INFOGEST static in vitro simulation of gastrointestinal food digestion.

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.

Satiety Impact of Different Potato Products Compared to Pasta Control.

OBJECTIVE: A variety of potato dishes are regularly consumed worldwide, but the satiety value of these foods is not well established. The primary objective of this study was to compare the satiating effects of 4 equi-energy meals containing different potato preparations with an equi-energy pasta control meal. METHODS: This study used a randomized crossover design to assess the impact of 4 equi-energy potato-based meals (fried French fries, baked potato, mashed potato, or potato wedges) on subjective satiety sensations (visual analogue scale [VAS] ratings) and subsequent energy intake (ad libitum meal [kcal]), compared to a control pasta-based meal. Thirty-three healthy nonobese men and women participated in the study. RESULTS: VAS ratings indicated that the meal containing fried french fries was perceived to be substantially more satiating than the equi-energy pasta control meal, with all other potato-based meals not differing overall from control. All test meals had a comparable effect on energy intake at a later ad libitum meal. CONCLUSIONS: Consumers reported higher levels of satiety following a meal where the principal carbohydrate source was fried french fries, compared to when they had consumed an energy-matched meal containing carbohydrate in the form of pasta. All other potato preparations had similar effects on satiety as pasta. It is concluded that participants perceived a meal with fried french fries as providing greater satiety than a pasta control meal.

Engineering Digestion: Multiscale Processes of Food Digestion.

Food digestion is a complex, multiscale process that has recently become of interest to the food industry due to the developing links between food and health or disease. Food digestion can be studied by using either in vitro or in vivo models, each having certain advantages or disadvantages. The recent interest in food digestion has resulted in a large number of studies in this area, yet few have provided an in-depth, quantitative description of digestion processes. To provide a framework to develop these quantitative comparisons, a summary is given here between digestion processes and parallel unit operations in the food and chemical industry. Characterization parameters and phenomena are suggested for each step of digestion. In addition to the quantitative characterization of digestion processes, the multiscale aspect of digestion must also be considered. In both food systems and the gastrointestinal tract, multiple length scales are involved in food breakdown, mixing, absorption. These different length scales influence digestion processes independently as well as through interrelated mechanisms. To facilitate optimized development of functional food products, a multiscale, engineering approach may be taken to describe food digestion processes. A framework for this approach is described in this review, as well as examples that demonstrate the importance of process characterization as well as the multiple, interrelated length scales in the digestion process.

Digestibility of gluten proteins is reduced by baking and enhanced by starch digestion.

SCOPE: Resistance of proteins to gastrointestinal digestion may play a role in determining immune-mediated adverse reactions to foods. However, digestion studies have largely been restricted to purified proteins and the impact of food processing and food matrices on protein digestibility is poorly understood. METHODS AND RESULTS: Digestibility of a total gliadin fraction (TGF), flour (cv Hereward), and bread was assessed using in vitro batch digestion with simulated oral, gastric, and duodenal phases. Protein digestion was monitored by SDS-PAGE and immunoblotting using monoclonal antibodies specific for celiac-toxic sequences (QQSF, QPFP) and starch digestion by measuring undigested starch. Whereas the TGF was rapidly digested during the gastric phase the gluten proteins in bread were virtually undigested and digested rapidly during the duodenal phase only if amylase was included. Duodenal starch digestion was also slower in the absence of duodenal proteases. CONCLUSION: The baking process reduces the digestibility of wheat gluten proteins, including those containing sequences active in celiac disease. Starch digestion affects the extent of protein digestion, probably because of gluten-starch complex formation during baking. Digestion studies using purified protein fractions alone are therefore not predictive of digestion in complex food matrices.

A mid-morning snack of almonds generates satiety and appropriate adjustment of subsequent food intake in healthy women.

PURPOSE: To assess the effect of consuming a mid-morning almond snack (28 and 42 g) tested against a negative control of no almonds on acute satiety responses. METHOD: On three test days, 32 healthy females consumed a standard breakfast followed by 0, 28 or 42 g of almonds as a mid-morning snack and then ad libitum meals at lunch and dinner. The effect of the almond snacks on satiety was assessed by measuring energy intake (kcal) at the two ad libitum meals and subjective appetite ratings (visual analogue scales) throughout the test days. RESULTS: Intake at lunch and dinner significantly decreased in a dose-dependent manner in response to the almond snacks. Overall, a similar amount of energy was consumed on all three test days indicating that participants compensated for the 173 and 259 kcals consumed as almonds on the 28 and 42 g test days, respectively. Subjective appetite ratings in the interval between the mid-morning snack and lunch were consistent with dose-dependent enhanced satiety following the almond snacks. However, in the interval between lunch and dinner, appetite ratings were not dependent on the mid-morning snack. CONCLUSION: Almonds might be a healthy snack option since their acute satiating effects are likely to result in no net increase in energy consumed over a day.

