Bioresource Technology 98 (2007) 29632967

Short Communication

Recovery of antioxidant phenolics from white vinication solid by-products employing water/ethanol mixtures
Dimitris P. Makris
a b

a,b,*

, George Boskou a, Nikolaos K. Andrikopoulos

Laboratory of Food ChemistryBiochemistryPhysical Chemistry, Department of Science of DieteticsNutrition, Harokopio University, 70, El. Venizelou Str., 17671 Kallithea, Athens, Greece Department of Food Quality Management and Chemistry of Natural Products, Mediterranean Agronomic Institute of Chania (MAICh), PO Box 85, 73100 Chania, Greece Received 9 May 2006; received in revised form 1 October 2006; accepted 1 October 2006 Available online 15 November 2006

Abstract Solid wastes from white vinication, including grape peels, seeds and stems, were used as raw material for the recovery of antioxidant polyphenols. Extractions were performed using non-toxic media composed of water/ethanol mixtures and hydrochloric, acetic or tartaric acid. Recovery eciency was assessed by monitoring the antioxidant potency of extracts and several indices related to their polyphenolic composition, including total polyphenol, total avonoid, total avanol and condensed tannin (proanthocyanidin) content. Among the by-products tested, seeds were shown to contain exceptional amounts of total polyphenols (13.76 g per 100 g dry weight), followed by stems (7.47 g per 100 g dry weight) and peels (0.97 g per 100 g dry weight). Extracts with the highest antioxidant activity from all byproducts were obtained with 57% ethanol. Acidication of this medium with 0.1% HCl improved polyphenol recovery and antiradical activity for stem extracts, but it was unfavourable for seed extraction. 2006 Elsevier Ltd. All rights reserved.

Keywords: Antioxidants; By-products; Polyphenols; Vinication; Wastes; Wine industry

1. Introduction Ecient, inexpensive and environmentally rational utilisation of agri-food industry wastes is of undisputed importance for higher protability and minimal environmental impact. One of the higher value options is the recovery of bioactive plant food constituents, which could be used in
Abbreviations: AAR, antiradical activity; CTE, catechin equivalents; CyE, cyaniding equivalents; DMACA, p-(dimethylamino)-cinnamaldehyde; GAE, gallic acid equivalents; MeOH, methanol; TFl, total avanols; TFd, total avonoids; TP, total polyphenols; SD, standard deviation; TRE, trolox equivalents. * Corresponding author. Address: Department of Food Quality Management and Chemistry of Natural Products, Mediterranean Agronomic Institute of Chania (MAICh), PO Box 85, 73100 Chania, Greece. Tel.: +30 28210 35056; fax: +30 28210 35001. E-mail address: dimitris@maich.gr (D.P. Makris). 0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.10.003

pharmaceutical, cosmetics and food industry. The most prominent class of such phytochemicals are polyphenols, which occur in large amounts in vinication by-products (Alonso et al., 2002). In this regard, the last years several studies have been carried out, with the aim to establish optimised conditions for polyphenol extraction. Most of these studies are focused on pomace deriving from red wine production, whereas other by-products, i.e. stems, as well as white vinication solid wastes have been disregarded or given much less attention. White grape peels and grape stems have been shown to contain a spectrum of potentially bioactive polyphenols (Lu and Foo, 1999; Souquet et al., 2000), and thus their examination merits a greater attention. The scope of the present study was an examination on the possibilities of using non-toxic, cheap, and readily available means of recovering phenolics from white

