Probing protein-tannin interactions by isothermal titration microcalorimetry

Isothermal titration microcalorimetry (ITC) has been applied to investigate protein-tannin interactions. Two hydrolyzable tannins were studied, namely myrabolan and tara tannins, for their interaction with bovine serum albumin (BSA), a model globular protein, and gelatin, a model proline-rich random coil protein. Calorimetry data indicate that protein-tannin interaction mechanisms are dependent upon the nature of the protein involved. Tannins apparently interact nonspecifically with the globular BSA, leading to binding saturation at estimated tannin/BSA molar ratios of 48:1 for tara- and 178:1 for myrabolan tannins. Tannins bind to the random coil protein gelatin by a two-stage mechanism. The energetics of the first stage show evidence for cooperative binding of tannins to the protein, while the second stage indicates gradual saturation of binding sites as observed for interaction with BSA. The structure and flexibility of the tannins themselves alters the stoichiometry of the interaction, but does not appear to have any significant affect on the overall binding mechanism observed. This study demonstrates the potential of ITC for providing an insight into the nature of protein-tannin interactions.     

Hydrolyzable tannin structures influence relative globular and random coil protein binding strengths

Binding parameters for the interactions of pentagalloyl glucose (PGG) and four hydrolyzable tannins (representing gallotannins and ellagitannins) with gelatin and bovine serum albumin (BSA) have been determined from isothermal titration calorimetry data. Equilibrium binding constants determined for the interaction of PGG and isolated mixtures of tara gallotannins and of sumac gallotannins with gelatin and BSA were of the same order of magnitude for each tannin (in the range of 10(4)-10(5) M(-1) for stronger binding sites when using a binding model consisting of two sets of multiple binding sites). In contrast, isolated mixtures of chestnut ellagitannins and of myrabolan ellagitannins exhibited 3-4 orders of magnitude greater equilibrium binding constants for the interaction with gelatin (approximately 2 x 10(6) M(-1)) than for that with BSA (approximately 8 x 10(2) M(-1)). Binding stoichiometries revealed that the stronger binding sites on gelatin outnumbered those on BSA by a ratio of at least approximately 2:1 for all of the hydrolyzable tannins studied. Overall, the data revealed that relative binding constants for the interactions with gelatin and BSA are dependent on the structural flexibility of the tannin molecule.     

Effects of protein-polyphenol interactions on beverage haze, stabilization, and analysis

The haze-forming activity of a polypeptide depends greatly on its proline content. Haze-forming polyphenols have at least two binding groups, each of which has at least two hydroxy groups on an aromatic ring. The protein/polyphenol ratio has a strong influence on the amount of haze formed; the largest amount occurs when the numbers of polyphenol binding ends and protein binding sites are nearly equal. This has important consequences for turbidimetric methods used to measure haze-active proteins and polyphenols in beverages. The ratio also influences the effectiveness of a number of stabilization procedures.     

Characterization of haze-active proteins in apple juice

The nature of the haze-active protein (HAP) in apple juice was investigated. Heat treatment removed protein indiscriminately while polyvinylpolypyrrolidone (PVPP) treatment was fairly specific for proteins of 15 and 28 kDa. Presumably, the PVPP bound to polyphenols, which in turn were complexed with protein. Three candidate apple HAPs were isolated. Two were extracted from juice with acetone and fractionated by hydrophobic interaction chromatography and solid phase extraction with C18 (HAP I) or SAX (HAP II) material. Hydroxyproline-rich protein was extracted from apple tissue (HAP III). The order of haze formation with tannic acid was gliadin > HAP III > HAP II > HAP I > bovine serum albumin, which shows increasing haze formation with increasing proline content. The sizes of HAP I, II, and III were 28, 15, and 12 kDa; the first two corresponded to the sizes of proteins removed by PVPP treatment and are involved in juice haze formation.     

