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.     

Influence of the glycosylation of human salivary proline-rich proteins on their interactions with condensed tannins

Binding of condensed tannins to salivary proteins is supposed to be involved in their astringency. First, complexes arising from the interaction of saliva from two individuals and tannins were studied. Then interaction mixture models containing purified saliva proteins were developed. The highest polymerized tannins predominantly precipitated together with the salivary proteins. Electrophoresis of proteins in combination with thiolysis analysis of tannins indicated proline-rich protein (PRP)-polyphenol complexes in precipitated fractions and also in the soluble ones with individual differences. Individual salivas exhibiting different protein patterns were discriminated with regard to their ability to interact with tannins. From binding studies with purified classes of salivary proteins, interactions were shown to depend on the nature of the protein, in particular on their glycosylation state. For low concentrations of tannins, glycosylated PRP-tannin interactions led to complexes that remained soluble, whereas those arising from nonglycosylated PRP-tannin interactions were precipitated. This finding could indicate that under physiological conditions, complexes involving glycosylated proteins maintain part of the lubrication of the oral cavity, whereas tannin trapping leads to a lower astringency perception.     

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.     

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.     

Binding affinity of hydrolyzable tannins to parotid saliva and to proline-rich proteins derived from it

Proline-rich proteins (PRP) in human parotid saliva have a high affinity for dietary polyphenolic compounds (tannins), forming stable complexes that may modulate the biological and nutritional properties of the tannin. The formation of such complexes may also have an important role in the modulation or promotion of the sensation of oral astringency perceived when tannin-rich foods and beverages are consumed. The major classes of PRP (acidic, basic, and glycosylated) have been isolated from human saliva, and the relative binding affinities of a series of hydrolyzable tannins, which are found in a number of plant-derived foods and beverages, to these PRP classes have been determined using a competition assay. All of the classes of PRP have a high capacity for hydrolyzable tannins. Within the narrow range of binding affinities exhibited, structure/binding relationships with the levels of tannin galloylation, hexahydroxydiphenoyl esterification, and degree of polymerization were identified. No individual class of human salivary PRP appears to have an exclusive affinity for a particular type of hydrolyzable tannin.     

Polyphenol oxidase, tannase and proteolytic activity in relation to tannin concentration in the soil organic horizon under silver birch and Norway spruce

Our aim was to compare enzyme activities (tannase, polyphenol oxidase and protease) with concentrations of tannins and their ability to precipitate proteins in the litter layer and the humus layer under silver birch (Betula pendula Roth.) and Norway spruce (Picea abies L.). We also estimated the influence of these enzymes on protein-tannin complexes and the influence of tannins on proteolytic activity. The study site was a tree species experiment in Eno, middle-eastern Finland, having three replicated plots dominated by 42-year-old silver birch and Norway spruce. Our hypotheses were (1) tree species and soil layer have an influence on tannin concentrations and enzyme activities, (2) that tannin and protein concentrations in soil organic horizon are positively correlated with enzyme activities and (3) that the enzymes studied have the ability to degrade tannin-protein complexes and that tannins can inhibit proteolytic activity. Concentrations of total tannins and hydrolysable tannins, and tannase and proteolytic activities were higher in the humus layer than in the litter layer. In general the highest values of concentrations of total tannins and hydrolysable tannins and enzyme activities were obtained for the birch humus layer, but the concentrations of condensed tannins and proteins were highest in the litter layer and under spruce. A strong correlation between substrate concentration and enzyme activity was found between hydrolysable tannins and tannase activity. Polyphenol oxidase showed similar activities in both layers. To study the influence of enzymes on protein-tannin complex we synthesized such complexes using bovine serum albumin and either condensed tannins from silver birch and Norway spruce needles or a hydrolysable tannin, tannic acid. Studies with commercial enzymes and enzymes extracted from the soil showed some decrease in tannin concentration of the tannin-protein complex over time, but surprisingly, only a negligible decrease in protein concentration. Complexes of protein with condensed tannins were more recalcitrant than tannic acid-protein complexes. Tannins, depending on the concentration and chemical structure, tended to inhibit proteolytic activity. Our results indicate that protein-tannin complexes are relatively recalcitrant since the enzymes studied here do not effectively release protein from the complexes. Also proteolytic activity and the concentration of extractable proteins seem to be low in soil. However, tannin-degrading enzymes showed high activities.     

