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.

In this study, the release of proteins and other biomolecules into an aqueous media from two red macroalgae (Sphaerococcus coronopifolius and Gelidium spinosum) was studied using eight different cell disruption techniques. The contents of carbohydrates, pigments, and phenolic compounds coextracted with proteins were quantified. In addition, morphological changes at the cellular level in response to the different pretreatment methods were observed by an optical microscope. Finally, the antioxidant capacity of obtained protein extracts was evaluated using three in vitro tests. For both S. coronopifolius and G. spinosum, ultrasonication for 60 min proved to be the most effective technique for protein extraction, yielding values of 3.46 ± 0.06 mg/g DW and 9.73 ± 0.41 mg/g DW, respectively. Furthermore, the highest total contents of phenolic compounds, flavonoids, and carbohydrates were also recorded with the same method. However, the highest pigment contents were found with ultrasonication for 15 min. Interestingly, relatively high antioxidant activities like radical scavenging activity (31.57–65.16%), reducing power (0.51–1.70, OD at 700 nm), and ferrous iron-chelating activity (28.76–61.37%) were exerted by the different protein extracts whatever the pretreatment method applied. This antioxidant potency could be attributed to the presence of polyphenolic compounds, pigments, and/or other bioactive substances in these extracts. Among all the used techniques, ultrasonication pretreatment for 60 min appears to be the most efficient method in terms of destroying the macroalgae cell wall and extracting the molecules of interest, especially proteins. The protein fractions derived from the two red macroalgae under these conditions were precipitated with ammonium sulfate, lyophilized, and their molecular weight distribution was determined using SDS-PAGE. Our results showed that the major protein bands were observed between 25 kDa and 60 kDa for S. coronopifolius and ranged from 20 kDa to 150 kDa for G. spinosum. These findings indicated that ultrasonication for 60 min could be sufficient to disrupt the algae cells for obtaining protein-rich extracts with promising biological properties, especially antioxidant activity.

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.

The fractionation of bioactive peptides from hydrolysate is a main challenge to produce efficient alternative for synthetic additives. In this work, electrodialysis with ultrafiltration membrane (EDUF) was proposed to increase the purity of one antimicrobial peptide from slaughterhouse by-product hydrolysate. This targeted-peptide, α137–141 (653 Da, TSKYR), inhibits a large spectrum of microbial growths and delays meat rancidity; therefore, if concentrated, it could be used as food antimicrobial. In this context, three pH values were investigated during EDUF treatment to increase the α137–141 purity: 4.7, 6.5, and 9. pH 9 showed the highest purity increase—75-fold compared to the initial hydrolysate. Although the whole hydrolysate contains more than 100 peptides, only six peptides were recovered at a significant concentration. In this fraction, the α137–141 peptide represented more than 50% of the recovered total peptide concentration. The EDUF α137–141-enriched fraction obtained in this optimized condition would be a promising natural preservative to substitute synthetic additives used to protect food.

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.

Hydrolysis of bovine hemoglobin (bHb), the main constituent of bovine cruor by-product, releases a natural antimicrobial peptide (NKT) which could present a major interest for food safety. To enrich this, tangential ultrafiltration can be implemented, but ultrafiltration conditions are mainly empirically established. In this context, the application of a simulation method for predicting the NKT yield and enrichment was investigated. Ultrafiltration performances were studied for decolored bHb hydrolysates at different degrees of hydrolysis (DH; 3%, 5%, 10% and 18%) and colored hydrolysates (3% and 5% DH) with 1 and 3 kg·mol⁻¹ regenerated cellulose membranes. The simulation method helped to identify the most promising hydrolysate (in terms of NKT enrichment, yield and productivity) as the 3% DH colored hydrolysate, and UF conditions (volumetric reduction factor of 5 and 3 with 1 and 3 kg·mol⁻¹ membrane, respectively) for higher antimicrobial recovery. A maximal enrichment factor of about 29 and NKT purity of 70% in permeate were observed. The results showed that the antimicrobial activity was in relation with the process selectivity and NKT purity. Finally, this reliable method, applied for predicting the ultrafiltration performances to enrich peptides of interest, is part of a global approach to rationally valorize protein resources from various by-products.