Protein hydrolysates from Pleurotus spp. mushrooms as a source of antioxidant peptides and their stability after gastrointestinal digestion

Pleurotus spp. (shimeji or oyster mushroom) is widely recognized for its medicinal properties, and has become an integral part of the diet for individuals seeking healthier lifestyles. Mushrooms contain numerous and highly bioactive compounds, and can also serve as an excellent source of protein. This study investigated the antioxidant properties of protein hydrolysates derived from Pleurotus spp., obtained using proteases (Alcalase™ 2.4 L, Flavourzyme™ 500 L, and Neutrase™ 0.8 L) applied individually or in binary/ternary combinations. The protein hydrolysates displayed trichloroacetic acid (TCA)-soluble proteins ranging from 59.81 % to 83.66 %, and higher antioxidant activities than the intact protein. The use of Neutrase™ 0.8 L resulted in protein hydrolysates containing 69.70 % of TCA-soluble proteins and exhibited the highest values for antioxidant properties: ABTS (125.01 μmol TE g⁻¹), DPPH (36.67 μmol TE g⁻¹) and FRAP (9.26 μmol TE g⁻¹). Protein hydrolysates with the lowest molecular weights (< 3 kDa) contributed to the highest ABTS and DPPH activities, while FRAP activity was favored by fractions containing molecular weights between 5 and 30 kDa. High content of hydrophobic amino acids (27.80 %) positively contribute to the antioxidant properties of the hydrolysates. Simulated in vitro digestion showed that the action of gastrointestinal enzymes potentiated the antioxidant activities by 126.10 % (ABTS), 150.13 % (DPPH) and 85.79 % (FRAP). Using liquid chromatography-mass spectrometry (LC-MS/MS), 31 peptides were identified, with the LTGPL fragment showing promising antioxidant activity based on in silico analysis. This study demonstrated that these mushrooms and their hydrolysates are a rich source of natural antioxidant compounds, highlighting their potential for use in nutraceutical products.