Carbohydrates inhibit salivary proteins precipitation by condensed tannins

Condensed tannins are a group of polyphenols that are associated with the astringency sensation, as they readily interact and precipitate salivary proteins. As this interaction is affected by carbohydrates, the aim of this work was to study the effect of some carbohydrates used in the food industry [arabic gum (AG), pectin, and poligalacturonic acid (PGA)] on the salivary proteins/grape seed procyanidins interaction. This was assessed monitoring the salivary proteins that remain soluble in the presence of condensed tannins with the addition of carbohydrates (HPLC) and analysis of the respective precipitates (SDS-PAGE). The results show that pectin was the most efficient in inhibiting protein/tannin precipitation, followed by AG and PGA. The results suggest that pectin and PGA exert their effect by formation of a ternary complex protein/polyphenol/carbohydrate, while AG competes with proteins for tannin binding (competition mechanism). The results also point out that both hydrophilic and hydrophobic interactions are important for the carbohydrate effects.     

Influence of wine pectic polysaccharides on the interactions between condensed tannins and salivary proteins

Alpha-amylase, a major human salivary protein, and IB8c, a representative of the proline-rich proteins, were obtained by isolation from saliva and by solid-phase synthesis, respectively. The interactions between these proteins and condensed tannins isolated from grape seeds were studied at different protein and tannin concentrations by measuring their aggregation. Pectic polysaccharides were isolated from wine, and their effect on protein tannin aggregation was assessed. The results presented in this study showed that the most acidic fractions of arabinogalactan proteins have the ability to inhibit the formation of aggregates between the grape seed tannins and the two different salivary proteins. Rhamnogalacturonan II has the same ability toward alpha-amylase but not IB8c under the conditions of the present study. Polysaccharides show effects at concentrations at which they are present in wine, which could mean an influence in wine astringency. The interaction between condensed tannins and alpha-amylase is differently affected by ionic strength when compared with IB8c.     

Quantitative analysis of polymeric procyanidins (Tannins) from grape (Vitis vinifera) seeds by reverse phase high-performance liquid chromatography

A reverse phase C(18) HPLC method with potential for high automated throughput has been developed for the quantitative analysis of polymeric procyanidins (tannins) in grape seed extracts. Chromatography gave rise to 13 distinct UV-absorbing peaks with good baseline separation. The UV-absorbing peak eluting last is distinct and therefore easily quantified. Biochemical analyses including ultrafiltration, protein precipitation, and Sephadex LH20 chromatography combined with electrospray mass spectrometric analyses establish that this peak predominantly contains polymeric procyanidins. The polymers, which appear to be galloylated to various degrees and seem to fragment in a characteristic manner during electrospray mass spectrometry, are well separated from catechins and procyanidin oligomers of up to 4 units. The recovery of polymeric grape seed tannins with this HPLC method was 86%, which is similar to the 89% recovery achieved with commercial quebracho tannins. The concentration of tannins in seeds from ripe Vitis vinifera cv. Shiraz grapes ranged from 1360 to 2830 mg/kg of berries.     

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.     

Tannin-protein complexes as radical scavengers and radical sinks

The 2,2'-azinobis(3-ethylbenzothiazoline 6-sulfonic acid) radical cation (ABTS(*)(+)) decolorization assay has been used to determine the antioxidant activity of the polyphenol epicatechin(16) (4 --> 8) catechin (procyanidin, PC) alone or in complex with the model proteins bovine serum albumin (BSA) or gelatin. PC had a molar antioxidant capacity of approximately 54, 92, or 108 radicals at pH values of 3.0, 4.9, or 7.4, respectively. Radical scavenging occurred via a rapid step followed by a slow step. Interaction with gelatin reduced the rate of rapid scavenging by 50% (PC-BSA mixtures reduced by 15%). Inhibition paralleled formation of precipitable PC-protein complexes over a range of protein/PC ratios. However, inhibition was virtually overcome in 10 min. Reaction with ABTS(*)(+) converted the PC-protein complexes from a dissociable form to a form resistant to dissociation by strong denaturants such as SDS. This study demonstrates that PC is a potent ABTS(*)(+) scavenger even when bound to protein and that the complexes may act as a radical sink within the gastrointestinal tract.     