Modelling of nutrient bioaccessibility in almond seeds based on the fracture properties of their cell walls.

The cell walls (dietary fibre) of edible plants, which consist of mainly non-starch polysaccharides, play an important role in regulating nutrient bioaccessibility (release) during digestion in the upper gastrointestinal tract. Recent studies have shown that structurally-intact cell walls hinder lipid release from the parenchyma cells of almond seeds. A theoretical model was developed to predict the bioaccessibility of lipid using simple geometry and data on cell dimensions and particle size for calculating the number of ruptured cells in cut almond cubes. Cubes (2 mm) and finely-ground flour of low and high lipid bioaccessibility, respectively, were prepared from almond cotyledons. The model predictions were compared with data from in vitro gastric and duodenal digestion of almond cubes and flour. The model showed that lipid bioaccessibility is highly dependent on particle size and cell diameter. Only a modified version of the model (the Extended Theoretical Model, ETM), in which the cells at the edges and corners were counted once only, was acceptable for the full range of particle sizes. Lipid release values predicted from the ETM were 5.7% for almond cubes and 42% for almond flour. In vitro digestion of cubes and flour showed that lipid released from ruptured cells was available for hydrolysis and resulted in lipid losses of 9.9 and 39.3%, respectively. The ETM shows considerable potential for predicting lipid release in the upper gastrointestinal tract. Further work is warranted to evaluate the efficacy of this model to accurately predict nutrient bioaccessibility in a broad range of edible plants.

Differential digestion of human milk proteins in a simulated stomach model.

A key element in understanding how human milk proteins support the health and development of the neonate is to understand how individual proteins are affected during digestion. In the present study, a dynamic gastric model was used to simulate infant gastric digestion of human milk, and a subsequent proteomic approach was applied to study the behavior of individual proteins. A total of 413 human milk proteins were quantified in this study. This approach demonstrated a high degree of variability in the susceptibility of human milk proteins to gastric digestion. Specifically this study reports that lipoproteins are among the class of slowly digested proteins during gastric processes. The levels of integral lysozyme C and partial lactadherin in milk whey increase over digestion. Mucins, ribonuclease 4, and macrophage mannose receptor 1 are also resistant to gastric digestion. The retention or enhancement in whey protein abundance can be ascribed to the digestive release of milk-fat-globule-membrane or immune-cell enclosed proteins that are not initially accessible in milk. Immunoglobulins are more resistant to digestion compared to total milk proteins, and within the immunoglobulin class IgA and IgM are more resistant to digestion compared to IgG. The gastric digestion of milk proteins becomes more apparent from this study.

Survival of Lactobacillus rhamnosus strains inoculated in cheese matrix during simulated human digestion.

Survival of probiotic bacteria during transit through the gastrointestinal (GI) tract is influenced by a number of environmental variables including stomach acidity, bile salts, digestive enzymes and food matrix. This study assessed survival of seven selected Lactobacillus rhamnosus strains delivered within a model cheese system to the human upper GI tract using a dynamic gastric model (DGM). Good survival rates for all tested strains were recorded during both simulated gastric and duodenal digestion. Strains H12, H25 and N24 demonstrated higher survival capacities during gastric digestion than L. rhamnosus GG strain used as control, with H12 and N24 continuing to grow during duodenal digestion. Strains L. rhamnosus F17, N24 and R61 showed adhesion properties to both HT-29 and Caco-2 cells. The ability to attach to the cheese matrix during digestion was confirmed by scanning electron microscopy, also indicating production of extracellular polysaccharides as a response to acid stress.

Natural almond skin reduced oxidative stress and inflammation in an experimental model of inflammatory bowel disease.

