Necrotic enteritis (NE), caused by Clostridium perfringens, is an emerging issue in poultry farming. New approaches, other than antibiotics, are necessary to prevent NE development and the emergence of multidrug-resistant bacteria. Enterococci are commensal microorganisms that can produce enterocins, antimicrobial peptides with activities against pathogens, and could be excellent candidates for protective cultures. This study aimed to screen and characterize Enterococcus strains of poultry origin for their inhibitory activity against C. perfringens. In total, 251 Enterococcus strains of poultry origin plus five bacteriocin-producing (BP+) E. durans strains of other origins were screened for antimicrobial activity against the indicator C. perfringens X2967 strain using the “spot on the lawn” method. We detected thirty-two BP+ strains (eleven Enterococcus faecium, nine E. gallinarum, eight E. faecalis, three E. durans, and one E. casseliflavus). We further studied the antimicrobial activity of the supernatants of these 32 BP+ strains using agar well diffusion and microtitration against a collection of 20 C. perfringens strains. Twelve BP+ enterococci that were found to exhibit antimicrobial activity against C. perfringens were characterized using whole genome sequencing. Among these, E. faecium X2893 and X2906 were the most promising candidates for further studies as protective cultures for poultry farming. Both strains belong to the sequence type ST722, harbor the genes encoding for enterocin A and enterocin B, do not possess acquired resistance genes, do not carry plasmids, and present the acm gene, which is implicated in host colonization. Further research is needed to determine the utility of these strains as protective cultures.

Porcine blood, a significant byproduct of the pork industry, represents a potential source of antimicrobial peptides (AMPs). AMPs offer a promising alternative to chemical antimicrobials, which can be used as natural preservatives in the food industry. AMPs can exhibit both antibacterial and/or antifungal properties, thus improving food safety and addressing the growing concern of antibiotic and antifungal resistance. The objective of this study was to evaluate the antimicrobial activity of potential AMPs previously identified from porcine cruor hydrolysates. To this end, a total of sixteen peptides were chemically synthesized and their antimicrobial activities (antibacterial, anti-mold, and anti-yeast) were evaluated using microtitration and agar well diffusion methods against a wide range of microorganisms. Five new peptide sequences demonstrated antifungal activity, with Pep5 (FQKVVAGVANALAHKYH), an alpha-helix peptide, exhibiting the most promising results. Pep5 demonstrated efficacy against nine of the eleven fungal isolates, exhibiting low minimum inhibitory concentrations (MICs) and a fungicidal effect against key spoilage fungi (Rhodotorula mucilaginosa, Debaryomyces hansenii, Candida guilliermondii, Paecilomyces spp., Eurotium rubrum, Mucor racemosus, Aspergillus versicolor, Penicillium commune, and P. chrysogenum). These findings illustrate the potential of porcine blood hydrolysates as a source of AMPs, particularly antifungal peptides, which are less known and less studied than the antibacterial ones. Among the tested sequences, Pep5 exhibited the most promising characteristics, including broad-spectrum activity, low MICs, and a fungicidal effect. It is, therefore, a promising candidate for further research and for potential applications in the porcine industry and beyond.

There is a growing interest in natural preservatives driven by consumer demand for clean-label products. In Canada, approximately 48 million liters of blood are produced annually during chicken slaughter, offering an opportunity to valorize cruor, the solid blood component rich in hemoglobin, for use in food preservation. This study investigated the hydrolysis of chicken cruor with pepsin at pH 2, 3, 4, and 5 for 180 min to produce antimicrobial peptides. The highest degree of hydrolysis (11.70 ± 0.77 %) was observed at pH 2, similar to pH 3 where the enzyme exhibited a zipper mechanism. Hydrolysates at pH 2 and 3 inhibited fungal strains (Paecilomyces spp., Rhodotorula mucilaginosa, and Mucor racemosus) with MIC: 0.63 mM, while no antibacterial activity was observed. Partial Least Square-Discriminant Analysis (PLS-DA) allowed the identification of 31 antifungal peptides, including LARKYH, active against R. mucilaginosa (MIC: 0.63 mM), highlighting chicken cruor's potential as a source of bio-preservatives.

In recent years, cruor from slaughterhouse blood has garnered growing interest as a potential source of antimicrobial peptides obtained through enzymatic hydrolysis. In addition, electrodialysis with ultrafiltration membrane (EDUF) represents a strategy for valorizing peptide-rich hydrolysates, enabling the selective separation and concentration of antimicrobial peptides, according to their size and charge. Hence, this study evaluated the potential of EDUF to fractionate, for the first time, calf cruor hydrolysate and explore its use as a novel source of antimicrobial peptides. The resulting peptide fractions were characterized to investigate the selectivity of peptide migration in relation to peptide physico-chemical characteristics and membrane properties and to finally assess their antimicrobial activity. High migration rates of 12.75 ± 2.17 g/m²h and 8.94 ± 0.38 g/m²h were observed for the cationic (P⁺) and anionic (P⁻) recovery fractions, respectively. These results suggested that peptide migration from calf cruor hydrolysate to both recovery fractions during EDUF was influenced by the combined effects of molecular weight, net charge, hydrophobicity, specific amino acid residues (L, Y), and peptide–membrane interactions. Furthermore, the initial and final hydrolysates as well as P⁺ fractions exhibited antifungal activities against Paecilomyces spp. and Rhodotorula mucilaginosa with minimum inhibitory concentrations (MIC) ranging from 0.312 to 0.615 mg/mL and minimum fungicidal concentrations (MFCs) ranging from 0.312 to 1.250 mg/mL. In contrast, the P⁻ fraction did not exhibit antifungal activity, but a slight anti-Listeria activity was detected, with a MIC of 10 mg/mL. These findings highlight the potential of upcycling calf blood into functional antifungal and antibacterial agents, supporting a circular economy approach and transforming waste streams into value-added ingredients that enhance food preservation.