Naima Nedjar-Arroume’s research while affiliated with Artois University and other places

In the actual context of food safety and circular economy, the separation of the α137–141 antimicrobial peptide (TSKYR, 653 Da) coming from the hydrolysis of bovine hemoglobin, a non-valorized slaughterhouse by-product, and its reuse as a preservative agent for food products during storage would be of major interest. However, depending on the degree of hydrolysis (DH) of the bovine hemoglobin, the TSKYR peptide environment into the hydrolysate will be different and would impact its recovery yield by electrodialysis with ultrafiltration membranes (EDUF), a hybrid and eco-friendly technology. In this context, five DHs (3, 5, 10, 13 and 18%) were investigated to study the influence of the peptide populations on the α137–141 selective separation by EDUF. It appeared from these results that the most appropriate DH was of 5% since a lower population of peptides between 500 and 1000 Da was present and the subunits of hemoglobin were already digested. Hence, an enrichment factor of about 13 folds for the α137–141 was obtained in comparison with the initial hydrolysate. At our knowledge, it was the first time that the effect of the hydrolysis degree of a protein is demonstrated to impact preferentially the separation of a specific peptide by EDUF. Graphic Abstract

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.

The porcine cruor represents a co-product from slaughterhouses. It mainly contains hemoglobin which is rich in bioactive peptides obtained by hydrolysis by porcine pepsin. The aim of this study is to valorize this new source rich of bioactive peptides to be used as feed additives. An in silico study on the potential of porcine cruor to contain bioactive peptides was studied by comparing it with peptides derived from bovine hemoglobin. The blast results showed a high similarity between porcine cruor hydrolysate peptides and bovine hemoglobin hydrolysates for both α and β chains with identities of 87% and 83% respectively. Cleavage sites of porcine cruor were also predicted using Peptide Cutter software and compared with bovine hemoglobin. Results reveal the preservation of most of peptides of porcine cruor hydrolysates comparing to bovine hemoglobin hydrolysates. The presence of bioactive peptides was then determined by mass spectrometry analysis. Results showed the presence of 37 bioactive peptides defined by their sequences found in previous study with mainly antibacterial and opioid peptides. The antibacterial activity was then verified by in vitro analysis against 4 bacterial strains. Results showed interesting MIC values comparing with tested synthetic peptides (4.53 µM for M.luteus and L.innocua, 35.94 µM for E.coli and 312 µM for S.newport). The antioxidant activity was also verified by studying DPPH•+ trapping and results showed an antiradical activity for porcine cruor hydrolysate greater than neokyotorphin which was previously validated as a natural preservative to substitute synthetic additives against food alteration (14% and 10% respectively). These similarities of porcine cruor hydrolysate in peptic profiles as well as biological activities make it a new interesting source of bioactive peptides.

La présente invention décrit l'obtention d'un isolat protéique de pois chiche suivi de la production d' hydrolysats enrichis en peptides antioxydants, sous l'action d'enzyme protéolytique (alcalase) et plus particulièrement l'invention rapporte la purification, l'identification et la caractérisation de 2 nouveaux peptides antioxydants. Selon le procédé de l'invention, l'obtention de peptides antioxydants repose sur une hydrolyse enzymatique contrôlée des protéines de pois chiche sous l'action d'une enzyme exogène, suivie de méthodes systématiques de fractionnement, de tests biologiques et d'identification. Dans le cadre de brevet, l'activité antioxydante a été mise en évidence au niveau de l'isolat protéique et ses hydrolysats à différentes concentrations. Selon la présente invention, les hydrolysats, ayant un degré d'hydrolyse de l'ordre de 14,67%, sont plus actifs et l'activité antioxydante est améliorée avec l'augmentation de la concentration. Les hydrolysats obtenus ont été fractionnés par filtration sur gel Sephadex G-25 puis par chromatographie liquide haute performance de phase inverse (RP-HPLC). Pour identifier et caractériser les peptides des fractions P3 et P8 sortie de la RP-HPLC, la technique de spectrométrie de masse dite en «tandem» ou MS/MS a été utilisée. Deux peptides Asp-His-Gly et Val-Gly-Asp-lle ont été identifiés. Des masses moléculaires de 327,33 et 402,49 Da ont été enregistrés pour les fractions P3 et P8, respectivement.

