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This study aimed to assess the effect of tannin molecular mass on nonbleachable pigment formation and subsequent stability under wine-like conditions. Tannin fractions of a defined molecular mass range were prepared from grape skins and seeds and reacted with malvidin-3-glucoside for 120 days in three media types: chemically defined wine media with or without acetaldehyde addition or model wine without acetaldehyde. Precipitation was observed after the reaction period and increased in response to both higher tannin molecular mass and acetaldehyde concentration. To confirm whether acetaldehyde-mediated condensation of tannin and anthocyanin modified the solubility of the nonbleachable pigments formed, HPLC-MS was used for the semiquantitative identification of vinyl derivatives and ethyl-linked adducts in soluble and precipitated materials. It was found that the proportion of vinyl derivatives and ethyl-linked anthocyanin was elevated in tannin precipitates relative to soluble pigmented material. Despite substantial losses of tannin due to precipitation, the resulting nonbleachable pigment concentration and color intensity were higher in wine media containing elevated acetaldehyde, when each tannin size category was considered independently. The results of this study indicated that the development of nonbleachable pigments from larger tannins may be limited when acetaldehyde-mediated condensation with anthocyanin predominates in wine, concomitant with precipitation.

BACKGROUND AND AIMS: Winemakers utilise various remediation strategies for decreasing ‘reductive’ characters in wine. Remediation strategies, such as the addition of diammonium phosphate (DAP) during fermentation, copper fining, the addition of fresh lees or lees products to wine, and aeration of the must during and after fermentation, are commonly employed in an effort to prevent the formation or to remove undesirable volatile sulfur compounds. In this study, the relative effectiveness of five strategies for remediation of ‘reductive’ aroma was compared in a Shiraz wine. METHODS AND RESULTS: At the onset of the development of ‘reductive’ aromas during Shiraz fermentation, each ferment was treated with a unique remediation strategy: DAP addition; macro‐oxygenation; copper fining; a combination of macro‐oxygenation and copper fining; or the addition of fresh lees from a donor wine. While a relatively small difference in volatile sulfur compounds was found between treatments, copper fining, lees and DAP addition produced wines with elevated ‘reductive’ characters at certain time‐points post‐bottling; while macro‐oxygenation or macro‐oxygenation with copper produced wines with a low sensory score for ‘reduction’‐related attributes. CONCLUSIONS: Macro‐oxygenation during fermentation was the most effective remediation strategy. Lees addition, and to a lesser extent, copper fining and DAP addition, diminished fruit attributes and produced wines with noticeable ‘reductive’ characters. SIGNIFICANCE OF THE STUDY: Macro‐oxygenation during fermentation produced wines with the lowest ‘reduction’‐related attributes while enhancing ‘fruity’ attributes. This work also highlighted the compositional effects of each type of remediation treatment.

Pinot noir fruit is often relatively low in the phenolics important for wine mouthfeel and color stabilization. Previous research has shown that leaf removal can influence the concentration and composition of Pinot noir fruit phenolics, but it is not clear to what extent these effects on grape composition are carried through the winemaking process. A novel thermal treatment, ‘Controlled Phenolic Release’ (CPR), has been demonstrated effective for increasing the phenolic concentration of Pinot noir wines, but there is currently limited information on the interaction between this process and viticultural practices. CPR is microwave heating of grape must to hasten phenolic extraction. This study applied viticultural treatments to Pinot noir (no leaf removal; leaf removal at post-flowering and pre-veraison) in two commercial vineyards (A and B), and winemaking treatments in replicated microvinifications (control; CPR with skin contact; CPR with early pressing) were applied to fruit from vineyard A. Effects on grape and wine phenolics were examined using UV-visible spectrophotometry. Wine tannin composition and polymerization were assayed by methyl cellulose precipitation (MCPT) and high-performance liquid chromatography (HPLC). Viticultural treatments applied produced significant effects in vineyard A but not vineyard B. Winemaking treatments impacted the concentration of phenolics in wines, but there was no interaction between viticultural and winemaking treatments; effects were additive. Of scientific and practical significance was the finding that grape phenolic concentration determined from homogenate extractions of fruit did not reliably predict Pinot noir wine phenolics. Further, in wines, variation in tannin composition, the extent of tannin polymerization and tannin size was found between treatments. The novel findings here are that the viticultural treatment and the winemaking treatments were both significant and, being independent of each other, offered two separate ways to adjust phenolics in Pinot noir wine. Viticultural and winemaking treatments were also shown to affect tannin composition, with potential mouthfeel and color stability implications.

Background and Aims Regulations in some wine‐producing countries allow water addition to grape must. The research presented compared the impact of water addition to must on wine phenolics and colour relative to that of wines at the same alcohol concentration, prepared from fruit harvested earlier. Methods and Results Shiraz grapes were harvested at three stages; earlier harvest at 13.4 and 14.4°Be (H‐1, H‐2) and a later harvest at 15.6°Be (H‐3). Water was then added to H‐3 must to match the sugar concentration of H‐1 and H‐2, respectively. Two modes of water addition were followed: (i) water was directly added to the must; or (ii) juice was substituted for water. Both water addition treatments significantly decreased the concentration of phenolics throughout the winemaking process. At the end of malolactic fermentation, the concentration of phenolics, tannin and non‐bleachable pigments, tannin molecular mass and wine colour density were lower in the water addition treatments compared with that of H‐3, but in all other compositional aspects were higher than that of H‐1 and H‐2. Interestingly, where the extent of alcohol reduction by water addition was equivalent, no difference in any of the measured wine attributes was found between the two modes of water addition. Conclusions Water addition to the H‐3 treatment led to higher wine tannin, colour and non‐bleachable pigment than were found in wines made from grapes harvested at a lower sugar concentration. Significance of the Study The study presents a meaningful comparison of potential outcomes in Shiraz wine phenolics and colour for producers who aim to lower alcohol using the approaches of either water addition or early harvest.

