AbstractThe applications of protein hydrolysates as food preservatives and nutraceutical ingredients have attracted much attention because of their beneficial effects. The interest in these ingredients has shifted toward their biological activities with benefits to human health. Bioactive peptides are known as antioxidant agents that could promote health‐promoting effects and prolong food shelf‐life beyond their basic nutritional value. Thus, the aim of this study was to investigate antioxidant, antimicrobial, and in vitro cytotoxic properties of corn pollen protein (CPP) hydrolysates obtained by different enzymes. Proteolytic activity in terms of degree of hydrolysis (DH) and SDS‐PAGE analysis was measured in pancreatin (H‐Pan), pepsin (H‐Pep), and trypsin (H‐Tri) hydrolysates. Amino acid composition, antioxidant and antimicrobial activities, and cytotoxicity of hydrolysates were evaluated. DH and SDS‐PAGE revealed higher proteolytic activity of pepsin compared to other enzymes. Amino acid analysis showed that the functional amino acids such as antioxidant types were most predominant in H‐Pep compared to two other samples. Antioxidant activity of hydrolysates was found to be affected by the type of enzyme and the concentration of hydrolysates. There was a significant difference (p< 0.05) between antioxidant activity of different hydrolysates. The highest antioxidant activity in terms of Trolox equivalent antioxidant capacity (0.23–2.75 mM), DPPH (33.3%–64.8%), and hydroxyl (33.7%–63.2%) radical scavenging activities, chelation of iron (33.2%–62.5%) and copper (30.2%–50.5%) metals, and total antioxidant activity (0.65–0.85) was obtained for H‐Pep followed by H‐Pan and H‐Tri samples. Antibacterial tests showed that pepsin‐hydrolyzed protein was not significantly (P> 0.05) effective againstE. coliat any concentrations, however, it showed significant (P< 0.05) concentration‐dependent effect againstS. aureus(with inhibition zones of 15–25 mm). Cytotoxicity results revealed that CPP, as a nonhydrolyzed protein, did not generally show antiproliferative activity, however, a significant (P< 0.05) ability of H‐Pep hydrolysate in decreasing HT‐29 colon cancer cell line viability was seen in a concentration‐dependent manner (the lowest cell viability of 32% at 5 mg/mL). Overall, investigating the application of protein‐based hydrolysates is one of the possible strategies that govern their applied intentions as preservatives and nutraceuticals in the food and pharmaceutical industries.