Impact of ultrasonic pretreatment on pumpkin seed protein: Effect on protease activities, protein structure, hydrolysis kinetics and functional properties

Abstract

Adding value to food by-products, such as pumpkin seeds, is an important strategy for the complete utilization of plant foods and advancing sustainability goals. This study aimed to maximize the production of bioactive peptides from pumpkin seed protein (PSP) by combining ultrasonic (US) pretreatment (40 kHz, 23.8 W/L) with enzymatic hydrolysis. The PSP's structure after sonication and its effects on the commercial proteases (Brauzyn®, Flavourzyme®, Neutrase®) activity and degree of hydrolysis were studied. The hydrolysis consequences regarding solubility and antioxidant activity of the resulting peptides were also evaluated. Sonication of PSP increased enzymatic activity by up to 21.3 % for Brauzyn®, 24.8 % for Flavourzyme® and 19.2 % for Neutrase®. Consequently, there was an increase in the degree of hydrolysis (up to 89 %) using sonicated PSP, particularly at 60 min/40 °C. These effects can be attributed to ultrasound-induced protein conformation changes, including increased intrinsic fluorescence intensity (<22 %), shifts in UV spectra, and alterations in FTIR amide bands, especially a decrease in β-sheet content (<7.14 %). Additionally, ultrasonic pretreatment reduced particle size (<43.9 %) and polydispersity index (<58 %), enhancing enzyme accessibility by fragmenting protein aggregates, as observed via scanning electron microscopy. As a result, the peptides obtained from the hydrolysis of sonicated PSP exhibited higher protein solubility (12 % to 49 % at pH 6.0) and improved antioxidant activity (5.6 % to 77 %). Overall, sonication of PSP for 60 min at 40 °C followed by hydrolysis with Neutrase® proved to be the most effective strategy for producing highly soluble peptides with enhanced antioxidant properties, highlighting the potential of ultrasound as a valuable tool for optimizing bioactive peptide production. Based on these results, the developed process is ready for scale-up by the food industry, aiming to obtain protein hydrolysates with improved functional and/or nutritional properties from a low-cost raw material. In parallel, further researches can focus on the potential application of these hydrolysates as ingredients in bakery, meet or dairy products.

Keywords: Antioxidant activity; Enzyme activation; Kinetic parameters; Solubility; Sonication.