Abstract

The objectives of this study were to evaluate the effects of the grape cultivar geographical location and extraction technique on the total phenolic compounds (TPC), antioxidant capacity (AC), oil yield and quality of Quebranta (Vitis vinifera) grape seeds. Seeds were defatted with supercritical CO₂ and the bioactive compounds were extracted with subcritical water and macerations with methanol, ethanol, and acetone. The differences in grape seed oil yield were not significant (p > 0.05). The most abundant fatty acid determined was linoleic (66.37-67.37%). The highest TPC corresponded to the extracts from zones A and B obtained with subcritical water, 167.56 ± 10.40 and 161.83 ± 4.95 mg GAE/g dw, respectively. The highest AC by DPPH was also achieved by the extracts from zones A and B (1,479.90 ± 12.86 and 1,628.15 ± 80.32 µmol TE/g dw, respectively) with subcritical water extraction. The highest AC by FRAP was observed in the subcritical water extracts from zones B and C, 1,429.29 ± 29.75 and 1,389.54 ± 7.46 µmol TE/g dw, respectively. Grape seed is a valuable source of nutritionally oil and bioactive compounds, which can be obtained from by-products of pisco production for potential use in the food and pharmaceutical industries.

Keywords:
grape seed oil; bagasse; unsaturated fatty acids; supercritical CO₂; subcritical water extraction; antioxidant capacity

1 Introduction

Pisco, an alcoholic beverage obtained by the distillation of fresh musts from recently fermented pisco grapes, is produced in the regions of Lima, Ica, Arequipa, Moquegua, and Tacna in Peru (Instituto Nacional de Defensa de la Competencia y de la Protección de la Propiedad Intelectual, 2017Instituto Nacional de Defensa de la Competencia y de la Protección de la Propiedad Intelectual – INDECOPI. (2017). Guía práctica de la denominación de origen Pisco. Lima: INDECOPI.). Ica is the most important region in the wine industry, which produces all varieties, with the Quebranta grape being the most productive (Mathis et al., 2017Mathis, A. C. G., Acevedo, F., & Aroca, G. (2017). Tequila and pisco. In A. Pandey, M. Á. Sanromán, G. Du, C. R. Soccol & C.-G. Dussap (Eds.), Current developments in biotechnology and bioengineering: food and beverages industry (pp. 469-486). Amsterdam: Elsevier. http://dx.doi.org/10.1016/B978-0-444-63666-9.00017-0.
http://dx.doi.org/10.1016/B978-0-444-636... ). Pisco production in Peru grows annually, reaching 4190, 4220, 5210 and 4964 m³in 2016, 2017, 2018 and 2019, respectively (Instituto Nacional de Estadística e Informática, 2021Instituto Nacional de Estadística e Informática – INEI. (2021). Series nacionales. Retrieved from http://webapp.inei.gob.pe:8080/sirtod-series/
http://webapp.inei.gob.pe:8080/sirtod-se... ). This growth, simultaneously, generates a large amount of solid residue because 6 to 7 kg of grapes are used to make 1 L of pisco (Programa de las Naciones Unidas para el Desarrollo, 2004Programa de las Naciones Unidas para el Desarrollo – PNUD. Centro de Innovación Tecnológica Vitivinícola – CITEvid. (2004). La uva y el pisco: potencialidades productivas. Lima: PNUD/CITEvid.). In Ica, solid waste is often burned or discarded in landfills near the food industries, creating environmental and health problems. The solid residue, called pomace, is made up of a mixture of husks, seeds and stems; and they are known to be a source of antioxidants such as phenolic acids and flavonoids (Cheng et al., 2012Cheng, V. J., Bekhit, A. E. D. A., McConnell, M., Mros, S., & Zhao, J. (2012). Effect of extraction solvent, waste fraction and grape variety on the antimicrobial and antioxidant activities of extracts from wine residue from cool climate. Food Chemistry, 134(1), 474-482. http://dx.doi.org/10.1016/j.foodchem.2012.02.103.
http://dx.doi.org/10.1016/j.foodchem.201... ). Among pomace components, seeds represent 38 to 52% based on the dry matter. In addition, the seeds stand out for being a source of oil, rich in polyunsaturated fatty acids, particularly linoleic acid (Maier et al., 2009Maier, T., Schieber, A., Kammerer, D. R., & Carle, R. (2009). Residues of grape (Vitis vinifera L.) seed oil production as a valuable source of phenolic antioxidants. Food Chemistry, 112(3), 551-559. http://dx.doi.org/10.1016/j.foodchem.2008.06.005.
http://dx.doi.org/10.1016/j.foodchem.200... ). Likewise, seeds have a high content of phenolic compounds such as flavonoids, anthocyanins, catechins, flavanol glycosides and phenolic acids (Porto & Natolino, 2017Porto, C., & Natolino, A. (2017). Supercritical fluid extraction of polyphenols from grape seed (Vitis vinifera): study on process variables and kinetics. The Journal of Supercritical Fluids, 130, 239-245. http://dx.doi.org/10.1016/j.supflu.2017.02.013.
http://dx.doi.org/10.1016/j.supflu.2017.... ; Lafka et al., 2007Lafka, T. I., Sinanoglou, V., & Lazos, E. S. (2007). On the extraction and antioxidant activity of phenolic compounds from winery wastes. Food Chemistry, 104(3), 1206-1214. http://dx.doi.org/10.1016/j.foodchem.2007.01.068.
http://dx.doi.org/10.1016/j.foodchem.200... ), resveratrol (Tian et al., 2017Tian, Y., Wang, Y., Ma, Y., Zhu, P., He, J., & Lei, J. (2017). Optimization of subcritical water extraction of resveratrol from Grape seeds by response surface methodology. Applied Sciences, 7(4), 321. http://dx.doi.org/10.3390/app7040321.
http://dx.doi.org/10.3390/app7040321... ) that provide beneficial biological effects for human health (Paladino & Zuritz, 2011Paladino, S. C., & Zuritz, C. A. (2011). Extracto de semillas de vid (Vitis vinifera L.) con actividad antioxidante: eficiencia de diferentes solventes en el proceso de extracción. Revista de la Facultad de Ciencias Agrarias, 43(1), 187-199.).

