Influence of Maceration Temperature in Red Wine Vinification on Extraction of Phenolics from Berry Skins and Seeds of Grape (Vitis vinifera)

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1 6628 (123) Biosci. Biotechnol. Biochem., 71, , 27 Influence of Maceration Temperature in Red Wine Vinification on Extraction of Phenolics from Berry Skins and Seeds of Grape (Vitis vinifera) Kazuya KOYAMA, y Nami GOTO-YAMAMOTO, and Katsumi HASHIZUME National Research Institute of Brewing, Kagamiyama, Higashihiroshima, Hiroshima , Japan Received November 7, 26; Accepted January 31, 27; Online Publication, April 7, 27 [doi:1.1271/bbb.6628] The extraction of phenolics from berry skins and seeds of the grape, Vitis vinifera cv. Cabernet Sauvignon, during red wine maceration and the influence of different temperature conditions (cold soak and/or heating at the end of maceration) were examined. Phenolics contained mainly in berry skins, viz., anthocyanin, flavonol, and epigallocatechin units within proanthocyanidins, were extracted during the early stage of maceration, whereas those in seeds, viz., gallic acid, flavan-3-ol monomers, and epicatechin-gallate units within proanthocyanidins, were gradually extracted. In addition to their localization, the molecular size and composition of the proanthocyanidins possibly influenced the kinetics of their extraction. Cold soak reduced the extraction of phenolics from the seeds. Heating at the end of maceration decreased the concentration of proanthocyanidins. Thus, modification of the temperature condition during maceration affected the progress of the concentration of phenolics, resulting in an alteration of their make-up in the finished wine. Key words: grape; red wine; phenolics; proanthocyanidins; extraction Phenolic compounds contribute greatly to the sensory characteristics of wine, and in particular, of red wine. Anthocyanins and their derivatives are predominant pigments in red wine. Flavonols and hydroxycinnamates are bitter, and proanthocyanidins, which are polymers of flavan-3-ol units and are also called condensed tannin, contribute bitterness and astringency. 1 3) In addition, these compounds have recently received much attention because of their potential contribution to human health due to their antioxidant, antimicrobial, antiviral, and anticarcinogenic characteristics. 4) Phenolic compounds are present mainly in the skins and seeds in red grape berries. 3,) Anthocyanins and flavonols are found in the skins. Hydroxycinnamates 3,) are found in the flesh and skins. On the other hand, flavan-3-ol monomers 6 8) and the majority of gallic acids 3,) in red wine are likely to originate in the seeds. Gallic acid is thought to be released by hydrolysis and heat breakdown from certain esters or more complex molecules, such as the epicatechin-gallate unit in proanthocyanidins, which are contained mainly in the seeds. Proanthocyanidins are extracted from both the skins and the seeds. Proanthocyanidins from the skins and seeds have been found to be organoleptically different. The seed proanthocyanins were found to be coarser than the skin proanthocyanidins. 1) Proanthocyanidins are multi-component polymers, and their structural features have not been thoroughly studied. However, an analytical method using acid depolymerization with nucleophilic agents has been developed, and it enabled us to determine the subunit composition and mean degree of polymerization (mdp) of proanthocyanidins and to evaluate their influence on the extraction of proanthocyanidins during maceration. 9,1) Analyses using this method revealed that skin proanthocyanidins differ from seed proanthocyanidins by the presence of prodelphinidins, their higher mdp, and their lower proportion of galloylated subunits. 1,7,9,11) These differences are expected to be the reason for their organoleptic differences. 1) Peyrot des Gachons and Kennedy 11) showed that extraction of skin and seed proanthocyanidins can be traced separately during fermentation due to their apparent differences. In addition to proanthocyanidins, analytical methods for other phenolic compounds using HPLC and HPLC- MS have been improved, and the concentrations of all classes of phenolics extracted from grape skins and seeds can be quantified. Winemaking practices influence the extraction of phenolic compounds. Changing the temperature during processing is an effective method that influences extraction because temperature affects the permeability of the cells and membranes in grape berries. The following methods are examples in which the tempery To whom correspondence should be addressed. Tel: ; Fax: ; koyama@nrib.go.jp Abbreviations: mdp, mean degree of polymerization; %P, percentage of epigallocatechin units within proanthocyanidins; %G, galloylation rate within proanthocyanidins; LMWP, low-molecular-weight proanthocyanidins; HMWP, high-molecular-weight proanthocyanidi

