Journal of Food Engineering 78 (2007) 793801 www.elsevier.

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Antioxidants from grape stalks and marc: Inuence of extraction procedure on yield, purity and antioxidant power of the extracts
Giorgia Spigno *, Dante Marco De Faveri
` Institute of Oenology and Food Engineering, Universita Cattolica Sacro Cuore, Via Emilia Parmense, 84, 29100 Piacenza, Italy Received 2 May 2005; accepted 17 November 2005 Available online 6 January 2006

Abstract This study was aimed to assess the feasibility of extracting antioxidant compounds from wine-making wastes (grape stalks and marc) by solvent extraction. Together with the type of raw material it was also investigated the inuence of some process parameters on nal antioxidants yields and extract purity: a degreasing pre-treatment, type of solvent (ethanol or a mixture ethylacetate:water/9:1), temperature (28 or 60 C) and length of maceration (5 or 24 h). Solvent and temperature were statistically inuent (p < 0.05), and the yields were higher with ethanol (but with lower purities) and at 60 C. Antioxidant power of the extracts was evaluated by dierent analytical methods. An accurate comparison of our data with many literature works about antioxidants recovery from dierent natural sources showed similar results and highlighted a general great variability in the extraction procedures. 2005 Elsevier Ltd. All rights reserved.
Keywords: Antioxidants; By-products; Grape; Marc; Polyphenols; Solvent extraction; Stalks

1. Introduction In the last few years diminishing the environmental impact of industrial wastes has been a subject of increasing concern. The adverse impact of many food industries is due to the presence in their wastes of residual phenols from the plant raw material they use. In industrial wastewaters, these compounds considerably increase biochemical and chemical oxygen demands, with detrimental eects on the ora and fauna of discharge zones, while in solid residues used as fertilizers, they may inhibit germination properties. Grapes are one of the worlds largest fruit crops, and even wine-making wastes such as marc (the residue after pressing for white wines or vinication for red wines) and stalks, are rich in phenols. Despite the above described pollutant character, grapes, wine, grape seeds and skins

Corresponding author. Tel.: +39 0523599181; fax: +39 0523 599232. E-mail address: giorgia.spigno@unicatt.it (G. Spigno).

extracts are reported to exert favourable eects on human health due to their phenolic content (Khanna et al., 2002; Shrikhande, 2000; van de Wiel, van Golde, & Hart, 2001): such as protection against cardiovascular disease, anti-inammatory activity and anti-carcinogenic eects. Furthermore, the activity of these compounds as food lipid antioxidants is well known. The addition of antioxidants is a method of increasing the shelf life, especially of fats, oil and fat containing food products. Since synthetic antioxidants, such as BHA and BHT have restricted use in foods due to their toxicological eects on various species and suspected carcinogenic potential, the search of natural and safe antioxidants, especially of plant origin, has greatly increased in recent years. Literature is so rich of examples of recovery of antioxidant compounds from natural sources, that it is not possible to report here a complete list of them. This abundance of literature is due to the fact that antioxidants are naturally present in many foods, so that they can be seen as potential sources of natural antioxidants: oilseeds, nuts, cereals, legumes, vegetables, fruits,

