A standardised static in vitro digestion method suitable for food - an international consensus.
Minekus M,Alminger M,Alvito P,Ballance S,Bohn T,Bourlieu C,Carrière F,Boutrou R,Corredig M,Dupont D,Dufour C,Egger L,Golding M,Karakaya S,Kirkhus B,Le Feunteun S,Lesmes U,Macierzanka A,Mackie A,Marze S,McClements D J,Ménard O,Recio I,Santos C N,Singh R P,Vegarud G E,Wickham M S J,Weitschies W,Brodkorb A
Food & function
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.
Gastrointestinal digestion of hazelnut allergens on molecular level: Elucidation of degradation kinetics and resistant immunoactive peptides using mass spectrometry.
Korte Robin,Bräcker Julia,Brockmeyer Jens
Molecular nutrition & food research
SCOPE:Allergy to hazelnut seeds ranks among the most prevalent food allergies in Europe. The aim of this study was to elucidate the gastrointestinal digestion of hazelnut allergens on molecular level. METHODS AND RESULTS:Hazelnut flour was digested in vitro following the Infogest consensus model. For six allergenic proteins, the time-dependent course of digestion was monitored by SDS-PAGE and HPLC-MS/MS, and degradation products were characterized by a bottom-up proteomics approach. Depending on the molecular structure, a specific biochemical fate was observed for each allergen, and degradation kinetics were traced back to the peptide level. 1183 peptides were characterized, including 130 peptides that carry known IgE-binding epitopes and may represent sensitizers for hazelnut allergy. The kinetics of peptide formation and degradation were determined by label-free quantification and follow a complex multi-stage mechanism. CONCLUSION:We present a comprehensive survey on the gastrointestinal digestion of a relevant allergenic food on level of the peptidome, including the first systematic characterization and quantification of degradation products. This provides information on the differential resistance of plant food allergens and their structural elements undergoing digestion and forms the basis for a deeper understanding of the molecular principles responsible for sensitization to food allergy.
Correlation between in vitro and in vivo data on food digestion. What can we predict with static in vitro digestion models?
Bohn T,Carriere F,Day L,Deglaire A,Egger L,Freitas D,Golding M,Le Feunteun S,Macierzanka A,Menard O,Miralles B,Moscovici A,Portmann R,Recio I,Rémond D,Santé-Lhoutelier V,Wooster T J,Lesmes U,Mackie A R,Dupont D
Critical reviews in food science and nutrition
During the last decade, there has been a growing interest in understanding food's digestive fate in order to strengthen the possible effects of food on human health. Ideally, food digestion should be studied in vivo on humans but this is not always ethically and financially possible. Therefore, simple in vitro digestion models mimicking the gastrointestinal tract have been proposed as alternatives to in vivo experiments. Thus, it is no surprise that these models are increasingly used by the scientific community, although their various limitations to fully mirror the complexity of the digestive tract. Therefore, the objective of this article was to call upon the collective experiences of scientists involved in Infogest (an international network on food digestion) to review and reflect on the applications of in vitro digestion models, the parameters assessed in such studies and the physiological relevance of the data generated when compared to in vivo data. The authors provide a comprehensive review in vitro and in vivo digestion studies investigating the digestion of macronutrients (i.e., proteins, lipids, and carbohydrates) as well as studies of the bioaccessibility and bioavailability of micronutrients and phytochemicals. The main conclusion is that evidences show that despite the simplicity of in vitro models they are often very useful in predicting outcomes of the digestion in vivo. However, this has relies on the complexity of in vitro models and their tuning toward answering specific questions related to human digestion physiology, which leaves a vast room for future studies and improvements.
In vitro Gastrointestinal Models for Prebiotic Carbohydrates: A Critical Review.
