Comprehensive probiogenomics analysis of the commensal Escherichia coli CEC15 as a potential probiotic strain.

BACKGROUND: Probiotics have gained attention for their potential maintaining gut and immune homeostasis. They have been found to confer protection against pathogen colonization, possess immunomodulatory effects, enhance gut barrier functionality, and mitigate inflammation. However, a thorough understanding of the unique mechanisms of effects triggered by individual strains is necessary to optimize their therapeutic efficacy. Probiogenomics, involving high-throughput techniques, can help identify uncharacterized strains and aid in the rational selection of new probiotics. This study evaluates the potential of the Escherichia coli CEC15 strain as a probiotic through in silico, in vitro, and in vivo analyses, comparing it to the well-known probiotic reference E. coli Nissle 1917. Genomic analysis was conducted to identify traits with potential beneficial activity and to assess the safety of each strain (genomic islands, bacteriocin production, antibiotic resistance, production of proteins involved in host homeostasis, and proteins with adhesive properties). In vitro studies assessed survival in gastrointestinal simulated conditions and adhesion to cultured human intestinal cells. Safety was evaluated in BALB/c mice, monitoring the impact of E. coli consumption on clinical signs, intestinal architecture, intestinal permeability, and fecal microbiota. Additionally, the protective effects of both strains were assessed in a murine model of 5-FU-induced mucositis. RESULTS: CEC15 mitigates inflammation, reinforces intestinal barrier, and modulates intestinal microbiota. In silico analysis revealed fewer pathogenicity-related traits in CEC15, when compared to Nissle 1917, with fewer toxin-associated genes and no gene suggesting the production of colibactin (a genotoxic agent). Most predicted antibiotic-resistance genes were neither associated with actual resistance, nor with transposable elements. The genome of CEC15 strain encodes proteins related to stress tolerance and to adhesion, in line with its better survival during digestion and higher adhesion to intestinal cells, when compared to Nissle 1917. Moreover, CEC15 exhibited beneficial effects on mice and their intestinal microbiota, both in healthy animals and against 5FU-induced intestinal mucositis. CONCLUSIONS: These findings suggest that the CEC15 strain holds promise as a probiotic, as it could modulate the intestinal microbiota, providing immunomodulatory and anti-inflammatory effects, and reinforcing the intestinal barrier. These findings may have implications for the treatment of gastrointestinal disorders, particularly some forms of diarrhea.

A mixture of milk and vegetable lipids in infant formula changes gut digestion, mucosal immunity and microbiota composition in neonatal piglets.

PURPOSE: Although composition of infant formula has been significantly improved during the last decade, major differences with the composition and structure of breast milk still remain and might affect nutrient digestion and gut biology. We hypothesized that the incorporation of dairy fat in infant formulas could modify their physiological impacts by making their composition closer to that of human milk. The effect of milk fat and milk fat globule membrane (MFGM) fragments in infant formulas on gut digestion, mucosal immunity and microbiota composition was evaluated. METHODS: Three formulas containing either (1) vegetable lipids stabilized only by proteins (V-P), (2) vegetable lipids stabilized by a mixture of proteins and MFGM fragments (V-M) and (3) a mixture of milk and vegetable lipids stabilized by a mixture of proteins and MFGM fragments (M-M) were automatically distributed to 42 newborn piglets until slaughter at postnatal day (PND) 7 or 28, and compared to a fourth group of sow's suckling piglets (SM) used as a breast-fed reference. RESULTS: At both PND, casein and ß-lactoglobulin digestion was reduced in M-M proximal jejunum and ileum contents compared to V-P and V-M ones leading to more numerous ß-Cn peptides in M-M contents. The IFNγ cytokine secretion of ConA-stimulated MLN cells from M-M piglets tended to be higher than in V-P ones at PND 7 and PND 28 and was closer to that of SM piglets. No dietary treatment effect was observed on IL-10 MLN cell secretion. Changes in faecal microbiota in M-M piglets resulted in an increase in Proteobacteria and Bacteroidetes and a decrease in Firmicutes phyla compared to V-P ones. M-M piglets showed higher abundances of Parabacteroides, Escherichia/Shigella and Klebsiella genus. CONCLUSIONS: The incorporation of both milk fat and MFGM fragments in infant formula modifies protein digestion, the dynamic of the immune system maturation and the faecal microbiota composition.

Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion.

Holder pasteurization (62.5°C, 30 min) ensures sanitary quality of donor's human milk but also denatures beneficial proteins. Understanding whether this further impacts the kinetics of peptide release during gastrointestinal digestion of human milk was the aim of the present paper. Mature raw (RHM) or pasteurized (PHM) human milk were digested (RHM, n = 2; PHM, n = 3) by an in vitro dynamic system (term stage). Label-free quantitative peptidomics was performed on milk and digesta (ten time points). Ascending hierarchical clustering was conducted on "Pasteurization × Digestion time" interaction coefficients. Preproteolysis occurred in human milk (159 unique peptides; RHM: 91, PHM: 151), mostly on ß-casein (88% of the endogenous peptides). The predicted cleavage number increased with pasteurization, potentially through plasmin activation (plasmin cleavages: RHM, 53; PHM, 76). During digestion, eight clusters resumed 1054 peptides from RHM and PHM, originating for 49% of them from ß-casein. For seven clusters (57% of peptides), the kinetics of peptide release differed between RHM and PHM. The parent protein was significantly linked to the clustering (p-value = 1.4 E-09), with ß-casein and lactoferrin associated to clusters in an opposite manner. Pasteurization impacted selectively gastric and intestinal kinetics of peptide release in term newborns, which may have further nutritional consequences.

Milk Polar Lipids Affect In Vitro Digestive Lipolysis and Postprandial Lipid Metabolism in Mice.

BACKGROUND: Polar lipid (PL) emulsifiers such as milk PLs (MPLs) may affect digestion and subsequent lipid metabolism, but focused studies on postprandial lipemia are lacking. OBJECTIVE: We evaluated the impact of MPLs on postprandial lipemia in mice and on lipid digestion in vitro. METHODS: Female Swiss mice were gavaged with 150 µL of an oil-in-water emulsion stabilized with 5.7 mg of either MPLs or soybean PLs (SPLs) and killed after 1, 2, or 4 h. Plasma lipids were quantified and in the small intestine, gene expression was analyzed by reverse transcriptase-quantitative polymerase chain reaction. Emulsions were lipolyzed in vitro using a static human digestion model; triglyceride (TG) disappearance was followed by thin-layer chromatography. RESULTS: In mice, after 1 h, plasma TGs tended to be higher in the MPL group than in the SPL group (141 µg/mL vs. 90 µg/mL; P = 0.07) and nonesterified fatty acids (NEFAs) were significantly higher (64 µg/mL vs. 44 µg/mL; P < 0.05). The opposite was observed after 4 h with lower TGs (21 µg/mL vs. 35 µg/mL; P < 0.01) and NEFAs (20 µg/mL vs. 32 µg/mL; P < 0.01) in the MPL group compared with the SPL group. This was associated at 4 h with a lower gene expression of apolipoprotein B (Apob) and Secretion Associated, Ras related GTPase 1 gene homolog B (Sar1b), in the duodenum of MPL mice compared with SPL mice (P < 0.05). In vitro, during the intestinal phase, TGs were hydrolyzed more in the MPL emulsion than in the SPL emulsion (decremental AUCs were 1750%/min vs. 180%/min; P < 0.01). MPLs enhance lipid intestinal hydrolysis and promote more rapid intestinal lipid absorption and sharper kinetics of lipemia. CONCLUSIONS: Postprandial lipemia in mice can be modulated by emulsifying with MPLs compared with SPLs, partly through differences in chylomicron assembly, and TG hydrolysis rate as observed in vitro. MPLs may thereby contribute to the long-term regulation of lipid metabolism.

Specificity of infant digestive conditions: some clues for developing relevant in vitro models.

Digestion of nutrients is an essential function of the newborn infant gut to allow growth and development and understanding infant digestive function is essential to optimize nutrition and oral drug delivery. Ethical considerations prohibit invasive in vivo trials and as a consequence in vitro assays are often conducted. However, the choice of in vitro model parameters are not supported by an exhaustive analysis of the literature and do not mimic precisely the digestive conditions of the infant. This review contains a compilation of the studies which characterized the gastroduodenal conditions in full-term or preterm infants of variable postnatal age from birth up to six months. Important data about healthy full-term infants are reported. The enzymatic (type of enzymes and level of activity) and nonenzymatic (milk-based diet, frequency of feeding, bile salt concentrations) conditions of digestion in infants are shown to differ significantly from those in adults. In addition, the interindividual and developmental variability of the digestive conditions in infants is also highlighted.

