RECOVERY OF ANTIOXIDANTS FROM GRAPE PRODUCTS BY USING SUPERCRITICAL FLUIDS AND MEMBRANE TECHNOLOGY

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1 ISSN: RECOVERY OF ANTIOXIDANTS FROM GRAPE PRODUCTS BY USING SUPERCRITICAL FLUIDS AND MEMBRANE TECHNOLOGY Sagrario Beltrán*, María Teresa Sanz, Belén Santamaría, Ruth Murga, and Gonzalo Salazar Area of Chemical Engineering. Department of Biotechnolog and Scinces of Foods. University of Burgos. Plaza Misael Bañuelos s/n Burgos. Spain. Tel , ABSTRACT Polyphenols are a wide variety of compounds that occur, not only in wine and grapes, but also in many other fruits and vegetables, tea, extra virgin olive oil, chocolate and other cocoa products, etc. Second only to carbohydrates, they are the most abundant type of compounds among plants. Their interest in nutrition and pharmacy has been widely reported and hence, the interest on their isolation has arisen. In this work, we review two technologies that have been studied in our laboratory to extract, isolate and purify different phenolic compounds found in grapes. The first of these technologies is supercritical fluid extraction. Carbon dioxide is the most widely used supercritical fluid. Specifically, it has also been the most studied supercritical solvent to extract phenolic compounds from different subtracts. However, most of the phenolic compounds are poorly solved in pure CO 2, and most of the work reported use co-solvents, such as low molecular weight alcohols, to improve the solubility of the solute in the supercritical phase, and therefore, the extraction efficiency. The main inconvenient of using co-solvents, usually liquids under atmospheric conditions, is that an additional stage is needed at the end of the process to separate the co-solvent from the solute. This could be avoided by using other supercritical solvents that, being also gases under atmospheric conditions, could dissolve polyphenols better than CO 2 (non-polar solvent) and therefore could lead to more efficient extraction processes. Some membrane technologies have also been studied with the aim to purify some of the phenolic compounds previously extracted by using conventional liquid solvents. Complex phenolic compounds, such as proanthocyanidins, are essentially polymer chains of flavonoids, mainly catechins. Therefore, technologies such as microfiltration, ultrafiltratrion and nanofiltration, can be used for separation by size, related to its molecular weight. The results show that separation of different phenolic compounds of different molecular weight can be performed efficiently by membrane technologies. KEYWORDS Complex phenols, grapes, grape seeds, SC-CO 2 extraction, membrane technologies.

2 INTRODUCTION Antioxidants have been shown to have a wide range of positive biological effects. During the last decades, many studies have shown that the dietary intake of natural antioxidants can neutralize the action of harmful free radicals and may be an important defense mechanism of our body against oxidative stress [1-2]. Grapes, wine and other grape products are rich sources of antioxidants with a high potential for the prevention and treatment of human malignancies. The key components responsible for these beneficial effects are widely considered to be complex phenols, which have been reported to possess various potent biological activities such as antioxidant, antiviral, enzyme inhibiting, antitumor and anti-hiv activities [3-5]. Complex phenols posses a high antioxidant activity and great ability as free radical scavengers [6-7]. Many of their biological actions have been attributed to their intrinsic reducing capabilities, although this concept appears now to be a simplistic way to conceive their activity, and the true mechanism is being investigated [2]. The properties mentioned above give to natural complex phenols a great potential as active principles in the pharmaceutical industry and as antioxidants in the food industry. There is, therefore, an increasing interest in isolating these compounds from their natural matrices. One of the separation technologies that are being explored to achieve such separation is supercritical fluid extraction (SFE). SFE with carbon dioxide is under consideration for separation of natural substances from complex materials at industrial scale due to the mild operation conditions needed and other environmental benefits [8-9]. Also membrane technologies have been shown to be suitable for separation of polyphenols from other not so interesting compounds and for fractionation of the different types of polyphenols [10-12]. Recovery of polyphenols from grapes or grape products by using supercritical fluids The efficient design of a supercritical fluid extraction (SFE) process requires the knowledge of the solubility of the solutes to be extracted in the fluid to be used as solvent under supercritical conditions. Taking this into account, the first work carried our in our laboratory consisted in a literature search of the solubility of different phenolic compounds in supercritical carbon dioxide (SC-CO 2 ), the most widely used supercritical solvent due to their mild SC conditions and the fact that is considered a green solvent. Since reported solubility data are very scarce [13], we experimentally determined the solubility of some representative components of the phenolic fraction found in grapes. The solubility of some naturally occurring phenolic compounds, with a medium molecular weight, was determined in the first place: the compounds selected were main components in grape seeds, i.e.: gallic acid, (+)-catechin, and (-)-epicatechin. The solubility in pure carbon dioxide was so low, that the solute could not be detected in the supercritical phase even the method used to determine solubilities was a dynamic-analytical method with long contacting times; therefore, the solubility of such compounds in the mixture carbon dioxide + ethanol at 20 MPa and 313 K was determined. The values obtained were fairly low, of the order of 10-6 weight fraction, even when the amount of ethanol in carbon dioxide was 10% (v/v) [14]. Additionally, the solubility of some low molecular weight phenolic compounds, in pure SC-CO 2, was determined in the temperature range from 313 K to 333 K and in the pressure range from 8.5 MPa and 50 MPa. These compounds were: 3,4-dihydroxy benzoic acid (protocatechuic acid), methyl 3,4,5-trihydroxybenzoate (gallic acid methyl ester or 3271

