Evolution of 49 Phenolic Compounds in Shortly-Aged Red Wines Made from Cabernet Gernischt (Vitis Vinifera L

Food Sci. Biotechnol. Vol. 18, No. 4, pp. 1001 ~ 1012 (2009)

Zheng Li, Qiu-Hong Pan, Zan-Min Jin, Jian-Jun He, Na-Na Liang, and Chang-Qing Duan* Center for Viticulture and Enology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, PR China

Abstract A total of 49 phenolic compounds were identified from the aged red wines made from Cabernet Gernischt (Vitis vinifera L. cv.) grapes, a Chinese characteristic variety, including 13 anthocyanins, 4 pryanocyanins, 4 flavan-3-ol monomers, 6 flavan-3-ol polymers, 7 flavonols, 6 hydroxybenzoic acids, 5 hydroxycinnamic acids, 3 stilbenes, and 1 polymeric pigment. Evolution of these compounds was investigated in wines aged 1 to 13 months. Variance analysis showed that the levels of most phenolics existed significant difference in between wines aged 3 and 9 months. Cluster analysis indicated that 2 groups could be distinguished, one corresponding to wines aged 1 to 3 months and the other to wines aged 4 to 13 months. It was thus suggested that there were 2 key-stages for the development of fine wine quality, at the aged 3 and 9 months, respectively. This work would provide some helpful information for quality control in wine production. Keywords: Cabernet Gernischt, aged wine, phenolic compound, evolution, high performance liquid chromatography coupled with tandem mass spectrometry

Introduction 3-ols in red wine and correlation with wine age (12), the evolutions of low molecular weight phenolic compounds Phenolic compounds in red wines mainly contain such as gallic acids and caffeic acids (13,14), as well as the anthocyanins, flavonols, flavan-3-ols, phenolic acids effects of oak barrel compounds and sorption behaviors on (including hydroxybenzoic acids and hydroxycinnamic evolution of phenolic compounds (15), have been reported. acids), and stilbenes. Except anthocyanins, the others are The previous studies are mainly paid to non-anthocyanin called as non-anthocyanin phenolics. All these compounds phenolics in white wines, for they are closely related to are extracted into wine during fermentation and closely browning of white wines (16-18). Non-anthocyanin phenolics related to the quality of wine. It has been known that also play important roles in constituting the organoleptic phenolic compounds not only contribute wine to color, properties of red wines. Baranac et al. (19) reported that flavor, astringency, and bitterness, which constitute essential flavonols are considered to be the best cofactors present in sensory prosperities, but also endow wine with abundant the phenolic composition of red wines. Darias-Martín et al. nutritional and healthy functions (1,2). (20) and Bloonfield et al. (21) also mentioned that flavan- Many factors can influence the phenolic composition 3-ols and hydroxycinamic acids (caffeic, ρ-coumaric, and and magnitude of wines, including grape variety, the ferulic acids) can act as cofactors for copigmentation. In technology applied in winemaking, and the reactions that the aged red wines, it has been known that a change of the take place during aging. Many published works have been color hue from purple-red to red, and then to yellow-red reported that phenolic profiles of wines differ from grape tonalities involves the formation of polymeric anthocyanins variety to variety (3-5). Moreover, Alamo et al. (6) from the covalent linking of monomeric anthocyanins reported that even for different aging containers, behavior (22,23). As mentioned above, previous studies involving of phenolic reactions in the aged wines also shows the non-anthocyanin phenolics in red wines mainly focus remarkable different, which will lead to generation of on the identification and content evolution of copigmentation- different organoleptic characteristics of wines. Therefore, related compounds, and on the fermentation process. Few for a wine made from special single variety, special were reported to date concerning the evolution of non- enological process and aging technology are required in anthocyanin phenolics during aging, especially the change order to fully exploit the characteristics of this variety. of non-copigmented phenolics. Phenolic compounds during wine aging are not always Cabernet Gernischt (Vitis vinifera L. cv.) is a Chinese changeless. Many previous studies have been reported on characteristic variety that might have been imported from the evolution of anthocyanins profiles, containing the Europe in 1894 and naturally selected in China. The red effects of copigmentation on wine color (7,8), the formation wines made from Cabernet Gernischt are getting popular of pyranoanthocyanins and their influence factors in wines with customers owing to attractive color and fine taste. The (9-11). Moreover, formation of ethylidene-bridged flavan- objective of the present study is to identify major phenolic compounds in aged Cabernet Gernischt wine by high *Corresponding author: Tel: +86-10-62737136; Fax: +86-10-62737136 performance liquid chromatography (HPLC)-coupled with E-mail: [email protected] tandem mass spectrometry (MS/MS) and to investigate the Received March 7, 2009; Revised April 21, 2009; evolutions of these phenolic compounds during the oak Accepted April 26, 2009 barrel aging. Through this work, we intended to uncover

