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Extraction of resveratrol from the pomace of Palomino fino grapes by supercritical carbon dioxide
Journal of Food Engineering 96 (2010) 304–308
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Extraction of resveratrol from the pomace of Palomino fino grapes by supercritical carbon dioxide L. Casas, C. Mantell *, M. Rodríguez, E.J. Martínez de la Ossa, A. Roldán, I. De Ory, I. Caro, A. Blandino Department of Chemical Engineering, Food Technology and Environmental Technologies, Faculty of Science, University of Cadiz, Box 40, 11510 Puerto Real, Cadiz, Spain
a r t i c l e
i n f o
Article history: Received 29 May 2009 Received in revised form 22 July 2009 Accepted 4 August 2009 Available online 9 August 2009 Keywords: Resveratrol Supercritical carbon dioxide extraction Palomino grape HPLC
a b s t r a c t Resveratrol is a phenolic compound that is present in grapes and has significant benefits for human health. The development of methods to obtain concentrates of this compound is currently a major challenge in the food industry. In the work described here, resveratrol from grape seeds, stems, skin and pomace of the Palomino fino grape variety was extracted by supercritical carbon dioxide extraction. The effect of pressure (100, 400 bar), temperature (35, 55 °C) and the addition of modifier (5% v/v of ethanol) was evaluated to identify optimal resveratrol extraction from this by-product. Extraction yields and concentrations of resveratrol in the extracts were determined. The best results were obtained on working at high pressure and low temperature using 5% v/v ethanol as a co-solvent. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Phenolic compounds represent an important group of micronutrients present in the plant world, and which form a part of the human food and animal feed. In particular, trans-resveratrol (3,5,40 -trihydroxystilbene) has gained significant worldwide attention due to its ability to inhibit or retard a wide variety of diseases in animals (Baur et al., 2006), including cardiovascular disease and cancer (Bradamante et al., 2004), and to increase stress resistance and lifespan (Baur et al., 2006; Valenzano et al., 2006). Resveratrol is commonly found in grape skins and seeds. Its content in wine depends on the grape variety, the mechanical pre-treatment (crushed and pressed) and the vinification process. Generally, the concentration is higher in red than in white wines, since the must is fermented with the skins (Romero-Pérez et al., 1996). Furthermore, significant differences can be found in the resveratrol content from one vintage to another (Threlfall et al., 1999), from different vineyard locations, and as a result of different weather patterns. Among the white wines, Greek and Portuguese wines are reported to contain the lowest levels of 0.03–0.14 mg/L (Dourtoglou et al., 1999) and 0.01–0.51 mg/L (De Revel et al., 1996) resveratrol, respectively. Resveratrol contents between 0.8 and 8 mg/L in Spanish white wines from ‘‘D.O. Rías Baixas” and the Galician wines are particularly notable (Rodríguez-Delgado et al., 2002; Feijóo et al., 2008).
* Corresponding author. Tel.: +34 956 016458; fax: +34 956 016411. E-mail address: [email protected] (C. Mantell). 0260-8774/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2009.08.002
In recent years, newer techniques such as extraction by supercritical fluids (SFE), extraction by pressurized liquids (PLE), and extraction assisted by microwave irradiation (MAE) have replaced conventional techniques like Soxhlet extraction for solid samples. These alternative techniques simplify the process, considerably reduce the consumption of solvents, and also increase the rate of the extraction process. Although SFE is limited to compounds of low or medium polarity, literature reports on extraction of polyphenols by SFE in the presence of organic solvent modifiers are available (Mantell et al., 2003). There are numerous reports on the recovery of polyphenolic compounds from different varieties of grapes by SFE. Palma and Taylor (1999) applied this technology to separate eight polyphenolic compounds on spiked inert matrices using ethyl acetate or methanol as a modifier. Tena et al. (1998) used CO2 modified with 5% (v/v) methanol and the extraction parameters were 50 °C and 350 bar. Pascual-Martí et al. (2000) investigated the supercritical fluid extraction process of resveratrol from grape skins of Vitis vinifera, and found extraction at 40 °C, 150 bar, with 7.5% (v/v) ethanol as modifier to be optimum. Berna et al. (2001) showed the influence of process conditions on the solubility of resveratrol in supercritical carbon dioxide and ethanol. These authors suggested the use of 7.5% ethanol as co-solvent and elevated pressure to carry out the extraction. Cho et al. (2006) used an ultrasonication-assisted method in order to search an effective extraction of resveratrol from grape. This methods exhibited more efficiency than the conventional solvent extraction with ethanol/water (80:20%, v/v) maintained at 60 °C for 30 min. In the new methods, the recovery of resveratrol
L. Casas et al. / Journal of Food Engineering 96 (2010) 304–308
increase by 24–30%, compared with the conventional solvent extraction. Grape pomace is an industrial waste from the wine process, and consists of grape seeds, skin and stems. Components such as resveratrol remain in the pomace at concentrations that are dependent on the wine manufacture process. de Campos et al. (2008) used supercritical fluid extraction to extract grape pomace from the production of Cabernet sauvignon vintage. These authors use as solvent SC-CO2 and analyze the effect of the addition of ethanol as co-solvent at 150 bar and 40 °C. The percents of co-solvent in the solvent were 10%, 15% and 20% w/w. The highest yields of the extracts were obtained by Soxhlet extraction using ethanol (13.2% w/w), butanol (12.2% w/w), and also by SFE with 15% ethanol (9.2% w/w). Three varieties of grape marc, native to Slovenia (Refošk, Merlot and Cabernet), were studied by Tünde et al. (2009). These authors found a mixture of organic solvent and water at 60 °C to be the most efficient in single-step extractions, and the pre-treatment with SC-CO2 (with or without ethanol as co-solvent) to improve the extraction of polyphenols from the grape marc. This method provides an alternative to the pre-treatment of the plant materials and replaces toxic organic solvents. Palomino grape represents 95% of grape production in the Jerez region (Cádiz, Spain) and this is dedicated to the production of generous wines ‘‘D.O. Jerez-Xerez-Sherry”. The pressing of this grape produces approximately 15–20% of pomace, and this is usually used as a fertilizer. Resveratrol has not been detected in the Jerez wines (Domínguez et al., 2001) or has been detected at very low concentrations (0.03 mg/L) (Martínez-Ortega et al., 2000). However, Roldán et al. (2003) detected its presence in the Palomino fino grape. Its content in the grape skins varies from 2.59 and 4.51 mg/ kg depending on the vintage, and is increased on infection of the grapes by the fungus, Botrytis cinerea. The present work looks into the viability of using SC-CO2 for the recovery of resveratrol from the pomace of the Palomino fino grape press process. Experiments were designed using different parts of the grape pomace, stems, skin and seeds as raw materials. The effect of temperature, pressure and modifier concentration of the SC-CO2 on the total extraction yield, and on the recovery of resveratrol in the extract was evaluated.