The extraction methods for the recovery of bioactive compounds, from the residues of the pisco industry, have been extensively reported. Some environmentally friendly methods that stand out for grape oil extraction are expanded CO₂ (Li et al., 2020Li, H., Fu, X., Deng, G., David, A., & Huang, L. (2020). Extraction of oil from grape seeds (Vitis vinifera L.) using recyclable CO2-expanded ethanol. Chemical Engineering and Processing - Process Intensification, 157, 108147. http://dx.doi.org/10.1016/j.cep.2020.108147.
http://dx.doi.org/10.1016/j.cep.2020.108... ), microwaves, ultrasound and supercritical CO₂ (Dimić et al., 2020Dimić, I., Teslić, N., Putnik, P., Kovačević, D. B., Zeković, Z., Šojić, B., Mrkonjić, Ž., Čolović, D., Montesano, D., & Pavlić, B. (2020). Innovative and conventional valorizations of grape seeds from winery by-products as sustainable source of lipophilic antioxidants. Antioxidants, 9(7), 568. http://dx.doi.org/10.3390/antiox9070568. PMid:32630185.
http://dx.doi.org/10.3390/antiox9070568... ). Non-conventional technologies used for the recovery of phenolic compounds from grape seed include subcritical water (Duba et al., 2015Duba, K. S., Casazza, A. A., Mohamed, H. B., Perego, P., & Fiori, L. (2015). Extraction of polyphenols from grape skins and defatted grape seeds using subcritical water: experiments and modeling. Food and Bioproducts Processing, 94, 29-38. http://dx.doi.org/10.1016/j.fbp.2015.01.001.
http://dx.doi.org/10.1016/j.fbp.2015.01.... ; Loarce et al., 2020Loarce, L., Oliver-Simancas, R., Marchante, L., Díaz-Maroto, M. C., & Alañón, M. E. (2020). Implementation of subcritical water extraction with natural deep eutectic solvents for sustainable extraction of phenolic compounds from winemaking by-products. Food Research International, 137, 109728. http://dx.doi.org/10.1016/j.foodres.2020.109728. PMid:33233297.
http://dx.doi.org/10.1016/j.foodres.2020... ) and pressurized liquid extraction (Allcca-Alca et al., 2021Allcca-Alca, E. E., León-Calvo, N. C., Luque-Vilca, O. M., Martínez-Cifuentes, M., Pérez-Correa, J. R., Mariotti-Celis, M. S., & Huamán-Castilla, N. L. (2021). Hot pressurized liquid extraction of polyphenols from the skin and seeds of Vitis vinifera L. cv. Negra Criolla pomace a Peruvian native pisco industry waste. Agronomy, 11(5), 866. http://dx.doi.org/10.3390/agronomy11050866.
http://dx.doi.org/10.3390/agronomy110508... ). Regarding the conventional methods, studies of grape seed extracts obtained by maceration with methanol (Porto et al., 2013Porto, C., Porretto, E., & Decorti, D. (2013). Comparison of ultrasound-assisted extraction with conventional extraction methods of oil and polyphenols from grape (Vitis vinifera L.) seeds. Ultrasonics Sonochemistry, 20(4), 1076-1080. http://dx.doi.org/10.1016/j.ultsonch.2012.12.002. PMid:23305938.
http://dx.doi.org/10.1016/j.ultsonch.201... ), and other solvents such as acetone, ethanol and methanol have been reported (Cheng et al., 2012Cheng, V. J., Bekhit, A. E. D. A., McConnell, M., Mros, S., & Zhao, J. (2012). Effect of extraction solvent, waste fraction and grape variety on the antimicrobial and antioxidant activities of extracts from wine residue from cool climate. Food Chemistry, 134(1), 474-482. http://dx.doi.org/10.1016/j.foodchem.2012.02.103.
http://dx.doi.org/10.1016/j.foodchem.201... ; Paladino & Zuritz, 2011Paladino, S. C., & Zuritz, C. A. (2011). Extracto de semillas de vid (Vitis vinifera L.) con actividad antioxidante: eficiencia de diferentes solventes en el proceso de extracción. Revista de la Facultad de Ciencias Agrarias, 43(1), 187-199.). However, there have been no studies assessing non-conventional extraction methods in Quebranta (Vitis vinifera) grape seeds.

Information about bioactive compounds in grape seeds, a by-product of the pisco industry, and their extraction techniques is abundant. On the contrary, more research is needed about the influence of the cultivar different locations and non-conventional extraction methods on those compounds or others of interest. Therefore, the objectives of this study were to evaluate the effects of the cultivar geographical location and extraction method on the TPC and AC of the defatted Quebranta (Vitis vinifera) grape seeds. Also, the influence of the cultivar location on the grape seed oil yield and fatty acids profile was investigated.

