pH-Dependent Changes in Structural Stabilities of Bt Cry1Ac Toxin and Contrasting Model Proteins following Adsorption on Montmorillonite.

The environmental fate of insecticidal Cry proteins, including time-dependent conservation of biological properties, results from their structural stability in soils. The complex cascade of reactions involved in biological action requires Cry proteins to be in solution. However, the pH-dependent changes in conformational stability and the adsorption-desorption mechanisms of Cry protein on soil minerals remain unclear. We used Derjaguin-Landau-Verwey-Overbeek (DLVO) calculation and differential scanning calorimetry to interpret the driving forces and structural stabilities of Cry1Ac and two contrasting model proteins adsorbed by montmorillonite. The structural stability of Cry1Ac is closer to that of the "hard" protein, α-chymotrypsin, than that of the "soft" bovine serum albumin (BSA). The pH-dependent adsorption of Cry1Ac and α-chymotrypsin could be explained by DLVO theory, whereas the BSA adsorption deviated from it. Patch-controlled electrostatic attraction, hydrophobic effects, and entropy changes following protein unfolding on a mineral surface could contribute to Cry1Ac adsorption. Cry1Ac, like chymotrypsin, was partly denatured on montmorillonite, and its structural stability decreased with an increase in pH. Moreover, small changes in the conformational heterogeneity of both Cry1Ac and chymotrypsin were observed following adsorption. Conversely, adsorbed BSA was completely denatured regardless of the solution pH. The moderate conformational rearrangement of adsorbed Cry1Ac may partially explain why the insecticidal activity of Bt toxin appears to be conserved in soils, albeit for a relatively short time period.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

Multimethod Approach for Extensive Characterization of Gallnut Tannin Extracts.

Gallotannins extracted from gallnuts are commonly added to wine to improve its properties. They consist of mixtures of galloylester derivatives of glucose. However, their composition and properties are not well-established. In this study, methods based on liquid chromatography coupled to ultraviolet-visible detection and mass spectrometry, size-exclusion chromatography, and one-dimensional (31P) and two-dimensional (1H diffusion ordered spectroscopy, 31P total correlated spectroscopy, and 1H/13C heteronuclear single-quantum correlation and heteronuclear multiple-bond correlation) nuclear magnetic resonance spectroscopies have been implemented for extensive chemical characterization of three commercial gallnut tannin extracts. Differences in the proportions of the different constituents (gallic, digallic, and trigallic acids and galloylglucose derivatives) and in the structure and molecular weight distributions of gallotannins were demonstrated between the three extracts, with chains containing 8.5, 12.2, and 12.4 galloyl groups on average for TAN A, TAN B1, and TAN B2, respectively. The antioxidant capacities of the extracts, evaluated using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) method, were similar and related mostly to their total tannin content, with only a limited impact of the tannin composition.

Interactions between flavan-3-ols and poly(L-proline) studied by isothermal titration calorimetry: effect of the tannin structure.

Interactions of proline-rich proteins (PRPs) with flavan-3-ols was studied using poly(L-proline) as a model protein by means of isothermal titration calorimetry (ITC). Several parameters were varied: (i) the galloylation and B-ring trihydroxylation of the flavan-3-ols (catechin, epicatechin, epicatechin gallate, and epigallocatechin gallate) and (ii) the degree of polymerization (monomers were compared to a mixture of oligomers with average degree of polymerization of 3.85). Large differences were observed between the flavan-3-ol monomers: no enthalpy change was measured when catechin and epicatechin were titrated by poly(L-proline), whereas thermodynamic parameters were determined in the case of galloylated monomers and mixture of oligomers. Stoichiometry ranged from 1 oligomer bound for each 12 proline units to 1 galloylated monomer for each 8 or 10 proline units. Association constants were in the range of 10(4)-10(5) M(-1), indicating a relatively high affinity of galloylated flavanols toward poly(L-proline), and the coexistence of both enthalpy- and entropy-driven phenomena was suggested. Finally, the binding of grape seed tannins to proteins was shown to be a cooperative process.

Interactions between a non glycosylated human proline-rich protein and flavan-3-ols are affected by protein concentration and polyphenol/protein ratio.

Interactions between salivary proline-rich proteins and tannins are involved in astringency, which is one of the most important organoleptic sensations perceived when drinking wine or tea. This work aimed to study interactions between a recombinant human salivary proline-rich protein, IB-5, and a flavan-3-ol monomer, epigallocatechin gallate (EGCG). IB-5 presented the characteristics of natively unfolded proteins. Interactions were studied by dynamic light scattering, isothermal titration microcalorimetry, and circular dichroism. The interaction mechanism was dependent on protein concentration. At low concentrations, a three-stage mechanism was evidenced. Saturation of the interaction sites (first stage) was followed by protein aggregation into metastable colloids at higher EGCG/protein ratios (second stage). Further increasing this ratio led to haze formation (third stage). At low ratios, a disorder-to-order transition of IB-5 structure upon binding was evidenced. At high protein concentrations, direct bridging between proteins and EGCG was observed, resulting in significantly lower aggregation and turbidity thresholds.

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.

Grape seed and apple tannins: emulsifying and antioxidant properties.

Tannins are natural antioxidants found in plant-based foods and beverages, whose amphiphilic nature could be useful to both stabilize emulsions and protect unsaturated lipids from oxidation. In this paper, the use of tannins as antioxidant emulsifiers was studied. The main parameters influencing the stability of emulsions (i.e. tannins structure and concentration, aqueous phase pH, and ionic strength) were identified and optimized. Oil in water emulsions stabilized with tannins were compared with those stabilized with two commercial emulsifying agents, poly(vinyl alcohol) (PVA) and polyoxyethylene hydrogenated castor oil. In optimized conditions, the condensed tannins allowed to obtain a stability equivalent to that of PVA. Tannins presented good antioxidant activity in oil in water emulsion, as measured by the conjugated autoxidizable triene (CAT) assay.

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.

Probing the micellar solubilisation and inter-micellar exchange of polyphenols using the DPPH· free radical.

Encapsulation of polyphenols can be used for improving their stability and targeting. We present here a spectrophotometric method to probe the micellar solubilisation and inter-micellar exchange of polyphenols using the 2,2-diphenyl-1-picrylhydrazyl (DPPH·) free radical as a visible probe. Our method relies on the partitioning of DPPH· into micelles, on the reduction of DPPH· by polyphenols, and on the change in absorbance of DPPH· when reduced/oxidised. Hence, an absorbance drop at 528 nm gives evidence of the co-localisation of polyphenols and DPPH· in micelles. Using catechin and sodium dodecyl sulfate (SDS) as model molecules, we have shown that the reduction stoichiometry increases up to the critical micelle concentration (CMC) of SDS, where it reaches a plateau: this is due to the solubilisation of catechin in pre-micellar aggregates and then in micelles. The initial rate of reduction increases with increasing SDS concentration up to the CMC and then decreases due to a dilution effect.

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.

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.