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.

Cytotoxicity and Identification of Antibacterial Compounds from Baillonella toxisperma Bark Using a LC-MS/MS and Molecular Networking Approach.

Baillonella toxisperma is a medicinal plant used in northern Gabon to treat microbial diseases. It is a plant well-known by local populations, but very few studies have focused on the molecules responsible for the antibacterial activities of B. toxisperma. This study proposes a dereplication strategy based on molecular networking generated from HPLC-ESI-Q/TOF data, allowing investigation of the molecules responsible for the antibacterial activity of B. toxisperma. From this strategy, eighteen compounds were putatively identified. All of these compounds belonged mainly to five families of natural compounds, including phenylpropanolamines, stilbenes, flavonoids, lignans and phenolic glycosides. The chemical study carried out from the bark of B. toxisperma allowed us to identify, for the first time, compounds such as resveratrol and derivatives, epicatechin, epigallocatechin and epigallocatechin gallate. In addition, antibacterial activity (diffusion method and microdilution) and cytotoxicity (Cell Counting Kit-8 (CCK-8 Assay)) in vitro were evaluated. The crude ethanolic extract, as well as the fractions of B. toxisperma, showed significant antibacterial activity. However, the ethanolic fractions F2 and F4 presented high antibacterial activity compared to the crude extract. Cytotoxicity studies on colon-cancer cells (Caco-2) and human keratinocyte cells (HaCaT) showed moderate cytotoxicity in both cell types. This study clearly shows the therapeutic potential of the ethanolic extract of the bark of B. toxisperma and provides information on the phytochemical composition and bioactive compounds of the plant.

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.

In vitro comparison of whey protein isolate and hydrolysate for their effect on glucose homeostasis markers.

Research on new strategies to regulate glucose homeostasis to prevent or manage type 2 diabetes is a critical challenge. Several studies have shown that protein-rich diets could improve glucose homeostasis. Whey protein hydrolysis allows the release of amino acids and bioactive peptides, which exert numerous well-documented bioactivities. This study evaluates and compares the hypoglycemic potential of a whey protein hydrolysate and a whey protein isolate after static in vitro simulated gastrointestinal digestion (SGID) using the INFOGEST protocol. The peptide molecular mass distributions of the digested samples were evaluated by size exclusion chromatography and show that after digestion, the whey hydrolysate is significantly more hydrolyzed. After SGID, the whey protein hydrolysate induces a significative greater secretion of GLP-1 after two hours of contact with the enteroendocrine STC-1 cell line than the whey protein after isolation. In addition, the digested whey hydrolysate increases preproglucagon (GCG) and pro-convertase-1 (PCSK1) expression. The digested hydrolysate also inhibits the DPP-IV activity after an intestinal barrier passage challenge using a Caco-2/HT29-MTX mixed-cell model. Our results highlight that the prehydrolysis of whey proteins modify the intestinal peptidome, leading to a potentially greater hypoglycemic effect. This study confirms the previously observed in vitro hypoglycemic effect of this hydrolysate and evidences the beneficial impact of the industrial hydrolysis process.

Synthetic Flavonoid BrCl-Flav-An Alternative Solution to Combat ESKAPE Pathogens.

ESKAPE pathogens are considered as global threats to human health. The discovery of new molecules for which these pathogens have not yet developed resistance is a high medical priority. Synthetic flavonoids are good candidates for developing new antimicrobials. Therefore, we report here the potent in vitro antibacterial activity of BrCl-flav, a representative of a new class of synthetic tricyclic flavonoids. Minimum inhibitory/bactericidal concentration, time kill and biofilm formation assays were employed to evaluate the antibacterial potential of BrCl-flav. The mechanism of action was investigated using fluorescence and scanning electron microscopy. A checkerboard assay was used to study the effect of the tested compound in combination with antibiotics. Our results showed that BrCl-flav displayed important inhibitory activity against all tested clinical isolates, with MICs ranging between 0.24 and 125 µg/mL. A total kill effect was recorded after only 1 h of exposing Enterococcus faecium cells to BrCl-flav. Additionally, BrCl-flav displayed important biofilm disruption potential against Acinetobacter baumannii. Those effects were induced by membrane integrity damage. BrCl-flav expressed synergistic activity in combination with penicillin against a MRSA strain. Based on the potent antibacterial activity, low cytotoxicity and pro-inflammatory effect, BrCl-flav has good potential for developing new effective drugs against ESKAPE pathogens.