2 Materials and methods

2.1 Sample preparation

Approximately, 60 kg of Quebranta (Vitis vinifera) grape pomace from the production of pisco, were used. The pomace came from 3 geographical areas: San Juan Bautista (14°0'12.01"S 75° 44'21.27"W), Subtanjalla (14°1'17.82"S 75°44'35.43"W) and Los Patos (14°2'56.22"S 75° 43'52.36"W) located in the Ica Valley (Ica-Peru). The pisco production process was similar in the three areas mentioned above. Briefly, the Quebranta grape clusters were placed in the crusher-destemmer machine. Then, the grape was pressed, the juice was obtained, and the pomace (peels and seeds) was separated. The must, fermented grape juice, was later distilled to obtain pisco. The samples, kept at 0 to 5 °C, were brought to the Instituto Tecnologico de la Produccion (ITP) (Callao, Peru). The pomace was dried in a cold air dryer (Asahi, CV-20AN, Japan) at 25 °C for 36 h until they reached 13% as the maximum moisture content. Then, it passed through a 7 mm sieve (KM Testing sieve, Japan) to separate the seeds that were dried in a forced convection oven (Venticell, USA) at 40 °C for 6 h until a maximum content of 7%, and ground in an analytical mill (A 11 Basic, IKA, USA). Finally, the dried and ground Quebranta grape seeds (QGS) were passed through 25 mesh (0.707 mm) and 35 mesh (0.500 mm) sieves (Retsch, Lima, Peru). The particles retained by both sieves were vacuum packed in polyethylene bags, protected from light and refrigerated at 5 ± 1 °C until later use.

2.2 Reagents

Methanol HPLC grade (JT Baker, USA), 99.9% absolute ethanol (Scharlau, Spain), acetone (Merck, USA), a 37-component fatty acid methyl ester (FAME) mixture (C4-C24) (Sigma-Aldrich, USA), gallic acid monohydrate ≥ 98.5% ACS (Sigma-Aldrich, China), sodium carbonate (≥ 99.9%) (Merck, USA), Folin Ciocalteu’s phenol reagent (2N) (Sigma-Aldrich, USA), DPPH (2,2-diphenyl- 1-picrylhydrazyl) (95%) (Alfa Aesar, Germany), Trolox (6-hydroxy-2,5,7,8-tetramethylchrome-2-carboxylic acid) (97%) (Sigma-Aldrich, China), TPTZ (Alfa Aesar, UK), hydrochloric acid (JT Baker, Canada), iron trichloride hexahydrate (Merck, Germany), deionized water supplied by the Barnstead water purification system (Barnstead, Model D11911, Germany), carbon dioxide 99.5% v/v liquefied gas (Linde, Peru), nitrogen atmosphere Ultrapuro (Linde, Peru).

2.3 Experimental design

A complete randomized design (CRD) of 1 x 3 with 4 repetitions was used to evaluate the effect of three cultivar geographical locations of Quebranta grape: (A) San Juan Bautista, (B) Subtanjalla and (C) Los Patos on yield and fatty acids profile of Quebranta grape seeds (QGS) oil extracted with supercritical CO₂. Subsequently, a 3 x 4 full factorial design with 3 repetitions was used to evaluate the effect of the three cultivar geographical locations and four extraction methods on TPC and ACin vitroof QGS. The levels corresponding to the extraction methods factor were: (a) subcritical water, maceration using as solvents (b) 70% ethanol (v/v), (c) methanol and (d) 50% acetone (v/v). The levels corresponding to the location factor were: (A) San Juan Bautista, (B) Subtanjalla and (C) Los Patos.

2.4 Oil extraction with supercritical CO₂

Dried Quebranta grape seeds (QGS) were defatted with supercritical CO₂ using a multi-solvent extractor equipment Model 2802.000 (Figure 1) (Top Industrie, Vaux le Pénil, France) with an extraction cell of 1 L capacity that had a volume reducer device (87 cm³, internal diameter = 2.8 cm, internal height = 14.1 cm). Approximately, 35 g of QGS and five alternating layers of 5 mm glass beads (5 g) were filled into the extraction cell (Figure 1a). Extractions were performed at 33.5 °C and 188 bar according to Sánchez et al. (2018)Sánchez, M. B., Huanca, A. C., & Gómez, Ó. T. (2018). Optimización del rendimiento de la extracción de aceite de semillas de Vitis vinifera con CO2 supercrítico. Revista de la Sociedad Química del Perú, 84(2), 217-227. http://dx.doi.org/10.37761/rsqp.v84i2.143.
http://dx.doi.org/10.37761/rsqp.v84i2.14... with modifications in the CO₂flow that was 47 g/min in this study. The grape seed oil yield was expressed on a dry basis according to Equation 1 as follows:

Where: W₁ is the oil mass and W₂ is QGS mass

2.5 Preparation of extracts for phenolic and antioxidant capacity assays

Four extraction methods were selected for phenolics extraction of defatted Quebranta grape seeds

(DQGS): subcritical water extraction (SWE) and by maceration with ethanol, methanol and acetone.

2.6 Subcritical Water Extraction (SWE)

The subcritical water extractions were carried out with a multisolvent extractor equipment (Top Industrie, series 2802.0000, Vaux le Pénil, France) without a volume reducer (Figure 1b), and deionized water as solvent, previously degasified for 30 min at 25 °C in a sonicator (VWR International, SymphonyTM, 97043-942, China). Approximately, 31 g of DQGS and five alternating layers of 5 mm glass beads (700 g) were filled into the extraction cell (709 cm³, internal diameter: 8 cm, internal height: 14.1 cm) as shown in Figure 1b. As described by Duba et al. (2015)Duba, K. S., Casazza, A. A., Mohamed, H. B., Perego, P., & Fiori, L. (2015). Extraction of polyphenols from grape skins and defatted grape seeds using subcritical water: experiments and modeling. Food and Bioproducts Processing, 94, 29-38. http://dx.doi.org/10.1016/j.fbp.2015.01.001.
http://dx.doi.org/10.1016/j.fbp.2015.01.... , extractions were performed at 120 °C and 100 bar. Then, 500 mL of water were added to the reactor and absorbed with the cosolvent pump at 15 mL/min for 40 min until the desired pressure was achieved. This condition was kept static for 3 h for a greater extraction of phenolic compounds. Finally, extracts were cooled in an ice bath for 10 min until full recovery of the extract from the system. Collected extracts were stored at 4 ± 1 °C until analysis. Extracts were analyzed in triplicate.

