The grape seeds were isolated from the pomace resulting from the wine making process, washed, dried for 24 hours (h) at room temperature, and ground. Seeds from the grape varieties of the 2015 harvest were investigated, namely four red brands, including Cabernet Sauvignon, Feteasca Neagra, Merlot, Pinot Noir and two white brands Columna and Riesling Italian. The material was supplied by the winery of Murfatlar, situated in Dobrogea, a south-eastern region of Romania.

The pectin lyase solution (Pectinex XXL, activity 10.000 U mL^-1 according to the supplier) was purchased from Novozymes A/S (Bagsvaerd, Denmark).

The 10-14% BF₃ solution in methanol was supplied by Merck (Darmstadt, Germany). Petroleum ether (b.p. 40-60 ºC), analytical grade methylene chloride, methanol, and 96% ethanol solvents were purchased from Sigma Aldrich and were used as delivered. The citric acid and disodium phosphate dihydrate for the buffer solution were supplied by Sigma Aldrich.

Portions of 10 g ground grape seeds were kept for certain amounts of time in a solution of 3 mL commercial enzyme and 37 mL buffer solution of pH 3.5 (0.1M citric acid and 0.2M Na₂HPO₄, 2.3/1 volume ratio), in 100 mL capped glass amber flasks, at room temperature (20 ºC), with gentle intermittent stirring. The optimal time for treatment was established based on the oil yield (see Figure 1). For each grape seed variety the experiments were conducted in triplicate. After treatment, the seeds were separated by filtration, washed and dried before extraction.

The residual solution was concentrated in a Heidolph rotary evaporator, in a water bath (50 ºC), and the residue was studied by NMR spectroscopy.

For checking the buffer effect on the grape seed a number of experiments were performed by keeping the seeds in the buffer solution (ratio w/v = 1/4) for 24 hours, at room temperature. The quantity of the oil from these samples was in the same range as the corresponding untreated oil.

The oil samples were obtained from the dried ground seeds (pre-treated or not with enzyme) by the Soxhlet extraction method with petroleum ether (b.p. 40-60 ºC), following the protocol ISO 659 (2009). The solvent was partially recovered by evaporation under atmospheric pressure, heated on a water bath at 65 ºC, using a Heidolph rotary evaporator. The weight of the oil was measured with an analytical balance (accuracy of ± 0.0001 g) and the corresponding volume was calculated using the density value for grape seed oil of 0.92 g/mL (Ceriani et al., 2008). The value of the density was confirmed by checking on a number of experimental samples. The experiments were performed in triplicate, the average volumes of oil being presented in Figure 2. The initial quantity of seeds was the same for both types of experiments (with or without pre-treatment) as measured before pre-treatment.

The grape seed oil FAPs were determined following the ISO 12966 (2015) protocol. The free fatty acids were obtained by treatment of 0.5 mg oil with 0.9 mL NaOH 0.5 M solution in methanol, at reflux, for 10 min. Then, they were esterified by treatment with 0.1 mL BF₃ solution and 0.8 mL methanol, at reflux, for another 10 min. After dilution with 4 mL distilled water, the fatty acid methyl esters (FAMEs) were extracted with methylene chloride and were submitted to a standard gas chromatographic (GC) analysis. GC analysis was performed on a highly polar capillary column Supelco SP^TM 2560 (100 m × 0.25 mm, 0.20 μm film) with a biscyanopropyl stationary phase. A certified reference material (Supelco^TM 37 Component FAME Mix) was used for the identification of the FAPs (see table 1). For each oil sample, three GC analyses were performed. The composition was expressed as a percentage based on the ratio between each peak area and the total area.

The CUPRAC assay (Apak et al., 2008) was applied to assess the total antioxidative capacity (TAC) of the oil. Samples of oil extracted by sonication in ethanol (1/10, v/v) were treated with 1 mL CuSO₄ 10^-2 M aqueous solution, 1 mL of 7.5 × 10^-3 M Neocuproine ethanolic solution, 1 mL CH₃COONH₄ buffer (pH 7) and 0.4 mL distilled water. After stirring for 30 min, at room temperature, the absorbance (Abs) at 450 nm was measured. TAC was expressed as a trolox equivalent (TE, μmol per 100 g oil) based on a previously drawn calibration curve (y = 0.058x; R² = 0.99). The average values obtained from three experiments for each oil sample are presented in Table 1.