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.

Evaluation of Antiradical and Antioxidant Activities of Lipopeptides Produced by Bacillus subtilis Strains.

This study investigated the antiradical and antioxidant potential of the three families of lipopeptides (i.e., surfactin, mycosubtilin, and plipastatin/fengycin) produced by Bacillus subtilis strains. The antiradical/antioxidant activities of highly purified lipopeptides were studied in acellular models using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide anion ( O 2 . - ), hydrogen peroxide, (H2O2) and hydroxyl radical (HO.). At a lipopeptide concentration of 500 mg.L-1, the maximum inhibition of DPPH reached 22.88% (obtained for plipastatin). Moreover, the scavenging effects of O 2 . - , H2O2, and HO. at the highest concentration tested (250 mg.L-1) were found to be 6, 21, and 3% for surfactin, 19, 9, and 15% for mycosubtilin, 21, 18, and 59% for plipastatin, 21, 31, and 61% for the mixture of surfactin/plipastatin, and 13, 16, and 15% for the mixture of surfactin/mycosubtilin, respectively. These results showed that plipastatin was the best candidate due to its antioxidant activities.

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.

Chicory: Understanding the Effects and Effectors of This Functional Food.

Industrial chicory has been the subject of numerous studies, most of which provide clinical observations on its health effects. Whether it is the roasted root, the flour obtained from the roots or the different classes of molecules that enter into the composition of this plant, understanding the molecular mechanisms of action on the human organism remains incomplete. In this study, we were interested in three molecules or classes of molecules present in chicory root: fructose, chlorogenic acids, and sesquiterpene lactones. We conducted experiments on the murine model and performed a nutrigenomic analysis, a metabolic hormone assay and a gut microbiota analysis, associated with in vitro observations for different responses. We have highlighted a large number of effects of all these classes of molecules that suggest a pro-apoptotic activity, an anti-inflammatory, antimicrobial, antioxidant, hypolipidemic and hypoglycemic effect and also an important role in appetite regulation. A significant prebiotic activity was also identified. Fructose seems to be the most involved in these activities, contributing to approximately 83% of recorded responses, but the other classes of tested molecules have shown a specific role for these different effects, with an estimated contribution of 23-24%.

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.

An Exploratory Study of the Role of Dietary Proteins in the Regulation of Intestinal Glucose Absorption.

Several studies have demonstrated that high protein diets improve glucose homeostasis. Nevertheless, the mechanisms underlying this effect remain elusive. This exploratory study aims to screen and compare the acute effects of dietary proteins from different sources on intestinal glucose absorption. Six dietary proteins from various sources were thus selected and digested thanks to the INFOGEST static gastrointestinal digestion protocol. The digested proteins were able to decrease intestinal glucose absorption in vitro and ex vivo. Moreover, acute ingestion of casein and fish gelatin led to improved glucose tolerance in Wistar rats without significant effect on insulin secretion. In parallel, GLUT2 mRNA expression in enterocytes was decreased following short-term incubation with some of the digested proteins. These results strengthen the evidence that digested protein-derived peptides and amino acids are key regulators of glucose homeostasis and highlight their role in intestinal glucose absorption.

Explorative Screening of Bioactivities Generated by Plant-Based Proteins after In Vitro Static Gastrointestinal Digestion.

