Biosci. Biotechnol. Biochem., 68 (12), 2623–2626, 2004
Note
Fatty Acid Compositions of Commercial Red Wines Keita Y UNOKI,1;2 Mikio T ANJI,1 Yukie M URAKAMI,1 Yoshihiro Y ASUI,3 Shuji H IROSE,3 and Masao O HNISHI1;2; y 1
Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan 2 The United Graduate School of Agricultural Science, Iwate University, Morioka, Iwate 020-8550, Japan 3 Tokachi-Ikeda Research Institute for Viticulture and Enology, Ikeda, Hokkaido 083-0002, Japan Received July 8, 2004; Accepted September 30, 2004
A determination was made of fatty acid compositions in twelve commercial red wines made from grapes differing in kind and vintage. Twelve fatty acids were identified, palmitic, myristic, and lauric acids being found predominant. Total acyls (3281 nmol/100 ml) differed considerably. Changes in fatty acid constituents in must from grape berries and wines according to the process of manufacture were also examined. Key words:
red wine; fatty acid; lipids; alcoholic fermentation; grape
Intensive research has been carried out to detect constituents in wine such as organic acids, flavorimparting compounds, and phenolics.1,2) Lipophilic constituents in wines have been found in trace amounts,3) but the details remain unclear. -Carotene and six related compounds, including xanthophylls, have recently been identified in wine, and changes in their amounts with the course of alcoholic fermentation were quantitatively determined.4) Cocito and Delfini detected fatty acids in wine by gas chromatography (GC) as trimethylsilyl ether derivatives of chloroform extracts,5) but no determination was made of fatty acid compositions. Thus no definitive data appear to be available regarding fatty acid constituents in wine, but the possibility of the presence in wine of certain lipids derived from grapes and microorganisms has been demonstrated. In Japanese sake-production, lipid components from substances such as milled rice have been shown to be essential for microorganism growth and ester formation.6) It follows that wine quality might bear some relation to the lipid constituents originally present in grapes. These some lipid constituents might also serve to distinguish different varieties of grapes. In this study, determination was initially made of the fatty acid components present in six domestic (Japanese) and six foreign (non-Japanese) wines, followed by clarification of changes in these components during the y
course of wine manufacture over a period of 25 months. The wines used appear in Table 1. They were obtained from a local source in May 2001. The lipophilic constituents were extracted immediately. At various periods during red wine production from Kiyomi grapes grown in Tokachi, Hokkaido, Japan in 2001, samples were taken: must from a free run of destemmed and crushed grapes prior to alcoholic fermentation, wine subsequent to first racking after alcoholic fermentation (at 1 month after alcoholic fermentation), wines before and after malolactic fermentation (at 4 and 6 months after alcoholic fermentation respectively) and wine after bottling, at 25 months following must. All samples obtained were processed at the Tokachi-Ikeda Research Institute for Viticulture and Enology, Ikeda, Hokkaido, Japan. Sample (700–1000 ml) concentration was carried