0260-8774/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2005.11.020

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herbs, spices and teas (Pokorny, Yanishlieva, & Gordon, 2001). Almost all the found references use solvent extraction for antioxidants recovery, even though supercritical uid extraction (Dapkevicius, Venskutonis, van Beek, & Linssen, 1998; Hadolin, Rizner Hras, Bauman, & Knez, 2004; Luengthanaphol et al., 2004) and ultrasonically assisted solvent extraction (Vinatoru, 2001) have been proposed. Traditionally, these natural antioxidant extracts are thought for a medical use, but since there are still many uncertainties about their eective bioavailability and metabolism (Astley, 2003; Hollman, 2001), it appears really interesting and promising their application in food systems. Some studies have investigated the addition of natural antioxidants to vegetables oils to inhibit oxidation during stor age (Bandoniene, Pukalskas, Venskutonis, & Gruzdiene, 2000; Bera, Lahiri, & Nag, 2004; Reahman, Habib, & Shah, 2004; Suja, Abraham, Thamizh, Jayalekshmy, & Arumughan, 2004) or during frying (Lalas & Dourtoglou, 2003; Lee, Kim, Park, & Choe, 2003). Natural antioxidants have also been added to inhibit lipid oxidation in meat (Barretto, Ida, Silva, Torres, & Shimokomaki, 2003; Sanchez` Escalante, Djenane, Torrescano, Beltran, & Roncales, 2003), biscuits (Reddy, Urooj, & Kumar, 2005) or to preserve fresh beef meat colour by incorporating them into packaging polyethylene lms (Moore et al., 2003). Then, phenolic compounds can be considered as high added-value by-products and the employment of low-cost industrial wastes could greatly reduce the production costs and increase the margin prot of the products. However, most of the cited references started from fresh raw material, rather than from wastes, as is the case for works about recovery of phenolic compounds from grape which utilize directly whole grape (Alcolea, Cano, Acosta, & Arnao, 2002; Revilla, Ryan, & Martin-Ortega, 1998), grape seeds or skins (Ahn et al., 2002; Baydar, Ozkan, & Sagdic, 2004; Jayaprakasha, Singh, & Sakariah, 2001; Palma & Taylor, 1999; Pirniyazov, Abdulladzhanova, Mavlyanov, Kamaev, & Dalimov, 2003; Santos-Buelga, Francia Aricha, & Escribano-Bailon, 1995; Souquet, Cheynier, Brossaud, & Moutounet, 1996). We have found only a few papers relating to the use of grape marc (Bonilla, Mayen, Merida, & Medina, 1999; Cruz, Domnguez, & Parajo, 2004; Gonzales-Paramas, Esteban-Ruano, Santos-Buelga, & Rivas-Gonzalo, 2004; Jayaprakasha, Selvi, & Sakariah, 2003; Lapornik, Prosek, & Wondra, 2005; Negro, Tommasi, & Miceli, 2003; Pekic, Kovac, Alonso, & Revilla, 1998; Torres et al., 2002), and the phenolic composition of grape stems (Delaunay, ` Castagnino, Cheze, & Vercauteren, 2002; Souquet, Labarbe, Le Guerneve, Cheynier, & Moutounet, 2000). As reported in Table 1, almost all the cited works used a solvent-extraction procedure, with the exception of Palma and Taylor (1999) who applied near critical CO2. The procedures are dierent and the choice of operating parameters is not generally motivated, even because many

researches were simply aimed to analyse the polyphenolic content. Only a few works have tried to optimise some process parameters. The most investigated factor is the type of solvent (Baydar et al., 2004; Pekic et al., 1998; Revilla et al., 1998; Jayaprakasha et al., 2001, 2003). Bonilla et al. (1999) investigated the inuence of crushing the grape marc and of extraction time on polyphenols yield; Gonzales-Paramas et al. (2004) evaluated the inuence of alcohol extraction and drying on residual polyphenols content and antioxidant activity in grape pomace and seeds; Lapornik et al. (2005) tried to optimise the extraction of polyphenols from grape marc using dierent solvents and times. Generally, we have noticed that literature lacks of systematic approaches to optimise the process in order to maximise the extract yield, purity and antioxidant power of the obtained extracts. Therefore, the aim of the present work was to: Investigate extraction of antioxidants from two winemaking wastes, marc and stems. Unlike marc, stalks have never been studied for this purpose. For better comparison marc and stalks were collected from the same grape variety and vintage. Simplify the extraction procedure. On the basis of published researches, we selected a solvent extraction method and studied the inuence of some process parameters on the nal extract yield and purity: degreasing pre-treatment of the material, extraction solvent, time and temperature. Summarise the state of the art about extraction of antioxidants from natural sources (in particular grape and grape by-product) in order to highlight the common used procedures and the obtained extract yields and purities.

2. Materials and methods 2.1. Materials Stalks and marc (by Barbera red grape) were kindly provided by a wine-making factory in Piacenza (northern Italy) in the vintage 2003. Stalks were collected after the operation of pressing/destemming, while marc was collected after devatting. Both the materials were oven dried at 60 C up to a moisture content of about 24% (determined by dry weight in oven at 105 C until constant weight) and milled in a commercial Moulinex mixer (Moulinette S) to obtain a ne powder. All chemicals were of analytical grade. b-carotene, linoleic acid BHA and Tween-40 were supplied by Fluka. 2.2. Solvent extraction We investigated the inuence of four process parameters (each one made varying on two levels) on extraction from both stalks and marc:

