Olivia Ménard

A standardised static in vitro digestion method suitable for food-an international consensus

Simulated gastro-intestinal digestion is widely employed in many fields of food and nutritional sciences, as conducting human trials are often costly, resource intensive, and ethically disputable. As a consequence, in vitro alternatives that determine endpoints such as the bioaccessibility of nutrients and non-nutrients or the digestibility of macronutrients (e.g. lipids, proteins and carbohydrates) are used for screening and building new hypotheses. Various digestion models have been proposed, often impeding the possibility to compare results across research teams. For example, a large variety of enzymes from different sources such as of porcine, rabbit or human origin have been used, differing in their activity and characterization. Differences in pH, mineral type, ionic strength and digestion time, which alter enzyme activity and other phenomena, may also considerably alter results. Other parameters such as the presence of phospholipids, individual enzymes such as gastric lipase and digestive emulsifiers vs. their mixtures (e.g. pancreatin and bile salts), and the ratio of food bolus to digestive fluids, have also been discussed at length. In the present consensus paper, within the COST Infogest network, we propose a general standardised and practical static digestion method based on physiologically relevant conditions that can be applied for various endpoints, which may be amended to accommodate further specific requirements. A frameset of parameters including the oral, gastric and small intestinal digestion are outlined and their relevance discussed in relation to available in vivo data and enzymes. This consensus paper will give a detailed protocol and a line-by-line, guidance, recommendations and justifications but also limitation of the proposed model. This harmonised static, in vitro digestion method for food should aid the production of more comparable data in the future.

Phospholipid, Sphingolipid, and Fatty Acid Compositions of the Milk Fat Globule Membrane are Modified by Diet

The phospholipid and sphingolipid composition of milk is of considerable interest regarding their nutritional and functional properties. The objective of this article was to determine the lipid composition of the milk fat globule membrane (MFGM) of milk from cows fed a diet rich in polyunsaturated fatty acids. The experiments were performed with 2 groups of 6 cows feeding on (i) maize silage ad libitum (+ grassland hay, mixture of cereals, soyabean meal) or (ii) the maize silage-based diet supplemented with extruded linseed (bringing a lipid proportion of 5% of dry matter). The phospholipid and sphingolipid composition of the MFGM was determined using HPLC/ELSD. The fatty acid (FA) composition of total lipids and phospholipids was determined using GC. As expected, the linseed-supplemented diet decreased the saturated FA and increased the unsaturated FA content in milk fat. MFGM in milk from cows fed the diet rich in polyunsaturated FA resulted in (i) a higher amount of phospholipids (+ 18%), which was related to a smaller size of milk fat globules (ii) an increase of 30% (w/w) of the concentration in sphingomyelin, (iii) a higher content in stearic acid (1.7-fold), unsaturated FA (1.36-fold), and C18:1 trans FA: 7.2 +/- 0.5% (3.7-fold). The MFGM contained a higher concentration of unsaturated FA (C18:1, C18:2, and C18:3) and very long-chain FA (C22:0, C23:0, C24:0, EPA, DHA) compared with total lipids extracted from milk. The technological, sensorial, and nutritional consequences of these changes in the lipid composition of the MFGM induced by dietary manipulation remain to be elucidated.

Human milk fat globules: polar lipid composition and in situ structural investigations revealing the heterogeneous distribution of proteins and the lateral segregation of sphingomyelin in the biological membrane.

Although human milk fat globules (MFG) are of primary importance since they are the exclusive lipid delivery carriers in the gastrointestinal tract of breast-fed infants, they remain the poorly understood aspect of milk. The objectives of this study were to investigate these unique colloidal assemblies and their interfacial properties, i.e. composition and structure of their biological membrane. In mature breast milk, MFG have a mean diameter of 4-5 microm, a surface area of about 2m(2)/g fat and an apparent zeta potential ζ=-6.7 ± 0.5 mV at 37°C. Human MFG contain 3-4mg polar lipids/g fat as quantified by HPLC/ELSD. The main polar lipids are sphingomyelin (SM; 36-45%, w/w), phosphatidylcholine (19-23%, w/w) and phosphatidylethanolamine (10-15%, w/w). In situ structural investigations of human MFG have been performed using light and confocal microscopy with adapted fluorescent probes, i.e. Nile Red, the extrinsic phospholipid Rh-DOPE, Fast Green and the lectin WGA-488. This study revealed a spatial heterogeneity in the human milk fat globule membrane (MFGM), with the lateral segregation of SM in liquid-ordered phase domains of various shapes and sizes surrounded by a liquid-disordered phase composed of the glycerophospholipids in which the proteins are dispersed. The glycocalyx formed by glycoproteins and cytoplasmic remnents have also been characterised around human MFG. A new model for the structure of the human MFGM is proposed and discussed. The unique composition and lateral organisation of the human MFGM components could be of metabolic significance and have health impact for the infants that need to be further explored.

