Comparison of the Bioactive Properties of Human and Bovine Hemoglobin Hydrolysates Obtained by Enzymatic Hydrolysis: Antimicrobial and Antioxidant Potential of the Active Peptide α137-141.

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.

Potential of Human Hemoglobin as a Source of Bioactive Peptides: Comparative Study of Enzymatic Hydrolysis with Bovine Hemoglobin and the Production of Active Peptide α137-141.

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 ß 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,ß31-40 LVVYPWTQRF,ß32-40, VVYPWTQRF), an ACE inhibitor (ß129-135 KVVAGVA), an anticancer agent (ß33-39 VVYPWTQ), and an antioxidant (α137-141 TSKYR). To the best of our knowledge, these peptides have never been found in human hemoglobin before.

In Vivo and In Vitro Comparison of the DPP-IV Inhibitory Potential of Food Proteins from Different Origins after Gastrointestinal Digestion.

Dipeptidyl-peptidase IV (DPP-IV) plays an essential role in glucose metabolism by inactivating incretins. In this context, food-protein-derived DPP-IV inhibitors are promising glycemic regulators which may act by preventing the onset of type 2 diabetes in personalized nutrition. In this study, the DPP-IV-inhibitory potential of seven proteins from diverse origins was compared for the first time in vitro and in vivo in rat plasma after the intestinal barrier (IB) passage of the indigested proteins. The DPP-IV-inhibitory potentials of bovine hemoglobin, caseins, chicken ovalbumin, fish gelatin, and pea proteins were determined in rat plasma thirty minutes after oral administration. In parallel, these proteins, together with bovine whey and gluten proteins, were digested using the harmonized INFOGEST protocol adapted for proteins. The DPP-IV half-maximal inhibitory concentration (IC50) was determined in situ using Caco-2 cells. The DPP-IV-inhibitory activity was also measured after IB passage using a Caco2/HT29-MTX mixed-cell model. The peptide profiles were analyzed using reversed-phase high-performance liquid chromatography tandem mass spectrometry (RP-HPLC-MS/MS) with MS data bioinformatics management, and the IC50 of the identified peptides was predicted in silico. The in vitro and in vivo DPP-IV-inhibitory activity of the proteins differed according to their origin. Vegetable proteins and hemoglobin yielded the highest DPP-IV-inhibitory activity in vivo. However, no correlation was found between the in vivo and in vitro results. This may be partially explained by the differences between the peptidome analysis and the in silico predictions, as well as the study complexity.

New Bioactive Peptides Identified from a Tilapia Byproduct Hydrolysate Exerting Effects on DPP-IV Activity and Intestinal Hormones Regulation after Canine Gastrointestinal Simulated Digestion.

Like their owners, dogs and cats are more and more affected by overweight and obesity-related problems and interest in functional pet foods is growing sharply. Through numerous studies, fish protein hydrolysates have proved their worth to prevent and manage obesity-related comorbidities like diabetes. In this work, a human in vitro static simulated gastrointestinal digestion model was adapted to the dog which allowed us to demonstrate the promising effects of a tilapia byproduct hydrolysate on the regulation of food intake and glucose metabolism. Promising effects on intestinal hormones secretion and dipeptidyl peptidase IV (DPP-IV) inhibitory activity were evidenced. We identify new bioactive peptides able to stimulate cholecystokinin (CCK) and glucagon-like peptide 1 (GLP-1) secretions, and to inhibit the DPP-IV activity after a transport study through a Caco-2 cell monolayer.

O-GlcNAcylation is a key modulator of skeletal muscle sarcomeric morphometry associated to modulation of protein-protein interactions.