The aim of the present study was to examine the effects of natural almond skin (NS) powder in mice subjected to experimental colitis. Colitis was induced in mice by intracolonic instillation of dinitrobenzene sulfonic acid (DNBS). NS powder was administered daily orally (30 mg/kg). Four days after DNBS administration, colon NF-κB and p-JNK activation was increased as well as TNF-α and IL-1ß productions. Neutrophil infiltration, by myeloperoxidase (MPO) activity, in the mucosa was associated with up-regulation of ICAM-1 and P-selectin. Immunohistochemistry for i-NOS, nitrotyrosine and poly (ADP-ribose) polymerase (PARP) showed an intense staining in the inflamed colon. Treatment with NS powder significantly reduced the appearance of diarrhea and body weight loss. This was associated with a significant reduction in colonic MPO activity. NS powder also reduced NF-κB and p-JNK activation, the pro-inflammatory cytokines release, the appearance of i-NOS, nitrotyrosine and PARP in the colon and reduced the up-regulation of ICAM-1 and the expression of P-selectin. The results of this study suggested that administration of NS powder may be beneficial for treatment of inflammatory bowel disease.

The effect of composition and gastric conditions on the self-emulsification process of ibuprofen-loaded self-emulsifying drug delivery systems: a microscopic and dynamic gastric model study.

PURPOSE: To investigate the physical processes involved in the emulsification of self-emulsifying drug delivery systems (SEDDSs) and the use of the Dynamic Gastric Model (DGM) as a characterisation tool. METHODS: SEDDSs based on soybean oil, Tween 80, Span 80 and ibuprofen were prepared and their equilibrium phase diagrams established. The emulsification behaviour in a range of media was studied using polarised light microscopy and particle sizing. The behaviour of the SEDDSs in the DGM and conventional testing equipment was assessed. RESULTS: A range of liquid crystalline mesophases was observed, enhanced in the presence of the drug. Polarised light microscopy showed different emulsification processes in the presence and absence of the drug, which was also manifest in different droplet sizes. The droplet size distribution varied between the DGM and the USP II dissolution apparatus. CONCLUSIONS: The model SEDDS displays complex liquid crystalline behaviour which may be intimately involved in the emulsification process, which in turn may alter particle size on emulsification, although there remains a question as to the in vivo significance of this effect. Furthermore, we demonstrate that the DGM represents a very promising new method of assessing the biological fate of SEDDSs.

Effect of carrot (Daucus carota) microstructure on carotene bioaccessibilty in the upper gastrointestinal tract. 1. In vitro simulations of carrot digestion.

Studies investigating carotene bioaccessibility (release from the food matrix to a solubilized form) directly from plant material during the process of digestion are scarce, mainly due to the difficulties associated with obtaining such material. Therefore, this paper examines the relationship between tissue microstructure and carotene bioaccessibility using an in vitro digestion model. Dietary oil provides a pool for the initial solubilization. Therefore, carotene partitioning into an emulsified oil phase was assessed using raw carrot tissue and carrot tissue subjected to various degrees of heating and particle size reduction and, in all cases, was found to be greatly reduced compared with juiced carrot. Carotene bioaccessibility was found to be greater from raw tissues than heated tissues of the same size. This is because heating increases the propensity for intact cells to separate, effectively encapsulating the carotene. Although the gross structure of the tissues was found to be relatively unaffected by in vitro digestion, at the cellular level some cell-wall swelling and cell death were observed, particularly close to the surfaces of the tissue. This study suggests that cell-wall rupture prior to digestion is an absolute requirement for carotene bioaccessibility in the upper intestine and that heating does not enhance carotene release from intact cells.

Immunomodulatory and antiviral activity of almond skins.

The elimination of a viral infection requires a proinflammatory host response (type 1 immunity), characterized by activation of mononuclear cells and production of proinflammatory cytokines, such as interferons (IFNs), tumor necrosis factor (TNF)-alpha and interleukin (IL)-12. On the other hand, IL-4 and IL-10 play a role in decreasing the inflammatory response supported by helper T (Th)1 cells. In this study we evaluated the effects of almond skins on the release of cytokines by peripheral blood mononuclear cells (PBMC), either infected or not with herpes simplex virus type 2 (HSV-2). Natural (NS) and blanched almond skins (BS) were subjected to simulated gastric and duodenal digestion and used at not cytotoxic concentrations. NS induced a significant decrease in HSV-2 replication, whereas extracts obtained from BS did not significantly influence the viral replication. High levels of cytokines production, such as IFN-alpha (38+/-5.3 pg/ml), IL-12 (215+/-17.1 pg/ml), IFN-gamma (5+/-0.7 IU/ml), TNF-alpha (3940+/-201.0 pg/ml), were detected. Moreover, IL-10 (210+/-12.2 pg/ml) and IL-4 (170+/-21.4 pg/ml), representative of Th2 responses, were found. Our data suggest that almond skins improve the immune surveillance of PBMC towards viral infection, both by triggering the Th1 and Th2 subsets.

