In the present study, seeds of the Cabernet Sauvignon, Shiraz, Hamburg Muscat, and Alfons Lavallee varieties (Vitis vinifera L.) from the 2018 harvest period, obtained from Tekirdağ Viticulture Research Institute, were used. After separating the grape seeds from the marc and removing foreign substances, they were dried in the sun and prepared for the cold pressing process to obtain oil. The grape seeds from the four different grape varieties were divided into three equal amounts.

The heat treatment was carried out in a home-type oven (Bosch) with adjustable temperature. No heat treatment was applied to the first batch, which was evaluated as a control sample. The second batch was heat treated in the oven at 40 °C for 30 minutes, and the third batch was heat treated in the oven at 80 °C for 30 minutes. All analyses were carried out in the same way for all three batches.

Grape seeds weighing 3-4 kg, free from foreign substances, were placed in a cold press machine (Model Ekotok-1, Tokul Agro Products Ind. and Trade Ltd. Co., Izmir, Turkey) and the extracted oil was stored in dark-colored bottles at +4 °C under refrigerator conditions.

Crude oil analysis of the grape seed samples was carried out according to the Soxhlet extraction principle using petroleum ether solvent (AOAC, 1990).

A viscosity analysis of the grape seed oil samples was conducted using a stress and temperature-controlled rheometer at 25 °C in the range of 1-100 shear rates. The results were given in mPa.s.

The L* (lightness/darkness), a* (redness/greenness), and b* (yellowness/blueness) color values of the grape seed oils were measured using a Minolta Chroma Meter CR 400 (Konica Minolta, Inc., Osaka, Japan). The device was calibrated before measurement, and color values were determined using the CIELab color scale. Readings were taken from three different points to obtain an average value for all samples.

The IUPAC method numbered 2.201 (IUPAC, 1987) was used to determine the free fatty acidity of the samples. Percent free fatty acidity is stated as the percentage of oleic acid of the total fatty acids that are not bound to the triglyceride structure in the oil.

The IUPAC method numbered 2.501 (IUPAC, 1987) was used to determine the peroxide number. The basis of this analysis is that potassium iodide is oxidized by peroxide oxygen in the oil, releasing iodine, and the amount of this free iodine is titrated with thiosulfate.

The total phenolic content of the extracted oil samples was measured using the Folin-Ciocalteu assay, as described by Waterhouse (2002). Total antioxidant scavenging activity was determined by the DPPH free radical scavenging capacity method, following the procedure reported by Garzón and Wrolstad (2009).

To prepare grape seed oil samples for analysis, the oil was mixed with methanol (1:1) in a vortex, and the methanolic part was separated. The remaining oil was mixed again with methanol (1:1) and the methanolic portion was separated again. The separated methanolic extract was used in the analysis.

For total phenolic content analysis, all results were expressed in terms of gallic acid equivalent (mg/kg). For antioxidant activity, different amounts of extracts (100, 200, 300 μL) were added to 1.95 mL of DPPH solution. After mixing by vortex, the mixture was left in the dark at room temperature for 30 minutes. Absorbance was read at 517 nm. Results were calculated in ‘μmol trolox/g oil’. Samples were analyzed in triplicate.

The fatty acid composition of the grape oil samples was determined by gas chromatography. The samples were converted into methyl ester derivatives with BF3-methanol according to the method Ce 2-66 of Araujo et al. (2008). The extracted oil samples were converted to their fatty acid methyl esters (FAME). The samples were injected into the gas chromatograph (Perkin-Elmer 8320B), equipped with a split injector (split ratio 1:40) (nitrogen was used as the carrier gas) and a flame ionization detector (FID). A silica capillary column (CP Sil 88, 50 m x 250 μm i.d., 0.20 μm film; Chrompack, Middelburg, Netherlands) coated with 100% cyanopropyl polysiloxane was used. The detector temperature was set at 250 °C. Helium was used as the carrier gas at a rate of 1 mL/min. Peak identification was performed by comparing the retention time of each fatty acid with a commercial standard mixture of FAMEs.

