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

peer-reviewedSimulated 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.COST action FA1005 Infogest22 (http://www.cost-infogest.eu/) is acknowledged for providing funding for travel, meetings and conferences

INFOGEST static in vitro simulation of gastrointestinal food digestion

peer-reviewedSupplementary information is available at http://dx.doi.org/10.1038/s41596-018-0119-1 or https://www.nature.com/articles/s41596-018-0119-1#Sec45.Developing a mechanistic understanding of the impact of food structure and composition on human health has increasingly involved simulating digestion in the upper gastrointestinal tract. These simulations have used a wide range of different conditions that often have very little physiological relevance, and this impedes the meaningful comparison of results. The standardized protocol presented here is based on an international consensus developed by the COST INFOGEST network. The method is designed to be used with standard laboratory equipment and requires limited experience to encourage a wide range of researchers to adopt it. It is a static digestion method that uses constant ratios of meal to digestive fluids and a constant pH for each step of digestion. This makes the method simple to use but not suitable for simulating digestion kinetics. Using this method, food samples are subjected to sequential oral, gastric and intestinal digestion while parameters such as electrolytes, enzymes, bile, dilution, pH and time of digestion are based on available physiological data. This amended and improved digestion method (INFOGEST 2.0) avoids challenges associated with the original method, such as the inclusion of the oral phase and the use of gastric lipase. The method can be used to assess the endpoints resulting from digestion of foods by analyzing the digestion products (e.g., peptides/amino acids, fatty acids, simple sugars) and evaluating the release of micronutrients from the food matrix. The whole protocol can be completed in ~7 d, including ~5 d required for the determination of enzyme activities.COST action FA1005 INFOGEST (http://www.cost-infogest.eu/ ) is acknowledged for providing funding for travel, meetings and conferences (2011-2015). The French National Institute for Agricultural Research (INRA, www.inra.fr) is acknowledged for their continuous support of the INFOGEST network by organising and co-funding the International Conference on Food Digestion and workgroup meeting

Transport of Particles in Intestinal Mucus under Simulated Infant and Adult Physiological Conditions: Impact of Mucus Structure and Extracellular DNA

The final boundary between digested food and the cells that take up nutrients in the small intestine is a protective layer of mucus. In this work, the microstructural organization and permeability of the intestinal mucus have been determined under conditions simulating those of infant and adult human small intestines. As a model, we used the mucus from the proximal (jejunal) small intestines of piglets and adult pigs. Confocal microscopy of both unfixed and fixed mucosal tissue showed mucus lining the entire jejunal epithelium. The mucus contained DNA from shed epithelial cells at different stages of degradation, with higher amounts of DNA found in the adult pig. The pig mucus comprised a coherent network of mucin and DNA with higher viscosity than the more heterogeneous piglet mucus, which resulted in increased permeability of the latter to 500-nm and 1-µm latex beads. Multiple-particle tracking experiments revealed that diffusion of the probe particles was considerably enhanced after treating mucus with DNase. The fraction of diffusive 500-nm probe particles increased in the pig mucus from 0.6% to 64% and in the piglet mucus from ca. 30% to 77% after the treatment. This suggests that extracellular DNA can significantly contribute to the microrheology and barrier properties of the intestinal mucus layer. To our knowledge, this is the first time that the structure and permeability of the small intestinal mucus have been compared between different age groups and the contribution of extracellular DNA highlighted. The results help to define rules governing colloidal transport in the developing small intestine. These are required for engineering orally administered pharmaceutical preparations with improved delivery, as well as for fabricating novel foods with enhanced nutritional quality or for controlled calorie uptake

Effect of cooling rate on the structural and moisture barrier properties of high and low melting point fats

E-mail [email protected] audienceThe effect of three cooling rates (rapid, intermediate and slow CR) on the moisture barrier properties and on the physical state of acetylated and high melting point hydrophobic self-supported moisture barriers has been investigated. The selected CR were representative of industrial processing conditions and the selected barrier materials of common effective GRAS substances (acetomonopalmitin, white beeswax, two commercial blends of beeswax and acetylated glycerides and a blend of palmitic/stearic acids). Variations of CR affected crystallisation kinetics, SFC in an extend depending on the fat chemical composition and degree of undercooling, crystal size and ratio of polymorphs present in the materials. It did not have major influence on the contact angles with water measured at the surface of the materials and on the mass-volume area properties of the material. The resultant effect on the macroscopic moisture barrier properties of the materials were evaluated using water vapour permeability (WVP) measurements. The CR had no significant effect on the WVP, except for one blend of acetylated fat and beeswax for which a slow CR may have favoured the healing of imperfections. The variations of WVP between all materials and CRs were mainly attributed to variation in materials polarity using multivariable analysi

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

International audienceHolder 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 onmilk 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 -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 -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 -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

Holder pasteurization impacts the proteolysis, lipolysis and disintegration of human milk under in vitro dynamic term newborn digestion

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

The structure of infant formulas modulate the lipolysis, the proteolysis and the disintegration of the matrices during in vitro gastric digestion

Milk lipids supply 50-60 % of the calories necessary for newborn growth under the form of maternal milk or infant formulas. Infant formulas have been optimized in terms of chemical composition but not with regards to the structure of the emulsion. An important difference remains between the native emulsions of milk fat globules (4 µm) as opposed to the processed submicronic emulsions (0.5 µm) of infant formulas. This structural difference may modify the kinetics of digestion and disintegration of the emulsion in the digestive tract of the newborns. Indeed the native emulsions develop an interfacial surface of 1.8 m2/g of lipid stabilized by a natural trilayered membrane based of phospholipids and proteins, whereas infant formulas present a much higher neoformed interfacial surface (30 m2/g of lipid) stabilized by dairy proteins. This higher interfacial surface is supposed to increase infant formula susceptibility to lipolysis but also to proteolysis through the higher accessibility of proteases to adsorbed proteins. To check this hypothesis, 3 matrices were formulated: M1 containing raw bovine milk fat globules dispersed in an infant formula-type serum phase, M2 similar to M1 with an additional high pressure homogenization treatment and M3 similar to M2 with the addition of a pasteurization treatment. The matrices were subjected to the semi-dynamic gastric phase of an in vitro digestion mimicking the conditions reported in newborns. The disintegration and the kinetics of lipolysis and proteolysis were monitored during the 180 minutes of digestion. The initial structure affected both the kinetics of the hydrolysis and the disintegration of the matrices. The initial rate of lipolysis of M1 was ~ 55 times lower than for M2 or M3 which was partially explained by its lower specific surface. Total proteolysis of caseins was observed at 30 minutes on M3 whereas it only occurred after 120 minutes for M1. The initial structure impacted the gastric destabilization with the formation of larger aggregates for M1 (mode at 190 µm at 30 min) than on the two other matrices. Since the free fatty acids and amino acids liberated in the gastric phase can act as second messenger and modulate the gastric emptying, this effect of structure may have important physiologic consequences. The gastric in vitro semi-dynamic model allowed rapid screening of matrices and limitations of consumption of digestive enzymes. In the future, it will be employed on maternal milk to determine the additional effect of the endogenous lipase on the digestion