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While there is a consensus that food structure affects food digestion, the underlying mechanisms remain poorly understood. A previous experiment in pigs fed egg white gels of same composition but different structures evidenced such effect on food gastric disintegration. In this study, we detailed the consequences on intra-gastric pH, pepsin concentration and proteolysis by sampling throughout the stomach over 6 h digestion. Subsequent amino acid absorption was investigated as well by blood sampling. While acidification was almost homogeneous after 6 h digestion regardless of the gel, pepsin distribution never became uniform. Pepsin started to accumulate in the pylorus/antrum region before concentrating in the body stomach beyond 4 h, time from which proteolysis really started. Interestingly, the more acidic and soft gel resulted in a soon (60 min) increase in proteolysis, an earlier and more intense peak of plasmatic amino acids, and a final pepsin concentration three times higher than with the other gels.

Background Saliva may play a role in digestion through its enzymatic content or physico-chemical impact on the food bolus or chyme. Salivary proteins also interact with tannins, which could counteract their effect on digestive enzymes. We here evaluated the impact of saliva on gastric proteolysis of solid or liquid dairy products, including one contaning tannins.MethodSwiss-type cheese (C), milk in water (M) or milk in black tea (T), all in presence of human saliva vs water, were digested using semi-dynamic (C) and static conditions (M,T), respectively. Proteolysis was monitored throughout gastric digestion by OPA assay in all samples. The degree of hydrolysis was also calculated for cheese using acid titration data.ResultsFor cheese, the presence of saliva delayed slightly the initiation of proteolysis. OPA measurements suggested a small proteolytic inhibition by saliva. For milk, the presence of tannins increased gastric proteolysis (possibly by denaturing the pepsin substrates) but saliva had no substantial impact on OPA measurements, whether the matrix contained tannins or not.Conclusion In the conditions used, saliva had a moderate, if any, impact on gastric digestion of dairy products. Due to the documented effect of saliva on emulsions stability, the study could be extended to lipolysis.

Fortification of human milk (HM) is often necessary to meet the nutritional requirements of preterm infants. This study sought to establish whether HM supplemented with an experimental donkey milk-derived fortifier (DMF) or a commercial bovine milk-derived fortifier (BMF) affected digestion, using an in vitro dynamic system at the preterm stage. Particle size in gastric phase was higher in DMF than in BMF, due to protein aggregates surrounding lipid globules. Before digestion, BMF, with its extensively hydrolysed proteins, had a higher degree of proteolysis (30%) than DMF (11%), which contained intact proteins. After digestion, this difference was reduced concomitantly to a similar net degree of proteolysis (33%). DMF, with a higher proportion of ω3, resulted in a lower ω6/ω3 free PUFA ratio than BMF throughout digestion, although the final degree of lipolysis was similar (54%). In summary, DMF could represent a better source of proteins and lipids for the preterm infant.

Nutritional dependencies, especially those regarding nitrogen sources, govern numerous microbial positive interactions. As for lactic acid bacteria (LAB), responsible for the sanitary, organoleptic, and health properties of most fermented products, such positive interactions have previously been studied between yogurt bacteria. However, they have never been exploited to create artificial cocultures of LAB that would not necessarily coexist naturally, i.e., from different origins. The objective of this study was to promote LAB positive interactions, based on nitrogen dependencies in cocultures, and to investigate how these interactions affect some functional outputs, e.g., acidification rates, carbohydrate consumption, and volatile-compound production. The strategy was to exploit both proteolytic activities and amino acid auxotrophies of LAB. A chemically defined medium was thus developed to specifically allow the growth of six strains used, three proteolytic and three nonproteolytic. Each of the proteolytic strains, Enterococcus faecalis CIRM-BIA2412, Lactococcus lactis NCDO2125, and CIRM-BIA244, was cocultured with each one of the nonproteolytic LAB strains, L. lactis NCDO2111 and Lactiplantibacillus plantarum CIRM-BIA465 and CIRM-BIA1524. Bacterial growth was monitored using compartmented chambers to compare growth in mono- and cocultures. Acidification, carbohydrate consumption, and volatile-compound production were evaluated in direct cocultures. Each proteolytic strain induced different types of interactions: strongly positive interactions, weakly positive interactions, and no interactions were seen with E. faecalis CIRM-BIA2412, L. lactis NCDO2125, and L. lactis CIRM-BIA244, respectively. Strong interactions were associated with higher concentrations of tryptophan, valine, phenylalanine, leucine, isoleucine, and peptides. They led to higher acidification rates, lower pH, higher raffinose utilization, and higher concentrations of five volatile compounds. IMPORTANCE Interactions of lactic acid bacteria (LAB) are often studied in association with yeasts or propionibacteria in various fermented food products, and the mechanisms underlying their interactions are being quite well characterized. Concerning interactions between LAB, they have mainly been investigated to test antagonistic interactions. Understanding how they can positively interact could be useful in multiple food-related fields: production of fermented food products with enhanced functional properties or fermentation of new food matrices. This study investigated the exploitation of the proteolytic activity of LAB strains to promote positive interactions between proteolytic and nonproteolytic strains. The results suggest that proteolytic LAB do not equally stimulate nonproteolytic LAB and that the stronger the interactions between LAB are, the more functional outputs we can expect. Thus, this study gives insight into how to create new associations of LAB strains and to guarantee their positive interactions.

Designing bacterial co-cultures adapted to ferment mixes of vegetal and animal resources for food diversification and sustainability is becoming a challenge. Among bacteria used in food fermentation, lactic acid bacteria (LAB) are good candidates, as they are used as starter or adjunct in numerous fermented foods, where they allow preservation, enhanced digestibility, and improved flavor. We developed here a strategy to design LAB co-cultures able to ferment a new food made of bovine milk and lupin flour, consisting in: (i) in silico preselection of LAB species for targeted carbohydrate degradation; (ii) in vitro screening of 97 strains of the selected species for their ability to ferment carbohydrates and hydrolyze proteins from milk and lupin and clustering strains that displayed similar phenotypes; and (iii) assembling strains randomly sampled from clusters that showed complementary phenotypes. The designed co-cultures successfully expressed the targeted traits i.e., hydrolyzed proteins and degraded raffinose family oligosaccharides of lupin and lactose of milk in a large range of concentrations. They also reduced an off-flavor-generating volatile, hexanal, and produced various desirable flavor compounds. Most of the strains in co-cultures achieved higher cell counts than in monoculture, suggesting positive interactions. This work opens new avenues for the development of innovative fermented food products based on functionally complementary strains in the world-wide context of diet diversification.