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vinication solid by-products. On such a basis, the solvent systems tested were composed of ethanol, a vinication coproduct that could be obtained after fermentation of the sugar-containing pomace and distillation. Tartaric acid can be obtained from must, or wine dregs and lees, while acetic acid can be produced through acetication of ethanol solutions or must/wine dregs. For all these products, there is also the possibility for recycling. Hydrochloric acid is a cheap, industrial, non-oxidative product. The implementation of similar techniques may potentially provide the basis for a sustainable process of integrated exploitation of vinication by-products. 2. Methods 2.1. Chemicals Folin-Ciocalteu reagent and ascorbic acid were from Fluka (Steinheim, Germany). Trolox, gallic acid, 2,2-diphenyl-picrylhydrazyl (DPPH) stable radical, p(dimethylamino)-cinnamaldehyde (DMACA) and catechin were from Sigma Chemical Co. (St. Louis, MO, USA). Sodium nitrite and aluminium chloride hexahydrate were from Merck (Darmstad, Germany). 2.2. Vinication by-products White vinication by-products were from Roditis cultivar (Vitis vinifera sp.), obtained from a winery in the region of Koropi (prefecture of Attica, Greece), and included pomace (peels and seeds) and stems. Seeds were manually separated from peels immediately after receipt and all byproducts were stored at 40 C until tested. 2.3. Extraction procedure A suitable quantity of tissue (approx. 4.5 g) was chopped into small pieces with a sharp, stainless steel cutter to facilitate extraction. The chopped tissue was ground with sea sand and a small portion of the extraction solvent, with a pestle and a mortar, and then left to macerate for 30 min in the dark. The paste formed was placed in a 100 mL conical ask with 25 mL of solvent (solvent-to-solid ratio %5.5) and extraction was performed under stirring at 700 rpm on a magnetic stirrer for 15 min. The extract was ltered through paper lter and this procedure was repeated twice more. The extracts were then combined in a 100 mL volumetric ask and made to the volume. All extracts were centrifuged at 4500 rpm prior to analyses. For control extractions, a solvent system consisted of 0.1% HCl in MeOH/acetone/water (6/3/1, v/v/v) was used. All other procedures were as aforementioned. 2.4. Assays Moisture was determined after drying the by-products in an air current-heated oven at 95 C for 48 h. Total

polyphenols analysis was carried out employing the Folin-Ciocalteu methodology (Arnous et al., 2002). Results were expressed as mg gallic acid equivalents (GAE) per 100 g dry weight. For total avonoids, a modied method of Kim et al. (2003) was used. A 0.1-mL aliquot of extract appropriately diluted was mixed with 0.4 mL distilled water in a 2-mL microcentrifuge tube, 0.03 mL 5% NaNO2 was added, and allowed to react for 5 min. Following this, 0.02 mL 10% AlCl3 was added and the mixture stood for further 5 min. Finally, to the reaction mixture 0.2 mL 1 M Na2CO3 and 0.25 mL distilled water were added, and the absorbance at 510 nm was obtained against blank prepared similarly, by replacing extract with distilled water. Total avonoid content was calculated from a calibration curve using catechin as standard, and expressed as mg catechin equivalents (CTE) per 100 g dry weight. Flavanols were determined after derivatisation with p-(dimethylamino)-cinnamaldehyde (DMACA), using the optimised protocol established by Nigel and Glories (1991). Extract (0.2 mL) suitably diluted with MeOH was introduced into a 2-mL microcentrifuge tube and 0.5 mL HCl (0.24 N in MeOH) and 0.5 mL DMACA solution (0.2% in MeOH) were added. The mixture was allowed to react for 5 min at room temperature, and the absorbance was obtained at 640 nm. Control sample was prepared by replacing sample with MeOH. Results were expressed as mg catechin equivalents (CTE) per 100 g dry weight. Proanthocyanidins were analysed by the method described by Waterman and Mole (1994). Butanol reagent was prepared by mixing 70 mg ferrous sulphate (FeSO4) with 5 mL concentrated HCl and made to 100 mL with n-butanol. An aliquot of 0.05 mL sample was mixed thoroughly in a 2-mL, screw-cup vial with 0.7 mL butanol reagent and heated at 95 C in a water bath for 45 min. Following this the sample was cooled, 0.25 mL n-butanol was added and the absorbance at 550 nm (A550) was measured. Results were expressed as cyaniding equivalents (CyE) per 100 g dry weight using as e = 26,900 and MW = 449.2. Antiradical activity (AAR) was determined by employing the procedure reported (Arnous et al., 2002). Each extract was diluted 1:20 with methanol immediately before the analysis. Sample (0.025 mL) was added to 0.975 mL DPPH solution (73 lM in MeOH), and the absorbance was read at t = 0 At0 and t = 30 min (At30 . Results 515 515 were expressed as Trolox equivalents (mM TRE) per g of dry weight using the following equation:  AAR  0:018 %DA 0:017 FD tw 1