1001 1002 Z. Li et al. the correlation of evolution of various phenolic compounds then were directly used for analysis of high performance during red wine aging and to elucidate possible mechanism liquid chromatography (HPLC)-coupled with tandem mass of wine quality development in aging wine. spectrometry (MS/MS) without dilution. For non-anthocyanin phenolics (including flavan-3-ols, Materials and Methods flavonols, hydroxybenzoic acids, hydroxycinnamic acids, and stilbenes), a total of 100 mL wine sample were diluted Winemaking Fully-ripen ‘Cabernet Gernischt’ grape with the same volume of purified water, and then extracted berries (Vitis vinifera L. cv.) were harvested from a thrice with 80 mL of ethyl acetate (analytical grade). The commercial vineyard in Changyu castle (Hebei, China) in ethyl acetate phase of the combined extracts was removed Oct. 2004 on the basis of similar size and absence of by a rotary evaporation at 28ºC and the remainder was physical injuries or infections. These berries were divided resolved in methanol (chromatography grade) up to a final into 3 groups, which were named as T1, T2, and T3 volume of 5 mL. The final samples were filtered by 0.22- respectively. Each group with about 10,000 kg berries was µm organic membranes prior to analysis by HPLC-MS/ applied for winemaking and all the treatments were MS. controlled under identical conditions. After stemming and Quantitative analyses by HPLC-MS/MS: For anthocyanins, crushing, a pectic enzyme (EX-V; Lallemand Co., Toulous, An Agilent 1100 series LC-MSD trap VL, equipped with France, 20 g/L) and an amount of 60 mg/L of SO2 were a G1379A degasser, a G1311A QuatPump, a G1313A added to the must with skin maceration. The fermentation ALS, a G1316A column, a G1315B DAD and a Kromasil- was then performed at 26-28ºC with a commercial C18 column (250×4mm, 6.5µm), was used. The solvents Saccharomyces cereviside yeast (BM; Lallemand Co., 0.20 were (A) 6%(v/v) acetonitrile containing 2%(v/v) formic kg/kL) and Oenococcus oeni lactic acid bacteria (Vitilactic- acid, and (B) 54%(v/v) acetonitrile containing 2%(v/v) D; Martia Vialatte, France. 10 g/kL). The wine-making formic acid. The gradient was from 10% B for 1 min, from strictly obeyed the manufacture techniques of red wine 10 to 25% B for 17 min, isocratic 25% B for 2 min, from made from ‘Carbernet Gernischt’ in China Changyu 25 to 40% for 10 min, from 40 to 70% for 5 min, from 70 Winery Co., Ltd. in 2004. After the end of fermentation to 100% for 5 min, at a flow rate of 1.0 mL/min. Injection (including alcohol and malolactic fermentations), some volumes were 30 µL, and the detection wavelength was conventional parameters were assessed and shown in Table 525 nm. The column temperature was 50ºC. MS conditions 1. The results indicated that these wines could reach the were as follows: electrospray ionization (ESI), positive ion standard and had similar physicochemical parameters. model; nebulizer, 35 psi; dry gas flow, 10 L/min; dry gas These wine samples were subsequently aged for a period temperature, 325ºC; Scan, 100-1,000 m/z. of 13 months in new French Allier MT+ oak barrels. The For non-anthocyanins phenolic compounds, an Agilent aging conditions were controlled at 16ºC and with a 1200 series, equipped with a G1322A degasser, a G1312B humidity of 80%. Wine aging samples to be analyzed were Bin pump, a G1367C HiP-ALS, a G1316B TCC, a taken about 500 mL from the barrels after 1, 2, 3, 4, 5, 9, G1314C VWD, and a ZORBAX SB-C18 column (3×50 and 13 months of aging, respectively, and were mm, 1.8 µm) were used. The solvents were (A) 10%(v/v) immediately used for the following analyses. acetic acid, and (B) 90%(v/v) acetonitrile containing 10%(v/v) acetic acid. The elution gradient was from 5 to Chemicals and standards The standards, malvidin-3-O- 8% B for 5 min, from 8 to 12% B for 2 min, from 12 to glucoside, catechin, quercetin, gallic aid, caffeic acid, and 18% for 5 min, from 18 to 22% for 5 min, from 22 to 35% resveratrol, were all purchased from Sigma-Aldrich (St. for 2 min, from 35 to 100% B for 2 min, 100% B for 4 min Louis, MO, USA). Methanol, formic acid, acetic acid, and and from 100 to 5% B for 2 min, as well as at a flow rate acetonitrile glacial acetic acid (HPLC grade) were obtained of 1.0 mL/min. Injection volumes were 2 µL, and the detection from Fisher Co. (Fairlawn, NJ, USA). Ethyl acetate wavelength was 280 nm. The column temperature was (analytical grade) and deionized water were from Beijing 25ºC. MS conditions were as follows: ESI, negative ion Chemical Reagent Plant (Beijing, China) and Wahaha Co. model; nebulizer, 35 psi; dry gas flow, 10 L/min; dry gas (Hangzhou, China), respectively. All the other chemicals temperature, 325ºC; scan, 100-1,000 m/z. were purchased from Sigma-Aldrich unless noted specially. Duplicate analysis was carried out for each wine sample following the preparation and analysis procedures mentioned Extraction and analyses of phenolic compounds from above. wine Quantification of phenolic compounds: Anthocyanins Preparation of sample: For anthocyanins, wine sample of were quantified using malvidin-3-O-glucoside as a standard, 2 mL was filtered by 0.45-µm inorganic membranes and and flavanols using catechin, flavonol using quercetin,