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Table 1 Comparison of dierent literature works on antioxidants extraction from natural sources (NR = not reported; EA = ethylacetate; MetOH = methanol; EtOH = ethanol; fw = on fresh weight) Raw material Red grape marc Extraction procedure 50 g powder (degreased or not), at 28/60 C, for 5/24 h, with 200 ml: EtOH EA:water (9:1) EA:water (9:1) 50 g degreased powder, 5 h at 60/70 C, twice with 150 ml EA:water (9:1) EA:water (17:3) EA:water (4:1) Polyphenols yield (%) (GAE) 0.27 0.15 0.33 (Catechins) 0.86 1.35 1.56 Red grape seeds White grape seeds Fresh grape seeds 100 g degreased powder, 8 h with 150 ml hot (T NR) Acetone:water:acetic acid (90:9.5:0.5) 10 g, 8 h with 40 ml EA:water (9:1), T NR 100 g degreased powder/200 ml acetone:water: acetic acid (90:9.5:0.5), 60 C, 8 h or EA:MetOH:water (60:30:10) 240 mg crushed seeds/10 ml MetOH:water (4:1), 16 h, Troom 1 h, T < 30 C, with ultrasonication 30 mg and near critical CO2 (Catechins) 2.5 (Catechins) 0.5 (GAE) 9.6 10.8 (Catechins) 0.066 0.063 0.078 (tannins) (GAE) 0.25 0.53 Ahn et al. (2002) Revilla et al. (1998) 43 54.00 50.5 46 47 Baydar et al. (2004) 62.8 66.8 Palma and Taylor (1999) Jayaprakasha et al. (2003) Pekic et al. (1998) Extract purity (%) 6.00 9.01 22.34 Jayaprakasha et al. (2001) References Present work

Red grape stems Red grape seeds

Fresh grape seeds

Grape seeds Fresh red grape skins

100 g powder with 100 ml 100% EtOH, 24 h Troom Skins of 100 grapes/60 ml solvent: MetOH 16 h/ 25 C + 80% MetOH 4 h/Troom + 50% MetOH 4 h/Troom + deionised water 16 h/ 25 C + 75% acetone 1 h/Troom MetOH/12 N HCl (99:1) 4 h/25 C + MetOH/12 N HCl (99:1) 12 h/25 C + MetOH/12 N HCl (99:1) 4 h/25 C 100 grapes/60 ml solvent (as above) 250 g (50 g d.m.)/1 L EA:water (1:1) 530 , T NR 1 g/3 mL 80% EtOH with 0.5% 0.1 N HCl, T and time NR 50 g/100 ml water or 70% EtOH or 70% MetOH, 11224 h, Troom 5 g freeze dried + NRml MetOH 75%, 15 0 ultrasonic bath (TNR), twice. Liquid concentrated, water dissolved and extracted 3 times with EA 2 g freeze-dried for seeds 1 kg washed and dried/8 kg solvent in autoclave: Water, 4% H2SO4, 130 C, 90 0 Water, 100 C 5 h Water, 130 C, 90 0 Followed always by EA extraction of liquors and freeze-drying 1 kg fresh extracted by reux with boiling MetOH or water (3 L) 1 h. Freeze drying. Purication by Amberlite resin and freeze-drying 60% EtOH, 60 h, 20 C 80% EtOH, 8 min, 20 C
0

0.24

Fresh grapes Red grape marc Red grape marc Seeds from marc Skins from marc Grape marc

0.430.51 0.11 (HPLC) (GAE) 0.59 2.4 0.37 (GAE) 0.0351.36 0.06 0.080.14 0.15 0.150.18 (GAE) 0.20 0.10 0.22 0.011 0.14 0.28 0.11 Bonilla et al. (1999) Negro et al. (2003)

Lapornik et al. (2005) avan-3ols Gonzales-Paramas et al. (2004)

Grape marc Distilled marc Grape seeds Distilled seeds Distilled grape marc

Cruz et al. (2004) 12.5 13.3 18.3

Lettuce, Chicory byproducts Black Currants

Tot phenols 0.16 (fw) 0.015 3.6793.956 3.553.802

Llorach et al. (2004) 5.02 15.20 (HPLC) Cacace and Mazza (2003) (continued on next page)

796 Table 1 (continued) Raw material Cherries (dierent parts) Aromatic herbs Fennel seeds Rosehips Grapefruits Flesh Skin Aromatic herbs Red beans Boldo leaves

