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.     

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.     

Phenolic Content,Antioxidant Activity,and Consumer Acceptability of Sorghum Cookies

Cookies were produced from different sorghum flours to determine their potential as vectors of antioxidants. Different sorghum cultivars and their flour extraction rates were evaluated for their effects on phenolic content and antioxidant activity of the cookies. Consumer acceptance of the sorghum cookies was compared with that of wheat flour cookies. For each sorghum cultivar, cookies of 100% extraction rate flours had two to three times more total phenolics compared with those of 70% extraction rate flours, while antioxidant activity was 22–90% higher. Cookies of the condensed tannin sorghum had two to five times more phenolics compared with those of condensed tannin‐free sorghum. Antioxidant activity was 145–227 μMol Trolox equivalents (TE)/g in cookies of condensed tannin sorghum compared with 10–102 μMol TE/g in those of condensed tannin‐free sorghum. The sorghum flours had slightly higher phenolic content and antioxidant activity values than their corresponding cookies. Cookies of the red tannin‐free sorghum flours (PAN 8564/8446) were equally liked as wheat flour cookies, except for texture. However, cookies of condensed tannin sorghum were least accepted compared with wheat flour cookies despite their high antioxidant activity.     

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.     

Macronutrients and metals released from soils by solutions of naturally occurring phenols

Phenolic compounds produced by plants enter the soil by leaching and litter decomposition. The goal of this work is to determine the effect of phenolic compounds on solubility of plant macronutrients and metals in agroforestry systems. Soils from forest and pasture systems were repeatedly extracted with water (control) or phenolic solutions and then compared to a Mehlich 3 reference. The phenolics were aqueous solutions of tannic acid or β –1,2,3,4,6‐penta‐O‐galloyl‐D‐glucose (PGG) (hydrolyzable tannins), procyanidin (condensed tannin), or small phenolics catechin, gallic acid, or methyl gallate. The concentration of the macronutrients Ca, Mg, K, P, and S, and the metals Fe, Al, Mn, and Zn in the supernatants was determined by inductively‐coupled plasma spectroscopy. Cumulative extraction of macronutrients was generally similar to or less than the amount obtained by the Mehlich 3 extraction with the lowest recoveries obtained with the water control, PGG, and procyanidin. Metals tended to be somewhat more extractable from forest soil, especially with gallic acid, tannic acid or PGG treatments. Three mechanisms affected extraction of analytes by phenol‐containing solutions: (1) pH‐driven dissolution (Ca and Mg), (2) chelation of the metal (Al) by the polyphenol, or (3) reduction of the metal (Fe and Mn). Relatively low extraction of nutrients by some polyphenols is attributed to the tendency of some phenols to sorb to soil. This study demonstrates that tannins and related compounds change the solubility of macronutrients and metals in soils by a complex process that is not easily predictable from simple chemical properties of the phenolics.     

Size-exclusion chromatography of tea tannins and intercepting potentials of peptides for the inhibition of trypsin-caseinolytic activity by tea tannins

Molecular-weight distribution and characterization of tea tannin were investigated by high-performance liquid chromatography and the equivalent preparative exclusion gel chromatography using Sephadex G-25. The characteristics of the fractions were studied regarding the amounts of terminal catechin, sugar, and gallic acid, the color reaction of the Folin-Chiocalteu reagent, the UV absorbance, and the inhibition activity for the trypsin-caseinolytic activity per weight. Furthermore, we investigated the intercepting activities of the inhibition by the amino acids, peptides, their analogues, poly(ethylene glycol)s (PEGs), and histatin 5 using the inhibition of trypsin-caseinolytic activity by tea. Arg, Lys, and their peptides had strong intercepting activities for the inhibition, but only a weak activity was detected in the Pro peptides or gelatin-like peptides of (Pro-Pro-Gly)(n) (n = 5 or 10). The guanidyl group of Arg and the amino methylene group of Lys were important for the intercepting activity, but the activity was weakly dependent upon the peptide bond formation. The intercepting activity of the peptides or PEG exponentially increased with the number of polymerizations. Histatin 5 did not have a remarkably strong intercepting activity considering the peptide length. The activity of the synthetic histatin 5 in which all of the Lys and Arg were substituted by Ala was at the same level as histatin 5.     

