Deciphering the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during soy juice fermentation using phenotypic and transcriptional analysis.

In the context of sustainable diet, the development of soy-based yogurt fermented with lactic acid bacteria is an attractive alternative to dairy yogurts. To decipher the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during soy juice (SJ) fermentation, the whole genome of the strain CIRM-BIA865 (Ld865) was sequenced and annotated. Then Ld865 was used to ferment SJ. Samples were analyzed throughout fermentation for their cell number, carbohydrate, organic acid, free amino acid, and volatile compound contents. Despite acidification, the number of Ld865 cells did not rise, and microscopic observations revealed the elongation of cells from 3.6 µm (inoculation) to 36.9 µm (end of fermentation). This elongation was observed in SJ but not in laboratory-rich medium MRS. Using transcriptomic analysis, we showed that the biosynthesis genes of peptidoglycan and membrane lipids were stably expressed, in line with the cell elongation observed, whereas no genes implicated in cell division were upregulated. Among the main sugars available in SJ (sucrose, raffinose, and stachyose), Ld865 only used sucrose. The transcriptomic analysis showed that Ld865 implemented the two transport systems that it contains to import sucrose: a PTS system and an ABC transporter. To fulfill its nitrogen needs, Ld865 probably first consumed the free amino acids of the SJ and then implemented different oligopeptide transporters and proteolytic/peptidase enzymes. In conclusion, this study showed that Ld865 enables fast acidification of SJ, despite the absence of cell division, leads to a product rich in free amino acids, and also leads to the production of aromatic compounds of interest. IMPORTANCE: To reduce the environmental and health concerns related to food, an alternative diet is recommended, containing 50% of plant-based proteins. Soy juice, which is protein rich, is a relevant alternative to animal milk, for the production of yogurt-like products. However, soy "beany" and "green" off-flavors limit the consumption of such products. The lactic acid bacteria (LAB) used for fermentation can help to improve the organoleptic properties of soy products. But metabolic data concerning LAB adapted to soy juice are lacking. The aim of this study was, thus, to decipher the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during fermentation of a soy juice, based on a multidisciplinary approach. This result will contribute to give tracks for a relevant selection of starter. Indeed, the improvement of the organoleptic properties of these types of products could help to promote plant-based proteins in our diet.

The use of 15N-labelled protein to account for the endogenous nitrogen contribution to in vitro protein digestibility measurement.

Protein digestibility, a key indicator of dietary protein quality for human nutrition, can be estimated using an in vitro digestion model, however its definition and determination remain variable across studies. The present study aimed to determine the contribution of the endogenous nitrogen (N) to the plant and animal protein digestibility values obtained in vitro. 15N-labelled gluten and caseins (4, 8 and 16 % of the model meal) were used to differentiate dietary and endogenous N and were digested using the INFOGEST in vitro digestion model with no oral phase. The dietary and endogenous N were measured before and during digestion after centrifugation and 10 kDa ultrafiltration. The proteolysis degree was measured by the OPA method. The endogenous and dietary N were determined by elemental analyser coupled with isotopic ratio mass spectrometry. Apparent and true digestibility were determined and values of 135, 92 and 71 % for apparent vs. 78, 69, 60 % for true digestibility were obtained for 4, 8 and 16 % dietary protein level, respectively, with a significant effect of protein level. Differences between apparent and true digestibility pointed out the important contribution of the endogenous nitrogen. Our results showed that 40 % of the N below 10 kDa, i.e., the digestible fraction, were from endogenous origin (i.e. from the pancreatin) and was even present before digestion. An average value of 27 % for pancreatin N autolysis was estimated independently of the protein levels or sources. The use of 15N-labelled protein to evaluate in vitro protein digestibility highlighted the important contribution of the endogenous N, in particular when low dietary protein solution (4 %) are digested. This gives new keys to overcome drawbacks of in vitro models for determining protein digestibility.

In vitro digestion of two protein-rich dairy products in the ageing gastrointestinal tract.

