# Protease Selection Influences Molecular Weight, In Vitro Antioxidant Activity and LO2 Cellular Protective Effects of Oyster Protein Hydrolysates

## TL;DR

This study shows that different proteases affect the breakdown of oyster proteins and the antioxidant properties of the resulting peptides.

## Contribution

The study reveals that protease choice and resulting peptide molecular weight significantly impact antioxidant activity and cellular protection.

## Key findings

- Alkaline protease produced the highest degree of hydrolysis and most peptides ≤1 kDa.
- Trypsin and papain-derived low-molecular-weight peptides showed stronger antioxidant activity than others.
- Low-molecular-weight fractions effectively protected LO2 cells from oxidative damage, with trypsin and papain showing the best results.

## Abstract

This study compared the effectiveness of alkaline protease, neutral protease, trypsin, and papain in hydrolyzing oyster proteins and evaluated the antioxidant activities of the resulting hydrolysates. Alkaline protease achieved the highest degree of hydrolysis (30.96%) and the highest proportion of peptides ≤1 kDa (64.23%). Papain showed the lowest hydrolysis degree (18.29%). After separation by Sephadex G-15 gel filtration chromatography, the resulting low-molecular-weight peptide fractions (≤1 kDa) from each hydrolysate exhibited higher in vitro antioxidant activity than the higher-molecular-weight fractions (>1 kDa). Notably, trypsin and papain-derived low-molecular-weight fractions (OPP-T2 and OPP-P2) demonstrated stronger DPPH radical scavenging and inhibition of linoleic acid autoxidation than those from alkaline and neutral proteases. Cell experiments revealed that all low-molecular-weight fractions effectively alleviated H2O2-induced oxidative damage in LO2 cells. OPP-T2 and OPP-P2 exhibited significantly stronger protection of cell membrane integrity and enhancement of superoxide dismutase (SOD) activity than OPP-A2 and OPP-N2 (p < 0.05). OPP-T2 also showed the most pronounced increase in glutathione peroxidase (GSH-Px) activity (p < 0.05). These findings demonstrate that protease selection critically influences hydrolysis efficiency and antioxidant activity, with molecular weight being a key determinant of peptide antioxidant capacity. This work provides a reference for the development and application of oyster peptides.