In vitro protein degradation of 38 sainfoin accessions and its relationship to tannin content by different assays

This study compared 38 sainfoin and 2 Lotus accessions to their respective tannin contents, N buffer solubility, and in vitro protein degradation. Tannin contents were measured by a protein precipitation method using either bovine serum albumin or Rubisco and by the colorimetric HCl/butanol method. Precipitation of bovine serum albumin and Rubisco was highly correlated (R(2) = 0.939). Correlations between the protein precipitation variants and the HCl/butanol method were relatively low (R(2) < 0.6). Protein degradation was measured at 4 h of incubation in an inhibited in vitro system and could not be explained by any of the tannin assays (R(2) < 0.03) and only partially by N buffer solubility (R(2) ≤ 0.433). Decisive factors other than the quantity of tannins or their ability to precipitate proteins must be considered. Resistance of soluble protein toward degradation can possibly be caused by tannin protein binding.     

Carbon respiration and nitrogen dynamics in Corsican pine litter amended with aluminium and tannins

We investigated the carbon (C) mineralisation and nitrogen (N) dynamics in litter from a Corsican pine forest in response to individual and combined additions of aluminium (Al), condensed tannin (extracted from fresh Corsican pine needles) and hydrolysable tannin (commercial tannic acid). Production rates of CO₂, NH₄⁺ and NO₃⁻ concentrations, tannin concentrations and Al speciation were determined at various time intervals during a 28-day incubation experiment. The addition of Al decreased CO₂ production and shut down nitrification. Exchangeable NH₄⁺ strongly increased in the Al-amended litter, likely due to (i) decreased microbial uptake of NH₄⁺, (ii) the inhibition of nitrification and (iii) competition for soil organic matter (SOM) binding sites by Al. Both tannin species affected C mineralisation and/or N dynamics, be it in different ways. Addition of tannic acid led to a strong increase of the C mineralisation rate and microbial uptake of N, caused by rapid degradation of this labile tannin and subsequent increased microbial nutrient demand. Net immobilisation of N occurred as long as one week after addition. Condensed tannin was not consumed but probably strongly bound to (nitrogenous) SOM compounds, forming recalcitrant complexes and decreasing net N mineralisation. Complexation of Al by tannins in solution before addition to the litter mitigated the Al-induced release of exchangeable NH₄⁺. In the case of condensed tannin with complexed Al, this was due to detoxification of Al through complexation. Increased microbial demand for N likely played a major role in decreased NH₄⁺ accumulation in the samples to which tannic acid with complexed Al was added. Nitrification was shut down despite of the complexation of Al by either condensed tannin or tannic acid.     

Interference of condensed tannin in lignin analyses of dry bean and forage crops

Legumes with high concentrations of condensed tannin (pinto bean [Phaseolus vulgaris L.], sainfoin [Onobrychis viciifolia Scop.], and big trefoil [Lotus uliginosus Hoff.]), were compared to a selection of forages, with low or zero condensed tannin (smooth bromegrass [ Bromus inermis Leyss], Lotus japonicus [Regel] K. Larsen, and alfalfa [Medicago sativa L.]), using four methods to estimate fiber or lignin. Protocols were validated by using semipurified condensed tannin polymers in adulteration assays that tested low-lignin tissue with polyphenolic-enriched samples. The effect on lignin assay methods by condensed tannin concentration was interpreted using a multivariate analysis. There was an overestimation of fiber or lignin in the presence of condensed tannin in the acid detergent fiber (ADF) and Klason lignin (KL) assays compared to that in the thioglycolic acid (TGA) and acid detergent lignin (ADL) methods. Sulfite reagents (present in TGA lignin method) or sequential acidic digests at high temperatures (ADF followed by ADL) were required to eliminate condensed tannin. The ADF (alone) and KL protocols are not recommended to screen nonwoody plants, such as forages, where condensed tannin has accumulated in the tissue.     

Repeated applications of tannins and related phenolic compounds are retained by soil and affect cation exchange capacity