The high-temperature short-time (HTST, 72 °C, 15 s) pasteurization of human milk (HM) has been proposed as an alternative to the Holder method (HoP, 62.5 °C, 30 min), to increase the preservation of bioactive compounds. We have investigated the impact of HTST and HoP pasteurization on the gastrointestinal kinetics of human milk, using a dynamic in vitro system in a preterm newborn model. An increased protein aggregation on the surface of fat globules following pasteurization, albeit to a lesser extent in HTST than in HoP, was observed. Despite relevant differences in the undigested milk samples, both pasteurization methods led to similar proteolytic patterns, while raw HM presented a higher native lactoferrin content throughout digestion. The slightly decreased amino acid release following HoP, with respect to HTST and raw HM, indicated that peptidomic analysis, which is currently underway, might provide interesting insights on the differential digestive kinetics of differently pasteurized HM.

International audience; Four different model infant formulae were compared under simulated conditions of the infant gastrointestinal tract using an in vitro dynamic digestion protocol. These formulae were based on whey protein isolate, whey protein isolate + lactoferrin, long chain triacylglycerols or long + medium chain triacylglycerols. In each of the four cases, the influence of protein and lipid composition on their subsequent structural disintegration, proteolysis and lipolysis during digestion was investigated. Structural characterization of the four types of infant formulae was done before and during simulated digestion using confocal microscopy and laser light scattering methods. Proteolysis was followed using molecular weight distribution methodology, primary amine quantification, amino acid bioaccessibility and peptide analyses. Lipolysis was determined by gas chromatography. During gastric digestion, the structure of infant formulae based on lactoferrin was barely affected by the physicochemical conditions in the gastric chamber and all the protein fractions were found to be resistant to gastric proteolysis, except for α-lactalbumin. The degree of lipolysis within the gastric and intestinal phase was influenced by the lipid composition, noting that those formulae containing medium chain triacylglycerols exhibited a higher extent of lipolysis. These results are relevant for the development of formulae enriched with functional ingredients that may provide bioactive peptides and faster energy delivery to the newborn baby.

International audience; Whey proteins present encrypted biofunctional peptides that need to be released from the native protein to exert their biological activity. Antihypertensive whey peptides are the most studied ones, which can be explained by high prevalence of this chronic degenerative disease. The present study investigated whether the molecular changes occurred during the gastrointestinal digestion of a whey protein hydrolysate could modulate its va-sorelaxant potential in rat aortic rings. Spectrophotometric data and SDS-PAGE gel showed a small degree of hydrolysis during the gastric phase and intense intestinal proteolysis. RP-HPLC revealed the formation of a large peptide profile. During the simulated digestion, 198 peptides were generated and identified and, left-shifted the concentration-response curve of the endothelium-dependent vasorelaxation, as recorded for the digested hy-drolysates. In conclusion, gastrointestinal digestion of the whey hydrolysate leads to the generation of bioactive peptides with enhanced vasodilatory potency, reinforcing the relevance of whey-derived products in blood pressure regulation.

International audience; Background & aims: It has been suggested that homogenization of Holder-pasteurized human milk (PHM) could improve fat absorption and weight gain in preterm infants, but the impact on the PHM digestive kinetics has never been studied. Our objective was to determine the impact of PHM homogenization on gastric digestion in preterm infants. Methods: In a randomized controlled trial, eight hospitalized tube-fed preterm infants were their own control to compare the gastric digestion of PHM and of homogenized PHM (PHHM). PHM was obtained from donors and, for half of it, was homogenized by ultrasonication. Over a six-day sequence, gastric aspirates were collected twice a day, before and 35, 60 or 90 min after the start of PHM or PHHM ingestion. The impact of homogenization on PHM digestive kinetics and disintegration was tested using a general linear mixed model. Results were expressed as means ± SD. Results: Homogenization leaded to a six-fold increase in the specific surface (P < 0.01) of lipid droplets. The types of aggregates formed during digestion were different between PHM and PHHM, but the lipid fraction kept its initial structure all over the gastric digestion (native globules in PHM vs. blend of droplets in PHHM). Homogenization increased the gastric lipolysis level (P < 0.01), particularly at 35 and 60 min (22 and 24% higher for PHHM, respectively). Homogenization enhanced the proteolysis of serum albumin (P < 0.05) and reduced the meal emptying rate (P < 0.001, half-time estimated at 30 min for PHM and 38 min for PHHM). The postprandial gastric pH was not affected (4.7 ± 0.9 at 90 min). Conclusions: Homogenization of PHM increased the gastric lipolysis level. This could be a potential strategy to improve fat absorption, and thus growth and development in infants fed with PHM; however, its gastrointestinal tolerance needs to be investigated further. This trial was registered at clinicaltrials.gov as NCT02112331.

Heat treatment is commonly used in dairy technology. Usually, milk is heated and cooled many times before to obtain the final product and milk components are then subject to cumulative heat treatments. In the present work, we investigated the heat induced aggregation (90 °C until 24 h) of two model proteins, β-lactoglobulin (β-Lg) and β-casein (β-CN), in the presence of glucose and subsequent consequences on simulated gastro-duodenal digestion. Protein aggregation and digestion were monitored using polyacrylamide gel electrophoresis, size exclusion chromatography, dynamic light scattering and transmission electron microscopy. Concomitant heating and protein glycation affect aggregation kinetics as well as protein sensitivity to enzymatic digestion. Spherical covalently linked aggregates were favored in the case of β-CN in the presence of glucose. Glucose limited the formation of twisted fibrils from β-Lg. We clearly showed that aggregates of both proteins formed in the presence of glucose were more resistant to enzymatic digestion. Those formed from β-Lg being highly resistant and still present at the end of simulated gastro-duodenal process. These findings underline the importance not only of the aggregation as such but also of the nature of formed aggregates on protein digestibility.

