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Volatile components of grape pomaces from different cultivars of Sicilian Vitis vinifera L.
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Bioresource Technology 99 (2008) 260–268
Volatile components of grape pomaces from different cultivars of Sicilian Vitis vinifera L. Giuseppe Ruberto b
a,*
, Agatino Renda a, Vincenzo Amico b, Corrado Tringali
b
a Istituto del C.N.R. di Chimica Biomolecolare, Via del Santuario 110, I-95028 Valverde, CT, Italy Dipartimento di Scienze Chimiche, Universita` degli Studi di Catania, Viale A. Doria 6, I-95125 Catania, Italy
Received 30 August 2006; received in revised form 22 December 2006; accepted 23 December 2006 Available online 23 February 2007 Dedicated to the memory of Dr. Gianfranco Borin.
Abstract The volatile components of grape pomace coming from the processing of some of the most important varieties of grape (Vitis vinifera L.) cultivated in Sicily, namely Nero d’Avola, Nerello Mascalese, Frappato and Cabernet Sauvignon, have been determined by gas-chromatography (GC) and gas-chromatography–mass spectrometry (GC–MS). According to the winemaking procedure that entails the removal of stalks before fermentation, two kinds of grape pomace are obtained. The first consists of skins, pulp residues and seeds, the proper grape pomace, which is partially used for grappa, a typical Italian spirit, and alcohol production, the second consists almost exclusively of stalks. On the whole, 38 components have been characterized in the samples of grape pomaces, with Frappato cv. showing the richest composition; instead, 88 components have been detected in the stalks of Frappato, Nero d’Avola, Nerello Mascalese and Cabernet Sauvignon varieties. In order to make a comparison between the grape varieties easier, the volatile components detected in the two sets of samples (grape pomaces and stalks) have been grouped in different classes. Significant differences among varieties have been detected and statistical treatment of data is also reported. This study is part of a wider project aimed at the possible exploitation of the main agro-industrial by-products. At the same time it is one of the first reports on the volatile components of this waste material. 2007 Elsevier Ltd. All rights reserved. Keywords: Vitis vinifera L.; Grape pomace; Stalks; Volatile components; GC–MS
1. Introduction Agro-industrial by-products present two rather conflicting aspects: on one hand their disposal represents a great economical and ecological problem, further complicated by legal restrictions, while on the other, they may be considered a promising and renewable source of useful compounds for their technological and nutritional properties (Laufenberg et al., 2003; Montgomery, 2004; Schieber et al., 2001). It is likely that the most immediate and economic use of this material is as feed and as fertilizer, even though some pre-treatments and composting procedures are necessary in *
Corresponding author. Tel.: +39 0957212136; fax: +39 0957212141. E-mail address: [email protected] (G. Ruberto).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.12.025
most cases. It also is true that these wastes are rich in several micronutrients such as carotenoids, polyphenols, tocopherols, vitamins, oligo elements and others, whose beneficial effects on human health are frequently highlighted, therefore this large amount of waste material could be a very cheap source of the aforesaid components. Of course, such potential exploitation would not avoid the disposal of a still substantial amount of waste, but the economic profit should make it more bearable. Grape (Vitis vinifera L.) is one of the world’s largest fruit crops, in excess of 60 million metric tons (www.fao.org), and is mainly grown for wine production. Grape pomace, the main by-product of wine production, consists of skins, seeds and stalks, reaching an estimated amount of 13% by weight of processed grape (Torres et al., 2002). In line with different winemaking procedures,
G. Ruberto et al. / Bioresource Technology 99 (2008) 260–268
two kinds of grape pomaces are obtained after the removal of stalks before maceration and successive fermentation steps. The first consists of skins, pulp residues and seeds, the proper grape pomace, which is partially used for grappa and alcohol production; the second consists almost exclusively of stalks. The chemical composition of grape pomace is rather complex: alcohols, acids, aldehydes, esters, pectins, polyphenols, mineral substances, sugars etc. are the most represented classes of compounds (Bonilla et al., 1999; Mantell et al., 2003; Murthy et al., 2002; Saquet et al., 2000). The evaluation of the qualitative aspects of a grape pomace is carried out in view of the production of high quality grappa; otherwise the grape pomace is used for alcohol distillation, or thrown away. The best grape pomaces are highly rich in vinous liquid, namely not exhaustively pressed, with a moisture degree ranging from 55% to 70%, which allows to exploit the raw material better and to extract the organoleptic characteristics of the native vine. Concerning the pool of the volatile components of a grape pomace which confer its particular aroma, few studies have so far been reported (Park et al., 1991; Silva et al., 1996; Hashizume and Samuta, 1997), if compared with the analogous studies made on wine (Aznar et al., 2001; Flamini, 2005). In our ongoing studies aimed at the possible exploitation of the main agro-industrial by-products obtained in Sicily, we wish to report here the results of a study of the volatile components of grape pomaces and stalks of the most important varieties of grape cultivated in Sicily for wine production, namely Nero d’Avola, Nerello Mascalese, Frappato and Cabernet Sauvignon. 2. Methods 2.1. Plant material Grape pomaces and stalks of Nero d’Avola and Frappato were donated by the ‘‘Valle dell’Acate’’ wine firm, Acate, RG, Italy – those from Nerello Mascalese and Cabernet Sauvignon were given by the ‘‘Emanuele Scammacca Barone del Murgo’’ wine firm, Santa Venerina, CT, Italy. The winemaking procedures were similar for all samples, namely grape clusters were crushed and destemmed using a destemmer-crusher. The crushed grapes were treated with sulphur dioxide (0.2–0.5% total mash) and with selected strains of Saccharomyces cerevisiae to start up the fermentation. After 6–8 days of maceration, when alcoholic fermentation was finished, the mash was pressed. Stalks coming from destemming procedure and grape pomace coming from the maceration procedure were subjected to the distillation procedures within 24 h of their collection. All materials were collected during the 2004 vintage. 2.2. Isolation and analysis of volatile components Fresh grape pomace and stalks (300 g each) were subjected to simultaneous steam distillation-extraction (SDE)
261
for 3 h with a modified Likens–Nickerson apparatus using hexane (1 ml) as the solvent (Koedman, 1987). The SDE procedure was also carried out after adjusting the initial pH of the grape pomace and stalks suspensions to 7, by adding the necessary amount of 2 N NaOH solution. The mixtures were immediately analysed on a Shimadzu gas chromatograph, Model 17-A equipped with a flame ionization detector (FID). Analytical conditions: DB-5 MS capillary column (30 m · 0.25 mm · 0.25 lm), helium as carrier gas. Injection in split mode (1:50), injected volume 1 ll, injector and detector temperature 250 and 280 C, respectively. Linear velocity in column 19 cm/s. The oven temperature was held at 60 C for 6 min, then programmed from 60 to 300 C at 2 C/min. Percentages of compounds were determined from their peak areas in the GC–FID profiles. Gas-chromatography–mass spectrometry (GC–MS) was carried out in the fast mode on a Shimadzu GC–MS mod. GCMS-QP5050A, ionization voltage 70 eV, electron multiplier 900 V, transfer line temperature 280 C. Analytical conditions: SPB-5 capillary column (15 m · 0.10 mm · 0.15 lm), helium as carrier gas. Injection in split mode (1:100), injected volume 1 ll, injector and detector temperature 250 C. Constant linear velocity in column 50 cm/s. The oven temperature was held at 60 C for 1 min, then programmed from 60 to 280 C at 10 C/min. Identification of components was based on GC retention indexes, computer matching with Wiley 275 and NIST (Versions 21 and 75) libraries, comparison of the fragmentation patterns with those reported in the literature and whenever possible, coinjection with authentic samples (Jennings and Shibamoto, 1980; Adams, 2001). Pure standards were purchased from Aldrich Chemical Co., Extrasynthese, France, and Fluka Chemie AG, Switzerland. 2.3. Statistical analysis SPSS software, 14.1 version, was used to carry out statistical analysis of the data. ANOVA and Duncan’s multiple range test were applied to the data to determine significant differences between the analysed volatile components; the model was statistically significant with a value of P 6 0.01. 3. Results and discussion 3.1. Volatile components of grape pomace Table 1 lists the 38 volatile components characterized in all samples of grape pomaces, whereas Fig. 1 shows a typical GC–FID profile. The Frappato cultivar showed the richest composition with 35 compounds; Nerello Mascalese and Nero d’Avola cultivars had a comparable composition with 21 and 19 components, respectively; finally the Cabernet Sauvignon cultivar gave the simplest mixture with only 17 identified components. The most represented class of