Retention of tannins, produced by plants, could be important for managing soil organic matter and nutrient cycling. However, we know little about the comparative retention of different classes of tannins and related compounds or if soils have a maximum storage capacity for them. To address these questions, forest, and pasture loam soils, collected at 0-5 cm (surface) and 10-20 cm (subsurface), were repeatedly treated with water (Control) or solutions containing condensed and hydrolyzable tannins or related phenolic subunits (10 mg g⁻¹ soil). Treatments included a polymeric flavonoid-based procyanidin from sorghum, catechin, tannic acid, β-1,2,3,4,6-penta-O-galloyl-d-glucose (PGG), gallic acid, and methyl gallate. After each application, soluble-C in supernatants was determined by oxidative-combustion infrared analysis and retention of treatment-carbon by soil was calculated as the difference between added and recovered soluble-C. An interaction between soil depth and treatment was evident through all applications with highest retention of both hydrophobic (PGG) and hydrophilic (procyanidin) tannins, compared to other phenolic compounds. For all treatments except gallic acid and methyl gallate, higher sorption occurred in surface soil, which contained more organic matter than subsurface soil. With each successive application, less additional treatment-C was retained by soil and the amount of C remaining in supernatants was correlated with the presence of phenolic substances. Cumulative retention by surface soil was more than 10.3, 8.5 and 6.4 mg C g⁻¹ soil for PGG, tannic acid, and procyanidin, several times higher than the other compounds. Soluble-C extracted from treated soil, with cool water (23 °C), was 1-2 orders of magnitude greater than Control samples and highly correlated with Prussian Blue (PB) phenolics, indicating some retained treatment-C was only weakly held on the soil. The final extraction, with hot water (80 °C), removed more soluble-C, particularly from surface samples, that contained fewer PB phenolics per unit soluble-C than cool water extracts. After all extractions more than 85% of sorbed procyanidin-C was retained by samples compared to 81% of methyl gallate, 79% of PGG, 74% of tannic acid, 50% of catechin, and 40% of the gallic acid. Total C, measured in soil after all extractions, was close to expected values, confirming tannins and phenolic compounds had remained in soil and were not otherwise lost. Cation exchange capacity was increased about 30% in subsurface and forest samples by PGG, a hydrolyzable tannin, but decreased by 30% and 35% in surface and pasture soil, respectively, by its monomer, gallic acid.     

Probing the interaction of polyphenols with lipid bilayers by solid-state NMR spectroscopy

Polyphenols are bioactive natural products that appear to act against a wide range of pathologies. Mechanisms of activity have not been established, but recent studies have suggested that some polyphenols bind to membranes. This study examined the interaction between lipid bilayers and three structurally diverse polyphenols. It was hypothesized that features of the polyphenols such as polarity, molecular size, molecular geometry, and number and arrangement of phenol hydroxyl groups would determine the tendency to interact with the bilayer. The examined compounds included a mixed polyphenol, (-)-epigallocatechin gallate (EGCg); a proanthocyanidin trimer comprising catechin-(4→8)-catechin-(4→8)-catechin (cat?; and a hydrolyzable tannin, 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG). These polyphenols were incorporated at different levels into 2H-labeled 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilamellar vesicles (MLVs). 31P and 2H solid-state NMR experiments were performed to determine the dynamics of the headgroup region and the hydrophobic acyl chain region of the lipid bilayer upon addition of polyphenols. The chemical shift anisotropy (CSA) width of the 31P NMR spectra decreased upon addition of polyphenols. Addition of PGG induces a dramatic reduction on the CSA width compared with the control lipid bilayer sample, whereas addition of cat? barely reduces the CSA width. The 2H quadupolar splitting of the lipids also decreased upon addition of polyphenols. At the same concentration, PGG substantially reduced the quadrupolar splitting, whereas cat? barely reduced it when compared with the control sample. From a calculation of the order parameters of the acyl chain region of the lipid bilayer, it was concluded that the hydrophobic part of the lipid bilayer was perturbed by PGG, whereas cat? did not cause large perturbations. The data suggest that the polarity of the polyphenols affects the interaction between tannins and membranes. The interactions may relate to the biological activities of polyphenols.     

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.     

Condensed tannins and flavonoids from the forage legume sulla (Hedysarum coronarium)

The condensed tannin concentrations and composition and the characterization of the phenolic constituents in the leaves of the forage legume sulla (Hedysarum coronarium), a biennial forage legume found in temperate agricultural regions, were studied. The colorimetric butanol-HCl assay was used for the quantitation of the seasonal condensed tannin concentrations in the leaves of sulla. Fractionation of extracts on Sephadex LH-20 using step elution with aqueous methanol, followed with aqueous acetone or gradient elution with water, aqueous methanol, and aqueous acetone, gave condensed tannin and flavonoid fractions. The chemical characteristics of the purified condensed tannin fractions were studied by acid-catalyzed degradation with benzyl mercaptan and electrospray ionization mass spectrometry (ESI-MS). Thiolysis revealed that epigallocatechin was the major extender unit (15-75%) while gallocatechin was the major terminal unit (50-66%), thus indicating the extractable sulla condensed tannin fraction as the prodelphinidin type. Condensed tannin oligomers to polymers obtained from Sephadex LH-20 gradient fractions ranged between 2.9 and 46 mDP. The homo- and heterogeneous oligomer ions in condensed tannin gradient fractions detected by ESI-MS ranged from 2 to 10 DP and are consistent with the values obtained by thiolysis (2.9-6.9 DP). Lower molecular weight phenolics, including flavonoids and phenolic acids, were characterized by liquid chromatography atmospheric pressure chemical ionization mass spectrometry (LC-APCI/MS) and ESI/MS/MS on a linear ion trap. The flavonoids extracted with aqueous acetone and methanol from sulla leaves and identified included kaempferol, rutin, quercetin-7-O-α-L-rhamnosyl-3-O-glucosylrhamnoside, quercetin-3-O-α-L-rhamnosyl-7-O-glucoside, kaempferol-3-O-β-D-glucoside-dirhamnoside, genistein-7-O-β-D-glucosyl-6″-O-malonate, formononetin-7-O-β-D-glucoside-6″-O-malonate, and afrormosin and the phenolic acid chlorogenic acid.     