Several studies demonstrated that fermented milks may provide a large number of bioactive peptides into the gastrointestinal tract. We previously showed that beta-casomorphin-7, an opioid-like peptide produced from bovine β-casein, strongly stimulates intestinal mucin production in ex vivo and in vitro models, suggesting the potential benefit of milk bioactive peptides on intestinal protection. In the present study, we tested the hypothesis that the total peptide pool (TPP) from a fermented milk (yoghurt) may act on human intestinal mucus-producing cells (HT29-MTX) to induce mucin expression. Our aim was then to identify the peptide(s) carrying the biological activity and to study its impact in vivo on factors involved in gut protection after oral administration to rat pups (once a day, 9 consecutive days). TPP stimulated MUC2 and MUC4 gene expression as well as mucin secretion in HT29-MTX cells. Among the four peptide fractions that were separated by preparative reversed-phase high-performance liquid chromatography, only the C2 fraction was able to mimic the in vitro effect of TPP. Interestingly, the sequence [94-123] of β-casein, present only in C2 fraction, also regulated mucin production in HT29-MTX cells. Oral administration of this peptide to rat pups enhanced the number of goblet cells and Paneth cells along the small intestine. These effects were associated with a higher expression of intestinal mucins (Muc2 and Muc4) and of antibacterial factors (lysozyme, rdefa5). We conclude that the peptide β-CN(94-123) present in yoghurts may maintain or restore intestinal homeostasis and could play an important role in protection against damaging agents of the intestinal lumen.

β-Casofensin is a milk-derived bioactive peptide that interacts with intestinal goblet cells. We aimed to determine whether β-casofensin could prevent intestinal dysfunctions induced by neonatal maternal separation (NMS) and whether it retains its protective effects when administered in a casein matrix. We also evaluated whether a casein matrix enriched in β-casofensin protects against intestinal enteritis induced by indomethacin. β-Casofensin abolished NMS-induced jejunal hyperpermeability and prevented the depletion of goblet and Paneth cells induced by NMS. In addition, β-casofensin maintained its effectiveness against NMS-induced intestinal barrier alterations when administered in a casein matrix. A casein matrix containing β-casofensin also reduced intestinal damages induced by indomethacin. A functional food containing β-casofensin may prevent both the deleterious effects of neonatal stress on the intestinal barrier and indomethacin-induced enteritis. These results suggest a promising application of β-casofensin as a gut barrier protector in the context of bioactive foods and clinical nutrition.

1- Introduction: Propionibacterium freudenreichii is an Actinobacterium widely used in the dairy industry as a ripening culture for Swiss-type cheeses, for vitamin B12 production and some strains display probiotic properties. It is reportedly a hardy bacterium, able to survive the cheese-making process and digestive stresses. 2- Objectives : The aim of the study was to decipher metabolic adaptations of P. freudenrechii responsible for long-term survival under gradual nutritional shortage. 3- Materials & methods: For 11 days, in a non-nutrient supplemented culture medium, eight strains were monitored by measuring their optical density, counting colony-forming units (CFU) and using LIVE/DEAD staining and microscopy observation. The strain with the highest survival rate was incubated for 11 days (9 days after entry into stationary phase) in Yeast Extract Lactate medium at 30 °C under microaerophilic conditions, without any adjunct during the incubation. Its physiological adaptation was investigated by RNA-seq analysis. The carbon and free amino acids sources available in the medium, and the organic acids produced by the strain, were monitored throughout growth and survival. 4- Results: Under these conditions, the eight studied strains displayed high survival rates, their culturability reaching more than 9 log 10 CFU/ml after 2 days. After 11 days, this value ranged from 7.8 to 8.2 log 10 CFU/ml depending on the strain, and at least 50% of the P. freudenreichii population displayed an intact envelope. P. freudenreichii with the highest survival rate was CIRM-BIA 138 (alias ITG P9). Although lactate (the preferred carbon source for P. freudenreichii) was exhausted three days after inoculation, the CIRM-BIA 138 strain sustained a high population level of 9.3 log 10 CFU/mL and revealed a complete disruption of metabolism at the entry into stationary phase as compared to exponential phase. 5- Conclusions: P. freudenreichii adapts its metabolism during entry into stationary phase by down-regulating oxidative phosphorylation, glycolysis, and the Wood-Werkman cycle, by exploiting new nitrogen (glutamate, glycine, alanine) sources, by down-regulating the transcription, translation and secretion of protein. Utilization of polyphosphates was suggested.