Conditional cell-wall mutants of Saccharomyces cerevisiae as delivery vehicles for therapeutic agents in vivo to the GI tract.

Strains of Saccharomyces cerevisiae capable of lysis upon conditional down-regulation of cell-wall biogenesis genes (SRB1 and PKC1) have been reported. Here, we show that they lyse and release recombinant protein not only under laboratory conditions, but (more importantly) under conditions found in the human stomach and duodenum. These findings provide proof that, in principle, such conditional lysis strains could be used as an integral part of a system for the oral delivery of therapeutic proteins. However, the current mechanism of conditional lysis is based on the use of the MET3 promoter which requires addition of methionine and cysteine for down-regulation of SRB1 and PKC1. This requirement makes it difficult to apply in vivo. We reasoned that promoters, suitable for in vivo down-regulation of lysis-inducing genes, could be identified amongst yeast genes whose transcript abundance is reduced under conditions found in the human gut. A microarray experiment identified a number of candidate genes with significantly reduced transcript levels under simulated human gut conditions. The greatest effects were seen with ANB1, TIR1, and MF(ALPHA)2), and we propose that their promoters have the potential to be used in vivo to achieve yeast lysis in the gut.

Adsorption of bile salts and pancreatic colipase and lipase onto digalactosyldiacylglycerol and dipalmitoylphosphatidylcholine monolayers.

It is increasingly recognized that changes in the composition of the oil-water interface can markedly affect pancreatic lipase adsorption and function. To understand interfacial mechanisms determining lipase activity, we investigated the adsorption behavior of bile salts and pancreatic colipase and lipase onto digalactosyldiacylglycerol (DGDG) and dipalmitoylphosphatidylcholine (DPPC) monolayers at the air-water interface. The results from Langmuir trough and pendant drop experiments showed that a DGDG interface was more resistant to the adsorption of bile salts, colipase, and lipase compared to that of DPPC. Atomic force microscopy (AFM) images showed that the adsorption of bile salts into a DPPC monolayer decreased the size of the liquid condensed (LC) domains while there was no visible topographical change for DGDG systems. The results also showed that colipase and lipase adsorbed exclusively onto the mixed DPPC-bile salt regions and not the DPPC condensed phase. When the colipase and lipase were in excess, they fully covered the mixed DPPC-bile salt regions. However, the colipase and lipase coverage on the mixed DGDG-bile salt monolayer was incomplete and discontinuous. It was postulated that bile salts adsorbed into the DPPC monolayers filling the gaps between the lipid headgroups and spacing out the lipid molecules, making the lipid hydrocarbon tails more exposed to the surface. This created hydrophobic patches suitable for the binding of colipase and lipase. In contrast, bile salts adsorbed less easily into the DGDG monolayer because DGDG has a larger headgroup, which has strong intermolecular interactions and the ability to adopt different orientations at the interface. Thus, there are fewer hydrophobic patches that are of sufficient size to accommodate the colipase on the mixed DGDG-bile salt monolayer compared to the mixed DPPC-bile salt regions. The results from this work have reinforced the hypothesis that the interfacial molecular packing of lipids at the oil-water interface influences the adsorption of bile salts, colipase, and lipase, which in turn impacts the rate of lipolysis.

In vitro evaluation of the prebiotic properties of almond skins (Amygdalus communis L.).

In this study we investigated the potential prebiotic effect of natural (NS) and blanched (BS) almond skins, the latter being a byproduct of the almond-processing industry. A full model of the gastrointestinal tract, including in vitro gastric and duodenal digestion, followed by colonic fermentation using mixed faecal bacterial cultures, was used. Both NS and BS significantly increased the population of bifidobacteria and Clostridium coccoides/Eubacterium rectale group, resulting in a prebiotic index (3.2 for BS and 3.3 for NS) that compared well with the commercial prebiotic fructo-oligosaccharides (4.2) at a 24-h incubation. No significant differences in the proportion of gut bacteria groups and in short-chain fatty acid production were detected between NS and BS, showing that polyphenols present in almond skins did not affect bacterial fermentation. In conclusion, we have shown that dietary fibre from almond skins altered the composition of gut bacteria and almond skins resulting from industrial blanching could be used as potential prebiotics.