Carbon and nitrogen dynamics in a forest soil amended with purified tannins from different plant species

Tannins are purported to be an important factor controlling nitrogen cycling in forest ecosystems, and the ability of tannins to bind proteins in protein-tannin complexes is thought to be the primary mechanism responsible for these effects. In this study, we examined the influence of well-characterized tannins purified from five different plant species on C and N dynamics of a forest soil A horizon. Tannic acid, a commonly used and commercially available hydrolyzable tannin (HT), and cellulose were also included for comparison. With the exception of tannins from huckleberry (Vaccinium ovatum), the amendments increased respiration 1.4-4.0 fold, indicating that they were acting as a microbial C source. Tannic acid was significantly more labile than the five purified tannins examined in this study. All treatments decreased net N mineralization substantially, through greater N immobilization and decreased mineralization. The six tannins inhibited gross ammonification rates significantly more than cellulose. This suggests that added tannins had effects in addition to serving as an alternative C source. Tannins purified from Bishop pine (Pinus muricata) were the only tannins that significantly inhibited potential gross nitrification rates, however, rates were low even in the control soil making it difficult to detect any inhibition. Differences in tannin structure such as condensed versus HTs and the hydroxylation pattern of the condensed tannin B-ring likely explain differences observed among the tannin treatments. Contrary to other studies, we did not find that condensed tannins were more labile and less inhibitory than HTs, nor that shorter chained tannins were more labile than longer chained tannins. In addition to supporting the hypothesis that reduced N availability in the presence of tannins is caused by complexation reactions, our data suggests tannins act as a labile C source leading to increased N immobilization.     

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.     

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。     

Insoluble condensed tannins of canola/rapeseed

The contents of soluble, SDS-extractable, and insoluble condensed tannins were determined in canola/rapeseed hulls from several varieties by utilizing the proanthocyanidin assay. The total amount of tannins in rapeseed/canola hulls ranged from 1913 to 6213 mg per 100 g of oil-free hulls. Insoluble tannins predominated in canola/rapeseed hulls and comprised from 70 to 95.8% of total tannins present. The amounts of SDS-extractable tannins were comparable to those of soluble tannins but constituted only 4.7-14. 1% of insoluble tannins present.     

Tannic acid and Norway spruce condensed tannins can precipitate various organic nitrogen compounds

There is increasing evidence that tannins affect degradation of soil organic matter and nitrogen cycling. It is assumed that the influence of tannins on biochemical processes is partly related to their ability to precipitate proteins. However, there is almost no information about precipitation of organic nitrogen-containing compounds other than proteins. A few studies indicate that tannins can precipitate arginine, choline or chitosan. In this study we used commercial tannic acid and condensed tannins extracted from Norway spruce (Picea abies (L.) Karst) needles to investigate precipitation of a wide range of organic nitrogen compounds including amino acids (all 20 proteinaceous), peptides (insulin, oxidized glutathion, reduced glutathion, AlaAla, GlyGlu, GlyPhe, GlyGlyGly), proteins (bovine serum albumin, Rubisco i.e. d-ribulose 1,5-diphosphate carboxylase), nitrogen bases, polyamines and aminosugars (N-acetyl-d-glucosamine, chitin and chitosan). Our results showed that tannins can precipitate a subset of these compounds - of the amino acids only arginine, of the peptides studied only insulin, all the proteins, polyamines, nitrogen bases, chitin and chitosan, but not N-acetyl-d-glucosamine. Concentrations of organic nitrogen compound and tannins affected amount of these compounds in precipitates. Moreover, pH value affected precipitation. The amount of precipitated organic nitrogen compound and the amount of precipitated tannins showed positive correlation across different pH. Precipitation of organic N-containing compounds other than protein by tannins can potentially affect reactions in all biochemical mixtures including tannins and these organic nitrogen compounds, and affect soil N cycling.     

Understanding the native Californian diet: Identification of condensed and hydrolyzable tannins in tanoak acorns (Lithocarpus densiflorus)

The tanoak (Lithocarpus densiflorus) acorn was a staple food in the Native American diet and is still used in traditional dishes. Acorns from the genus Quercus have been shown to contain a large range of hydrolyzable tannins. However, neither hydrolyzable nor condensed tannins have been characterized in tanoak acorns. The aim of this study was to identify the full range of hydrolyzable and condensed tannins in extracts of tanoak acorns using liquid chromatography/electrospray ionization-mass spectrometry/mass spectrometry. Condensed tannins were identified as B type oligomers of (epi)-catechin (procyanidins) with a degree of polymerization up to six. Oligomers up to and including tetramers were identified by UV spectra and MS detection whereas pentamers and hexamers were detected only by MS. The total concentration of condensed tannins was 464 mg/100 g acorn pericarp. The concentration of propocyanidin monomers, dimers, trimers, and tetramers in acorn pericarp (mg/100 g acorn pericarp) were 95 +/- 10.9, 148 +/- 35.0, 90 +/- 17.9, and 131 +/- 1.9, respectively. No procyanidins were found in the acorn cotyledon tissue. A total of 22 hydrolyzable tannins were identified in methanolic extracts of acorn cotyledon tissue. Gallic acid derivatives predominated and included galloylated esters of glucose, hexahydrodiphenoyl esters of glucose, and methylated gallates. Galloylated esters of glucose were present as isomers of galloyl glucose, digalloyl glucose, and trigalloyl glucose. Mass spectral fragmentation patterns indicate the presence of one gallic acid-galloyl glucose isomer and two gallic acid-digalloyl-glucose isomers. No isomers of tetragalloyl glucose and pentagalloyl glucose were identified. Ellagic acid and ellagic acid pentoside were also identified.     