To explore the effect of pectolytic enzyme application on polysaccharide size and colloidal interactions, rosé wines were prepared from pressed Shiraz grape juice, and red wines were made from the same juice by the addition of skins and seeds from fresh pressings. A pectolytic enzyme preparation containing primarily polygalacturonase and side-activities of arabinase and pectin lyase was used. Enzyme treatment enhanced the contribution of high molecular weight (≈ 200 kDa) polysaccharides rich in mannose and glucose (mannoproteins) and removed arabinose-rich polysaccharides of intermediate (≈ 40 kDa) size. Enzyme application reduced the molecular weight average of a class of smaller polysaccharides of approximately 6 kDa (rhamnogalacturonan II). All wines had similar particle sizes, but enzyme treatment markedly reduced particle concentration in wines, particularly in rosé wines. Wine polysaccharides were purified and when reconstituted in model wine, showed reduced particle concentration in response to enzyme treatment. Aggregation of polysaccharide in the presence of seed tannin was also markedly reduced by enzyme treatment. The results indicated that changes in structure introduced by the enzyme affected wine colloidal properties, potentially increasing polysaccharide solubility.

Polymeric pigments formed via ethyl linkages between grape tannins and anthocyanins are important to the development of stable red wine color. To determine the effect of tannin structure on the stability and color properties of ethyl-linked polymeric pigments, tannin fractions with average polymerization between 4 and 43 units were prepared from grape skins and seeds and combined with malvidin-3-glucoside (M3G) in model wine containing acetaldehyde. As tannin molecular mass increased, the reaction rate with M3G increased. Compared with skin tannins of comparable molecular mass, seed tannins reacted more rapidly with M3G but were prone to precipitation. This resulted in a loss of polymeric pigments formed from seed tannins, which was greater as tannin molecular mass increased. Aggregation occurred following the reaction of seed tannin with M3G, concomitant with precipitation. The aggregation-precipitation phenomenon was not observed for skin tannin-derived pigments, indicating a greater stability in solution than those formed from seed tannins.

Protein is reportedly negligible in most red wines, due to its loss following co-precipitation with phenolic substances. A method for protein quantification in red wine was developed which overcame analytical interference from phenolic substances, based on ethanol precipitation, followed by acid-hydrolysis and amino acid quantification. Protein concentration was surveyed in a range of red wines produced from V. vinifera and interspecific (Vitis spp) hybrids, revealing higher than expected concentrations, ranging from 23 mg/L ± 2.57 to 380 mg/L ± 16. The results showed that tannin extracted from grapes remains soluble in wine in the presence of protein even at high protein (100 mg/L) and tannin (500 mg/L) concentrations. As a further consequence of this, the particle size and concentration of colloids within high- and low-protein wines were similar, independent of protein or tannin concentration. Higher wine tannin concentration was also correlated with increased heat stability of wine protein.

When bushfires occur near grape growing regions, vineyards can be exposed to smoke, and depending on the timing and duration of grapevine smoke exposure, fruit can become tainted. Smoke-derived volatile compounds, including volatile phenols, can impart unpleasant smoky, ashy characters to wines made from smoke-affected grapes, leading to substantial revenue losses where wines are perceivably tainted. This study investigated the potential for post-harvest ozone treatment of smoke-affected grapes to mitigate the intensity of smoke taint in wine. Merlot grapevines were exposed to smoke at ~7 days post-veraison and at harvest grapes were treated with 1 or 3 ppm of gaseous ozone (for 24 or 12 h, respectively), prior to winemaking. The concentrations of smoke taint marker compounds (i.e., free and glycosylated volatile phenols) were measured in grapes and wines to determine to what extent ozonation could mitigate the effects of grapevine exposure to smoke. The 24 h 1 ppm ozone treatment not only gave significantly lower volatile phenol and volatile phenol glycoside concentrations but also diminished the sensory perception of smoke taint in wine. Post-harvest smoke and ozone treatment of grapes suggests that ozone works more effectively when smoke-derived volatile phenols are in their free (aglycone) form, rather than glycosylated forms. Nevertheless, the collective results demonstrate the efficacy of post-harvest ozone treatment as a strategy for mitigation of smoke taint in wine.

Changes to Australian regulations now allow the limited addition of water to high-sugar musts pre-fermentation. In light of these changes, this study explored how water addition affects Shiraz wine composition and sensory properties. Wines were made from grapes at &asymp, 13.5, 14.5 and 15.5&deg, Baume. Water was added to musts from the ripest fruit by direct addition, or by using a juice substitution (run-off and replace) approach. To compare the effect of juice run-off independently, saigne&eacute, treatments were included. Wines made from the fruit that was harvested earlier generally had a lower &ldquo, opacity&rdquo, and higher &ldquo, red fruit&rdquo, aroma as the defining sensory attributes. Undiluted wines made from riper fruit had higher phenolics, and were characterised by &ldquo, dark fruit&rdquo, and &ldquo, dried fruit&rdquo, attributes, and &ldquo, spice&rdquo, a &ldquo, brown colour&rdquo, These attributes were accentuated in wines from the same fruit which received saigne&eacute, treatments and reduced in all of the water addition treatments. In particular, higher levels of water addition without juice substitution increased the &ldquo, cooked vegetable&rdquo, drain&rdquo, attributes in the wines. This indicates possible negative effects of larger water additions, such that a low to moderate adjustment in Shiraz winemaking is suggested.

Accentuated Cut Edges (ACE) is a recently developed grape must extraction technique, which mechanically breaks grape skins into small fragments but maintains seed integrity. This study was the first to elucidate the effect of ACE on Shiraz wine&rsquo, s basic chemical composition, colour, phenolic compounds, polysaccharides and sensory profiles. A further aim was to investigate any potential influence provided by ACE on the pre-fermentation water addition to must. ACE did not visually affect Shiraz wine colour, but significantly enhanced the concentration of tannin and total phenolics. Wine polysaccharide concentration was mainly increased in response to the maceration time rather than the ACE technique. ACE appeared to increase the earthy/dusty flavour, possibly due to the different precursors released by the greater skin breakage. The pre-fermentation addition of the water diluted the wine aromas, flavours and astringency profiles. However, combining the ACE technique with water addition enhanced the wine textural quality by increasing the intensities of the crucial astringent wine quality sub-qualities, adhesive and graininess. Furthermore, insights into the chemical factors influencing the astringency sensations were provided in this study. This research indicates that wine producers may use ACE with pre-fermentation water dilution to reduce the wine alcohol level but maintain important textural components.