as determined from linear regression, after plotting %DA515 of known solutions of Trolox against concentration; where %DA515
At0 At30 515 515 At0 515

100, tw the dry weight (g),

and FD the dilution factor (20).

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5.94 0.02 6.64 0.17 8.55 0.49a 7.61 0.55 5.98 0.22 132.0 8.9 1.98 0.09b 12,727 891 10,884 260 3352.7 63.9b 480.3 1.2 6.72 0.07

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456.8 2.3 4.57 0.07a 4.8 0.3 0.31 0.03 7128 134b 4734 27 57.0 403 37 297 21 31.0 4.9 1665.1 7.5 Values represent means of triplicate determination (SD). Superscripted greek letters a and b denote dierence at a 99% and 95% signicance level, respectively. a Total polyphenols (mg GAE per 100 g dw). b Total avonoids (mg CTE per 100 g dw). c Total avanols (mg CTE per 100 g dw). d Proanthocyanidins (mg CyE per 100 g dw). 217.4 4.1 2.51 0.26 12,513 630 12,402 809 3844.2 21.5

2.5. Statistical analyses All determinations were carried out at least in triplicate and values were averaged and given along the standard deviation (SD). Correlations were established using regression analysis at a 95%, 99%, and 99.9% signicance level. Dierences among polyphenol indices and antiradical activity values were calculated using one-sample T-test at 95% and 99% signicance level. For all statistics, SPSSTM 10 was used.
AAR PC Seeds TP AAR PC TFd TFl

3. Results Previous studies on grape pomace extraction showed that a solvent system composed of acetone/MeOH/water and acidied with 0.1% HCl was the most ecient in extracting polyphenols from grape pomace (Ju and Howard, 2003). Such a solvent system was employed for extraction of the by-products (Table 1) and the extracts obtained served as control samples. The rst step in the optimisation of an ecient solvent system was the examination of the eect of ethanol content. Ethanol percentage in the solvent systems used varied from 28.5 to 85.5, a region that has been previously shown to provide high yield for grape seed extraction (Shi et al., 2003; Yilmaz and Toledo, 2006). Table 1 shows that control extraction of peels gave unsurpassed values for all polyphenol indices and AAR. For stems, 57% EtOH yielded TP amounts comparable to control, but addition of 0.1% HCl greatly improved the recoveries of all polyphenolic classes, with a concomitant impact to the AAR. Similarly, 57% EtOH gave seed extracts with the highest TP and TFd concentration, and AAR. Addition of any of the acidifying agents, however, was unfavourable at improving polyphenol recovery and AAR. In order to evaluate the contribution of the individual polyphenolic classes to the antioxidant eciency of extracts, simple linear regression analysis was carried out. For all classes determined, the correlation coecients found were particularly high and statistically signicant (P < 0.001), the most important link being with the TFl content (r2 = 0.9107).