2.7 Extraction by maceration with ethanol, methanol or acetone

The extraction of phenolics from DQGS was done with ethanol, methanol or acetone as solvent.

Approximately, 4 g of DQGS was weighed into a 100 mL glass bottle and extracted with 80 mL of ethanol/water (70 : 30 v/v), methanol or acetone/water (50 : 50 v/v) (Sánchez et al., 2018Sánchez, M. B., Huanca, A. C., & Gómez, Ó. T. (2018). Optimización del rendimiento de la extracción de aceite de semillas de Vitis vinifera con CO2 supercrítico. Revista de la Sociedad Química del Perú, 84(2), 217-227. http://dx.doi.org/10.37761/rsqp.v84i2.143.
http://dx.doi.org/10.37761/rsqp.v84i2.14... ). The bottles were vortexed with a magnetic stirrer (IKA, RT 10, Germany) at 60 RPM for 3 h at 25 °C. Finally, the extracts were filtered through Whatman No. 4 (20-30 µm) filter paper and stored at 4 ± 1 °C in airtight glass bottles until analysis. Extracts were analyzed in triplicate.

2.8 Chemical analysis

Fatty acid profile of grape seed oil

The fatty acid profile was determined as described by Prevot & Mordret (1976)Prevot, G., & Mordret, M. (1976). Utilisation des colonnes capillaires de yerre pour l’analyse des cords gras par chromatographie en phase gazeose. Revue Francaise des Corps Gras, 23, 7-8. as follows.

A gas chromatograph with an FID detector (Autosystem XL, Perkin Elmer, USA) equipped with a Supelcowax 10 column (Merck, Germany) (30 m × 0.25 mm id; film thickness: 0.25 μm) was used. Hydrogen was used as the carrier gas at 5 psi. The injector and detector temperatures were 250 °C and 270 °C, respectively. A volume of 2 µL was injected at a split ratio 100 : 1. The fatty acid peaks were identified by comparison with the retention times of the Fatty Acid Methyl Ester Mix C4-C14 (Sigma-Aldrich, USA). The area of ​​the peaks was calculated using the TotalChrom Navigator software (v. 6.2.0) (Perkin Elmer, USA). The percentage of each fatty acid was calculated by comparing the individual area of ​​each peak with the fatty acids total area. Oil samples were analyzed in quadruplicate.

Total Phenolic Content (TPC)

TPC was determined according to Singleton et al. (1999)Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods in Enzymology, 299, 152-178. http://dx.doi.org/10.1016/S0076-6879(99)99017-1.
http://dx.doi.org/10.1016/S0076-6879(99)... . A gallic acid standard curve was prepared with concentrations of 50, 100, 150, 200 and 400 mg/L. The readings were carried out at 750 nm with a UV-VIS spectrophotometer (Perkin Elmer, Perkin Elmer®, LAMBDA 950, USA). The results were expressed in mg of gallic acid equivalent (GAE) per g of DQGS (dw).

2.9 Antioxidant capacity assays

DPPH

The antioxidant capacity of DQGS was determined according to Kim et al. (2002)Kim, J.-K., Noh, J.-H., Lee, S.-E., Choi, J.-S., Suh, H.-S., Chung, H.-Y., Song, Y.-O., & Choi, W.-C. (2002). The first total synthesis of 2, 3, 6-tribromo-4, 5-dihydroxybenzyl methyl ether (TDB) and its antioxidant activity. Bulletin of the Korean Chemical Society, 23(5), 661-662. http://dx.doi.org/10.5012/bkcs.2002.23.5.661.
http://dx.doi.org/10.5012/bkcs.2002.23.5... . A calibration curve was generated with Trolox standard solutions of 50, 100, 250, 500, 750 and 1000 µM. The absorbance was measured in a UV-VIS spectrophotometer at 518 nm. The percentage of inhibition of different concentrations of DQGS extract against the DPPH radical was calculated. The concentration necessary to inhibit 50% of DPPH radicals was expressed in µg extract/mL (IC₅₀). Also, IC₅₀for Trolox was determined to express the AC for Trolox with respect to the AC of DQGS extract which was expressed in mmol Trolox Equivalent (TE) per g sample (dw).

FRAP

The AC of DQGS samples was determined according to the methodology of Benzie & Strain (1996)Benzie, I. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Analytical Biochemistry, 239(1), 70-76. http://dx.doi.org/10.1006/abio.1996.0292. PMid:8660627.
http://dx.doi.org/10.1006/abio.1996.0292... . Appropriate diluted samples were added to the FRAP reagent (acetate buffer pH 3.6, TPTZ (2,4,6-tripyridyl-s-triazine) and FeCl3 6H2O in a ratio 25 : 2.5 : 2.5). The absorbance was measured with UV-VIS spectrophotometer (Perkin Elmer, Lambda 850, USA) at 595 nm. A calibration curve was prepared using Trolox standard solutions of 50, 150, 300, 400, 500 and 600 µM. FRAP values were expressed as µmol of Trolox Equivalent (TE) per g sample (dw).

2.10 Statistical analysis

Experimental results were analyzed for significant differences using a one-way analysis of variance (ANOVA). Tukey's HSD multiple comparisons of means were determined at the 0.05 confidence level. The Pearson test was performed to obtain correlation (r) values ​​between TPC and AC (DPPH and FRAP). All analyzes were carried out with SPSS statistic software v. 26 (IBM, Peru). Data are reported as mean ± standard deviation (SD) and were calculated with Excel 2016 (Microsoft, USA).