Table 1. Conventional analytical data of the red wines at the end of malolactic fermentation (n=3) Alcohol Residul sugar Total acids Volatile acids Free SO Total SO Dry extracts Tank No. 2 2 (% v/v) (g/L) (g/L) (g/L) (mg/L) (mg/L) (g/L) T1 11.6a1) 3.3a 6.1a 0.30a 17a 28a 34.8a T2 11.7a 3.6a 5.6a 0.35a 12a 28a 33.6a T3 12.0a 2.6a 5.7a 0.30a 14a 34a 33.9a 1)Different letters are significantly different at the 0.05 level according to ANOVA. Evolution of Phenolic Compounds in Cabernet Gernischt Shortly-aged Wines 1003 hydroxybenzoic acids using gallic acid, hydroxycinnamic Evolutions of phenolic compounds in aged Cabernet acids using caffeic acid and stilbenes using resveratrol. Gernischt wines Anthocyanins: As seen from Table 4, 15 anthocyanins Analyses of conventional parameters Wine conventional were identified from T1 wine sample and 16 anthocyanins parameters were determined at the end of malolactic from T2 and T3 wine samples. Some anthocyanins such as fermentation, according to China Official Methods (GB/T petunidin-3-O-(6-O-coumaryl)-glucoside, trans-peonidin- 15037-94). These parameters include alcohol degree, 3-O-(6-O-coumaryl)-glucoside, malvidin-3-O-glucoside- residual sugar, total acids, volatile acids, free SO2, total acetaldehyde, and dephinidin-3-O-(6-O-acetyl)-glucoside, SO2, and dry extracts. Three independent analyses were showed trace or did not be detected within experimental performed for each sample. aging periods. Corresponding to each anthocyanin compound, the ratio of the concentration of individual to Statistical analysis Statistical analyses were performed total anthocyanins always kept changeless throughout the by means of SPSS 11.5 software (Chicago, IL, USA). One- aged study. The concentration of malvidins-3-O-glucoside way analysis of variance (ANOVA) was applied to all wine accounted for about 60% of total anthocyanins at various samples to verify significant changes (significant at 5% aging periods and malvidin-3-O-(6-O-acetyl)-glucoside for level) according to aging time. For each phenolic compound, about 20%. Accompanying with aging in oak barrels, the a total of 6 data were used for ANOVA. Hierarchical concentration of total anthocyanins showed statistically cluster analysis was carried out to see data trends and to significant and continuous decrease, and at the aged 13 ascertain the reference points of wine quality development months, the concentration was only approximately 40% of in the course of aging. the initial concentration. ANOVA result revealed that such a decrease in the concentration of total anthocyanins Results and Discussion resulted from significant degradation of most monomeric anthocyanins detected, except dephinidin-3-O-glucoside Identification of phenolic compounds in aged Cabernet which showed progressive decrease, but not statistically Gernischt wine by HPLC-MS/MS Seventeen anthocyanins, significant, in the process of aging. Interestingly, a fluctuant 31 non-anthocyanin phenolics, and 1 anthocyanin-flavanol change trend was present for 4 pyranoanthocyanins, of pigment were effectively separated. According to the which 2 vitisin-type pyranoanthocyanins (malvidin-3-O- HPLC-MS/MS information library of standard phenolic glucoside-pyruvic acid and malvidin-3-O-glucoside-4- compounds developed in our previous work (24,25), these acetaldehyde) showed a slight increase and then a decrease, 49 phenolic compounds were well identified from the aged while quite the contrary for other 2 vinylphenol-type Cabernet Gernischt wine. These compounds included 17 pyranoanthocyanins (malvidin-3-O-glucoside-4-vinylphenol anthocyanins, 10 flavan-3-ols, 7 flavonols, 6 hydroxybenzoic and malvidin-3-O-(6-O-acetyl)-glucoside-4-vinylphenol). acids, 5 hydroxycinnamic acids, 3 stilbenes, and 1 It has been well known that monomeric anthocyanins anthocyanin-flavanol pigment (malvidin-3-O-glocoside- significantly decrease, and even disappeared during red (C2-O-C7, C4-C8)-(epi)catechin). The detailed HPLC-UV- wine aging (7,8). The present results in the aged Cabernet MS/MS spectral data were all listed in Table 2 and 3. Gernischt wines also demonstrated the existence of this

Table 2. Anthocyanins identified from aged Cabernet Gernischt wine by HPLC-MS Peak No. Compound name RT1) (min) [M·H]+ (frag. m/z) 1 Dephinidin-3-O-glucoside 04.412 465(303) 2 Petunidin-3-O-glucoside 07.373 479(317) 3 Peonidin-3-O-glucoside 10.069 463(301) 4 Malvidin-3-O-glucoside 11.089 493(331) 5 Dephinidin-3-O-(6-O-acetyl)-glucoside 12.410 507(303) 6 Malvidin-3-O-glucoside-4-pyruvic acid 13.665 561(399) 7 Malvidin-3-O-glucoside-4-acetaldehyde 15.806 491(287) 8 Cyanidin-3-O-(6-O-acetyl)-glucoside 15.918 517(355) 9 Petunidin-3-O-(6-O-acetyl)-glucoside 17.314 521(317) 10 Peonidin-3-O-(6-O-acetyl)-glucoside 21.164 505(301) 11 Malvidin-3-O-(6-O-acetyl)-glucoside 22.123 535(331) 12 Petunidin-3-O-(6-O-coumaryl)-glucoside 25.184 625(317) 13 cis-Malvidin-3-O-(6-O-coumaryl)-glucoside 27.834 639(331) 14 trans-Peonidin-3-O-(6-O-coumaryl)-glucoside 29.401 609(301) 15 trans-Malvidin-3-O-(6-O-coumaryl)-glucoside 29.884 639(331) 16 Malvidin-3-O-glucoside-4-vinylphenol 32.423 609(447) 17 Malvidin-3-O-(6-O-acetyl)-glucoside-4-vinylphenol 34.669 651(447) 1)Retention time. 1004 Z. Li et al.