It is still unclear if changes in protein digestibility and absorption kinetics in old age may affect the anabolic effect of high-protein foods. The objective of this study was to investigate the digestion of two high-protein (10% w/w) dairy products in vitro: a fermented dairy product formulated with a ratio of whey proteins to caseins of 80 to 20% (WBD) and a Skyr containing mainly caseins. The new static in vitro digestion model adapted to the general older adult population (≥65 years) proposed by the INFOGEST international consortium was implemented to investigate the digestion of these products and compared with the standard version of the protocol. Kinetics of proteolysis was compared between both models for each product, in the gastric and intestinal phases of digestion. Protein hydrolysis was studied by the OPA method, SDS-PAGE, and LC-MS/MS, and amino acids were quantified by HPLC. Protein hydrolysis by pepsin was slower with the older adult model than with the young adult model, and consequently, in spite of a longer gastric phase duration, the degree of proteolysis (DH) at the end of the gastric phase was lower. Two different scenarios were observed depending on the type of dairy product studied: -10 and -40% DH for Skyr and WBD, respectively. In the intestinal phase, lower concentrations of free leucine were observed in older adult conditions (approx. -10%), but no significant differences in proteolysis were observed overall between the models. Therefore, the digestion conditions used influenced significantly the rate and extent of proteolysis in the gastric phase but not in the intestinal phase.

Protein ingredient quality of infant formulas impacts their structure and kinetics of proteolysis under in vitro dynamic digestion.

Infant formula (IF) is a complex matrix requiring numerous ingredients and processing steps. The objective was to understand how the quality of protein ingredients impacts IF structure and, in turn, their kinetics of digestion. Four powdered IFs (A/B/C/D), based on commercial whey protein (WP) ingredients, with different protein denaturation levels and composition (A/B/C), and on caseins with different supramolecular organisations (C/D), were produced at a semi-industrial level after homogenization and spray-drying. Once reconstituted in water (13 %, wt/wt), the IF microstructure was analysed with asymmetrical flow field-flow fractionation coupled with multi-angle light scattering and differential refractometer, transmission electron microscopy and electrophoresis. The rehydrated IFs were subjected to simulated infant in vitro dynamic digestion (DIDGI®). Digesta were regularly sampled to follow structural changes (confocal microscopy, laser-light scattering) and proteolysis (OPA, SDS-PAGE, LC-MS/MS, cation-exchange chromatography). Before digestion, different microstructures were observed among IFs. IF-A, characterized by more denatured WPs, presented star-shaped mixed aggregates, with protein aggregates bounded to casein micelles, themselves adsorbed at the fat droplet interface. Non-micellar caseins, brought by non-micellar casein powder (IF-D) underwent rearrangement and aggregation at the interface of flocculated fat droplets, leading to a largely different microstructure of IF emulsion, with large aggregates of lipids and proteins. During digestion, IF-A more digested (degree of proteolysis + 16 %) at 180 min of intestinal phase than IF-C/D. The modification of the supramolecular organisation of caseins implied different kinetics of peptide release derived from caseins during the gastric phase (more abundant at G80 for IF-D). Bioactive peptide release kinetics were also different during digestion with IF-C presenting a maximal abundance for a large proportion of them. Overall, the present study highlights the importance of the structure and composition of the protein ingredients (WPs and caseins) selected for IF formulation on the final IF structure and, in turn, on proteolysis. Whether it has some physiological consequences remains to be investigated.

Impact of process and composition of formulas for elderly on in vitro digestion using the dynamic DIDGI® model.

Due to the lower efficiency of the elderly digestion system, new formulations are needed in order to increase the bioaccessibility of macronutrients. The aim of the work was to evaluate the effect of the process of protein sources production using either liquid (F2) vs spray dried milk proteins (F1/F3) and the source of lipids (vegetable oil (F1) vs mix of vegetable oil + bovine milk cream (F2/F3)) ingredients on the macronutrient digestion of three experimental elderly formulas. The dynamic in vitro digestion model DIDGI®, was adapted to simulate the digestive conditions of the elderly. An exhaustive review of the literature was carried out in order to simulate as closely as possible the elderly digestive parameters and constituted the starting point towards a consensus in vitro digestion model that will be proposed soon by the INFOGEST scientific network. The three experimental formulas (F1/F2/F3) differing by the composition and process applied were submitted to the DIDGI® dynamic in vitro digestion over four hours using parameters adapted to the elderly. The three formulas were compared in terms of proteolysis and lipolysis. A slight impact of the process (liquid vs spray-dried) on the degree of proteolysis at the end of digestion was observed with 50.8% for F2 compared to 56.8% for F1 and 52.9% for F3 with<5% of difference between the 3 formulas. Concerning the degree of lipolysis, the addition of bovine cream led to a lesser extent of lipolysis with 63.7 and 60.2% for F2 and F3 respectively versus 66.3% for F1 (containing only vegetable oil). Our results highlighted the beneficial input of the milk fat with a higher level of phospholipids and a lower ω6/ω3 PUFA ratio and can be a good alternative to the use of the vegetable fat in drinks for elderly people.

