Enzyme-acid pretreatment conditions for enhancing yield and phenolic content in cold pressed grape seed oil

Grape seed oil (GSO) is a valuable source of unsaturated fatty acids and bioactive compounds; however, the efficiency of cold pressing is often limited by low oil recovery and restricted transfer of antioxidant constituents into the oil phase. This study investigated the effects of acid, enzyme, and combined acid-enzyme pretreatments applied at three concentration levels (1, 2.5, and 4%, w/w) and three incubation periods (12, 24, and 36 h) on the yield and quality characteristics of cold-pressed GSO. Oil yield increased from 8.13% in the untreated control to a maximum of 9.68% following a 4% acid pretreatment for 36 h. The highest TPC (97.8 mg GAE/kg oil) and DPPH radical scavenging activity (90.18%) were obtained under moderate pretreatment conditions, whereas prolonged incubation generally reduced antioxidant quality. FFA values ranged from 0.24% to 3.42%, with enzymatic treatments exerting the strongest influence on hydrolytic changes. Peroxide value, p- anisidine value, and oxidative stability were also significantly affected by pretreatment conditions, indicating that both the formation and decomposition of oxidation products were altered by pretreatment conditions. Three-way ANOVA, coupled with effect size analysis, revealed that incubation time was the dominant factor affecting oil yield (η 2 =0.458) and TPC (η 2 =0.849), whereas pretreatment type exerted the greatest influence on FFA (η 2 =0.726). Antioxidant activity, color characteristics, and secondary oxidation products were primarily governed by interactions among pretreatment type, concentration, and incubation time. Overall, shorter incubation periods (12-24 h), particularly at moderate pretreatment concentrations (2.5% for acid and acid-enzyme pretreatments), provided a more favorable balance between phenolic retention, antioxidant activity, and oil quality than prolonged incubation. The findings contribute to the valorization of grape-processing by-products and provide practical guidance for the production of high-quality cold-pressed GSO.