3 Results and discussion

3.1 Oil yield and fatty acid profile of Quebranta grape seed oil

Quebranta grape seed oil yields are shown in Table 1. No significant differences (p > 0.05) were observed due to the effect of the cultivar geographical location. The QGS oil yield from areas A, B and C were higher than the values obtained through supercritical CO₂ extraction by Jokić et al. (2016)Jokić, S., Bijuk, M., Aladić, K., Bilić, M., & Molnar, M. (2016). Optimisation of supercritical CO2 extraction of grape seed oil using response surface methodology. International Journal of Food Science & Technology, 51(2), 403-410. http://dx.doi.org/10.1111/ijfs.12986.
http://dx.doi.org/10.1111/ijfs.12986... from Croatian grape seeds (14.49%) and Souza et al. (2020)Souza, R. D. C., Machado, B. A. S., Barreto, G. A., Leal, I. L., Anjos, J. P., & Umsza-Guez, M. A. (2020). Effect of experimental parameters on the extraction of grape seed oil obtained by low pressure and supercritical fluid extraction. Molecules, 25(7), 1634. http://dx.doi.org/10.3390/molecules25071634. PMid:32252316.
http://dx.doi.org/10.3390/molecules25071... from Vitis viniferagrape seed (Brazil) (12.54%). Lower yields in the range of 12.0 to 12.7% were also reported byCoelho et al. (2018)Coelho, J. P., Filipe, R. M., Robalo, M. P., & Stateva, R. P. (2018). Recovering value from organic waste materials: supercritical fluid extraction of oil from industrial grape seeds. The Journal of Supercritical Fluids, 141, 68-77. http://dx.doi.org/10.1016/j.supflu.2017.12.008.
http://dx.doi.org/10.1016/j.supflu.2017.... in grape seeds from Portugal. These differences may be due to different cultivars as determined byWen et al. (2016)Wen, X., Zhu, M., Hu, R., Zhao, J., Chen, Z., Li, J., & Ni, Y. (2016). Characterisation of seed oils from different grape cultivars grown in China. Journal of Food Science and Technology, 53(7), 3129-3136. http://dx.doi.org/10.1007/s13197-016-2286-9. PMid:27765984.
http://dx.doi.org/10.1007/s13197-016-228... , who found significant differences among extraction yields of various grape seed cultivars that ranged from 13.71 to 15.92%.

The fatty acid composition of QGS oils from areas A, B and C is shown inTable 1. Nine kinds of fatty acids were detected in grape seed oil samples. Results revealed that grape seed oil was mainly composed of polyunsaturated fatty acids (PUFA) which account for 66.67-67.68% of total fatty acids followed by monounsaturated fatty acids (MUFA). No significant differences (p > 0.05) were found between PUFA of grape seed oil from areas A and C. Also, no significant differences were found between MUFA of grape seed oil for the 3 areas under study. On the contrary, significant differences were found in saturated fatty acids (SFA) with oil from area B showing the greatest amount (12.35%). The most abundant fatty acid was linoleic acid (C18:2) ranging from 66.37-67.37%, then oleic acid (C18:1) (19.55-20.01%), palmitic acid (C16:0) (7.02-7.19%) and stearic acid (C18:0) (4.35-4.89%) as also reported by Wen et al. (2016)Wen, X., Zhu, M., Hu, R., Zhao, J., Chen, Z., Li, J., & Ni, Y. (2016). Characterisation of seed oils from different grape cultivars grown in China. Journal of Food Science and Technology, 53(7), 3129-3136. http://dx.doi.org/10.1007/s13197-016-2286-9. PMid:27765984.
http://dx.doi.org/10.1007/s13197-016-228... on several grape varieties. No significant differences (p > 0.05) were observed between the linoleic acid content of grape seed oil from areas A and C but significant differences (p < 0.05) were found between areas B and C with the latter area showing the greatest content (67.37%). These results were in accordance with those reported forVitis viniferaseeds from Italy and Mexico by Fiori et al. (2014)Fiori, L., Lavelli, V., Duba, K. S., Harsha, P. S. C. S., Mohamed, H. B., & Guella, G. (2014). Supercritical CO2 extraction of oil from seeds of six grape cultivars: modeling of mass transfer kinetics and evaluation of lipid profiles and tocol contents. The Journal of Supercritical Fluids, 94, 71-80. http://dx.doi.org/10.1016/j.supflu.2014.06.021.
http://dx.doi.org/10.1016/j.supflu.2014.... andFranco-Mora et al. (2015)Franco-Mora, O., Salomon-Castaño, J., Morales, A. A., Castañeda-Vildózola, Á., & Rubí-Arriaga, M. (2015). Acidos grasos y parámetros de calidad del aceite de semilla de uva silvestre (Vitis spp.). Scientia Agropecuaria, 6(4), 271-278. http://dx.doi.org/10.17268/sci.agropecu.2015.04.04.
http://dx.doi.org/10.17268/sci.agropecu.... , respectively, who used supercritical CO₂for oil extraction. These results were also verified by Coelho et al. (2018)Coelho, J. P., Filipe, R. M., Robalo, M. P., & Stateva, R. P. (2018). Recovering value from organic waste materials: supercritical fluid extraction of oil from industrial grape seeds. The Journal of Supercritical Fluids, 141, 68-77. http://dx.doi.org/10.1016/j.supflu.2017.12.008.
http://dx.doi.org/10.1016/j.supflu.2017.... who reported range values for linoleic, oleic, palmitic, and stearic acids from grape (Vitis vinifera L.) seeds from the center of Portugal, similar to the ranges reported in this study. Also, comparable results were obtained byBada et al. (2015)Bada, J. C., León-Camacho, M., Copovi, P., & Alonso, L. (2015). Characterization of grape seed oil from wines with protected denomination of origin (PDO) from Spain. Grasas y Aceites, 66(3), 1-6.in grape seed oil from Spain extracted with hexane. Conversely, Souza et al. (2020)Souza, R. D. C., Machado, B. A. S., Barreto, G. A., Leal, I. L., Anjos, J. P., & Umsza-Guez, M. A. (2020). Effect of experimental parameters on the extraction of grape seed oil obtained by low pressure and supercritical fluid extraction. Molecules, 25(7), 1634. http://dx.doi.org/10.3390/molecules25071634. PMid:32252316.
http://dx.doi.org/10.3390/molecules25071... reported lower values of linoleic and oleic fatty acids while higher values of stearic and palmitic acids from a Brazilian grape variety. Preharvest and processing parameters are the main factors that influence the quality of fruit seed oil (Kaseke et al., 2020Kaseke, T., Opara, U. L., & Fawole, O. A. (2020). Fatty acid composition, bioactive phytochemicals, antioxidant properties and oxidative stability of edible fruit seed oil: effect of preharvest and processing factors. Heliyon, 6(9), e04962. http://dx.doi.org/10.1016/j.heliyon.2020.e04962. PMid:32995635.
http://dx.doi.org/10.1016/j.heliyon.2020... ). Differences in grape seed oil fatty acid composition may be caused by different cultivars of grape seed, cultivation conditions, cultivar geographical location and the oil extraction method used.