Glomalin-related soil protein: Assessment of current detection and quantification tools

Despite the widely acknowledged importance of arbuscular mycorrhizal fungi (AMF) in soil ecology, quantifying their biomass and presence in field soils is hindered by tedious techniques. Hence biochemical markers may be useful, among which glomalin-related soil protein (GRSP) could show a particular promise. Presently GRSP is operationally defined, its identification resting solely on the methods used to extract it from soil (citric acid buffer and autoclaving) and the assays (Bradford/enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody) utilized to detect it. The current assumption is that most non-heat stable soil proteins except glomalin are destroyed during the harsh extraction procedure. However, this critical assumption has not been tested. The purpose of this research was to challenge the GRSP extraction process to determine the accuracy of the Bradford method as a measure of glomalin; and to provide some assessment of the specificity of the ELISA monoclonal antibody. In two studies we spiked soil samples either with known quantities of a glycoprotein (BSA: bovine serum albumin) or with leaf litter from specific sources. After extraction 41-84% of the added BSA was detected with the Bradford method. This suggests that the currently used extraction procedure does not eliminate all non-glomalin proteins. Also, ELISA cross-reactivity against BSA was limited, ranging from 3% to 14%. Additions of leaf litter also significantly influenced GRSP extraction and quantification suggesting that plant-derived proteins, as would occur in the field, had a similar effect as BSA. Litter additions decreased the immunoreactive protein values, suggesting interference with antibody recognition. We conclude that the use of GRSP, especially Bradford-based detection, in the assessment of AMF-derived substances within field soils is problematic, it may be inappropriate in situations of significant organic matter additions.     

Characterization of a calcium-soluble protein fraction from yellow mustard (Sinapis alba) seed meal with potential application as an additive to calcium-rich drinks

A calcium-soluble protein isolate (CSPI) was prepared from the supernatant obtained after addition of 0.75 M calcium chloride to a pH 5.0 aqueous extract of yellow mustard (Sinapis alba) seed meal. Total amino acid analysis showed that the CSPI has significantly higher (p < 0.05) contents of glutamic acid + glutamine, cysteine, and proline when compared to the precipitated, calcium-insoluble proteins. Peptide mass fingerprinting of tryptic peptides of the major polypeptides by mass spectrometry indicated that the CSPI is composed mainly of cruciferin proteins with a contribution from napins (the major allergenic proteins of S. alba). The S. alba CSPI had significantly higher (p < 0.05) protein solubility and emulsion formation ability in the presence of 0.75 M calcium chloride when compared to similar isolates prepared from Brassica juncea (brown mustard) and soybean seed meals. We suggest that the S. alba CSPI could be used to prepare calcium-fortified high protein liquid products. However, the presence of allergenic proteins in this extract may limit its widespread food use.     

Evidence for protein degradation by Botrytis cinerea and relationships with alteration of synthetic wine foaming properties

Botrytis cinerea is an important fungal pathogen particularly dreaded in the cool climate vineyard. It is responsible for important damage, especially the decrease in foamability of sparkling wines, such as Champagne. Different studies have shown that proteins are largely involved in the stabilization of Champagne foam despite their low concentration. Other works demonstrated changes in the electrophoretic characteristics of must proteins originating from botrytized grapes, although the cause of such alterations was never explained. In the first part of this study, results showed the release by B. cinerea of 3.5 mg/L total proteins in a synthetic liquid medium. Among these proteins, the presence of a protease activity on bovine serum albumin (BSA) and must proteins was demonstrated by using a colorimetric method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the model wine, the Bradford method showed a BSA loss of 66% after 24 h and a loss of 96% after 120 h. In the same model wine, the soluble must protein concentration decreased by 35% after 1 week and by 53% after 2 weeks while the control showed no protein loss. B. cinerea proteases were then able to degrade BSA and must proteins and were above all active at must and wine pH and in the presence of ethanol and SO(2). The second part of this work was dedicated to the relationship between the presence of B. cinerea proteases and its effects on the synthetic wine foaming properties. The addition of a B. cinerea culture medium (1/33 v/v) to the synthetic wine containing 21 mg/L soluble grape proteins induced a decrease in foamability by 60% after 1 week. For BSA in the model wine, the foamability decreased by 32% after 24 h and by 95% after 120 h, as shown by the colorimetric method. These experiments demonstrate for the first time the relationship between B. cinerea protease activity and the decrease in wine foaming properties.     