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.     

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.     

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.     

Interactions between wine polyphenols and aroma substances. An insight at the molecular level

Control of the organoleptic quality of wine or grape-derived beverages requires the study of the interactions between flavor volatiles and polyphenols. The influence of catechin and a wine highly condensed tannin fraction on the volatility of aroma substances was investigated using a dynamic headspace technique. In a hydroalcoholic solution, isoamyl acetate, ethyl hexanoate, and benzaldehyde appeared to be more retained than limonene at low catechin concentrations (0-5 g/L). The tannin fraction induced a slight decrease of benzaldehyde volatility and a salting out of limonene and had no effect on the two esters. Furthermore, investigations at the molecular level were conducted using (1)H NMR spectroscopy. Chemical shift changes registered upon addition of a ligand to a substrate kept at constant concentration allowed the determination of the dissociation constant in a 1:1 binding model. Complexation with catechin was evaluated to be similarly weak for benzaldehyde and the two esters. In addition, catechin and epicatechin displayed a higher affinity for benzaldehyde than for 3, 5-dimethoxyphenol, supporting the hypothesis of a hydrophobic driving force.     

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.     

Germplasm Sources for Improvement of Forage Quality in Lotus corniculatus L. and L. uliginosus Schkuhr (Fabaceae)

Extensive germplasm resources are present in collections of Lotus corniculatus and L. uliginosus, two forage species that are valuable in situations where other legumes are not persistent. Naturalised populations in many regions are the primary gene pool for genetic improvement of these species and interspecific hybrids are an important source of variation for agronomic and forage quality traits. Although previous research has provided evidence that selection for optimal levels of condensed tannins and herbage productivity are feasible breeding objectives, less is known about correlations of condensed tannin with other parameters of nutritive value. A germplasm collection of 38 accessions in both species from 9 geographic regions was used as a data base to determine these correlations. Forage of each accession was sampled in two successive years from a field site in south-eastern Australia and analysed for condensed tannin, in vitro digestible dry matter, nitrogen, acid detergent fibre and neutral detergent fibre. In both species condensed tannin was negatively correlated with in vitro digestible dry matter and nitrogen. This result helped to identify potentially valuable accessions with low condensed tannin (<4% of dry weight) and high in vitro digestible dry matter (>70%). One of these was an interspecific hybrid of L. uliginosus and L. corniculatus, confirming the view that interspecific hybridization would be a valuable avenue for improvement of agronomic and forage quality characters in Lotus species.     

A high-throughput method for the quantification of proanthocyanidins in forage crops and its application in assessing variation in condensed tannin content in breeding programmes for Lotus corniculatus and Lotus uliginosus

Lotus corniculatus and Lotus uliginosus are agronomically important forage crops used in ruminant livestock production. The condensed tannin (CT) content, dry matter (DM) production, and persistence of these species are key characteristics of interest for future exploitation of these crops. Here we present field data on 19 varieties of L. corniculatus, 2 varieties of L. uliginosus and, additionally, a glasshouse experiment using 6 varieties of L. corniculatus and 2 varieties of L. uliginosus. Current methods for the quantification of condensed tannins in crop species are slow and labor intensive and are generally based upon polymer hydrolysis following the extraction of chlorophyll in a liquid phase. Presented here is a high-throughput protocol for condensed tannin quantification suitable for microtiter plates based upon the precipitation of condensed tannin polymers in complex with bovine serum albumin (BSA) with subsequent hydrolysis of precipates using butan 1-ol/ hydrochloric acid.