2. Materials and methods 2.1. Sampling and chemicals White grape pomace from the Jerez-Xerez-Sherry area (Palomino fino variety) was used as the raw material. Samples of freshly pressed white grape were collected from a local wine cellar and placed in an oven for 48 h at 60 °C to obtain a dry solid. The resulting solid was manually separated into four fractions: whole grape pomace, seeds, stems and skin. Each fraction was pulverizing with hammer mill to obtain a particle size distribution adequate. Subsequently, each sample was vacuum-packed using household vacuum equipment (Professional Family, Orved, Italy). The mean particle size of each fraction was as follows: 0.165 ± 0.102 mm for stem, 0.261 ± 0.164 mm for skins and 0.319 ± 0.239 mm for seeds. The pomace sample showed the following distribution of weight fractions: 47.9% of skin, 43.7% of seeds, 8.4% of stem and mean particle size of 0.408 ± 0.375 mm. The packed samples were stored at room temperature under darkness until used. The carbon dioxide (99.995%) used was provided by Carburos Metalicos (Barcelona, Spain). Standard of trans-resveratrol (99%) was provided by Sigma–Aldrich and the other reagents (ethanol, methanol, hydrochloric acid and acetic acid, all with a purity of HPLC gradient grade) by Panreac.
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2.2. Extraction at high pressure The extractions were carried out in an Isco extractor (Nebraska, USA, model SFX220). The equipment consisted of one extractor with a maximum capacity of 10 mL and 2 lm filters at the inlet and outlet to avoid haulage of the sample. The extractor was also fitted with a thermostatic system that allowed the extraction to be carried out at a constant temperature. The solvent was introduced by syringe pumps (Isco, Nebraska, USA, model 260D and 100DX), which allowed a constant pressure. The samples left the vessel through a micrometric valve, which was thermostated to avoid obstructions at the exit due to the solidification of CO2. The automatic control of the equipment make possible to work with the pumps at different programs. In this case, a consolvent program is used that make possible to add a constant percent of flow-rate of one pump regarding to the other pump. The flow-rate was automatically measured by the program based in the movement of the piston inside of the syringe pump. This flow-rate was measured in volumetric units at operation pressure and 20 °C of temperature. The extraction cartridge was loaded with approximately 4.6 g of sample which had previously been homogenized to maintain a constant apparent density in all experiments, and then introduced into the extractor to reach the operating temperature. The pumps were loaded with carbon dioxide and ethanol until the operating pressure was reached. The automatic decompression valves of the extractor were then closed. The valves connecting the pumps were opened so as to open the extractor. The extractor was then pressurized with CO2 and ethanol. When a balanced state was attained, the micrometric valve was opened until a constant flow of 0.8 g/min was achieved. In order to achieve complete extraction of the substances in question, a relatively long extraction time was used (3 h). The extracts were collected in glass tubes containing methanol and analyzed by high-performance liquid chromatography (HPLC). Extreme conditions of pressure were tested, i.e. 100 and 400 bar. Experiments were carried out at relatively low temperatures between 35 and 55 °C because, according to many authors (Chafer et al., 2005; Pascual-Martí et al., 2001; Romero-Pérez et al., 2001), the yields of extraction of resveratrol decreases at higher temperatures and prolonged extraction times. Experiments were also carried out with 5% volume of ethanol as co-solvent, since this is the most commonly used in the SC-CO2 extraction of natural products. The results shown are averages of these two independent experiments with a reproducibility of approximately 7.8% CV (coefficient of variation). 2.3. Conventional extraction The methodology described by Roldán et al. (2003) was used for the conventional extraction. Samples of 25 g were macerated with 50 mL of a mixture of methanol/HCl (0.1%) for 30 min in an ultrasonic bath. After the extraction, the sample was centrifuged and filtered [0.45 lm Teflon syringe filters (Millex-LCR, Milipore, Bedford, MA)] prior to analysis by direct injection on HPLC. 2.4. Determination by HPLC The HPLC equipment consisted of an Agilent Technologies 1100 Series chromatograph, with UV–visible detector, auto sampler and PC software for the control and processing of the chromatographic data. The method used was based in the published by PascualMartí et al. (2000). This method is adequate for quantification of cis- and trans- isomers of resveratrol and their glucosides. The column (250 mm 4.6 mm) was a C18 Hypersil ODS (5 lm particle size) (Supelco). The solvent used was water:methanol:acetic acid
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(75:20:5). The flow-rate was set to 1.5 mL/min and 20 lL of filtered extract was injected for each sample. Detection was carried out at a wavelength of 306 nm. Trans-resveratrol was identified by comparison of its retention times with that of the commercial standard (Sigma) and was quantified by means of a calibration curve. The calibration curve was as follow:
A ¼ 55:176C þ 31:192
ð1Þ