Table 3. Non-anthocyanin phenolics identified from aged Cabernet Gernischt wine by HPLC-MS Peak No. Compound name RT1) (min) [M·H]+ (frag. m/z) 1 Gallic acid 0.528 169(125) 2 Gallocatechin 1.025 305(179,125,219,261) 3 Dihydroxyphenylethanol 1.113 153(123) 4 Caffeic acid 1.282 179(135) 5 Dihydroxyphenylethanol 1.390 153(123) 6 Methyl gallate 1.762 183(169,139) 7 ρ-Coumaric acid 2.075 163(119) 8 Epigallocatechin 2.610 305(179,125,137,165,203,221) 9 Catechin 2.840 289(245,205,179,125) 10 Ferutaric acid 3.119 325(193,163,113) 11 Procyanidin trimer 3.843 865(577,695,407,543,713,739) 12 Procyanidin trimer 4.031 865(577,695,407,543,713,739) 13 Syringic acid 4.738 197(181,153) 14 Procyanidin dimer 5.173 577(425,407,451,289,559) 15 Ethly gallate 5.531 197(169,125) 16 Epicatechin 5.996 289(245,205,179,125) 17 Procyanidin dimer-3-O-gallate 7.077 729(407,559) 18 Dihydroquercetin-O-hexoside 7.932 465(301,339,151,193,447) 19 Procyanidin dimer 8.856 577(425,407,451,289,559) 20 trans-Piceid 10.524 389(227) 21 Malvidin-3-O-glucoside-(C2-O-C7, C4-C8)-(epi)catechin 11.777 781(493,583,289,407,629) 22 Dihydroquercetin-3-O-rhamnoside 12.363 449(285,303,151) 23 Quercetin-3-O-glucoside 12.865 463(301) 24 Kaempferol-3-O-glucoside 14.739 447(285,255,327) 25 cis-Piceid 15.139 389(227) 26 Myricetin 15.375 317(179,151) 27 Procyanidin dimer-3-O-gallate 16.509 729(407,559) 28 Ethyl caffeate 17.241 207(179,135) 29 trans-Resveratrol 18.596 227(185,159) 30 Rutin 19.051 609(301,179,151) 31 Quercetin 19.423 301(151,179) 32 Ethyl p-coumarate 21.533 191(163) 1)Retention time. phenomenon. Two reasons could have been concluded. One aged study. These polymers contained 3 groups, is condensation reactions between monomeric anthocyanins procyanidin trimers (peak no. 11 and 12), procyanidin leading to formation of polymeric anthocyanins. Schwarz dimers (peak no. 14 and 19), and procyanidin dimer-3-O- et al. (9,10) and Rentzsch et al. (11) have reported that gallates (peak no. 17 and 27). In the aged wines, the relatively higher concentrations of pyranoanthocyanins concentrations of flavan-3-ol polymers such as procyanidin were present in wines at the beginning of aging and dimer-3-O-gallate (peak no.17), procyanidin trimer (peak steadily decline in further course of aging, which has a no. 12), and procyanidin dimer (peak no. 14) were much good consistence with our present observation. higher than their precursor flavan-3-ol monomers, but there Pyranoanthocyanins have been demonstrated by Bakker were no significant changes in the concentrations of these and Timberlake (26) that more stable than their original 3 polymers within aging period. The concentrations of anthocyanins and can protect from degradation caused by other polymers, procyanidin dimer (peak no. 19) and SO2 and oxidation. The other is from the sorption of oaks procyanidin dimer-3-O-gallate (peak no. 27), showed a on phenolic compounds as Barrera-García et al. (15) gradual increase. Except epicatechin that significantly mentioned. increased during aging, other flavan-3-ol monomers all Flavan-3-ols: It was observed that 4 flavan-3-ol monomers, showed first an increase and then a decrease. As a result of 6 flavan-3-ol polymers, and 1 polymeric pigment [malvidin- these changes, total flavan-3-ols also behaved an increase 3-O-glucoside-(C2-O-C7,C4-C8)-(epi)catechin] were and following decrease in the process of aging. identified from the aged Cabernet Gernischt wines (Table The flavan-3-ols are the most abundant phenolics in 5). Malvidin-3-O-glucoside-(C2-O-C7, C4-C8)-(epi)catechin wines. According to previous studies, Fernández-Zurbano could be detected at the onset of aging from all aged wines, et al. (16) and Simpson (18) reported that the decrease in whereas no significant change was observed within the the concentrations of flavan-3-ol monomers and polymers Evolution of Phenolic Compounds in Cabernet Gernischt Shortly-aged Wines 1005

Table 4. Evolution of anthocyanins in shortly-aged red wines made from Cabernet Gernischt Aging (month) Anthocyanins1) (mg/L) Tank No. 12345913 T1 3.31 3.09 3.08 3.42 2.70 3.03 2.76 Dephinidin-3-O-glucoside T2 2.10 2.53 2.12 2.09 2.12 1.80 2.06 T3 5.17 5.12 4.69 4.86 4.50 3.36 3.07 ANOVA a2) aaaaaa T1 7.35 5.98 6.07 6.76 4.37 5.44 4.30 Petunidin-3-O-glucoside T2 4.66 5.05 3.89 3.55 3.39 2.79 3.18 T3 10.55 11.23 9.69 9.03 8.01 5.57 4.47 ANOVA a a ab ab ab ab b T1 4.03 3.71 3.81 4.03 2.78 3.26 2.71 Peonidin-3-O-glucoside T2 4.11 3.73 3.39 2.83 2.62 2.27 2.56 T3 4.28 5.99 5.84 4.07 3.68 2.73 2.39 ANOVA a a a ab bc bc c T1 168.82 134.98 135.51 147.55 112.23 77.76 76.35 Malvidin-3-O-glucoside T2 161.19 148.78 134.48 98.25 77.68 71.11 65.82 T3 180.41 206.48 196.82 153.00 130.24 92.79 74.82 ANOVA a a ab bc cd de e T1 1.93 1.87 1.79 1.84 1.66 1.79 TR3) Dephinidin-3-O-(6-O-acetyl)-glucoside T2 ND ND ND ND ND ND ND3) T3 ND ND ND ND ND ND ND ANOVA a a ab a b ab -- T1 1.42 1.50 1.55 1.57 1.59 1.89 1.32 Malvidin-3-O-glucoside-4-pyruvic acid T2 1.42 1.49 1.40 1.67 1.87 1.54 1.46 T3 1.58 1.68 1.65 1.66 1.66 1.96 1.48 ANOVA d cd cd bc ab a d T1 TR TR TR TR ND ND ND Malvidin-3-O-glucoside-4-acetaldehyde T2 TR TR TR 1.31 1.53 TR TR T3 TR TR TR TR TR ND ND ANOVA ------T1 2.28 2.01 1.97 1.97 1.43 1.52 TR Cyanidin-3-O-(6-O-acetyl)-glucoside T2 2.39 2.16 2.08 1.61 TR TR TR T3 2.45 2.61 2.44 1.93 1.70 TR TR ANOVA a a a b bc c -- T1 3.26 2.73 2.71 2.85 1.94 2.20 1.79 Petunidin-3-O-(6-O-acetyl)-glucoside T2 2.53 2.51 2.32 1.90 1.64 1.54 1.72 T3 3.94 4.19 4.06 3.24 2.83 1.90 1.70 ANOVA a a a ab bc c c T1 3.83 3.25 3.25 3.45 2.04 2.54 1.50 Peonidin-3-O-(6-O-acetyl)-glucoside T2 3.84 3.72 3.16 2.42 2.07 1.58 1.96 T3 4.45 4.84 4.51 3.52 3.09 1.45 TR ANOVA a a ab b c c c T1 58.02 45.00 44.70 47.80 23.77 30.91 20.96 Malvidin-3-O-(6-O-acetyl)-glucoside T2 62.07 55.92 50.71 35.22 26.31 22.76 17.34 T3 63.43 70.19 65.41 48.59 39.66 21.64 15.91 ANOVA a a a b c cd d T1 TR TR TR TR ND ND ND Petunidin-3-O-(6-O-coumaryl)-glucoside T2 2.49 2.12 1.99 1.59 TR TR TR T3 1.80 1.90 1.83 1.54 TR TR TR ANOVA a a a b ------1006 Z. Li et al.

