As described above, grape pomace is considered a valuable source of polyphenols. The biological and functional properties of these compounds are intensively studied. Over the years, researchers of different disciplines have investigated the mechanisms of chemoprevention, anticardiovascular disease and other disease prevention activities of grape
polyphenols, revealing that these compounds indeed possess appealing properties [20-23]. Therefore, grape pomace has a great potential to serve as a source of pharmaceutical compounds as well as of functional food ingredients.
The composition as well as the quality of grape pomace extracts are strongly dependant on the extraction technique, the solvent used, the origin of the raw material, its storage conditions and the pre-treatment applied [24-26]. The quality of an extract can be represented by its properties, i.e. its biological activities. Despite the generally recognized antioxidant ability of substances present in grapes, specific studies on the biological activities of grape pomace extracts obtained by SFE are still needed.
Comparing the properties of extracts obtained with different extraction methods is very useful for assessing the optimal extraction conditions, able to extract valuable compounds while maintaining their biological activity. Oliveira and coworkers studied the global extraction yield, antimicrobial activity and composition profile of grape pomace extracts (Merlot and Syrah varieties) obtained with different extraction methods and conditions: SFE with pure SCCO2 and with the addition of ethanol, at pressures up to 300 bar and
temperatures of 50 and 60°C, and Soxhlet extraction [27]. The main components of the extracts, analyzed by HPLC, were gallic acid, p-OH-benzoic acid, vanillic acid and epicatechin. All extracts were tested to assess their antimicrobial and anifungal activity on four bacterial strains (Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas
aeruginosa) and on three fungi strains (Candida albicans, Candida parapsilosis, Candida krusei). All extracts obtained by SFE were effective against S. aureus (inhibition zone >9
mm). Among the microorganisms studied, P. aeruginosa was the most resistant and only one SF extract (obtained at 250 bar and 60°C) gave a positive result (halo size >9 mm). Such extract was the only one effective against all bacteria tested (both Gram-positive and Gram- negative). Besides that, the extract obtained by SCCO2 at 150 bar and 60°C was also very
effective against the bacteria tested, except P. aeruginosa (7 mm halo size), while also being effective against the fungus C. albicans with a halo size of 21 mm. The extracts obtained by Soxhlet extraction, on the other hand, were effective only against ona bacterial strain, B.
cereus, with an inhibition zone of 14 mm. Moreover, SFE is performed in the absence of air
and light, which protects the bioactivity of the extracts [28]. The best performance of SFE in comparison with other extraction methods was also reported by Kitzberger et al. [29] for shiitake extracts and by Palma et al. for grape seed extracts [30].
The extracts were also submitted to the test of microdilution in culture broth to determine the minimum concentration able to inhibit the growth of certain microorganism, i.e., the minimum inhibition concentration (MIC). The supercritical extracts obtained from Merlot pomace by SFE at 300 bar and 50°C and at 150 bar and 50°C showed the lowest MIC values against S. aureus, 625±375 and 750 ± 250 g/mL, respectively. The extracts were more effective (lowest MIC values) against Gram-positive bacteria, mainly S. aureus, comparing to Gram-negative ones (E. coli and P. aeruginosa). Only the supercritical extract obtained from Merlot by SCCO2 (50 ◦C, 300 bar) showed moderate activity against E. coli and P.
aeruginosa, inhibiting their growth with a MIC value of 1000 g/mL, while all other extracts
behaved as weak inhibitors against the Gram-negative bacteria (MIC above 1600 g/mL). Thus, in general, supercritical extracts from grape pomace could be classified as moderate inhibitors against Gram-positive bacteria (S. aureus and B. cereus).
The best antifungal activity was presented by the SCCO2/200 bar/50°C extract,
were considered relevant in terms of quality aspects: the Merlot extract by SCCO2 at 200 bar
and 50°C, which presented medium inhibition power against all three fungi and the two Gram-positive bacteria tested; and the Merlot extract by SCCO2 at 300 bar and 50°C, a
moderate inhibitor against all bacteria tested. The antimicrobial activity could be related to the plant cultivation conditions since the active components are usually synthesized as a response to stress such as microorganisms attack or strong UV radiation [31-32]. Thus, the better antimicrobial results provided by the selected Merlot SF extracts (50°C at 200 bar and 300 bar) may be attributed to the phenolic substances identified such as gallic, p-OH-benzoic and vanillic acids, whose antimicrobial abilities are well documented [33-34]. Besides the above mentioned components, other bioactive substances may have participated as antimicrobial agents. In fact, natural extracts may be more beneficial than isolated constituents, because of the synergic positive interactions among compounds [35].