The polyphenols (PPs) were quantified using the known Folin-Ciocalteu (FC) method (Ballus et al., 2015). Dried seeds (2 g), before and after the oil extraction, were extracted with 9 mL ethanol/water (2/1, v/v). Extract samples (0.1 mL) were mixed with the FC reagent (0.5 mL), distilled water (1.5 mL) and, after 5 min, with 20% Na₂CO₃ solution (1.5 mL). The mixture was incubated for 2 h, at room temperature, in the dark. The Abs at 750 nm was measured vs. a blank. The analyses were performed in triplicate, for seeds with or without enzymatic pre-treatment, before and after oil extraction. The TPPC was expressed as gallic acid equivalents (GAE, mg per 100 g dried seeds) derived from a previously obtained calibration curve (y = 0.061x; R² = 0.98) (Table 2).

One of the most important features of lipids is their fatty acid profile (FAP). The previous research involving enzymatic pre-treatment (Passos et al., 2009; Marousek et al., 2015b) did not check this aspect for the oil applications. Thus, the extracted oils were analyzed by a GC standard method (see paragraph 2.2.3). The FAPs of the oil samples are presented in Table 1.

The main component of these oils was linoleic acid, a representative polyunsaturated fatty acid (PUFA). The saturated fatty acid (SFA) content was around 10%. Similar FAPs were revealed for Portuguese grape varieties (Fernandes et al., 2013) and Spanish wines with protected denomination of origin (Bada et al., 2015). Due to their low SFAs and high linoleic acid contents these oils present good nutritional qualities (Alsharari et al., 2017). According to the experimental results the pre-treatment changed the FAPs only slightly (≤ 0.4%).

As expected, the statistical analysis revealed a close correlation between PUFA and linoleic acid (y = 1.004x+0.1003; R² = 0.99). The grape variety showed an impact on the content in fatty acids in oil. Principal component analysis (PCA) applied to FAPs disclosed the differences or similarities of the analyzed oils (Figure 3). The PCA analysis revealed the following eigenvalues: F1: 2.067, F2: 1.375, F3: 0.558, F4: 0.000. Thus, the variance values of the principal components (PCs) were 51.66% (F1), 34.39% (F2), 13.95% (F3) and 0.003% (F4). The cumulative percentage for PC1 and PC2 coordinates was over 86.00% so only these 2 PCs had to be considered.

The oils from Columna and Riesling (with a higher content in oleic acid) as well as Merlot and Cabernet Sauvignon (with higher content in linoleic acid) showed a good discrimination on the F1 (PC1) direction; whereas Feteasca Neagra and Cabernet Sauvignon (with high content in SFA) exhibited good discrimination on the F2 (PC2) direction.

A comparison with oils from other seeds seemed of interest. According to the literature the average values for PUFAs (%) (Chira et al., 2011) for oils obtained from sunflower (59.77 ± 4.80), soybean (55.02 ± 2.88) and rapeseed (25.95 ± 1.70) were by far lower than those for the grape seed oil (75.00 ± 2.40%). Thus, this oil stands out for its high content in healthy PUFAs.

Besides FAP, the antioxidant capacity of vegetable oils is essential for their applications (Chambre et al., 2019). The method used to establish the TAC was the CUPRAC assay. This assay is recommended due to its simplicity, reduced cost and reduced reaction time (Apak et al., 2008). It gives an accurate estimation of the TAC of the analyzed sample. The experimental results obtained for the studied oils are presented in Table 1. The oils obtained after the enzymatic treatments of seeds are richer in antioxidants, with the TAC values being 1.7-2.4 times higher compared to those of oils from untreated seeds (see Table 1). The enzymatic treatment destroyed not only the interactions of oil with the cell walls but also that of the antioxidants (Chamorro et al., 2012), increasing the quantities of extracted antioxidants. As expected, the oils obtained from the red grape varieties had higher antioxidant contents and improved health benefits.

A comparison of the TAC of grape seed oils (see Table 1) with other vegetable oil TACs is of interest. Thus, from the literature data the TAC values, expressed as TE (mmol kg^-1) are as follows: 1.79 for extra virgin olive oil, 2.20 (soybean), 1.29 (corn), 1.17 (sunflower) (Pellegrini et al., 2003). Due to the enzymatic treatment the related grape seed oils are ranked in better positions by their TE values.