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G. Ruberto et al. / Bioresource Technology 99 (2008) 260–268
Table 1 Volatile components of Frappato (Fr), Nerello Mascalese (NM), Nero d’Avola (NA) and Cabernet Sauvignon (CS) grape pomacesa Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
c
Cyclohexane 3-Methylhexanec Heptanec 1,1-Diethoxy ethane 2-Methylbutan-1-olc Octanec 1-Butoxy-1-ethoxy ethane 1-Butanol, 3-methyl acetate 1-Butanol, 2-methyl acetate Nonanec 1,1-Diethoxy-2-methyl butane 1-Ethoxy-1-pentoxy ethane 2-Pentyl furane Ethyl hexanoatec Limonenec Terpinolenec Ethyl octanoatec a-Ylangene Ethyl 9-decenoated Ethyl decanoate 3,7-Guaiadiene Aromadendrenec 3-Methyl butyl octanoate 2-Methyl butyl octanoate c-Muurolene c-Amorphene c-Cadinene Ethyl dodecanoatec 3-Methyl butyl decanoate 2-Methyl butyl decanoate Ethyl tetradecanoatec 3-Methyl butyl dodecanoate Ethyl 9-hexadecenoated Ethyl hexadecanoatec Manoyl oxide Ethyl linoleatec Ethyl 9-octadecenoated Ethyl octadecanoatec
KIb
Fr
NA
NM
CS
647 669 697 724 729 797 868 872 875 897 954 973 989 997 1027 1086 1195 1366 1385 1394 1423 1438 1444 1447 1492 1506 1517 1591 1641 1644 1793 1844 1971 1993 2005 2159 2166 2192
– – t 28.59 – 1.11 0.23 0.48 0.13 0.21 0.10 0.72 0.28 0.67 0.44 0.18 7.46 0.35 0.66 18.83 0.10 0.17 0.63 0.17 0.33 0.28 0.23 10.01 0.54 0.17 1.33 0.14 0.72 7.02 0.28 1.47 1.06 0.37
– – – 6.07 1.19 0.29 – 0.29 – – – 0.23 – 1.08 – – 11.63 – 1.75 29.23 – – 0.68 0.16 – – – 15.38 0.91 – 1.94 0.31 1.16 6.43 0.10 3.05 2.07 0.39
– – – 10.59 1.12 0.52 – t – – – 0.31 – 0.43 – – 8.57 – – 25.55 – – 0.52 – – – – 14.67 0.74 – 3.13 0.31 0.75 11.06 0.40 4.50 2.62 0.55
0.50 0.34 2.24 21.95 0.93 – – – – – – 0.32 – 0.38 – – 3.41 – – 9.87 – – – – – – – 6.01 0.30 – 1.60 – 0.68 7.22 – 2.23 1.54 0.28
(1.15)A (0.14)A (0.07) (0.06)A (0.05) (0.05) (0.02) (0.03)A (0.01) (0.07)B (0.03) (0.01) (0.11)C (0.05) (0.04)B (0.13)C (0.02) (0.02) (0.02)A (0.02)A (0.02) (0.04) (0.04) (0.21)C (0.05)C (0.02) (0.03)D (0.02)B (0.04)B (0.03)B (0.02)B (0.05)D (0.04)D (0.02)B
(0.05)D (0.06)A (0.09)C (0.06)B
(0.02)C (0.02)A
(0.06)A (0.04)A (0.10)A
(0.04)A (0.05)A
(0.08)A (0.02)A (0.05)B (0.02)A (0.04)A (0.04)C (0.02)C (0.04)B (0.04)B (0.02)B
(0.06)C (0.05)A (0.01)B
(0.02)B (0.05)C
(0.12)B
(0.72)B
(0.03)B
(0.07)B (0.06)B (0.03)A (0.04)A (0.04)B (0.19)A (0.05)A (0.04)A (0.11)A (0.04)A
(0.02) (0.02) (0.04) (0.26)B (0.04)B
(0.04)B (0.02)C
(0.05)D
(0.06)D
(0.09)D (0.01)D (0.05)C (0.01)B (0.09)B (0.08)C (0.07)C (0.02)C
a The numbering refers to elution order, values (area percent) represent averages of three determinations and standard deviation (±SD) is given in parentheses (t = traces < 0.05%), different capital letter in the same row represents significant difference at P 6 0.01 by Duncan’s multiple range test. b Retention indexes relative to C6–C22 n-alkanes on DB-5 column. c Co-elution with pure component. d Correct isomer not identified.
components in all four cultivars was that of ethyl esters of aliphatic acids with a linear chain from 6 to 18 carbon atoms, both saturated and unsaturated, with the former predominating. The main components were ethyl octanoate, ethyl decanoate, ethyl dodecanoate and hexyl hexadecanoate. Other common compounds of the four cultivars were represented by some acetals, particularly 1,1-diethoxy ethane, which is the main component of Cabernet Sauvignon and Frappato. As previously mentioned, the grape pomace of the Frappato cultivar stood out from the others, both for the higher number of components and, notably, for the presence of nine terpenes, which account, unlike the previously mentioned components, for a more significant varietal differentiation of the examined cultivars. Two monoterpenes:
limonene and terpinolene – six sesquiterpenes: a-ylangene, 3,7-guaiadiene, aromadendrene, c-muurolene, c-amorphene, c-cadinene – and one diterpene: manoyl oxide have been characterised. However, it should be underlined that the total amount of these compounds is to be considered very low. In the Nero d’Avola and Nerello Mascalese only manoyl oxide was detected, whereas no terpene has been detected in the Cabernet Sauvignon. Statistically significant differences were found between the average content of most components in the four cultivars by ANOVA and Duncan’s multiple range test (Table 1). In particular, the most important components in differentiating samples were, in that order, ethyl octanoate, ethyl dodecanoate, ethyl 9-decenoate, ethyl linoleate, ethyl decanoate, ethyl hexadecanoate and ethyl tetradecanoate.
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Fig. 1. GC–FID profile of volatile components of Nerello Mascalesce grape pomace (for the numbering see Table 1).
3.2. Volatile components of stalks The composition of volatile components isolated from the stalks of Frappato, Nero d’Avola, Nerello Mascalese and Cabernet Sauvignon proved much richer and more complex, as exemplified by the GC–FID profile in Fig. 2. Table 2 lists the 88 components, 77 of which were fully identified. They have been separated in two classes, namely non-terpenoid compounds and terpenes, to make a comparison of the four varieties easier. Unlike the volatile components of the grape pomaces (Table 1), greater amounts of the terpenes were present in the stalks and numerically comparable to non-terpenoid derivatives, the last all of acetogeninic nature. Furthermore, whilst the composition of the grape pomaces showed substantial compositional uniformity, even though with the previously described differences, the composition of the stalks of the four cultivars was quite different from each other. These differences are emphasized if the comparison is carried out throughout specific classes of compounds, in which it is possible to further subdivide the two categories listed in Table 2. With this in mind, hydrocarbons, alcohols, aldehydes, and carboxylic acids and derivatives among the non-terpenoid compounds have been analysed in detail. A substantial difference among the four cultivars was observed with the regards to the hydrocarbons. Nero d’Avola contained the highest number of these components, cyclohexane and heptane being the principal compounds. Heptane was practically the only component of
this class detected in the Frappato. In the Nerello Mascalese variety, only three compounds with relatively high molecular weight, namely pentadecane, 9-(Z)-tricosene and tricosane, have been detected; the level of hydrocarbons was almost insignificant in the Cabernet Sauvignon. Concerning the aliphatic alcohols, again Nero d’Avola showed the most complex composition with the 3-methylbutan-1-ol and hexanol as the main compounds. Cabernet Sauvignon and Nerello Mascalese showed an identical qualitative profile due to 3-(E)-hexen-1-ol, hexanol and 3-ethyl-4-methyl-pentan-1-ol. Almost negligible was the amount of these components in the Frappato variety represented exclusively by hexanol. The qualitative composition of aldehydes was fairly similar in all varieties; however whereas Frappato and Nerello Mascalese cvv. showed detectable presence of 10 aldehydes, in Nero d’Avola and Cabernet Sauvignon many aldehydes were below the detection limit. It should be pointed out that in each cvv. 2-(E)-hexenal, hexanal and nonanal were the main components. The last class of examined compounds concerned the carboxylic acids and their derivatives. The most represented compounds were esters, whereas only one anhydride and one free carboxylic acid have been determined. Among the esters, the Nero d’Avola showed the richest and most complex composition with 11 components, being the bphenylethyl acetate the main one, this compound was absent in the other two varieties. The esters content of the Cabernet Sauvignon was represented exclusively by
264
G. Ruberto et al. / Bioresource Technology 99 (2008) 260–268
Fig. 2. GC–FID profile of volatile components of Nero d’Avola stalks (for the numbering see Table 2).