Table 4. Continued Aging (month) Anthocyanins (mg/L) Tank No. 12345913 T1 2.57 2.03 2.02 2.03 1.45 1.60 1.43 cis-Malvidin-3-O-(6-O-coumaryl)-glucoside T2 2.42 2.41 2.18 1.67 1.44 1.41 1.52 T3 3.36 3.63 3.19 2.72 2.40 1.74 1.59 ANOVA a ab ab bc cd cd d T1 TR TR TR TR TR ND ND trans-Peonidin-3-O-(6-O-coumaryl)-glucoside T2 1.77 1.68 1.47 TR TR TR TR T3 2.45 2.48 2.32 1.81 1.47 TR TR ANOVA a aaaa---- T1 15.20 9.89 9.53 9.65 4.39 4.71 3.17 trans-Malvidin-3-O-(6-O-coumaryl)-glucoside T2 15.94 12.84 10.72 6.40 4.76 3.35 4.04 T3 22.14 23.45 20.81 13.84 10.57 4.77 3.76 ANOVA a a ab bc cd d d T1 2.37 1.75 1.89 1.99 1.61 2.14 2.27 Malvidin-3-O-glucoside-4-vinylphenol T2 2.61 2.66 2.27 2.29 2.47 2.14 2.75 T3 1.95 2.21 2.04 2.01 1.83 2.47 2.59 ANOVA ab ab b ab b ab a T1 1.55 1.30 1.31 1.36 1.19 1.40 1.39 Malvidin-3-O-(6-O-acetyl)-glucoside-4-vinylphenol T2 1.67 1.65 1.49 1.53 1.55 1.45 1.63 T3 1.31 1.41 1.35 1.32 1.25 1.42 1.48 ANOVA a ab ab ab b ab a T1 275.93 219.09 219.20 236.27 163.14 140.21 119.95 Total anthocyanins T2 271.21 249.26 223.66 164.33 129.45 113.74 107.50 T3 309.28 347.43 326.64 253.12 212.89 141.80 113.25 ANOVA a a ab b c cd d 1)The content of each anthocyanin in this table is equivalent to that of malvidin-3-O-glucoside and the detection limit of malvidin-3-O-glucoside in methanol is 0.13 mg/L in the present study. 2)Different letters are significantly different at the 0.05 level according to ANOVA. 3)TR and ND represent trace and not-detected, respectively. during wine aging may be reasonably ascribed to oxidative change was present for the concentration of total flavonols. processes; Drinkine et al. (12) suggested that the decrease Cantos et al. (3) mentioned that difference in cultivars of of flavan-3-ols are followed by condensation and subsequent red wine grapes can be appraised on the basis of the precipitation in the oak barrels. On the other hand, the flavonol profiles, as well as the anthocyanins fingerprints. concentrations of some flavan-3-ols show a short increase To our knowledge, this is the first report concerning the and then a decrease, which result from the release of oak flavonol profiles of Cabernet Gernischt wines. Indeed, it wood compounds (15) or the hydrolysis of the oligomers in needs to be determined whether the flavonol profiles can wines like gallocatechin and epigallocatechin (27,28). be used for differentiation of Carbenet Gernscht wines Singleton and Trousdale (27) suggested that epigallocatechin, from other variety red wines. Baranac et al. (19) reported as the galloylated precursors of epicatechin, was hydrolyzed that flavonols are considered to be one of the best cofactors during aging, which may explain the decrease in and participate in the copigmentation in young and shortly epigallocatechin concentration and the increase in aged red wines, which might be main cause for low level epicatechin concentration in the course of aging. of flavonols. During aging in oak barrel, the flavonol Flavonols: In the aged Cabernet Gernischt wines, the glycosides and their aglycones can be invoked into such concentrations of flavonols were very low (Table 6). Only reactions as condensation, oxidation, and copigmentation trace amount of quercetin was detected in all the detected and thus their concentrations markedly decline, as shown samples. Myricetin and dihydroquercetin-3-O-rhamnoside in Table 6. also showed very low levels, which gradually increased at Hydroxybenzoic acids: Six hydroxybenzoic acids were the beginning of aging and subsequently decreased. In the identified from the aged Cabernet Gernischt wines. There process of aging, significant degradation was observed for were 2 groups of dihydroxyphenylethanol detected (peak 3-glucosides of both quercetin and kaempferol that no. 3 and 5), of which the compound at peak 5 showed a accounted for almost 60% in total flavonols in the aged significant increase at the aged 13 months (Table 7). The Cabernet Gernischt wine, and quercetin-3-glucoside almost total hydroxybenzoic acids showed a significant increase disappeared in 13 month-aged wines. No significant within the aged 2 months and a significant decrease at aged Evolution of Phenolic Compounds in Cabernet Gernischt Shortly-aged Wines 1007