The antioxidant properties of natural extracts are currently considered the most appealing thanks to possible large scale applications in the food and cosmetic industries. In a recent work, the extraction of proanthocyanidins from grape pomace was conducted using SCCO2
[36]. The effect of pressure, flow rate and quantity of co-solvent were analyzed. The performance of the extractions was evaluated in terms of phenolic yield, proanthocyanidins content and antioxidant activity. SFE was compared with conventional SLE with methanol as the solvent. The highest total antioxidant activity for grape pomace extracts was obtained with SFE, employing a CO2 flow rate of 6 kg/h and 10% cosolvent (a ethanol-water mixture at
57% v/v ethanol). In such conditions, the total antioxidant activity, evaluated by the total free radical scavenger ability, was 8703 mgα tocopherol /100 g dried matter, increasing by 20% compared to the conditions employing a flow rate of 4 kg/h and 7.5% of cosolvent (7187 mgα tocopherol /100 g dried matter), and it was about 13-folds that of the methanol extract (678 mgα tocopherol /100 g dried matter). This finding suggests that different CO2 flow rate and
percentage of cosolvent, as well as the extraction methods, affect the extraction of phenols responsible for the antioxidant activity of the extracts. Both SF extracts presented a high level of total proanthocyanidins (703.7 and 630.2 mgcatechin /100 gdried matter respectively), compared to the methanol extract (159.0 mgcatechin /100 gdried matter). Moreover, it is interesting to note that flow rate and cosolvent amount have an influence on the proanthocyanidins fraction that is extracted (monomeric, oligomeric and polymeric fractions). Polymeric fractions of proanthocyanidins always showed the highest antioxidant activity. This can be attributed to the structure of polymeric flavan-3-ols characterized by the presence of several hydroxyl functions exhibiting a higher ability to donate a hydrogen atom and to support the unpaired electron as compared to the low molecular weight phenols [37]. Comparing SFE to SLE, proanthocyanidins could account for about 97% of the total antioxidant activity of SL extracts but only for about 60% in SF extracts. This indicates that SFE could extract not only selectively the proanthocyanidins, but a great amount of other antioxidant compounds, not extractable with conventional methods.
Such findings are in agreement with other works that compare different extraction methods [38-39]. Overall, SFE affords richer extracts, with higher number of separated compounds; the use of EtOH as co-solvent increases the antioxidant potential of the extract, if compared with pure CO2 extraction, due to the increase in solvent polarity; the supercritical
fluid is able to extract important compounds not detected in conventional extracts, such as oleic acid and phytol, used in cosmetic products.
In a recent work, grape pomace SF extracts were added to samples of fish oil, and experiments of accelerated oxidation were monitored by formation of dienes and trienes [40]. Fish oils are often using for enriching soup, milks, and other products because of their high content in ω-3 polyunsaturated fatty acids. For this reason, oils are readily oxidized and the addition of natural antioxidants as rosemary extracts, catechins from green tea or polyphenols is common [41-42]. SF extracts were able to prevent significantly trienes formation (and dienes formation to a minor extent), being promising for substituting the synthetic preservatives in food processing and manufacture. Results indicated that the most polar molecules were more effective for preventing fish oil oxidation, due to the fact that they are disposed in the active air/oil interphase.
C
ONCLUSIONThe wine industry is rapidly changing, with new regulations being applied, increasing worldwide demand but also new challenges due to the emergence of new producers on the global market. Residues of the wine industry have so far been considered mostly as a waste management issue, while in the future they will more and more be seen as valuable natural sources for biologically active compounds such as antioxidants, whose demand not only in the pharmaceutical sector, but also in the food and cosmetic industry, is steadily increasing. SFE is a promising methodology for the production of high-quality extracts. It is a green and safe technique that, although requiring initially high investments, could provide valuable alternatives to conventional extraction methods. Some parameters have already been identified as crucial for the quality of SF extracts (i.e. pressure, amount and nature of cosolvent), that, though the works studying their properties are still limited, are generally richer and of higher quality if compared to those obtained by conventional techniques.
R
EFERENCES[1] State of World Vitiviniculture situation, 37th World Congress of Vine and Wine. [2] Silva, ML; Macedo, AC; Malcata, FX. Steam distilled spirits from fermented grape
pomace. Food Sci. Technol. Int. 2000, 6, 285−300.
[3] Kammerer, D; Claus, A; Carle, R; Schieber, A. Polyphenol screening of pomace from red and white grape varieties (Vitis vinifera L.) by HPLC-DAD-MS/MS. J. Agric. Food
Chem. 2004, 52, 4360−4367.
[4] Kammerer, DR; Schieber, A; Carle, R. Characterization and recovery of phenolic compounds from grape pomace − a review. J. Appl. Bot. Food Qual. 2005, 79, 189−196.
