Physicochemical Properties and In Vitro Antioxidant Activity Characterization of Protein Hydrolysates Obtained from Pumpkin Seeds Using Conventional and Ultrasound-Assisted Enzymatic Hydrolysis.

Pumpkin seed proteins (PSPs) are a promising resource for obtaining bioactive peptides but their low solubility hinders enzymatic hydrolysis, reducing yield and bioactivity. In addition, enzymatic processes require specific conditions and long processing times; improving the efficiency of this process is essential to expand its industrial applications. In this context, using a high-frequency, low-intensity ultrasound (US) has proven to be an effective strategy for optimizing the hydrolysis of plant protein. This study evaluated the US-assisted (38 W/L, 40 kHz) and conventional hydrolysis of pumpkin seed proteins (PSPs) for 180 min at 25 °C, 40 °C, and at the optimum temperature condition for each enzyme studied (60 °C for Brauzyn, 55 °C for Flavourzyme, and 50 °C for Neutrase), as well as the impact of this process on the macrostructural and functional characteristics of the hydrolysates obtained. The degree of hydrolysis (DH) was significantly higher in US-assisted reactions, reaching increases of up to 57.7% with Neutrase at 40 °C. The US also positively influenced the protein solubility of the hydrolysates, especially at pH levels close to the isoelectric point, with improvements of up to 100%, compared to the hydrolysates obtained from the conventional reaction. The antioxidant activity was also enhanced by the US, compared to the conventional reaction, emphasizing the hydrolysates obtained through the action of Flavourzyme, which showed increases of 52.4% and 42.6% in the scavenging of DPPH and ABTS radicals, respectively. The analysis of the mean particle size revealed significant reductions with the US (<26.2%). Consequently, the polydispersity index (PDI) demonstrated greater uniformity in the particles obtained from the US-assisted reactions (reductions of up to 20.3%). UV-Vis spectroscopy and intrinsic fluorescence also indicated possible alterations in the tertiary structure of the peptides obtained, mainly in US-assisted reactions. Therefore, US-assisted PSP hydrolysis resulted in better enzymatic performance and produced protein hydrolysates with bioactive potential for food applications.