3.2 Total Phenolic Content (TPC)

The TPC of DQGS is shown in Table 2. TPC ranged from 27.89 ± 2.24 mg GAE/g dw to 167.56 ± 10.40 mg GAE/g (dw). The cultivar geographical location, the extraction method and their interaction had a significant influence (p < 0.05) on the TPC (Table 3). The highest TPC was obtained with subcritical water (SWE), 167.56 and 161.83 mg GAE/g dw, in DQGS from areas A and B, respectively, and no significant differences (p > 0.05) were found between the values. Those values were ​​close to those reported byBozan et al. (2008)Bozan, B., Tosun, G., & Ozcan, D. (2008). Study of polyphenol content in the seeds of red grape (Vitis vinifera L.) varieties cultivated in Turkey and their antiradical activity. Food Chemistry, 109(2), 426-430. http://dx.doi.org/10.1016/j.foodchem.2007.12.056. PMid:26003368.
http://dx.doi.org/10.1016/j.foodchem.200... in seeds ofVitis viniferavariety Papaz Karazi (Turkey).Ordoñez et al. (2019)Ordoñez, E., Leon-Arevalo, A., Rivera-Rojas, H., & Vargas, E. (2019). Cuantificación de polifenoles totales y capacidad antioxidante en cáscara y semilla de cacao (Theobroma cacao L.), tuna (Opuntia ficus indica Mill), uva (Vitis Vinífera) y uvilla (Pourouma cecropiifolia). Scientia Agropecuaria, 10(2), 175-183. http://dx.doi.org/10.17268/sci.agropecu.2019.02.02.
http://dx.doi.org/10.17268/sci.agropecu.... reported lower TPC in Vitis viniferaBlack grape seeds extracted with methanol (9.07 g GAE/100 g dry sample). A similar scenario was also described by Orellana et al. (2019)Orellana, D. E. C., Solorzano, R. A. A., & Ticlayauri, E. A. M. (2019). Actividad antioxidante de extractos de semillas de uvas recuperadas del residuo sólido de actividades vitivinícolas en el Valle de Cañete, Perú. Functional Food Science and Technology Journal, 1(1), 73-89. in Quebranta grape seeds where lower TPC values were found (97.26 mg GAE/g dry sample) when using an acidified methanol/water solution as the extraction method.

TPC of DQGS from area A extracted by SWE ​​was higher than the values determined by conventional methods with different solvents (70% ethanol, methanol and 50% acetone). This may be due to the subcritical state of the water that facilitates the diffusion of the analyte, the decrease in viscosity, surface tension and dielectric constant (Turner & Ibañez, 2012Turner, C., & Ibañez, E. (2012). Pressurized hot water extraction and processing. In N. Lebovka, E. Vorobiev & F. Chemat (Eds.). Enhancing extraction processes in the food industry (pp. 223-253). New York: CRC Press.; Zhang et al., 2020Zhang, J., Wen, C., Zhang, H., Duan, Y., & Ma, H. (2020). Recent advances in the extraction of bioactive compounds with subcritical water: a review. Trends in Food Science & Technology, 95, 183-195. http://dx.doi.org/10.1016/j.tifs.2019.11.018.
http://dx.doi.org/10.1016/j.tifs.2019.11... ). In addition, the thermal energy supplied with subcritical water decreases the activation energy required for desorption process which can interrupt cohesive (solute-solute) and adhesive (solute-matrix) interaction (Teo et al., 2010Teo, C. C., Tan, S. N., Yong, J. W. H., Hew, C. S., & Ong, E. S. (2010). Pressurized hot water extraction (PHWE). Journal of Chromatography. A, 1217(16), 2484-2494. http://dx.doi.org/10.1016/j.chroma.2009.12.050. PMid:20060531.
http://dx.doi.org/10.1016/j.chroma.2009.... ).Duba et al. (2015)Duba, K. S., Casazza, A. A., Mohamed, H. B., Perego, P., & Fiori, L. (2015). Extraction of polyphenols from grape skins and defatted grape seeds using subcritical water: experiments and modeling. Food and Bioproducts Processing, 94, 29-38. http://dx.doi.org/10.1016/j.fbp.2015.01.001.
http://dx.doi.org/10.1016/j.fbp.2015.01.... reported a TPC of 124 mg GAE/g in grape seeds from Italy extracted with subcritical water. Our results were higher than those reported by the aforementioned authors possibly due to the variety of grape and growing area, as well as the solvent/sample ratio in the extraction process and other parameters, as reported byRavber et al. (2015)Ravber, M., Knez, Ž., & Škerget, M. (2015). Simultaneous extraction of oil-and water-soluble phase from sunflower seeds with subcritical water. Food Chemistry, 166, 316-323. http://dx.doi.org/10.1016/j.foodchem.2014.06.025. PMid:25053062.
http://dx.doi.org/10.1016/j.foodchem.201... . Lachman et al. (2009)Lachman, J., Šulc, M., Faitová, K., & Pivec, V. (2009). Major factors influencing antioxidant contents and antioxidant activity in grapes and wines. International Journal of Wine Research, 1(1), 101-121. http://dx.doi.org/10.2147/IJWR.S4600.
http://dx.doi.org/10.2147/IJWR.S4600... reported significant differences among vineyard regions and varieties in total polyphenol content in grape skins. Further, they found significant differences in polyphenolic antioxidants of red and white Spanish wines of different geographical origins.