All characteristics examined in the experiment, which was set up according to four grape varieties and three temperature applications, were subjected to variance analysis according to the “Random Plot Trial Design”. Difference groupings of their means are presented in separate tables. All characteristics in which variety, temperature, and variety x temperature interactions were found to be significant were subjected to the LSD (Least Significant Difference) test at the 0.05 level.

The crude oil ratios of the grape seeds of the four grape varieties are presented in Table 1. As shown Table 1, the variety with the highest oil content is Hamburg Muscat (9.29%) and it is followed by Alfons Lavallee (8.50%), Shiraz (7.65%) and Cabernet Sauvignon (6.04%). The changes in the oil content of grape seeds depending on the variety were found to be statistically significant (p < 0.01). The oil contents of grape seeds (6.04-9.29%) detected in the present study (Table 1) are almost similar to grape seeds (6.93-8.80%) reported by Koç et al. (2019) and lower than that reported by Apaydin et al. (2017) for Cabernet Franc and Shiraz grape seed samples (11.73-14.77%) and by Juhaimi et al. (2017) for Cabernet Sauvignon (10.06 %). The crude oil content of oilseeds and oily fruits depends on the type and variety of seeds (Juhaimi et al., 2017; Apaydın et al., 2017; Taşeri et al., 2018), seed-growing region (Koç et al., 2018), harvest period (Özcan et al., 2017) and post-harvest storage conditions.

Some physicochemical properties of heat-treated grape seeds at different temperatures are presented in Table 2. As seen in Table 2, viscosity values of the control group grape varieties were determined to be between 44.78-52.80 mPa.s. In the control group grape varieties, the highest viscosity was determined in the Alfons Lavallee variety (52.80 mPa.s), while the lowest viscosity was detected in the Cabernet Sauvignon variety (44.78 mPa.s). When examining the changes in the viscosity values of grape seed oils according to temperature applications, it was determined that the viscosity value increased at both temperatures (40 and 80 °C) applied to the Shiraz variety. It was determined that the viscosity values of the Cabernet Sauvignon and Alfons Lavallee varieties first increased and then decreased depending on the applied temperature, while the viscosity value of the Hamburg Muscat variety first decreased and then increased depending on the temperature increase. All these changes in the viscosity values of seed oils due to heat treatment were found to be statistically significant (p < 0.01). Previous studies show that viscosity does not always experience a constant increase or decrease with the application of heat to the seeds (Şimşek et al., 2015) . It can be said that this situation is due to the fatty acid composition changing under the influence of temperature. The increase in oil viscosity according to temperature increase may be the result of the formation of polymers, polymerization, and the formation of high molecular weight compounds, including carbon-carbon and carbon-oxygen-carbon bridges between fatty acids.

The effects of heat treatment on the free fatty acid and peroxide values of the oil samples are presented in Table 2. The free fatty acid value of control group grape seeds ranged between 0.20 and 0.68% in the present study. The lowest free fatty acidity value was determined at the control temperature in the oils of the Hamburg Muscat variety (0.20%). The peroxide value varied between 7.72 and 9.88 meq O₂/kg for the control group grape seed oils in this study and is compliant with Turkish Food Codex, which permits a maximum of 15 meq O₂/kg for peroxide value and a maximum of 2% in terms of oleic acid for free fatty acidity. Similar results were reported by Koç et al. (2019) who found the free fatty acid and peroxide values of grape seed oils obtained by the cold press method to be 0.67-2.74% and 10.45-22.03 meq O₂/kg, respectively. Similarly, Fu et al. (2018) determined the free fatty acid value (%) in the Cabernet Sauvignon variety control group grape seeds as 0.55%.