Current pharmaceutical design
BACKGROUND:In the last decade, various consortia and companies have created standardized digestion protocols and gastrointestinal simulators, such as the protocol proposed by the INFOGEST Consortium, the simulator SHIME, the simulator simgi®, the TIM, etc. Most of them claim to simulate the entire human gastrointestinal tract. However, few results have been reported on the use of these systems with potential prebiotic carbohydrates. METHODS:This critical review addresses the existing data on the analysis of prebiotic carbohydrates by different in vitro gastrointestinal simulators, the lack of parameters that could affect the results, and recommendations for their enhancement. RESULTS:According to the reviewed data, there is a lack of a realistic approximation of the small intestinal conditions, mainly because of the absence of hydrolytic conditions, such as the presence of small intestinal brush border carbohydrases that can affect the digestibility of different carbohydrates, including prebiotics. CONCLUSION:There is a necessity to standardize and enhance the small intestine simulators to study the in vitro digestibility of carbohydrates.
Mass spectrometry data of and pig digestion of skim milk powder.
Egger Lotti,Schlegel Patrick,Baumann Christian,Stoffers Helena,Guggisberg Dominik,Brügger Cédric,Dürr Desirée,Stoll Peter,Vergères Guy,Portmann Reto
Data in brief
The data in this article are related to the research article entitled "Physiological comparability of the harmonized INFOGEST digestion method to pig digestion" (Egger et al., 2012). In this article, proteins identified in the different sections of pig skim milk powder (SMP) digestion are presented. In addition to the exemplary β-casein profiles of the paper, the peptide patterns of the other most abundant milk proteins during digestion in individual pigs are shown as heatmaps and line graphs. These data clearly reveal the digestion resistant protein regions and illustrate the variability between the pigs in the different sampling sections. Moreover, peptide patterns of the same SMP proteins comparing the harmonized digestion (IVD) with pig digestion show the physiological relevance of the IVD protocol. Finally, correlation coefficients were calculated to indicate similarities between pig sampling sections and gastric and intestinal IVD endpoints.
Bioaccessibility and cellular uptake by Caco-2 cells of carotenoids and chlorophylls from orange peels: A comparison between conventional and ionic liquid mediated extractions.
Murador Daniella C,De Souza Mesquita Leonardo M,Neves Bruna V,Braga Anna R C,Martins Paula L G,Zepka Leila Q,De Rosso Veridiana V
Native extracts from orange peels were obtained by a conventional method using acetone and, an alternative method using ionic liquid (1-butyl-3-methylimidazolium chloride ([Cmim]Cl)). The bioaccessibilities and cellular uptakes of carotenoids, esters and chlorophylls were evaluated, since the influence of esterification on bioaccessibility and bioavailability is not well established. For this, the extracts were emulsified, submitted to in vitro simulated digestion model according to the INFOGEST protocol, followed by uptake by Caco-2 cells. Compounds were separated, identified and quantified by HPLC-PDA-MS/MS. After digestion, 22.0% and 26.2% of the total carotenoids and 45.9% and 68.7% of the chlorophylls were bioaccessible from the acetone and [Cmim]Cl extracts, respectively. The bioaccessibilities of xanthophylls and carotenes were significantly higher than those of the mono- and diesters. The uptake by Caco-2 cells varied from 130.2 to 131.9 ng/mg cell protein for total carotenoids and from 243.8 to 234.2 ng/mg cell protein for chlorophylls in the acetone and [Cmim]Cl extracts, respectively. In general, xanthophylls and esters were better absorbed than carotenes.
Digestion of micellar casein in duodenum cannulated pigs. Correlation between in vitro simulated gastric digestion and in vivo data.
Miralles B,Sanchón J,Sánchez-Rivera L,Martínez-Maqueda D,Le Gouar Y,Dupont D,Amigo L,Recio I
Correlation and validation of the results of simulated gastrointestinal digestion of food compounds towards in vivo data is essential. The objective of this work was to monitor the digestion of milk micellar casein in the porcine upper intestinal tract and to match the outcome with the gastric in vitro digestion following the Infogest harmonized protocol. In pig duodenum, small amounts of intact caseins were present in all samples, while caseins were observed up to 60 min of gastric in vitro digestion. The peptide profile generated after in vitro and in vivo digestion showed clear similarities with specific overrepresented regions rich in proline and other hydrophobic residues. The statistical comparison of the in vivo and in vitro peptidome resulted in satisfactory correlation coefficients, up to 0.8. Therefore, the in vitro protocol used was a robust and simple model that provides a similar peptide profile than that found in porcine duodenum.
