Production of Demineralized Antibacterial, Antifungal and Antioxidant Peptides from Bovine Hemoglobin Using an Optimized Multiple-Step System: Electrodialysis with Bipolar Membrane.

Numerous studies have shown that bovine hemoglobin, a protein from slaughterhouse waste, has important biological potential after conventional enzymatic hydrolysis. However, the active peptides could not be considered pure since they contained mineral salts. Therefore, an optimized multi-step process of electrodialysis with bipolar membranes (EDBM) was carried out to produce discolored and demineralized peptides without the addition of chemical agents. The aim of this study was to test the antibacterial, antifungal and antioxidant activities of discolored and demineralized bovine hemoglobin hydrolysates recovered by EDBM and to compare them with raw and discolored hydrolysates derived from conventional hydrolysis. The results demonstrate that discolored-demineralized hydrolysates recovered from EDBM had significant antimicrobial activity against many bacterial (gram-positive and gram-negative) and fungal (molds and yeast) strains. Concerning antibacterial activity, lower MIC values for hydrolysates were registered against Staphylococcus aureus, Kocuria rhizophila and Listeria monocytogenes. For antifungal activity, lower MIC values for hydrolysates were registered against Paecilomyces spp., Rhodotorula mucilaginosa and Mucor racemosus. Hemoglobin hydrolysates showed fungicidal mechanisms towards these fungal strains since the MFC/MIC ratio was ≤4. The hydrolysates also showed a potent antioxidant effect in four different antioxidant tests. Consequently, they can be considered promising natural, low-salt food preservatives. To the best of our knowledge, no previous studies have identified the biological properties of discolored and demineralized bovine hemoglobin hydrolysates.

Bovine Hemoglobin Enzymatic Hydrolysis by a New Eco-Efficient Process-Part II: Production of Bioactive Peptides.

Bovine cruor, a slaughterhouse waste, was mainly composed of hemoglobin, a protein rich in antibacterial and antioxidant peptides after its hydrolysis. In the current context of food safety, such bioactive peptides derived from enzymatic hydrolysis of hemoglobin represent potential promising preservatives for the food sector. In this work, the hemoglobin hydrolysis to produce bioactive peptides was performed in a regulated pH medium without the use of chemical solvents and by an eco-efficient process: electrodialysis with bipolar membrane (EDBM). Bipolar/monopolar (anionic or cationic) configuration using the H+ and OH- generated by the bipolar membranes to regulate the pH was investigated. The aim of this study was to present and identify the bioactive peptides produced by EDBM in comparison with conventional hydrolysis and to identify their biological activity. The use of the EDBM for the enzymatic hydrolysis of hemoglobin has allowed for the production and identification of 17 bioactive peptides. Hydrolysates obtained by EDBM showed an excellent antimicrobial activity against six strains, antioxidant activity measured by four different tests and for the first time anti-fungal activities against five yeasts and mold strains. Consequently, this enzymatic hydrolysis carried out in regulated pH medium with bipolar membranes could provide bioactive peptides presenting antibacterial, antifungal and antioxidant interest.

Bovine Hemoglobin Enzymatic Hydrolysis by a New Ecoefficient Process-Part I: Feasibility of Electrodialysis with Bipolar Membrane and Production of Neokyotorphin (α137-141).

Neokyotorphin (α137-141) is recognized as an antimicrobial peptide and a natural meat preservative. It is produced by conventional enzymatic hydrolysis of bovine hemoglobin, a major component of cruor, a by-product of slaughterhouses. However, during conventional hydrolysis, chemical agents are necessary to adjust and regulate the pH of the protein solution and the mineral salt content of the final hydrolysate is consequently high. To produce this peptide of interest without chemical agents and with a low salt concentration, electrodialysis with bipolar membrane (EDBM), an electromembrane process recognized as a green process, with two different membrane configurations (cationic (MCP) and anionic (AEM) membranes) was investigated. Hydrolysis in EDBM showed the same enzymatic mechanism, "Zipper", and allowed the generation of α137-141 in the same concentration as observed in conventional hydrolysis (control). EDBM-MCP allowed the production of hydrolysates containing a low concentration of mineral salts but with fouling formation on MCP, while EDBM-AEM allowed the production of hydrolysates without fouling but with a similar salt concentration than the control. To the best of our knowledge, this was the first time that EDBM was demonstrated as a feasible and innovative technology to produce peptide hydrolysates from enzymatic hydrolysis.