According to Table 2, free fatty acid, as a sign of free fatty acid hydrolysis, and peroxide values, indicative of oxidation in grape seeds, increased significantly (p < 0.01) in direct proportion to the increase in temperature. The highest increase in free fatty acid among the varieties was detected in the Shiraz variety after 80 °C heat treatment. The free fatty acid value increased from 0.23 to 0.71% in Shiraz grape seed oil. Likewise, as shown in Table 2, the peroxide number increased from 8.19 to 14.87 meq O₂/kg in Shiraz as a result of 80 °C temperature application. Maximum peroxide numbers in all grape varieties were observed after the 80 °C temperature application, and these values are in agreement with the Turkish Food Codex. It is inferred that the increase in temperature affected lipid oxidation, resulting in the formation of hydroperoxides. Similar to our findings, Herchi et al. (2016) investigated the effect of heat treatment (110 °C) on some quality characteristics of flaxseed hull oil and stated that the free fatty acid, initially determined as 0.9%, increased to 1.7% after heat treatment. Şimşek et al. (2015) also reported that peroxide and free fatty acid values increased with an increase in roasting temperatures. The increase in free fatty acid value could be attributed to lipid oxidation and hydrolysis, which produce free fatty acids. Additionally, the increase in peroxide numbers indicates that this oil was unstable to oxidative degradation.

Color, one of the physical properties of oils, is an important quality indicator. Color values are expressed as L* (lightness/darkness), a* (red/green), and b* (yellow/blue). The measurements of the L*, a*, and b* color values of the oils from grape varieties after heat treatment are shown in Table 2. The highest L* value in all varieties was observed at the control temperature. On the other hand, the highest a* values in the oils of Cabernet Sauvignon and Alfons Lavallee varieties were determined at the control temperature, 40 °C in Shiraz, and 80 °C in Hamburg Misket. When examining the changes in the a* values of grape seed oils depending on temperature applications, it was determined that the a* value increased at both temperature levels (40 and 80 °C) applied to the Hamburg Marble variety. When b* values are examined, the highest b* values in the oils of Cabernet Sauvignon, Alfons Lavallee and Hamburg Misket varieties were measured at the control temperature, and the highest b* value in the oils of the Shiraz variety was measured at 40 °C. While changes in the L* and a* values of the seed oils were found to be statistically significant (p < 0.01), the changes in the b* value were not statistically significant.

The antioxidant activity and total phenolic content of grape seed oils were determined and are shown in Table 2. The results of the total phenolic contents were expressed as mg equivalents of gallic acid/kg oil, and the results of the antioxidant capacity were shown as μmol trolox/g oil. The phenolic contents of the control grape seed oil samples were found to be 453.57, 418.43, 524.77, and 259.67 mg GAE/kg grape seed for Cabernet Sauvignon, Alfons Lavallee, Shiraz, and Hamburg Muscat, respectively. The antioxidant activity of the control grape seed oil samples was found to be 1.82, 1.39, 1.26, and 0.64 μmol trolox/g grape seed oil for Cabernet Sauvignon, Alfons Lavallee, Shiraz, and Hamburg Muscat, respectively. These values are in accordance with reports in the literature. (Seçen, 2017; Göktürk Baydar et al., 2007). The antioxidant activity results obtained from our study are slightly higher than the results obtained by Koç et al. (2019) but are compatible with that study. The minor differences between the polyphenol contents of grape seed oils are due to the variety as well as climatic conditions, maturity, and growing region of the grapes. Literature reviews have revealed that the antioxidant activity in raw materials is highly related to the presence of phenolic compounds. When examining the changes in the total phenolic content of grape seed oils depending on temperature applications, it was determined that the highest total phenolic content values for all varieties were detected at the control temperature and decreased after both heat treatment applications. Similar to total phenolic content, the antioxidant activity of the grape seed oils decreased as the temperature applied increased. All these changes were found to be statistically significant (p < 0.01). When examining literature studies, it was determined that in some studies (Konuk and Korel, 2015; Perez et al., 2015) the total phenolic content decreased with the effect of temperature, while in others (Kim et al., 2006) it increased. This difference may be due to the variety, the degree of heat treatment applied, and the presence of phenolic substances in different bound forms in the product.

