Characterization of fatty acid, antioxidant, and polyphenol content of grape seed oil from different Vitis vinifera L. varieties ☆

– In this study, we examined the yield and oil quality of eight different grape varieties. For the experiments, the grape seeds were obtained from the Benedictine Pannonhalma Archabbey in the northwestern region of Hungary. The aim of the studies was to determine the oil yield obtained by extraction and to examine the differences between the fatty acid composition, antioxidant capacity, and total polyphenol content of the oils of different grape varieties. Based on the results, the oil content of the grape seeds varied between 99.91 g/kg and 126.74 g/kg. The grape seed oils analysed contained signi ﬁ cant amounts of stearic acid (3.42 – 9.93%), palmitic acid (7.81 – 10.66%), oleic acid (14.29 – 19.92%), and linoleic acid (66.85 – 72.47%). Besides, the grape seed oils tested contained several other fatty acids in small amounts. There were signi ﬁ cant differences in the total antioxidant and total polyphenol content of the oils. Total polyphenol content ranged from 0.24 to 1.13 mg GAE/g, while the total antioxidant content changed between 0.12 and 0.78 m g TEAC/g. The results show that the studied varieties are suitable for the production of table grape seed oil based on their oil yield, and the oils have favourable, health-protecting properties in terms of their quality.


Introduction
Grape (Vitis vinifera L.) is one of the most widely grown fruit in the world. Nearly 80 million tons of grapes are harvested annually (OIV, 2019). Much of this is consumed in the form of fresh fruit, juice or raisins. The other part of the harvested grapes is used for winemaking, it also shows that wine is one of the most popular alcoholic beverages (Kallithraka et al., 2006;Teixeira et al., 2014). The most important wine regions are in Europe (Italy, Spain, France, Germany and Portugal), North and South America (United States, Argentina, Chile) Asia (China) (OIV, 2019), Australia and South Africa. However, the cultivation and processing of grapes also involves the production of large quantities of byproducts such as grape marc, grape seed, grape skin, grape stem and grape leaf. Most of these by-products contain large amounts of physiologically important phenolic compounds. Grape marc is the main by-product that remains after pressing; which is about 20% of all grapes processed in wineries (Goula et al., 2016;Bordiga et al., 2019). Grape marc is a basic byproduct of wine production, which also includes 20-26% grape seed with the valuable oil and proteins it contains . Thirty-five thousand tonnes of grape seed oil are produced worldwide each year, of which 10 000 tonnes come from French factories (Pierron, 2017).
The grapes should ideally contain 4 seeds, which are formed from the four seed buds in the seed coat. In exceptional cases, more seeds may be formed, but usually less than four seeds are typical, 13% of the total weight of grapes is grape seed. The complete absence of seeds can be a characteristic of the grape variety, this is an ideal property in the case of table grapes and raisins. The nutrition of grape seed is physiologically important as it contains roughly 32-43 m/m% dietary fiber, 7-17 m/m% protein and about 5-8 m/m% complex phenolic compound, as well as sugars, mineral salts, etc. (Fantozzi, 1981;Shi et al., 2003;Campos et al., 2008;Tangolar et al., 2009;. In addition, grape seeds contain 8-20% oil, which is rich in essential fatty acids. The amount of oil that can be extracted depends on the grape variety and the extraction process used (Bail et al., 2008). The chemical composition of the extracted oil is influenced by the degree of ripeness and species of the seeds, the environmental parameters, the cultivation works and to a small extent the seed extraction protocol (Shinagawa et al., 2015;Garavaglia et al., 2016;Martin et al., 2020). Despite these factors, unlike grapes, grape seeds and their extracts, especially grape seed oil, contain large amounts of lipophilic molecules, among which are several important biocomponents.
The most common of these molecules are fatty acids. In general, about 90% of the total amount of grapeseed oil is mono-and polysaturated fatty acids. It contains in the highest amount of linoleic acid (58-78%), oleic acid (3-15%), and to a lesser extent other saturated fatty acids (10%) (Bail et al., 2008;Rombaut et al., 2015;Konuskan et al., 2019).
The second largest group of lipophilic molecules in grape seeds are vitamins. Phytosterols are also important lipophilic molecules in grape seed oil, but their amount is influenced by harvest conditions and oil extraction methods. The biological importance of phytosterols lies in their antioxidant activity and their role in cholesterol metabolism (Shinagawa et al., 2015;Garavaglia et al., 2016).
Phenols are natural molecules with antioxidant effects and are present in grapes, especially in grape seeds and their extracts. However, hydrophilic phenols make up only a small proportion of grape seed oil (Fernandes et al., 2013;Khurana et al., 2013), but properly selected oil extraction processes can increase the phenol content of oils (Maier et al., 2009;Rombaut et al., 2015). Among the phenolic compounds, grape seed oil contains mainly gallic acid, catechin, epicatechin, procyanidins and proanthocyanidins or condensed tannins, which are known for their antioxidant activity (Garavaglia et al., 2016).
In this study, grape seed oil was produced from eight different grape varieties using Soxhlet extraction, which was grown in the northwestern region of Hungary by the Benedictine Archabbey of Pannonhalma. The aim of the studies was to determine the oil yield obtained by solvent extraction and to investigate the differences between the fatty acid composition, antioxidant capacity and total polyphenol content of the oils of different grape varieties.