1977.0 77.6b 1008.4 23.0 1047.1 10.3 900.9 8.0b 2074.6 57.2b 922 62a 197.2 3.0a 31.2 1.2a 1.74 0.09a 33 0b 14.9 0.8 4.9 0.5 0.25 0.02 232 27 19.4 3.4 4.4 0.6 0.31 0.02 264 73 41.6 0.9 4.1 0.0 0.22 0.01 386 35 43.0 1.0 5.5 0.2 0.31 0.00 f 28.5 57.0 85.5 57.0 970 15a 191 28 265 4 271 20 429 35 5798 178 3120 60a 5618 408 4513 242 7468 143b 5399 499 3043 62 2073 151b 4394 264 6181 334b

215.1 2.7 55.5 1.0a 154.7 3.8 107.4 0.7 255.7 3.1b

3.17 0.06b 2.24 0.02 2.84 0.09 2.06 0.12 3.58 0.33a

11,108 909 11,090 511 4605.1 203.1a 7957 602a 7870 238a 3107.5 20.8a 13,756 849b 13,305 802b 4231.2 25.4 12,242 272 11,638 932 3671.0 142.1 12,278 212 9394 635 4193.3 17.7

446.9 4.7 146.5 0.8a 508.9 6.7 466.3 9.0 464.3 9.7

Table 1 Polyphenolic indices and antiradical activity of the by-product extracts obtained by employing various extracting media

PCd

TFlc

4. Discussion The outcome of the investigation showed that peels contain by far less important amounts of polyphenols compared with stems and seeds, and as a result peel extracts exhibit low antioxidant potency. In particular, peels contain 60% higher moisture levels than seeds, but only 8.7% of their TP content. This nding is of prime importance, given that moisture plays a crucial role regarding by-product preservation and drying costs. Thus proper handling of white grape pomace would minimise drying time, increase preservation period and reduce the amount of solvent required for optimal polyphenol recovery.

%EtOH Acidication Peels

57.0

0.1% HCl (pH 1.50) 1% AcOH (pH 3.28) 1% TA (pH 2.69)

337 13 313 40

TPa

TFdb

38.2 4.6

4.8 0.3 0.33 0.02 4699 382 2289 185b 1034.4 2.8

Stems

AARe

TP

TFd

TFl

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Table 2 Comparative data illustrating the eciency of polyphenol extraction from grape seeds using the condition described in this study Solvent system 50% aq. ethanol Conditions Two-stage extraction 65 C 7.5:1 liquid-to-solid ratio 1.5 h extraction duration for each stage Two-stage extraction 65 C 5:1 liquid-to-solid ratio 1.5 h extraction duration for each stage Ultraltration (0.22 lm pore size) One-stage extraction Room temperature Sonication 10:1 liquid-to-solid ratio 30 min extraction duration Three-stage extraction Room temperature Grinding 5.5:1 liquid-to-solid ratio 15 min extraction duration for each stage Yield (% w/w)a 3.9 (GAE) Reference Shi et al. (2003)

50% aq. ethanol

11.4 (GAE)

Nawaz et al. (2006)

60% aq. ethanol

2.8 (GAE)

Yilmaz and Toledo (2006)

57% aq. ethanol

13.8 (GAE)

This study

Values are expressed on a dry weight basis.

Ecient extraction of all by-products was achieved employing 57% aqueous ethanol. Further increase in extracting eciency was brought about by acidication, but the trend was not the same for all by-products and for all polyphenolic classes. Concerning grape seeds, which is the richest and the most well studied vinication by-product, the highest yield was reached when 57% ethanol was used as the solvent system, whereas acidication with any of the agents used did not aord any increase in yield. For a critical assessment of the extraction eciency, comparative data are given in Table 2. Evidently the implementation of the method described herein gave much higher yields than those of other studies that used aqueous ethanol with similar composition. At this point, however, it should be emphasised the fact that extraction yield also depends on the polyphenol content of seeds, which may vary widely among grape varieties. On the other hand, dierences among samples originating from dierent grape type (white or red) are statistically insignicant (Guendez et al., 2005a). Apart from the total polyphenol content, the composition of individual substances is also critical, in terms of expressing antiradical activity. Evidence suggested that grape seed extracts rich in procyanidin B1 might be more active (Guendez et al., 2005b). In this examination it was demonstrated that the TFl content is prominently associated with the antiradical activity (P < 0.001), and this is another parameter that should be taken into consideration when assessing the eciency of a method for the extraction of active phenolics. In most cases the TP content is taken as the sole criterion, but such a unilateral evaluation may bring about misleading results.