G. Spigno, D.M. De Faveri / Journal of Food Engineering 78 (2007) 793801

Extraction procedure 10 g powder/20 ml Acetone, ultrasound 10 0 ; 10 ml acetone:water (7:3) twice, 80 ml Chloroform, T NR 15 g powder/900 ml acetone, 6 h in Soxhlet apparatus 25 g/500 ml boiling water 15 0 25 g/100 ml ethanol 5 times 100 mg freeze-dried powder/2 ml EtOH 50%, 4 C, 24 h 50 mg freeze-dried/5 ml 80% MetOH/water, 90 C, 3 h 5 ml 1.2 M HCl in 80% MetOH/water, 90 C, 3 h 610 g milled/50 ml (twice) MetOH 99.5%, 2 h, T NR 100 g powder/500 ml 50% EtOH, 75 C 3 h. Filtration and freeze drying. 20 g degreased powder/MetOH (in Soxhlet), dried, extracted with water/60 C/48 h; aqueous phase extracted with CHCl3 and EA 20 g frozen berries/20 ml MetOH:HCl 2% (95:5 v/v), 60 min (twice) 25 g liquid nitrogen powdered sample/25 ml Acetone: water (7:3) + acetone: chloroform (1:2)

Polyphenols yield (%)

Extract purity (%) (GAE) 0.050.5 1.864.77 13.09 82.19 612 (fw) 0.130.17 0.150.26

References (Chaovanalikit & Wrolstad, 2004) Bandoniene et al. (2000) Oktay et al. (2003) Gao et al. (2000) Gorinstein et al. (2004)

(GAE) 2.12 (GAE) 9.0 (GAE)

(GAE) (GAE) (avonoids) 1.4 0.140.88

0.43.8 1.6

Miliauskas et al., 2004 (Chou, Chao, & Chung, 2003) Quezada et al. (2004)

Black berries Blackberry Whole Only pulp Only seeds

(GAE/fresh weight) (GAE)

Benvenuti et al. (2004) Siriwoharn and Wrolstad (2004)

0.820.84 0.800.96 1.461.75

(1) a degreasing pre-treatment in a Soxhlet apparatus with hexane (as suggested by Jayaprakasha et al., 2001) for 6 h in the case of stalks, and up to 24 h in the case of marc (levels: yes or no); (2) solvent: ethanol or ethyl acetate:water/9:1; (3) extraction time: 5 or 24 h; (4) extraction temperature: 28 C or 60 C. All the 32 resulting combinations were performed in triplicates. The mixture ethylacetate:water was selected since being reported as one of the best solvent for extrac tion of polyphenols from grape seeds (Pekic et al., 1998), because it preferentially extracts those phenols that are readily dissolved in the lipid fraction of the food, because its low boiling point facilitates its removal and reuse and, nally, because any possible residue is scarcely toxic since at levels around mg/l it is a typical component of fermented drinks (Bonilla et al., 1999). Ethanol was chosen since alcohols are the most used solvents in antioxidants extraction works and, furthermore, it is the natural solvent of these compounds in the wine-making process. 50 g of sample (degreased or not) were extracted with 200 ml of solvent in a shaking incubator at the temperature and for the time established. The extracts were centrifuged (at 5000 rpm for 5 0 ), dried over anhydrous Na2SO4, and solvent was removed by evaporation under reduced pressure at 40 C. Petroleum ether was added to the concentrated (in ratio 5:1) to form a precipitate which was separated by centrifu gation (8000 rpm for 5 0 ) (Pekic et al., 1998; Pirniyazov