out with a rotary evaporator at 45 C followed by lipophilic constituent extraction by the method of Bligh and Dyer.7) Briefly, 100 ml chloroform and 200 ml methanol were added to 80 ml concentrated sample or must followed by intense shaking. At 1 h, 100 ml chloroform and 100 ml distilled water were added so as to partition the two phases. The lower chloroform layer was washed twice with chloroform-methanol-water (3:48:47, v/v) and evaporated to dryness to provide the total lipid yield. Total lipids (40 mg to 5 mg each of wine and must) were methanolyzated with 2 ml methanolic 5% HCl for 2 h at 95 C and fatty acid methyl esters were extracted three times with 2 ml n-hexane subsequent to the addition of 1 ml water. The combined extracts were applied onto thin-layer chromatography on silica gel using hexane–diethyl ether (85:15, v/v) as solvent to purify fatty acid methyl esters, which were then analyzed by capillary GC and GC–MS, as described previously.8) Tridecanoic acid served as the internal standard for quantitative analysis. The lipid constituent concentrations ranged from 27 to 96 mg/100 ml for the six domestic wines and 31 to 56 mg/100 ml for the six foreign brands. Twelve fatty
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Table 1. Characteristics of Fatty Acid Constituents in Commercial Red Wines Sample No. 1 2 3
5 6 7 8 9 10 11 12
12:0 Japan (Hokkaido) Japan Kiyomai (Hokkaido) Japan Zweigeltrebe (Hokkaido) Japan Muscat Bailey A (Yamagata) Japan Melrot (Nagano) Cabernet Japan Sauvignon (Yamanashi) Cabernet France Sauvignon Cabernet Chile Sauvignon Cabernet U.S.A. Sauvignon Tempranillo Spain Pinot noir France Syrah Australia Kiyomi
Total acylsa SFA/UFAb
Harvest area Vintage 14:0
16:0
16:1(7)
16:1(9)
18:0
18:1(9)
18:1(11)
18:2
18:3
20:0
22:0
1997
15:1 2:9 22:9 5:7 33:6 7:0
3:3 0:2 1:3 0:5
7:2 3:5
6:8 1:7 0:1 0:2
7:7 1:3 1:3 0:6 0:4 0:4 0:3 0:6 54 3:7
3.90
1996
14:4 3:1 21:5 4:0 33:5 2:4
6:3 0:8 2:8 0:4
9:1 1:1
5:9 1:5 0:2 0:1
4:3 1:7 0:9 0:3 0:7 0:5 0:4 0:1 32 3:4
3.91
1997
14:3 2:4 20:9 2:4 34:3 1:5
7:7 4:5 1:5 1:1
9:1 2:6
6:5 0:9 0:3 0:2
3:6 0:8 0:8 0:5 0:4 0:2 0:6 0:2 38 1:2
3.90
2000
13:2 5:8 17:8 1:4 32:9 2:8
4:1 2:3 2:4 0:9
6:9 0:4
8:0 0:9 0:7 0:4 10:5 1:1 2:4 0:4 0:4 0:1 0:7 0:7 52 9:7
2.56
1998
14:0 2:8 18:0 3:0 30:5 1:8
4:5 0:1 1:8 0:2
6:1 0:5
6:9 0:8 0:4 0:7 14:1 1:8 2:8 1:9 0:2 0:1 0:7 0:4 51 8:1
2.27
2000
15:3 4:9 20:3 4:7 34:0 3:7
6:7 0:4 2:3 0:5
9:7 2:3
4:8 1:2 1:4 0:2
3:3 1:8 1:0 0:7 0:5 0:0 0:7 0:6 41 12:6
4.13
1998
8:4 6:4 12:6 4:2 47:0 3:4
2:8 3:1 1:9 0:8
6:1 2:9
13:4 0:7 0:2 0:4
4:5 0:8 2:6 0:5 0:3 0:5 0:2 0:7 81 4:6
2.94
1999
15:5 3:2 21:2 1:7 29:9 3:2
7:7 0:2 2:0 0:9
7:9 0:7
7:3 3:4 1:0 0:0
5:5 0:8 1:2 3:8 0:3 0:1 0:5 0:1 48 19:3
3.04
1997
13:3 4:6 16:6 1:4 38:5 4:8
5:2 2:1 2:3 1:7
8:8 2:8
1999 1999 1998
15:3 1:6 19:9 3:5 37:2 5:8 2:7 2:6 2:6 0:2 7:9 2:4 11:7 1:4 14:1 4:2 45:2 11:9 3:0 1:9 1:2 2:8 12:7 1:0 14:7 2:7 17:1 1:7 40:8 7:9 2:1 0:5 2:1 0:5 7:9 10:6
Data expressed as means standard deviation in triplicate analyses. a nmol/100 ml wine. b ( saturated fatty acid)/( saturated fatty acid).
4:9 1:6 0:8 1:1
6:4 4:0 2:1 0:5 0:7 0:2 0:4 0:1 50 9
3.61
6:1 0:7 1:1 0:1 7:1 3:0 0:2 1:1 4:2 5:3 0:3 0:1
5:1 2:5 0:9 0:7 0:5 0:1 0:7 0:3 36 15:8 3:2 4:3 0:8 0:4 0:3 0:1 0:5 0:2 57 15:5 7:3 1:3 2:1 0:7 0:5 0:1 0:9 0:4 79 7:5
