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This study focuses on the enzymatic hydrolysis of hemoglobin, the main component of cruor that gives blood its red color in mammals. The antibacterial and antioxidant potentials of human hemoglobin hydrolysates were evaluated in comparison to bovine hemoglobin. The results showed strong antimicrobial activity of the peptide hydrolysates against six bacterial strains, independent of the initial substrate concentration level. The hydrolysates also showed strong antioxidant activity, as measured by four different tests. In addition, the antimicrobial and antioxidant activities of the human and bovine hemoglobin hydrolysates showed little or no significant difference, with only the concentration level being the determining factor in their activity. The results of the mass spectrometry study showed the presence of a number of bioactive peptides, the majority of which have characteristics similar to those mentioned in the literature. New bioactive peptides were also identified in human hemoglobin, such as the antibacterial peptides PTTKTYFPHF (α37-46), FPTTKTYFPH (α36-45), TSKYR (α137-141), and STVLTSKYR (α133-141), as well as the antioxidant TSKYR (α137-141). According to these findings, human hemoglobin represents a promising source of bioactive peptides beneficial to the food or pharmaceutical industries. [ABSTRACT FROM AUTHOR]

Cruor, the main component responsible for the red color of mammalian blood, contains 90% haemoglobin, a protein considered to be a rich source of bioactive peptides. The aim of the present study is to assess the potential of human hemoglobin as a source of bioactive peptides, compared with bovine hemoglobin, which has been extensively studied in recent years. More specifically, the study focused on the α137–141 fragment of bovine haemoglobin (TSKYR), a small (653 Da) hydrophilic antimicrobial peptide. In this work, the potential of human hemoglobin to contain bioactive peptides was first investigated in silico in comparison with bovine hemoglobin-derived peptides using bioinformatics tools. The blast results showed a high identity, 88% and 85% respectively, indicating a high similarity between the α and β chains. Peptide Cutter software was used to predict cleavage sites during peptide hydrolysis, revealing major conservation in the number and location of cleavage sites between the two species, while highlighting some differences. Some peptides were conserved, notably our target peptide (TSKYR), while others were specific to each species. Secondly, the two types of hemoglobin were subjected to similar enzymatic hydrolysis conditions (23 °C, pH 3.5), which showed that the hydrolysis of human hemoglobin followed the same reaction mechanism as the hydrolysis of bovine hemoglobin, the 'zipper' mechanism. Concerning the peptide of interest, α137–141, the RP-UPLC analyses showed that its identification was not affected by the increase in the initial substrate concentration. Its production was rapid, with more than 60% of the total α137–141 peptide production achieved in just 30 min of hydrolysis, reaching peak production at 3 h. Furthermore, increasing the substrate concentration from 1% to 10% (w/v) resulted in a proportional increase in α137–141 production, with a maximum concentration reaching 687.98 ± 75.77 mg·L−1, approximately ten-fold higher than that obtained with a 1% (w/v) concentration. Finally, the results of the UPLC-MS/MS analysis revealed the identification of 217 unique peptides in bovine hemoglobin hydrolysate and 189 unique peptides in human hemoglobin hydrolysate. Of these, 57 peptides were strictly common to both species. This revealed the presence of several bioactive peptides in both cattle and humans. Although some had been known previously, new bioactive peptides were discovered in human hemoglobin, such as four antibacterial peptides (α37–46 PTTKTYFPHF, α36–45 FPTTKTYFPH, α137–141 TSKYR, and α133–141 STVLTSKYR), three opioid peptides (α137–141 TSKYR,β31–40 LVVYPWTQRF,β32–40, VVYPWTQRF), an ACE inhibitor (β129–135 KVVAGVA), an anticancer agent (β33–39 VVYPWTQ), and an antioxidant (α137–141 TSKYR). To the best of our knowledge, these peptides have never been found in human hemoglobin before. [ABSTRACT FROM AUTHOR]

Protein hydrolysates are, in general, mixtures of amino acids and small peptides able to supply the body with the constituent elements of proteins in a directly assimilable form. They are therefore characterised as products with high nutritional value. However, hydrolysed proteins display an unpleasant bitter taste and possible off-flavours which limit the field of their nutrition applications. The successful identification and characterisation of bitter protein hydrolysates and, more precisely, the peptides responsible for this unpleasant taste are essential for nutritional research. Due to the large number of peptides generated during hydrolysis, there is an urgent need to develop methods in order to rapidly characterise the bitterness of protein hydrolysates. In this article, two enzymatic hydrolysis kinetics of micellar milk caseins were performed for 9 h. For both kinetics, the optimal time to obtain a hydrolysate with appreciable organoleptic qualities is 5 h. Then, the influence of the presence or absence of peptides and their intensity over time compared to the different sensory characteristics of hydrolysates was studied using heat maps, random forests and regression trees. A total of 22 peptides formed during the enzymatic proteolysis of micellar caseins and influencing the bitterness the most were identified. These methods represent simple and efficient tools to identify the peptides susceptibly responsible for bitterness intensity and predict the main sensory feature of micellar casein enzymatic hydrolysates. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]