Degradation of Proteins From Colostrum and Mature Milk From Chinese Mothers Using an Infant Digestion Model.
Elwakiel Mohèb,Boeren Sjef,Wang Wendan,Schols Henk A,Hettinga Kasper A
Frontiers in nutrition
This study provided insights into the degradation of human milk proteins in an infant digestion model by comparing colostrum (week 1) and mature milk (week 4) of 7 Chinese mothers individually. In this study, we adapted the exiting INFOGEST model, to conditions representative to infants (0 to 3 month-old). The level of undigested proteins was analyzed by LC-MS/MS after gel-electrophoretic separation and in-gel digestion. The BCA protein assay showed that the total undigested milk protein content decreased from the start to the end of digestion with variations between mothers, especially in the gastric phase (25-80%). Undigested proteins could also still be found after the intestinal phase, ranging from 0.5 to 4.2% of initial protein content. Based on LC-MS/MS analysis, milk protein digestion varied between the mothers individually, especially during the gastric phase. No differences could be observed between protein digestion from colostrum and mature milk after the intestinal phase. The highest levels of proteins remaining after intestinal digestion can be linked to the group immune-active proteins, for all mothers. The level of protease inhibitors and total protein content in the milk did not correlate with the overall proteolysis during digestion. The results also showed that milk serum proteins partly remained after the gastric phase, with 33% remaining from colostrum and 37% remaining from mature milk. More than 40 milk serum proteins were detected after the intestinal phase. Some of the highly abundant milk serum proteins (lactoferrin, serum albumin, bile salt-activated lipase, immunoglobulins, α-antichymotrypsin) were still partially present intact after the intestinal phase, for all mothers. Caseins were also not completely digested in the gastric phase, with 35% remaining from colostrum and 13% remaining from mature milk. Caseins, on the other hand, were almost completely digested after the intestinal phase. The complete degradation of caseins into peptides might be related to their structural features. Overall, this study showed that digestion differed for the various human milk proteins by adapting an digestion model to infant physiological conditions, with the main differences between digestion of the milk from individual mothers being observed after gastric digestion.
Heat treatment of β-lactoglobulin affects its digestion and translocation in the upper digestive tract.
Deng Ying,Govers Coen,Tomassen Monic,Hettinga Kasper,Wichers Harry J
Heat treatment is a commonly applied unit operation in the processing of β-lactoglobulin containing products. This does, however, influence its structure and thereby impacts its activity and digestibility. We describe how various heat-treatments of β-lactoglobulin change the digestibility using a modified version of the current consensus INFOGEST protocol. Additionally, protein was investigated for its translocation over the intestinal epithelial barrier, which would bring them in contact with immune cells. The extent of gastric digestibility was higher when the protein structure was more modified, while the influence of glycation with lactose was limited. Translocation studies of protein across Caco-2 cell monolayers showed a lower translocation rate of protein heated in solution compared to the others. Our study indicates that structural modifications after different heat-treatments of β-lactoglobulin increase in particular gastric digestibility and the translocation efficiency across intestinal epithelial cells.
Proteomic modelling of gluten digestion from a physiologically relevant food system: A focus on the digestion of immunogenic peptides from wheat implicated in celiac disease.
Ogilvie Olivia,Roberts Sarah,Sutton Kevin,Domigan Laura,Larsen Nigel,Gerrard Juliet,Demarais Nicholas
Celiac disease is an autoimmune illness activated by gluten peptides produced during gastrointestinal digestion. A simulated in vitro digestion of gluten was conducted to define the profile and kinetic release pattern of immunogenic gluten peptides in a physiologically relevant food matrix. White bread was digested using the INFOGEST in vitro standardised digestion protocol from 0 to 240 min and subsequently analysed by SDS-PAGE, quantitative LC-MS/MS, untargeted LC-MS/MS and ELISA. The release profile of six gluten peptides was defined by quantitative LC-MS/MS; none were detected in the gastric phase, but rapidly peaked in the intestinal phase. These results were corroborated by the ELISA analysis. Untargeted proteomics identified 83 immunogenic peptides. Their qualitative concentrations were defined throughout digestion, demonstrating complex relationships through proteolysis. This analysis suggests immunogenic gluten may peak within the intestinal duodenum and gives new insights into the complexity of gluten digestion from a physiologically relevant food matrix.