Oil extraction from grape seeds 2.3.1 Soxhlet-extraction
The milled grape seeds (10 g) were extracted with 170 mL petroleum ether for 3 h at a maximum temperature of 70°C in a Soxhlet apparatus. After extraction was completed, petroleum ether was evaporated by a rotary evaporator (Bibby, RE 100).
The oils obtained were weighed and the yields were calculated. Grape seed oils were weighed into 4 mL screw cap vials and stored in an ultra-freezer at À55°C for further analysis.

GC-MS method 2.4.1 Sample preparation
Six mL of 0.5 M NaOH was added into a round bottom flask containing 25 mg grape seed oil. Samples were extracted for 6 min at 70°C using a laboratory water bath equipment. After the oil droplets had dissolved, the solution was heated with 70 mL of boron trifluoride in methanol (14%) for 2.5 minutes at 70°C. Finally, 4 mL n-hexane and 6 mL saturated NaCl solution were added and vortexed. For analysis, the methyl-esterified sample was taken from the upper hexane phase, which was first removed and concentrated under nitrogen.

GC-MS analysis
For the determination of fatty acid composition of the oils a Shimadzu (Kyoto, Japan) GCMS-QP2010 SE type equipment was used. Fatty acids were separated on a Zebron BPX-70 (30 m Â 0.25 mm Â 0.25 mm) column (Phenomenex, USA). The applied temperature program was 60°C-120°C with a heating rate of 13°C/min, then 120°C-240°C with a heating rate of 2°C/min, and finally 240°C for 8 minutes. One mL of sample solutions was transferred to the 220°C injector of the GC-MS in split mode (split ratio 40). Helium was used as carrier gas with a linear flow rate (1 mL/min).

Determination of total antioxidant and polyphenol content 2.5.1 Sample preparation
To determine the antioxidant and polyphenol content, grape seed oils were extracted by solvent extraction technique as follows. Briefly, a mass of 1 g of oil was extracted with 5 mL of ethanol/water (70:30 v/v%) at 60°C for 5 min by means of an RF-120F ultrasonic bath (Realsonic, Hungary). The oily extracts were filtered through a filter paper, and the filtrate was used for further analysis.

FRAP assay
Two hundred mL of extracted sample, 3 mL of FRAP solution, and 100 mL of water were pipetted into a test tube. The finished solutions were placed in a dark place for 5 min and then their absorbance was measured with a Spectroquant Pharo 100 spectrophotometer (Merck, Germany) at a wavelength of 593 nm against the blank. Trolox was used as a standard (1-30 ug/mL) and the results were expressed as ug Trolox equivalent antioxidant capacity (TEAC)/g oil.