International audience; This study explored plasma levels and urinary and fecal excretion of metabolites and microbial-derived catabolites over a 24 h period following the ingestion of red wine (RWP) or grape seed (GSP) proanthocyanidin-rich extracts by rats. In total, 35 structurally-related (epi)catechin metabolites (SREMs) and 5-carbon side chain ring fission metabolites (5C-RFMs) (phenyl-γ-valerolactones and phenylvaleric acids), and 50 phenolic acid and aromatic catabolites were detected after intakes of both extracts. The consumption of the RWP extract, but not the GSP extract, led to the appearance of a ∼200 nmol L-1 peak plasma concentration of SREMs formed from flavan-3-ol monomers. In contrast, ingestion of the GSPs, but not the RWPs, resulted in a substantial increase in microbiota-derived 5-carbon side chain ring fission metabolites (5C-RFMs) in plasma. 5C-RFMs, along with low molecular weight phenolic catabolites were detected in urine after ingestion of both extracts. The GSP and RWP extracts had respective mean degrees of polymerisation 5.9 and 6.5 subunits, and the RWP extract had an upper polymer size of 21 subunits compared to 44 subunits for the GSP extract. The differences in plasma metabolite profiles might, therefore, be a consequence of this polydispersity impacting on the microbiota-mediated rates of cleavage of the proanthocyanidin subunits and their subsequent metabolism and absorption. Urinary excretion of phenolic catabolites indicated that 11% of RWPs and 7% for GSPs were subjected to microbial degradation. In all probability these figures, rather than representing the percentage of proanthocyanidins that are completely degraded, indicate partial cleavage of monomer subunits producing a much higher percentage of shortened proanthocyanidin chains. Obtaining more detailed information on the in vivo fate of proanthocyanidins is challenging because of the difficulties in analysing unabsorbed parent proanthocyanidins and their partially degraded flavan-3-ol subunit chains in feces. Further progress awaits the development of improved purification and analytical techniques for proanthocyanidins and their use in feeding studies, and in vitro fecal and bacterial incubations, with radio and/or stable isotope-labelled substrates.

This study investigated the color, phenolic, polysaccharide, volatile and sensory profiles of Cabernet Sauvignon wines made from flash detente (FD) treated musts fermented at different temperatures (16, 24 or 32 °C), with and without suspended grape solids. Low fermentation temperature and low solids content increased the concentration of esters, whereas the opposite conditions increased the concentration of fusel alcohols, polysaccharides and glycerol. Higher fermentation temperatures also increased linalool concentration independent of solids content. Traditional maceration fermentation conditions gave the highest concentration of fusel alcohols and 1-hexanol relative to FD treatments. Pre-fermentation removal of grape solids from FD juice created wines with increased red fruit and confectionery attributes, whereas inclusion of 3.5% grape solids increased dark fruit notes. In comparison, control wines had significantly higher green and savory attributes compared to wines from FD treatments. Research findings demonstrated the potential for FD to be used to create differentiated red wine styles.

Producing wines within an acceptable range of astringency is important for quality and consumer acceptance. Astringency can be modified by fining during the winemaking process and the use of vegetable proteins (especially potato proteins) as fining agents has gained increasing interest due to consumers&rsquo, requirements. The research presented was the first to investigate the effect of a potato protein dose on the kinetics of tannin and phenolic removal compared to gelatin for two unfined Cabernet Sauvignon wines. To further understand the results, the influence of the wine matrix and fining parameters (including pH, ethanol concentration, sugar concentration, temperature, and agitation) were tested according to a fractional 25-1 factorial design on one of the Cabernet Sauvignon wines using potato proteins. The results from the factorial design indicate that potato protein fining was significantly influenced by wine pH, ethanol concentration, fining temperature as well as an interaction (pH &times, ethanol) but not by sugar content or agitation. Insights into the steps required for the optimisation of fining were gained from the study, revealing that potato protein fining efficiency could be increased by treating wines at higher temperatures (20 &deg, C, rather than the conventional 10&ndash, 15 &deg, C), and at both a lower pH and/or alcohol concentration.

A changing climate has led to winegrapes being harvested with increased sugar levels and at greater risk of berry shrivel. A suggested easy-to-adopt strategy to manage the associated rising wine alcohol levels is the pre-fermentative substitution of juice with either "green harvest wine" or water. Our study investigates the effects of this approach on Vitis vinifera L. cv. Cabernet Sauvignon wine quality attributes. Wines were also made from fruit collected at consecutive earlier harvest time points to produce wines comparable in alcohol to the substituted wines. Tannin concentrations and colour did not change significantly in the wines with modified alcohol content even at higher juice substitution rates. Differences in polysaccharide and tannin composition indicated variability in extraction dynamics according to substitution rate and type of blending component. In scenarios where berry shrivel is inevitable, the incorporation of water in particular offers much promise as part of a strategy to manage wine alcohol content.

Background and Aims The choice of yeast strain(s) to conduct the fermentation can greatly affect wine chemical and sensory profile. Even though the use of non‐Saccharomyces co‐inocula to build complexity and diversify styles is increasingly in vogue, a limited number of such products are available to date, and more research is required to guide their use in the wine industry. This study evaluates the potential of commercial yeast inocula to modulate the quality of Shiraz wines at two maturity levels. Methods and Results Vinification outcomes of eight yeast treatments were compared in earlier (24°Brix) and later (29°Brix) harvested Shiraz fruit. Yeast treatments included five non‐Saccharomyces products with sequentially inoculated Saccharomyces cerevisiae, a commercial blend of non‐Saccharomyces and S. cerevisiae strains, and a S. cerevisiae inoculum. Fermentation monitoring, and comprehensive analytical profiling in terms of basic chemistry, volatile composition, phenolic measurements and descriptive sensory analysis, allowed for the comparison of the resulting wines. Both harvest date and yeast inoculation treatments had a significant impact on a range of compositional and, in turn, sensory parameters of the wines. Conclusions Certain non‐Saccharomyces sequential inoculation treatments led to increased appeal of earlier harvest wines compared to the S. cerevisiae Control. These treatments, however, were related to an increased risk of arrested fermentation in higher ripeness conditions. Significance of the Study This study contributes to a better understanding of yeast inoculum‐derived modulation of Shiraz wine quality parameters at different maturity levels.