Role of the bolus degree of structure on the protein digestibility during in vitro digestion of a pea protein-fortified sponge cake chewed by elderly.
Assad-Bustillos Melissa,Palier Juliette,Rabesona Hanitra,Choiset Yvan,Della Valle Guy,Feron Gilles
Journal of texture studies
This study investigated the digestibility of proteins in a pea protein-fortified sponge cake, as well as the impact of the degree of structure of the bolus produced by elderly subjects on the digestibility of proteins by combining ex vivo and in vitro approaches via the standardized protocol INFOGEST. The sponge cakes were consumed by a group of 20 elderly subjects with contrasting physiology, their boli were recovered just before swallowing, and their apparent viscosity was measured to delineate the bolus degree of structure. According to this criterion, two pools were formed with boli from subjects selected at the extremes: low viscosity and high viscosity, with apparent viscosity values (at 120 s ) of 124 ± 18 and 208 ± 19 Pa s, respectively. The sponge cakes and the two pools underwent in vitro digestion. Protein hydrolysis kinetics was followed by measuring the released primary amino groups (NH ) and by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis at different time points. For all samples, the representative bands of pea proteins disappear gradually during digestion, accompanied by the appearance of bands indicating the presence of proteins with M < 15 kDa. In addition, the NH concentrations increase over time and do not differ between sponge cake and pea protein isolate. Moreover, the degree of structure of the food bolus has no significant effect on the concentration of NH released. These results showed that pea proteins in a fortified sponge cake are bioaccessible under standardized conditions and that the degree of structure of the bolus did not influence protein digestibility for these foods.
Application of an in vitro digestion model to study the metabolic profile changes of an herbal extract combination by UHPLC-HRMS.
Thumann Timo A,Pferschy-Wenzig Eva-Maria,Aziz-Kalbhenn Heba,Ammar Ramy M,Rabini Sabine,Moissl-Eichinger Christine,Bauer Rudolf
Phytomedicine : international journal of phytotherapy and phytopharmacology
BACKGROUND:STW 5 is a fixed herbal combination containing extracts from nine medicinal plants: bitter candytuft, greater celandine, garden angelica roots, lemon balm leaves, peppermint leaves, caraway fruits, licorice roots, chamomile flowers, and milk thistle fruit. STW 5 is a clinically proven treatment for functional dyspepsia and irritable bowel syndrome. PURPOSE:Using a static in vitro method, we simulated oral, gastric, and small intestinal digestion and analyzed the metabolic profile changes by UHPLC-HRMS to determine the impact of oro-gastro-intestinal digestion on STW 5 constituents. STUDY DESIGN AND METHODS:STW 5 was incubated according to the InfoGest consensus method. Samples of each digestive phase were analyzed by UHPLC-HRMS in ESI positive and negative modes. After data processing, background subtraction, and normalization, the peak areas of detectable compounds were compared to untreated reference samples and recovery ratios were calculated to monitor the metabolic profile of STW 5 during simulated digestion. RESULTS:Although the levels of some constituents were reduced, we did not observe complete degradation of any of the constituents of STW 5 upon in vitro digestion. We did not detect any new metabolites beyond increased levels of caffeic acid and liquiritigenin due to degradation of progenitor compounds. Changes observed in intestinal bioaccessibility ratios were mainly a result of isomerization, hydrolysis, protein binding, and low water solubility. CONCLUSION:The majority of STW 5 constituents are stable towards simulated in vitro digestion and can reach the colon to interact with gut microbiota if they remain unabsorbed in the upper intestinal tract.
Hydrolysis of plant proteins at the molecular and supra-molecular scales during in vitro digestion.
Reynaud Yohan,Lopez Michel,Riaublanc Alain,Souchon Isabelle,Dupont Didier
Food research international (Ottawa, Ont.)