核桃酚类物质和蛋白间的相互作用及其抗氧化活性

酚类物质和蛋白质之间存在多种相互作用力,因此难以被充分提取。该研究以核桃蛋白为研究对象,用不同量的尿素和氯化钠定向破坏核桃酚类物质和核桃蛋白之间的离子键、氢键和疏水作用力,以研究核桃酚类物质和核桃蛋白之间的非共价键结合机制。结果显示在pH值3.0时,77.7%的酚类物质与蛋白以共价键结合,而有34.6%的缩合单宁与核桃蛋白以共价键结合。在pH值8.0时,有65.4%酚类物质和59.4%的缩合单宁与核桃蛋白以非共价键结合。其中疏水作用力是主要的非共价键结合方式。对以不同非共价键与核桃蛋白相结合的酚类物质的抗氧化活性进行测定,结果显示以离子键与蛋白结合的酚类物质的抗氧化活性最强。其中,在pH值3.0条件下与WPI（walnut protein isolates）以离子键结合的酚类物质的DPPH自由基清除能力为5.7%,Fe2+螯合率为5.9%,在pH值8.0条件下与WPI以离子键结合的酚类物质的DPPH自由基清除能力为10.9%, Fe2+螯合率为11.5%。另外,该研究还探讨了在pH值3.0和8.0时,70%(v/v)乙醇和60%(v/v)甲醇提取核桃蛋白中酚类物质的能力,结果表明这两种提取剂不能显著提高酚类物质的提取率,且不能有效得到具有较强抗氧化能力的酚类物质。     

Proteomic analysis of high protein soybean (Glycine max) accessions demonstrates the contribution of novel glycinin subunits

Limited biochemical information is available on soybean accessions that have seed protein content greater than 45% of the seed dry weight. SDS-PAGE analysis of seed proteins from nine soybean accessions revealed significantly higher amount of seed storage proteins in these accessions when compared with that of soybean cultivar Williams 82. High-resolution two-dimensional gel electrophoretic analysis of seed proteins revealed significant differences among several seed storage protein components in these accessions. A total of 51 protein spots were identified using peptide mass fingerprinting (MALDI-TOF MS). The contribution of these proteins to the overall protein content of the accessions was quantified using Delta2D image analysis software. Results showed that among the majority of the nine accessions, the largest difference in higher protein quantity was within the seed 11S storage globulins. The high protein trait from PI407788A was successfully transferred to an experimental line, LG99-469, demonstrating that this trait was transferable and robust.     

Haze formation in model beer systems

The interaction of a haze-active protein (gliadin) and a haze-active polyphenol (tannic acid) was studied in a model beer system in order to investigate the principle mechanisms of haze formation at low temperatures. Low concentrations (g/L) of tannic acid, high concentrations of gliadin, and comparatively high temperatures lead to maximum haze values. When considered on a molar basis, the greatest haze levels are displayed at an approximate 1:1 equivalence of polyphenol and protein. The greater part of haze formation was completed within 0.5 h, irrespective of the concentration of gliadin, the concentration of tannic acid, and the temperature of the model solution.     

Interactions of grape seed tannins with salivary proteins

To evaluate the amount and type of condensed tannins binding salivary proteins, which are supposed to be involved in astringent sensation, model systems allowing further analyses of proteins and condensed tannins were developed. The precipitates formed after addition of grape seed tannins to salivary proteins indicate that a binding interaction occurs. Dissociation of insoluble complexes was achieved by sodium dodecyl sulfate treatment. Thiolysis reaction allowed the quantification and characterization of proanthocyanidins on both the resulting pellet and the supernatant. Binding proteins were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The higher polymerized tannins predominantly precipitated together with the salivary proteins. The condensed tannins remaining in solution were low molecular weight polymers.     

