Bioresource Technology 98 (2007) 2963–2967
Short Communication
Recovery of antioxidant phenolics from white vinification solid by-products employing water/ethanol mixtures Dimitris P. Makris
a,b,*
, George Boskou a, Nikolaos K. Andrikopoulos
a
a
b
Laboratory of Food Chemistry–Biochemistry–Physical Chemistry, Department of Science of Dietetics–Nutrition, 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 vinification, 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 efficiency was assessed by monitoring the antioxidant potency of extracts and several indices related to their polyphenolic composition, including total polyphenol, total flavonoid, total flavanol 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. Acidification 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; Vinification; Wastes; Wine industry
1. Introduction Efficient, inexpensive and environmentally rational utilisation of agri-food industry wastes is of undisputed importance for higher profitability 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 flavanols; TFd, total flavonoids; 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:
[email protected] (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 vinification 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 vinification 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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vinification solid by-products. On such a basis, the solvent systems tested were composed of ethanol, a vinification 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 acetification 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 vinification 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. Vinification by-products White vinification 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 flask 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 filtered through paper filter and this procedure was repeated twice more. The extracts were then combined in a 100 mL volumetric flask 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 flavonoids, a modified 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 flavonoid 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 t¼30 was read at t = 0 ðAt¼0 515 Þ and t = 30 min (A515 Þ. Results were expressed as Trolox equivalents (mM TRE) per g of dry weight using the following equation: AAR ¼
0:018 %DA þ 0:017 FD tw
ð1Þ
as determined from linear regression, after plotting %DA515 of known solutions of Trolox against concentration; where %DA515 ¼
At¼0 At¼30 515 515 At¼0 515
100, tw the dry weight (g),
and FD the dilution factor (20).
456.8 ± 2.3 4.57 ± 0.07a 217.4 ± 4.1 2.51 ± 0.26 12,513 ± 630 12,402 ± 809 3844.2 ± 21.5 Values represent means of triplicate determination (±SD). Superscripted greek letters a and b denote difference at a 99% and 95% significance level, respectively. a Total polyphenols (mg GAE per 100 g dw). b Total flavonoids (mg CTE per 100 g dw). c Total flavanols (mg CTE per 100 g dw). d Proanthocyanidins (mg CyE per 100 g dw).
1665.1 ± 7.5 4.8 ± 0.3 0.31 ± 0.03 7128 ± 134b 4734 ± 27 31.0 ± 4.9 403 ± 37 297 ± 21 57.0
AAR
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 4.8 ± 0.3 0.33 ± 0.02 4699 ± 382 2289 ± 185b 1034.4 ± 2.8 38.2 ± 4.6 57.0
0.1% HCl (pH 1.50) 1% AcOH (pH 3.28) 1% TA (pH 2.69)
337 ± 13 313 ± 40
446.9 ± 4.7 146.5 ± 0.8a 508.9 ± 6.7 466.3 ± 9.0 464.3 ± 9.7
PC TFl TFd
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 3.17 ± 0.06b 2.24 ± 0.02 2.84 ± 0.09 2.06 ± 0.12 3.58 ± 0.33a 215.1 ± 2.7 55.5 ± 1.0a 154.7 ± 3.8 107.4 ± 0.7 255.7 ± 3.1b 1977.0 ± 77.6b 1008.4 ± 23.0 1047.1 ± 10.3 900.9 ± 8.0b 2074.6 ± 57.2b 5399 ± 499 3043 ± 62 2073 ± 151b 4394 ± 264 6181 ± 334b 5798 ± 178 3120 ± 60a 5618 ± 408 4513 ± 242 7468 ± 143b 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
Seeds
TP AAR PC TFl TFd
Stems
TP AARe PCd TFlc TFdb TPa
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 finding 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 Acidification Peels
4. Discussion
Table 1 Polyphenolic indices and antiradical activity of the by-product extracts obtained by employing various extracting media
Previous studies on grape pomace extraction showed that a solvent system composed of acetone/MeOH/water and acidified with 0.1% HCl was the most efficient 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 first step in the optimisation of an efficient solvent system was the examination of the effect 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 efficiency of extracts, simple linear regression analysis was carried out. For all classes determined, the correlation coefficients found were particularly high and statistically significant (P < 0.001), the most important link being with the TFl content (r2 = 0.9107).
970 ± 15a 191 ± 28 265 ± 4 271 ± 20 429 ± 35
3. Results
–
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% significance level. Differences among polyphenol indices and antiradical activity values were calculated using one-sample T-test at 95% and 99% significance level. For all statistics, SPSSTM 10 was used.
–f 28.5 57.0 85.5 57.0
2.5. Statistical analyses
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5.94 ± 0.02 6.64 ± 0.17 8.55 ± 0.49a 7.61 ± 0.55 5.98 ± 0.22
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Table 2 Comparative data illustrating the efficiency of polyphenol extraction from grape seeds using the condition described in this study Solvent system
Conditions
Yield (% w/w)a
Reference
50% aq. ethanol
• • • •
Two-stage extraction 65 C 7.5:1 liquid-to-solid ratio 1.5 h extraction duration for each stage
3.9 (GAE)
Shi et al. (2003)
50% aq. ethanol
• • • • •
Two-stage extraction 65 C 5:1 liquid-to-solid ratio 1.5 h extraction duration for each stage Ultrafiltration (0.22 lm pore size)
11.4 (GAE)
Nawaz et al. (2006)
60% aq. ethanol
• • • • •
One-stage extraction Room temperature Sonication 10:1 liquid-to-solid ratio 30 min extraction duration
2.8 (GAE)
Yilmaz and Toledo (2006)
57% aq. ethanol
• • • • •
Three-stage extraction Room temperature Grinding 5.5:1 liquid-to-solid ratio 15 min extraction duration for each stage
13.8 (GAE)
This study
a
Values are expressed on a dry weight basis.
Efficient extraction of all by-products was achieved employing 57% aqueous ethanol. Further increase in extracting efficiency was brought about by acidification, 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 vinification by-product, the highest yield was reached when 57% ethanol was used as the solvent system, whereas acidification with any of the agents used did not afford any increase in yield. For a critical assessment of the extraction efficiency, 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, differences among samples originating from different grape type (white or red) are statistically insignificant (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 efficiency 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 vinification solid by-products that contained significant amounts of antioxidant polyphenols. Peels contained relatively high moisture levels and low phenolic burden. 2. A solvent system consisting of 57% aqueous ethanol was efficient for seed polyphenol recovery. Optimal results for stems, assessed on the basis of polyphenol yield and antiradical activity, were obtained after acidification of 57% ethanol with 0.1% HCl. 3. The preparation of extracts with improved antioxidant potency from vinification by-products should be based on the estimation of other polyphenolic classes, in addition to total polyphenol content, since the overall antioxidant effect may be defined by the relative amounts of the most active components. The results showed that flavanols (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 financial support in the form of post-doctoral scholarship. References Alonso, A.M., Guille´n, D.A., Barroso, C.G., Puertas, B., Garcı´a, A., 2002. Determination of antioxidant activity of wine byproducts and its
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