Keren A. Bindon

Interaction between Grape-Derived Proanthocyanidins and Cell Wall Material. 1. Effect on Proanthocyanidin Composition and Molecular Mass

Insoluble cell wall material was prepared from the skin and flesh of commercially ripe Vitis vinifera L. cv. Shiraz berries and then combined in suspension with preveraison skin and seed proanthocyanidin containing solutions. Analysis of proanthocyanidins before and after fining with cell wall material by phloroglucinolysis provided information on recovery by mass, subunit composition, and mean degree of polymerization, whereas proanthocyanidin molecular mass distribution was determined by gel permeation chromatography. Cell wall material from flesh showed the highest affinity for proanthocyanidin, binding up to 47% and 57% w/w of total seed and skin proanthocyanidin respectively. Comparison of the molecular mass distribution of skin or seed proanthocyanidin before and after fining indicated that affinity of cell walls for proanthocyanidin increased with increasing proanthocyanidin molecular mass. Initial results of subunit composition of skin and seed proanthocyanidin mixtures following fining with cell wall material showed that the % galloylation decreased, suggesting a preference for seed-derived proanthocyanidins. Subsequent experiments suggest that fining with insoluble cell wall material is size-based and does not have a specific affinity for seed-derived proanthocyanidins.

Interaction between grape-derived proanthocyanidins and cell wall material. 2. Implications for vinification.

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.

Relationships between harvest time and wine composition in Vitis vinifera L. cv. Cabernet Sauvignon 1. Grape and wine chemistry

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.

Ripening-induced changes in grape skin proanthocyanidins modify their interaction with cell walls.

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.

Relationships between harvest time and wine composition in Vitis vinifera L. cv. Cabernet Sauvignon 2. Wine sensory properties and consumer preference

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.

Factors Affecting Skin Tannin Extractability in Ripening Grapes

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.

Optimization of a Method for the Extraction and Quantification of Carotenoids and Chlorophylls during Ripening in Grape Berries (Vitis vinifera cv. Merlot)

An extraction method to identify and quantify the carotenoid and chlorophyll profile of lyophilized tissue from unripe (green) to ripe (red) Merlot grape berries was developed. The RP-HPLC method baseline separated all of the carotenoids and chlorophylls and their derivatives. Problems encountered during sample storage and extraction are discussed as well as possible alternative methods. This study confirmed that carotenoids and chlorophylls decreased on a per berry (microg/berry) and concentration (microg/g) basis from veraison to harvest over two growing seasons. The carotenoid 5,8-epoxy-beta-carotene was quantified for the first time in grapes and represents a significant amount of the total carotenoids present at harvest. All the carotenoids and chlorophylls except beta-carotene appeared to be sensitive to seasonal variation in climatic conditions. Lutein and beta-carotene were found to be the most abundant carotenoids present in Merlot grape berries together with chlorophyll a for both seasons studied.

Comparison of the affinity and selectivity of insoluble fibres and commercial proteins for wine proanthocyanidins.

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.

Tissue-Specific and Developmental Modifications of Grape Cell Walls Influence the Adsorption of Proanthocyanidins

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.

Selective extraction of polysaccharide affects the adsorption of proanthocyanidin by grape cell walls

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.