Identification of novel peptides for lipid oxidation inhibition through enzymatic hydrolysis

The increasing demand for natural and sustainable food preservatives has led to a growing interest in antioxidant peptides as safer alternatives to synthetic additives like EDTA. Peptides are being extensively studied, as those derived from food proteins have gained significant attention due to their diverse functional properties, particularly their antioxidant activities, metal chelation capabilities, and lipid peroxidation inhibition. The objective of this study is to optimize the enzymatic hydrolysis process using various enzymes under different conditions to prevent lipid oxidation and enhance the natural preservative properties of soybean cultivar protein. Soy protein was isolated from a selected soybean cultivar (R98-1821) and hydrolyzed with different endo- and exo-proteases such as Alcalase, papain, protease from Aspergillus oryzae, and Flavorzyme. The hydrolysates were evaluated for functional properties, lipid oxidation inhibition, and ferrous ion chelation.  Mass spectrometry (MS) and size-exclusion chromatography (SEC) were used to determine peptide profiles, molecular weight distributions, and key sequences. For lipid oxidation, the most effective treatment was a two-stage hydrolysis, with 1-hour Alcalase followed by 1-hour protease, resulting in 92% inhibition. In comparison, protease from A. oryzae demonstrated better lipid oxidation inhibition than Alcalase, while Alcalase showed superior metal chelation ability. Ferrous ion chelation activity of 80% was achieved with a combination of protease followed by Alcalase at a concentration of 0.5 mg/mL. Enzymatic hydrolysis enhanced emulsion stability, foaming, and gelation, especially with endo-protease preceding exo-protease. SEC analysis revealed low-molecular-weight peptides associated with enhanced bioactivity, and MS identified key peptide sequences responsible for lipid oxidation inhibition. This study confirmed that enzymatic hydrolysis conditions play a key role in determining peptide quality and characteristics. Optimizing the enzymatic hydrolysis process is crucial for preventing lipid oxidation, supporting safer, eco-friendly food preservation, and meeting the demand for clean labels.