BACKGROUND: The sarcomere structure of skeletal muscle is determined through multiple protein-protein interactions within an intricate sarcomeric cytoskeleton network. The molecular mechanisms involved in the regulation of this sarcomeric organization, essential to muscle function, remain unclear. O-GlcNAcylation, a post-translational modification modifying several key structural proteins and previously described as a modulator of the contractile activity, was never considered to date in the sarcomeric organization. METHODS: C2C12 skeletal myotubes were treated with Thiamet-G (OGA inhibitor) in order to increase the global O-GlcNAcylation level. RESULTS: Our data clearly showed a modulation of the O-GlcNAc level more sensitive and dynamic in the myofilament-enriched fraction than total proteome. This fine O-GlcNAc level modulation was closely related to changes of the sarcomeric morphometry. Indeed, the dark-band and M-line widths increased, while the I-band width and the sarcomere length decreased according to the myofilament O-GlcNAc level. Some structural proteins of the sarcomere such as desmin, αB-crystallin, α-actinin, moesin and filamin-C have been identified within modulated protein complexes through O-GlcNAc level variations. Their interactions seemed to be changed, especially for desmin and αB-crystallin. CONCLUSIONS: For the first time, our findings clearly demonstrate that O-GlcNAcylation, through dynamic regulations of the structural interactome, could be an important modulator of the sarcomeric structure and may provide new insights in the understanding of molecular mechanisms of neuromuscular diseases characterized by a disorganization of the sarcomeric structure. GENERAL SIGNIFICANCE: In the present study, we demonstrated a role of O-GlcNAcylation in the sarcomeric structure modulation.

Tissue Non-specific Alkaline Phosphatase (TNAP) in Vessels of the Brain.

The microvessels of the brain represent around 3-4 % of the brain compartment but constitute the most important length (400 miles) and surface of exchange (20 m(2)) between the blood and the parenchyma of brain. Under influence of surrounding tissues, the brain microvessel endothelium expresses a specific phenotype that regulates and restricts the entry of compounds and cells from blood to brain, and defined the so-called blood-brain barrier (BBB). Evidences that alkaline phosphatase (AP) is a characteristic feature of the BBB phenotype that allows differentiating capillary endothelial cells from brain to those of the periphery have rapidly emerge. Thenceforth, AP has been rapidly used as a biomarker of the blood-brain barrier phenotype. In fact, brain capillary endothelial cells (BCECs) express exclusively tissue non-specific alkaline phosphatase (TNAP). There are several lines of evidence in favour of an important role for TNAP in brain function. TNAP is thought to be responsible for the control of transport of some compounds across the plasma membrane of the BCECs. Here, we report that levamisole-mediated inhibition of TNAP provokes an increase of the permeability to Lucifer Yellow of the endothelial monolayer. Moreover, we illustrate the disruption of the cytoskeleton organization. Interestingly, all observed effects were reversible 24 h after levamisole removal and correlated with the return of a full activity of the TNAP. This reversible effect remains to be studied in details to evaluate the potentiality of a levamisole treatment to enhance the entry of drugs in the brain parenchyma.

Characterization of Bacillus velezensis 32a metabolites and their synergistic bioactivity against crown gall disease.

Crown gall disease caused by Agrobacterium tumefaciens is considered to be the main bacterial threat of stone fruit plants in Mediterranean countries. In a previous study, Bacillus velezensis strain 32a was isolated from Tunisian rhizosphere soil and revealed high antagonistic potential against A. tumefaciens strains. In order to better characterize the antagonistic activity of this strain against this important plant pathogen, the production of secondary metabolites was analyzed using liquid chromatography coupled with mass spectrometry. The results revealed the production of different compounds identified as surfactins, fengycins, iturins and bacillibactin belonging to the lipopeptide group, three polyketides (macrolactins, oxydifficidin and bacillaenes), bacilysin and its chlorinated derivative; chlorotetaine. The involvement of lipopeptides in this antagonistic activity was ruled out by performing agar and broth dilution tests with pure molecules. Thus, the construction of B. velezensis 32a mutants defective in polyketides and bacilysin biosynthesis and their antagonistic activity was performed and compared to a set of derivative mutants of a comparable strain, B. velezensis GA1. The defective difficidin mutants (△dfnA and △dfnD) were unable to inhibit the growth of A. tumefaciens, indicating the high-level contribution of difficidin in the antagonism process. While the macrolactin deficient mutant (∆mlnA) slightly decreased the activity, suggesting a synergetic effect with difficidin. Remarkably, the mutant △dhbC only deficient in bacillibactin production showed significant reduction in its capacity to inhibit the growth of Agrobacterium.Taken collectively, our results showed the strong synergetic effect of difficidin and macrolactins and the significant implication of siderophore to manage crown gall disease.