Dynamic digestion of a high protein beverage based on amaranth: Structural changes and antihypertensive activity.

An amaranth beverage (AB) was subjected to a simulated process of dynamic gastrointestinal digestion DIDGI®, a simple two-compartment in vitro dynamic gastrointestinal digestion system. The structural changes caused to the proteins during digestion and the digesta inhibitory capacity of the angiotensin converting enzyme (ACE) were investigated. In gastric compartment the degree of hydrolysis (DH) was 14.7 ± 1.5 % and in the intestinal compartment, proteins were digests in a greater extent (DH = 60.6 ± 8.4 %). Protein aggregation was detected during the gastric phase. The final digesta obtained both at the gastric and intestinal level, showed ACE inhibitory capacity (IC50 80 ± 10 and 140 ± 20 µg/mL, respectively). Purified fractions from these digesta showed even greater inhibitory capacity, being eluted 2 (E2) the most active fraction (IC50 60 ± 10 µg/mL). Twenty-six peptide sequences were identified. Six of them, with potential antihypertensive capacity, belong to A. hypochondriacus, 3 agglutinins and 3 encrypted sequences in the 11S globulin. Results obtained provide new and useful information on peptides released from the digestion of an amaranth based beverage and its ACE bioactivity.

In vitro gastrointestinal digestion of cow's and sheep's dairy products: Impact of species and structure.

Sheep's milk (SM) is known to differ from cow's milk (CM) in nutritional composition and physicochemical properties, which may lead to different digestion behaviours. This work aimed to investigate the impact of the species (cow vs sheep) and the structure (milk vs yogurt) on the digestion of dairy products. Using an in vitro static gastrointestinal digestion model, CM, SM, cow's milk yogurt (CY) and sheep's milk yogurt (SY) were compared on particle size evolution, microscopic observations, degree of lipolysis, degree of proteolysis, specific protein degradation and calcium bioaccessibility. Species and structure affected particle size evolution during the gastric phase resulting in smaller particles for yogurts compared to milks as well as for CM products compared to SM products. Species impacted lipid composition and lipolysis, with SM products presenting higher short/medium-chain fatty acids content and higher intestinal degree of lipolysis. Proteolysis was influenced by structure, with milks showing higher intestinal degree of proteolysis compared to yogurts. Caseins were digested faster in CM, ⍺-lactalbumin was digested faster in SM despite its higher concentration, and during gastric digestion ß-lactoglobulin was more degraded in CM products compared to SM products and more in yogurts compared to milks. Lastly, SM products released more bioaccessible calcium than CM products. In conclusion, species (cow vs sheep) impacted more the digestion compared to the structure (milk vs yogurt). In fact, SM was different from CM mainly due to a denser protein network that might slow down the accessibility of the enzyme to its substrate which induce a delay of gastric disaggregation and thus lead to slower the digestion of the nutrients.

Gastric devolution of transglutaminase-induced acid and rennet-induced casein gels using dynamic DIDGI® and static COST action INFOGEST protocols.

Limited studies in the literature have compared in vitro dynamic and in vitro static protocols for modelling the gastric digestive process of food systems. This experiment explores the differences between two different in vitro approaches to the devolution of a transglutaminase-induced acid gel (TG, pH 5.1-5.3) and rennet-induced gel (RG, pH 6.5-6.7). Gels were exposed to a simulated oral phase, followed by either the dynamic DIDGI® or static COST action INFOGEST protocol to simulate gastric conditions. Protein hydrolysis was evident from 15 min onwards for TG exposed to the dynamic protocol where levels continued to increase at a steady rate. In contrast, RG exhibited a notable lag-phase before levels increased from around 60 min onwards. Under the static protocol, protein hydrolysis was observed for both TG and RG upon exposure to the gastric environment which continued to increase over time. Despite these differences, similar levels of protein hydrolysis were found for TG and RG at the gastric endpoint using either protocol demonstrating that both the dynamic DIDGI® and static COST action INFOGEST methods provide a suitable and comparable environment for the in vitro digestion of casein protein under simulated gastric conditions.