3 methyl gallate), 3,4-dihydroxy benzaldehyde (protocatechualdehyde), 4-hydroxycinnamic acid (p-coumaric acid), 3,4-dihydroxycinnamic acid (caffeic acid), 4-hydroxy-3- methoxycinnamic acid (ferulic acid), 4-hydroxy-3,5-dimethoxybenzoic acid (syringic acid) and 4-hydroxy-3-methoxybenzoic acid (vanillic acid) [15-17]. Their chemical structure is reported in Table 1. Table 1.- Chemical structure of the low molecular weight phenolic compounds whose solubility in SC-CO 2 has been experimentally determined. Solute Formula R 1 -group R 2 -group R 3 -group Vanillic acid C 8 H 8 O 4 H COOH OCH 3 Ferulic acid C 10 H 10 O 4 H CH=CH-COOH O-CH 3 Protocatechualdehyde C 7 H 6 O 3 H CHO OH Syringic acid C 9 H 10 O 5 OCH 3 COOH OCH 3 Methyl gallate C 8 H 8 O 5 OH COO-CH 3 OH p-coumaric acid C 9 H 8 O 3 H CH=CH-COOH H Protocatechuic acid C 7 H 6 O 4 H COOH OH Caffeic acid C 9 H 8 O 4 H CH=CH-COOH OH Chemical structure of the different solutes All the compounds are solids and their solubility was experimentally determined by the dynamic analytical method in an apparatus that has been previously described [15]. Sample analysis was carried out offline by HPLC equipped with a diode array detector (DAD, Hewlett-Packard 1100 series). The coupling of chromatography and DAD would allow the detection of the compounds degradation that could take place, or some of the impurities of the pure solute that could be preferentially solved by the SC CO 2. The experimental solubility was determined by carrying out at least 5 experiments in which the total amount of SC-CO 2 that flowed through the cell was varied, hence varying the total amount of solute dissolved. The plot of the amount of solute dissolved vs. the amount of CO 2 used for its dissolution is a linear function whose slope is the solubility at the temperature and pressure at which the operation was carried out. All the data were thus obtained with a standard error that is represented for each experimental data in Figure 1. As can be observed in Figure 1, the solubility was found to be of the order of 10-5 to 10-7 molar fraction, depending on the nature of the compound, pressure and temperature. The solubility increased with pressure, at constant temperature, in all cases. The effect of temperature, however, was more complex and retrograde solubility behaviour (crossover pressure around MPa) was observed for all the compounds except for caffeic acid [15-17]. The solubility increases with temperature at pressures above the crossover pressure, while at pressures below the crossover pressure, the solubility decreases with increasing temperature. Observing these fairly low solubility data, it can be concluded that SFE of phenolic compounds from grapes, will only be efficient if co-solvents are used together with carbon dioxide. However, it would be much more convenient to avoid the use of co-solvents: when co-solvents are used, they have to be removed from the extract at the end of the process, as when performing extraction with liquid solvents. This way, part of the most interesting advantages of SFE is lost. 3272