where A is the area expressed in mAu and C is the concentration expressed in mg/L. The correlation coefficient (R) was 0.9999. The experiments on each extraction were carried out in triplicate in order to evaluate the variability of the measurements. The results are shown as the average of all the independent analyses with a reproducibility of approximately 2.5% CV (coefficient of variation). A typical chromatogram of the samples is included in Fig. 1. 3. Results and discussion In SFE, the solvating power of the fluids can be manipulated by changing pressure and/or temperature so as to achieve a remarkably high selectivity. This tuneable solvating power of SFE is particularly useful for the extraction of complex samples such as plant materials. SC-CO2 is not very suitable for the extraction of polar analytes. This problem is often tackled by use of modifiers. Depending on the type of sample matrix and the affinity of the analyte for the matrix, the modifier may influence the extraction in three different ways: (1) increase the solubility of the analyte in the supercritical fluid as a result of analyte–modifier interactions in the fluid phase; (2) facilitate analyte desorption – the molecules of polar modifiers are able to interact with the matrix and compete efficiently with the analyte for the active sites in the matrix; (3) distort the matrix–analyte diffusion process and favour penetration of the supercritical fluid into the matrix when the modifier swells the matrix. The results for extraction yields are presented in Tables 1–3. The maximum extraction yields of pomace and its individual components were obtained on addition of 5% volume of ethanol as cosolvent to the SC-CO2. This is attributed to the modification of the properties of the carbon dioxide – a change that enables this fluid to extract the lipophilic and the hydrophilic compounds. The fluid pressure is an essential parameter in SC-CO2 extraction, since fluid density is directly related to pressure. It can be observed from Table 1 that at constant temperature, an increase in the pressure increased the extraction yield. The density of carbon dioxide is higher as higher pressure, and consequently, the solvency of SC-CO2 also increases. Berna et al. (2001) also suggest
the extraction at elevated pressure to be advantageous. These results are consistent with those reported in the bibliography by other authors (Pascual-Martí et al., 2000 and Palma and Taylor, 1999). Temperature also has a significant effect on the recovery of components in SC-CO2. According to the rules of kinetics, if the other variables are constant, an increase in temperature is related to a more intensive thermal motion of solutes in the active sites of the matrix. This situation is beneficial for the solutes to overcome the adsorbing energy forces on the matrix and to be desorbed more efficiently from the active sites by SC-CO2 at higher temperature. From a thermodynamic point of view, the saturated vapour pressure increases with a corresponding increase in the temperature, thus enabling the solutes to dissolve in SC-CO2 more easily. Moreover, the density of SC-CO2 decreases with an increase in temperature, thus decreasing the solvency of SC-CO2. These three effects compete with one another during the extraction process. The effect of temperature on the extraction yield also is shown in Table 1. At 100 bar, an increase in the temperature from 35 to 55 °C did not have a significant effect on the extraction yield. In some cases, a decrease in yield was observed. This was seen in SC-CO2, both with and without the addition of co-solvent. This behaviour is attributed to compensation between factors with an increase in temperature. Nevertheless, at a higher pressure of 400 bar, an increase in the temperature was beneficial in the extraction. This effect is more marked when co-solvent is not added to the system, with increases in the extraction yields of 153%, 60%, 150% and 250% for seed, stem, skin and grape pomace, respectively, compared to the yields obtained at 100 bar. The resveratrol concentration in the extracts obtained (mg resveratrol/g extract) are shown in Table 2. It is evident that the use of ethanol as co-solvent with SC-CO2 is more selective than the process using SC-CO2 alone. This is due to the influence of the co-solvent on the solubility of the solute, and therefore on the extraction yield of the process. On the other hand, pressure and temperature appeared to have a less marked influence on resveratrol content. Nevertheless, the use of high pressure (400 bar) and low temperature (35 °C) was desirable for the extraction. Best results were obtained with grape skins as compared to the other raw materials. Table 3 clearly shows the recovery of resveratrol from the raw material to be optimum at 400 bar, 35 °C and 5% Ethanol. SC-CO2 without the addition of co-solvent at low pressure (100 bar) was not selective for resveratrol. In fact, resveratrol was not detected under all the conditions analyzed at this pressure (35, 55 °C). An increase in the pressure to 400 bar improved the extraction of resveratrol. However, it is necessary to add a co-solvent in order to increase the extraction yields significantly.
Fig. 1. Chromatograms of the sample, the retention time were 19.487 and 21.652 min for trans-resveratrol and cis-resveratrol, respectively.
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L. Casas et al. / Journal of Food Engineering 96 (2010) 304–308 Table 1 Total extraction yields expressed as mg of extract/100 g of dry sample. Extraction conditions CO2
100 bar 400 bar
CO2 + EtOH
100 bar 400 bar
Seed
Stem
Skin
Grape pomace
35 °C 55 °C 35 °C 55 °C
132 90 650 1648
129 80 320 513
123 70 590 1445
165 90 430 1514
35 °C 55 °C 35 °C 55 °C
1223 1190 1390 2122
489 500 800 1026
1556 1600 2300 2805
1288 760 1500 2176
Table 2 Extraction yields of resveratrol expressed as mg of resveratrol/100 g of dry sample. Extraction conditions CO2
100 bar 400 bar
CO2 + EtOH
100 bar 400 bar
Seed
Stem
Skin
Grape pomace
35 °C 55 °C 35 °C 55 °C
– – 0.2 0.3
– – 0.6 0.6
– – 0.5 0.4
– – 0.4 0.3
35 °C 55 °C 35 °C 55 °C
6.1 5.0 8.3 11.1 –
0.7 – 0.9 0.7 1.7
13.1 14.0 49.1 45.5 3.1
10.1 7.0 16.1 19.2 0.9
Conventional extraction (–) Not detectable.
Table 3 Concentration of resveratrol in the extract (mg resveratrol/g extractor). Extraction conditions CO2
100 bar 400 bar
CO2 + EtOH
100 bar 400 bar
Seed
Stem
Skin
Grape pomace
35 °C 55 °C 35 °C 55 °C
– – 0.31 0.18
– – 1.87 1.17
– – 0.84 0.27
– – 0.93 0.19
35 °C 55 °C 35 °C 55 °C
4.99 4.20 5.97 5.23
1.43 – 1.12 0.68
8.42 8.75 21.35 16.22
7.84 9.21 10.73 8.82
(–) Not detectable.