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.

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.

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.

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.

Higher microbial diversity in raw than in pasteurized milk Raclette-type cheese enhances peptide and metabolite diversity after in vitro digestion.

Numerous bacteria are responsible for hydrolysis of proteins during cheese ripening. The raw milk flora is a major source of bacterial variety, starter cultures are needed for successful acidification of the cheese and proteolytic strains like Lactobacillus helveticus, are added for flavor improvement or acceleration of ripening processes. To study the impact of higher bacterial diversity in cheese on protein hydrolysis during simulated human digestion, Raclette-type cheeses were produced from raw or heat treated milk, with or without proteolytic L. helveticus and ripened for 120 days. Kinetic processes were studied with a dynamic (DIDGI®) in vitro protocol and endpoints with the static INFOGEST in vitro digestion protocol, allowing a comparison of the two in vitro protocols at the level of gastric and intestinal endpoints. Both digestion protocols resulted in comparable peptide patterns after intestinal digestion and higher microbial diversity in cheeses led to a more diverse peptidome after simulated digestion.

Addition of Dairy Lipids and Probiotic Lactobacillus fermentum in Infant Formulas Modulates Proteolysis and Lipolysis With Moderate Consequences on Gut Physiology and Metabolism in Yucatan Piglets.

Breast milk is the gold standard in neonatal nutrition, but most infants are fed infant formulas in which lipids are usually of plant origin. The addition of dairy lipids and/or milk fat globule membrane extracts in formulas improves their composition with beneficial consequences on protein and lipid digestion. The probiotic Lactobacillus fermentum (Lf) was reported to reduce transit time in rat pups, which may also improve digestion. This study aimed to investigate the effects of the addition of dairy lipids in formulas, with or without Lf, on protein and lipid digestion and on gut physiology and metabolism. Piglets were suckled from postnatal days 2 to 28, with formulas containing either plant lipids (PL), a half-half mixture of plant and dairy lipids (DL), or this mixture supplemented with Lf (DL+Lf). At day 28, piglets were euthanized 90 min after their last feeding. Microstructure of digesta did not differ among formulas. Gastric proteolysis was increased (P < 0.01) in DL and DL+Lf (21.9 ± 2.1 and 22.6 ± 1.3%, respectively) compared with PL (17.3 ± 0.6%) and the residual proportion of gastric intact caseins decreased (p < 0.01) in DL+Lf (5.4 ± 2.5%) compared with PL and DL (10.6 ± 3.1% and 21.8 ± 6.8%, respectively). Peptide diversity in ileum and colon digesta was lower in PL compared to DL and DL+Lf. DL and DL+Lf displayed an increased (p < 0.01) proportion of diacylglycerol/cholesterol in jejunum and ileum digesta compared to PL and tended (p = 0.07) to have lower triglyceride/total lipid ratio in ileum DL+Lf (0.019 ± 0.003) as compared to PL (0.045 ± 0.011). The percentage of endocrine tissue and the number of islets in the pancreas were decreased (p < 0.05) in DL+Lf compared with DL. DL+Lf displayed a beneficial effect on host defenses [increased goblet cell density in jejunum (p < 0.05)] and a trophic effect [increased duodenal (p = 0.09) and jejunal (p < 0.05) weights]. Altogether, our results demonstrate that the addition of dairy lipids and probiotic Lf in infant formula modulated protein and lipid digestion, with consequences on lipid profile and with beneficial, although moderate, physiological effects.

Colloidal transport of lipid digesta in human and porcine small intestinal mucus.