2 K. KOYAMA et al. ature during maceration is changed: fermentation under high temperature, cold soak, must or grape freezing, heating the must at the end of maceration, and thermovinification ) Heating at the end of maceration and cold soak are usually applied at more moderate temperatures than those used for thermovinification or must freezing. Gerbaux et al. 13,17) showed that higher anthocyanin content, color intensity, total polyphenols, and total sensory score were obtained in Pinot noir wine by heating at the end of maceration than by control vinification. Heating at about 4 C for 1 d after fermentation was preferable, as this temperature had a siginificant effect on phenolic extraction without producing the large quantity of volatile acids or oxidized off-flavor that are often observed under high temperature, for example, during a thermovinification process at 8 C. Gerbaux did not observe any improvement in phenolic extraction as a result of cold soak, 13) a vinification method in which wine musts are kept at low temperature for several days before alcohol fermentation. However, in another study, cold soak at 1 C for 3 d enhanced the extraction of both anthocyanins and skin-derived proanthocyanidins. 2) In a previous study with Cabernet Sauvignon, cold soak extracted more color than was obtained in the control, but there was no significant difference in the total polyphenols between the finished wine of the control and that obtained from the cold soak. 1) These discrepancies might be due to the differences in the conditions of the cold soak process. Comprehensive analysis of the kinetics of extraction of all the wine phenolics during maceration should provide important information for understanding the observed effects of temperature on the extraction of phenolics from berry skins and seeds. The objective of this study was to examine the kinetics of the extraction of all classes of phenolics from seeds and skins during red wine maceration, which is not thoroughly understood, and to then evaluate the effects of cold soak and heating at the end of maceration on the extraction of phenolics in red wine. Materials and Methods Materials. All chromatographic solvents were HPLC grade. ( )-Epicatechin, ( )-epicatechin-3-o-gallate, and ( )-epigallocatechin were purchased from Kurita Water Industries (Tokyo). (+)-Catechin and gallic acid were obtained from Sigma (St. Louis, MO). Malvidin-3- glucoside, quercetin, and caffeic acid were purchased from Extrasynthese (Genay, France). Winemaking. Grapes of Vitis vinifera cv. Cabernet Sauvignon (vintage 24, 18:3 :3% (w/v) of sugars, :49 :3% (w/v) of titratable acidity as tartaric acid, and ph 3:46 :3) harvested in Yamanashi Prefecture, Japan, were used for four batches of vinification. A -kg lot of grapes was destemmed and crushed into a stainless steel tank. Sucrose was added to 23% (w/v) of the final sugar concentration in the juice. Six hours after musts were sulfited to 7 mg/l, wine yeast (L2323, Lallemand, Rexdale, Canada) was inoculated. The caps were punched down twice a day. The temperature was controlled by chillers and heaters under four different treatments during the 1-d maceration period (Fig. 1). The glucose and fructose concentrations in the musts were checked with a D-glucose/D-fructose kit (Boehringer Mannheim, Darmstadt, Germany) to ensure that fermentation had ceased. For heating at the end of maceration, the must temperature was raised to 42 C after fermentation (on the 9th day) and gradually decreased to 2 C for 1 d. After maceration, the wine was pressed in a hydraulic press. Samples from each must were taken every 2 d during the maceration period. At the 1th day of maceration, samples were taken from the wines immediately after