The recovery of resveratrol using SC-CO2 was higher than conventional extraction with a solvent mixture at atmospheric pressure [Methanol:HCl (0.1%)]. SC-CO2 extraction enabled resveratrol to be obtained from seeds, which was not possible by conventional methods. This behaviour can be attributed to the higher diffusion properties of SC fluids regarding conventional solvents. This fact allows to the SC solvent to reach some active sites of the solid matrix inside the seed that the conventional solvent cannot reach. These diffusional problems do not happen in the rest of the parts of the pomace where the solute is accessible to the conventional solvent. The extract obtained from the skins had higher levels of resveratrol than that obtained from seeds and stems. The amount of resveratrol in grape pomace, therefore, depends on the proportion of skin, seeds and stems in the whole by-product. Although literature on the recuperation of resveratrol from Palomino fino grape pomace has not been published previously, that from other varieties of grape skins, seeds and pomace have been analyzed. For example, Pezet and Cuenat (1999) found resveratrol at 0.68 and 0.39 mg/100 g of fresh weight in the skin and grape pomace, respectively. The values in the Muscadine variety were 1.17 and 5.32 mg/100 g of dry weight in the skin and pomace, respectively (Ector et al., 1996). In the case of red varieties, Sun et al. (2006) reported resveratrol levels of 1455, 68 and 656 mg/100 g of dry weight in stems, seeds and skin, respectively. Pascual-Martí et al. (2000) confirmed that the res-
veratrol content in the skins of red grape varieties to be higher than in white grape varieties and found 356 and 1706 mg/ 100 g of fresh weight in the skins of Chardonnay and Tempranillo varieties, respectively. It is important to note that in our work, the extraction of resveratrol was carried out on the by-product of Jerez-Xerez-Sherry wine production process. Hence, the resveratrol content would depend on the pressure applied in this press process. During this step, a large amount of the grape compounds, essentially from the skins, is transported to the must and this increases on increasing the pressure of the press process. In the production of Jerez-Xerez-Sherry wines, the press process was continued until the pomace was exhausted. Consequently, high levels of compounds are transferred to the must. Nevertheless, the amount of resveratrol obtained with SC-CO2 is comparable to those obtained by other authors. The values obtained from the skins and the pomace are higher than the results obtained by Ector et al. (1996) and Pascual-Martí et al. (2000) for the Chardonnay variety, and by Tünde et al. (2009) using conventional methods and SC-CO2 extraction.
4. Conclusion Grape pomace of Palomino fino is a potential source of resveratrol. SC-CO2 extraction under optimized conditions ensures its
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extraction, thus allowing the final by-product to be reused for other activities. Acknowledgements The authors thank the Junta de Andalucia for financial support (Project PAI05-TEP-00231), which enabled this work to be carried out. References Baur, J.A., Pearson, K.J., Price, N.L., Jamieson, H.A., Lerin, C., Kalra, A., Prabhu, V.V., Allard, J.S., Lopez-Lluch, G., Lewis, K., Pistell, P.J., Poosala, S., Becker, K.G., Boss, O., Gwinn, D., Wang, M., Ramaswamy, S., Fishbein, K.W., Spencer, R.G., Lakatta, E.G., Le Couteur, D., Shaw, R.J., Navas, P., Puigserver, P., Ingram, D.K., de Cabo, R., Sinclair, D.A., 2006. Resveratrol improves health and survival of mice on a highcalorie diet. Nature 444, 337–342. Berna, A., Cháfer, A., Montón, J.B., 2001. High-pressure solubility data of the system resveratrol (3) + ethanol (2) + CO2 (1). J. Supercrit. Fluids 19, 133–139. Bradamante, S., Barenghi, L., Villa, A., 2004. Cardiovascular protective effects of resveratrol. Cardiovasc. Drug Rev. 22, 169–188. Chafer, A., Pascual-Martí, M., Salvador, A., Berna, A., 2005. Supercritical fluid extraction and HPLC determination of relevant polyphenolic compounds in grape skin. J. Sep. Sci. 28, 2050–2056. Cho, Y., Hong, J., Chun, H., Lee, S., Min, H., 2006. Ultrasonication-assisted extraction of resveratrol from grapes. J. Food Eng. 77, 725–730. de Campos Luanda, M.A.S., Fernanda, V.L., Rozangela, C.P., Sandra, R.S., 2008. Free radical scavenging of grape pomace extracts from Cabernet sauvingnon (Vitis vinifera). Bioresour. Technol. 99 (17), 8413–8420. De Revel, G., Hogg, T., Santos, C., 1996. Analyse du cis- and trans-resveratrol dans les vins produits au Portugal. J. Int. Sci. Vigne Vin. 30, 31–37. Domínguez, C., Guillén, D.A., Barroso, C.G., 2001. Automated solid-phase extraction for sample preparation followed by high-performance liquid chromatography with diode array and mass spectrometric detection for the analysis of resveratrol derivatives in wine. J. Chromatogr. A. 918, 303–310. Dourtoglou, V.G., Makris, D.P., Bois-Dounas, F., Zonas, Ch., 1999. trans-Resveratrol concentration in wines produced in Greece. J. Food Comp. Anal. 12, 227–233. Ector, B.J., Magee, J.B., Hegwood, C.P., Coign, M.J., 1996. Resveratrol concentration in Muscadine berries, juice, pomace, purees, seeds and wines. Am. J. Enol. Vitic. 47 (1), 57–62. Feijóo, O., Moreno, A., Falqué, E., 2008. Content of trans- and cis-resveratrol in Galician white and red wines. J. Food Comp. Anal. 21, 608–613.