4.42 5.45 4.54
K. YUNOKI et al.
4
Fatty acid (mol%) Variety
Fatty Acids in Red Wine
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Table 2. Changes in Fatty Acid Compositions (mol%) in 2001 Kiyomi Red Wines during Wine Manufacture Fatty acid
Must (0)
After AFa (1)
Before MLFb (4)
After MLFb (6)
After bottling (25)
10:0 12:0 14:0 16:0 16:1(7) 16:1(9) 18:0 18:1(9) 18:1(11) 18:2 18:3 20:0 22:0 23:0 24:0 25:0 26:0
— 0:3 0:1 0:7 0:2 25:7 4:1 0:2 0:1 0:4 0:1 2:6 0:7 5:2 1:4 1:2 0:6 42:9 3:3 13:7 3:7 1:0 0:3 2:6 0:4 0:5 0:1 1:3 0:5 0:7 0:3 1:0 0:4
1:4 1:0 3:0 1:1 1:7 0:4 28:5 1:3 0:2 0:1 0:5 0:1 6:4 1:0 4:8 0:3 0:7 0:0 36:8 0:9 8:1 0:5 1:0 0:2 2:6 1:1 0:7 0:4 2:0 1:2 0:3 0:3 1:3 0:7
— 1:1 0:1 1:6 0:0 29:2 0:5 0:4 0:2 0:5 0:2 8:6 0:4 6:3 0:1 0:8 0:0 35:7 0:6 6:7 0:1 1:4 0:1 3:8 0:9 0:7 0:0 1:7 0:4 0:3 0:1 1:2 0:3
— 1:4 0:2 2:0 0:2 28:4 1:1 0:4 0:1 1:1 0:2 7:6 1:4 5:4 0:3 1:1 0:4 36:4 1:9 6:7 0:4 0:9 0:3 3:1 0:5 0:7 0:4 2:6 1:3 0:8 0:5 1:4 0:5
— 0.7 1.1 33.6 0.2 0.6 10.3 4.5 0.8 33.1 5.4 1.1 3.1 1.7 2.1 0.2 1.5
4;460 1340
3;550 180
3;710 130
1,430
0.96 1.05
0.99 0.99
0.96 1.01
1.25 0.88
Total acylsc SFA/UFAd UIe
106;000 9150 0.57 1.34
Data expressed as means standard deviation in triplicate analyses, except for sample after bottling, which value is expressed as average of two independent experiments. Month(s) following start of wine manufacture indicated in parentheses. a Alcoholic fermenation. b Malolactic fermenation. c nmol/100 ml wine. d ( saturated fatty acid)/( unsaturated fatty acid). e unsaturation index; (mi ni)/(100), where mi is percentage of each fatty acid and ni, the number of C-C double bonds of its fatty acid i.
acids with carbon lengths from 12 to 22 were generally present in the red wines, palmitic acid (16:0) being the most abundant at 29.9% to 47.0% (Table 1). Mediumchain fatty acids such as lauric (12:0) and myristic acids (14:0) were also present as major components at 8.4% to 15.5% and 12.6% to 22.9% respectively. Saturated fatty acid to unsaturated fatty acid ratios (SFA/UFA) ranged from 2.27 to 5.45. Among the domestic wines (nos. 1–6), major fatty acids were present in essentially the same compositions. SFA/UFA for samples 1–3 and 6 ranged from 3.90 to 4.13, while in samples 4 and 5 it was 2.56 and 2.27 respectively, owing to the greater amounts of linoleic acid (18:2) in these two samples. Total acyls (total fatty acids) were present at 32 to 54 nmol/100 ml and slightly high in samples 4 and 5. The three wines from Hokkaido had basically the same fatty acid compositions, though slight differences were noted in total acyl content. The foreign wine fatty acid compositions were the same as noted for the domestic samples (Table 1). Major fatty acids in the foreign wines (nos. 8–10 and 12) were found in essentially the same proportions as in the domestic wines. Fatty acid compositions and SFA/UFA values (4.42 and 4.54) were the same for the most part for samples 10 and 12, even though grape variety and production site differed. Samples 7 and 11 generally possessed more palmitic acid than any of the other samples. SFA/UFA was highest in sample 11 (5.45) and lowest in sample 7 (2.94) among the foreign wines,
owing to a greater oleic acid [18:1(9)] content in sample 7. Oleic acids in samples 6, 8, and 9, from Cabernet Sauvignon, were present at 4.8% to 7.3%, even though sample 7 was made from the same variety. Linoleic acid content differed slightly in the foreign samples and ranged from 3.2% to 7.3%. Total acyls in the foreign wines were found at 36 to 81 nmol/100 ml. The sample 7 and 12 concentrations exceeded those in the domestic wines. Total acyl content showed no apparent relation to that of lipophilic compounds extracted with a mixture of chloroform and methanol (data not shown) or to SFA/UFA. The fatty acid compositions from pre-alcoholic fermentation to the final product of bottled wine at 25 months are presented in Table 2. Total acyls in must were present at 106 103 nmol/100 ml, 2,000 times as much as in the commercial wines. Fatty acid concentrations following alcoholic fermentation dropped to 1/24 of that in must (4,460 nmol/100 ml). Fatty acid constituents continued to decrease slightly (3,550 nmol/ 100 ml) for 4 months after alcoholic fermentation, but following malolactic fermentation remained at the values noted prior to fermentation (3,710 nmol/ 100 ml). Fatty acid in bottled wine (1,430 nmol/ 100 ml) at 4.5 months after bottling (25 months after must) decreased to half that subsequent to malolactic fermentation, and was higher in the commercial wines. Must was found to contain considerable amounts of polyunsaturated fatty acids such as linoleic and linolenic
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K. YUNOKI et al.