Digested casein phosphopeptides impact intestinal calcium transport in vitro.

Calcium is the most abundant mineral in the human body and is involved in critical physiological and cellular processes. It is essential for the development, maintenance, and integrity of bone tissue throughout life. Identifying new natural food-grade chelating agents to improve calcium uptake is of increasing interest. Casein phosphopeptides (CPPs), highly phosphorylated peptides obtained after enzymatic hydrolysis of caseins, represent promising calcium-chelating candidates. The aim of this study was to investigate, using cell culture models, the ability of a digested milk matrix enriched in CPPs to regulate calcium transport through the intestinal barrier and elucidate the involved mechanisms. To this end, a CPP-preparation underwent in vitro static digestion and was subsequently incubated with an intestinal barrier model to monitor calcium uptake and transport. Our results demonstrated that the digested CPP preparation enhanced the trans-epithelial calcium transport via paracellular pathways and that CPPs, identified by peptidomics, crossed the intestinal barrier in the same time.

Glial-cell-mediated re-induction of the blood-brain barrier phenotype in brain capillary endothelial cells: a differential gel electrophoresis study.

In the neurovascular unit, brain microvascular endothelial cells develop characteristic barrier features that control the molecular exchanges between the blood and the brain. These characteristics are partially or totally lost when the cells are isolated for use in in vitro blood-brain barrier (BBB) models. Hence, the re-induction of barrier properties is crucial for the relevance of BBB models. Although the role of astrocyte promiscuity is well established, the molecular mechanisms of re-induction remain largely unknown. Here, we used a DIGE-based proteomics approach to study endothelial cellular proteins showing significant quantitative variations after BBB re-induction. We confirm that quantitative changes mainly concern proteins involved in cell structure and motility. Furthermore, we describe the possible involvement of the asymmetric dimethylarginine pathway in the BBB phenotype re-induction process and we discuss asymmetric dimethylarginine's potential role in regulating endothelial function (in addition to its role as a by-product of protein modification). Our results also suggest that the intracellular redox potential is lower in the in vitro brain capillary endothelial cells displaying re-induced BBB functions than in cells with limited BBB functions.

Impact of Bioinformatics Search Parameters for Peptides' Identification and Their Post-Translational Modifications: A Case Study of Proteolysed Gelatines from Beef, Pork, and Fish.

Bioinformatics software, allowing the identification of peptides by the comparison of peptide fragmentation spectra obtained by mass spectrometry versus targeted databases or directly by de novo sequencing, is now mandatory in peptidomics/proteomics approaches. Programming the identification software requires specifying, among other things, the mass measurement accuracy of the instrument and the digestion enzyme used with the number of missed cleavages allowed. Moreover, these software algorithms are able to identify a large number of post-translational modifications (PTMs). However, peptide and PTM identifications are challenging in the agrofood field due to non-specific cleavage sites of physiological- or food-grade enzymes and the number and location of PTMs. In this study, we show the importance of customized software programming to obtain a better peptide and PTM identification rate in the agrofood field. A gelatine product and one industrial gelatine hydrolysate from three different sources (beef, pork, and fish), each digested by simulated gastrointestinal digestion, MS-grade trypsin, or both, were used to perform the comparisons. Two main points are illustrated: (i) the impact of the set-up of specific enzyme versus no specific enzyme use and (ii) the impact of a maximum of six PTMs allowed per peptide versus the standard of three. Prior knowledge of the composition of the raw proteins is an important asset for better identification of peptide sequences.

Differential effects of milk proteins on amino acid digestibility, post-prandial nitrogen utilization and intestinal peptide profiles in rats.