the ethyl esters of octanoic and decanoic acids, as previously observed for the grape pomace. In the Frappato the esters were represented by hexyl acetate, ethyl octanoate, as well as ethyl esters of C16 and C18 acids. Finally, the esters in the Nerello Mascalese were the ethyl derivatives of fatty acids with 16 and 18 carbon atoms. The anhydride and the free carboxylic acid have been identified, respectively, as 2-n-hexyl-3-methyl maleic anhydride (Fig. 3) present in all cultivars, and palmitic acid present only in the Frappato and Nerello Mascalese and at trace level in the Nero d’Avola. The tentative characterization of the cited anhydride was not so straightforward. In fact, according to the matching procedure using the available database (Wiley and NIST) the best result was represented by the 2-carboxymethyl-3-n-hexyl maleic anhydride (Fig. 3), a natural compound isolated from some strains of an Aspergillus sp. (Weidenmuller et al., 1972), whose molecular weight is 240 instead of 196 determined for the compound in hand. A careful examination of the fragmentations in the mass spectra of the unknown compound, also compared with those of known anhydride, allowed us to establish that the difference was attributed to the presence of a methyl group instead of a carboxymethyl group, the main fragmentations (168, 140, 126, 98 m/z) being common to the two compounds (Fig. 3). Finally, analysis of literature data evidenced that 2-nhexyl-3-methyl maleic anhydride has been characterised among the volatile components of a seedless grape
(Thompson variety) (Buttery et al., 1981), and also detected in the essential oil of the bark of an Australian cupressacea, Callitris intratropica (Collins, 2002). The content of terpenes is quite complex, if compared with that of grape pomaces (Table 1). Here too, the compounds have been grouped in three classes, namely monoterpenes, sesquiterpenes and diterpenes, to render comparison of the cultivars easier. Six monoterpenes have been identified, five of which oxygenated, and only one hydrocarbon: limonene. Nero d’Avola had the highest number (only geraniol was absent) of these components. In the Frappato, linalool, geraniol and limonene were detected, and only linalool and geraniol in the Nerello Mascalese; the monoterpenes content was almost negligible in the Cabernet Sauvignon. The sesquiterpene class was instead represented by 34 components, 24 of which fully characterized. The Nerello Mascalese cv. showed all 34 compounds; the Cabernet Sauvignon sample showed 27 components; the Frappato content was distributed over 22 components. Finally, the Nero d’Avola cv. showed the lowest content of sesquiterpenes, distributed over 20 components, none of which exceeded 1%. Most of identified sesquiterpenes were hydrocarbons (C15H24), the main components being a-ylangene, 3,7guaiadiene, aromadendrene, germacrene D, epizonarene, d-cadinene, a-cadinene and germacrene B (Table 2). Amongst the four oxygenated components, those present in highest amount were epi-a-cadinol, in all cultivars, and
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Table 2 Volatile components of Frappato (Fr), Nerello Mascalese (NM), Nero d’Avola (NA) and Cabernet Sauvignon (CS) stalksa Compound
1 2 3 4 5 6 12 44 57 87 88
Non-terpenoid components Hydrocarbons 3,3-Dimethylpentanec Cyclohexanec 3-Methylhexanec 1,3-Dimethylcyclopentane 1,2-Dimethylcyclopentane Heptanec 2-Methylheptanec Tetradecanec Pentadecanec 9-(Z)-Tricosenec Tricosanec
10 11 16 17 23
Alcohols 3-Methyl-butan-1-olc 2-Methyl-butan-1-olc 3-(E)-Hexen-1-olc Hexanolc 3-Ethyl-4-methyl-pentan-1-ol
14 15 18 19 26 30 33 31 36 38 79
Aldehydes Hexanalc 2-(E)-Hexenalc Heptanalc 2-(E)-Heptenalc Phenylacetaldehydec Nonanalc Decanalc 2-(E)-Nonenalc 2-(E)-Decenalc 2-(E)-4-(Z)-Decadienalc Palmitic aldehyde
7 8 21 22 32 35 44 53 68 80 81 82 85 86
Carboxilyc acid derivatives Ethyl propionatec Propyl acetatec 3-(Z)-Hexenyl acetatec Hexyl acetatec Ethyl octanoatec b-Phenyl-ethyl acetate Ethyl decanoatec 2-Hexyl-3-methyl maleic anhydride Ethyl dodecanoatec Palmitic acidc Ethyl 9-hexadecenoated Ethyl hexadecanoatec Ethyl linoleatec Ethyl linolenatec
KIb
Fr
NA B
NM A
651 664 672 685 691 699 759 1396 1495 2277 2297
58.37 (0.31) 16.19 (0.30)B – – – – – 16.19 (0.30)B – – – t t
71.65 36.85 0.52 12.19 3.04 0.95 1.26 18.14 0.42 0.33 – – –
(0.39) (0.26)A (0.01) (0.07) (0.05) (0.03) (0.07) (0.24)A (0.02) (0.05)
727 730 858 860 1019
0.66 (0.03)D t – t 0.66 (0.03)D –
15.19 6.73 2.52 0.26 4.40 1.28
791 845 896 954 1042 1099 1200 1157 1258 1293 1814
8.22 3.07 1.21 0.43 0.08
(0.22)C (0.05)B (0.03)D (0.03)C (0.01)B
1.85 0.41 0.50 0.36 0.31 –
(0.16)C (0.02)A (0.05)A (0.02)A (0.02)
33.30 – – t 0.12 0.71 – – 14.42 – 16.87 t 0.30 0.49 0.39
(0.20)A
708 710 1005 1012 1197 1254 1393 1480 1603 1965 1970 1991 2158 2165
24 25 27 28 29 34
Terpenes Monoterpenes Limonenec 1,8-Cineolec trans-Linalool oxide cis-Linalool oxide Linaloolc Geraniolc
1027 1030 1071 1086 1096 1252
15.25 1.91 0.18 – – – 1.33 0.40
(0.28)C (0.06)B (0.01)B
37 39 40
Sesquiterpenes Vitispirane Cyclosativenec a-Ylangene
1273 1352 1366
12.77 0.51 0.31 1.23
t
CS D
40.97 1.26 – – – – – – – – 0.42 0.47 0.37
(0.18) (0.02)C
(0.23)B (0.20) (0.01) (0.03)C (0.12)B (0.04)C
5.63 – – 0.76 3.34 1.53
(0.04)C
3.08 (0.02)D 0.83 (0.03)C 1.53 (0.03)C
12.92 3.10 3.89 1.22 0.31 t 2.34 0.28 0.44 0.39 – 0.95
(0.10)B (0.10)B (0.10)B (0.05)B (0.04)A
t – t 0.72 (0.02)D t t t – –
(0.03)B (0.03)B (0.05)A (0.02)A (0.04) (0.13)B
(0.26)D (0.11)A (0.04)A (0.06) (0.01) (0.02) (0.04)B
38.42 1.68 – – – – 0.62 1.06
(0.06)A (0.07)C
(0.05)A (0.03)B
9.94 3.39 0.27 0.48 0.40 1.60 0.64 –
(0.35)C (0.02)A (0.05)B (0.02)C
6.55 (0.16)D 0.44 (0.04)B – 0.82 (0.10)D
34.89 0.38 0.50 1.86
(0.05)A (0.02)B (0.04)A (0.06)B
(0.22)A (0.17)A (0.03)B (0.11)B (0.03)B
(0.04)C (0.01) (0.01) (0.02) (0.04)A (0.04)C (0.04) (0.05)B (0.04)C
(0.02)B (0.02)C (0.03)B
21.16 – – – t – – – 11.36 – 8.44 0.26 0.34 0.32 0.44
(0.02)B (0.02)B
16.53 0.70 0.45 0.30 0.44 0.21 6.04 0.62 5.16 t t 0.66 0.39 0.77 0.79
(0.01) (0.01) (0.02)
(0.03)A (0.03)A (0.01)A (0.03)A
(0.04)B (0.06)B (0.05)B (0.04)AB (0.06)B (0.01)B
(0.02)B (0.03)A
44.72 (0.21)C t t – – – t – t – t t t 16.96 – – 4.40 5.72 6.84
(0.02)A
19.61 5.30 7.36 2.01 – 0.87 4.07 t t t – t
(0.10)A (0.06)A (0.04)A (0.04)A
8.15 t t – – 1.09 – 3.12 3.94 t – – – – –
(0.11)D
(0.11)A (0.10)A (0.03)A
(0.03) (0.07)A
(0.07)A (0.03)A (0.02)D
30.34 (0.25)B t t – – – t t 30.34 (0.25)B – – 3.48 (0.05)A (continued on next page)
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G. Ruberto et al. / Bioresource Technology 99 (2008) 260–268
Table 2 (continued) Compound
KIb
Fr
NA
NM
41 42 46 47 48 49 50 51 52 54 55 56 57 59 60 61 62 63 64 65 66 67 69 70 71 72 73 74 75 76 77
a-Copaenec b-Bourbonene N.I.e MW 204 (189, 161, 133, 105, 91) N.I. MW 204 (161, 133, 119, 105, 91) a-trans-Bergamotene 3,7-Guaiadiene Aromadendrenec Muurola-4(14),5-diene Germacrene-D d-Selinene c-Muurolene Epizonarene a-Muurolene c-Cadinene d-Cadinene a-Cadinene N.I. MW 204 (189, 149, 122, 107, 91) a-Calacorene Germacrene-B Nerolidolc N.I. MW 204 (161, 135, 121) N.I. MW 220 (202, 177, 159, 123, 81) N.I. MW 204 (177, 135, 94, 43) N.I. MW 204 (189, 161, 119, 105, 93) N.I. MW 204 (161, 119, 105, 93) N.I. MW 222 (204, 161, 121, 105, 95) d-Cadinol Epi-a-Cadinol N.I. MW 220 (202, 159, 105, 91) N.I. MW 220 (202, 187, 159, 105, 91) Eudesm-7(11)-en-4-ol
1370 1378 1411 1421 1430 1437 1442 1470 1474 1484 1487 1492 1493 1499 1506 1517 1521 1536 1549 1559 1579 1587 1609 1617 1624 1634 1639 1647 1656 1667 1686
– t t t – 0.86 1.04 – 0.16 t t 1.53 t 1.46 1.20 – 0.84 0.38 0.58 – 0.81 t – – – – 0.99 0.87 – – –
– – t t – 0.34 0.54 – 0.47 t – 0.94 t t 0.95 0.72 0.46 t 0.42 t t – – – – 0.26 – 0.19 – – –