et al., 2003). The residue (crude extract) was dissolved in ethanol for freeze-storage and analysis. 2.3. Extracts analysis The total phenols content was evaluated by determining ` the absorbance at 280 nm (Ribereau-Gayon, 1970) and expressed as equivalent of gallic acid (GAE) by construction of a calibration curve. GAE percent yield was calculated as (g GAE/100 g dry starting material). The purity of the extracts was determined as the percent amount of GAE in the crude extract weighed after precipitation with petroleum ether and centrifugation. The antioxidant activity of the extracts was evaluated in a b-carotene-linoleate system (Jayaprakasha et al., 2001). 0.2 mg of b-carotene, 20 mg of linoleic acid and 200 mg of Tween-40 were mixed in 0.5 ml of chloroform. After removing of chloroform at 40 C under vacuum, the mixture was diluted with 50 ml of oxygenated water and well mixed. Aliquots of this emulsion (5 ml) were transferred into dierent test tubes containing 0.2 ml of extracts in ethanol (200 ppm) or 0.2 ml of ethanol (control), or 0.2 ml of BHA (200 ppm; GRAS regulations limit BHA to 200 ppm of the fat or oil content of the food product). Absorbances of all the samples were read at 470 nm (holding the samples at 50 C) at 15 min intervals. All determinations were carried out in triplicate. An emulsion prepared as above without b-carotene served as blank. The antioxidant activity index (AAI) was evaluated in terms of bleaching of the

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b-carotene and calculated as [(DAE DAC)/ (DAB DAC)] * 100, where DAB is the variation in absorbance (from time 0 to 150 0 ) of the sample containing BHA (with assigned AAI = 100); DAE that of the sample containing the extract; DAC that of the blank (with assigned AAI = 0). Antioxidant power was also assessed in some preliminary tests by the Shaal Oven Test (or accelerated resistance test) which simply consists in monitoring the increase of peroxides value (PV; AOCS, 1989) in an oil samples kept in a thermostatic oven at 60 C. The addition of antioxidants should avoid or, at least, delay, the peroxides formation. Sunower oil was bought at a local market. As suggested by Bandoniene et al. (2000), calculated amounts of the extracts were mixed with 4 ml of absolute ethanol and added to 25 g of oil, and alcohol was evaporated during stirring. Oil samples (25 g), without and with antioxidants (200 ppm BHA or 200 ppm extract) were placed in open asks and PV measurements were done every two days according to the AOCS method Cd 2393 (Duh & Yen, 1997). The antioxidant power, AOP, was calculated as (1 DPVE/DPVC) * 100, where DPV is the increase in the PV of oil with antioxidant (E), or without (control), when PV reached its maximum. 2.4. Statistical analysis Data were analysed according to according to a factorial analysis of variance using the Generalised Linear Model procedure of SPSS (v. 11.5.), considering main eects and their two-way interactions. Probability of P < 0.05 was considered statistically signicant. The same SPSS software was used to calculate the correlation coecient (R) to determine the existence of any relationship between AAI and phenols concentration. 3. Results and discussion 3.1. Solvent extraction It must be said that ethanol extraction from grape stalks (but not from marc, and not with ethylacetate) gave a

crude extract that was partly ethanol and partly water soluble, that is to say two dierent extracts were obtained. If the GAE yield for stalks is calculated by the sum of these two extracts, the inuence of starting material (marc or stalks) on nal GAE yield was not signicant (Fig. 1; Table 2). However, considering separately the two stalks extracts, marc gave always statistically higher GAE recovery, while the purity was higher for stalks extracts (Table 2, Fig. 2). This could be due to the fact that stems are a lignocellulosic material, whose main components lignin, cellulose and hemicelluloses can be separated only after strong acid and alkaline hydrolyses (Spigno, Fusca, & De Faveri, 2004). The inuence of the other process parameters was then evaluated for marc and stalks separately (Figs. 1 and 2, Table 3). Degreasing reduced GAE yield and improved only slightly purity of stalks-extracts. Type of solventwas always highly signicant. Purity was higher by using ethylacetate, in particular with stalks and, in fact, it is reported that the use of methanol, ethanol, acetone and their mixtures with water in dierent proportions generally yields a signicant co-extraction of concomitant substances, which makes the procedure of extract purication more dicult and decreases the yield of target antioxidants (Pekic et al., 1998), while ethylacetate exhibits signicant selectivity in respect of natural products. On the other hand ethanol allowed for higher yields. Time: was never inuent. This is not in agreement with some literature results. That is Lapornik et al. (2005) found out that in water-extracts made of grape the yield
Table 2 Signicance of material eect on GAE yield and extract purity, considering for ethanol extraction from stalks: the total extract (A), the ethanol soluble extract (B), the water soluble extract (C) Comparison A F GAE yield Purity 0.137 Sig 0.713 Comparison B F 248.04 106.70 Sig 0.000 0.000 Comparison C F 16.19 50.34 Sig 0.000 0.000

%GAE Yield Ethanol

Stalks (E)

Stalks (W)