The digestion of plant protein is highly dependent on multiple factors, with two of the most important being the protein source and the food matrix. The present study investigated the effects of these two factors on the digestion of seitan (a wheat-based food), tofu, soya juice, and a homemade emulsion of soy oil and water that was stabilised with pea protein. The four plant matrices and their respective protein isolates/concentrates (wheat gluten, soya protein, pea protein) were subjected to in vitro static digestion following the INFOGEST consensus protocol. We monitored the release of α-amino groups during digestion. We found that food matrix had a strong influence on protein digestion: soya juice was more hydrolysed than fresh tofu (51.1% versus 33.1%; P = 0.0087), but fresh tofu was more hydrolysed than soya protein isolate (33.1% versus 17.9%; P < 0.0001). Likewise, the pea-protein emulsion was better hydrolysed than the pea-protein isolate (P = 0.0033). Differences were also detected between the two solid foods investigated here: a higher degree of hydrolysis was found for tofu compared to seitan (33.1% versus 11.8%), which was perhaps a function of the presence of numerous dense protein aggregates in the latter but not the former. Furthermore, freeze-drying more than doubled the final degree of hydrolysis of seitan (P < 0.0001), but had no effect on tofu (P = 1.0000). Confocal microscopy revealed that protein networks in freeze-dried seitan were strongly altered with respect to the fresh product; instead, protein networks in freeze-dried and fresh tofu were largely similar. Finally, we found that the protease:protein ratio had a strong effect on the kinetics of proteolysis: a 3.7-fold increase in the concentration of the soya protein isolate with respect to that of the soya juice decreased the final degree of hydrolysis from 50.3 to 17.9% (P = 0.0988).
In vitro digestibility of goat milk and kefir with a new standardised static digestion method (INFOGEST cost action) and bioactivities of the resultant peptides.
Nehir El Sedef,Karakaya Sibel,Simsek Sebnem,Dupont Didier,Menfaatli Esra,Eker Alper Tolga
Food & function
The hydrolysis degrees of goat milk and kefir during simulated gastrointestinal digestion and some bioactivities of the resulting peptides after fermentation and digestion were studied. A static in vitro digestion method by the COST FA1005 Action INFOGEST was used and goat milk and kefir were partially hydrolyzed during the gastric phase and had above 80% hydrolysis after duodenal digestion. There were no differences between the digestibility of goat milk and kefir (p > 0.05). Goat milk and kefir displayed about 7-fold antioxidant activity after digestion (p < 0.05). Fermentation showed no effect on the calcium-binding capacity of the samples (p > 0.05), however, after in vitro digestion calcium-binding capacity of the goat milk and kefir increased 2 and 5 fold, respectively (p < 0.05). Digested goat milk and kefir showed a higher dose-dependent inhibitory effect on α-amylase compared to undigested samples (p < 0.05). α-Glucosidase inhibitory activities and in vitro bile acid-binding capacities of the samples were not determined at the studied concentrations.
Physiologically Based Modeling of Food Digestion and Intestinal Microbiota: State of the Art and Future Challenges. An INFOGEST Review.
Feunteun Steven Le,Al-Razaz Ahmed,Dekker Matthijs,George Erwin,Laroche Beatrice,van Aken George
Annual review of food science and technology
This review focuses on modeling methodologies of the gastrointestinal tract during digestion that have adopted a systems-view approach and, more particularly, on physiologically based compartmental models of food digestion and host-diet-microbiota interactions. This type of modeling appears very promising for integrating the complex stream of mechanisms that must be considered and retrieving a full picture of the digestion process from mouth to colon. We may expect these approaches to become more and more accurate in the future and to serve as a useful means of understanding the physicochemical processes occurring in the gastrointestinal tract, interpreting postprandial in vivo data, making relevant predictions, and designing healthier foods. This review intends to provide a scientific and historical background of this field of research, before discussing the future challenges and potential benefits of the establishment of such a model to study and predict food digestion and absorption in humans. Expected final online publication date for the , Volume 12 is March 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
The relevance of a digestibility evaluation in the allergenicity risk assessment of novel proteins. Opinion of a joint initiative of COST action ImpARAS and COST action INFOGEST.