OBJECTIVE: The aim of this study was to analyze the protein digestibility and postprandial metabolism in rats of milk protein matrices obtained by different industrial processes. MATERIAL AND METHODS: The study was conducted on Wistar rats that consumed a meal containing different 15N-labeled milk proteins. Four milk matrices were tested: native micellar caseins (C1), caseins low in calcium (C2 low Ca2+), a matrix containing a ratio 63:37 of caseins and whey proteins (CW2) and whey proteins alone (W). Blood and urine were collected during the postprandial period and rats were euthanized 6 h after meal intake to collect digestive contents and organs. RESULTS: Orocaecal digestibility values of amino acids ranged between 96.0 ± 0.2% and 96.6 ± 0.4% for C1-, C2 low Ca2+- and W-matrices, while this value was significantly lower for CW2 matrix (92.4 ± 0.5%). More dietary nitrogen was sequestered in the splanchnic area (intestinal mucosa and liver) as well as in plasma proteins after ingestion of W matrix, especially compared to the C1- and C2 low Ca2+-matrices. Peptidomic analysis showed that more milk protein-derived peptides were identified in the caecum of rats after the ingestion of the matrices containing caseins compared to W matrix. CONCLUSION: We found that demineralization of micellar caseins did not modify its digestibility and postprandial metabolism. The low digestibility of the modified casein-to-whey ratio matrix may be ascribed to a lower accessibility of the protein to digestive enzymes due to changes in the protein structure, while the higher nitrogen splanchnic retention after ingestion of whey was probably due to the fast assimilation of its protein content. Finally, our results showed that industrial processes that modify the structure and/or composition of milk proteins influence protein digestion and utilization.

Multivariate analysis of chemical and genetic diversity of wild Humulus lupulus L. (hop) collected in situ in northern France.

The hop plant (Humulus lupulus L.) has been exploited for a long time for both its brewing and medicinal uses, due in particular to its specific chemical composition. These last years, hop cultivation that was in decline has been experiencing a renewal for several reasons, such as a craze for strongly hopped aromatic beers. In this context, the present work aims at investigating the genetic and chemical diversity of fifty wild hops collected from different locations in Northern France. These wild hops were compared to ten commercial varieties and three heirloom varieties cultivated in the same sampled geographical area. Genetic analysis relying on genome fingerprinting using 11 microsatellite markers showed a high level of diversity. A total of 56 alleles were determined with an average of 10.9 alleles per locus and assessed a significant population structure (mean pairwise FST = 0.29). Phytochemical characterization of hops was based on volatile compound analysis by HS-SPME GC-MS, quantification of the main prenylated phenolic compounds by UHPLC-UV as well as untargeted metabolomics by UHPLC-HRMS and revealed a high level of chemical diversity among the assessed wild accessions. In particular, analysis of volatile compounds revealed the presence of some minor but original compounds, such as aromadendrene, allo-aromadendrene, isoledene, ß-guaiene, α-ylangene and ß-pinene in some wild accessions; while analysis of phenolic compounds showed high content of ß-acids in these wild accessions, up to 2.37% of colupulone. Genetic diversity of wild hops previously observed was hence supported by their chemical diversity. Sample soil analysis was also performed to get a pedological classification of these different collection sites. Results of the multivariate statistical analysis suggest that wild hops constitute a huge pool of chemical and genetic diversity of this species.

A multi-centre peptidomics investigation of food digesta: current state of the art in mass spectrometry analysis and data visualisation.

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.

Coculture of Trichoderma harzianum and Bacillus velezensis Based on Metabolic Cross-Feeding Modulates Lipopeptide Production.

Cocultures have been widely explored for their use in deciphering microbial interaction and its impact on the metabolisms of the interacting microorganisms. In this work, we investigate, in different liquid coculture conditions, the compatibility of two microorganisms with the potential for the biocontrol of plant diseases: the fungus Trichoderma harzianum IHEM5437 and the bacterium Bacillus velezensis GA1 (a strong antifungal lipopeptide producing strain). While the Bacillus overgrew the Trichoderma in a rich medium due to its antifungal lipopeptide production, a drastically different trend was observed in a medium in which a nitrogen nutritional dependency was imposed. Indeed, in this minimum medium containing nitrate as the sole nitrogen source, cooperation between the bacterium and the fungus was established. This is reflected by the growth of both species as well as the inhibition of the expression of Bacillus genes encoding lipopeptide synthetases. Interestingly, the growth of the bacterium in the minimum medium was enabled by the amendment of the culture by the fungal supernatant, which, in this case, ensures a high production yield of lipopeptides. These results highlight, for the first time, that Trichoderma harzianum and Bacillus velezensis are able, in specific environmental conditions, to adapt their metabolisms in order to grow together.