Marc
a

%GAE Yield Ethylacetate

Stalks

Marc
b

0.4

0.4

0.2

0.2

0
5h 28C 24h 28C 5h 60C 24h 60C 5h 28C Deg 24h 28C Deg 5h 60C Deg 24h 60C Deg

0
5h 28 C 24h 28 C 5h 60 C 24h 60 C 5h 28 C Deg 24h 28 C Deg 5h 60 C Deg 24h 60 C Deg

Fig. 1. Polyphenols yields obtained with ethanol (a) and ethylacetate (b) extraction from both grape and stalks (E: ethanol soluble extract; W: water soluble extract; Deg: degreased sample).

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PURITY (%) Ethanol

25 20 15 10 5 0
5h 28C 24h 28C 5h 60C 24h 60C

Stalks (W)

Marc

Stalks (E)
a

PURITY(%) Ethylacetate
25 20 15 10 5 0

Marc

Stalks
b

5h 28C Deg

24h 5h 28 C 60C Deg Deg

24h 60C Deg

5h 28C

24h 28C

5h 60C

24h 60C

5h 28C Deg

24h 28C Deg

5h 60C Deg

24h 60C Deg

Fig. 2. Purity of the extracts obtained with ethanol (a) and ethylacetate (b) extraction from both grape and stalks (E: ethanol soluble extract; W: water soluble extract; Deg: degreased sample).

Table 3 Signicance of main eects and their interactions on GAE yield and extract purity, considering for ethanol extraction from stalks: the total extract (A), the ethanol soluble extract (B), the water soluble extract (C) Marc F GAE yield Solvent Degreasing Time Temperature Solv * time Solv * Temp Purity Solvent Degreasing Time Temperature Solv * Temp Time * Temp 30.10 10.04 2.14 34.65 0.84 5.79 50.42 1.07 0.92 0.02 Sig 0.000 0.003 0.153 0.000 0.365 0.022 0.000 0.17 0.34 0.90 Stalks A F 580.61 1.17 14.05 124.70 9.80 120.49 Sig 0.000 0.000 0.001 0.000 0.004 0.000 Stalks B F 37.89 2.42 0.59 23.61 0.15 20.29 161.79 11.27 10.27 1.20 8.75 5.15 Sig 0.000 0.000 0.447 0.000 0.705 0.000 0.000 0.002 0.003 0.167 0.006 0.030 Stalks C F 214.87 0.61 11.39 65.17 7.81 63.32 375.81 6.93 10.72 2.18 13.49 5.94 Sig 0.000 0.440 0.002 0.000 0.009 0.000 0.000 0.013 0.003 0.151 0.001 0.021

of polyphenols gently increased with the time, while in the case of alcohol-extracts it strongly increased with the longer time of extraction. Pekic et al. (1998) report that the kinetics curves of proanthocyanidins yield are of parabolic shape with the initial part being linear (up to 8 h), whereas their second parts show a slower increase and an asymptotic ending. In most of the cited references the eect of time is not an investigated factor, with a length of extraction varying from less than 1 h to 48 h. Temperature was strongly inuent on GAE yields for both marc and stalks, but not on purity, probably because temperature increase favours extraction by increasing solubility and diusion coecient of any compounds, not only of antioxidants. However, it is also reported that polyphenols are heat sensible, so 60 C was chosen as the upper limit (Cacace & Mazza, 2003). Eects interactions: the interaction Solvent * Temperature was always signicant for GAE yield: temperature was inuent with ethanol but not with ethylacetate. Purity of stalk extracts decreased with temperature with ethylacetate but not with ethanol. The interactions Solvent * Time and Time * Temperature were weakly

signicant and only in some cases. All the other possible two-way interactions were not important. At this point we tried to compare our results with some literature works reporting yield and purity data (Table 1). Considering the references about grape, the highest values are relative to the use of only seeds, the part of the fruit richest in polyphenols (average tannin content of 36% on fresh weight and 411% on d.w.). Baydar et al. (2004) recovered an extremely high amount of polyphenols, while the other authors obtained from 0.15% to 2.5% yield and purities above 40%. The few studies regarding grape marc show data similar to our, with 0.060.22% yields and 14 18.3% purity. Globally the found methods are comparable for the used ratio raw material/solvent, but not for the other parameters time and temperature, solvent (which sometimes are even not specied) and analysis and expression of results. From Table 1 it could be derived that antioxidants could be quite easily extracted even with simple procedures. It must be underlined that GAE yield and purity of the present work could have been underestimated for two reasons. First of all samples could not be concentrated to a