Verhoeckx Kitty,Bøgh Katrine Lindholm,Dupont Didier,Egger Lotti,Gadermaier Gabriele,Larré Colette,Mackie Alan,Menard Olivia,Adel-Patient Karine,Picariello Gianluca,Portmann Reto,Smit Joost,Turner Paul,Untersmayr Eva,Epstein Michelle M
Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association
The current allergenicity assessment of novel proteins is based on the EFSA GMO guidance. Recently, EFSA launched a new guidance document on allergenicity assessment of GM plants (2017). This document describes, amongst other topics, the new scientific and regulatory developments on in vitro protein digestibility tests. The EFSA GMO Panel stated that for in vitro protein digestibility tests, additional investigations are needed before any additional recommendation in the form of guidance can be provided. To this end, an interim phase is considered necessary to evaluate the revisions to the in vitro gastrointestinal digestion test, proposed by EFSA. This prompted the establishment of a joint workshop through two COST Action networks: COST Action ImpARAS and COST Acton INFOGEST. In 2017, a workshop was organised to discuss the relevance of digestion in allergenicity risk assessment and how to potentially improve the current methods and readouts. The outcome of the workshop is that there is no rationale for a clear readout that is predictive for allergenicity and we suggest to omit the digestion test from the allergenicity assessment strategy for now, and put an effort into filling the knowledge gaps as summarized in this paper first.
Rice in vitro digestion: application of INFOGEST harmonized protocol for glycemic index determination and starch morphological study.
Fernandes Jean-M,Madalena Daniel A,Pinheiro Ana C,Vicente António A
Journal of food science and technology
Starch is the main sugar source present in staple foods. Understanding starch hydrolysis during digestion and the resulting glucose release can be important to strategically modulate starch digestion and glucose absorption. In vitro digestion methodologies are fundamental to evaluate starch hydrolysis length and rate, but the lack of uniformity between protocols prevent the comparison of results. In this context, three different Carolino rice varieties (i.e., Carolino white-Cw, Carolino brown-Cb and Carolino Ariete brown-CAb) were submitted to the INFOGEST harmonized in vitro digestion protocol for the evaluation of starch hydrolysis and subsequent glycemic index (GI) determination, and starch granules morphological study. Samples of Carolino rice presented total starch percentages between 64.52 (for Cb) to 71.52% (for Cw) with low amylose content (16.19-19.95%, varying in the following order Cb < Cab ≈ Cw). During digestion, between 39.43 (for CAb) to 44.48% (for Cb) of starch was hydrolyzed, classifying samples as medium GI foods (61.73-69.17). Starch hydrolysis was accompanied by a decrease of starch granules dimensions. For all samples, area decrease was higher than 59%, perimeter decrease was higher than 37%, feret diameter decrease was higher than 39% and minimum feret diameter decrease was higher than 32%. This work provides new insights to describe, both qualitatively and quantitatively, the fate of rice during digestion, and allowed establishing a comparative basis for the development of rice-based recipes with a lower GI.
Compared digestibility of plant protein isolates by using the INFOGEST digestion protocol.
Santos-Hernández Marta,Alfieri Fabio,Gallo Veronica,Miralles Beatriz,Masi Paolo,Romano Annalisa,Ferranti Pasquale,Recio Isidra
Food research international (Ottawa, Ont.)