Introduction et but de l’étude : Assurer une croissance et un développement optimal chez les nouveau-nés prématurés est une priorité (WHO, 2015). Contrairement aux formules infantiles, le lait maternel permet de limiter les entérocolites nécrosantes, mais doit être supplémenté pour répondre aux besoins nutritionnels du prématuré. Cette stratégie est largement débattue. Une alternative pourrait être l’homogénéisation du lait maternel pasteurisé afin d’augmenter sa digestibilité lipidique (Thomaz, Goncalves, & Martinez, 1999). L’homogénéisation, qui fragmente les globules gras en gouttelettes, conduit à l’augmentation de la surface disponible pour les lipases (Bourlieu et al., 2015). L’objectif était d’évaluer l’impact de l’homogénéisation de lait pasteurisé sur ses cinétiques d’hydrolyse gastrique et sa déconstruction en phase gastrique chez le prématuré. Matériel et méthodes : 8 enfants prématurés hospitalisés nourris par sonde gastrique ont participé à cette étude randomisée (NCT02112331). Sur 6 jours consécutifs, chaque enfant recevait chaque jour du lait maternel pasteurisé (LMP) et pasteurisé homogénéisé (LMP+H). Les effluents gastriques étaient collectés à jeun puis à 35, 60 ou 90 min, chaque temps étant collecté en double. Après mesure du volume et pH, les effluents étaient analysés par granulométrie laser et microscopie confocale, puis par électrophorèse (SDS-PAGE) pour le suivi des protéines et par chromatographie pour le suivi lipidique. Concernant les statistiques (logiciel R), un modèle linéaire mixte était utilisé avec comme facteurs : temps, repas, sujet et sujet x repas, et éventuellement après sélection de modèle : jour de prélèvement ou poids de l’enfant, teneur en lipides ou protéines. Résultats et Analyse statistique : L’homogénéisation du lait maternel pasteurisé a conduit à une réduction significative de taille des particules d’un facteur 10 [mode de la distribution (moy ± ET) : 7.0 ± 0.9 (LMP) vs. 0.8 ± 0.3 – 0.16 ± 0 µm (LMP+H)] et une augmentation significative de la surface spécifique d’un facteur 6 [4.1 ± 1.2 (LMP) vs 25.5 ± 3.8 m²/g de lipide (LMP+H)]. L’homogénéisation accélérait significativement la lipolyse instantanée (LMP+H : 10.9 ± 1.8, 12.7 ± 2.3 et 15.0 ± 7.1 % ; LMP : 8.5 ± 2.7, 9.6 ± 2.6 et 13.8 ± 5.2 % à 35, 60 et 90 min, respectivement) mais n’avait pas d’impact sur la protéolyse. Un ralentissement significatif de la vidange gastrique du repas était observé pour LMP+H avec un temps de demi-vidange de 38 min contre 30 min pour LMP. Il n’y avait pas de différence significative d’acidification gastrique entre LMP et LMP+H. Conclusion : L’impact de l’homogénéisation du lait maternel pasteurisé sur l’augmentation du degré de lipolyse gastrique a été démontré in vivo, en parallèle d’un ralentissement de la vidange gastrique. L’impact global sur la croissance du prématuré reste à évaluer dans une étude clinique randomisée plus large.

International audience; When the mother’s own human milk is unavailable or limited, pasteurized human milk from milk banks is preferentially administered instead of infant formula, especially for vulnerable hospitalized neonates. Holder pasteurization (62.5 °C, 30 min) may alter human milk composition and structure, which may modulate its digestive behavior. An in vitro dynamic system was set up to simulate the gastrointestinal digestion of term newborns in order to compare the kinetics of lipolysis, proteolysis and structural disintegration of raw versus pasteurized human milk. Human milk from 5 donors was pooled. Half of the pool was either administrated raw (RHM) or pasteurized (PHM). Digestions were conducted at least in duplicate for RHM and PHM. Heat-induced protein aggregation was observed in PHM. During gastric digestion, β-casein was proteolyzed significantly faster for PHM than for RHM (p < 0.05), whereas lactoferrin tended to be proteolyzed slower (p = 0.07) for PHM. Pasteurization selectively affected the intestinal release of some amino acids. At any time of the gastrointestinal digestion, the lipolysis of PHM was significantly lower than that of RHM, but no impact was observed on the profile of released fatty acids. RHM presented a structural destabilization after 60 min of gastric digestion, while there was no large variation for PHM. In the intestinal phase, the evolution of the particle sizes was rather similar. Overall, Holder pasteurization impacted the proteolysis, lipolysis and disintegration of human milk. However, this impact was limited and the physiologic and metabolic consequences remain to be investigated.

The sensitivities of the R25-I26 bond on bovine beta-casein and on its N-terminal fragment beta(1-105) to trypsin digestion were compared by monitoring the liberation of the beta(1-25) product. It was shown that this peptide bond was poorly and slowly hydrolysed on beta(1-105), while it is highly susceptible to trypsin attack when whole protein is used as substrate. The marked resistance of beta(1-105) is linked to its inhibitory effect on trypsin activity (apparent K'i = 1.2 x 10(-6) M), as demonstrated by using a related chromogenic substrate. Indeed, a preincubation step of trypsin with beta(1-105) leads to a more pronounced inhibitory effect. The progress curves obtained with and without preincubation show that beta(1-105) acts as a slow binding inhibitor on trypsin activity. These findings promise further insight into the action and the regulation of proteolytic enzymes.

We recently reported the identification of a peptide from yoghurts with promising potential for intestinal health: the sequence (94-123) of bovine β-casein. This peptide, composed of 30 amino acid residues, maintains intestinal homoeostasis through production of the secreted mucin MUC2 and of the transmembrane-associated mucin MUC4. Our study aimed to search for the minimal sequence responsible for the biological activity of β-CN(94-123) by using several strategies based on (i) known bioactive peptides encrypted in β-CN(94-123), (ii) in silico prediction of peptides reactivity and (iii) digestion of β-CN(94-123) by enzymes of intestinal brush border membranes. The revealed sequences were tested in vitro on human intestinal mucus-producing HT29-MTX cells. We demonstrated that β-CN(108-113) (an ACE-inhibitory peptide) and β-CN(114-119) (an opioid peptide named neocasomorphin-6) up-regulated MUC4 expression whereas levels of the secreted mucins MUC2 and MUC5AC remained unchanged. The digestion of β-CN(94-123) by intestinal enzymes showed that the peptides β-CN(94-108) and β-CN(117-123) were present throughout 1·5 to 3 h of digestion, respectively. These two peptides raised MUC5AC expression while β-CN(117-123) also induced a decrease in the level of MUC2 mRNA and protein. In addition, this inhibitory effect was reproduced in airway epithelial cells. In conclusion, β-CN(94-123) is a multifunctional molecule but only the sequence of 30 amino acids has a stimulating effect on the production of MUC2, a crucial factor of intestinal protection.

The native whey proteins have been intensively used in a multitude of food applications due to their high nutritional, biological, and versatile technofunctional properties. The range of applications of whey proteins has further been extended in the last decades by the use of whey protein aggregates offering new technofunctional properties. These properties are directly dependent on the structure of whey protein aggregates, i.e., their size, shape, density, surface properties, and the type of bonds maintaining proteins together in the aggregates. In this review, after a brief description of the major whey proteins, we examine the most important advances reported to date pertaining to the available approaches to modify the structure of whey proteins for specific functionalities. Our laboratory, Science and Technology of Milk and Eggs, has contributed significantly to the advancement of knowledge on the structure-function relationships of whey proteins either in the native or denatured/aggregated forms. Our expertise and research approaches are highlighted throughout some selected results accumulated during the last decade in comparison with results from the literature.