Table 5. Evolution of flavan-3-ols and their derivatives in shortly-aged red wines made from Cabernet Gernischt Flavan-3-ols1) Aging (month) Tank No. (mg/L) 12345913 T1 8.70 9.70 9.39 8.39 7.04 8.58 8.95 Gallocatechin T2 8.00 9.74 8.45 9.97 7.26 8.55 6.77 T3 9.02 10.89 11.12 10.35 9.84 9.33 8.80 ANOVA bc2) aababccbc T1 24.17 25.08 24.69 24.97 23.15 21.67 17.62 Epigallocatechin T2 28.02 26.10 23.06 23.97 22.99 20.30 17.23 T3 12.15 27.03 23.07 20.01 19.47 15.97 13.38 ANOVA ab a ab ab ab bc c T1 7.23 9.06 9.01 10.80 8.24 10.74 9.99 Catechin T2 9.74 11.80 10.66 13.78 9.30 12.25 8.03 T3 8.36 12.16 9.74 10.08 9.91 11.62 9.77 ANOVA c ab bc a c a c T1 1.68 2.00 2.33 1.58 1.28 1.92 1.71 Procyanidin trimer T2 2.97 3.90 1.65 2.36 0.93 2.19 2.07 T3 2.95 3.30 2.70 2.05 1.65 TR3) TR ANOVA ab a b bc c bc bc T1 27.65 32.42 30.36 28.62 24.66 36.20 40.26 Procyanidin trimer T2 42.82 51.26 47.19 51.18 52.01 60.56 45.68 T3 39.50 46.76 45.67 39.45 34.74 34.05 22.79 ANOVA a a a a a a a T1 19.66 18.72 21.10 18.99 20.07 23.09 13.44 Procyanidin dimer T2 28.40 28.03 25.68 27.97 20.36 21.63 11.76 T3 22.94 25.49 28.45 26.09 22.74 25.54 22.68 ANOVA a a a a a a b T1 5.30 6.64 6.86 6.63 5.70 8.64 8.29 Epicatechin T2 3.87 6.57 5.60 8.09 6.81 8.44 8.48 T3 4.93 5.87 5.72 6.48 6.86 8.91 10.58 ANOVA c b b b b a a T1 52.57 58.91 56.65 52.21 52.33 55.09 48.22 Procyanidin dimer-3-O-gallate T2 45.17 48.97 46.81 50.38 53.18 52.53 49.15 T3 24.74 34.22 37.31 35.29 33.48 46.80 45.65 ANOVA a a a a a a a T1 5.39 6.62 6.42 5.92 4.83 7.00 7.23 Procyanidin dimer T2 6.42 6.38 6.30 6.99 4.86 5.66 5.22 T3 4.69 5.59 5.85 4.95 4.62 6.45 6.73 ANOVA b a a a ab a a T1 3.77 4.11 3.59 3.05 2.19 2.36 2.36 Malvidin-3-O-glucoside-(C2-O-C7, C4-C8)-(epi)catechin T2 4.97 5.29 4.34 4.39 3.02 3.37 3.22 T3 6.53 8.06 7.79 6.56 5.77 6.04 5.52 ANOVA a a a a a a a T1 TR TR TR TR TR TR TR Procyanidin dimer-3-O-gallate T2 1.15 0.90 0.77 0.95 1.07 1.25 1.73 T3 0.55 0.86 0.78 0.80 0.71 1.14 1.60 ANOVA bc bc c bc bc b a T1 156.12 173.26 170.40 161.19 149.49 175.27 158.07 Total flavan-3-ols T2 181.53 198.92 180.50 200.02 181.80 196.73 159.34 T3 136.36 180.24 178.21 162.11 149.80 165.83 147.50 ANOVA cd a abc abcd bcd ab d 1)The content of each flavan-3-ol in this table is equivalent to that of catechin and the detection limit of catechin in methanol is 0.075 mg/L in the present study. 2)Different letters are significantly different at the 0.05 level according to ANOVA. 3)Trace. 1008 Z. Li et al.

Table 6. Evolution of flavonols in shortly-aged red wines made from Cabernet Gernischt

Flavonols1) Aging (month) Tank No. (mg/L) 12345913 T1 TR TR TR TR TR TR TR Dihydroquercetin-O-hexoside T2 0.37 0.34 0.37 0.43 0.22 0.28 0.33 T3 0.57 0.87 0.98 1.51 1.26 1.81 1.23 ANOVA a2) aaaaaa T1 ND3) ND ND TR3) TR TR TR Dihydroquercetin-3-O-rhamnoside T2 0.17 0.22 0.23 0.13 TR TR TR T3 0.20 0.37 0.39 0.30 0.37 0.40 0.28 ANOVA c abc abc bc ab a abc T1 0.19 0.27 0.21 0.17 TR TR TR Quercetin-3-O-glucoside T2 0.27 0.26 0.15 0.30 TR TR TR T3 0.38 0.51 0.42 0.29 0.20 0.13 0.10 ANOVA ab a ab ab ab c c T1 1.85 2.44 2.19 2.22 1.85 1.96 1.89 Kaempferol-3-O-glucoside T2 2.44 2.19 2.09 2.05 1.50 1.38 1.26 T3 4.14 4.61 3.98 3.01 3.05 2.79 2.55 ANOVA ab a ab ab ab ab b T1 1.06 1.11 1.08 1.18 0.68 0.49 0.49 Myricetin T2 1.11 1.06 0.99 1.13 0.95 0.69 0.55 T3 0.44 0.46 0.54 1.36 0.22 0.76 0.89 ANOVA b b b a b b b T1 0.65 0.70 0.68 0.58 0.40 0.81 0.80 Rutin T2 0.48 0.40 0.32 0.38 0.19 0.49 0.61 T3 0.53 0.41 0.47 0.36 0.32 0.28 0.24 ANOVA a ab ab ab b ab a T1 TR TR TR TR TR TR TR Quercetin T2 TR TR TR TR TR TR TR T3 TR TR TR TR TR TR TR ANOVA ------T1 4.04 4.53 4.16 4.15 2.93 3.26 3.18 Total flavonols T2 4.84 4.48 4.16 4.42 2.86 2.84 2.74 T3 6.26 7.24 6.78 6.83 5.43 6.18 5.29 ANOVA a a a a a a a 1)The content of each flavonol in this table is equivalent to that of quercetin and the detection limit of quercetin in methanol is 0.050 mg/L in this study. 2)Different letters are significantly different at the 0.05 level according to ANOVA. 3)TR and ND represent trace and not-detected, respectively.