Screening of Antimicrobial Activities and Lipopeptide Production of Endophytic Bacteria Isolated from Vetiver Roots.

The exploration of certain microbial resources such as beneficial endophytic microorganisms is considered a promising strategy for the discovery of new antimicrobial compounds for the pharmaceutical industries and agriculture. Thirty-one endophytic bacterial strains affiliated with Bacillus, Janthinobacterium, Yokenella, Enterobacter, Pseudomonas, Serratia, and Microbacterium were previously isolated from vetiver (Chrysopogon zizanioides (L.) Roberty) roots. These endophytes showed antifungal activity against Fusarium graminearum and could be a source of antimicrobial metabolites. In this study, in particular, using high-throughput screening, we analyzed their antagonistic activities and those of their cell-free culture supernatants against three species of Fusarium plant pathogens, a bacterial strain of Escherichia coli, and a yeast strain of Saccharomyces cerevisiae, as well as their capacity to produce lipopeptides. The results showed that the culture supernatants of four strains close to B. subtilis species exhibited antimicrobial activities against Fusarium species and E. coli. Using mass spectrometry analyses, we identified two groups of lipopeptides (surfactins and plipastatins) in their culture supernatants. Whole-genome sequencing confirmed that these bacteria possess NRPS gene clusters for surfactin and plipastatin. In vitro tests confirmed the inhibitory effect of plipastatin alone or in combination with surfactin against the three Fusarium species.

Identification, production and bioactivity of casein phosphopeptides - A review.

Milk and dairy products are significant sources of proteins and peptides impacting human health. In this way, the interest in CPPs, bioactive phosphorylated peptides resulting from the hydrolysis of caseins, has grown in the past years. CPPs were mainly studied for their capacity to chelate and increase the bioavailability of essential minerals involved in multiple physiological processes. Moreover, CPPs harbour interesting antioxidant and anti-inflammatory properties. Recent in vivo and in vitro studies demonstrated that these different roles are strongly linked to the intrinsic properties of CPPs and CPP concentrate preparations. This review first comments on the different methods of CPP analytical characterization, focusing on recent techniques. Then, the CPP release occurring during the gastrointestinal digestion was reviewed, followed by the different CPP obtention processes and their impact on their physicochemical characteristics. Finally, the different bioactive roles attributed to CPPs, including mineral chelating properties, are discussed. We show that CPPs have a promising role in treating various pathologies, notably to compensate for deficiencies in certain nutrients and an anti-oxidant and anti-inflammatory role. Nevertheless, the mechanisms by which CPPs exert their role remain to be elucidated, and this requires precise characterization of CPPs. This work highlights the key parameters to be considered to study and produce CPPs and the different ways to be investigated in the future to elucidate their roles in vivo and characterize their potential for human health.

Structure of Lacticaseicin 30 and Its Engineered Variants Revealed an Interplay between the N-Terminal and C-Terminal Regions in the Activity against Gram-Negative Bacteria.