they were pressed. The samples were centrifuged at 1; g for 1 min, and the supernatants were then filtered in a glass filter (GF/D, Whatman International, Maidstone, UK) and stored at 3 C until use. Extraction of skin and seed phenolics. Skin and seed phenolics were extracted from berries of Cabernet Sauvignon with a modification of the method of Downey et al. 21) After peeling and deseeding, the berry skins and seeds of the grapes were immediately frozen in liquid nitrogen and stored at 8 C. Frozen samples were ground to a fine powder using a Multi-Beads Shocker (Yasui Kikai, Osaka, Japan), and extracted with 2:1 acetone/water for 24 h. The extracts were used for phloroglucinolysis after fractionation. Spectrophotometric determinations. The color density (CD) and tint were determined by the method of Somers and Evans 22) using a UV visible spectrophotometer with a 2-mm path-length cuvette. The values were converted to those obtained with a 1-mm light-path cuvette. The absorbance of the must at 2 nm was divided into three fractions, viz., polymeric pigment, copigmentation, and free anthocyanins, by the method of Boulton et al. 23) Reversed phase HPLC analysis of monomeric phenolics. Wine and must samples were filtered through.4 mm PTFE syringe-tip filters (CR13 mm, Pall Gelman Laboratory, Ann Arbor, MI) before analysis of reversed phase HPLC by a modification of the method of Ritchey and Waterhouse. 24) The equipment used for HPLC analysis was an Agilent (Wilmington, DE) 11 series with a four-solvent system and a diode-array detector coupled to Chemstation. Zorbax SB C18 (2:1 1 mm, mm particle size) kept at 3 C was used with a flow rate of.3 ml/min. The solvents used for the separation were A ¼ 2 mm ammonium formate adjusted to ph 2.6 with formic acid; B ¼ 2% A with 8% acetonitrile; and C ¼ 2:3 M formic acid (ph 1.). A modified solvent gradient condition is shown in Table 1.

3 Table 1. Solvent Gradient Conditions for HPLC Analysis of Monomeric Phenolics Time (min) Solvent A (%) Solvent B (%) Solvent C (%) Extraction of Phenolics from Berry Skins and Seeds of Grape Specific gravity Temperature ( C) Compounds were identified on the basis of their UV spectra, and the m=z value obtained with HPLC/ESI-MS (LCQ Advantage, Thermo Electron, San Jose, CA) analysis operated in negative mode. For quantification, standard curves were prepared using caffeic acid for cinnamates at 31 nm, catechin for flavan-3-ol monomers at 28 nm, quercetin for flavonols at 36 nm, and malvidin-3-glucoside for anthocyanins at 2 nm. For gallates, the concentration of gallic acid, which was a predominant gallate, was determined at 28 nm. Fractionation of phenolic compounds by C18 seppack cartridges. For proanthocyanidin analysis, wine and must samples and extracts of grape skins and seeds were fractionated by the method of Sun et al. 2) Dealcoholized (deacetonized) samples adjusted to ph 7. were applied to preconditioned Sep-Pack cartridges (Waters, Milford, MA) connected in series (.9 g tc18,.84 g C18), followed by a washing with water. After the samples were dried with an N 2 stream, flavanol monomers were eluted with diethyl ether and oligomers and polymers were recovered by the elution of methanol. Fractions of proanthocyanidin oligomers and polymers were used in the analyses described below. Fig. 1. Fermentation Profiles of Wines under Four Temperature Conditions. The time course of specific gravity (solid lines with closed symbols) and temperature (dotted lines with open symbols) in the musts., control;, cold soak;, heating at the end of maceration (heat treatment);, cold soak and heating at the end of maceration (cold soak and heat treatment). Acid catalysis in the presence of excess phloroglucinol (phloroglucinolysis). Phloroglucinolysis was performed under the condition of Kennedy and Jones. 9) The samples were combined with 1. volumes of.2 N HCl in methanol containing 12 g/l phloroglucinol and 2 g/ l ascorbic acid. After the reaction was performed at C for 2 min, volumes of 4 mm aqueous sodium acetate were added to stop it. The concentrations of flavan-3-ols (terminal units) and phloroglucinol adducts (extension units) generated by depolymerization of proanthocyanidins were determined by HPLC analysis under a modification of the conditions of Kennedy and Jones. 