Study of wine tannin oligomers by on-line liquid chromatography electrospray ionization mass spectrometry.

Thiolysis of a wine tannin fraction yielded trihydroxylated flavanol units (as previously observed in grape skins) in addition to the well-known procyanidins (dihydroxylated units), usually described in the literature for grape condensed tannins. To determine how they occur in condensed tannins, the wine fraction was analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry. Thus, various series of ion peaks containing a variable number of trihydroxylated units were detected as monocharged ions from dimers up to pentamers. From pentamers, oligomers were found as doubly charged ions. Heptamer species corresponded to the highest mass detected. These results showed that wine condensed tannins consist of, besides procyanidins, mixed tri- and dihydroxylated flavanol units and also of pure trihydroxylated flavanol units. These new data should be taken into account to interpret organoleptic properties of wines.     

NMR, HPLC-ESI-MS, and MALDI-TOF MS analysis of condensed tannins from Delonix regia (Bojer ex Hook.) Raf. and their bioactivities

The structures of the condensed tannins isolated from leaf, fruit, and stem bark of Delonix regia (Bojer ex Hook.) Raf. have been investigated with (13)C nuclear magnetic resonance ((13)C NMR) and high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) coupled with thiolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses. The results showed that these condensed tannins from D. regia possessed structural heterogeneity in monomer units and degree of polymerization. Propelargonidin (PP) and procyanidin (PC) were found in the leaf, fruit, and stem bark of D. regia, while prodelphinidin (PD) was found only in the leaves. The polymer chain lengths of condensed tannins from leaf and fruit organs were detected to be trimers to hexadecamers but from trimers to tridecamers for stem bark. B-type linkages were present in all these compounds. Condensed tannins from different parts of D. regia can be explored as tyrosinase inhibitors and food antioxidants because of their potent antityrosinase and antioxidant activities. The inhibitor concentration leading to 50% enzyme activity (IC(50)) was estimated to be 38 ± 1, 73 ± 2, and 54 ± 1.5 μg/mL for the condensed tannins of leaf, fruit, and stem bark. Condensed tannins extracted from stem bark exhibited the highest antioxidant activity; the DPPH scavenging activity (IC(50)) and the FRAP values were 90 ± 2 μg/mL and 5.42 ± 0.09 mmol AAE/g, respectively.     

Interaction of tannin with human salivary histatins

The ability of all major human salivary histatins to precipitate condensed tannin was demonstrated, and it was found that histatins 3 and 5 share the same condensed tannin-binding region but less tannin bound to histatin 1. The condensed tannin-binding region of histatin 5 includes both the N- and the C-terminal parts, although more tannin binding occurs in the C-terminal region. Epigallocatechin gallate (EGCG) showed similar binding characteristics as condensed tannin, but much less EGCG was precipitated. Pentagalloyl glucose (PGG) was precipitated equally well by histatins 1, 3, and 5 and bound equally well to the N- and C-terminal regions of histatin 5. In contrast to condensed tannin, cleaving histatin 5 into N- and C-terminal fragments increased their ability to precipitate PGG. Together, these results show a number of differences in the nature of interaction of histatins with condensed tannin, EGCG, and PGG. Most of the condensed tannin-protein complexes remained insoluble under conditions similar to those in the stomach and the small intestine, suggesting that histatins may act as a defense against dietary tannin in humans.     

Effects of different organic farming methods on the concentration of phenolic compounds in sea buckthorn leaves

The effects of different cultivation methods on the amount of phenolic compounds in leaves of 1-year-old seedlings of two Finnish sea buckthorn (Hippophae rhamnoides L. ssp. rhamnoides) cultivars 'Terhi' and 'Tytti' were studied in a field experiment established at coastal area in Merikarvia, western Finland. The cultivation methods included different fertilizers (suitable for organic cultivation), mulches (organic and plastic), and land contours (flat vs low hill surface). Two experiments were conducted. The first allowed the estimation of the effects of cultivar, fertilizer, surface contour, and all their interactions, while the other allowed the estimation of the effects of mulches, land contours, and their interactions for the cultivar 'Tytti'. Eleven different hydrolyzable tannins, pentagalloylglucose, and 14 other phenolic compounds were detected by chemical analysis with high-performance liquid chromatography (HPLC). The amount of phenolic compounds varied between different land contours and mulches. The concentrations of gallic acid, pentagalloylglucose, quercetin-3-rhamnoside, monocoumaroyl astragalin A, total hydrolyzable tannins, and condensed tannins were significantly higher on the flat surface than on the low hill surface. The plastic mulch decreased the concentration of gallic acid, hydrolyzable tannins, and condensed tannins compared to the other mulches used. These results suggest ways to cultivate sea buckthorn to produce large amounts of valuable chemicals, especially tannins in the leaves.     