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powder, but to a viscous and dense paste (probably due to the presence of sugars and fats), so in the future polyphenols will be measured before concentration; secondly after addition of petroleum ether to marc extracts we did not separate a solid precipitate. Looking at antioxidant extractions from natural sources other than grape, again dierent methods have been used, the ratios material/solvent were generally higher, and good results were obtained from fennel seeds and blackcurrants, but not for the extract purity. 3.2. Antioxidant power

vegetable oil, but addition of our extracts showed a prooxidant eect. It was visually observed that extracts did not well solubilize into the oil but formed a reddish layer on the glass at the interface oil/air. This is not in agreement with other works which found a certain (not always high) protection of oil oxidation by dierent natural extracts (Bandoniene et al., 2000; Bera et al., 2004; Suja et al., 2004). However these works do not report a detailed description of the solubilisation procedure, and Nwuha, Nakajima, Tong, and Ichikawa (1999) report about the problems of solubility of green tea extracts into edible oils. 4. Conclusions

The AAI ranged from 40 to 80 with no inuence of solvent, time, temperature and degreasing, while stalks extracts showed a slightly higher power than marc ones (Fig. 3). Jayaprakasha et al. (2001) found a range of 12 80 for grape seeds extracts with a 10100 mg/l catechins equivalents. Correlation between our AAI and GAE concentration was positive and signicant (P < 0.01), but weak (Pearson correlation coecient of 0.42). Positive and variable coecients (0.230.96) are reported also by other authors (Jayaprakasha et al., 2001; Parejo et al., 2003). In general, even using other assays for the antioxidant power, it is observed that the protection linearly increases with the antioxidants concentration up to a certain value, above which the increase is slower or absent. Furthermore the degree of correlation depends on the class of compounds and is generally higher for total polyphenols than for anthocyanins, avonoids or avonols (Fukumoto & Mazza, 2000; Gao, Bjork, Trajkowski, & Uggla, 2000; Oktay, Gulcin, & Kufreviodlu, 2003; Quezada, Asencio, ` Del Valle, Aguilera, & Gomez, 2004). b-carotene test is simple and can give a screening evaluation of the antioxidant power, but it gives poorly reproducible results (due to variations in b-carotene bleaching reaction), it is not specic (being subject to interference from oxidation and reducing agents in crude extracts, and linoleic acid is not representative of typical food lipids. Thats why we decided to test our extracts directly into a

This paper showed that grape stalks could be a good low-cost antioxidants source as well as grape marc. The simple procedure chosen (one solvent-one step extraction) gave results comparable to other literature results obtained with similar, longer or more complicated systems. There are many papers about recovery of antioxidants from natural sources, but rarely a systematic approach for optimisation of the process in order to maximise nal yields and purity is followed. We tried to optimize some parameters, nding out some suggestions and aspects that should be further investigated: Degreasing does not seem a useful pre-treatment to enhance extract purity. 60 C is better than 28 C, but it should be veried if it could be possible to obtain the same results (or even better in case a certain degree of thermal degradation occurred at 60 C) at lower temperatures in order to reduce the energy cost of the process. There is no signicant dierence between 5 and 24 h, but it should be demonstrated if there is an intermediate maximum after which antioxidants degradation takes place. Extracts could not be concentrated to powder by vacuum-drying, so freeze-drying should be applied. A complete characterisation of the extracts composition will help understanding the dierent nature of marc and stalks extract. Further purication of extracts (to obtain highly pure preparations) by quite a simple and economic method, such as employment of colloidal gas aphrons (Jauregi & Varley, 1999) should be investigated. Application of extracts into dierent foods has to be studied, since so far antioxidant power has been mostly estimated in model systems not representative of the complex nature of dierent foodstu.

100 75 IAA150' 50 25 0 0 100 200 300 GAE mg/L 400 500 600

Acknowledgements Authors thank Dr. L. Tramelli and Dr. S. Trigila for their precious contribution and Prof. A. Silva for her important suggestions about polyphenols chemistry.

Marc

Stalks

Fig. 3. Antioxidant power of the extracts as a function of polyphenols concentration and origin material.

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