0.17 0.56 0.81 0.54 0.49 1.50 1.35 0.99 3.07 0.90 0.60 1.31 0.53 0.96 1.66 2.27 1.00 0.74 2.05 0.73 0.56 0.58 0.82 0.85 0.52 1.78 0.89 2.23 0.43 0.80 0.46
83 84
Diterpenes Manoyl oxide Neophytadiene
2005 2110
0.57 (0.06)B 0.57 (0.06)B –
– – –
1.85 (0.03)A 0.79 (0.02)A 1.06 (0.02)
t t –
9 13 20 43 79
Others 1,1-Diethoxy ethane N.I. MW 114 (85, 73, 56, 43) 2-Pentylfuranec Biphenyl oxide 6,10,14-Trimethyl-pentadecan-2-onec
725 768 988 1379 1844
0.49 (0.02)C – – 0.49 (0.02)B – –
2.01 (0.06)A 1.18 (0.07) 0.83 (0.02) – – t
0.74 (0.03)B – – 0.39 (0.04)C t 0.35 (0.02)
2.11 (0.02)A t – 1.01 (0.01)A 1.10 (0.02) t
(0.04)C (0.05)C (0.03)D
(0.06)A (0.03)A (0.03)C (0.03)C (0.07)B (0.06)C (0.06)A
(0.19)A (0.02)C
(0.04)D (0.05)D (0.03)C
(0.03)C
(0.01)D (0.03)C (0.02)D (0.02)D
(0.03)C (0.03)D
CS (0.02) (0.04) (0.03)B (0.03) (0.02) (0.01)B (0.04)B (0.01)B (0.05)A (0.07)B (0.01) (0.05)B (0.02) (0.02)B (0.02)B (0.04)A (0.02)B (0.04)A (0.02)B (0.02) (0.02)B (0.03)B (0.02) (0.04) (0.02) (0.06)A (0.01)A (0.02)A (0.03) (0.02) (0.04)
t t 1.14 t t 1.87 1.84 1.29 2.43 1.04 t 1.48 t t 1.81 1.83 1.18 0.74 2.17 – t 3.73 – t t 1.56 1.00 1.75 – – –
(0.03)A
(0.06)A (0.05)A (0.06)A (0.07)B (0.02)A (0.04)A
(0.04)A (0.21)B (0.03)A (0.03)A (0.03)A
(0.10)A
(0.03)B (0.04)A (0.02)B
a The numbering refers to elution order, values (area percent) represent averages of three determinations and standard deviation (±SD) is given in parentheses (t = traces < 0.05%), different capital letter in the same row represents significant difference at P 6 0.01 by Duncan’s multiple range test. b Retention indexes relative to C6–C22 n-alkanes on DB-5 column. c Co-elution with pure component. d Correct isomer not identified. e N.I. = unidentified.
d-cadinol identified in the Frappato, Nerello Mascalese and Cabernet Sauvignon samples (Table 2). Finally, the sole diterpenes were manoyl oxide, a tricyclic ether, in the Nerello Mascalese and Frappato varieties, and, at trace level, in the Cabernet Sauvignon; neophytadiene, a linear hydrocarbon, was present only in the Nerello Mascalese. Also in this case statistically significant differences were found between the average content of most components in the four cultivars by ANOVA and Duncan’s multiple range test (Table 2). Among the non-terpenoid compounds hydrocarbons and carboxilyc acid derivatives showed the
most differentiating contribute, the same held true for sesquiterpenes among the terpene components. A last aspect that has been considered in this study concerned the pH values of the aqueous suspensions of grape pomace and stalks submitted to the SDE process. In fact, in both cases the pH values ranged between 4.20 and 4.40, and it is well known that several kinds of compounds are not stable when heated in aqueous acidic solution giving rise to degradation and/or rearrangement products. In order to establish the role of the acidity of the aqueous suspension of grape pomace and stalks on the chemical profile of volatile components during the distillation
G. Ruberto et al. / Bioresource Technology 99 (2008) 260–268
267
Fig. 3. MS spectra of 2-n-hexyl-3-methyl maleic anhydride (a) and 2-carboxymethyl-3-n-hexyl maleic anhydride (b).
processes, the initial and the final pH, after any SDE process, has been measured, observing that in the case of the grape pomace it decreased by 0.3–0.4 units from the initial value of 4.3, whereas with stalks the final pH (4.4) was unchanged or showed a slight increasing trend (0.1–0.2 units). Successively, we have carried out the SDE process adjusting to 7 the pH values of the acidic aqueous suspensions of grape pomace and stalks. In both cases a decrease (1–2 units) of pH values has been observed however, the GC–FID and GC–MS profiles of the pH 7 adjusted and non-adjusted suspensions, were substantially superimposable, showing that, in the experimental conditions here used, the initial acidity did not affect the volatile composition of both grape pomaces and stalks. In conclusion, it is still premature to establish if some kind of exploitation of these volatile components is feasible, for example in the food field; further studies are necessary, such as the analysis of other varieties also comprising the white cultivars, as well as the application of alternative extraction methodologies. However, knowledge of the aromatic profile of grape pomace seems useful as a further parameter for the control of maceration and successive fermentation steps of the wine-making procedure, as well as establishing a much more finalized use of the grape pomace for producing high quality distillates. Acknowledgements The authors are grateful to Dr. Carmen Parisi for her valuable contribution to this work whilst preparing of her degree thesis. Thanks are also due to Mr. Antonio Giuff-
rida (ISA-CRA, Acireale CT) for his helpful assistance in statistical analyses, to Mr. Sebastiano Nolasco (Oenologist) for his explanations in winemaking procedures and to Mr. Salvatore Cristaldi (ICB-CNR Valverde CT) for his help in collecting and stocking grape pomace samples. This work was financially supported by Consiglio Nazionale delle Ricerche (C.N.R. – Rome, Italy) and Ministero della Pubblica Istruzione, Universita` e Ricerca Scientifica (PRIN 2003, Rome, Italy), University of Catania (Progetti di Ricerca di Ateneo, Catania, Italy). References Adams, R.P., 2001. Identification of Essential Oil Components by Gas Chromatography–Quadrupole Mass Spectroscopy. Allured Publ. Corp., Carol Stream IL. Aznar, M., Lopez, R., Cacho, J.F., Ferreira, V., 2001. Identification and quantification of impact odorants of age red wines from Rijoa. GC– Olfactometry, quantitative GC–MS, and odor evaluation of HPLC fraction. J. Agr. Food Chem. 49, 2829–2924. Bonilla, F., Mayene, M., Merida, J., Mulina, M., 1999. Extraction of phenolic compounds from red grape marc for use as food lipid antioxidant. Food Chem. 66, 209–215. Buttery, R.G., Seifert, R.M., Ling, L.C., Soderstrom, E.I., Yerington, A.P., 1981. Raisin and dried fig volatile components: possible insect attractants. In: ACS Symposium Series No. 170 (Qual. Sel. Fruits Veg. North Am.), Washington, DC, pp. 29–41. Collins, V.J., 2002. Methods of producing essential oils from species of the genus Callitris. Pat. Specif. Australia 2002, application: AU 98-75075 199980708. Flamini, R., 2005. Some advances in the knowledge of grape, wine and distillates chemistry as achieved by mass spectrometry. J. Mass Spectrom. 40, 705–713. Food and Agriculture Organization of United Nations, FAO,.
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Hashizume, K., Samuta, T., 1997. Green odorants of grape stem and their ability to cause a wine stemmy flavour. J. Agr. Food Chem. 45, 1333– 1337. Jennings, W., Shibamoto, T., 1980. Qualitative Analysis of Flavor and Fragrances Volatiles by Capillary Gas Chromatography. Academic Press, New York. Koedman, A., 1987. Some aspect of essential oil preparation. In: Sandra, P., Bicchi, C. (Eds.), Capillary Gas Chromatography in Essential Oil Analysis. Huethig, Heidelberg, pp. 13–27. Laufenberg, G., Kunz, B., Nystroem, M., 2003. Transformation of vegetable waste into value added products: (A) the upgrading concepts; (B) practical implementations. Bioresource Technol. 87, 167–198. Mantell, C., Rodriguez, M., de la Ossa, E.M., 2003. A screening analysis of the high-pressure extraction of anthocyanins from red grape pomace with carbon dioxide and cosolvent. Eng. Life Sci. 3, 38–42. Montgomery, R., 2004. Development of biobased products. Bioresource Technol. 91, 1–29. Murthy, K.N.C., Singh, R.P., Jayaprakasha, G.K., 2002. Antioxidant activities of grape (Vitis vinifera) pomace extracts. J. Agr. Food Chem. 50, 5909–5914.