acids (18:3), which came from the grapes. Following alcoholic fermentation, capric acid (10:0) was newly detected along with increased levels of lauric acid and myristic acid. This might have been due to de novo synthesis by yeast for alcoholic fermentation, and the medium-chain fatty acids would be present mainly as ethyl esters.9) After alcoholic fermentation, saturated fatty acids like stearic acid (18:0) increased, while linoleic and linolenic acids decreased with consequently greater SFA/UFA and a reduced unsaturation index (UI). Pre- and post-malolactic fermentation fatty acid compositions were essentially the same as after alcoholic fermentation, linoleic and palmitic acids generally being predominant. Fatty acid constituents were thus found not to change during malolactic fermentation. In bottled wine 19 months after malolactic fermentation, SFA/UFA was greater than 1 and the unsaturation index less than after malolactic fermentation. Linoleic acid was still detected at 33%. Four saturated fatty acids with carbon lengths of more than 23 were found in small amounts in must and all samples at every stage. The lipid content of wine was previously found to be 5–10 mg/100 ml, with oleic acid and myristic acid present as fatty acids.3) Lipid constituents in this study were detected at 27 to 96 mg/100 ml, possibly owing to incomplete removal of other constituents during lipid extraction. Ten other fatty acids were found in the red wine samples, whose major constituents in most cases were palmitic acid, myristic acid, and lauric acid. Three domestic brands (samples 1–3), all produced in the same manner from grapes from Hokkaido, had basically the same fatty acids, but the fatty acid compositions of red wines made with Cabernet Sauvignon (samples 6–9) all differed clearly. Differences in production method and cultivation site would thus appear to be the determinants of the fatty acid constituents of red wine. Fatty acids in wine have been reported likely to derive not only from yeast but grape seeds as well.3) The predominant myristic and lauric acids in commercial red wines were found in this study to increase following alcoholic fermentation, indicating that wine yeast is the factor responsible for fatty acid production. The significant amounts of fatty acids in must greatly decreased after alcoholic fermentation, accompanied by notable decreases in polyunsaturated fatty acids. Fatty acids, particularly those that are polyunsaturated, may thus be considered selectively used by yeast during alcoholic fermentation as carbon source or membrane lipids. But most of fatty acids in must were assumed to be no longer present on unsolubilization with phenolic compounds and yeast cells subsequent to racking (data not shown). Fatty acid constituents were found to undergo no change during malolactic fermentation, indicating that fatty acid de novo synthesis might be inhibited in the lactic acid bacteria responsible for
malolactic fermentation, since these bacteria use external polyunsaturated fatty acids as membrane lipid components. For bottled and commercial wines (sample 1 in Table 1) made in 1997 with the same grape variety but in different years, long-term aging was found further to decrease the fatty acid content, particularly that of polyunsaturated fatty acids, possibly as a result of oxidation, with consequently greater saturated fatty acid amounts. The Kiyomi vintage 1999 total fatty acid was 296 nmol/100 ml with linoleic acid present at 22% (data not shown). These parameters for sample 1 (Kiyomi in 1997) were 54 nmol/100 ml and 7.7% (Table 1), suggesting that fatty acids, especially those polyunsaturated in wines produced by the same methods, possibly decrease with vintage aging. Information on the fatty acid constituents of wines might be of use for determining grape variety, climatic variation in harvest areas, and wine quality. For this purpose, more detailed research should be directed to the fatty acids in wine.
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