In relation to areas B and C, no significant differences in TPC were observed between SWE and the extraction method with acetone. No significant differences were found in TPC of grape samples from area A extracted by the conventional methods. DQGS from area A extracted with 70% ethanol, methanol and 50% acetone showed the lowest TPC values, 27.89, 32.40 and 40.43 mg GAE/g dw, respectively, when compared with TPC of the grape samples from areas B and C extracted with the same methods. These values ​​were higher than those reported byPaladino & Zuritz (2011)Paladino, S. C., & Zuritz, C. A. (2011). Extracto de semillas de vid (Vitis vinifera L.) con actividad antioxidante: eficiencia de diferentes solventes en el proceso de extracción. Revista de la Facultad de Ciencias Agrarias, 43(1), 187-199.in Cabernet Sauvignon (Argentina) grape seed extracts obtained with water, ethanol, methanol and acetone. They were also higher than the values reported by Laos et al. (2020)Laos, F. S., Paucar, H. A., Gamboa, W. Q., Ceccarelli, J. G., & Campos, M. V. (2020). Determinación de polifenoles totales y actividad antioxidante de extracto de semillas de uvas residuos de la producción de Piscos. Revista de la Sociedad Química del Perú, 86(2), 123-131. http://dx.doi.org/10.37761/rsqp.v86i2.282.
http://dx.doi.org/10.37761/rsqp.v86i2.28... in Quebranta grape seed extracts obtained by an ultrasound bath with ethanol: water: acetic acid (90/9.5/0.5). Differences in the results between this study and the literature could be attributed to the extraction method as reported by Rababah et al. (2008)Rababah, T. M., Ereifej, K. I., Al-Mahasneh, M. A., Ismaeal, K., Hidar, A. G., & Yang, W. (2008). Total phenolics, antioxidant activities, and anthocyanins of different grape seed cultivars grown in Jordan. International Journal of Food Properties, 11(2), 472-479. http://dx.doi.org/10.1080/10942910701567521.
http://dx.doi.org/10.1080/10942910701567... , variety of grape cultivar o seasonal influences among others (Yilmaz et al., 2015Yilmaz, Y., Göksel, Z., Erdoğan, S. S., Öztürk, A., Atak, A., & Özer, C. (2015). Antioxidant activity and phenolic content of seed, skin and pulp parts of 22 grape (Vitis vinifera L.) cultivars (4 common and 18 registered or candidate for registration). Journal of Food Processing and Preservation, 39(6), 1682-1691. http://dx.doi.org/10.1111/jfpp.12399.
http://dx.doi.org/10.1111/jfpp.12399... ).

3.3 Antioxidant Capacity (AC)

The AC of DQGS by DPPH and FRAP is shown in Table 2. The cultivation area and the extraction technique had a significant effect on the DPPH and FRAP values, as well as a combined effect on those values ​​(Table 3). Likewise, the Pearson analysis showed a correlation at a level of 0.01 between TPC and DPPH (R = 0.7544), TPC and FRAP (R = 0.8582) and DPPH with FRAP (R = 0.6444). As pointed out by Lachman et al. (2009)Lachman, J., Šulc, M., Faitová, K., & Pivec, V. (2009). Major factors influencing antioxidant contents and antioxidant activity in grapes and wines. International Journal of Wine Research, 1(1), 101-121. http://dx.doi.org/10.2147/IJWR.S4600.
http://dx.doi.org/10.2147/IJWR.S4600... , TPC is mainly correlated with the antioxidant potency and the antiradical activity. Thus, a positive correlation between TPC and its antioxidant power was confirmed.