4 Figure 1.- Experimental solubility (expressed as solute mol fraction, y) of some low molecular weight phenolic compounds contained in grapes, at 333 K. The error bars represent the standard error of each solubility datum. Continuous lines represent the solubility isotherms calculated with the Peng Robinson equation of state. Once the solubility behaviour of complex phenols in supercritical carbon dioxide was known, some experimental work on SC-CO 2 extraction of complex phenols from natural matrices was carried out in our laboratory. The natural subtract used was defatted milled grape seeds (DMGS) supplied by a Spanish company that produces grape seed oil (Aceites Borges Pont S.A.). The results obtained showed that most of the complex phenols of interest contained in grape seeds can be extracted with a 15% (v/v) methanol [14]. Quantitative results are presented in Table 2. Table 2.- Phenolic profile of the original defatted milled grape seeds (mg of component / g of grape seeds) and amount of the individual phenols extracted by SFE at 313 K and 20 or 30 MPa. The supercritical fluid was a mixture of CO 2 with 5, 10 and 15% volume of methanol. mg of component extracted / 100 g of DMGS P = 20 MPa P = 30 MPa Rt, DMGS 5% 10% 15% 5% 10% 15% Compound name min mg/g (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) 11.1 Gallic acid Protocatechuic acid Monogalloyl glucose* Protocatechualdehyde (+)-Catechin (-)-Epicatechin * Tentative compound. Rt = Retention time in HPLC elution. 3273

5 The analytical procedure to quantify the polyphenols contained in a complex extract obtained from natural sources has been largely studied [18-19]. In this work, where the extract obtained by SFE was a multicomponent mixture of a large variety of polyphenols, the analysis was carried out with a HPLC equipped with MSD and DAD (Hewlett Packard, 1100 series) and the method used has been previously reported elsewhere [14]. Other possibility to recover the phenolic compounds contained in grapes, specifically in grape seeds, could be to extract grape seed oil by SFE. Grape seed oil presents a solubility in pure SC-CO 2 of 0.1 weight fraction [13], and it is efficiently extracted with SC-CO 2. The expectation was that the phenolic fraction could be co-extracted with the oil contained in grape seeds to obtain an oil rich with antioxidants. However, an evaluation of the phenolic content of the oil extracted by SFE, showed that such content was negligible. Again, extraction using co-solvents would surely extract both, oil and phenolic compounds. Recovery of polyphenols from grapes or grape products by using membrane technologies Since SFE with pure SC-CO 2 seems not to be an efficient procedure for extracting the most complex phenols contained in grapes and some co-solvents should be used, we studied the direct extraction with conventional liquid solvents, specifically a hydroalcoholic solution (methanol + water, 80/20) that extracted mainly the oligomeric proanthocyanidins (PAS), more interesting from the nutritional point of view than highly polymerized PAS. Proanthocyanidins are essentially polymer chains of flavonoids such as catechins, Figure 2, and their structure can be easily modified, being hydrolyzed when temperature increases. Due to this behaviour, conventional techniques, such as evaporation, are not suitable to concentrate them (thermal degradation may occur) and membrane technologies, such as microfiltration (MF), ultrafiltration (UF) and nanofiltration (NF) can be good alternative processes [11-12]. The recovery of proanthocyanidins is interesting due to their properties. Nutritionally, because of their interactions with proteins modify food adsorption and act as reinforcing structural proteins. Pharmacologically, because proanthocyanidins are natural antioxidants that protect against reactive oxygen species involved in arthritis and cardiovascular diseases [6-7]. Figure 2.- Chemical structure of the basic units that form the polymeric proanthocyanidins. There are several works on fractionation of vegetal extracts by membrane technology [12, 20-21], including some patents [22]; However, so far, fractionation of PAS from vegetal sources has only been carried out by elution of different solvents through a chromatographic column [23]. 3274

