of anthocyanins and tannins, is needed to better relate grape and wine polyphenol composition. © 2020 Society of Chemical Industry.

Wine Thermosensitive Proteins Adsorb First and Better on Bentonite during Fining: Practical Implications and Proposition of Alternative Heat Tests.

Bentonite fining is the most popular treatment used to remove proteins in white and rosé wines. The usual heat test used to adjust the bentonite dose consists of heating the wine during 30 min at 80 °C. At this temperature, all of the proteins are unfolded, and this can lead to an overestimation of the dose. We have shown that proteins adsorb on bentonite in a specific order and, more importantly, that the proteins responsible for haze formation adsorb first. Fluorescence spectroscopy showed that this is due to the structural properties of proteins, which can be classified as hard and soft proteins. Alternative heat tests were performed at a lower temperature (40 °C) and showed a better correlation with accelerated aging. These tests were also less dependent upon the wine pH.

Fate of Anthocyanins and Proanthocyanidins during the Alcoholic Fermentation of Thermovinified Red Musts by Different Saccharomyces cerevisiae Strains.

UV-visible spectrophotometry, size exclusion chromatography, and UHPLC-QqQ-MS were combined to evaluate the respective impacts of chemical changes and adsorption by yeasts on both proanthocyanidins (PA) and anthocyanins during the fermentation of a thermovinified red must by four yeast strains. Results evidenced a sharp decrease in anthocyanins (∼45%) and color intensity (∼50%), along with the formation of pyranoanthocyanins and flavanol-anthocyanin dimers related to yeast metabolism. However, the latter only accounted for 10% of anthocyanin losses. Comparison of spectrophotometry and phloroglucinolysis data underlined the involvement of PAs in chemical changes, related to yeast metabolites but also to direct reactions. Color losses during fermentation in this case study were mostly related to the formation of colorless compounds and to a decrease of copigmentation. Adsorption represented only a small proportion of pigment losses (∼5%) but a major part of those corresponding to total oligomeric/polymeric species, which were specifically involved. Only small differences could be evidenced between the four studied strains.

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.

Interactions of Condensed Tannins with Saccharomyces cerevisiae Yeast Cells and Cell Walls: Tannin Location by Microscopy.

Interactions between grape tannins/red wine polyphenols and yeast cells/cell walls was previously studied within the framework of red wine aging and the use of yeast-derived products as an alternative to aging on lees. Results evidenced a quite different behavior between whole cells (biomass grown to elaborate yeast-derived products, inactivated yeast, and yeast inactivated after autolysis) and yeast cell walls (obtained from mechanical disruption of the biomass). Briefly, whole cells exhibited a high capacity to irreversibly adsorb grape and wine tannins, whereas only weak interactions were observed for cell walls. This last point was quite unexpected considering the literature and called into question the real role of cell walls in yeasts' ability to fix tannins. In the present work, tannin location after interactions between grape and wine tannins and yeast cells and cell walls was studied by means of transmission electron microscopy, light epifluorescence, and confocal microscopy. Microscopy observations evidenced that if tannins interact with cell walls, and especially cell wall mannoproteins, they also diffuse freely through the walls of dead cells to interact with their plasma membrane and cytoplasmic components.

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.

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).

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: ß-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.

Characterization of oxidized tannins: comparison of depolymerization methods, asymmetric flow field-flow fractionation and small-angle X-ray scattering.

Condensed tannins are a major class of plant polyphenols. They play an important part in the colour and taste of foods and beverages. Due to their chemical reactivity, tannins are not stable once extracted from plants. A number of chemical reactions can take place, leading to structural changes of the native structures to give so-called derived tannins and pigments. This paper compares results obtained on native and oxidized tannins with different techniques: depolymerization followed by high-performance liquid chromatography analysis, small-angle X-ray scattering (SAXS) and asymmetric flow field-flow fractionation (AF4). Upon oxidation, new macromolecules were formed. Thioglycolysis experiments showed no evidence of molecular weight increase, but thioglycolysis yields drastically decreased. When oxidation was performed at high concentration (e.g., 10 g L(-1)), the weight average degree of polymerization determined from SAXS increased, whereas it remained stable when oxidation was done at low concentration (0.1 g L(-1)), indicating that the reaction was intramolecular, yet the conformations were different. Differences in terms of solubility were observed; ethanol being a better solvent than water. We also separated soluble and non-water-soluble species of a much oxidized fraction. Thioglycolysis showed no big differences between the two fractions, whereas SAXS and AF4 showed that insoluble macromolecules have a weight average molecular weight ten times higher than the soluble ones.

Effects of ionic strength and sulfate upon thermal aggregation of grape chitinases and thaumatin-like proteins in a model system.

Consumers expect white wines to be clear. During the storage of wines, grape proteins can aggregate to form haze. These proteins, particularly chitinases and thaumatin-like proteins (TL-proteins), need to be removed, and this is done through adsorption by bentonite, an effective but inefficient wine-processing step. Alternative processes are sought, but, for them to be successful, an in-depth understanding of the causes of protein hazing is required. This study investigated the role played by ionic strength (I) and sulfate toward the aggregation of TL-proteins and chitinases upon heating. Purified proteins were dissolved in model wine and analyzed by dynamic light scattering (DLS). The effect of I on protein aggregation was investigated within the range from 2 to 500 mM/L. For chitinases, aggregation occurred during heating with I values of 100 and 500 mM/L, depending on the isoform. This aggregation immediately led to the formation of large particles (3 µm, visible haze after cooling). TL-protein aggregation was observed only with I of 500 mM/L; it mainly developed during cooling and led to the formation of finite aggregates (400 nm) that remained invisible. With sulfate in the medium chitinases formed visible haze immediately when heat was applied, whereas TL-proteins aggregated during cooling but not into particles large enough to be visible to the naked eye. The data show that the aggregation mechanisms of TL-proteins and chitinases are different and are influenced by the ionic strength and ionic content of the model wine. Under the conditions used in this study, chitinases were more prone to precipitate and form haze than TL-proteins.

Tannin oxidation: intra- versus intermolecular reactions.

Grape and apple condensed tannin fractions were autoxidized at high concentrations (5 g/L) in aqueous solutions and analyzed by thiolysis (depolymerization followed by HPLC analysis) and small angle X-ray scattering (SAXS). Structural parameters of native (unoxidized) tannin polymers were derived from SAXS according to the wormlike chain model: the length per monomer is 15 A, the length of the statistical segment 17 A, and the cross section of the macromolecule has a radius within the range 3-4.5 A. The rather short length of the statistical segment is an effect of the different location of interflavanol linkages, which cause a loss of orientational correlation between successive monomers. Oxidation created new bonds that were resistant to thiolysis, and, according to thiolysis, some of these new bonds were intramolecular. However, according to SAXS, oxidation at high tannin concentration caused the weight average degree of polymerization to increase, indicating that intermolecular reactions took place as well, creating larger macromolecules. In the case of the smaller grape seed tannins, these intermolecular reactions took place "end to end" leading to the formation of longer linear macromolecules, at least in the earlier stages of oxidation. In the case of the larger apple tannins, the SAXS patterns were characteristic of larger branched macromolecules. Accordingly, the intermolecular reactions were mainly "end to middle". This is in agreement with the higher probabilities of "end to middle" reactions arising from a higher ratio extension unit/terminal unit in the latter case.

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.