The AC values ​​by DPPH ranged from 174.74 ± 26.18 to 1628.15 ± 80.32 µmol TE/g dw and were higher than those reported byCoklar (2017)Coklar, H. (2017). Antioxidant capacity and phenolic profile of berry, seed, and skin of Ekşikara (Vitis vinifera L) grape: influence of harvest year and altitude. International Journal of Food Properties, 20(9), 2071-2087. http://dx.doi.org/10.1080/10942912.2016.1230870.
http://dx.doi.org/10.1080/10942912.2016.... inVitis viniferaseeds from Turkey. The greatest AC values by DPPH were found in DQGS from areas A and B extracted with SWE, 1,479.90 ± 12.86 and 1,628.15 ± 80.32 µmol TE/g dw, respectively. No significant differences were found between those values. The highest values obtained could be due to the structural and molecular modifications because of SWE treatment which improves the biological activity of antioxidants (Getachew & Chun, 2017Getachew, A. T., & Chun, B. S. (2017). Molecular modification of native coffee polysaccharide using subcritical water treatment: structural characterization, antioxidant, and DNA protecting activities. International Journal of Biological Macromolecules, 99, 555-562. http://dx.doi.org/10.1016/j.ijbiomac.2017.03.034. PMid:28283450.
http://dx.doi.org/10.1016/j.ijbiomac.201... ). For the DQGS from area C, no significant differences were found on AC by DPPH of extracts obtained with SWE, methanol and 50% acetone. The lowest AC values by DPPH were observed for the extracts from areas A and B when 70% ethanol and methanol extraction methods were applied. DQGS from area B extracted with 50% acetone showed higher AC values ​​by DPPH when compared to 70% ethanol and methanol extractions.

Regarding the AC by FRAP, the values ​​ranged from 200.39 ± 19.86 to 1,429.29 ± 29.75 µmol TE/g dw. The greatest AC values by FRAP of DQGS from areas B and C extracted with SWE were 1,429.29 ± 29.75 and 1,389.54 ± 7.46 µmol TE/g dw, respectively. No significant differences were found between those values. The extract from area A showed a lower AC by FRAP when the SWE was used (845.13 ± 95.32 µmol TE/g dw).

The non-conventional extraction technologies, such as SWE, currently underused because of the lack of data on the profitability of the investment, offer great opportunities and challenges. However, high capital investment, high running cost, training, maintenance cost, etc. increase limitations to scale-up green extraction methods. Thus, a cost assessment analysis could provide an understanding of the cost-benefit related to the utilization of those “green techniques”. In addition, there is a great necessity to work on some parameters such as replacement of solvents with emerging green alternatives for efficient extraction (Belwal et al., 2020Belwal, T., Chemat, F., Venskutonis, P. R., Cravotto, G., Jaiswal, D. K., Bhatt, I. D., Devkota, H. P., & Luo, Z. (2020). Recent advances in scaling-up of non-conventional extraction techniques: learning from successes and failures. Trends in Analytical Chemistry, 127, 115895. http://dx.doi.org/10.1016/j.trac.2020.115895.
http://dx.doi.org/10.1016/j.trac.2020.11... ; Picot-Allain et al., 2021Picot-Allain, C., Mahomoodally, M. F., Ak, G., & Zengin, G. (2021). Conventional versus green extraction techniques—a comparative perspective. Current Opinion in Food Science, 40, 144-156. http://dx.doi.org/10.1016/j.cofs.2021.02.009.
http://dx.doi.org/10.1016/j.cofs.2021.02... ).

In the case of AC by FRAP of DQGS from area B, no significant differences were found among the extracts obtained with the conventional methods. The lowest AC values by FRAP were observed for the extracts from area A when 70% ethanol and methanol extraction methods were applied as was also observed with DPPH. Garrido (2016)Garrido, M. M. (2016). Aprovechamiento de subproductos de vinificación. Evaluación del potencial biológico de la semilla de uva. (Undergraduate thesis). Universidad de Sevilla, Sevilla. analyzed Pedro Ximénez grape variety from Spain and found an AC by FRAP of 249.83 ± 62.69 µmol TE/g dw similar to the value determined in this study when using DQGS methanol and ethanol extracts from area A. On the other hand, our AC by FRAP determined in all extracts from areas B and C were higher than the value obtained by Garrido (2016)Garrido, M. M. (2016). Aprovechamiento de subproductos de vinificación. Evaluación del potencial biológico de la semilla de uva. (Undergraduate thesis). Universidad de Sevilla, Sevilla..

Furthermore, the extracts from area A obtained with 50% acetone presented higher AC values ​​by DPPH and FRAP methods compared to 70% ethanol extracts. No significant differences (p > 0.05) were observed between AC by DPPH and FRAP of DQGS from area C extracted with 50% acetone and methanol. Margraf et al. (2016)Margraf, T., Santos, É. N. T., Andrade, E. F., van Ruth, S. M., & Granato, D. (2016). Effects of geographical origin, variety and farming system on the chemical markers and in vitro antioxidant capacity of Brazilian purple grape juices. Food Research International, 82, 145-155. http://dx.doi.org/10.1016/j.foodres.2016.02.003.
http://dx.doi.org/10.1016/j.foodres.2016... evaluated purple grape juice and concluded the geographical origin and variety of grapes have an important role in distinguishing Brazilian purple grape juices according to the free-radical scavenging activity (ABTS) and reducing capacity (FRAP).

4 Conclusions

This study reported the effects of the cultivar geographical area and extraction methods on the TPC and AC of DQGS obtained from the pisco industry in Ica-Peru. Also, the influence of the cultivar location on grape seed oil yield and fatty acids profile was reported. The QGS oil, from the three areas under study, showed a high content of PUFA with linoleic acid present in the highest amount. In comparing extraction methods, subcritical water treatment, a suitable environmentally friendly technique, achieved the highest average value ​​of TPC in DQGS (area A), and AC determined by DPPH and FRAP methods in DQGS (area B). These results show the advantages of green technologies such as subcritical water for extraction of bioactive compounds with antioxidant properties from Pisco production waste. Therefore, further studies should explore the application of QGS oil and extracts obtained with subcritical water as food ingredients and in the pharmaceutical industry. In addition, it could be also concluded that geographical location had a significant effect on TPC and AC of Quebranta grapes. Thus, as future perspective, study of conditions such as temperature, soil type, availability of nutrients and other environmental factors may help to maximize health benefits of bioactive compounds.

References

Publication Dates

History