The gastrointestinal digestion of food proteins can generate peptides with a wide range of biological activities. In this study, we screened various potential bioactivities generated by plant-based proteins. Whey protein as an animal protein reference, five grades of pea protein, two grades of wheat protein, and potato, fava bean, and oat proteins were submitted to in vitro SGID. They were then tested in vitro for several bioactivities including measures on: (1) energy homeostasis through their ability to modulate intestinal hormone secretion, to inhibit DPP-IV activity, and to interact with opioid receptors; (2) anti-hypertensive properties through their ability to inhibit ACE activity; (3) anti-inflammatory properties in Caco-2 cells; (4) antioxidant properties through their ability to inhibit production of reactive oxygen species (ROS). Protein intestinal digestions were able to stimulate intestinal hormone secretion by enteroendocrine cells, to inhibit DPP-IV and ACE activities, to bind opioid receptors, and surprisingly, to decrease production of ROS. Neither pro- nor anti-inflammatory effects have been highlighted and some proteins lost their pro-inflammatory potential after digestion. The best candidates were pea, potato, and fava bean proteins.

In Vitro Assessment of the Impact of Industrial Processes on the Gastrointestinal Digestion of Milk Protein Matrices Using the INFOGEST Protocol.

The goal of this study was to determine the impact of industrial processes on the digestion of six milk protein matrices using the harmonized INFOGEST in vitro static digestion protocol. First, this method was optimized to simple protein matrices using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) to compare the intestinal protein hydrolysis obtained with increasing quantities of pancreatin. Similar results were achieved with the originally required pancreatin amount (trypsin activity of 100 U.mL-1) and with a quantity of pancreatin equivalent to a trypsin activity of 27.3 U.mL-1, which was thus used to perform the in vitro digestion of the milk matrices. Molecular weight profiles, peptide heterogeneity from LC-MS/MS data, calcium, free amino acid, and peptide concentrations were determined in the gastric and intestinal phases to compare the milk protein digests. Results showed that the industrial process affected not only the protein distribution of the matrices but also most likely the protein structures. Indeed, differences arose in terms of peptide populations generated when the caseins were reticulated or when their calcium concentrations were reduced.

Dataset on phenotypic characterization, on protein and genome analysis of three fluorescent Pseudomonas strains from mid-mountain water.

The identification of non-fermentative Gram negative bacilli from run-off and spring water, including fluorescent Pseudomonas is very complex and investigations are needed to contribute to the systematic of these bacteria. In this dataset, the phenotypical profiles of three strains isolated from Vosges mountains first identified as Pseudomonas fluorescens were determined using APIⓇ 50 CH galleries. Then, the identification of their proteins released directly into water was carried out using tandem/mass spectrometry after separating proteins on native two-dimensional polyacrylamide gels. Finally, genotypic analysis data is presented, that illustrates biodiversity in this fluorescent bacterial group. This data is referred by a research article entitled "Fluorescent Pseudomonas strains from mid-mountain water able to release antioxidant proteins directly into water".

Fluorescent Pseudomonas strains from mid-mountain water able to release antioxidant proteins directly into water.

Little is known about fluorescent Pseudomonas and investigations are needed to help us better understand how their species work. The aim was here to mimic what naturally occurs in environmental water containing strains isolated from mid-mountain water samples and identified as Pseudomonas fluorescens by conventional biochemical techniques. Three strains were cultured before being directly inoculated into distilled water. Surprisingly, the three cell-less extracts obtained after spinning the bacterial suspensions showed strong in vitro anti-oxidative effects against superoxide anion and hydroxyl radical but with discrepancies. The extracts obtained were found to contain antioxidant proteins among other stress proteins that were released by viable bacteria. They were identified using tandem/mass spectrometry and showed different profiles in sodium-dodecyl sulfate polyacrylamide gel electrophoresis. Bacterial identification was deepened using 16S ribonucleic acid and genome sequencing analyses to explain the differences observed between strains.

Antimicrobial Activity of Lipopeptide Biosurfactants Against Foodborne Pathogen and Food Spoilage Microorganisms and Their Cytotoxicity.