Inhibition of DPP-IV may improve glycemic control in diabetics by preventing the rapid breakdown and there by prolonging the physiological action of incretin hormones. Barbel muscle protein hydrolysate (BMPH) was noted to exhibit DPP-IV inhibitory activity, with an IC50 value of 1.94mg/mL. It was fractionated into five major fractions (FI-FV) by size exclusion chromatography using a Superdex peptide. The FIII fraction was noted to display the highest inhibitory activity, with an IC50 value of 1.23mg/mL, and was, therefore, further fractionated by RP-HPLC. Four major peptide sub-fractions were selected. The results revealed that the SF4 sub-fraction showed the highest DPP-IV inhibitory activity, with an IC50 value of 0.21mg/mL. This sub-fraction was submitted to RP-HPLC, ESI-MS, and ESI-MS/MS analyses. The findings indicated that SF4 consisted of two peptides (IC50=96μg/mL), namely PP1 and PP2, whose structures were identified as Trp-Ser-Gly (330Da) and Phe-Ser-Asp (349Da), respectively. This is the first report of these sequences from barbel proteins. The structural modelling through docking simulations results with DPP-IV showed that the Trp-Ser-Gly peptide bound to DPP-IV with high affinity. Overall, the results suggested that BMPH can be considered as a promising natural source of DPP-IV inhibitory peptides.

This study aimed to identify novel ACE inhibitory peptides from the muscle of cuttlefish. Proteins were hydrolyzed and the hydrolysates were then subjected to various types of chromatography to isolate the active peptides. Nine ACE inhibitory peptides were isolated and their molecular masses and amino acid sequences were determined using ESI-MS and ESI-MS/MS, respectively. The structures of the most potent peptides were identified as Val-Glu-Leu-Tyr-Pro, Ala-Phe-Val-Gly-Tyr-Val-Leu-Pro and Glu-Lys-Ser-Tyr-Glu-Leu-Pro. The first peptide displayed the highest ACE inhibitory activity with an IC50 of 5.22μM. Lineweaver-Burk plots suggest that Val-Glu-Leu-Tyr-Pro acts as a non-competitive inhibitor against ACE. Furthermore, antihypertensive effects in spontaneously hypertensive rats (SHR) also revealed that oral administration of Val-Glu-Leu-Tyr-Pro can decrease systolic blood pressure significantly (p<0.01). These results suggest that the Val-Glu-Leu-Tyr-Pro would be a beneficial ingredient for nutraceuticals and pharmaceuticals acting against hypertension and its related diseases.

There is great interest in the development of antimicrobial peptides as a potentially novel class of antimicrobial agents. In this study we evaluated the mode of action of new peptides (Gly-Val-His, Trp-His-Arg, Trp-His-Phe, Pro-Pro-Ser-Ser, Ala-Ala-Ala-Leu, Ala-Ala-Gly-Gly-Val, Ala-Ala-Val-Lys-Met, Ala-Ser-Ser-Ser), previously characterized, from barbel protein hydrolysates against Listeria monocytogenes via a membrane damage mechanism. The minimum inhibitory concentrations (MIC) of these peptides toward six strains and their hemolytic activity toward bovine erythrocytes were determined. Prediction of peptide secondary structure indicated that these peptides should have random coil structures and high content of hydrophobic amino acids. In addition, results of the determination of extracellular potassium, which is considered a good marker of membrane permeability, revealed that treatment with pure barbel peptides could cause morphological changes of L. monocytogenes and destruction of the cell integrity via irreversible membrane damage. The results could provide information for investigating the antibacterial model of antibacterial peptides derived from fish protein hydrolysates.