6 In this work, we have developed a sequence to purify different fractions of PAS, enriched in different molecular weight molecules, employing several cross-flow filtration sequences with polymeric membranes of different cut-off, Figure 3. I. Feed Diafiltration DS-5 (180 Da) Diafiltration TP = 14 bar J = 20 L/h m 2 II. Low molecular weight phenolic compounds YM100 (100 kda) TP = 0.7 bar J = 140 L/h m 2 Diafiltration III. Proteins, polysaccharides and highly condensed tannins YC05 (500 Da) Diafiltration TP = 7 bar J = 25.2 L/h m 2 Concentration MRC-1 (1 kda) DS-5 (180 Da) TP = 3.5 bar J = L/h m 2 TP = 20 bar J = L/h m 2 V. Oligomeric proanthocyanidins VI. Dimmer proanthocyanidins IV. Catechins Solvent I II VI III V IV Figure 3.- Fractions obtained by purification, fractionation and concentration of defatted milled grape seeds by membrane technology according to the sequence depicted above. The raw material used as feed was a hydroalcoholic extract from defatted milled grape seeds (DMGS). Several transmembrane pressures, concentrations and filtration designs (with or without diafiltration) were tested in plate and frame membrane modules to obtain the highest purity in the fractionated streams. Operational problems, as oxidation of the products obtained, were observed, thus, processing under inert atmosphere was developed to avoid oxidation of the extracts. A final sequence with four different membranes at pilot-plant scale was achieved. As indicated in Table 3, the different fractions where rich, in more than 90 %, in the different types of components. As an example, the chromatogram and composition of fraction II are presented in Table 4. Table 3 also shows that the polyphenols total content, as determined by the Folin Ciocalteau analysis, is largest in the fractions that contain largely polymerized polyphenols. The antioxidant capacity (Trolox equivalent) was also evaluated obtaining the highest value for the fraction concentrated in monomer catechins [20]. 3275

7 Table 3.- Yield, polyphenolic total content and antioxidant capacity of the different streams obtained through the membrane sequence depicted in Figure 3 Fraction obtained in the sequence depicted in Figure 3 mg mg mg dried equivalent Trolox / g fraction / gallic acid / g dried g DMGS dried fraction fraction I. Feed (whole phenolic extract) II. Low molecular weight phenolic compounds (acids and aldehydes) III. Proteins, polysaccharides and highly condensed tannins IV. Catechins (monomers) V. Oligomeric proanthocyanidins VI. Dimmer proanthocyanidins Table 4.- Composition and chromatogram of fraction II, mainly containing low molecular weight phenolic compounds (acids and aldehydes). mg / g of Phenolic compound detected components Rt* (min) 4.9 Gallic acid Protocatechuic acid Protocatechualdehyde Caffeic acid Syringic acid p-coumaric acid 76.9 Total acids and aldehydes quercetina kaemferol 8.2 Total Flavonols 35.7 Non identified components 25.8 * Rt: retention time in HPLC elution CONCLUSIONS Two technologies have been explored to isolate complex phenols from their natural matrices. Extraction, using supercritical carbon dioxide as solvent, has been essayed in the first place as a substitute of extraction with conventional solvents. The results showed that, due to the low solubility of complex phenols in supercritical carbon dioxide, supercritical extraction with pure carbon dioxide is not a good alternative to conventional solvent extraction. The addition of some co-solvent is necessary in order to obtain reasonable extraction yields. In a second try, conventional solvents were used to obtain whole extracts that were then fractionated by using membrane technologies. As conclusion, it can be stated that membrane technologies seem to be a good procedure for separation of the polyphenols contained in grape seeds to obtain fractions with different molecular weight, different polyphenolic content and different antioxidant activity. Fractionation by type of compounds and degree of polymerization seems also to be feasible. Polyphenols are rapidly oxidized and their processing should be carefully performed under inert atmosphere. The yield of this process indicated that it could be feasible to process the defatted milled grape seeds that are actually a waste of the seed oil industry. 3276

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