Staphylococcus aureus is a gram-positive bacterium responsible for a wide range of infections. Host cell cycle alteration is a sophisticated mechanism used by pathogens to hijack the defense functions of host cells. We previously demonstrated that S. aureus MW2 (USA400) bacteria induced a G2/M phase transition delay in HeLa cells. We demonstrate here that this activity is triggered by culture supernatant compounds. Using size exclusion chromatography of the MW2 supernatant, followed by mass spectroscopy analysis of corresponding peaks, we identified phenol-soluble modulin α (PSMα) peptides as the likely candidates for this effect. Indeed, synthetic PSMα1 and PSMα3 caused a G2/M phase transition delay. The implication of PSMα in cell cycle alteration was confirmed by comparison of S. aureus Los Angeles County clone (LAC) wild-type with the isogenic mutant LAC∆psmα, which lacks the psmα operon encoding PSMα1–4. PSMα-induced G2/M transition delay correlated with a decrease in the defensin genes expression suggesting a diminution of antibacterial functions of epithelial cells. By testing the supernatant of S. aureus human clinical isolates, we found that the degree of G2/M phase transition delay correlated with PSMα1 production. We show that PSMs secreted by S. aureus alter the host cell cycle, revealing a newly identified mechanism for fostering an infection.—Deplanche, M., Filho. R. A. E.–A., Alekseeva, L., Ladier, E., Jardin, J., Henry, G., Azevedo, V., Miyoshi, A., Beraud, L., Laurent, F., Lina, G., Vandenesch, F., Steghens, J.-P., Le Loir, Y., Otto, M., Götz, F., Berkova, N. Phenol-soluble modulin α induces G2/M phase transition delay in eukaryotic HeLa cells.

Digestion of proteins leads to the release of numerous peptides in the gastrointestinal tract, among them several bioactive peptides. This review compiles, organises and critically analyses the information available on bioactive peptides present in vivo in the digestive tract of adult humans and animals. It focuses on milk proteins, which are recognised as one of the main sources of bioactive peptides with different biological functions. To date, few studies have been performed on the presence of milk-derived peptides in the intestinal tract in vivo. The diversity of these studies is remarkable in terms of the milk products that have been tested, the animal models selected and the segment of the digestive tract considered. Altogether, 69 bioactive peptides identified in vivo are reviewed. Most of them are released from casein, and especially from β-casein, to which 38 bioactive peptides are attributed. These sequences can be classified mainly as angiotensin-converting enzyme (ACE) inhibitors, opioids and antimicrobial peptides. Particular attention is paid to β-casomorphins and caseinomacropeptide-derived peptides. The function of caseinophosphopeptides as a mineral carrier is also discussed. Few bioactive peptides derive from whey proteins., The authors are involved in the Food and Agriculture COST (European Cooperation in Science and Technology) Action FA1005 “Improving health properties of food by sharing our knowledge on the digestive process” (INFOGEST)

International audience; This study aimed at identifyingmilk peptides released in the gastrointestinal tract ofmini-pigs during the digestion of dairy matrices and determining the effect of the matrix structure on the gut lumen peptidome. Six dairy matrices (raw and heated milk, the corresponding rennet gels, and a non-stirred or a stirred acid gel prepared from the heated milk) of similar composition but with different structures were ingested by four mini-pigs. Duodenal effluentswere collected over a 5 h-period and analyzed by tandemmass-spectrometry.More than 16,000 peptideswere sequenced and unambiguously identified. The structure of dairy products had only little influence on the location of cleavage sites on the protein sequences.However, it strongly impacted the number of identified peptides andmore especially for rennet gels which led to about 3 times less detected peptides than for the other matrices. This effect was attributed to greater extents of dilution by digestive secretions associated with longer gastric retentions for the rennet gels. Potential bioactive peptides were also produced over time and are all reported to contribute to document the literature originating from in vivo experiments. Our results indicate that the structure of dairymatrices strongly affects the kinetics ofmilk protein digestion in vivo, more than themechanism of proteolysis itself.

Goat milk is characterized by a very low heat stability that could be attributed, in part, to the covalent interaction between whey proteins and casein micelles. However, the formation of such a complex in goat milk has never been evidenced. This study was designed to assess whether heat-induced covalent interaction occurs between purified casein micelles and beta-lactoglobulin. We used a multiple approach of ultracentrifugation of heated mixture, chromatographic fractionation of resuspended pellets, sequential enzyme digestion of disulfide-linked oligomers, and identification of disulfide-linked peptides by on-line liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), and tandem MS. We identified three different types of disulfide links: (1) expected intermolecular bridges between beta-Lg molecules; (2) disulfide bond involving two kappa-casein molecules; and (3) a disulfide bond between two peptides, one from beta-Lg and the other from kappa-casein. The involved sites in this last bond were Cys(160) of beta-Lg and Cys(88) of kappa-casein. Although the identified heterolinkage is possibly only one of several different types, the results of this study constitute the first direct evidence of the formation of a covalent complex between casein micelles and beta-lactoglobulin derived from goat milk.

Binding iron to the phosphorylated beta(1-25) peptide derived from beta-casein improves iron bioavailability in the rat. The aim of the present work was to learn how injected beta(1-25) and iron-beta(1-25) complex behave in the duodenum of rats using the technique of intestinal ligation in situ and reversed-phase (RP)-high performance liquid chromatography-electrospray mass spectrometry analysis of the lumen contents. The results demonstrate that beta(1-25) is sensitive to digestive enzymes including proteases/peptidases and phosphatases during duodenal transit. The lumen contents of rats perfused with iron free beta(1-25) contained all peptidic sequences derived from beta(1-25). In contrast, the phosphorylated part of beta(1-25) [i.e., beta(15-24)] was not detected in lumen of rats perfused with iron-beta(1-25) complex.