Contents of total protein, L-canavanine and condensed tannins of the one-flowered vetch (Vicia articulata Hornem.) collection of the Bank of Plant Germplasm of Cuenca (Spain)

A total of 125 accessions of one-flowered vetch (Vicia articulata Hornem.) mostly from the Iberian Peninsula have been analysed for total protein, L-canavanine and condensed tannins. It was observed a high variability in the composition: the protein content ranged from 18.20 to 30.07%, L-canavanine from 0.27 to 0.67%, and condensed tannins from 0.13 to 0.35%, which indicates a scarce domestication of the crop. The inclusion of the seeds into a diet to non-ruminants should be limited to marginal percentages because of the contents of L-canavanine and it suggests that a reduction of this compound by breeding should be encouraged. The protein and the L-canavanine contents showed a low and positive significant correlation (r = 0.55, P < 0.001), which complicates the selection of seeds having simultaneously low L-canavanine and high protein contents. On the other hand, no correlation between protein and condensed tannins or between L-canavanine and condensed tannins was observed. There was a poor relationship between the origin and the composition of the accessions and only the protein content was able to discriminate among the regions.     

Inhibitory effects of apple polyphenols and related compounds on cariogenic factors of mutans streptococci

The inhibitory effects of apple polyphenols (APP) on the synthesis of water-insoluble glucans by glucosyltransferases (GTF) of streptococci of the mutans group and on the sucrose-dependent adherence of the bacterial cells were examined in vitro. APP markedly inhibited the activity of GTF purified from the cariogenic bacterial cells. However, APP showed no significant effect on the growth of the cariogenic bacteria. The strongest GTF inhibitors in APP were apple condensed tannins (ACT), a mixture of procyanidins. The 50% inhibitory doses of ACT against the GTF of S. sobrinus and that of S. mutans were 1.5 microgram/mL and 5 microgram/mL, respectively. The ACT efficacy largely depended upon the degree of polymerization. Interestingly, while the other polyphenols known to inhibit GTF such as tannic acid markedly inhibited salivary alpha-amylase activity, APP and ACT only scarcely inhibited that enzyme activity. This means that APP and ACT might selectively inhibit the bacterial GTF activity under oral conditions.     

Interactions between yeast lees and wine polyphenols during simulation of wine aging. II. Analysis of desorbed polyphenol compounds from yeast lees

In the first part of this work, the analysis of the polyphenolic compounds remaining in the wine after different contact times with yeast lees during simulation of red wine aging was undertaken. To achieve a more precise view of the wine polyphenols adsorbed on lees during red wine aging and to establish a clear balance between adsorbed and remnant polyphenol compounds, the specific analysis of the chemical composition of the adsorbed polyphenolic compounds (condensed tannins and anthocyanins) after their partial desorbtion from yeast lees by denaturation treatments was realized in the second part of the study. The total recovery of polyphenol compounds from yeast lees was not complete, since a rather important part of the initial wine colored polyphenols, especially those with a dominant blue color component, remained strongly adsorbed on yeast lees, as monitored by color tristimulus and reflectance spectra measurements. All anthocyanins were recovered at a rather high percentage (about 62%), and it was demonstrated that they were not adsorbed in relation with their sole polarity. Very few monomeric phenolic compounds were extracted from yeast lees. With the use of drastic denaturing treatments, the total recovery of condensed tannins reached 83%. Such tannins extracted from yeast lees exhibited very high polymeric size and a rather high percentage of galloylated residues by comparison with initial wine tannins, indicating that nonpolar tannins were preferentially desorbed from yeast lees by the extraction treatments.     

Investigations into the biochemical basis for nematode resistance in roots of three musa cultivars in response to Radopholus similis infection

The Musa cultivars, Dwarf Cavendish, Yangambi Km5 and Kunnan, exhibit considerable differences in resistance to Radopholus similis. Infection resulted in significant increases in condensed tannins and flavan-3,4-diols in roots (P < 0.001). The highly resistant cultivar Kunnan had the highest levels of condensed tannins before and after infection. The preinfection levels were similar to the postinfection levels of the two other cultivars. Tannins had mostly procyanidin character, but Kunnan also contained propelargonidins; these compounds may be involved in the resistance mechanism. It is suggested that the butanol/HCl assay be used as a rapid test in screening for resistance to R. similis.