9) Seven major peaks were identified by HPLC/ESI-MS analysis. These compounds were quantified using calibration curves made from flavan-3-ol standards ((+)-catechin, ( )- epicatechin, ( )-epicatechin gallate, and ( )-epigallocatechin). The mdp, galloylation rate (%G), and percentage of epigallocatechin units (%P) were calculated as the molar ratio of extention units to terminal units, the molar ratio of galloylated units to total units, and the molar ratio of epigallocatechin units to total units respectively. The average molecular mass (amm) was estimated based on the proportional composition and mdp of the proanthocyanidins. The sums of the concentrations of these degradation products, from which the weights of the phloroglucinol moiety were subtracted, were converted to proanthocyanidin concentrations in the musts. Normal-phase HPLC. Molecular mass distribution of proanthocyanidins was analyzed by normal-phase HPLC by a modification of the method of Kennedy and Waterhouse. 26) The chromatographic system was calibrated using cacao proanthocyanidins extracted from cacao beans kindly provided by Dr. M. Natsume (Meiji Seika Kaisha Ltd., Saitama, Japan). Proanthocyanidins detected at 28 nm were divided into two groups, lowmolecular-weight proanthocyanidins (LMWP) and highmolecular-weight proanthocyanidins (HMWP), on the basis of the retention time of the dimer to the tetramer, and over that of the tetramer from the cacao bean standard, respectively. Area values were converted to equivalents of epicatechin. Statistical analysis. A t-test was used to evaluate significant differences in the mean concentration or percentage of phenolics in the musts and the wines after maceration with and without cold soak. To examine the influence of heating at the end of maceration, the differences in the mean rate of change from the 8th d to the 1th d with heat treatment and without heat treatment were evaluated by the same method.

4 K. KOYAMA et al. Table 2. Chemical Composition of the Wines under Four Different Temperature Conditions after Pressing Fraction (%) of color due to Total Alcohol Extract Polymeric Copigme Free phenol b Gelatin (% v/v) (% w/v) ph CD ac Tint d a A 2 nm pigment n- tation anthocyanins a A 28 nm (mg/l) index Control Cold soak Heat treatment Cold soak and heat treatment a Absorbance unit. b Measured by the Folin-Ciocalteu analytical method. c Color density, A 42 nm +A 2 nm. d Tint, A 42 nm /A 2 nm. Results Influence of temperature on fermentation and wine composition The start of fermentation, measured by the decrease in specific gravity, was retarded by the cold soak, since the temperature was maintained at about 13 C for the first 2 d (Fig. 1). Although the fermentation progress was different, on the 8th day, the specific gravity ceased decreasing under all conditions. The end of fermentation was also confirmed by the fact that no glucose or fructose was detected in the must. The alcohol concentrations of the wines after maceration under four temperatures were almost the same. Heating at the end of maceration slightly increased the wine ph (Table 2). Changes in monomeric phenolic concentrations in musts The musts and wines contained six classes of phenolic compounds, viz., cinnamates, gallates, flavonols, flavan- 3-ol monomers, anthocyanins, and proanthocyanidins. These phenolics, except for proanthocyanidins, were monomeric, and direct HPLC separation was used for quantification. Changes in their concentrations in the musts under different temperatures are shown in Fig. 2. The concentration of anthocyanins increased rapidly to a maximum during the early stage of maceration and decreased gradually thereafter throughout the remainder of fermentation (Fig. 2A), which is consistent with other studies. 