Folin-Ciocalteu assay
To 200 mL of grape seed oil extract, 1.5 mL of high purity water was pipetted and the reagents were added. First 2.5 mL of 10% Folin-Ciocalteu reagent, then 2 mL of 7.5% Na 2 CO 3 . The test tubes containing the mixture were placed in a dark place for 90 min, and then the absorbance was measured at 725 nm versus the blank. Gallic acid was used as a standard (25-100 mg/mL).

Data analysis
The total antioxidant and polyphenol contents of grape seed oils were determined in Microsoft Office Excel from the absorbance values measured for grape seed oils using the equation of the second-order least squares analytical curve fitted to the measurement solutions using the nonlinear leastsquares method. All the results are expressed as means (n = 3) ± standard deviation.

Oil yield
During the Soxhlet extraction, the oil content of each grape seed sample was determined gravimetrically. During the evaluation, we present our results in terms of g/kg dry matter (Fig. 1).
As shown in Figure 1, the grape seed cultivars we studied provided similar results in oil yield. Significant differences were found, the difference between the highest and lowest oil yield was 26.83 g/kg dry matter. The highest amount of oil was extracted from "Pinot Noir" (126.74 g/kg) and the lowest amount from "Lemberger" (99.91 g/kg) grape seed meal.

Fatty acid composition of grape seed oils
Grape seed oil is rich in polyunsaturated fatty acids. The fatty acid composition of the 8 types of grape seed oil tested is shown in Table 1. For all cultivars, the major fatty acids were stearic acid (C18:0), palmitic acid (C16:0), oleic acid (C18:1), and linoleic acid (C12:2). Linoleic acid was the most abundant fatty acid in all samples, contributing between 66.8% and 72.47% of total fatty acids. The Italian Riesling seeds had the lowest content of linoleic acid (66.85%), with "Merlot" containing the highest amount of 72.47%. The next fatty acid in the row is oleic acid, ranging from 14.29% (Cabernet Franc) to 19.92% (Italian Riesling). The seeds also contained significant palmitic acid. The smallest amount was detected in "Italian Riesling" (7.81%) and the highest amount in "Cabernet Franc" (10.66%). Grape seed oil samples still contained small but significant amounts of stearic acid, the smallest amount of "Merlot" (3.42%), and the largest amount of "Rhine Riesling" (9.93%) contained among the examined grape varieties. The minor fatty acids included caprylic acid, capric acid, lauric acid, myristic acid, pentadecanoic acid, palmitoleic acid, heptadecanoic acid, heptadecenoic acid, linolenic acid, arachidic acid, eicosenoic acid, docosanoic acid, land ignoceric acid (all at < 0.3%) We found significant differences in the fatty acid composition of the oils of each grape variety, a finding supported by several studies (Beveridge et al., 2005;Crews et al., 2006;Pardo et al., 2009;Sabir et al., 2012;Konuskan et al., 2019).

Polyphenol content of grape seed oils
Phenolic compounds are poorly soluble in the oily phases, but small amounts are transferred to the oil from the solid matrix during extraction. The method used was able to detect polyphenols from all types of grape seed oil.

Conclusion
The oil yields of the studied grape varieties showed significant differences and ranged from 9 to 13%. These values fit into the 8-20% oil yields reported in the literature. The fatty acid composition of eight different grape seed oils was determined by GC-MS. As shown by the results obtained, the fatty acid profile of oils was similar. In spite of this, sometimes, we found a significant difference between the fatty acid content of different grape varieties. Our study also revealed that the total polyphenol and antioxidant content of grape seed oil is much lower than that of grape seed, but its consumption can still have a beneficial effect on human health. Our result supported that the analyzed grape varieties are suitable for the production of edible grape seed oil. It is important to note that if we want to press oil under industrial conditions from grape seeds for consumption, we need a large amount of grape seed. In addition to the 8-20% oil yield mentioned in the literature, that means 5-13 kg of dried grape seeds are required to produce 1 liter of grape seed oil.