Casein structures differently affect postprandial amino acid delivery through their intra-gastric clotting properties.

We aimed to assess if casein structure affects its digestion and its subsequent amino acid delivery kinetic. Higher nitrogen levels were recovered in dialysates after in vitro digestions of sodium caseinate (SC, formed of small aggregates) compared to micellar casein (MC, native form of casein) and calcium caseinate (CC, intermediate structure). Likewise, plasma indispensable amino-acid concentration peak was higher after SC compared to MC or CC ingestion in healthy volunteers in a randomized, double blind, cross-over study. In pigs, gamma-scintigraphy using labelled meals revealed that SC was mainly localized in the proximal part of the stomach whereas MC was distributed in the whole gastric cavity. Caseins were found in both solid and liquid phases and partly hydrolyzed casein in the solid phase shortly after SC drink ingestion. These data support the concept of slow (MC) and rapid (SC) casein depending of casein structure, likely due to their intra-gastric clotting properties.

Ileal Digestibility of Nitrogen and Amino Acids in Human Milk and an Infant Formula as Determined in Neonatal Minipiglets.

BACKGROUND: Infant formula (IF) has to provide at least the same amount of amino acids (AAs) as human milk (HM). AA digestibility in HM and IF was not studied extensively, with no data available for tryptophan digestibility. OBJECTIVES: The present study aimed to measure the true ileal digestibility (TID) of total nitrogen and AAs in HM and IF to estimate AA bioavailability using Yucatan mini-piglets as an infant model. METHODS: Twenty-four 19-day-old piglets (males and females) received either HM or IF for 6 days or a protein-free diet for 3 days, with cobalt-EDTA as an indigestible marker. Diets were fed hourly over 6 h before euthanasia and digesta collection. Total N, AA, and marker contents in diets and digesta were measured to determine the TID. Unidimensional statistical analyses were conducted. RESULTS: Dietary N content was not different between HM and IF, while true protein was lower in HM (-4 g/L) due to a 7-fold higher non-protein N content in HM. The TID of total N was lower (P < 0.001) for HM (91.3 ± 1.24%) than for IF (98.0 ± 0.810%), while the TID of amino acid nitrogen (AAN) was not different (average of 97.4 ± 0.655%, P = 0.272). HM and IF had similar (P > 0.05) TID for most of the AAs including tryptophan (96.7 ± 0.950%, P = 0.079), except for some AAs (lysine, phenylalanine, threonine, valine, alanine, proline, and serine), with small significant difference (P < 0.05). The first limiting AA was the aromatic AAs, and the digestible indispensable AA score (DIAAS) was higher for HM (DIAASHM = 101) than for IF (DIAASIF = 83). CONCLUSION: HM, compared to IF, had a lower TID for total N only, whereas the TID of AAN and most AAs, including Trp, was high and similar. A larger proportion of non-protein N is transferred to the microbiota with HM, which is of physiological relevance, although this fraction is poorly considered for IF manufacturing.

Encapsulation of DHA oil with heat-denatured whey protein in Pickering emulsion improves its bioaccessibility.

This study compared the bioaccessibility of docosahexaenoic acid (DHA) provided encapsulated or unencapsulated within a food matrix. DHA oil was composed of DHA-enriched triacylglycerols prepared as Pickering emulsion by encapsulation with heat-denatured whey protein isolate particles and then incorporated into homogenized liquid egg to get omelets. The effect of encapsulation was analyzed by using a static in vitro digestion model of the adult, which digestive fluid enzymes have also been characterized by proteomics. First, the size of lipid droplets was shown to be smaller and uniformly dispersed in omelets with encapsulated-DHA oil compared to non-encapsulated-DHA oil. Distribution of droplets was more regular with encapsulated-DHA oil as well. As a consequence, we showed that encapsulating DHA oil promoted the hydrolysis by pancreatic lipase during the intestinal phase. A larger proportion of DHA enriched-triacylglycerols was hydrolyzed after two hours of digestion, leading to a greater release in free DHA. Thus, only 32% of DHA remained esterified in the triacylglycerols with encapsulated-DHA oil, compared to 43% with non-encapsulated-DHA oil. The DHA in free form ultimately represented 52% of the total DHA with encapsulated-DHA oil, compared to 40% with non-encapsulated-DHA oil. Finally, our results showed that as much DHA was released after one hour of intestinal digestion when the DHA oil was encapsulated as after two hours when the DHA oil was not encapsulated. Therefore, DHA bioaccessibility was significantly improved by encapsulation of DHA oil in omelets.