Park, S.M., Morrison, J.C., Adams, D.O., Noble, A.C., 1991. Distribution of free and glycosidically bound monoterpenes in the skin and mesocarp of Muscat of Alexandria grapes during development. J. Agr. Food Chem. 39, 514–518. Saquet, J.M., Labarbe, B., Le Guerneve`, C., Cheynier, V., Moutounet, M., 2000. Phenolic composition of grape stems. J. Agr. Food Chem. 48, 1076–1080. Schieber, A., Stintzing, F.C., Carle, R., 2001. By-products of plant food processing as a source of functional compounds – recent developments. Trends Food Sci. Technol. 12, 401–413. Silva, M.L., Malcata, F.X., De Revel, G., 1996. Volatile contents of grape marcs in Portugal. J. Food Comp. Anal. 9, 72–80. Torres, J.B., Varela, M.T., Garcia, J., Carilla, C., Matito, J.J., Centelles, M., Cascante, X., Sort, R., Bobet, A., 2002. Valorization of grape (Vitis vinifera) by products. Antioxidant and biological properties of polyphenolic fractions differing in procyanidin composition and flavonol content. J. Agr. Food. Chem. 50, 7548–7555. Weidenmuller, H.-L., Cavagna, F., Fehlhaber, H.-W., Prave, P., 1972. 2Carboxymethyl-3-n-hexyl-maleic acid anhydride, a novel metabolite from an Aspergillus. Tetrahedron Lett., 3519–3522.
Bioresource Technology 99 (2008) 260–268
Volatile components of grape pomaces from different cultivars of Sicilian Vitis vinifera L. Giuseppe Ruberto b
a,*
, Agatino Renda a, Vincenzo Amico b, Corrado Tringali
b
a Istituto del C.N.R. di Chimica Biomolecolare, Via del Santuario 110, I-95028 Valverde, CT, Italy Dipartimento di Scienze Chimiche, Universita` degli Studi di Catania, Viale A. Doria 6, I-95125 Catania, Italy
Received 30 August 2006; received in revised form 22 December 2006; accepted 23 December 2006 Available online 23 February 2007 Dedicated to the memory of Dr. Gianfranco Borin.
Abstract The volatile components of grape pomace coming from the processing of some of the most important varieties of grape (Vitis vinifera L.) cultivated in Sicily, namely Nero d’Avola, Nerello Mascalese, Frappato and Cabernet Sauvignon, have been determined by gas-chromatography (GC) and gas-chromatography–mass spectrometry (GC–MS). According to the winemaking procedure that entails the removal of stalks before fermentation, two kinds of grape pomace are obtained. The first consists of skins, pulp residues and seeds, the proper grape pomace, which is partially used for grappa, a typical Italian spirit, and alcohol production, the second consists almost exclusively of stalks. On the whole, 38 components have been characterized in the samples of grape pomaces, with Frappato cv. showing the richest composition; instead, 88 components have been detected in the stalks of Frappato, Nero d’Avola, Nerello Mascalese and Cabernet Sauvignon varieties. In order to make a comparison between the grape varieties easier, the volatile components detected in the two sets of samples (grape pomaces and stalks) have been grouped in different classes. Significant differences among varieties have been detected and statistical treatment of data is also reported. This study is part of a wider project aimed at the possible exploitation of the main agro-industrial by-products. At the same time it is one of the first reports on the volatile components of this waste material. 2007 Elsevier Ltd. All rights reserved. Keywords: Vitis vinifera L.; Grape pomace; Stalks; Volatile components; GC–MS
1. Introduction Agro-industrial by-products present two rather conflicting aspects: on one hand their disposal represents a great economical and ecological problem, further complicated by legal restrictions, while on the other, they may be considered a promising and renewable source of useful compounds for their technological and nutritional properties (Laufenberg et al., 2003; Montgomery, 2004; Schieber et al., 2001). It is likely that the most immediate and economic use of this material is as feed and as fertilizer, even though some pre-treatments and composting procedures are necessary in *
Corresponding author. Tel.: +39 0957212136; fax: +39 0957212141. E-mail address: [email protected] (G. Ruberto).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.12.025
most cases. It also is true that these wastes are rich in several micronutrients such as carotenoids, polyphenols, tocopherols, vitamins, oligo elements and others, whose beneficial effects on human health are frequently highlighted, therefore this large amount of waste material could be a very cheap source of the aforesaid components. Of course, such potential exploitation would not avoid the disposal of a still substantial amount of waste, but the economic profit should make it more bearable. Grape (Vitis vinifera L.) is one of the world’s largest fruit crops, in excess of 60 million metric tons (www.fao.org), and is mainly grown for wine production. Grape pomace, the main by-product of wine production, consists of skins, seeds and stalks, reaching an estimated amount of 13% by weight of processed grape (Torres et al., 2002). In line with different winemaking procedures,
G. Ruberto et al. / Bioresource Technology 99 (2008) 260–268
two kinds of grape pomaces are obtained after the removal of stalks before maceration and successive fermentation steps. The first consists of skins, pulp residues and seeds, the proper grape pomace, which is partially used for grappa and alcohol production; the second consists almost exclusively of stalks. The chemical composition of grape pomace is rather complex: alcohols, acids, aldehydes, esters, pectins, polyphenols, mineral substances, sugars etc. are the most represented classes of compounds (Bonilla et al., 1999; Mantell et al., 2003; Murthy et al., 2002; Saquet et al., 2000). The evaluation of the qualitative aspects of a grape pomace is carried out in view of the production of high quality grappa; otherwise the grape pomace is used for alcohol distillation, or thrown away. The best grape pomaces are highly rich in vinous liquid, namely not exhaustively pressed, with a moisture degree ranging from 55% to 70%, which allows to exploit the raw material better and to extract the organoleptic characteristics of the native vine. Concerning the pool of the volatile components of a grape pomace which confer its particular aroma, few studies have so far been reported (Park et al., 1991; Silva et al., 1996; Hashizume and Samuta, 1997), if compared with the analogous studies made on wine (Aznar et al., 2001; Flamini, 2005). In our ongoing studies aimed at the possible exploitation of the main agro-industrial by-products obtained in Sicily, we wish to report here the results of a study of the volatile components of grape pomaces and stalks of the most important varieties of grape cultivated in Sicily for wine production, namely Nero d’Avola, Nerello Mascalese, Frappato and Cabernet Sauvignon. 2. Methods 2.1. Plant material Grape pomaces and stalks of Nero d’Avola and Frappato were donated by the ‘‘Valle dell’Acate’’ wine firm, Acate, RG, Italy – those from Nerello Mascalese and Cabernet Sauvignon were given by the ‘‘Emanuele Scammacca Barone del Murgo’’ wine firm, Santa Venerina, CT, Italy. The winemaking procedures were similar for all samples, namely grape clusters were crushed and destemmed using a destemmer-crusher. The crushed grapes were treated with sulphur dioxide (0.2–0.5% total mash) and with selected strains of Saccharomyces cerevisiae to start up the fermentation. After 6–8 days of maceration, when alcoholic fermentation was finished, the mash was pressed. Stalks coming from destemming procedure and grape pomace coming from the maceration procedure were subjected to the distillation procedures within 24 h of their collection. All materials were collected during the 2004 vintage. 2.2. Isolation and analysis of volatile components Fresh grape pomace and stalks (300 g each) were subjected to simultaneous steam distillation-extraction (SDE)
261
for 3 h with a modified Likens–Nickerson apparatus using hexane (1 ml) as the solvent (Koedman, 1987). The SDE procedure was also carried out after adjusting the initial pH of the grape pomace and stalks suspensions to 7, by adding the necessary amount of 2 N NaOH solution. The mixtures were immediately analysed on a Shimadzu gas chromatograph, Model 17-A equipped with a flame ionization detector (FID). Analytical conditions: DB-5 MS capillary column (30 m · 0.25 mm · 0.25 lm), helium as carrier gas. Injection in split mode (1:50), injected volume 1 ll, injector and detector temperature 250 and 280 C, respectively. Linear velocity in column 19 cm/s. The oven temperature was held at 60 C for 6 min, then programmed from 60 to 300 C at 2 C/min. Percentages of compounds were determined from their peak areas in the GC–FID profiles. Gas-chromatography–mass spectrometry (GC–MS) was carried out in the fast mode on a Shimadzu GC–MS mod. GCMS-QP5050A, ionization voltage 70 eV, electron multiplier 900 V, transfer line temperature 280 C. Analytical conditions: SPB-5 capillary column (15 m · 0.10 mm · 0.15 lm), helium as carrier gas. Injection in split mode (1:100), injected volume 1 ll, injector and detector temperature 250 C. Constant linear velocity in column 50 cm/s. The oven temperature was held at 60 C for 1 min, then programmed from 60 to 280 C at 10 C/min. Identification of components was based on GC retention indexes, computer matching with Wiley 275 and NIST (Versions 21 and 75) libraries, comparison of the fragmentation patterns with those reported in the literature and whenever possible, coinjection with authentic samples (Jennings and Shibamoto, 1980; Adams, 2001). Pure standards were purchased from Aldrich Chemical Co., Extrasynthese, France, and Fluka Chemie AG, Switzerland. 2.3. Statistical analysis SPSS software, 14.1 version, was used to carry out statistical analysis of the data. ANOVA and Duncan’s multiple range test were applied to the data to determine significant differences between the analysed volatile components; the model was statistically significant with a value of P 6 0.01. 3. Results and discussion 3.1. Volatile components of grape pomace Table 1 lists the 38 volatile components characterized in all samples of grape pomaces, whereas Fig. 1 shows a typical GC–FID profile. The Frappato cultivar showed the richest composition with 35 compounds; Nerello Mascalese and Nero d’Avola cultivars had a comparable composition with 21 and 19 components, respectively; finally the Cabernet Sauvignon cultivar gave the simplest mixture with only 17 identified components. The most represented class of