Lipopeptide biosurfactants produced by Bacillus sp. were assessed regarding their antimicrobial activity against foodborne pathogenic and food spoilage microorganisms. Both Gram-positive and Gram-negative bacteria were found not to be susceptible to these lipopeptides. However, mycosubtilin and mycosubtilin/surfactin mixtures were very active against the filamentous fungi Paecilomyces variotti and Byssochlamys fulva, with minimum inhibitory concentrations (MICs) of 1-16 mg/L. They were also active against Candida krusei, MIC = 16-64 mg/L. Moreover it was found that the antifungal activity of these lipopeptides was not affected by differences in isoform composition and/or purity. Furthermore their cytotoxicity tested on two different cell lines mimicking ingestion and detoxification was comparable to those of approved food preservatives such as nisin. Overall, for the first time here mycosubtilin and mycosubtilin/surfactin mixtures were found to have high antifungal activity against food relevant fungi at concentrations lower than their toxicity level hence, suggesting their application for extending the shelf-life of products susceptible to these moulds. In addition combining nisin with mycosubtilin or mycosubtiliin/surfactin mixtures proved to be an effective approach to produce antimicrobials with broader spectrum of action.

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.

Chicory Roots for Prebiotics and Appetite Regulation: A Pilot Study in Mice.

The objectives of this work are to address the prebiotic effects of chicory ( Cichorium intybus) together with its possible role in appetite control. We compared nine chicory genotypes in order to determine if variations in the content of metabolites in the roasted roots would lead to modifications in release of satiety hormones and in composition of gut microbiota. To this aim, a 5-week dietary-intervention study was achieved using mice fed with distinct chicory-based preparations. A 16S rRNA gene-based metagenetic analysis of fecal microbiota was performed. In vitro gastrointestinal digestions were performed in order to study the effect of chicory intestinal digests on gut hormone regulation in enteroendocrine cells. Firmicutes/Bacteroidetes ratio and gut bacterial groups, such as Alloprevotella, Blautia, Alistipes, and Oscillibacter, were found to be modulated by chicory. On the other hand, CCK and GLP-1 satiety hormones were demonstrated to be significantly increased by chicory in vitro.

Food-Derived Hemorphins Cross Intestinal and Blood-Brain Barriers In Vitro.

A qualitative study is presented, where the main question was whether food-derived hemorphins, i.e., originating from digested alimentary hemoglobin, could pass the intestinal barrier and/or the blood-brain barrier (BBB). Once absorbed, hemorphins are opioid receptor (OR) ligands that may interact with peripheral and central OR and have effects on food intake and energy balance regulation. LLVV-YPWT (LLVV-H4), LVV-H4, VV-H4, VV-YPWTQRF (VV-H7), and VV-H7 hemorphins that were previously identified in the 120 min digest resulting from the simulated gastrointestinal digestion of hemoglobin have been synthesized to be tested in in vitro models of passage of IB and BBB. LC-MS/MS analyses yielded that all hemorphins, except the LLVV-H4 sequence, were able to cross intact the human intestinal epithelium model with Caco-2 cells within 5-60 min when applied at 5 mM. Moreover, all hemorphins crossed intact the human BBB model with brain-like endothelial cells (BLEC) within 30 min when applied at 100 µM. Fragments of these hemorphins were also detected, especially the YPWT common tetrapeptide that retains OR-binding capacity. A cAMP assay performed in Caco-2 cells indicates that tested hemorphins behave as OR agonists in these cells by reducing cAMP production. We further provide preliminary results regarding the effects of hemorphins on tight junction proteins, specifically here the claudin-4 that is involved in paracellular permeability. All hemorphins at 100 µM, except the LLVV-H4 peptide, significantly decreased claudin-4 mRNA levels in the Caco-2 intestinal model. This in vitro study is a first step toward demonstrating food-derived hemorphins bioavailability which is in line with the growing body of evidence supporting physiological functions for food-derived peptides.

Protein Digestion-Derived Peptides and the Peripheral Regulation of Food Intake.

The gut plays a central role in energy homeostasis. Food intake regulation strongly relies on the gut-brain axis, and numerous studies have pointed out the significant role played by gut hormones released from enteroendocrine cells. It is well known that digestive products of dietary protein possess a high satiating effect compared to carbohydrates and fat. Nevertheless, the processes occurring in the gut during protein digestion involved in the short-term regulation of food intake are still not totally unraveled. This review provides a concise overview of the current data concerning the implication of food-derived peptides in the peripheral regulation of food intake with a focus on the gut hormones cholecystokinin and glucagon-like peptide 1 regulation and the relationship with some aspects of glucose homeostasis.