Enotannin and mannoprotein additives are applied to achieve protein, cold, or color stability in wine, or to modify wine sensory properties. In most cases, the manufacturer only provides basic compositional information and a proposed effect in wine. In this study, 14 grape-based enotannins and eight mannoproteins were sourced from the Australian market, and their composition and molecular size distribution were determined. Diverse product composition was observed for both categories, suggesting that a range of effects could potentially be achieved by applying different products. Moreover, some products showed good agreement between product composition and their designated material of origin, while others showed significant differences.

In this work, the interaction of wine macromolecules with a bovine serum albumin (BSA) was investigated using fluorescence correlation spectroscopy (FCS). FCS offers the opportunity to study molecular and macromolecular aggregation without disturbing the wine by introducing only very small amounts of fluorescently labelled molecules to the system. It was observed that the diffusion coefficient of fluorescently labelled BSA varies with the addition of wine macromolecules, indicating changes in the protein conformation and the formation of complexes and aggregates. The addition of a wine polysaccharide rhamnogalacturonan II-enriched fraction led to aggregation, while addition of a mannoprotein-enriched fraction exhibited a protective effect on protein aggregation. Proteins strongly interacted with tannins, leading to the precipitation of protein-tannin complexes, while the presence of polysaccharides prevented this precipitation. Finally, the application of FCS was demonstrated in real wines, to investigate the problem of protein haze formation through live monitoring of heat-induced aggregation in wine Refereed/Peer-reviewed

Background and Aims The study aimed to determine how fresh grape mesocarp modifies the extraction and composition of tannin under wine-like conditions. A further aim was to observe whether addition of anthocyanin during extraction exerted an effect on the concentration of recovered tannin. Methods and Results Fresh red grape berries were dissected into skin, mesocarp and seed. Different combinations of the berry components were extracted in model wine or fermented. Initial results indicated that mesocarp material bound seed tannin more selectively than skin tannin. Further experiments pointed towards a more complex system, whereby adsorption of tannin by mesocarp facilitated ongoing extraction of tannin from the skins. The effect exerted by mesocarp was potentially to increase the concentration gradient between skins and the liquid medium by the continuous removal of tannin. Under these conditions, both skin and seed tannin could be removed from extracts or wines by suspended mesocarp material. White grape skins or mesocarp were also extracted with the addition of a variable concentration of purified anthocyanin and found to facilitate the extraction of otherwise non-extractable tannin. This effect was observed to be greater for grape mesocarp than for white grape skins. Conclusions The concentration and composition of tannin were shown to be modified by the presence of mesocarp material. A role for anthocyanin in enhancing the extraction of tannin was confirmed. Significance of the Study The results show that multiple factors interact to influence tannin extraction and retention from grapes.

The use of bentonite fining in the global wine industry causes losses in the range of US$1 billion per year. This work explores for the first time the potential use of natural zeolites as an alternative to bentonite to remove haze proteins from white wines. Wines treated with zeolites were compared to those obtained after fining using two commercial bentonites (Vitiben and Pluxcompact). Semillon wine was fully stabilized by applying 4 g/L of zeolite, whereas Sauvignon Blanc and Chardonnay required 6 g/L dosage of zeolite. Analysis of metal content showed that zeolites reduced potassium concentration by more than 30% in the treated wines, which could lead to improved tartrate stability. Importantly, compared to bentonites, zeolites cause much less wine loss, and they can be potentially reused as soil amendments in agriculture. The results of this study indicate that natural zeolites can offer winemakers an alternative to bentonite for haze protein removal from white wines. Refereed/Peer-reviewed

Red wine color is linked to wine quality and therefore has been the subject of much research over decades. Anthocyanins are red pigments produced in grape skins during ripening and their concentration and composition varies with grape variety as well as viticulture factors including grape ripeness, sunlight or UV exposure, plant water status and pathogens. Extraction of anthocyanins during winemaking triggers numerous reactions that lead to changes in the chemical structure, color and stability of these pigments to produce anthocyanin adducts such as pyranoanthocyanins or pigmented polymers. Winemaking factors influence the impact of such reactions on wine color and include oxygen exposure, maceration, concentration of SO 2 , presence of copigments, and the addition of fining agents. Measuring overall wine color is typically achieved using spectroscopic methods, including CEILAB, Glories or Somers measures. Anthocyanin compositions and concentrations can be measured by LC-MS techniques whereas characterization of pigmented polymers is best achieved after isolation, depolymerisation and subsequent characterization of monomeric subunits. Investigating the chemical composition of wine pigments and the factors influencing their formation enables the effective production of quality red wines.

The tannin and polysaccharide profiles and therefore sensory properties of wine are influenced by fruit maturity at harvest, and practices employed during winemaking. This study investigated the extent to which commercial winemaking supplements (skin and seed tannins, and mannoprotein (MP)) can enhance the mouthfeel properties of red wine, in particular, wine made from grapes harvested before commercial ripeness (early-harvest). Supplements were added to wines made from Shiraz grapes harvested at 20.8 and 24.5 &deg, Brix. The chemical composition and mouthfeel properties of wines were then determined by high performance liquid chromatography and descriptive analysis (DA), respectively. Wines made from riper grapes had higher levels of tannin than wines made from early-harvest grapes, but similar polysaccharide levels were observed. The addition of seed oenotannin yielded higher tannin levels than addition of skin oenotannin, particularly for wines made from early-harvest grapes. The DA panel perceived sensory differences between H1 and H2 wines, but could not perceive any effect of supplementation on wine mouthfeel properties, with the exception of a minor increase in sweetness, attributed to mannoprotein addition to H1 wines, even when MP was added to wines at 2.5 times the level recommended for use in Australia.