[5] Pinelo, M; Arnous, A; Meyer, AS. Upgrading of grape skins: significance of plant cell- wall structural components and extraction techniques for phenol release. Trends Food
Sci. Technol. 2006, 17, 579−590.
[6] Iacopini, P; Baldi, M; Storchi, P; Sebastiani, L. Catechin, epicatechin, quercetin, rutin and resveratrol in red grape: content, in vitro antioxidant activity and interactions. J.
[7] Sánchez-Alonso, I; Jim nez-Escrig, A; Saura-Calixto, F; Borderías, AJ. Antioxidant protection of white grape pomace on restructured fish products during frozen storage.
LWT−Food Sci. Technol. 2008, 41, 42−50.
[8] Yu, J; Ahmedna, M. Functional components of grape pomace: their composition, biological properties and potential applications. Int. J. Food Sci. Technol. 2013, 48, 221−237.
[9] Spencer, JPE; Abd El Mohsen, MM; Minihane, AM; Mathers, JC. Biomarkers of the intake of dietary polyphenols: strengths, limitations and application in nutrition research. British Journal of Nutrition 2008, 99, 12–22.
[10] Rockenbach, II; Rodrigues, E; Gonzaga, LV; Caliari, V; Genovese, MI; de Souza Schmidt Gonçalves, AE; Fett, R. Phenolic compounds content and antioxidant activity in pomace from selected red grapes (Vitis vinifera L. and Vitis labrusca L.) widely produced in Brazil. Food Chemistry 2011, 127, 174–179.
[11] Xia, E; Deng, G; Guo, Y; Li, H. Biological activities of polyphenols from grapes.
International Journal of Molecular Science 2010, 11, 622–646.
[12] Montealegre, RR; Peces, RR; Vozmediano, JLC; Gascuen, JM; Romero, EG. Phenolic compounds in skins and seeds of ten grape Vitis vinifera varieties grown in a warm climate. Journal of Food Composition Analysis 2006, 19, 687–693.
[13] Arranz, S; Silvan, JM; Saura-Calixto, F. Non extractable polyphenols, usually ignored, are the major part of dietary polyphenols: a study on the Spanish diet. Molecular
Nutrition and Food Research 2010, 54, 1–13.
[14] Llobera, A; Canellas, J. Dietary fibre content and antioxidant activity of Manto Negro red grape (Vitis vinifera), pomace and stem. Food Chemistry 2007, 101, 659–666. [15] Beveridge, THJ; Girard, B; Kopp, T; Drover, JCG. Yield and composition of grape
seed oils extracted by supercritical carbon dioxide and petroleum ether: varietal effects.
Journal of Agricultural and Food Chemistry 2005, 53, 1799–1804.
[16] Rubio, M; Alvarez-Ort , M; Fernández, E; Pardo, JE. Characterization of oil obtained from grape seeds collected during berry development. Journal of Agricultural and Food
Chemistry 2009, 57, 2812–2815.
[17] Baydar, NG; Akkurt, M. Oil content and oil quality properties of some grape seeds.
Turk Journal of Agricultural Forum 2001, 25, 163–168.
[18] Goni, I; Martın, N; Saura-Calixto, F. In vitro digestibility and intestinal fermentation of grape seed and peel. Food Chemistry 2005, 90, 281–286
[19] Knez, Z; Markocic, E; Leitgeb, M; Primozic, M; Knez Hrncic, M; Skerget, M. Industrial applications of supercritical fluids: A review. Energy 2014, 77, 235-243. [20] Karthikeyan, K; Sarala Bai, BR; Devaraj, SN. Cardioprotective effect of grape seed
procyanidins on isoproernol-induced myocardial injury in rats. Journal of
Cardiovascular pharmacology 2009, 53, 109–115.
[21] Singh, T; Sharma, SD; Katiyar, SK. Grape proanthocyanidins induce apoptosis by loss of mitochondrial membrane potential of human non-small cell lung cancer cells in vitro and in vivo. PLoS ONE 2011, 6, e27444.
[22] Zhang, F; Shi, JS; Zhou, H; Wilson, B; Hong, JS; Gao, HM. Resveratrol protects dopamine neurons against lipopolysaccharide-induced neurotoxicity through its anti- inflammatory actions. Molecular Pharmacology 2010, 78, 466–477.
[23] Alvarez, E; Rodino-Janeiro, BK; Jerez, M; Ucieda-Somoza, R; Nunez, MJ; Gonzalez- Juanatey, JR. Procyanidins from grape pomace are suitable inhibitors of human endothelial NADPH oxidase. Journal of Cell Biochemistry 2012, 113, 1386–1396. [24] Moure, A; Cruz, JM; Franco, D; Domínguez, JM; Sineiro, J; Domínguez, H; Núnez,
MJ; Parajó, JC. Natural antioxidants from residual sources. Food Chemistry 2001, 72, 145–171.