The heat treatment and the gelation are strong determinants of the kinetics of milk proteins digestion and of the peripheral availability of amino acids

This study aimed to determine the kinetics of milk protein digestion and amino acid absorption after ingestion of four dairy matrices by six minipigs: unheated or heated skim milk and corresponding rennet gels. Digestive contents and plasma samples were collected over a 7 h-period after meal ingestion. Gelation of milk slowed down the outflow of the meal from the stomach and the subsequent absorption of amino acids, and decreased their bioavailability in peripheral blood. The gelled rennet matrices also led to low levels of milk proteins at the duodenum. Caseins and β-lactoglobulin, respectively, were sensitive and resistant to hydrolysis in the stomach with the unheated matrices, but showed similar digestion with the heated matrices, with a heat-induced susceptibility to hydrolysis for β-lactoglobulin. These results suggest a significant influence of the meal microstructure (resulting from heat treatment) and macrostructure (resulting from gelation process) on the different steps of milk proteins digestion.

Validation of a new in vitro dynamic system to simulate infant digestion.

Understanding the mechanisms of infant formula disintegration in the infant gastrointestinal tract is a key step for developing new formulas with health benefits for the neonate. For ethical reasons, the access to in vivo data obtained on infants is limited. The use of animal models can be an alternative but these experiments are labour intensive, expensive and results obtained show high inter-individual variability, making their interpretation difficult. The aim of this work was to develop a simple in vitro dynamic gastrointestinal digestion system, for studying infant formula digestion, and to validate it by comparing the kinetics of proteolysis obtained in vitro with in vivo data collected from piglets. Results showed a good correlation between in vitro and in vivo data and confirmed the rapid hydrolysis of caseins in gastric conditions, whereas whey proteins appeared more resistant to digestion.

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.

The structure of infant formulas impacts their lipolysis, proteolysis and disintegration during in vitro gastric digestion

Milk lipids supply most of the calories necessary for newborn growth in maternal milk or infant formulas. The chemical composition of infant formulas has been optimized but not the structure of the emulsion. There is still a major difference between the native emulsions of milk fat globules and processed submicronic emulsions in infant formulas. This difference may modify the kinetics of digestion of emulsions in newborns and influence lipid metabolism. To check this, semi-dynamic gastric in vitro digestions were conducted on three matrices: a standardized milk emulsion containing native milk fat globules referred to as minimally-processed emulsion and two processed model infant formulas (homogenized or homogenized/pasteurized). Gastric conditions mimicked those reported in newborns. The minimally-processed emulsion was lipolyzed and proteolyzed slower than processed formulas. The difference in initial structure persisted during digestion. The surface of the droplets was the key parameter to control gastric lipolysis kinetics, the pattern of released fatty acids and proteolysis by faster hydrolysis of adsorbed proteins.

Acid and rennet gels exhibit strong differences in the kinetics of milk protein digestion and amino acid bioavailability

This study aimed at determining the kinetics of milk protein digestion and amino acid absorption after ingestion by six multi-canulated mini-pigs of two gelled dairy matrices having the same composition, similar rheological and structural properties, but differing by their mode of coagulation (acidification/renneting). Duodenal, mid-jejunal effluents and plasma samples were collected at different times during 7h after meal ingestion. Ingestion of the acid gel induced a peak of caseins and β-lactoglobulin in duodenal effluents after 20min of digestion and a peak of amino acids in the plasma after 60min. The rennet gel induced lower levels of both proteins in the duodenum (with no defined peak) as well as much lower levels of amino acids in the plasma than the acid gel. Plasma ghrelin concentrations suggested a potentially more satiating effect of the rennet gel compared to the acid gel. This study clearly evidences that the gelation process can significantly impact on the nutritive value of dairy products.

Impact of the Dairy Matrix Structure on Milk Protein Digestion Kinetics: Mechanistic Modelling Based on Mini-pig In Vivo Data

Beyond the individual content in nutrients, it is now established that the matrix structure is also to consider when evaluating the nutritional properties and possible health effects of a food material. The objective of this study was to gain knowledge on the effect of the structure of dairy products on the digestion of milk proteins as inferred from a mathematical modelling of mini-pig in vivo data. Six dairy matrices of the same composition but differing by their physicochemical and structural properties were investigated. They were manufactured using technological processes commonly used in the industry (heat treatment, rennet gelation, acid gelation and mixing). The experimental results cover a 7-h postprandial period and consist of plasmatic amino acid concentrations as well as dry matter contents and chromium concentrations (a marker of the liquid phase of the meal) of samples collected at the stomach exit. The model developed not only accounts for the main digestive events but also for phenomena that can occur within the stomach (milk clotting and aggregate syneresis). It provides a good fitting of all the experimental data and allows estimating parameter values that can be explained by considering the properties of the matrices investigated. The model has also been used to estimate quantities that cannot be observed experimentally (stomach volumes, endogenous secretions, gastric emptying half-time, etc.) in order to recover a better picture of all the results and validate the model predictions against the literature. It even appears that our simulations of gastric emptying and aminoacidemia superimpose very well with previously published data obtained using similar matrices and the same mini-pig species. This study shows that the great differences in the kinetics of amino acid absorption that were observed experimentally can be fully understood by considering the behaviour of the dairy matrices within the stomach. It therefore offers interesting perspectives for the integration of food structure parameters, and more particularly for dairy products, in the comprehensive view of the nutritional quality of food products.

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.