Int J Mol Sci

Porcine intestinal mucosa hydrolysates (PIMHs) are by-products of heparin production obtained through a specific enzymatic hydrolysis process, which can theoretically generate bioactive peptides (BAPs). This study aimed to identify, characterize, and quantify BAPs in two Palbio products manufactured by Bioiberica S.A.U. (Palafolls, Spain), which are PIMH protein sources used for animal feed: Palbio® HP (PHP) and Palbio® 62 SP® (P62). Using mass spectrometry (MS)-based peptidomics, we analyzed three samples from each product, fractionated based on molecular weight (<3 kDa, 3 to 10 kDa, and >10 kDa). The <3 kDa fraction was analyzed directly, while the other two fractions were enzymatically digested before MS analysis. The workflow identified 961 peptides in PHP and 1134 in P62. Subsequent bioinformatic analysis using public databases (APD2, StraPep, AHTPDB, and BIOPEP-UWM) led to the identification of six significant BAPs in both PHP and P62, with respective quantified amounts (pg peptide/μg sample): DAVEDLESVGK (0.1626, 0.1939), EGIPPDQQRLIFAGK (0.2637, 0.1852), TITLEVEPSDTIENVK (0.3594, 0.4327), TNVPRASVPDGFLS (1.4596, 0.1898), TNVPRASVPDGFLSEL (8.0500, 0.9224), and VHVVPDQLMAF (0.0310, 0.0054). The first three BAPs are related to antimicrobial activity, while the latter three are associated with cytokine/growth factor-like, antioxidant, and immunomodulatory activities. These bioactivities align with previously reported in vivo benefits observed in animal nutrition using Palbio products. Our findings demonstrate that PHP and P62 are valuable sources of BAPs, supporting their potential role in improving animal health and performance.