The fatty acid composition of the oils from the grape varieties used in this study is presented in Table 3. According to Table 3, the dominant fatty acid in all varieties is linoleic acid, followed by oleic, palmitic, and stearic acids, respectively. While the linoleic acid content of the control group grape seed oils ranges from 71.10 to 72.09%, the oleic acid and palmitic acid contents vary between 14.55 and 16.64% and 7.45 and 8.64%, respectively. It was determined that these fatty acid values in the control samples of all grape varieties did not differ significantly. All four varieties (Cabernet Sauvignon, Alfons Lavallee, Shiraz, and Hamburg Muscat) of grape seed oil were rich in linoleic, oleic, palmitic, and stearic acids. These fatty acids are well known for their health benefits and determine the health-promoting and nutritional features of oils. Other fatty acids were found at low levels. Our findings were similar to previous results reported by other authors, who also identified linoleic acid as the dominant fatty acid in grape seed oils. Ustun Argon et al. (2020) investigated the specific characteristics of cold-pressed grape seed oil in terms of chemical properties, production practices, nutritional profile, alternative usages, health effects, and adulteration. They stated that linoleic, palmitic, stearic, and oleic acids are the main fatty acids in cold-pressed grape seed oil (Ustun Argon et al., 2020). Similarly, Khan et al. (2020) reported that the major fatty acids in the Manjari Medika variety grape seed oil included linoleic acid (60-70%), oleic acid (8-13%), palmitic acid (4.5-8%) and stearic acid (4-6%). Apaydin et al., (2017) obtained values for linoleic acid content at 72.19 and 61.35% for seed oils of Cabernet Franc and Shiraz varieties, respectively. Similarly, Dabetic et al., (2021) indicated that the major fatty acid found in six different grape varieties was linoleic acid, reporting that linoleic acid ranged from 61.42 to 67.59%.

The saturated and unsaturated fatty acid values of the oils from the grape varieties used in the study are shown in Table 3. While the saturated fatty acid (SFA) contents of the control group grape seed oils range from 11.65 to 12.86%, the content of polyunsaturated fatty acids (PUFA) and monounsaturated fatty acids (MUFA) vary between 71.37 -72.42% and 14.96 -16.89%, respectively. This is in accordance with literature reports which generally classify the fatty acids in the grape seed oil of the control groups as PUFA > MUFA > SFA (Dabetic et al., 2021; Khan et al., 2020; Apaydin et al., 2017; Juhaimi et al., 2017). Linoleic acid, which is associated with a high rate of unsaturated fatty acids, was the key fatty acid in grape seed oils of all grape varieties. Moreover, the high amount of unsaturated fatty acids indicates that grape seed oil has a high nutritional value, making it indispensable for human health. The fatty acid composition of grape seed oil is affected by grape variety, climatic conditions, ripening time, and the fruit-growing region (Odabaşioglu et al. 2023; Dabetic et al., 2021).

As well as genetic factors and grape variety, the processes applied also affect the fatty acid composition of grape seed oil (Dabetic et al., 2021). After heat treatment, the fatty acid profiles of the oils changed slightly, and this change was observed more clearly in the Shiraz variety compared to other varieties. Regarding the degree to which varieties are affected by heat treatment, the highest change in fatty acids was detected in Shiraz oils, and the lowest change was detected in oils belonging to the Alfons Lavallee variety. Considering the effect of heat treatment on the major fatty acids, the increase in temperature caused a slight decrease in linoleic acid values and a more significant increase in oleic acid values. Therefore, while the PUFA value of oils decreased, the MUFA value increased, leading to an increase in UFA, which is the total unsaturation value. Grape variety and applied temperatures were effective in all these statistically significant changes in quality parameters. Similar to our findings, Herchi et al., (2016) stated that the value of polyunsaturated fatty acids of flaxseed hull oil decreased as a result of heat treatment.