Reactivity of human salivary proteins families toward food polyphenols

Tannins are well-known food polyphenols that interact with proteins, namely, salivary proteins. This interaction is an important factor in relation to their bioavailability and is considered the basis of several important properties of tannins, namely, the development of astringency. It has been generally accepted that astringency is due to the tannin-induced complexation and/or precipitation of salivary proline-rich proteins (PRPs) in the oral cavity. However, this complexation is thought to provide protection against dietary tannins. Neverthless, there is no concrete evidence and agreement about which PRP families (acidic, basic, and glycosylated) are responsible for the interaction with condensed tannins. In the present work, human saliva was isolated, and the proteins existing in saliva were characterized by chromatographic and proteomic approaches (HPLC-DAD, ESI-MS, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and MALDI-TOF). These approaches were also adapted to study the affinity of the different families of salivary proteins to condensed tannins by the interaction of saliva with grape seed procyanidins. The results obtained when all the main families of salivary proteins are present in a competitive assay, like in the oral cavity, demonstrate that condensed tannins interact first with acidic PRPs and statherin and thereafter with histatins, glycosylated PRPs, and bPRPs.     

Mechanism and characteristics of protein release from lactitol-based cross-linked hydrogel

Lactitol-based cross-linked hydrogel was synthesized, and model proteins (alpha-chymotrypsin, beta-lactoglobulin, bovine serum albumin (BSA), and gamma-globulin) were incorporated into the cross-linked hydrogel. The larger-molecular-weight proteins have lower diffusivity (D(e)) in the hydrogel. Increasing temperature accelerated the diffusion rate of proteins; however, the diffusion did not follow the Arrhenius equation at temperatures above 37 degrees C. The swelling ratio of the hydrogel was slightly decreased after heating for 2 h at 37 and 45 degrees C, and significantly reduced after 1 h at 60 degrees C. Therefore, diffusion of beta-lactoglobulin and BSA may be decreased by hydrogel shrinking at temperature over 37 degrees C. The model proteins have high affinities to buffer solution compared to the hydrogel network structure, resulting in high partition coefficients (K > 1) which do not affect the calculation of D(e) values. Incorporated protein release follows the theory of hindered diffusion.     

Effect of wine inhibitors on free pineapple stem bromelain activity in a model wine system

The influence of potential inhibitors, naturally present in wine, on the activity of stem bromelain was investigated in order to evaluate the applicability of this enzyme for protein stabilization in white wine. Bromelain proteolytic activity was tested against a synthetic substrate (Bz-Phe-Val-Arg-pNA) in a model wine system after adding ethanol, sulfur dioxide (SO(2)), skin, seed, and gallic and ellagic tannins at the average range of their concentration in wine. All the inhibitors of stem bromelain activity tested turned out to be reversible. Ethanol was a competitive inhibitor with a rather limited effect. Gallic and ellagic tannins have no inhibitory effect on stem bromelain activity, while both seed and skin tannins were uncompetitive inhibitors. The strongest inhibition effect was revealed for sulfur dioxide, which was a mixed-type inhibitor for the enzyme activity. This study provides useful information relative to a future biotechnological application of stem bromelain in winemaking.     

氨基酸对明胶膜结构及性能的影响

为了探索氨基酸作为明胶膜增塑剂的潜力,本试验研究了不同氨基酸(脯氨酸、亮氨酸、谷氨酸、丝氨酸和甲硫氨酸)对明胶膜厚度、机械性能、水蒸气透过率、透光率、颜色以及结构等的影响。结果表明,与对照组相比,除了甲硫氨酸外,其余的氨基酸都能增加明胶膜柔韧性;除了脯氨酸外,其余的氨基酸都增加了明胶膜的黄度值,并降低了明胶膜透光率和透明度;而添加脯氨酸的明胶膜的柔韧性最好、具有更好的表观特征,表面光滑紧致,同时具有更高的透明度、透光率和柔韧性。根据傅里叶变换红外光谱和二级结构分析发现,添加脯氨酸、丝氨酸和甲硫氨酸能通过氢键与明胶相互作用,降低明胶的无规则卷曲结构含量,增加β-折叠和β-转角结构含量。综合分析认为,脯氨酸可以作为明胶膜的天然增塑剂。本研究结果为增塑剂在明胶膜中的应用提供了新视角。