How to induce selective separation of antimicrobial peptides by electrodialysis with filtration membrane ?
Aurore Cournoyer1,2, Gaétan Daigle3, Mathieu Bazinet4, Jacinthe Thibodeau1,2 et Laurent Bazinet1,2

Department of Food Science and Laboratoire de Transformation Alimentaire et Procédés ÉlectroMembranaires (LTAPEM, Laboratory of Food Processing and Electromembrane Process), Université Laval, Québec, G1V 0A6, Canada

2 Institute of Nutrition and Functional Foods (INAF), Université Laval, Québec, G1V 0A6, Canada

Department of Mathematics and Statistics, Faculty of Sciences and Engineering, Université Laval, Québec, QC, Canada, G1V 0A6

Department of computer science and software engineering, Faculty of Sciences and Engineering, Université Laval, Québec, QC, Canada, G1V 0A6

Porcine blood is a major by-product from slaughterhouses. Its precipitated part after centrifugation, cruor, contained an interesting protein called hemoglobin. After enzymatic hydrolysis of hemoglobin, a wide variety of peptides is obtained, especially antimicrobial peptides. To produce fractions concentrated in active peptides, electrodialysis with ultrafiltration membrane (EDUF) was applied. The process already demonstrated the capacity to concentrate peptide, like TSKYR. Moreover, the present study aimed to evaluate the impacts of different current conditions (pulsed electric field (PEF) and polarity reversal (PR)) on peptide migration selectivity. In the present study, EDUF was used in a cationic configuration, with a 50 kDa molecular weight cut-off UF membrane. Hence, the direct current (DC) condition was compared with combination of pulse/pause duration corresponding to ratios of 1 and 10 for PEF and PR. The peptide populations in the recovery compartments were analyzed using UPLC-MS/MS and peptide relative abundances were compared between conditions. A principal component analysis (PCA) and a hierarchical cluster heatmap were performed to determine potential groups of peptides and to evaluate the impact of current conditions on peptide population. It occurred that PR ratio 1 generated the most significant differences concerning the peptides population migrated while PEF ratio 1 was the second most different. PEF and PR ratio 10, and DC had similar migrated peptide populations. A linear discriminant analysis (LDA) based on the different groups formed and 6 physicochemical characteristics of the peptides allowed to conclude that the main differences were explained by the charge at pH 9, the molecular mass, and the mass/charge ratio. Indeed, DC, PEF and PR at ratio 10 allow the migration of mainly cationic peptides as expected, while PR ratio 1 allows the migration of some anionic peptides with high molecular masses due to the short polarity reversal. For PEF ratio 1 the peptide population, mainly cationic peptide, was explained in a less important manner by their mass and their mass/charge. Preliminary tests in artificial intelligence via a decision tree approach made it possible to discriminate more precisely the impact of the physical characteristics of the peptides on their migration according to the current conditions. From all these results, it appears that the current condition as well as the pulse/pause combination strongly affect the selectivity of migration during EDUF and consequently may impact the final bioactivity. The next steps of the present study will be to evaluate the antimicrobial activities of the fractions produced in the different conditions of current.