Mantell, C., Rodríguez, M., Martínez de la Ossa, E., 2003. A screening analysis of the high-pressure extraction of anthocyanins from red grape pomace with carbon dioxide + co-solvent. Eng. Life Sci. 3 (1), 38–42. Martínez-Ortega, M.V., Carcí-Parrilla, M.C., Troncoso, A.M., 2000. Resveratrol content in wines and musts from the south of Spain. Nahrung 44 (4), 253–256. Palma, M., Taylor, T., 1999. Statistical design for optimization of extraction of polyphenols from an inert matrix using carbon dioxide-based fluids. Anal. Chim. Acta 391, 321–329. Pascual-Martí, M.C., Salvador, A., Chafer, A., Berna, A., 2000. Supercritical fluid extraction of resveratrol from grape skin of Vitis vinifera and determination by HPLC. Talanta 54, 735–740. Pascual-Martí, M., Salvador, A., Chafer, A., Berna, A., 2001. Supercritical fluid extraction of resveratrol from grape skin of Vitis vinifera and determination by HPLC. Talanta 54, 735–740. Pezet, R., Cuenat, P.H., 1999. Resveratrol in wine: extraction from skin during fermentation and post-fermentation standing of must from Gamay grapes. Am. J. Enol. Vitic. 47, 287–290. Rodríguez-Delgado, M.A., González, G., Pérez-Trujillo, J.P., García-Montelongo, F.J., 2002. trans-Resveratrol in wines from the Canary Islands (Spain). Analysis by high performance liquid chromatography. Food Chem. 47, 2666–2670. Roldán, A., Palacios, V., Caro, I., Pérez, L., 2003. Resveratrol content of Palomino fino grapes: influence of vitage and fungal infection. J. Agric. Food Chem. 51, 1464– 1468. Romero-Pérez, A.I., Lamuela-Raventós, R.M., Buxaderas, S., de la Torre-Boronat, M.C., 1996. Resveratrol and piceid as varietal markers of white wines. J. Agric. Food Chem. 44 (8), 1975–1978. Romero-Pérez, A., Lamuela-Raventós, R.M., Andrés-Lacueva, C., de la Torre-Boronat, M.C., 2001. Method for the quantitative extraction of resveratrol and piceid isomers in grape berry skins. Effect of powdery mildew on the stilbene content. J. Agric. Food Chem. 49, 210–215. Sun, B., Ribes, A.M., Leandro, M.C., Belchior, A.P., Spranger, M.I., 2006. Stilbenes: quantitative extraction from grape skins, contribution of grape solids to wine and variation during wine maturation. Anal. Chim. Acta 563, 382–390. Tena, M.T., Rios, A., Valcarcel, M., 1998. Supercritical fluid extraction of t-resveratrol and other phenolics from a spiked solid. Fresenius J. Anal. Chem. 361, 143–148. Threlfall, R.T., Morris, J.R., Mauromoustakeos, A., 1999. Effect of variety, ultraviolet light exposure, and enological methods on the trans-resveratrol level of wine. Am. J. Enol. Vitic. 50, 57–64. Tünde, V., Mojca, S., Zˇeljko, K., 2009. Extraction of phenolic compounds from elder berry and different grape marc varieties using organic solvents and/or supercritical carbon dioxide. J. Food Eng. 90 (2), 246–254. Valenzano, D., Terzibasi, E., Genade, T., Cattaneo, A., Domenici, L., Cellerino, A., 2006. Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate. Curr. Biol. 16, 296–300.
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Extraction of resveratrol from the pomace of Palomino fino grapes by supercritical carbon dioxide L. Casas, C. Mantell *, M. Rodríguez, E.J. Martínez de la Ossa, A. Roldán, I. De Ory, I. Caro, A. Blandino Department of Chemical Engineering, Food Technology and Environmental Technologies, Faculty of Science, University of Cadiz, Box 40, 11510 Puerto Real, Cadiz, Spain
a r t i c l e
i n f o
Article history: Received 29 May 2009 Received in revised form 22 July 2009 Accepted 4 August 2009 Available online 9 August 2009 Keywords: Resveratrol Supercritical carbon dioxide extraction Palomino grape HPLC
a b s t r a c t Resveratrol is a phenolic compound that is present in grapes and has significant benefits for human health. The development of methods to obtain concentrates of this compound is currently a major challenge in the food industry. In the work described here, resveratrol from grape seeds, stems, skin and pomace of the Palomino fino grape variety was extracted by supercritical carbon dioxide extraction. The effect of pressure (100, 400 bar), temperature (35, 55 °C) and the addition of modifier (5% v/v of ethanol) was evaluated to identify optimal resveratrol extraction from this by-product. Extraction yields and concentrations of resveratrol in the extracts were determined. The best results were obtained on working at high pressure and low temperature using 5% v/v ethanol as a co-solvent. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Phenolic compounds represent an important group of micronutrients present in the plant world, and which form a part of the human food and animal feed. In particular, trans-resveratrol (3,5,40 -trihydroxystilbene) has gained significant worldwide attention due to its ability to inhibit or retard a wide variety of diseases in animals (Baur et al., 2006), including cardiovascular disease and cancer (Bradamante et al., 2004), and to increase stress resistance and lifespan (Baur et al., 2006; Valenzano et al., 2006). Resveratrol is commonly found in grape skins and seeds. Its content in wine depends on the grape variety, the mechanical pre-treatment (crushed and pressed) and the vinification process. Generally, the concentration is higher in red than in white wines, since the must is fermented with the skins (Romero-Pérez et al., 1996). Furthermore, significant differences can be found in the resveratrol content from one vintage to another (Threlfall et al., 1999), from different vineyard locations, and as a result of different weather patterns. Among the white wines, Greek and Portuguese wines are reported to contain the lowest levels of 0.03–0.14 mg/L (Dourtoglou et al., 1999) and 0.01–0.51 mg/L (De Revel et al., 1996) resveratrol, respectively. Resveratrol contents between 0.8 and 8 mg/L in Spanish white wines from ‘‘D.O. Rías Baixas” and the Galician wines are particularly notable (Rodríguez-Delgado et al., 2002; Feijóo et al., 2008).
