The complexity of condensed tannin binding to bovine serum albumin--An isothermal titration calorimetry study.

Isothermal titration calorimetry was applied to study the binding of purified proanthocyanidin oligomers to bovine serum albumin (BSA). The molecular weight of the proanthocyanidin oligomer had a major impact on its binding to BSA. The calculated change in enthalpy (ΔH) and association constant (Ka) became greater as the oligomer size increased then plateaued at the heptameric oligomer. These results support a model for precipitation of proteins by proanthocyanidin where increased oligomer size enhanced the opportunity for cross linkages between proteins ultimately forming sediment-able complexes. The authors suggest tannin binding to proteins is opportunistic and involves multiple sites, each with a different Ka and ΔH of binding. The ΔH of binding comprises both an endothermic hydrophobic interaction and exothermic hydrogen bond component. This suggests the calculated entropy value (ΔS) for tannin-protein interactions is subject to a systematic error and should be interpreted with caution.

Impact of condensed tannin size as individual and mixed polymers on bovine serum albumin precipitation.

Condensed tannins composed of epicatechin from monomer to octamer were isolated from cacao (Theobroma cacao, L.) seeds and added to bovine serum albumin (BSA) individually and combined as mixtures. When added to excess BSA the amount of tannin precipitated increased with tannin size. The amount of tannin required to precipitate BSA varied among the polymers with the trimer requiring the most to precipitate BSA (1000 µg) and octamer the least (50 µg). The efficacy of condensed tannins for protein precipitation increased with increased degree of polymerisation (or size) from trimers to octamers (monomers and dimers did not precipitate BSA), while mixtures of two sizes primarily had an additive effect. This study demonstrates that astringent perception is likely to increase with increasing polymer size. Further research to expand our understanding of astringent perception and its correlation with protein precipitation would benefit from sensory analysis of condensed tannins across a range of polymer sizes.

A role for anthocyanin in determining wine tannin concentration in Shiraz.

Four wines were made to investigate the effect of different anthocyanin and tannin fruit concentrations on wine phenolics and colour. Wines that were made from fruit with high anthocyanin concentration had high tannin concentrations regardless of the concentration of tannin in fruit, while wines made from fruit with low anthocyanin also had low tannin concentration. It was found that fruit anthocyanin concentration correlated with wine tannin concentration, wine colour and polymeric pigment formation. Anthocyanin concentration might be a key component for increasing tannin solubility and extraction into wine and the formation of polymeric pigments. Industry implications include managing tannin and anthocyanin fruit concentration for targeting tannin extraction and polymeric pigment formation in wine.

Analysis of free and bound phenolics in wine and grapes by GC-MS after automated SPE.

The results of validation of a method for the analysis of free and bound phenolics in wine and grapes are presented. Wine and grape extracts are fractionated by automated solid-phase extraction on Bond Elut PPL cartridges to give free and bound phenolic fractions. Bound fractions are subjected to acid hydrolysis, and the phenolics released are recovered by solid-phase extraction on Bond Elut PPL cartridges. The fractions are further purified by automated solid-phase extraction on Bond Elut silica cartridges. After derivatisation to form trimethylsilyl ethers, the phenolics are determined by gas chromatography-mass spectrometry with selected ion monitoring. The method is suitable for robust, high-throughput monitoring of the concentrations of phenolics that can affect the palatability of wine.

Glucose, fructose and sucrose increase the solubility of protein-tannin complexes and at high concentration, glucose and sucrose interfere with bisulphite bleaching of wine pigments.

Wines were modified with increasing sugar concentrations and decreasing tannin concentrations and analysed by a combination of protein precipitation and bisulphite bleaching. Increasing sugar concentration decreased the precipitation of tannin and protein-precipitable polymeric pigments (PPP). The use of a hydrogen bond disruptor (urea) to reduce protein-tannin and protein-pigment complex formation showed that the effect of sugar concentration occurred by increasing the solubility of the tannin-protein complex, not by interfering with protein-tannin complex formation. By increasing the solubility of pigment-protein complexes, non-protein-precipitable polymeric pigments (nPPP) appeared to increase. There was also an increase in total polymeric pigments at each tannin concentration with increasing glucose and sucrose concentration, indicating that sugar concentration might also affect bisulphite bleaching of wine pigments. While a significant effect of sugar concentration on tannin-protein complex solubility was observed, these effects were greatest at sugar concentrations far in excess of normal wine making conditions. Under normal wine making conditions, sugar concentration will have a negligible effect on protein-precipitable tannin, PPP and nPPP concentrations.

Effects of urbanization on prevalence of Baylisascaris procyonis in intermediate host populations.

Baylisascaris procyonis is an intestinal parasite of raccoons (Procyon lotor) that can also infect humans and a wide range of wildlife species. Prevalence of B. procyonis in raccoon populations appears to decrease as the landscape urbanizes, but less is known about prevalence in the small-mammal intermediate hosts of the parasite. We measured prevalence of B. procyonis in populations of mice (Peromyscus spp.) in forest preserves along a gradient of urbanization in Illinois. Prevalence in the mouse intermediate host exhibited a trend opposite raccoons: prevalence increased as surrounding human population density increased. This counterintuitive result may be due to higher overall environmental loads of B. procyonis in urban areas due to higher raccoon densities. Our results emphasize the need to understand fully the transmission dynamics of B. procyonis in its definitive and intermediate hosts in order to design and implement effective strategies to mitigate zoonotic risks to humans.