The use of ingredients based on plant protein isolates is being promoted due to sustainability and health reasons. However, it is necessary to explore the behaviour of plant protein isolates during gastrointestinal digestion including the profile of released free amino acids and the characterization of resistant domains to gastrointestinal digestion. The aim of the present study was to monitor protein degradation of four legume protein isolates: garden pea, grass pea, soybean and lentil, using the harmonized Infogest in vitro digestion protocol. In vitro digests were characterized regarding protein, peptide and free amino acid content. Soybean was the protein isolate with the highest percentage of insoluble nitrogen at the end of the digestion (12%), being this fraction rich in hydrophobic amino acids. Free amino acids were mainly released during the intestinal digestion, comprising 21-24% of the total nitrogen content, while the percentage of nitrogen corresponding to peptides ranged from 66 to 76%. Legume globulins were resistant to gastric digestion whereas they were hydrolysed into peptides and amino acids during the intestinal phase. However, the molecular weight (MW) distribution demonstrated that all intestinal digests, except those from soybean, contained peptides with MW > 4 kDa at the end of gastrointestinal digestion. The profile of free amino acids released during digestion supports legume protein isolates as an excellent source of essential amino acids to be used in protein-rich food products. Peptides released during digestion matched with previously reported epitopes from the same plant species or others, explaining the ability to induce allergic reactions and cross-linked reactivity.
Factors impacting lipid digestion and nutraceutical bioaccessibility assessed by standardized gastrointestinal model (INFOGEST): oil.
Tan Yunbing,Zhang Zhiyun,Liu Jinning,Xiao Hang,McClements David Julian
Food & function
The oil droplets in commercial emulsified foods have dimensions that vary widely, from hundreds of nanometers to tens of micrometers. Previously, the size of the droplets in oil-in-water emulsions has been shown to impact their gastrointestinal behavior, which may influence their physiological effects. In this study, we analyzed the impact of oil droplet diameter (0.16, 1.1 and 8.2 μm) on lipid digestion and nutraceutical bioaccessibility using a widely used standardized gastrointestinal tract model: the INFOGEST method. The emulsions used consisted of corn oil droplets stabilized using a food-grade non-ionic surfactant (Tween 20), and the droplet size was controlled by preparing them with a microfluidizer (small), sonicator (medium), or high-shear blender (large). The surfactant-coated oil droplets were relatively resistant to size changes in the mouth and stomach, due to the strong surface activity and steric stabilization mechanism of the non-ionic surfactant used. As expected, the kinetics of lipid digestion were enhanced for smaller droplets because of their greater specific surface area. The degree of lipid digestion fell from 117% to 78% (p < 0.001) as the initial droplet diameter was raised from 0.16 to 8.2 μm. In addition, there was a reduction in β-carotene bioaccessibility from 83 to 15% (p < 0.001) with increasing droplet diameter. This result was ascribed to several effects: (i) some carotenoids were trapped inside the undigested oil phase; (ii) fewer mixed micelles were produced to internalize the carotenoids; and, (iii) a fraction of the carotenoids crystallized and sedimented. Our results underline the critical importance of considering droplet size when developing emulsified foods loaded with carotenoids. The results obtained by the INFOGEST method are consistent with those found using other in vitro methods in earlier studies.
Factors impacting lipid digestion and nutraceutical bioaccessibility assessed by standardized gastrointestinal model (INFOGEST): Emulsifier type.
Tan Yunbing,Zhang Zhiyun,Muriel Mundo Jorge,McClements David Julian
Food research international (Ottawa, Ont.)
This paper is part of a series examining the impact of the main factors influencing lipid digestion and nutraceutical bioaccessibility in β-carotene-loaded oil-in-water emulsions using the harmonized INFOGEST simulated gastrointestinal model. Here, the impact of emulsifier type was examined since food emulsions and nutraceutical delivery systems are often stabilized by various types of emulsifier. The INFOGEST method was adopted to investigate the in vitro gastrointestinal fate of emulsions stabilized by five kinds of food-grade emulsifier representing different classes: synthetic surfactants (Tween 20); natural surfactants (quillaja saponin); proteins (caseinate); polysaccharides (gum arabic); and phospholipids (soy lysolecithin). Microfluidization produced emulsions with small droplet sizes for all emulsifiers, except soy lysolecithin. Within the gastrointestinal model, the caseinate-coated oil droplets had the worst gastric stability, with severe droplet flocculation and coalescence occurring in the stomach. The fraction of the lipid phase that had been digested by the end of the gastrointestinal model was considerably lower for the emulsions stabilized by soy lysolecithin (93%) or caseinate (93%), than those stabilized by gum arabic (99%), quillaja saponin (111%) or Tween 20 (117%). This effect was attributed to lower surface area of lipids available for lipase to attach to for the lysolecithin and caseinate emulsions. The overall bioaccessibility of the β-carotene increased in this order: lysolecithin (25%) < gum arabic (51%) < caseinate (55%) < quillaja saponin (56%) < Tween 20 (62%). The impact of emulsifier type on carotenoid bioaccessibility was ascribed to various factors: (i) some emulsifiers inhibited lipid digestion and so a fraction of the β-carotene remained inside the undigested droplets and the mixed micelle phase had less solubilization capacity, i.e., lysolecithin, and caseinate; (ii) some emulsifiers protected β-carotene from chemical degradation, i.e., lysolecithin and caseinate; and (iii) some emulsifiers promoted sedimentation of the β-carotene-loaded micelles, i.e., lysolecithin. These results suggest that food emulsion behavior in the human gut may be influenced by the nature of the emulsifier employed, which is important knowledge when creating functional food and beverage products.