Precipitates were prepared from two compositionally different Pinot noir wines with addition of excess ethanol, and contained primarily polysaccharide, tannin and protein. The ethanol-soluble material was further fractionated into polymeric (tannin) and monomeric phenolics. Tannin associated with precipitates was of a higher molecular mass than that remaining in ethanolic solution. Wine fractions were reconstituted at the ratios of the original wine and analyzed using nanoparticle tracking analysis. The average particle size of the tannin fraction was 75-89 nm, and increased when combined with the precipitate (≅ 200 nm). Addition of the monomeric fraction to the tannin-precipitate complex increased both the incidence and concentration of smaller particles, reducing the average particle size. The formation of aggregates occurred in all fractions and only minor differences in particle size distribution were found between wines. Differences in particle concentration between wines appear to be due to differences in the total concentration of macromolecules rather than compositional differences.

This study developed, optimized and validated an ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) method to identify and quantify metabolites and microbial-derived catabolites in urine, plasma and feces of rats following ingestion of 50 mg of a red wine proanthocyanidin-rich extract. The method was validated for specificity, linearity, limit of detection (LD) and quantification (LQ), intra-day and inter-day precision, recovery and matrix effects, which were determined for 34 compounds in the three biological matrices. After method validation, three parent flavan-3-ols, four 5-carbon side chain ring fission metabolites, and 27 phenolic acid and aromatic catabolites were quantified in plasma, urine and feces after red wine proanthocyanidin intake. These results establish the value of the UHPLC-HRMS protocol in obtaining a detailed picture of proanthocyanidin metabolites and their microbial-derived catabolites, along with their phase II metabolites, in biological fluids of rat, and potentially in human clinical studies designed to evaluate the bioavailability of dietary flavan-3-ols.

This review summarises key findings from research on the impact of winemaking practices on the concentration and composition of tannins in red wines. The impact from the point of crushing onwards is summarised from our research and recent literature taking into account the effect of maceration, yeast selection, addition of mannoproteins, addition of oenotannins, fining by animal and plant proteins, filtration, oxygen exposure, barrel treatment, bottling and accelerated-ageing techniques. A sufficient body of evidence has developed in certain areas to make generalisations, but in several other areas tannins continue to show large variability of response to winemaking interventions depending on the grapes and their particular methods of treatment. Progress has been made in determining the underpinning mechanisms for some of the effects, but significant further research is required to understand why many of the effects occur. Knowledge gaps remaining in the area and proposals for future research are identified that will provide the opportunity for improved management of vineyards and winemaking to optimise tannins in grapes and wine, and for increased capacity to meet wine specification, consumer expectations and profitability.

Berry tissue components (skin, seed, and pulp) were isolated from Pinot noir grapes to determine the tannin content of each component. In addition, the role of each berry component in determining the phenolic profile of the wine was investigated by omitting or doubling each berry tissue in the must and fermenting these using submerged cap micro-vinification. Monomeric anthocyanin, total tannin and non-bleachable pigment concentrations in juice and wine were examined at seven time intervals from yeast inoculation (day 0) to 12 months bottle age (day 400). When included in the must, berry pulp was associated with reduced wine tannin concentration, an effect that was more selective for seed tannin. Consequently the tannin composition of wines made with whole-berry ferments most closely reflected that of skin tannin. The formation of non-bleachable pigments was greater in treatments where seeds were present during fermentation. However, when the contribution of seed tannin in wine was increased by doubling the complement of seeds in the must, anthocyanin concentration remained constant and non-bleachable pigments did not increase during ageing. Treatments with a double complement of grape skins were found to enhance both non-bleachable pigment and tannin in aged wines.

Shiraz ferments were injected with different levels of oxygen (20% and 40% O 2 as gas mixtures) to assess the impact of early oxygen exposure on wine tannins and colour measures after fermentation, and after 2 and 12 months bottle-ageing under either Saranex™ or Saran Tin™ screw caps. The O 2 -treated ferments contained lower concentrations of anthocyanins and tannins, and tannin composition showed lower molecular masses, conversion yields, and proportions of prodelphinidin subunits than those from the control ferments. The colour measures and tannin characteristics of the control wines after 12 months resembled those of the O 2 -treated wines at 2 months bottle-ageing, and oxygen exposure during fermentation had a greater impact on tannin structure than closure type. Early oxygen exposure during winemaking may result in wines with phenolic compositions that are characteristic of more aged wines, which may reduce the need for extended wine ageing.

Cell wall material was isolated from two Vitis vinifera grape varieties, Cabernet Sauvignon and Shiraz, following a buffered phenol extraction method. Using sequential fractionation in chelating agent, then increasing the molarity of aqueous potassium hydroxide, polysaccharide classes were selectively extracted from cell walls to produce fractions of defined polysaccharide composition. Following the application of phloroglucinolysis and a modified HCl-butanol colourimetric assay to cell wall fractions, more than 54% of cell wall-bound proanthocyanidin was localised within the chelator-soluble (pectic) fraction. Model adsorption experiments with a purified skin proanthocyanidin confirmed that the removal of pectic polysaccharides by chelator most significantly reduced the adsorption of proanthocyanidin by cell walls. Nevertheless, cell wall hemicellulosic fractions retained a high binding capacity for proanthocyanidin, although lower than that observed when pectin was present. Following removal of hemicelluloses by fractionation, the primarily lignocellulosic residue had a significantly reduced affinity for proanthocyanidin. With the exception of lignocellulose, a greater selectivity of adsorption for higher molecular mass proanthocyanidins was observed by the respective cell wall fractions.