13 months, which resulted from the evolutions of gallic and ethylgallate was observed in the aged Carbenet acids and ethyl gallate that accounted for about 40 and Gernscht wines, which may be due to the esterification and 45% of total hydroxybenzoic acids, respectively. ANOVA actylation of gallic acid in the latter stage of aging as indicated that gallic acids had a significant decrease at the Benítez et al. (31) studied. On the other hand, Miller et al. aged 13 months and ethyl gallate had a significant increase (29) and Fabios et al. (32) suggested that the oxidation in at the aged 2 months. Besides, syringic acid also showed a aging oak barrels can lead to loss of phenolic acids. Owing significant increase at the aged 2 months. to these above, the levels of total hydroxybenzoic acids Higher levels of gallic acid and ethylgallate in the aged steadily increased and subsequently decreased, as shown in wines may be originated from the releases of gallocatechin Table 7. and epigallocatechin as Singleton and Trousdale (27) Hydroxycinnamic acids: Five hydroxycinnamic acids described, and Miller et al. (29) and Fernández de Simón were identified from the aged Cabernet Gernischt wines et al. (30) reported that extractions from oak wood components (Table 8). Among the hydroxycinnamic acids detected, also can lead to the increase of hydroxybenzoic acids. caffeic acid contained the lowest concentration, followed Along with aging time prolonged, significantly negative by ρ-coumaric acid. Throughout the experimental aging correlation between evolution of the content of gallic acid periods, no significant change was found for most of Evolution of Phenolic Compounds in Cabernet Gernischt Shortly-aged Wines 1009

Table 7. Evolution of hydroxybenzoic acids in shortly-aged red wines made from Cabernet Gernischt

Hydroxybenzoic acids1) Aging (month) Tank No. (mg/L) 12345913 T1 19.12 21.89 21.08 19.02 18.43 23.86 14.62 Gallic acid T2 32.20 36.96 32.74 36.01 32.59 29.68 17.67 T3 31.97 40.83 42.96 38.75 35.05 32.01 17.72 ANOVA a2) aaaaab T1 0.63 1.10 3.19 6.04 4.57 0.98 0.12 Dihydroxyphenylethanol T2 0.95 2.87 2.35 3.19 TR3) TR TR T3 3.10 3.68 2.46 0.90 0.70 1.34 1.54 ANOVA ab ab ab a ab b b T1 1.11 1.41 1.34 1.20 0.84 1.35 1.79 Dihydroxyphenylethanol T2 1.62 1.92 1.74 1.96 1.06 1.51 2.37 T3 0.68 0.90 0.91 0.74 0.62 1.29 1.59 ANOVA bc b bc bc c b a T1 1.41 1.68 1.73 1.59 1.12 1.70 1.51 Methyl gallate T2 1.11 1.55 1.27 2.09 0.95 0.93 1.08 T3 1.47 1.17 1.57 1.20 1.63 1.41 1.41 ANOVA a a a a a a a T1 2.04 2.51 2.11 2.07 1.21 2.08 1.98 Syringic acid T2 2.32 2.49 2.17 1.92 0.99 1.96 1.37 T3 1.51 2.15 1.89 1.60 1.40 1.38 1.17 ANOVA b a ab b d bc cd T1 21.22 24.87 25.23 24.45 24.10 27.09 25.38 Ethly gallate T2 20.85 23.75 22.22 24.50 23.71 22.16 23.13 T3 21.01 26.38 27.21 25.48 23.91 28.87 28.65 ANOVA b a a a a a a T1 45.52 53.47 54.66 54.37 50.26 57.06 45.39 Total hydroxybenzoic acids T2 59.04 69.56 62.48 69.67 59.30 56.24 45.62 T3 59.74 75.12 76.98 68.66 63.31 66.30 52.08 ANOVA bc a a ab ab ab c 1)The content of each hydroxybenzic acid in this table is equivalent to that of gallic acid and the detection limit of gallic acid in methanol is 0.065 mg/L in the present study. 2)Different letters are significantly different at the 0.05 level according to ANOVA. 3)Trace. hydroxycinnamic acids, except caffeic acid that showed a (33) reported that piceid was considered to be the major significant increase. In addition, a fluctuant decrease was component in the grape juices. However, cis-piceid was present for ethyl caffeate and ethyl ρ-coumarate. quantified only in T3 wine sample. Total stilbenes had no Many studies have been reported that hydroxycinnamic significant change during the aged study. acids can react with anthocyanins to form pyranoanthocyanins, participate in the polymerization reactions (9-11) and Cluster analyses In an attempt to search a reference point oxidative reactions (13,16). However, in our study there of development of wine quality in the process of aging, may be two ways affecting evolution of hydroxycinnamic cluster analysis was carried out on the data of the identified acids in the aged Cabernet Gernischt wines. One possible compounds according to aging time. The dendrogram explanation is that their respective ester hydrolyzed to form obtained was shown in Fig. 1. The squared Eulidean them during aging due to the concentrations of ethyl distance was taken as a measure of proximity between 2 caffeate and ρ-coumarate expectedly decreased during wine samples from different aged time, and Ward’s method aging. Another is, during aging, oak compounds provided was used as the linkage rule. As observed in this figure, hydroxycinnamic acids, leading to the increase in aged there were 2 groups wine samples, one corresponding to wine. wines aged 1 to 3 months and the other to wines aged 4 to Stilbenes: Three stilbenes, trans-piceid, cis-piceid, and 13 months. Within the latter group, 2 new subgroups could trans-resveratrol, were identified from various aged be distinguished: one of wines aged 4 to 5 months, and one Cabernet Gernischt wines (Table 9). Trans-piceid, which of wines aged 9 to 13 months. The present results seemed was from glycosylation of resveratrol, showed the highest to elucidate that there was a key reference point, at the concentration during whole aging. Romero-Pérez et al. aged 3 months, for the development of wine quality during 1010 Z. Li et al.