* Corresponding author. Tel.: +34 956 016458; fax: +34 956 016411. E-mail address: [email protected] (C. Mantell). 0260-8774/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2009.08.002
In recent years, newer techniques such as extraction by supercritical fluids (SFE), extraction by pressurized liquids (PLE), and extraction assisted by microwave irradiation (MAE) have replaced conventional techniques like Soxhlet extraction for solid samples. These alternative techniques simplify the process, considerably reduce the consumption of solvents, and also increase the rate of the extraction process. Although SFE is limited to compounds of low or medium polarity, literature reports on extraction of polyphenols by SFE in the presence of organic solvent modifiers are available (Mantell et al., 2003). There are numerous reports on the recovery of polyphenolic compounds from different varieties of grapes by SFE. Palma and Taylor (1999) applied this technology to separate eight polyphenolic compounds on spiked inert matrices using ethyl acetate or methanol as a modifier. Tena et al. (1998) used CO2 modified with 5% (v/v) methanol and the extraction parameters were 50 °C and 350 bar. Pascual-Martí et al. (2000) investigated the supercritical fluid extraction process of resveratrol from grape skins of Vitis vinifera, and found extraction at 40 °C, 150 bar, with 7.5% (v/v) ethanol as modifier to be optimum. Berna et al. (2001) showed the influence of process conditions on the solubility of resveratrol in supercritical carbon dioxide and ethanol. These authors suggested the use of 7.5% ethanol as co-solvent and elevated pressure to carry out the extraction. Cho et al. (2006) used an ultrasonication-assisted method in order to search an effective extraction of resveratrol from grape. This methods exhibited more efficiency than the conventional solvent extraction with ethanol/water (80:20%, v/v) maintained at 60 °C for 30 min. In the new methods, the recovery of resveratrol
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increase by 24–30%, compared with the conventional solvent extraction. Grape pomace is an industrial waste from the wine process, and consists of grape seeds, skin and stems. Components such as resveratrol remain in the pomace at concentrations that are dependent on the wine manufacture process. de Campos et al. (2008) used supercritical fluid extraction to extract grape pomace from the production of Cabernet sauvignon vintage. These authors use as solvent SC-CO2 and analyze the effect of the addition of ethanol as co-solvent at 150 bar and 40 °C. The percents of co-solvent in the solvent were 10%, 15% and 20% w/w. The highest yields of the extracts were obtained by Soxhlet extraction using ethanol (13.2% w/w), butanol (12.2% w/w), and also by SFE with 15% ethanol (9.2% w/w). Three varieties of grape marc, native to Slovenia (Refošk, Merlot and Cabernet), were studied by Tünde et al. (2009). These authors found a mixture of organic solvent and water at 60 °C to be the most efficient in single-step extractions, and the pre-treatment with SC-CO2 (with or without ethanol as co-solvent) to improve the extraction of polyphenols from the grape marc. This method provides an alternative to the pre-treatment of the plant materials and replaces toxic organic solvents. Palomino grape represents 95% of grape production in the Jerez region (Cádiz, Spain) and this is dedicated to the production of generous wines ‘‘D.O. Jerez-Xerez-Sherry”. The pressing of this grape produces approximately 15–20% of pomace, and this is usually used as a fertilizer. Resveratrol has not been detected in the Jerez wines (Domínguez et al., 2001) or has been detected at very low concentrations (0.03 mg/L) (Martínez-Ortega et al., 2000). However, Roldán et al. (2003) detected its presence in the Palomino fino grape. Its content in the grape skins varies from 2.59 and 4.51 mg/ kg depending on the vintage, and is increased on infection of the grapes by the fungus, Botrytis cinerea. The present work looks into the viability of using SC-CO2 for the recovery of resveratrol from the pomace of the Palomino fino grape press process. Experiments were designed using different parts of the grape pomace, stems, skin and seeds as raw materials. The effect of temperature, pressure and modifier concentration of the SC-CO2 on the total extraction yield, and on the recovery of resveratrol in the extract was evaluated.
2. Materials and methods 2.1. Sampling and chemicals White grape pomace from the Jerez-Xerez-Sherry area (Palomino fino variety) was used as the raw material. Samples of freshly pressed white grape were collected from a local wine cellar and placed in an oven for 48 h at 60 °C to obtain a dry solid. The resulting solid was manually separated into four fractions: whole grape pomace, seeds, stems and skin. Each fraction was pulverizing with hammer mill to obtain a particle size distribution adequate. Subsequently, each sample was vacuum-packed using household vacuum equipment (Professional Family, Orved, Italy). The mean particle size of each fraction was as follows: 0.165 ± 0.102 mm for stem, 0.261 ± 0.164 mm for skins and 0.319 ± 0.239 mm for seeds. The pomace sample showed the following distribution of weight fractions: 47.9% of skin, 43.7% of seeds, 8.4% of stem and mean particle size of 0.408 ± 0.375 mm. The packed samples were stored at room temperature under darkness until used. The carbon dioxide (99.995%) used was provided by Carburos Metalicos (Barcelona, Spain). Standard of trans-resveratrol (99%) was provided by Sigma–Aldrich and the other reagents (ethanol, methanol, hydrochloric acid and acetic acid, all with a purity of HPLC gradient grade) by Panreac.