Wine quality can be significantly affected by tannin and polysaccharide composition, which can in turn be influenced by grape maturity and winemaking practices. This study explored the impact of three commercial wine additives, a maceration enzyme, an enotannin, and a mannoprotein, on the composition and sensory properties of red wine, in particular, in mimicking the mouthfeel associated with wines made from riper grapes. Shiraz grapes were harvested at 24 and 28 °Brix and the former vinified with commercial additives introduced either individually or in combination. Compositional analyses of finished wines included tannin and polysaccharide concentration, composition and size distribution by high-performance liquid chromatography, whereas the sensory profiles of wines were assessed by descriptive analysis. As expected, wines made from riper grapes were naturally higher in tannin and mannoprotein than wines made from grapes harvested earlier. Enzyme addition resulted in a significantly higher concentration and average molecular mass of wine tannin, which increased wine astringency. Conversely, mannoprotein addition reduced tannin concentration and astringency. Addition of enotannin did not meaningfully influence wine composition or sensory properties.

A series of five Vitis vinifera L. cv Cabernet Sauvignon wines were produced from sequentially-harvested grape parcels, with alcohol concentrations between 12% v/v and 15.5% v/v. A multidisciplinary approach, combining sensory analysis, consumer testing and detailed chemical analysis was used to better define the relationship between grape maturity, wine composition and sensory quality. The sensory attribute ratings for dark fruit, hotness and viscosity increased in wines produced from riper grapes, while the ratings for the attributes red fruit and fresh green decreased. Consumer testing of the wines revealed that the lowest-alcohol wines (12% v/v) were the least preferred and wines with ethanol concentration between 13% v/v and 15.5% v/v were equally liked by consumers. Partial least squares regression identified that many sensory attributes were strongly associated with the compositional data, providing evidence of wine chemical components which are important to wine sensory properties and consumer preferences, and which change as the grapes used for winemaking ripen.

Cabernet Sauvignon and Shiraz grapes were sourced from different regions within Australia, and microvinified with a skin contact period of 6 days. Grape samples were extracted using two protocols: a 15% v/v ethanol, 10 g/L tartaric acid extract of gently crushed berries (wine-like, WL) and a 50% v/v ethanol, pH 2 extract of grape berry homogenate. It was found that in WL extracts, grape tannin and anthocyanin concentrations were strongly related to wine tannin, anthocyanin and color density achieved during the skin contact period. No relationship was observed for grape tannin concentration analyzed in homogenate extracts and wine tannin, but a strong, positive relationship was found for anthocyanin concentration. When the data obtained from homogenate extraction was treated separately by grape variety, a stronger relationship between grape and wine tannin concentration was observed. Tannin compositional analysis in wines indicated that higher tannin concentrations were due to the extraction of tannin of higher molecular mass during fermentation, most likely from grape skins.

Red wines injected with nitrogen or oxygen during fermentation were used to identify the effect of gas exposure on tannin structure and reactivity with poly-l-proline. Tannin was purified from wine after fermentation and after three years of bottle storage. Tannin from nitrogen-treated wine had a lower percentage of galloylation and were less pigmented than tannin from oxygen-exposed wine. Self-aggregation of tannin was measured by nanoparticle tracking analysis and a larger particle size was observed for the oxidized treatment. The interaction of tannin and poly-l-proline was measured by isothermal titration calorimetry, and involved more hydrogen bonding than hydrophobic interactions in the case of nitrogen-treated wine tannin. Conversely, oxidized tannin was more hydrophobic and the association with poly-l-proline was entropy-driven due to a change of solvation. The results show meaningful changes in the structure and reactivity of tannin as a result of oxygen exposure during fermentation, which may impact astringency perception.

The application of animal-derived proteins as wine fining agents has been subject to increased regulation in recent years. As an alternative to protein-based fining agents, insoluble plant-derived fibers have the capacity to adsorb red wine tannins. Changes in red wine tannin were analyzed following application of fibers derived from apple and grape and protein-based fining agents. Other changes in wine composition, namely, color, monomeric phenolics, metals, and turbidity, were also determined. Wine tannin was maximally reduced by application of an apple pomace fiber and a grape pomace fiber (G4), removing 42 and 38%, respectively. Potassium caseinate maximally removed 19% of wine tannin, although applied at a lower dose. Fibers reduced anthocyanins, total phenolics, and wine color density, but changes in wine hue were minor. Proteins and apple fiber selectively removed high molecular mass phenolics, whereas grape fibers removed those of both high and low molecular mass. The results show that insoluble fibers may be considered as alternative fining agents for red wines.

For better understanding of the factors that impact proanthocyanidin (PA) adsorption by insoluble cell walls or interaction with soluble cell wall-derived components, application of a commercial polygalacturonase enzyme preparation was investigated to modify grape cell wall structure. Soluble and insoluble cell wall material was isolated from the skin and mesocarp components of Vitis vinifera Shiraz grapes. It was observed that significant depolymerization of the insoluble grape cell wall occurred following enzyme application to both grape cell wall fractions, with increased solubilization of rhamnogalacturonan-enriched, low molecular weight polysaccharides. However, in the case of grape mesocarp, the solubilization of protein from cell walls (in buffer) was significant and increased only slightly by the enzyme treatment. Enzyme treatment significantly reduced the adsorption of PA by insoluble cell walls, but this effect was observed only when material solubilized from grape cell walls had been removed. The loss of PA through interaction with the soluble cell wall fraction was observed to be greater for mesocarp than skin cell walls. Subsequent experiments on the soluble mesocarp cell wall fraction confirmed a role for protein in the precipitation of PA. This identified a potential mechanism by which extracted grape PA may be lost from wine during vinification, as a precipitate with solubilized grape mesocarp proteins. Although protein was a minor component in terms of total concentration, losses of PA via precipitation with proteins were in the order of 50% of available PA. PA-induced precipitation could proceed until all protein was removed from solution and may account for the very low levels of residual protein observed in red wines. The results point to a dynamic interaction of grape insoluble and soluble components in modulating PA retention in wine.