Table 8. Evolution of hydroxycinnamic acids in shortly-aged red wines made from Cabernet Gernischt Hydroxycinnamic acids1) Aging (month) Tank No. (mg/L) 12345913 T1 TR2) 0.18 0.17 0.12 0.07 0.12 0.19 Caffeic acid T2 TR 0.12 0.12 0.22 0.26 0.29 0.26 T3 TR 0.11 0.06 TR 0.12 0.31 0.33 ANOVA d3) cccbcaba T1 1.03 1.16 1.09 0.17 0.72 1.10 1.09 ρ-Coumaric acid T2 1.61 0.78 1.33 1.10 1.51 0.76 0.81 T3 0.43 1.22 2.01 2.09 1.07 1.84 1.72 ANOVA a a a a a a a T1 9.06 10.63 10.29 10.06 9.99 11.23 11.40 Ferutaric acid T2 9.59 10.50 9.43 10.60 9.14 9.96 10.59 T3 12.67 15.24 15.85 13.61 12.49 14.79 13.78 ANOVA a a a a a a a T1 2.85 3.03 2.80 2.35 1.92 2.10 1.95 Ethyl caffeate T2 1.79 1.71 1.53 1.71 1.33 1.66 1.47 T3 2.86 2.66 3.36 2.41 2.13 2.14 2.01 ANOVA ab ab a ab b ab b T1 3.18 3.30 3.11 3.19 2.88 3.28 2.76 Ethyl-ρ-coumarate T2 2.89 2.94 2.65 2.95 2.41 2.56 2.52 T3 3.69 3.64 3.78 3.37 3.02 3.55 3.17 ANOVA ab a ab ab b ab ab T1 16.12 18.29 17.46 15.89 15.58 17.84 17.39 Total hydroxycinnamic acid T2 15.88 16.05 15.07 16.58 14.65 15.24 15.64 T3 19.64 22.87 25.06 21.48 18.83 22.64 21.01 ANOVA a a a a a a a 1)The content of each hydroxycinnamic acid in this table is equivalent to that of caffeic acid and the detection limit of caffeic acid in methanol is 0.050 mg/L in the present study. 2)Different letters are significantly different at the 0.05 level according to ANOVA. 3)Trace.

Table 9. Evolution of stilbenes in shortly-aged red wines made from Cabernet Gernischt Stilbenes1) Aging (month) Tank No. (mg/L) 12345913 T1 0.88 0.88 0.85 0.86 0.60 0.89 0.89 trans-Piceid T2 0.49 0.51 0.47 0.49 0.32 0.51 0.56 T3 0.35 0.43 0.43 0.38 0.36 0.37 0.34 ANOVA a2) aaaaaa T1 TR3) TR TR TR ND3) ND ND cis-Piceid T2TRTRTRTRNDNDND T3 0.10 0.13 0.14 0.29 0.04 0.17 0.15 ANOVA c bc bc a d b b T1 0.51 0.56 0.48 0.35 0.34 0.34 0.33 trans-Resveratrol T2 0.58 0.61 0.49 0.50 0.37 0.34 0.34 T3 1.23 1.35 1.45 1.18 1.04 1.00 1.04 ANOVA a a a a a a a T1 1.39 1.44 1.33 1.21 0.94 1.23 1.23 Total stilbenes T2 1.07 1.13 0.96 0.99 0.69 0.86 0.90 T3 1.67 1.92 2.02 1.85 1.44 1.54 1.52 ANOVA a a a a a a a 1)The content of each compound in this table is equivalent to that of resveratrol and the detection limit of resveratrol in methanol is 0.050 mg/L in the present study. 2)Different letters are significantly different at the 0.05 level according to ANOVA. 3)TR and ND represent trace and not-detected, respectively. Evolution of Phenolic Compounds in Cabernet Gernischt Shortly-aged Wines 1011

Fig. 1. Dendrogram resulting from applying cluster analysis to the concentrations of 49 phenolic compounds (shown in Table 4-9) according to aging time. In terms of each compound, the data used for cluster analysis are from the means of a total of 6 values of triplicate wine samples. M1, M2, M3, M4, M5, M9, and M13 represent wine samples aged 1, 2, 3, 4, 5, 9, and 13 months, respectively. experimental aging period. According to the data of all the in used oak barrels and in the bottle. J. Agr. Food Chem. 48: 4613- detected phenolic compounds, the levels of 2 vitisin-type 4618 (2000) pyranoanthocyanins, most favan-3-ol monomers and 7. Eiro MJ, Heinonen M. Anthocyanin color behavior and stability polymers, most phenolic acids increased to different extent, during storage: Effect of intermolecular copigmentation. J. Agr. within 1 to 3 months of wine aging in oak barrels, whereas Food Chem. 50: 7461-7466 (2002) 8. Hermosín-Gutiérrez I, Lorenzo ES, Espinosa AV. Phenolic other detected anthocyanins indistinctively declined (Table composition and magnitude of copigmentation in young and shortly 4-9). After 4 months of aging, especially more than 9 aged red wines made from the cultivars, Cabernet Sauvignon, months of aging, different ranges of decrease were present Cencibel, and Syrah. Food Chem. 92: 269-283 (2005) for almost all the anthocyanins, major flavan-3-ol monomers 9. Schwarz M, Wabnitz TC, Winterhalter P. Pathway leading to the and polymers (epicatechin excepted), major flavonols and formation of anthocyanin-vinyphenol adducts and related pigments gallic acid (Table 4-9). ANVOA results revealed that the in red wines. J. Agr. Food Chem. 51: 3682-3687 (2003) 10. Schwarz M, Hofmann G, Winterhalter P. Investigations on phenolic compounds with significant variation in between anthocyanins in wines from Vitis vinifera cv. Pinotage: Factors wines aged 3 and 9 months included 11 anthocyanins, 3 influencing the formation of pinotin A and its correlation with wine flavan-3-ol monomers, and 1 polymer, a flavovol compound age. J. Agr. Food Chem. 52: 498-504 (2004) (quercetin-3-O-glucoside). Apparently, these compounds 11. Rentzsch M, Schwarz M, Winterhalter P, Hermosín-Gutiérrez I. were all key components of chemical reactions involving Formation of hydroxyphenyl-pyranoanthocyanins in Grenache wines: in color and mouthfeel in the aged wines. Based on these Precursor levels and evolution during aging. J. Agr. Food Chem. 55: results, it was thus suggested that 2 important milestones 4883-4888 (2007) 12. Drinkine J, Lopes P, Kennedy JA, Teissedre P, Saucier C. for the development of fine wine quality were at the aged Ethylidene-bridged flavan-3-ols in red wine and correlation with 3 and 9 months, respectively. This suggestion could wine age. J. Agr. 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