An in vitro study was conducted to investigate the sensitivity of lactose-β-lactoglobulin conjugates to β-galactosidase from Kluyveromyces lactis. The hydrolysis was monitored by ion exchange chromatography. Compared to free lactose or lactulose, which were rapidly hydrolysed, protein-bound lactose was not hydrolysed by β-galactosidase even after an extended hydrolysis time. Tryptic digestion of the conjugates before addition of β-galactosidase improved the substrate accessibility and led to the release of about 50% of the linked lactose. The results strongly suggest that the resistance of protein bound lactose is linked to the globular and compact structure of lactose-β-lactoglobulin conjugates.

y Since temperature and water activity are among the most important parameters that affect the Maillard reaction, the glycation sites in pure, native bovine β-lactoglobulin were determined after a mild heat treatment (60°C) in an aqueous solution and after a treatment under a restricted water environment (50°C, 65% relative humidity). In both systems, the results obtained underlined the structural heterogeneity of β-lactoglobulin (β-LG) glycoforms with respect to the number of lactose residues linked per protein molecule and to the binding sites involved. Subsequently, the effect of the glycation conditions on both the association behaviour and the conformational changes of the glycated β-LG were characterised by proteolytic susceptibility, binding of the fluorescent probe 8-anilino-1-naphtalene-sulfonic acid, SDS-PAGE and size exclusion chromatography. The results showed that dry-way glycation did not significantly alter the native-like behaviour of the protein while the treatment in solution led to important structural changes. These changes resulted in a specific denatured β-LG monomer, which covalently associated via the free thiol group. The homodimers thus formed and the expanded monomers underwent subsequent aggregation to form high molecular weight species, via non-covalent interactions. The use of monoclonal antibodies with defined epitopes, raised against native β-LG. confirmed that the protein conformation was much more modified when glycation was performed in a solution while the structural changes induced during dry-way treatment were limited to the AB loop region of the protein.

Direct monitoring of the Amadori product formation between lactose and β-lactoglobulin was performed by electrospray ionisation mass spectrometry. As expected, the glycation reaction was faster at low water activity than in aqueous system. Heterogeneous β-lactoglobulin glycoforms were observed, with different number of lactose bound: populations of β-lactoglobulin with 1 to 7 and 2 to 11 linked lactose were detected, respectively, after 10 and 22 h of reaction under 65% relative humidity and 50°C. Trypsinolysis of glycated proteins, followed by reverse-phase HPLC coupled to tandem mass spectrometry in the neutral loss scanning mode, indicated that all potential reactive amino groups, except Lys101, were involved in sugar binding. The resulting order of reactivity, as a function of reaction time, was as follows: Lys47,91>α-amino-group, Lys15,70,100>Lys60,69,75,77,83,135,138>Lys8,141.

The behavior of β-lactoglobulin (β-Lg) during heat treatments depends on the environmental conditions. The influence of the presence or absence of a reducing sugar, namely, glucose, on the modification of the protein during heating has been studied using fluorescence, polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), and transmission electron microscopy. Glycated products were formed during heating 24 h at 90 °C and pH 7. The fluorescence results revealed an accumulation of the advanced Maillard products and the formation of aggregates during heating. PAGE and SEC data suggested that the products in the control samples were essentially composed of covalently linked fibrillar aggregates and that their formation was faster than that for glycated samples. We showed that glucose affected the growing step of covalent aggregates but not the initial denaturation/aggregation step of native protein. Glucose-modified proteins formed a mixture of short fibrils and polydisperse aggregates. Our results revealed that β-Lg forms fibrils at neutral pH after heating and that glucose slows the formation of these fibrils.

The precipitation of peptides derived from tryptic hydrolysis of caseins was studied as a function of the pH (3.5 to 7.5), ionic strength (from 0 to 1 m ), and the chemical nature of the salt [NaCl (NH4)2 SO4] at 25°C. Precipitation occurred only in the acidic pH range. The precipitated fraction, analyzed by RP-HPLC, was constituted by a specific hydrophobic peptide group. The nine major peptides were identified as κ-CN (35–63), αs1-CN (91–100), αs1-CN (125–151), αs1-CN (152–193), β-CN (49–97), β-CN (108–169), β-CN (114–169), β-CN (184–202), and β-CN (184–209). The optimum precipitation was found to be at pH 3.5 and 0.25 m NaCl except for β-CN (184–202) and αs1-CN (125–151), which required a higher ionic strength. The results showed that pH had a greater effect on the precipitation of peptides than salt. However, neither pI nor hydrophobicity alone could have explained the salting-out behavior; probably a combination of the two properties is responsible.

International audience; Chronic exposure of advanced glycation end products (AGEs) is associated with development of insulin resistance in mammals and in L6 skeletal muscle cells. AGEs increase oxidative stress and inflammation through receptor for AGEs (RAGE). We hypothesized that a chronic intake of dietary high AGEs may affect skeletal muscle protein metabolism and muscle mass in aging. To elucidate this hypothesis, phosphorylation of anabolic signaling pathway (Akt, mTOR, p70S6K, 4E-BP1) and RAGE expression were measured by Western-blot in tibialis in young (5month old), adult (13month old) and old (25month old) rats fed a diet containing heated casein (high-AGEs) or non heated casein (low-AGE) for 3 months. Insulin sensitivity was also evaluated by OGTT. Muscle loss and insulin resistance were observed in old (P

BackgroundNormal mammary gland contains an extravascular population of B lymphoblasts, precursors of the immunoglobulin plasma cells that play a key role in the passive protection of neonates by secreting immunoglobulins to colostrum and milk. We investigated the presence of chemoattractants in the milk by analysing the chemoattractant activity of various fractions of this secretion. Milk chemoattractants are potentially involved in the recruitment of lymphocytes from the maternal bloodstream in lactating mammary glands.ResultsThe dilution-related lymphoid cell chemoattraction of whey was associated with a < 10 kDa ultrafiltrate. Active fractions were purified by reverse-phase high performance liquid chromatography. Two peptides of 2.7 kDa (DMREANYKNSDKYFHARGNYDAA) and 1 kDa (RPPGLPDKY) were identified as fragments of the SAA protein family, tentatively identified as SAA2. Only the 2.7 kDa synthetic peptide displayed chemotactic activity, at two different optimal concentrations. At the lower concentration (3.7 nM), it attracted B-cell lymphoblasts, whereas at the higher (3.7 μM), it attracted B lymphocytes. Then, the SAA mRNA expression was analysed and we observed more SAA transcripts during lactation than gestation.ConclusionThese data are consistent with the SAA23–45fragment being involved in preplasma B-cell recruitment to the mammary gland and resultant benefit to the neonate.