Exposure of grapes to smoke of vegetation with varying lignin composition and accretion of lignin derived putative smoke taint compounds in wine.

Smoke taint in wines from bushfire smoke exposure has become a concern for wine producers. Smoke taint compounds are primarily derived from pyrolysis of the lignin component of fuels. This work examined the influence of the lignin composition of pyrolysed vegetation on the types of putative smoke taint compounds that accrue in wines. At veraison, Merlot vines were exposed to smoke generated from five vegetation types with differing lignin composition. Smoke was generated under pyrolysis conditions that simulated bushfire temperature profiles. Lignin and smoke composition of each fuel type along with putative smoke taint compounds in wines were determined. The results showed that, regardless of fuel type, the commonly reported guaiacyl lignin derived smoke taint compounds, guaiacol and 4-methylguaiacol, represented about 20% of the total phenols in wines. Quantitatively, syringyl lignin derived compounds dominated the total phenol pools in both free and bound forms. The contributions of p-hydroxyphenyls were generally similar to the guaiacyl sources. A further unexpected outcome of the study was that pine smoke affected wines had significantly elevated levels of syringols compared to the controls although pine fuel and its smoke emission lacked syringyl products.

Detailed characterization of proanthocyanidins in skin, seeds, and wine of Shiraz and Cabernet Sauvignon wine grapes (Vitis vinifera).

The distribution of proanthocyanidin (PA) polymer lengths, proanthocyanidin concentration at each polymer length, and polymer composition were determined in the seed, skin, and wine of Shiraz and Cabernet Sauvignon grape berries grown in southeast Australia. PA was fractionated by semipreparative high performance liquid chromatography (HPLC) and analyzed by phloroglucinolysis and HPLC to report the degree of polymerization (DP), concentration, and composition at 11 DP values in seed and wine and 21 DP values in skin. In skin, the highest PA concentration was observed at a DP of 31 in Shiraz and 29 in Cabernet Sauvignon representing 15% of the total PA in both varieties. The distribution of seed PA had the highest concentration at a DP of 7 in Shiraz and 6 in Cabernet Sauvignon representing around 30% of the total PA. In the wine PA distribution, the highest concentration was observed at a DP of 11 in Shiraz and 9 in Cabernet Sauvignon representing around 26 and 32% of the distribution, respectively. A second peak in wine PA concentration was observed at the largest DP of 18 in Shiraz and 15 in Cabernet Sauvignon representing around 20% of the distribution. The composition in wine did not vary at different DP, but the proportion of epicatechin gallate varied in seed PA less than 4 DP. The proportion of epigallocatechin increased with increasing DP in skin PA. Wine PA had a DP range and composition similar to the distribution of skin PA between DP 4 and 18 suggesting that larger skin PAs are not extracted into wine. This study provides information that could be used to target the important PA fractions in grapes that need to be measured to understand (or predict) PA extraction into wine and eventual mouthfeel.

Reducing Baylisascaris procyonis roundworm larvae in raccoon latrines.

Baylisascaris procyonis roundworms, a parasite of raccoons, can infect humans, sometimes fatally. Parasite eggs can remain viable in raccoon latrines for years. To develop a management technique for parasite eggs, we tested anthelmintic baiting. The prevalence of eggs decreased at latrines, and larval infections decreased among intermediate hosts, indicating that baiting is effective.

Simultaneous separation by reversed-phase high-performance liquid chromatography and mass spectral identification of anthocyanins and flavonols in Shiraz grape skin.

A limitation of large-scale viticultural trials is the time and cost of comprehensive compositional analysis of the fruit by high-performance liquid chromatography (HPLC). In addition, separate methods have generally been required to identify and quantify different classes of metabolites. To address these shortcomings a reversed-phase HPLC method was developed to simultaneously separate the anthocyanins and flavonols present in grape skins. The method employs a methanol and water gradient acidified with 10% formic acid with a run-time of 48 min including re-equilibration. Identity of anthocyanins and flavonols in Shiraz (Vitis vinifera L.) skin was confirmed by mass spectral analysis.

Partial shading of Cabernet Sauvignon and Shiraz vines altered wine color and mouthfeel attributes, but increased exposure had little impact.

Few studies have investigated the impact of vine shading on the sensory attributes of the resultant wine. This study examines the effects of canopy exposure levels on phenolic composition plus aroma, flavor, and mouthfeel aspects in wine. Wines were made from Cabernet Sauvignon and Shiraz grapes (Vitis vinifera L.) subjected to different levels of canopy exposure in a commercial vineyard in the Sunraysia region, Victoria, Australia. Canopy exposure treatments included control (standard vineyard practice), exposed (achieved with a foliage wire 600 mm above the top cordon), highly exposed (using a foliage wire with leaf plucking in the fruit zone), and shaded treatment (using 70% shade-cloth). Spectral and descriptive analyses showed that levels of anthocyanins, other phenolics, and perceived astringency were lower in wines made from shaded fruit; however, the reverse was generally not observed in wines of exposed and highly exposed fruit. Descriptive analysis also showed wines from the shaded fruit were different from other treatments for a number of flavor and aroma characters. These findings have implications for vineyard management practices.

Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves.

Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.