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2.2. Extraction at high pressure The extractions were carried out in an Isco extractor (Nebraska, USA, model SFX220). The equipment consisted of one extractor with a maximum capacity of 10 mL and 2 lm filters at the inlet and outlet to avoid haulage of the sample. The extractor was also fitted with a thermostatic system that allowed the extraction to be carried out at a constant temperature. The solvent was introduced by syringe pumps (Isco, Nebraska, USA, model 260D and 100DX), which allowed a constant pressure. The samples left the vessel through a micrometric valve, which was thermostated to avoid obstructions at the exit due to the solidification of CO2. The automatic control of the equipment make possible to work with the pumps at different programs. In this case, a consolvent program is used that make possible to add a constant percent of flow-rate of one pump regarding to the other pump. The flow-rate was automatically measured by the program based in the movement of the piston inside of the syringe pump. This flow-rate was measured in volumetric units at operation pressure and 20 °C of temperature. The extraction cartridge was loaded with approximately 4.6 g of sample which had previously been homogenized to maintain a constant apparent density in all experiments, and then introduced into the extractor to reach the operating temperature. The pumps were loaded with carbon dioxide and ethanol until the operating pressure was reached. The automatic decompression valves of the extractor were then closed. The valves connecting the pumps were opened so as to open the extractor. The extractor was then pressurized with CO2 and ethanol. When a balanced state was attained, the micrometric valve was opened until a constant flow of 0.8 g/min was achieved. In order to achieve complete extraction of the substances in question, a relatively long extraction time was used (3 h). The extracts were collected in glass tubes containing methanol and analyzed by high-performance liquid chromatography (HPLC). Extreme conditions of pressure were tested, i.e. 100 and 400 bar. Experiments were carried out at relatively low temperatures between 35 and 55 °C because, according to many authors (Chafer et al., 2005; Pascual-Martí et al., 2001; Romero-Pérez et al., 2001), the yields of extraction of resveratrol decreases at higher temperatures and prolonged extraction times. Experiments were also carried out with 5% volume of ethanol as co-solvent, since this is the most commonly used in the SC-CO2 extraction of natural products. The results shown are averages of these two independent experiments with a reproducibility of approximately 7.8% CV (coefficient of variation). 2.3. Conventional extraction The methodology described by Roldán et al. (2003) was used for the conventional extraction. Samples of 25 g were macerated with 50 mL of a mixture of methanol/HCl (0.1%) for 30 min in an ultrasonic bath. After the extraction, the sample was centrifuged and filtered [0.45 lm Teflon syringe filters (Millex-LCR, Milipore, Bedford, MA)] prior to analysis by direct injection on HPLC. 2.4. Determination by HPLC The HPLC equipment consisted of an Agilent Technologies 1100 Series chromatograph, with UV–visible detector, auto sampler and PC software for the control and processing of the chromatographic data. The method used was based in the published by PascualMartí et al. (2000). This method is adequate for quantification of cis- and trans- isomers of resveratrol and their glucosides. The column (250 mm 4.6 mm) was a C18 Hypersil ODS (5 lm particle size) (Supelco). The solvent used was water:methanol:acetic acid
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(75:20:5). The flow-rate was set to 1.5 mL/min and 20 lL of filtered extract was injected for each sample. Detection was carried out at a wavelength of 306 nm. Trans-resveratrol was identified by comparison of its retention times with that of the commercial standard (Sigma) and was quantified by means of a calibration curve. The calibration curve was as follow:
A ¼ 55:176C þ 31:192
ð1Þ
where A is the area expressed in mAu and C is the concentration expressed in mg/L. The correlation coefficient (R) was 0.9999. The experiments on each extraction were carried out in triplicate in order to evaluate the variability of the measurements. The results are shown as the average of all the independent analyses with a reproducibility of approximately 2.5% CV (coefficient of variation). A typical chromatogram of the samples is included in Fig. 1. 3. Results and discussion In SFE, the solvating power of the fluids can be manipulated by changing pressure and/or temperature so as to achieve a remarkably high selectivity. This tuneable solvating power of SFE is particularly useful for the extraction of complex samples such as plant materials. SC-CO2 is not very suitable for the extraction of polar analytes. This problem is often tackled by use of modifiers. Depending on the type of sample matrix and the affinity of the analyte for the matrix, the modifier may influence the extraction in three different ways: (1) increase the solubility of the analyte in the supercritical fluid as a result of analyte–modifier interactions in the fluid phase; (2) facilitate analyte desorption – the molecules of polar modifiers are able to interact with the matrix and compete efficiently with the analyte for the active sites in the matrix; (3) distort the matrix–analyte diffusion process and favour penetration of the supercritical fluid into the matrix when the modifier swells the matrix. The results for extraction yields are presented in Tables 1–3. The maximum extraction yields of pomace and its individual components were obtained on addition of 5% volume of ethanol as cosolvent to the SC-CO2. This is attributed to the modification of the properties of the carbon dioxide – a change that enables this fluid to extract the lipophilic and the hydrophilic compounds. The fluid pressure is an essential parameter in SC-CO2 extraction, since fluid density is directly related to pressure. It can be observed from Table 1 that at constant temperature, an increase in the pressure increased the extraction yield. The density of carbon dioxide is higher as higher pressure, and consequently, the solvency of SC-CO2 also increases. Berna et al. (2001) also suggest
the extraction at elevated pressure to be advantageous. These results are consistent with those reported in the bibliography by other authors (Pascual-Martí et al., 2000 and Palma and Taylor, 1999). Temperature also has a significant effect on the recovery of components in SC-CO2. According to the rules of kinetics, if the other variables are constant, an increase in temperature is related to a more intensive thermal motion of solutes in the active sites of the matrix. This situation is beneficial for the solutes to overcome the adsorbing energy forces on the matrix and to be desorbed more efficiently from the active sites by SC-CO2 at higher temperature. From a thermodynamic point of view, the saturated vapour pressure increases with a corresponding increase in the temperature, thus enabling the solutes to dissolve in SC-CO2 more easily. Moreover, the density of SC-CO2 decreases with an increase in temperature, thus decreasing the solvency of SC-CO2. These three effects compete with one another during the extraction process. The effect of temperature on the extraction yield also is shown in Table 1. At 100 bar, an increase in the temperature from 35 to 55 °C did not have a significant effect on the extraction yield. In some cases, a decrease in yield was observed. This was seen in SC-CO2, both with and without the addition of co-solvent. This behaviour is attributed to compensation between factors with an increase in temperature. Nevertheless, at a higher pressure of 400 bar, an increase in the temperature was beneficial in the extraction. This effect is more marked when co-solvent is not added to the system, with increases in the extraction yields of 153%, 60%, 150% and 250% for seed, stem, skin and grape pomace, respectively, compared to the yields obtained at 100 bar. The resveratrol concentration in the extracts obtained (mg resveratrol/g extract) are shown in Table 2. It is evident that the use of ethanol as co-solvent with SC-CO2 is more selective than the process using SC-CO2 alone. This is due to the influence of the co-solvent on the solubility of the solute, and therefore on the extraction yield of the process. On the other hand, pressure and temperature appeared to have a less marked influence on resveratrol content. Nevertheless, the use of high pressure (400 bar) and low temperature (35 °C) was desirable for the extraction. Best results were obtained with grape skins as compared to the other raw materials. Table 3 clearly shows the recovery of resveratrol from the raw material to be optimum at 400 bar, 35 °C and 5% Ethanol. SC-CO2 without the addition of co-solvent at low pressure (100 bar) was not selective for resveratrol. In fact, resveratrol was not detected under all the conditions analyzed at this pressure (35, 55 °C). An increase in the pressure to 400 bar improved the extraction of resveratrol. However, it is necessary to add a co-solvent in order to increase the extraction yields significantly.