Small intestinal mucus transport of food-derived particulates has not been extensively studied, despite mucus being a barrier nutrients need to cross before absorption. We used complex dispersions of digesta obtained from simulated, dynamic gastrointestinal digestion of yogurt to examine the penetrability of human and porcine mucus to the particles formed of lipolysis products. Quantitative, time-lapse confocal microscopy revealed a sieve-like behaviour of the pig jejunal and ileal mucus. The digesta diffusivity decreased significantly over the first 30 min of mucus penetration, and then remained constant at ca. 5 × 10-12 m2 s-1 (approx. 70% decrease from initial values). A non-significantly different penetrability was recorded for the ileal mucus of adult humans. The digesta diffusion rates in neonatal, jejunal mucus of 2 week old piglets were 5-8 times higher than in the three different types of adult mucus. This is the first report that validates the mucus of fully-grown pigs as a human-relevant substitute for mucus permeation studies of nutrients/bio-actives and/or complex colloidal dispersions (e.g., post-digestion food particulates, orally-administrated delivery systems).

Are Faba Bean and Pea Proteins Potential Whey Protein Substitutes in Infant Formulas? An In Vitro Dynamic Digestion Approach.

Infant formulas (IFs) are used as substitutes for human milk and are mostly based on cow milk proteins. For sustainability reasons, animal protein alternatives in food are increasingly being considered, as plant proteins offer interesting nutritional and functional benefits for the development of innovative IFs. This study aimed to assess how a partial substitution (50%) of dairy proteins with faba bean and pea proteins influenced the digestibility of IFs under simulated dynamic in vitro digestion, which were set up to mimic infant digestion. Pea- and faba bean-based IFs (PIF and FIF, respectively) have led to a faster aggregation than the reference milk-based IF (RIF) in the gastric compartment; that did not affect the digesta microstructure at the end of digestion. The extent of proteolysis was estimated via the hydrolysis degree, which was the highest for FIF (73%) and the lowest for RIF (50%). Finally, it was apparent that in vitro protein digestibility and protein digestibility-corrected amino acid score (PDCAAS)-like scores were similar for RIF and FIF (90% digestibility; 75% PDCAAS), but lower for PIF (75%; 67%). Therefore, this study confirms that faba bean proteins could be a good candidate for partial substitution of whey proteins in IFs from a nutritional point of view, provided that these in vitro results are confirmed in vivo.

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.

Human milk pasteurisation reduces pre-lipolysis but not digestive lipolysis and moderately decreases intestinal lipid uptake in a combination of preterm infant in vitro models.

Donor human milk, pasteurised for safety reasons, is the first alternative for feeding preterm infants when mothers' own milk is unavailable. Breastmilk pasteurisation impact on lipid digestion and absorption was evaluated by a static in vitro digestion model for preterm infants coupled with intestinal absorption using Caco-2/TC7 cells. Lipid absorption was quantified by digital image analysis of lipid droplets, by measurement of basolateral triglyceride concentration and by analysing the expression of major genes involved. After in vitro digestion, lipolysis extent was 13% lower in pasteurised human milk (PHM) than in raw human milk (RHM). In Caco-2/TC7 cells, the number of lipid droplets was identical for both milk types, while the mean droplet area was 17% smaller with PHM. Altogether, pasteurisation decreased the pre-lipolysis of human milk. This initial difference in free fatty acid amount was only partially buffered by the subsequent processes of in vitro digestion and cellular lipid absorption.

Physico-chemical behaviors of human and bovine milk membrane extracts and their influence on gastric lipase adsorption.

Milk fat globule membrane conditions the reactivity and enzymatic susceptibility of milk lipids. The use of bovine membrane extracts to make infant formulas more biomimetic of human milk has been suggested recently. A comparison of the physico-chemical behavior of human and bovine milk membrane extracts and their interaction with gastric lipase is here undertaken using biophysical tools. Milk membrane extracts (70% of polar lipids) were obtained either pooling of mature human milk (n = 5) or bovine buttermilk. Human extract contained more anionic glycerophospholipids, less phosphatidylethanolamine and more unsaturated fatty acids (57% versus 46%) than bovine extract. Human extract presented a higher compressibility, with slower increase of surface pressure, than bovine extract. Micronic liquid condensed (LC) domains were evidenced in both extracts at 10 mN/m, but the evolution differs upon compression. Upon gastric lipase addition, an adsorption preference for liquid expanded phase (LE) was observed for both extracts. However, insertion was more homogeneous in terms of height level in human extract and impacted less its lipid lateral organization than in bovine extract. Both membrane extracts share close physico-chemical properties, however human membrane higher compressibility may favour gastric lipase insertion and higher interfacial reactivity in gastric conditions.