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G. Ruberto et al. / Bioresource Technology 99 (2008) 260–268
Table 1 Volatile components of Frappato (Fr), Nerello Mascalese (NM), Nero d’Avola (NA) and Cabernet Sauvignon (CS) grape pomacesa Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
c
Cyclohexane 3-Methylhexanec Heptanec 1,1-Diethoxy ethane 2-Methylbutan-1-olc Octanec 1-Butoxy-1-ethoxy ethane 1-Butanol, 3-methyl acetate 1-Butanol, 2-methyl acetate Nonanec 1,1-Diethoxy-2-methyl butane 1-Ethoxy-1-pentoxy ethane 2-Pentyl furane Ethyl hexanoatec Limonenec Terpinolenec Ethyl octanoatec a-Ylangene Ethyl 9-decenoated Ethyl decanoate 3,7-Guaiadiene Aromadendrenec 3-Methyl butyl octanoate 2-Methyl butyl octanoate c-Muurolene c-Amorphene c-Cadinene Ethyl dodecanoatec 3-Methyl butyl decanoate 2-Methyl butyl decanoate Ethyl tetradecanoatec 3-Methyl butyl dodecanoate Ethyl 9-hexadecenoated Ethyl hexadecanoatec Manoyl oxide Ethyl linoleatec Ethyl 9-octadecenoated Ethyl octadecanoatec
KIb
Fr
NA
NM
CS
647 669 697 724 729 797 868 872 875 897 954 973 989 997 1027 1086 1195 1366 1385 1394 1423 1438 1444 1447 1492 1506 1517 1591 1641 1644 1793 1844 1971 1993 2005 2159 2166 2192
– – t 28.59 – 1.11 0.23 0.48 0.13 0.21 0.10 0.72 0.28 0.67 0.44 0.18 7.46 0.35 0.66 18.83 0.10 0.17 0.63 0.17 0.33 0.28 0.23 10.01 0.54 0.17 1.33 0.14 0.72 7.02 0.28 1.47 1.06 0.37
– – – 6.07 1.19 0.29 – 0.29 – – – 0.23 – 1.08 – – 11.63 – 1.75 29.23 – – 0.68 0.16 – – – 15.38 0.91 – 1.94 0.31 1.16 6.43 0.10 3.05 2.07 0.39
– – – 10.59 1.12 0.52 – t – – – 0.31 – 0.43 – – 8.57 – – 25.55 – – 0.52 – – – – 14.67 0.74 – 3.13 0.31 0.75 11.06 0.40 4.50 2.62 0.55
0.50 0.34 2.24 21.95 0.93 – – – – – – 0.32 – 0.38 – – 3.41 – – 9.87 – – – – – – – 6.01 0.30 – 1.60 – 0.68 7.22 – 2.23 1.54 0.28
(1.15)A (0.14)A (0.07) (0.06)A (0.05) (0.05) (0.02) (0.03)A (0.01) (0.07)B (0.03) (0.01) (0.11)C (0.05) (0.04)B (0.13)C (0.02) (0.02) (0.02)A (0.02)A (0.02) (0.04) (0.04) (0.21)C (0.05)C (0.02) (0.03)D (0.02)B (0.04)B (0.03)B (0.02)B (0.05)D (0.04)D (0.02)B
(0.05)D (0.06)A (0.09)C (0.06)B
(0.02)C (0.02)A
(0.06)A (0.04)A (0.10)A
(0.04)A (0.05)A
(0.08)A (0.02)A (0.05)B (0.02)A (0.04)A (0.04)C (0.02)C (0.04)B (0.04)B (0.02)B
(0.06)C (0.05)A (0.01)B
(0.02)B (0.05)C
(0.12)B
(0.72)B
(0.03)B
(0.07)B (0.06)B (0.03)A (0.04)A (0.04)B (0.19)A (0.05)A (0.04)A (0.11)A (0.04)A
(0.02) (0.02) (0.04) (0.26)B (0.04)B
(0.04)B (0.02)C
(0.05)D
(0.06)D
(0.09)D (0.01)D (0.05)C (0.01)B (0.09)B (0.08)C (0.07)C (0.02)C
a The numbering refers to elution order, values (area percent) represent averages of three determinations and standard deviation (±SD) is given in parentheses (t = traces < 0.05%), different capital letter in the same row represents significant difference at P 6 0.01 by Duncan’s multiple range test. b Retention indexes relative to C6–C22 n-alkanes on DB-5 column. c Co-elution with pure component. d Correct isomer not identified.
components in all four cultivars was that of ethyl esters of aliphatic acids with a linear chain from 6 to 18 carbon atoms, both saturated and unsaturated, with the former predominating. The main components were ethyl octanoate, ethyl decanoate, ethyl dodecanoate and hexyl hexadecanoate. Other common compounds of the four cultivars were represented by some acetals, particularly 1,1-diethoxy ethane, which is the main component of Cabernet Sauvignon and Frappato. As previously mentioned, the grape pomace of the Frappato cultivar stood out from the others, both for the higher number of components and, notably, for the presence of nine terpenes, which account, unlike the previously mentioned components, for a more significant varietal differentiation of the examined cultivars. Two monoterpenes:
limonene and terpinolene – six sesquiterpenes: a-ylangene, 3,7-guaiadiene, aromadendrene, c-muurolene, c-amorphene, c-cadinene – and one diterpene: manoyl oxide have been characterised. However, it should be underlined that the total amount of these compounds is to be considered very low. In the Nero d’Avola and Nerello Mascalese only manoyl oxide was detected, whereas no terpene has been detected in the Cabernet Sauvignon. Statistically significant differences were found between the average content of most components in the four cultivars by ANOVA and Duncan’s multiple range test (Table 1). In particular, the most important components in differentiating samples were, in that order, ethyl octanoate, ethyl dodecanoate, ethyl 9-decenoate, ethyl linoleate, ethyl decanoate, ethyl hexadecanoate and ethyl tetradecanoate.
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Fig. 1. GC–FID profile of volatile components of Nerello Mascalesce grape pomace (for the numbering see Table 1).
3.2. Volatile components of stalks The composition of volatile components isolated from the stalks of Frappato, Nero d’Avola, Nerello Mascalese and Cabernet Sauvignon proved much richer and more complex, as exemplified by the GC–FID profile in Fig. 2. Table 2 lists the 88 components, 77 of which were fully identified. They have been separated in two classes, namely non-terpenoid compounds and terpenes, to make a comparison of the four varieties easier. Unlike the volatile components of the grape pomaces (Table 1), greater amounts of the terpenes were present in the stalks and numerically comparable to non-terpenoid derivatives, the last all of acetogeninic nature. Furthermore, whilst the composition of the grape pomaces showed substantial compositional uniformity, even though with the previously described differences, the composition of the stalks of the four cultivars was quite different from each other. These differences are emphasized if the comparison is carried out throughout specific classes of compounds, in which it is possible to further subdivide the two categories listed in Table 2. With this in mind, hydrocarbons, alcohols, aldehydes, and carboxylic acids and derivatives among the non-terpenoid compounds have been analysed in detail. A substantial difference among the four cultivars was observed with the regards to the hydrocarbons. Nero d’Avola contained the highest number of these components, cyclohexane and heptane being the principal compounds. Heptane was practically the only component of
this class detected in the Frappato. In the Nerello Mascalese variety, only three compounds with relatively high molecular weight, namely pentadecane, 9-(Z)-tricosene and tricosane, have been detected; the level of hydrocarbons was almost insignificant in the Cabernet Sauvignon. Concerning the aliphatic alcohols, again Nero d’Avola showed the most complex composition with the 3-methylbutan-1-ol and hexanol as the main compounds. Cabernet Sauvignon and Nerello Mascalese showed an identical qualitative profile due to 3-(E)-hexen-1-ol, hexanol and 3-ethyl-4-methyl-pentan-1-ol. Almost negligible was the amount of these components in the Frappato variety represented exclusively by hexanol. The qualitative composition of aldehydes was fairly similar in all varieties; however whereas Frappato and Nerello Mascalese cvv. showed detectable presence of 10 aldehydes, in Nero d’Avola and Cabernet Sauvignon many aldehydes were below the detection limit. It should be pointed out that in each cvv. 2-(E)-hexenal, hexanal and nonanal were the main components. The last class of examined compounds concerned the carboxylic acids and their derivatives. The most represented compounds were esters, whereas only one anhydride and one free carboxylic acid have been determined. Among the esters, the Nero d’Avola showed the richest and most complex composition with 11 components, being the bphenylethyl acetate the main one, this compound was absent in the other two varieties. The esters content of the Cabernet Sauvignon was represented exclusively by
264
G. Ruberto et al. / Bioresource Technology 99 (2008) 260–268
Fig. 2. GC–FID profile of volatile components of Nero d’Avola stalks (for the numbering see Table 2).