Cell wall material from Vitis vinifera L. cv. Cabernet Sauvignon grape skin and flesh was isolated at different stages of grape maturity to determine whether developmental changes in cell wall composition in different tissue types influence the binding of proanthocyanidins (PAs). Trends in cell wall adsorption of, and selectivity for, PAs were determined using two skin PAs that differed in their average molecular masses. Flesh cell walls consistently bound a higher amount of PA than those from skin. Key structural differences that reduced PA adsorption in skin cell walls by comparison with flesh cell walls were endogenously higher concentrations of insoluble PA, Klason lignin, and lower cell wall-bound protein. These differences may confer reduced flexibility and porosity of skin cell walls relative to flesh cell walls. Analysis of skin and flesh cell wall properties revealed that the onset of ripening was associated with a loss of type I arabinogalactan and galacturonic acid, which indicated a degradation of pectin within the cell wall. Flesh cell walls consistently bound PAs of larger molecular mass, and changes in PA adsorption properties after the onset of ripening were minor. For skin cell walls, adsorption of PA was lowest immediately following solubilization of galacturonic acid, and high molecular mass PAs were poorly bound. As ripening progressed, PAs of higher molecular mass were selectively adsorbed by skin cell walls, which indicates that ongoing cell wall remodeling during ripening may confer an increased porosity within the skin cell wall matrix, resulting in a greater adsorption of PA within a permeable structure.

Background and Aims: This study investigated flavonoid composition and C13-norisoprenoids (β-damascenone and β-ionone) in Shiraz grapes and wines, their relationships and links to wine sensory properties. Methods and Results: Differences in the grape berry flavonoid profile were created by exposing bunches to varying levels of sunlight intensity through canopy manipulation. Grapes were harvested at similar maturity and three replicate wines were made for each treatment in both vintages. Grapes produced under shaded canopy conditions had reduced anthocyanins and skin tannins, but little effect on seed tannins was observed. Pigmented polymers and tannins in wines were related to berry flavonoid composition (anthocyanins, skin and seed tannins, and their ratios). In grapes and wines, no significant effects were observed in response to canopy manipulation for two hydrolytically released C13-norisoprenoids, β-damascenone and β-ionone. Relationships were established for wine flavonoid composition, wine colour density, sensory perception of the astringency-related mouth-feel attributes and a quality scale. A positive relationship between wine quality score and hydrolytically released β-damascenone in both berries and wines was found, but not for free β-damascenone or any quantified forms of β-ionone. Conclusion: Higher concentrations of anthocyanins and skin tannins in berries, coupled with a lower concentration of seed tannins were associated with higher wine quality. The ratio anthocyanins*skin tannins/seed tannins is proposed as an indicator of wine flavonoid composition, wine colour and wine quality. Excessive canopy shade was detrimental to berry and wine composition and intensified sensory detection of ‘straw’ and ‘herbaceous’ characters in the wines. Significance of the Study: This study increases the understanding of the balance and composition of flavonoid compounds and C13-norisoprenoids in berries and their relationship with wine composition and wine sensory properties, but also highlights the importance of a canopy microclimate assessment.

Proanthocyanidins (PAs) were isolated from the skins, seeds and flesh of commercially ripe grapes, and from wine and marc produced from the same source. In the grape berry, skin PAs accounted for 54% of the total extractable PA, while seed and flesh-derived PA accounted for 30% and 15% of the total, respectively. Following fermentation, 25% of the fruit PA was found in the wine, while 27% was found in the pericarp isolated from marc, and 48% was unaccounted for (either remaining in the seed or adsorbed to lees). To investigate the role that cell wall material (CWM) has on PA extraction during fermentation, CWM isolated from skin and flesh were combined with PA in model suspensions. In general, the affinity of flesh CWM for PA increased with increasing PA molecular mass (MM); however, this relationship was not observed for the interaction of skin CWM with skin PA. Subsequent experiments suggest that the differences in the interaction of flesh and skin CWM with PA of higher MM (>15000 g/mol) may be limited by the structure of the CWM. Observed variations in the composition between skin and flesh CWM may explain the differences in PA interaction at high MM. Among wine-derived PA, no higher MM material was detected, suggesting that, during vinification, higher MM PA are nonextractable and/or are removed from the wine by interaction with CWM.

The aim of this study was to determine how changes in grape composition brought about by artificial shading (sunlight exclusion) influence wine properties including colour, flavonoid composition and sensory attributes. Prior to flowering, bunches of Shiraz grapes were enclosed in boxes designed to eliminate light without altering bunch temperature and humidity. This artificial bunch shading had little effect on berry ripening and accumulation of sugar but at harvest the shaded bunches had smaller berries and higher seed weight, juice pH and titratable acidity. The amount of anthocyanins in the fruit was not changed significantly but anthocyanin composition in the shaded berries was shifted towards dioxygenated anthocyanins (the glucosides of cyanidin and peonidin derivatives). Shaded fruit had increased seed tannins and decreased skin tannins but the largest relative change in flavonoids was a marked decrease in flavonols in the shaded fruit, similar to previous studies. Wines made from shaded fruit had lower wine colour density, total phenolics, anthocyanins and tannins when the wine was bottled and after ageing for up to three years. Analysis of potential flavour compounds following acid hydrolysis indicated that the wines made from shaded fruit had decreased levels of glycosides of β-damascenone and 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN). Sensory analysis of the wines indicated no significant difference in aroma attributes but the wines made from shaded fruit were rated lower for astringency, fruit flavour and flavour persistence in-mouth sensory attributes. The results indicate that extreme shading of Shiraz fruit can decrease wine colour, anthocyanins and tannins as well as altering sensory attributes.