S-carboxymethylated (SCM) kappa-casein forms in vitro fibrils that display several characteristics of amyloid fibrils, although the protein is unrelated to amyloid diseases. In order to get insight into the processes that prevent the formation of amyloid fibrils made of kappa-caseins in milk, we have characterized in detail the reaction and the roles of its possible effectors: glycosylation and other caseins. Given that native kappa-casein occurs as a heterogeneous mixture of carbohydrate-free and carbohydrate-containing chains, kinetics of fibril formation were performed on purified glycosylated and unglycosylated SCM kappa-caseins using the fluorescent dye thioflavin T in conjunction with transmission electron microscopy and Fourier transform infrared spectroscopy for morphological and structural analyses. Both unglycosylated and glycosylated SCM kappa-caseins have the ability to fibrillate. Kinetic data indicate that the fibril formation rate increases with SCM kappa-casein concentration but reaches a plateau at high concentrations, for both the unglycosylated and glycosylated forms. Therefore, a conformational rearrangement is the rate-limiting step in fibril growth of SCM kappa-casein. Transmission electron microscopy images indicate the presence of 10- to 12-nm spherical particles prior to the appearance of amyloid structure. Fourier transform infrared spectroscopy spectra reveal a conformational change within these micellar aggregates during the fibrillation. Fibrils are helical ribbons with a pitch of about 120-130 nm and a width of 10-12 nm. Taken together, these findings suggest a model of aggregation during which the SCM kappa-casein monomer is in rapid equilibrium with a micellar aggregate that subsequently undergoes a conformational rearrangement into a more organized species. These micelles assemble and this leads to the growing of amyloid fibrils. Addition of alpha(s1)-and beta-caseins decreases the growth rate of fibrils. Their main effect was on the elongation rate, which became close to that of the limiting conformation change, leading to the appearance of a lag phase at the beginning of the kinetics.

Clusters of phosphoserine residues in cow milk caseins bind iron (Fe) with high affinity. Casein inhibits Fe absorption in humans, but protein hydrolysis lessens this effect. Phosphopeptides from different caseins gave conflicting results on Fe absorption; release of phosphate residues by intestinal alkaline phosphatase could be a key point of that metabolism. The objectives of this study were to compare the absorption of Fe complexed to caseinophosphopeptides (CPP) of the main cow milk caseins β-casein (β-CPP) and αs-caseins (αs1-CPP) and to assess the role of alkaline phosphatase on this absorption. Two experimental models were used: an in vivo perfused rat intestinal loop and an in vitro Caco-2 cell culture model. In addition, we determined the effect of an intestinal phosphatase inhibitor on these various forms of Fe. Gluconate Fe was used as control. In both models, uptake and net absorption of Fe complexed to CPP from αS1-caseins were significantly lower than from Fe complexed to β-CPP. Inhibition of the intestinal phosphatase significantly increased the uptake and the absorption of Fe complexed to β-CPP without effect on the other forms of Fe. These results confirm the enhancing effect of β-casein and its CPP on Fe absorption. The differences between CPP could be explained by their structural and/or conformational features: binding Fe to αS1-CPP could impair access to digestive enzymes, whereas β-CPP-bound Fe is better absorbed than its free form. The differences in protein composition between cow and breast milk, which does not contain α-casein, could explain some of their differences in Fe bioavailability. Abbreviations: CPP, caseinophosphopeptides Fe, iron Iron (Fe) deficiency is one of the most common nutritional problems around the world, both in developed and in developing countries. It is responsible for a long-lasting impairment of neurologic development in infants; Fe is also essential for immunity and growth (1,2). Cow milk products and cow milk-based infant formulas are two main components of infant diet; however, Fe absorption from cow milk is low compared with breast milk (3); the high calcium concentration and the kind of proteins of cow milk could explain these differences (4,5). The main cow milk proteins are caseins, which bind cations, including Fe, by clusters of phosphoserine (6,7). This strong binding keeps Fe soluble at the alkaline gut pH but prevents its release in a free form available for absorption by the duodenal mucosa (8). Hydrolysis of whole caseins improves Fe absorption (5), but caseinophosphopeptides (CPP) released by the enzyme digestion of different caseins give conflicting results; the two main cow milk caseins are αS- and β-caseins; binding Fe to CPP of β-casein or to hydrolyzed β-casein improves its absorption and its bioavailability in rats and in humans (9-11); however, CPP derived from the hydrolysis of whole casein or fractions enriched with αS-casein CPP inhibit Fe absorption (12-14). One of the key steps of the absorption of CPP-bound Fe seems to be the release of phosphoserine residues by brush border phosphatase: it could be assumed that releasing free Fe could increase its absorption from a poorly absorbed form; however, releasing Fe from a stable, efficiently absorbed complex, such as Fe-βCPP (1-25) could impair its absorption by increasing the amount of free Fe subject to gut interactions. These functional differences between caseins and casein-derived CPP could give some clues to improve the bioavailability of cow milk Fe. The objectives of this study were to evaluate the absorption of Fe complexed to CPP purified either from β-casein or from αs1-casein and to determine the effect of an inhibitor of intestinal alkaline phosphatase (Na2WO4) (12,15). The study used two models, the in vivo perfused rat intestinal loop and a human colon adenocarcinoma cell line culture (Caco-2), which is predictive of human Fe bioavailability (16).