14,27) The cold soak limited the initial rise in the anthocyanin concentrations. At the 4th d, the anthocyanin concentrations in the cold-soaked musts were significantly lower than those in the control. However, the maximum was reached on the 6th d, and cold soak did not reduce the concentration at maximum and thereafter. Contrary to expectations, heating the must at the end of maceration did not increase the anthocyanin concentration. Flavonols showed an extraction curve similar to anthocyanins (Fig. 2B). Cold soak and heat treatment also affected their concentrations in a way similar to that of anthocyanins. The maximum was reached on the 8th d in the cold-soaked musts, which was later than in the case of anthocyanins. The concentrations of cinnamates reached a maximum on the 4th d under all conditions, followed by a reduction to a level 32 to 44% of the maximum after maceration (Fig. 2C). This high decrease might be related with the high susceptibility of compounds in this class to enzymic oxidation in the must. 28) The extraction patterns in flavan-3-ol monomers and gallic acid showed clear differences from those of the others (Fig. 2D, E). Flavan-3-ol monomers were extracted mostly during the late stage of maceration, and the concentrations continued to increase during maceration (Fig. 2D). Cold soak retarded their extraction significantly. Similarly, the concentration of gallic acid also increased continuously, although the extraction curves were approximately linear (Fig. 2E). Cold soak limited its rise, and the concentration in the wine after maceration was significantly lower than that in the control without cold soak. In all cases, heat treatment did not increase their concentrations, and rather decreased their levels. Gallic acid was the only compound which did not decrease under heat treatment. Extraction of proanthocyanidins %P, %G, and mdp of the proanthocyanidins isolated from the berries used for winemaking were 34., 1.6, and 18.1 for the skins and, 16., and 7.8 for the seeds respectively. These differences in the subunit composition and mdp between skins and seeds are consistent with reports by other groups. 1,7,9,11) The total proanthocyanidin concentrations in the must during maceration determined by phloroglusinolysis (Fig. 3A) were similar to those determined by vanillin assay (data not shown). Concentrations increased until the 8th d and then decreased. Cold soak significantly retarded the increases. Heating at the end of maceration significantly decreased the concentrations from those of the controls without heat treatment. %P of proanthocyanidins rapidly increased during the early stage of maceration to reach a maximum percentage of 18.6% on the 4th d and decreased thereafter during the remainder of maceration (Fig. 3C). On the other hand, %G progressively increased (Fig. 3D). Thus, the skin proanthocyanidins were extracted more rapidly than the seed proanthocyanidins, which is consistent with a report from Peyrot des Gachons and Kennedy. 11) These extraction profiles of skin and seed proanthocya-

5 A B Anthocyanin (mg/l) Flavonol (mg/l) nidins are similar to those of skin and seeds monomeric phenolics in the musts. Cold soak limited the initial rise in %P during the early period of maceration. At day 4, %P in the cold-soaked must was significantly lower than that in the control. The date of maximum %P was later than that in the control. However, no clear difference in %P was observed afterwards. Cold soak also retarded the rise in %G significantly during maceration. Heat treatment decreased %G beyond that of the control without heat treatment. mdp in the must proanthocyanidins increased initially toward stabilization. In the late stage of maceration, it decreased gradually (Fig. 3B). Cold soak retarded the initial increase in reaching the maximum, but no clear difference was observed on the 1th d. On the other hand, heat treatment significantly made the decrease sharper than that of the control without heat treatment. The molecular mass distribution of proanthocyanidins in wine was analyzed by normal-phase HPLC. As shown in Fig. 4, heat treatment decreased the concentration of the high polymeric fraction. Table 3 shows quantitatively that heat treatment decreased the HMWP concentration significantly (Table 3), which was consistent with the decrease in mdp (Fig. 3B) obtained from phloroglucinolysis. C Cinnamic acid (mg/l) D E Flavan-3-ols (mg/l) Gallic acid (mg/l) Fig. 2. Changes in the Concentrations of Monomeric Phenolics in the Musts under Four Temperature Conditions during Maceration. A, Anthocyanin. B, Flavonol. C, Cinnamic acid. D, Flavan-3-ols. E, Gallic acid. Symbols indicate four temperatures during maceration., control;, cold soak;, heat treatment;, cold soak and heat treatment. Musts with heat treatment are shown by dotted lines, in contrast to those without heat treatment, which are shown by solid lines. Indicates