Fig. 1. Chromatograms of the sample, the retention time were 19.487 and 21.652 min for trans-resveratrol and cis-resveratrol, respectively.
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L. Casas et al. / Journal of Food Engineering 96 (2010) 304–308 Table 1 Total extraction yields expressed as mg of extract/100 g of dry sample. Extraction conditions CO2
100 bar 400 bar
CO2 + EtOH
100 bar 400 bar
Seed
Stem
Skin
Grape pomace
35 °C 55 °C 35 °C 55 °C
132 90 650 1648
129 80 320 513
123 70 590 1445
165 90 430 1514
35 °C 55 °C 35 °C 55 °C
1223 1190 1390 2122
489 500 800 1026
1556 1600 2300 2805
1288 760 1500 2176
Table 2 Extraction yields of resveratrol expressed as mg of resveratrol/100 g of dry sample. Extraction conditions CO2
100 bar 400 bar
CO2 + EtOH
100 bar 400 bar
Seed
Stem
Skin
Grape pomace
35 °C 55 °C 35 °C 55 °C
– – 0.2 0.3
– – 0.6 0.6
– – 0.5 0.4
– – 0.4 0.3
35 °C 55 °C 35 °C 55 °C
6.1 5.0 8.3 11.1 –
0.7 – 0.9 0.7 1.7
13.1 14.0 49.1 45.5 3.1
10.1 7.0 16.1 19.2 0.9
Conventional extraction (–) Not detectable.
Table 3 Concentration of resveratrol in the extract (mg resveratrol/g extractor). Extraction conditions CO2
100 bar 400 bar
CO2 + EtOH
100 bar 400 bar
Seed
Stem
Skin
Grape pomace
35 °C 55 °C 35 °C 55 °C
– – 0.31 0.18
– – 1.87 1.17
– – 0.84 0.27
– – 0.93 0.19
35 °C 55 °C 35 °C 55 °C
4.99 4.20 5.97 5.23
1.43 – 1.12 0.68
8.42 8.75 21.35 16.22
7.84 9.21 10.73 8.82
(–) Not detectable.
The recovery of resveratrol using SC-CO2 was higher than conventional extraction with a solvent mixture at atmospheric pressure [Methanol:HCl (0.1%)]. SC-CO2 extraction enabled resveratrol to be obtained from seeds, which was not possible by conventional methods. This behaviour can be attributed to the higher diffusion properties of SC fluids regarding conventional solvents. This fact allows to the SC solvent to reach some active sites of the solid matrix inside the seed that the conventional solvent cannot reach. These diffusional problems do not happen in the rest of the parts of the pomace where the solute is accessible to the conventional solvent. The extract obtained from the skins had higher levels of resveratrol than that obtained from seeds and stems. The amount of resveratrol in grape pomace, therefore, depends on the proportion of skin, seeds and stems in the whole by-product. Although literature on the recuperation of resveratrol from Palomino fino grape pomace has not been published previously, that from other varieties of grape skins, seeds and pomace have been analyzed. For example, Pezet and Cuenat (1999) found resveratrol at 0.68 and 0.39 mg/100 g of fresh weight in the skin and grape pomace, respectively. The values in the Muscadine variety were 1.17 and 5.32 mg/100 g of dry weight in the skin and pomace, respectively (Ector et al., 1996). In the case of red varieties, Sun et al. (2006) reported resveratrol levels of 1455, 68 and 656 mg/100 g of dry weight in stems, seeds and skin, respectively. Pascual-Martí et al. (2000) confirmed that the res-
veratrol content in the skins of red grape varieties to be higher than in white grape varieties and found 356 and 1706 mg/ 100 g of fresh weight in the skins of Chardonnay and Tempranillo varieties, respectively. It is important to note that in our work, the extraction of resveratrol was carried out on the by-product of Jerez-Xerez-Sherry wine production process. Hence, the resveratrol content would depend on the pressure applied in this press process. During this step, a large amount of the grape compounds, essentially from the skins, is transported to the must and this increases on increasing the pressure of the press process. In the production of Jerez-Xerez-Sherry wines, the press process was continued until the pomace was exhausted. Consequently, high levels of compounds are transferred to the must. Nevertheless, the amount of resveratrol obtained with SC-CO2 is comparable to those obtained by other authors. The values obtained from the skins and the pomace are higher than the results obtained by Ector et al. (1996) and Pascual-Martí et al. (2000) for the Chardonnay variety, and by Tünde et al. (2009) using conventional methods and SC-CO2 extraction.
4. Conclusion Grape pomace of Palomino fino is a potential source of resveratrol. SC-CO2 extraction under optimized conditions ensures its
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