Hydrolysis of proteins could lessen their inhibiting effect on the poor absorption of cow's milk iron (Fe), which is responsible for the high incidence of Fe deficiency worldwide. When bound to Fe, caseinophosphopeptides (CPP) derived from milk proteins resist luminal digestion, enhance Fe solubility and could improve its bioavailability; brush border enzyme alkaline phosphatase activity could influence iron absorption by releasing free Fe; this study assessed its role in the absorption of CPP-bound Fe. Rat duodenal loops were perfused with Fe gluconate or Fe bound to the CPP of beta casein [beta-CN (1-25)], with or without the addition of an inhibitor of alkaline phosphatase, Na2WO4. The uptake of Fe-beta-CN (1-25) was greater than Fe gluconate. Na2WO4 enhanced the uptake of Fe-beta-CN (1-25) and not of Fe gluconate. So the release of free, insoluble Fe, by alkaline phosphatase seems to be prevented by providing Fe in the Fe-beta-CN (1-25) complex form. Its good disappearance rate makes beta-CN (1-25)-bound Fe a candidate for food fortification.

Under denaturing conditions, copper was shown to induce dimerization of bovine β-lactoglobulin (β-Lg). The occurrence of this chemical reaction and the characterization of the species formed were investigated under various experimental conditions. When β-Lg solution was heated or treated with a denaturant in the presence of copper, covalent dimerization through oxidation of SH groups was observed. The rate of dimer formation increased with increased concentration of both copper and denaturant. The copper-dependent conversion rate of native protein into the covalently- linked dimer was found to be seven to ten-fold faster for variant B than for variant A. The functional and biological implications of the copper-modified β-Lg are discussed., En conditions dénaturantes, nous avons observé que la présence du cuivre conduit à la formation de dimères de β-lactoglobuline (β-Lg) bovine. Nous avons étudié cette réaction chimique et caractérisé l’espèce moléculaire formée dans différentes conditions expérimentales. En présence d’un agent dénaturant ou après traitement thermique, la réaction de dimérisation a lieu par oxydation des groupements thiols de la protéine. La cinétique de dimérisation augmente avec les concentrations du cuivre et de l’agent dénaturant. Cette cinétique est par ailleurs sept à dix fois plus rapide pour le variant B de la β-Lg par rapport à celle du variant A. L’implication biologique éventuelle de cette réaction, favorisée par les atomes de cuivre, est présentée.

Caseinomacropeptide (CMP) is the product of milk κ-casein hydrolysis by chymosin. This macropeptide with a molecular weight of about 7000 g mol −1 contains several sequences of amino acids which show promising nutritional or biological properties. We have previously reported the production of such sequences (peptides) from CMP by trypsin as well as the reaction mechanism involved. In this study, we have focused on the study of the hydrolytic reaction and on the retention by the membrane of the peptides generated by the reaction, both in a batch system and in a continuous stirred reactor. Factors influencing the operation of the membrane reactor such as the substrate concentration, ionic strength, transmembrane pressure are studied and discussed. Conclusions on the design of a membrane reactor are then drawn.

The solubility of lactose-beta-lactoglobulin conjugates at pH 4.6, after heating near neutral pH in phosphate buffer/0.116 M NaCI, was investigated by size exclusion chromatography and compared with unmodified protein. Heated conjugates in the temperature range 65-90 degrees C showed greater solubility at pH 4.6. The proportion of soluble protein increased with the number of bound lactose molecules. Total solubility was obtained for conjugates with nine lactose residues attached per monomer of beta-lactoglobulin. The protective effect of bound sugar toward precipitation was associated with the formation of soluble disulfide cross-linked dimers, highly accessible to trypsin digestion. These results suggested that bound lactose, through steric hindrance and high surface hydrophilicity, prevents the thiol-disulfide exchange reactions of the polymerization-aggregation process of lactose-beta-lactoglobulin conjugates.

The kinetics of the tryptic release of bioactive peptides from caseinomacropeptide was investigated in both batch and continuous mode in an enzymatic membrane reactor. The hydrolysis of the three susceptible bonds, Lys 111 –Lys 112 , Lys 112 –Asn 113 , and Lys 116 –Thr 117 , was monitored by quantitative determination of the released products. A kinetic study in the batch system showed that the overall catalytic process follows a sequential mechanism where the Lys 116 –Thr 117 bond was only cleaved on the intermediary products resulting from the cleavage of the Lys 111 –Lys 112 and Lys 112 –Asn 113 bonds. When the reaction was performed in the continuous enzymatic membrane reactor, it was found that the enzyme preference toward the Lys 116 –Thr 117 bond depended on the relative concentrations of both the caseinomacropeptide and the intermediary products accumulated at steady state. Such concentrations were controlled by the enzyme and substrate concentrations and the substrate feeding flow rate; hence, by control of the operating parameters and with the understanding of the reaction mechanism, the enzyme action toward various peptidic bonds can be oriented in the continuous mode, offering the possibility of better control of the type of product recovered in the reactor output.

International audience; Continuous hydrolysis of sodium caseinate by alcalase was investigated in a recycle bioreactor coupled to an inorganic M5 membrane module. The effects of various substrate concentrations and the role of an ultrafiltration membrane on conversion rate were reported. Although a high level of conversion was obtained in the retentate side at a steady state, only part of the products formed was transmitted through the inorganic membrane. Degree of hydrolysis and product concentration in the reactor seem to be the main factors limiting product output during the continuous hydrolysis.

Transmission of β-CN (193–209), a hydrophobic peptide derived from c-terminal part of bovine β-casein, during crossflow ultrafiltration through M5 Carbosep membrane (carbon-zirconium) was studied as a function of pH, ionic strength and in the presence of small peptides mixture used as adsorption competitors. It was shown that part of the high apparent rejection observed seems to be due to the hydrophobic interactions between peptide and membrane support. Hence, using three membrane types: M5 Carbosep (carbonZrO2, 10 kDa (MWCO)), M2 Carbosep (carbonZrO2, 15 kDa) and Kerasep (Al2O3ZrO2, 15 kDa), the influence of the supports was investigated in relation to the effects they produce on β-CN (193–209) transmission and adsorption. At steady state of the process, the transmissions obtained were 6, 53 and 66% while the percent of the peptide adsorbed were 41, 89 and 0 % with M5, M2 and Kerasep membranes, respectively. These data indicate that, in the case of Carbosep membranes, the combination of peptide retention within the membrane pores and/or on the upper membrane surface as well as the adsorption of the solute on the carbon support were at the origin of the low unexpected β-CN (193–209) concentration in the permeate side.