Lacticaseicin 30 is one of the five bacteriocins produced by the Gram-positive Lacticaseibacillus paracasei CNCM I-5369. This 111 amino acid bacteriocin is noteworthy for being active against Gram-negative bacilli including Escherichia coli strains resistant to colistin. Prediction of the lacticaseicin 30 structure using the Alphafold2 pipeline revealed a largely helical structure including five helix segments, which was confirmed by circular dichroism. To identify the structural requirements of the lacticaseicin 30 activity directed against Gram-negative bacilli, a series of variants, either shortened or containing point mutations, was heterologously produced in Escherichia coli and assayed for their antibacterial activity against a panel of target strains including Gram-negative bacteria and the Gram-positive Listeria innocua. Lacticaseicin 30 variants comprising either the N-terminal region (amino acids 1 to 39) or the central and C-terminal regions (amino acids 40 to 111) were prepared. Furthermore, mutations were introduced by site-directed mutagenesis to obtain ten bacteriocin variants E6G, T7P, E32G, T33P, T52P, D57G, A74P, Y78S, Y93S and A97P. Compared to lacticaseicin 30, the anti-Gram-negative activity of the N-terminal peptide and variants E32G, T33P and D57G remained almost unchanged, while that of the C-terminal peptide and variants E6G, T7P, T52P, A74P, Y78S, Y93S and A97P was significantly altered. Finally, the N-terminal region was further shortened to keep only the first 20 amino acid part that was predicted to include the first helix. The anti-Gram-negative activity of this truncated peptide was completely abolished. Overall, this study shows that activity of lacticaseicin 30, one of the rare Gram-positive bacteriocins inhibiting Gram-negative bacteria, requires at least two helices in the N-terminal region and that the C-terminal region carries amino acids playing a role in modulation of the activity. Taken together, these data will help to design forthcoming variants of lacticaseicin 30 as promising therapeutic agents to treat infections caused by Gram-negative bacilli.

Assessment of Lipopeptide Mixtures Produced by Bacillus subtilis as Biocontrol Products against Apple Scab (Venturia inaequalis).

Apple scab is an important disease conventionally controlled by chemical fungicides, which should be replaced by more environmentally friendly alternatives. One of these alternatives could be the use of lipopeptides produced by Bacillus subtilis. The objective of this work is to study the action of the three families of lipopeptides and different mixtures of them in vitro and in vivo against Venturia inaequalis. Firstly, the antifungal activity of mycosubtilin/surfactin and fengycin/surfactin mixtures was determined in vitro by measuring the median inhibitory concentration. Then, the best lipopeptide mixture ratio was produced using Design of Experiment (DoE) to optimize the composition of the culture medium. Finally, the lipopeptides mixtures efficiency against V. inaequalis was assessed in orchards as well as the evaluation of the persistence of lipopeptides on apple. In vitro tests show that the use of fengycin or mycosubtilin alone is as effective as a mixture, with the 50-50% fengycin/surfactin mixture being the most effective. Optimization of culture medium for the production of fengycin/surfactin mixture shows that the best composition is glycerol coupled with glutamic acid. Finally, lipopeptides showed in vivo antifungal efficiency against V. inaequalis regardless of the mixture used with a 70% reduction in the incidence of scab for both mixtures (fengycin/surfactin or mycosubtilin/surfactin). The reproducibility of the results over the two trial campaigns was significantly better with the mycosubtilin/surfactin mixture. The use of B. subtilis lipopeptides to control this disease is very promising.

Sensopeptidomic Kinetic Approach Combined with Decision Trees and Random Forests to Study the Bitterness during Enzymatic Hydrolysis Kinetics of Micellar Caseins.

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.

Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry.

The identification of phosphopeptides is currently a challenge when they are part of a complex matrix of peptides, such as a milk protein enzymatic hydrolysate. This challenge increases with both the number of phosphorylation sites on the phosphopeptides and their amino acid length. Here, this paper reports a four-phase strategy from an enzymatic casein hydrolysate before a mass spectrometry analysis in order to enhance the identification of phosphopeptides and phosphosites: (i) the control protein hydrolysate, (ii) a two-step enzymatic dephosphorylation of the latter, allowing for the almost total dephosphorylation of peptides, (iii) a one-step enzymatic dephosphorylation, allowing for the partial dephosphorylation of the peptides and (iv) an additional endoGluC enzymatic hydrolysis, allowing for the cleavage of long-size peptides into shorter ones. The reverse-phase high-pressure liquid chromatography-tandem mass spectrometry (RP-HPLC-MS/MS) analyses of hydrolysates that underwent this four-phase strategy allowed for the identification of 28 phosphorylation sites (90%) out of the 31 referenced in UniprotKB/Swiss-Prot (1 June 2021), compared to 17 sites (54%) without the latter. The alpha-S2 casein phosphosites, referenced by their similarity in the UniProt database, were experimentally identified, whereas pSer148, pThr166 and pSer187 from a multiphosphorylated long-size kappa-casein were not. Data are available via ProteomeXchange with identifier PXD027132.