The influence of irrigation strategy on grape berry carotenoids and C13-norisoprenoid precursors was investigated for Vitis vinifera L. cv. Cabernet Sauvignon. Two irrigation treatments were compared, one in which vines received reduced irrigation applied alternately to either side of the vine (partial rootzone drying, PRD) and a second control treatment in which water was applied to both sides of the vine. Over the two years of the experiments, PRD vines received on average 66% of the water applied to the controls. Initially, the PRD treatment did not alter midday leaf (psiL) and stem (psiS) water potential relative to the control, but decreased stomatal conductance (gs). Continued exposure to the PRD treatment resulted in treated grapevines experiencing hydraulic water deficit relative to the control treatment and induced lowered midday psiL and psiS, which was also reflected in decreased berry weight at harvest. In both irrigation treatments, the most abundant grape berry carotenoids, beta-carotene and lutein, followed the developmental pattern typical of other grape varieties, decreasing post-veraison. At certain points in time, as the fruit approached maturity, the concentration of these carotenoids was increased in fruit of PRD-treated vines relative to the controls. This effect was greater for lutein than for beta-carotene. PRD consistently caused increases in the concentration of hydrolytically released C13-norisoprenoids beta-damascenone, beta-ionone, and 1,1,6-trimethyl-1,2-dihydronaphthalene in fruit at harvest (24 degrees Brix) over two seasons. The effect of the PRD treatment on the concentration of hydrolytically released C13-norisoprenoids was greater in the second of the two seasons of the experiment and was also reflected in an increase in total C13-norisoprenoid content per berry. This suggests that the increases in the concentration of the C13-norisoprenoids in response to PRD were independent of water deficit induced changes in berry size and were not the result of an altered berry surface area to volume ratio.

To determine the optimum methods for determining condensed tannin (CT) content in grape marc, butanol-hydrochloric acid assays and phloroglucinolysis were adapted for use, applied to a range of grape marc types, and the methods compared. Porter's assay (butanol-HCl) was found to give unreliable results due to nonlinear color responses to grape skin and seed tannin concentrations, whereas the modification to include acetone (Grabber's assay) overcame this. Differences between skin and seed tannin responses highlighted the need to adequately select the correct grape tannin standard, and the formation of pH-dependent color was accounted for through acidification of blank samples. For phloroglucinolysis, the inability to remove highly bound tannins from cell wall material was highlighted, although a measure of tannins remaining post-phloroglucinolysis (Grabber's assay) showed a trend with the level of exposure to oxidative storage or processing conditions. The comparison of CT concentrations from phloroglucinolysis and Grabber's assay gave poor correlation coefficients.

To explore the effect of tannin composition on pigment formation, model ferments of purified 3-O-monoglucoside anthocyanins (ACN) were conducted either alone or in the presence of two different tannins. Tannins were isolated from grape seeds (Sd) or skins (Sk) following exhaustive extraction in 70% v/v acetone. The Sd and Sk tannin fractions had a mean degree of polymerization of 5.2 and 25.6, respectively. The Sd fraction was highly galloylated, at 22%, but galloylation was

The acetone-extractable (70% v/v) skin tannin content of Vitis vinifera L. cv. Cabernet Sauvignon grapes was found to increase during late-stage ripening. Conversely, skin tannin content determined following ethanol extraction (10, 20, and 50% v/v) did not consistently reflect this trend. The results indicated that a fraction of tannin became less extractable in aqueous ethanol during ripening. Skin cell walls were observed to become more porous during ripening, which may facilitate the sequestering of tannin as an adsorbed fraction within cell walls. For ethanol extracts, tannin molecular mass increased with advancing ripeness, even when extractable tannin content was constant, but this effect was negligible in acetone extracts. Reconstitution experiments with isolated skin tannin and cell wall material indicated that the selectivity of tannin adsorption by cell walls changed as tannin concentration increased. Tannin concentration, tannin molecular mass, and cell wall porosity are discussed as factors that may influence skin tannin extractability. Refereed/Peer-reviewed

The study aimed to quantify the effects of grape maturity on wine alcohol, phenolics, flavour compounds and polysaccharides in Vitis vinifera L. cv Cabernet Sauvignon. Grapes were harvested at juice soluble solids from 20 to 26 °Brix which corresponded to a range of wine ethanol concentrations between 12% and 15.5%. Grape anthocyanin and skin tannin concentration increased as ripening progressed, while seed tannin declined. In the corresponding wines, monomeric anthocyanin and wine tannin concentration increased with harvest date, consistent with an enhanced extraction of skin-derived phenolics. In wines, there was an observed increase in yeast-derived metabolites, including volatile esters, dimethyl sulfide, glycerol and mannoproteins with harvest date. Wine volatiles which were significantly influenced by harvest date were isobutyl methoxypyrazine, C 6 alcohols and hexyl acetate, all of which decreased as ripening progressed. The implications of harvest date for wine composition is discussed in terms of both grape composition and yeast metabolism.

The fining action of commercial proteins and insoluble fibres for wine proanthocyanidin (PA) were compared. Fibres were prepared from fresh apple and grape sources, and their corresponding pomaces. PA removal by fibre was via adsorption, and required a higher dose to achieve a fining effect comparable with proteins. A principal component analysis data model revealed that PA molecular mass was significant in defining the fining response, and reflected changes in the proportion of the dominant terminal PA subunits catechin and epicatechin, but not epicatechin-3-O-gallate. For PA extension subunits, changes in epigallocatechin were inversely correlated with epicatechin and epicatechin-3-O-gallate. Generally, the application of proteins and fibres reduced PA molecular mass. Selectivity for PAs by subunit composition was variable between treatments, but differences were minor. This work demonstrates the potential use of fibres as an alternative to proteins in winemaking. Benefits, and possible limitations of such an approach are discussed.

Proanthocyanidins were isolated from the skins of Cabernet Sauvignon grapes at different stages of grape development in order to study the effect of proanthocyanidin modification on the interaction with grape cell wall material. After veraison, the degree of proanthocyanidin polymerization increased, and thereafter was variable between 24 and 33 subunits as ripening progressed. Affinity of skin cell wall material for proanthocyanidin decreased with proanthocyanidin ripeness following veraison. A significant negative relationship (R2=0.93) was found for average proanthocyanidin molecular mass and the proportion of high molecular mass proanthocyanidin adsorbed by skin cell wall material. This indicated that as proanthocyanidin polymerization increased, the affinity of a component of high molecular mass proanthocyanidins for skin cell wall material declined. This phenomenon was only associated with skin proanthocyanidins from colored grapes, as high molecular mass proanthocyanidins of equivalent subunit composition from colorless mutant Cabernet Sauvignon grapes had a higher affinity for skin cell wall material.