Supplementing human milk with a donkey or bovine milk derived fortifier: Consequences on proteolysis, lipolysis and particle structure under in vitro dynamic digestion.

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.

Spatial-temporal mapping of the intra-gastric pepsin concentration and proteolysis in pigs fed egg white gels.

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.

Positive Interactions between Lactic Acid Bacteria Promoted by Nitrogen-Based Nutritional Dependencies.

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.

Function-Driven Design of Lactic Acid Bacteria Co-cultures to Produce New Fermented Food Associating Milk and Lupin.

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.

Differential impact of Holder and High Temperature Short Time pasteurization on the dynamic in vitro digestion of human milk in a preterm newborn model.

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.

Gastrointestinal digestion enhances the endothelium-dependent vasodilation of a whey hydrolysate in rat aortic rings.

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 vasorelaxant 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 hydrolysates. 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.

Impact of homogenization of pasteurized human milk on gastric digestion in the preterm infant: A randomized controlled trial.

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.

Adaptation of Propionibacterium freudenreichii to long-term survival under gradual nutritional shortage.

BACKGROUND: 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. RESULTS: During this study, P. freudenreichii CIRM-BIA 138 (alias ITG P9), which has a generation time of five hours in Yeast Extract Lactate medium at 30 °C under microaerophilic conditions, was incubated for 11 days (9 days after entry into stationary phase) in a culture medium, without any adjunct during the incubation. 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. Although lactate (the preferred carbon source for P. freudenreichii) was exhausted three days after inoculation, the strain sustained a high population level of 9.3 log10 CFU/mL. Its physiological adaptation was investigated by RNA-seq analysis and revealed a complete disruption of metabolism at the entry into stationary phase as compared to exponential phase. 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.

Impact of pasteurization of human milk on preterm newborn in vitro digestion: Gastrointestinal disintegration, lipolysis and proteolysis.

Human milk feeding is an important recommendation for preterm newborns considering their vulnerability and digestive immaturity. Holder pasteurization (62.5°C, 30min) applied in milk banks modifies its biological quality and its microstructure. We investigated the impact of pasteurization of preterm human milk on its gastrointestinal kinetics of lipolysis, proteolysis and structural disintegration. An in vitro dynamic system was set up to simulate the gastrointestinal digestion of preterm newborns. A pool of preterm human milk was digested as raw or after Holder pasteurization. Pasteurization impacted the microstructure of undigested human milk, its gastrointestinal disintegration and tended to limit the intestinal lipolysis. Furthermore, the gastrointestinal bioaccessibility of some fatty acids was decreased by pasteurization, while the intestinal bioaccessibility of some amino acids was selectively modulated. The impact of pasteurization on the digestion of human milk may have nutritional relevance in vivo and potentially modulates preterm development and growth.

Phenol-soluble modulin α induces G2/M phase transition delay in eukaryotic HeLa cells.

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.

ß-Casein(94-123)-derived peptides differently modulate production of mucins in intestinal goblet cells.

We recently reported the identification of a peptide from yoghurts with promising potential for intestinal health: the sequence (94-123) of bovine ß-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 ß-CN(94-123) by using several strategies based on (i) known bioactive peptides encrypted in ß-CN(94-123), (ii) in silico prediction of peptides reactivity and (iii) digestion of ß-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 ß-CN(108-113) (an ACE-inhibitory peptide) and ß-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 ß-CN(94-123) by intestinal enzymes showed that the peptides ß-CN(94-108) and ß-CN(117-123) were present throughout 1·5 to 3 h of digestion, respectively. These two peptides raised MUC5AC expression while ß-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, ß-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.

A novel bioactive peptide from yoghurts modulates expression of the gel-forming MUC2 mucin as well as population of goblet cells and Paneth cells along the small intestine.

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 ß-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 ß-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 ß-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.