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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.

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.

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)