that the average concentration with cold soak and that without cold soak were significantly different at p < :. Extraction of Phenolics from Berry Skins and Seeds of Grape Wine color and phenol compound indices in wine The changes in wine color (absorbance at 2 nm) during maceration (Fig. ) were similar to those in the concentrations of anthocyanins (Fig. 2A). Wine color and phenolic indices are shown in Table 2. The rate of polymeric pigment was significantly lower with cold soak, but was not affected by heat treatment (Table 2). Discussion Extraction of skin and seed phenolics This study indicates that phenolics contained in berry skins, viz., anthocyanin, flavonol, and skin proanthocyanidins, were rapidly extracted during the early stage of maceration, whereas phenolics contained mainly in seeds, viz., gallic acid, flavan-3-ol monomers, and seed proanthocyanidins, were gradually extracted and not stabilized within the maceration period (Fig. 2, Fig. 3C, D). These results confirm other reports on anthocyanin 14,27) and proanthocyanidin 11) extraction. In addition, these results indicate that the extraction of the various classes of phenolics in berry skins had similar extraction profiles, and so did that in seeds. Similarly, Canals et al. 29) have reported that the total phenol (absorbance at 28 nm) from skins was rapidly extracted within 4 d, whereas that from seeds was extracted progressively. Thus the difference in the tissue structure between berry skins and seeds probably causes the difference in phenolic extraction between them. In addition to localization, the difference in the chemical structures of proanthocyanidins contained even

6 K. KOYAMA et al. A Proanthocyanidins (mg/l) B mdp C 2 D %P %G Fig. 3. Changes in the Composition and Mean Degree of Polymerization of Proanthocyanidins in the Musts under Four Temperature Conditions during Maceration. A, Concentration of total proanthocyanidins. B, Mean degree of polymerization (mdp). C, Percentage of prodelphinidin unit (%P). D, Percentage of galloylation (%G). Symbols and lines are shown in Fig. 2. Indicates that the average concentration with cold soak and that without cold soak were significantly different at p < :. yindicates that the average rate of the change from the 8th d to the 1th d under heat treatment and that without heat treatment were significantly different at p < :. 1 Table 3. Proanthocyanidins in the Wine after Pressing Absorbance (mau) Time (min) LMWP ad HMWP bd HMWP/ LMWP d amm c Control Cold soak Heat treatment Cold soak and heat treatment a Low-molecular-weight polymers expressed by mg epicatechin equivalents (ECE)/L. b High-molecular-weight polymers expressed by mg ECE/L. c Average molecular mass calculated from the data obtained by phloroglucinolysis. d Fractionated by normal-phase HPLC. Indicates that the mean concentrations in paired samples of the wines under heat treatment and their controls without heat treatment are significantly different at p < :. Fig. 4. Normal-Phase HPLC Chromatograms of Proanthocyanidins in Wine Heated at the End of Maceration (Solid Line) and the Control (Broken Line). The retention times for the cacao proanthocyanidin standard are shown as the string-of-dots line in the order of the degree of polymerization from monomer to decamer (from left to right). in the same tissue (skin or seed) might alter their extractability. For example, %P, which represents the extraction of skin proanthocyanidins, initially increased to reach a maximum on the 4th d (Fig. 3C), whereas mdp increased during the early stage of maceration until the 6th d (Fig. 3B), although mdp in berry skins was higher than in seeds. These results indicate that proanthocyanidins in skins with low mdp were extracted more rapidly than those in skins with high mdp. Similarly, the increase in %P during the early stage of maceration indicates that proanthocyanidins with low %P were extracted more rapidly than those with high %P among skin proanthocyanidins. Thus, chemical properties, such as hydrophobicity and the number of hydroxyl residues in proanthocyanidins, likely influenced the kinetics of extraction. In the course of their diffusion into the must, proanthocyanidins are thought to be repeatedly trapped