Comparative resistance of food proteins to adult and infant in vitro digestion models.

IgE-mediated allergy to milk and egg is widespread in industrialised countries and mainly affects infants and young children. It may be connected to an incomplete digestion of dietary proteins causing an inappropriate immune response in the gut. In order to study this, a biochemical model of infant gastroduodenal digestion has been developed, which has reduced levels of protease (eightfold for pepsin and tenfold for trypsin and chymotrypsin), phosphatidylcholine and bile salts, compared with the adult model. This model has been used to study the behaviour of three characterised food-relevant proteins (bovine beta-lactoglobulin (beta-Lg), beta-casein (beta-CN) and hen's egg ovalbumin), all of which are relevant cows' milk and hens' egg allergens. Digestion products were characterised using electrophoresis, immunochemical techniques and MS. These showed that ovalbumin and beta-CN were digested more slowly using the infant model compared with the adult conditions. Resistant fragments of beta-CN were found in the infant model, which correspond to previously identified IgE epitopes. Surprisingly, beta-Lg was more extensively degraded in the infant model compared with the adult one. This difference was attributed to the tenfold reduction in phosphatidylcholine concentration in the infant model limiting the protective effect of this phospholipid on beta-Lg digestion.

Physiological phosphatidylcholine protects bovine beta-lactoglobulin from simulated gastrointestinal proteolysis.

We have investigated the effect of phosphatidylcholine (PC) on the resistance of bovine beta-lactoglobulin (beta-Lg) to simulated in vitro gastrointestinal proteolysis. Whilst addition of PC did not affect the resistance of beta-Lg to gastric pepsinolysis, it protected the protein from subsequent degradation under duodenal conditions. The effect was dependent on the ratio of PC to beta-Lg, 16% of the protein remaining intact in the presence of an equimolar ratio of PC/protein, which increased to 62% when a 60-fold molar excess of PC was included. PC also altered the pattern of digestion products observed by SDS-PAGE. Thermal denaturation of beta-Lg abolished this effect showing that it was dependent on the native folded structure of the protein. Since neither of the beta-Lg ligands retinol or palmitate exerted a protective effect, it is unlikely that PC is mediating its effect by occupying the central calyx. An alternative explanation may be that the lipids bind to a secondary fatty acid binding site in beta-Lg, thus blocking the action of proteases for steric reasons. These data indicate how biomolecular interactions between proteins and lipids may alter patterns of proteolysis and need to be taken into consideration in any in vitro model of digestion.

Modulating pancreatic lipase activity with galactolipids: effects of emulsion interfacial composition.

It is widely known that the interfacial quality of lipid emulsion droplets influences the rate and extent of lipolysis. The aim of this work was to investigate the effect of two galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), adsorbed at the interface on in vitro digestibility of olive oil by porcine pancreatic lipase. The experiments were performed under simulated duodenal conditions in the presence of phosphatidylcholine (lecithin) and bile salts. It was found that emulsions prepared with DGDG had a longer lag phase prior to lipase activation with a decrease in lipolysis rate. In contrast, no inhibitory effect on lipase kinetics was observed in emulsions prepared with MGDG. We postulated that the larger headgroup and more tightly packed molecular organization of DGDG at the interface gave rise to steric hindrance that retarded colipase and lipase adsorption onto the substrate surfaces and hence delayed and reduced lipolysis. It was noted that the lag phase and lipolysis rate strongly depended on the DGDG/lecithin molar ratio in the systems: the higher the molar ratio, the longer the lag phase followed by a reduced lipolysis rate. The ability of DGDG to inhibit bile salt adsorption/displacement was also investigated. The results showed that bile salts did not completely displace DGDG from the interface, explaining the reason why DGDG still possessed inhibitory activity even in the presence of bile salts at a physiological relevant concentration. The results provide interesting insights into the influence of the galactolipid headgroup and lecithin on the emulsion interfacial quality which in turn regulates the lipolysis. The findings potentially could lead to the production of generic foods and drugs designed for regulating dietary fat absorption in the prevention and treatment of obesity and related disorders.