the ethyl esters of octanoic and decanoic acids, as previously observed for the grape pomace. In the Frappato the esters were represented by hexyl acetate, ethyl octanoate, as well as ethyl esters of C16 and C18 acids. Finally, the esters in the Nerello Mascalese were the ethyl derivatives of fatty acids with 16 and 18 carbon atoms. The anhydride and the free carboxylic acid have been identified, respectively, as 2-n-hexyl-3-methyl maleic anhydride (Fig. 3) present in all cultivars, and palmitic acid present only in the Frappato and Nerello Mascalese and at trace level in the Nero d’Avola. The tentative characterization of the cited anhydride was not so straightforward. In fact, according to the matching procedure using the available database (Wiley and NIST) the best result was represented by the 2-carboxymethyl-3-n-hexyl maleic anhydride (Fig. 3), a natural compound isolated from some strains of an Aspergillus sp. (Weidenmuller et al., 1972), whose molecular weight is 240 instead of 196 determined for the compound in hand. A careful examination of the fragmentations in the mass spectra of the unknown compound, also compared with those of known anhydride, allowed us to establish that the difference was attributed to the presence of a methyl group instead of a carboxymethyl group, the main fragmentations (168, 140, 126, 98 m/z) being common to the two compounds (Fig. 3). Finally, analysis of literature data evidenced that 2-nhexyl-3-methyl maleic anhydride has been characterised among the volatile components of a seedless grape
(Thompson variety) (Buttery et al., 1981), and also detected in the essential oil of the bark of an Australian cupressacea, Callitris intratropica (Collins, 2002). The content of terpenes is quite complex, if compared with that of grape pomaces (Table 1). Here too, the compounds have been grouped in three classes, namely monoterpenes, sesquiterpenes and diterpenes, to render comparison of the cultivars easier. Six monoterpenes have been identified, five of which oxygenated, and only one hydrocarbon: limonene. Nero d’Avola had the highest number (only geraniol was absent) of these components. In the Frappato, linalool, geraniol and limonene were detected, and only linalool and geraniol in the Nerello Mascalese; the monoterpenes content was almost negligible in the Cabernet Sauvignon. The sesquiterpene class was instead represented by 34 components, 24 of which fully characterized. The Nerello Mascalese cv. showed all 34 compounds; the Cabernet Sauvignon sample showed 27 components; the Frappato content was distributed over 22 components. Finally, the Nero d’Avola cv. showed the lowest content of sesquiterpenes, distributed over 20 components, none of which exceeded 1%. Most of identified sesquiterpenes were hydrocarbons (C15H24), the main components being a-ylangene, 3,7guaiadiene, aromadendrene, germacrene D, epizonarene, d-cadinene, a-cadinene and germacrene B (Table 2). Amongst the four oxygenated components, those present in highest amount were epi-a-cadinol, in all cultivars, and
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Table 2 Volatile components of Frappato (Fr), Nerello Mascalese (NM), Nero d’Avola (NA) and Cabernet Sauvignon (CS) stalksa Compound
1 2 3 4 5 6 12 44 57 87 88
Non-terpenoid components Hydrocarbons 3,3-Dimethylpentanec Cyclohexanec 3-Methylhexanec 1,3-Dimethylcyclopentane 1,2-Dimethylcyclopentane Heptanec 2-Methylheptanec Tetradecanec Pentadecanec 9-(Z)-Tricosenec Tricosanec
10 11 16 17 23
Alcohols 3-Methyl-butan-1-olc 2-Methyl-butan-1-olc 3-(E)-Hexen-1-olc Hexanolc 3-Ethyl-4-methyl-pentan-1-ol
14 15 18 19 26 30 33 31 36 38 79
Aldehydes Hexanalc 2-(E)-Hexenalc Heptanalc 2-(E)-Heptenalc Phenylacetaldehydec Nonanalc Decanalc 2-(E)-Nonenalc 2-(E)-Decenalc 2-(E)-4-(Z)-Decadienalc Palmitic aldehyde
7 8 21 22 32 35 44 53 68 80 81 82 85 86
Carboxilyc acid derivatives Ethyl propionatec Propyl acetatec 3-(Z)-Hexenyl acetatec Hexyl acetatec Ethyl octanoatec b-Phenyl-ethyl acetate Ethyl decanoatec 2-Hexyl-3-methyl maleic anhydride Ethyl dodecanoatec Palmitic acidc Ethyl 9-hexadecenoated Ethyl hexadecanoatec Ethyl linoleatec Ethyl linolenatec
KIb
Fr
NA B
NM A
651 664 672 685 691 699 759 1396 1495 2277 2297
58.37 (0.31) 16.19 (0.30)B – – – – – 16.19 (0.30)B – – – t t
71.65 36.85 0.52 12.19 3.04 0.95 1.26 18.14 0.42 0.33 – – –
(0.39) (0.26)A (0.01) (0.07) (0.05) (0.03) (0.07) (0.24)A (0.02) (0.05)
727 730 858 860 1019
0.66 (0.03)D t – t 0.66 (0.03)D –
15.19 6.73 2.52 0.26 4.40 1.28
791 845 896 954 1042 1099 1200 1157 1258 1293 1814
8.22 3.07 1.21 0.43 0.08
(0.22)C (0.05)B (0.03)D (0.03)C (0.01)B
1.85 0.41 0.50 0.36 0.31 –
(0.16)C (0.02)A (0.05)A (0.02)A (0.02)
33.30 – – t 0.12 0.71 – – 14.42 – 16.87 t 0.30 0.49 0.39
(0.20)A
708 710 1005 1012 1197 1254 1393 1480 1603 1965 1970 1991 2158 2165
24 25 27 28 29 34
Terpenes Monoterpenes Limonenec 1,8-Cineolec trans-Linalool oxide cis-Linalool oxide Linaloolc Geraniolc
1027 1030 1071 1086 1096 1252
15.25 1.91 0.18 – – – 1.33 0.40
(0.28)C (0.06)B (0.01)B
37 39 40
Sesquiterpenes Vitispirane Cyclosativenec a-Ylangene
1273 1352 1366
12.77 0.51 0.31 1.23
t
CS D
40.97 1.26 – – – – – – – – 0.42 0.47 0.37
(0.18) (0.02)C
(0.23)B (0.20) (0.01) (0.03)C (0.12)B (0.04)C
5.63 – – 0.76 3.34 1.53
(0.04)C
3.08 (0.02)D 0.83 (0.03)C 1.53 (0.03)C
12.92 3.10 3.89 1.22 0.31 t 2.34 0.28 0.44 0.39 – 0.95
(0.10)B (0.10)B (0.10)B (0.05)B (0.04)A
t – t 0.72 (0.02)D t t t – –
(0.03)B (0.03)B (0.05)A (0.02)A (0.04) (0.13)B
(0.26)D (0.11)A (0.04)A (0.06) (0.01) (0.02) (0.04)B
38.42 1.68 – – – – 0.62 1.06
(0.06)A (0.07)C
(0.05)A (0.03)B
9.94 3.39 0.27 0.48 0.40 1.60 0.64 –
(0.35)C (0.02)A (0.05)B (0.02)C
6.55 (0.16)D 0.44 (0.04)B – 0.82 (0.10)D
34.89 0.38 0.50 1.86
(0.05)A (0.02)B (0.04)A (0.06)B
(0.22)A (0.17)A (0.03)B (0.11)B (0.03)B
(0.04)C (0.01) (0.01) (0.02) (0.04)A (0.04)C (0.04) (0.05)B (0.04)C
(0.02)B (0.02)C (0.03)B
21.16 – – – t – – – 11.36 – 8.44 0.26 0.34 0.32 0.44
(0.02)B (0.02)B
16.53 0.70 0.45 0.30 0.44 0.21 6.04 0.62 5.16 t t 0.66 0.39 0.77 0.79
(0.01) (0.01) (0.02)
(0.03)A (0.03)A (0.01)A (0.03)A
(0.04)B (0.06)B (0.05)B (0.04)AB (0.06)B (0.01)B
(0.02)B (0.03)A
44.72 (0.21)C t t – – – t – t – t t t 16.96 – – 4.40 5.72 6.84
(0.02)A
19.61 5.30 7.36 2.01 – 0.87 4.07 t t t – t
(0.10)A (0.06)A (0.04)A (0.04)A
8.15 t t – – 1.09 – 3.12 3.94 t – – – – –
(0.11)D
(0.11)A (0.10)A (0.03)A
(0.03) (0.07)A
(0.07)A (0.03)A (0.02)D
30.34 (0.25)B t t – – – t t 30.34 (0.25)B – – 3.48 (0.05)A (continued on next page)
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Table 2 (continued) Compound
KIb
Fr
NA
NM
41 42 46 47 48 49 50 51 52 54 55 56 57 59 60 61 62 63 64 65 66 67 69 70 71 72 73 74 75 76 77
a-Copaenec b-Bourbonene N.I.e MW 204 (189, 161, 133, 105, 91) N.I. MW 204 (161, 133, 119, 105, 91) a-trans-Bergamotene 3,7-Guaiadiene Aromadendrenec Muurola-4(14),5-diene Germacrene-D d-Selinene c-Muurolene Epizonarene a-Muurolene c-Cadinene d-Cadinene a-Cadinene N.I. MW 204 (189, 149, 122, 107, 91) a-Calacorene Germacrene-B Nerolidolc N.I. MW 204 (161, 135, 121) N.I. MW 220 (202, 177, 159, 123, 81) N.I. MW 204 (177, 135, 94, 43) N.I. MW 204 (189, 161, 119, 105, 93) N.I. MW 204 (161, 119, 105, 93) N.I. MW 222 (204, 161, 121, 105, 95) d-Cadinol Epi-a-Cadinol N.I. MW 220 (202, 159, 105, 91) N.I. MW 220 (202, 187, 159, 105, 91) Eudesm-7(11)-en-4-ol