5. Conclusions From the results, it could be concluded that: 1. Seeds and stems were white vinication solid by-products that contained signicant amounts of antioxidant polyphenols. Peels contained relatively high moisture levels and low phenolic burden. 2. A solvent system consisting of 57% aqueous ethanol was ecient for seed polyphenol recovery. Optimal results for stems, assessed on the basis of polyphenol yield and antiradical activity, were obtained after acidication of 57% ethanol with 0.1% HCl. 3. The preparation of extracts with improved antioxidant potency from vinication by-products should be based on the estimation of other polyphenolic classes, in addition to total polyphenol content, since the overall antioxidant eect may be dened by the relative amounts of the most active components. The results showed that avanols (monomeric and oligo/polymeric) could play an important role in this regard. Acknowledgement Dr. D.P. Makris wishes to thank the Greek Scholarships Foundation (I.K.Y.) for the nancial support in the form of post-doctoral scholarship. References
Alonso, A.M., Guillen, D.A., Barroso, C.G., Puertas, B., Garca, A., 2002. Determination of antioxidant activity of wine byproducts and its

D.P. Makris et al. / Bioresource Technology 98 (2007) 29632967 correlation with polyphenolic content. J. Agric. Food Chem. 50, 5832 5836. Arnous, A., Makris, D.P., Kefalas, P., 2002. Correlation of pigment and avanol content with antioxidant properties in selected aged regional wines from Greece. J. Food Compos. Anal. 15, 655665. Guendez, R., Kallithraka, S., Makris, D.P., Kefalas, P., 2005a. An analytical survey of the polyphenols of seeds of varieties of grape (Vitis vinifera sp.) cultivated in Greece: implications for exploitation as a source of value-added phytochemicals. Phytochem. Anal. 16, 1723. Guendez, R., Kallithraka, S., Makris, D.P., Kefalas, P., 2005b. Determination of low molecular weight polyphenolic constituents in grape (Vitis vinifera sp.) seed extracts: correlation with antiradical activity. Food Chem. 89, 19. Ju, Z.Y., Howard, L.R., 2003. Eects of solvent and temperature on pressurized liquid extraction of anthocyanins and total phenolics from dried red grape skin. J. Agric. Food Chem. 51, 52075213. Kim, D.O., Chun, O.K., Kim, Y.J., Moon, H.Y., Lee, C.Y., 2003. Quantication of polyphenolics and their antioxidant capacity in fresh plums. J. Agric. Food Chem. 51, 65096515.

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Lu, Y., Foo, L.Y., 1999. The polyphenol constituents of grape pomace. Food Chem. 65, 18. Nawaz, H., Shi, J., Mittal, G.S., Kakuda, Y., 2006. Extraction of polyphenols from grape seeds and concentration by ultraltration. Separ. Purif. Tech. 48, 176181. Nigel, C.W., Glories, Y., 1991. Use of a modied dimethylaminocinnamaldehyde reagent for analysis of avanols. Am. J. Enol. Vitic. 42, 364366. Shi, J., Yu, J., Pohorly, J., Young, J.C., Bryan, M., Wu, Y., 2003. Optimization of the extraction of polyphenols from grape seed meal by aqueous ethanol solution. J. Food Agric. Environ. 1, 4247. Souquet, J.M., Labarbe, B., Le Guerneve, C., Cheynier, V., Moutounet, M., 2000. Phenolic composition of grape stems. J. Agric. Food Chem. 48, 10761080. Waterman, P.G., Mole, S., 1994. Analysis of Phenolic Plant Metabolites. Blackwell Scientic Publ., Oxford, pp. 8391. Yilmaz, Y., Toledo, R.T., 2006. Oxygen radical absorbance capacities of grape/wine industry byproducts and eect of solvent type on extraction of grape seed polyphenols. J. Food Compos. Anal. 19, 4148.