7 Absorbance (2 nm) Fig.. Changes in Absorbance at 2 nm during Maceration. The symbols and lines are as in Fig. 2. in and released from internal components of the cells, such as cell wall polysaccharides, soluble pectins, glycoprotein, etc., after the partial collapse of the cell membrane structure. The hydrogen bond and hydrophobic interactions were hypothesized to be dominant in the interactions between proanthocyanidins and purified cell walls or proteins as fining agents. 3,31) Similarly, proanthocyanidins with higher hydrophobicity and a higher number of hydroxyl groups, indicative of higher mdp, %P, and %G, might be selectively trapped and released into the must later with the aid of increased ethanol concentrations. Alternatively, the different cellular localization of proanthocyanidins with different compositions might explain this selectivity. 8,32,33) The decrease in the concentrations of skin phenolics after reaching a maximum (Fig. 2A, B, C) might be due to the adsorption of extracted polyphenols by yeast lees and/or grape solids, degradation, oxidation, or condensation. In the case of anthocyanins, the condensation reaction with proanthocyanidins has been assumed to contribute more or less to their decrease during the late stage of fermentation. 27) Influence of cold soak on the concentration of phenolics Cold soak retarded the initial rise in the concentrations of all classes of phenolics except for cinnamates. During maceration, cold soak reduced the extraction of flavan-3-ol monomers and gallic acid, which mainly come from the seeds, and of proanthocyanidins (Fig. 2, Fig. 3A). The retardation in the extraction was probably due to their lower ethanol concentration during the early stage of maceration. Canals et al. 29) found a significant influence of the ethanol concentration on the extractability of anthocyanins and, in particular, of proanthocyanidins from skins and seeds. In the case of skin phenolics, although cold soak retarded their extraction, it did not reduce the maximum concentrations. During the late stage of maceration, the skin monomeric phenolic concentrations in the coldsoaked musts were either similar to or rather higher than those in the control. Similarly, cold soak increased the extraction of proanthocyanidins from the skins and decreased that from the seeds in other study. 2) In Extraction of Phenolics from Berry Skins and Seeds of Grape general, an increase in the skin phenolics is considered to have some advantages for the organoleptic properties of the wine. For example, skin proanthocyanidin is considered to be softer and to have higher quality than seed proanthocyanidins. 2) Cold soak increased the ratio of anthocyanin to proanthocyanidins (a 67% increase) in the wine after maceration, suggesting a possible increase in the proportion of the anthocyanin-proanthocyanidin adducts against total polymers, which must affect the quality of the resultant wine after long-term storage. 2,2,34) Influence of heating at the end of maceration on the concentration of phenolics Heat treatment is generally assumed to damage grape cell membranes, which results in an increased extaction of phenolic compounds. 13,14,17) However, in this study, heating at the end of maceration did not increase phenolic extraction and, moreover, decreased the concentration of proanthocyanidins and mdp (Fig. 2, Fig. 3A, B). This decrease might be due to oxidation, degradation, and/or adsorption of proanthocyanidins, in particular, those with higher molecular weights. 3,3) Further studies are necessary to determine the reasons for the different results obtained in our study and others ,16,17) Potential variables, e.g., grape variety, berry maturity, heat conditions, and/or fermentation scale, might have affected the results. References 1) Vidal, S., Francis, L., Guyot, S., Marnet, N., Kwiatkowski, M., Gawel, R., Cheynier, V., and Waters, E. J., The mouth-feel properties of grape and apple proanthocyanidins in a wine-like medium. J. Sci. Food Agric., 83, (23). 2) Cheynier, V., Flucrand, H., Brossaud, F., Asselin, C., and Moutounet, M., Phenolic composition as related to red wine flavor. In Chemistry of Wine Flavor, eds. Waterhouse, A., and Ebeler, S., American Chemical Society, Washington, DC, pp (1999). 3) Cheynier, V., Polyphenols in foods are more complex than often thought. Am. J. Clin. 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