1370 1378 1411 1421 1430 1437 1442 1470 1474 1484 1487 1492 1493 1499 1506 1517 1521 1536 1549 1559 1579 1587 1609 1617 1624 1634 1639 1647 1656 1667 1686
– t t t – 0.86 1.04 – 0.16 t t 1.53 t 1.46 1.20 – 0.84 0.38 0.58 – 0.81 t – – – – 0.99 0.87 – – –
– – t t – 0.34 0.54 – 0.47 t – 0.94 t t 0.95 0.72 0.46 t 0.42 t t – – – – 0.26 – 0.19 – – –
0.17 0.56 0.81 0.54 0.49 1.50 1.35 0.99 3.07 0.90 0.60 1.31 0.53 0.96 1.66 2.27 1.00 0.74 2.05 0.73 0.56 0.58 0.82 0.85 0.52 1.78 0.89 2.23 0.43 0.80 0.46
83 84
Diterpenes Manoyl oxide Neophytadiene
2005 2110
0.57 (0.06)B 0.57 (0.06)B –
– – –
1.85 (0.03)A 0.79 (0.02)A 1.06 (0.02)
t t –
9 13 20 43 79
Others 1,1-Diethoxy ethane N.I. MW 114 (85, 73, 56, 43) 2-Pentylfuranec Biphenyl oxide 6,10,14-Trimethyl-pentadecan-2-onec
725 768 988 1379 1844
0.49 (0.02)C – – 0.49 (0.02)B – –
2.01 (0.06)A 1.18 (0.07) 0.83 (0.02) – – t
0.74 (0.03)B – – 0.39 (0.04)C t 0.35 (0.02)
2.11 (0.02)A t – 1.01 (0.01)A 1.10 (0.02) t
(0.04)C (0.05)C (0.03)D
(0.06)A (0.03)A (0.03)C (0.03)C (0.07)B (0.06)C (0.06)A
(0.19)A (0.02)C
(0.04)D (0.05)D (0.03)C
(0.03)C
(0.01)D (0.03)C (0.02)D (0.02)D
(0.03)C (0.03)D
CS (0.02) (0.04) (0.03)B (0.03) (0.02) (0.01)B (0.04)B (0.01)B (0.05)A (0.07)B (0.01) (0.05)B (0.02) (0.02)B (0.02)B (0.04)A (0.02)B (0.04)A (0.02)B (0.02) (0.02)B (0.03)B (0.02) (0.04) (0.02) (0.06)A (0.01)A (0.02)A (0.03) (0.02) (0.04)
t t 1.14 t t 1.87 1.84 1.29 2.43 1.04 t 1.48 t t 1.81 1.83 1.18 0.74 2.17 – t 3.73 – t t 1.56 1.00 1.75 – – –
(0.03)A
(0.06)A (0.05)A (0.06)A (0.07)B (0.02)A (0.04)A
(0.04)A (0.21)B (0.03)A (0.03)A (0.03)A
(0.10)A
(0.03)B (0.04)A (0.02)B
a The numbering refers to elution order, values (area percent) represent averages of three determinations and standard deviation (±SD) is given in parentheses (t = traces < 0.05%), different capital letter in the same row represents significant difference at P 6 0.01 by Duncan’s multiple range test. b Retention indexes relative to C6–C22 n-alkanes on DB-5 column. c Co-elution with pure component. d Correct isomer not identified. e N.I. = unidentified.
d-cadinol identified in the Frappato, Nerello Mascalese and Cabernet Sauvignon samples (Table 2). Finally, the sole diterpenes were manoyl oxide, a tricyclic ether, in the Nerello Mascalese and Frappato varieties, and, at trace level, in the Cabernet Sauvignon; neophytadiene, a linear hydrocarbon, was present only in the Nerello Mascalese. Also in this case statistically significant differences were found between the average content of most components in the four cultivars by ANOVA and Duncan’s multiple range test (Table 2). Among the non-terpenoid compounds hydrocarbons and carboxilyc acid derivatives showed the
most differentiating contribute, the same held true for sesquiterpenes among the terpene components. A last aspect that has been considered in this study concerned the pH values of the aqueous suspensions of grape pomace and stalks submitted to the SDE process. In fact, in both cases the pH values ranged between 4.20 and 4.40, and it is well known that several kinds of compounds are not stable when heated in aqueous acidic solution giving rise to degradation and/or rearrangement products. In order to establish the role of the acidity of the aqueous suspension of grape pomace and stalks on the chemical profile of volatile components during the distillation
G. Ruberto et al. / Bioresource Technology 99 (2008) 260–268
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Fig. 3. MS spectra of 2-n-hexyl-3-methyl maleic anhydride (a) and 2-carboxymethyl-3-n-hexyl maleic anhydride (b).
processes, the initial and the final pH, after any SDE process, has been measured, observing that in the case of the grape pomace it decreased by 0.3–0.4 units from the initial value of 4.3, whereas with stalks the final pH (4.4) was unchanged or showed a slight increasing trend (0.1–0.2 units). Successively, we have carried out the SDE process adjusting to 7 the pH values of the acidic aqueous suspensions of grape pomace and stalks. In both cases a decrease (1–2 units) of pH values has been observed however, the GC–FID and GC–MS profiles of the pH 7 adjusted and non-adjusted suspensions, were substantially superimposable, showing that, in the experimental conditions here used, the initial acidity did not affect the volatile composition of both grape pomaces and stalks. In conclusion, it is still premature to establish if some kind of exploitation of these volatile components is feasible, for example in the food field; further studies are necessary, such as the analysis of other varieties also comprising the white cultivars, as well as the application of alternative extraction methodologies. However, knowledge of the aromatic profile of grape pomace seems useful as a further parameter for the control of maceration and successive fermentation steps of the wine-making procedure, as well as establishing a much more finalized use of the grape pomace for producing high quality distillates. Acknowledgements The authors are grateful to Dr. Carmen Parisi for her valuable contribution to this work whilst preparing of her degree thesis. Thanks are also due to Mr. Antonio Giuff-
rida (ISA-CRA, Acireale CT) for his helpful assistance in statistical analyses, to Mr. Sebastiano Nolasco (Oenologist) for his explanations in winemaking procedures and to Mr. Salvatore Cristaldi (ICB-CNR Valverde CT) for his help in collecting and stocking grape pomace samples. This work was financially supported by Consiglio Nazionale delle Ricerche (C.N.R. – Rome, Italy) and Ministero della Pubblica Istruzione, Universita` e Ricerca Scientifica (PRIN 2003, Rome, Italy), University of Catania (Progetti di Ricerca di Ateneo, Catania, Italy). References Adams, R.P., 2001. Identification of Essential Oil Components by Gas Chromatography–Quadrupole Mass Spectroscopy. Allured Publ. Corp., Carol Stream IL. Aznar, M., Lopez, R., Cacho, J.F., Ferreira, V., 2001. Identification and quantification of impact odorants of age red wines from Rijoa. GC– Olfactometry, quantitative GC–MS, and odor evaluation of HPLC fraction. J. Agr. Food Chem. 49, 2829–2924. Bonilla, F., Mayene, M., Merida, J., Mulina, M., 1999. Extraction of phenolic compounds from red grape marc for use as food lipid antioxidant. Food Chem. 66, 209–215. Buttery, R.G., Seifert, R.M., Ling, L.C., Soderstrom, E.I., Yerington, A.P., 1981. Raisin and dried fig volatile components: possible insect attractants. In: ACS Symposium Series No. 170 (Qual. Sel. Fruits Veg. North Am.), Washington, DC, pp. 29–41. Collins, V.J., 2002. Methods of producing essential oils from species of the genus Callitris. Pat. Specif. Australia 2002, application: AU 98-75075 199980708. Flamini, R., 2005. Some advances in the knowledge of grape, wine and distillates chemistry as achieved by mass spectrometry. J. Mass Spectrom. 40, 705–713. Food and Agriculture Organization of United Nations, FAO,
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