[25] Louli, V; Ragoussis, N; Magoulas, K. Recovery of phenolic antioxidants from wine industry by-products. Bioresource Technology 2004, 92, 201–208.
[26] Chronopoulou, L; Agatone, AC; Palocci, C. Supercritical CO2 extraction of oleanolic acid from grape pomace. International Journal of Food Science and Technology 2013, 48, 1854-1860.
[27] Oliveira, DA; Salvador, AA; Smania Jr., A; Smania, EFA; Maraschin, M; Ferreira, SRS. Antimicrobial activity and composition profile of grape (Vitis vinifera) pomace extracts obtained by supercritical fluids. Journal of Biotechnology 2013, 164, 423-432. [28] Palma, M; Taylor, LT; Varela, RM; Cutler, SJ; Cutler, HG. Fractional extraction of
compounds from grape seeds by supercritical fluid extraction and analysis for antimicrobial and agrochemical activities. Journal of Agricultural and Food Chemistry 1999, 47, 5044–5048.
[29] Kitzberger, CSG; Smânia Jr., A; Pedrosa, RC; Ferreira, SRS. Antioxidant and antimicrobial activities of shiitake (Lentinula edodes) extracts obtained by organic solvents and supercritical fluids. Journal of Food Engineering 2007, 80, 631–638. [30] Palma, M; Taylor, LT; Varela, RM; Cutler, SJ; Cutler, HG. Fractional extraction of
compounds from grape seeds by supercritical fluid extraction and analysis for antimicrobial and agrochemical activities. Journal of Agricultural and Food Chemistry 1999, 47, 5044–5048.
[31] Berna, A; Cháfer, A; Montón, JB. High-pressure solubility data of the system resveratrol (3) + ethanol (2) + CO2 (1). Journal of Supercritical Fluids 2001, 19, 133– 139.
[32] Filip, V; Plocková, M; Smidrkal, J; Spicková, Z; Melzoch, K; Schmidt, S. Resveratrol and its antioxidant and antimicrobial effectiveness. Food Chemistry 2003, 83, 585–593. [33] Shoko, T; Soichi, T; Megumi, MM; Eri, F; Jun, K; Michiko, W. Isolation and
identification of an antibacterial compound from grape and its application to foods.
Nippon Nogeikagaku Kaishi 1999, 73, 125–128.
[34] Soni, MG; Carabin, IG; Burdock, GA. Safety assessment of esters of phydrozybenzoic acid (parabens). Food and Chemical Toxicology 2005, 43, 985–1015.
[35] Borchers, AT; Keen, CL; Gerstiwin, ME. Mushrooms, tumors, and immunity: an update. Experimental Biology and Medicine 2004, 229, 393–406.
[36] Da Porto, C; Natolino, A; Decorti, D. Extraction of proanthocyanidins from grape marc by supercritical fluid extraction using CO2 as solvent and ethanol-water mixture as co- solvent. Journal of Supercritical Fluids 2014, 87, 59-64.
[37] Saint-Cricq de Gaulejac, N; Provost, C; Vivas, N. Comparative study of polyphenols scavenging activities assessed by different methods. J. Agriculture and Food Chemistry 1999, 47, 425–431.
[38] de Campos, LMAS; Leimann, FV; Pedrosa, RC; Ferreira, SRS. Free radical scavenging of grape pomace extracts from Cabernet sauvingnon (Vitis vinifera). Bioresource
[39] Da Porto, C; Decorti, D; Natolino, A. Water and ethanol as co-solvent in supercritical fluid extraction of proanthocyanidins from grape marc: A comparison and a proposal. J.
of Supercritical Fluids 2014, 87, 1–8.
[40] Pinelo, M; Ruiz-Rodriguez, A; Sineiro, J; Senorans, FJ; Reglero, G; Nunez, MJ. Supercritical fluid and solid–liquid extraction of phenolic antioxidants from grape pomace: a comparative study. Eur Food Res Technol 2007, 226, 199–205.
[41] Medina, I; Gonzalez, MJ; Pazos, M; della Medaglia, D; Sacchi, R; Gallardo, JM. Activity of plant extracts for preserving functional food containing n-3-PUFA. Eur
Food Res Technol 2003, 217, 301–307.
[42] Wanasundara, VM; Shahidi, F. Stabilization of marine oils with flavonoids. J Food
Editor: Jason P. Owen © 2015 Nova Science Publishers, Inc.
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