Impact of gastric pH profiles on the proteolytic digestion of mixed βlg-Xanthan biopolymer gels.
Dekkers B L,Kolodziejczyk E,Acquistapace S,Engmann J,Wooster T J
Food & function
The understanding of how foods are digested and metabolised is essential to enable the design/selection of foods as part of a balanced diet. Essential to this endeavour is the development of appropriate biorelevant in vitro digestion tools. In this work, the influence of gastric pH profile on the in vitro digestion of mixtures of β-lactoglobulin (βlg) and xanthan gum prior to and after heat induced gelation was investigated. A conventional highly acidic (pH 1.9) gastric pH profile was compared to two dynamic gastric pH profiles (initial pH of 6.0 vs. 5.2 and H(+) secretion rates of 60 vs. 36 mmol h(-1)) designed to mimic the changes in gastric pH observed during clinical trials with high protein meals. In moving away from the pH 1.9 model, to a pH profile reflecting in vivo conditions, the initial rate and degree of protein digestion halved during the first 45 minutes. After 90 minutes of gastric digestion, all three pH profiles caused similar extents of protein digestion. Given that 50% gastric emptying times of (test) meals are in range of 30-90 min, it would seem highly relevant to use a dynamic pH gastric model rather than a pH 1.9 (USP) or pH 3 model (INFOGEST) in assessing the impact of food structuring approaches on protein digestion. The impact that heat induced gelation had on the degree of gel digestion by pepsin was also investigated. Surprisingly, it was found that heat induced gelation of βlg-xanthan mixtures at 70-90 °C for 20 minutes lead to a considerable decrease in the rate of proteolysis, which contrasts many studies of dispersed aggregates and gels of βlg alone whose heating accelerates pepsin activity due to unfolding. In the present case, the formation of a dense protein network created a fine pore structure which restricted pepsin access into the gel thereby slowing proteolysis. This work not only has implications for the in vitro assessment of protein digestion, but also highlights how protein digestion might be slowed, learnings that might have an influence on the design of foods as part of a satisfying balanced diet.
β-lactoglobulin micro- and nanostructures as bioactive compounds vehicle: In vitro studies.
Simões Lívia S,Martins Joana T,Pinheiro Ana C,Vicente António A,Ramos Oscar L
Food research international (Ottawa, Ont.)
β-Lactoglobulin (β-Lg) is known to be capable to bind hydrophilic and hydrophobic bioactive compounds. This research aimed to assess the in vitro performance of β-Lg micro- (diameter ranging from 200 to 300 nm) and nano (diameter < 100 nm) structures associated to hydrophilic and hydrophobic model compounds on Caco-2 cells and under simulated gastrointestinal (GI) conditions. Riboflavin and quercetin were studied as hydrophilic and hydrophobic model compounds, respectively. Cytotoxicity experiment was conducted using in vitro cellular model based on human colon carcinoma Caco-2 cells. Moreover, the digestion process was simulated using the harmonized INFOGEST in vitro digestion model, where samples were taken at each phase of digestion process - oral, gastric and intestinal - and characterized in terms of particle size, polydispersity index (PDI), surface charge by dynamic light scattering (DLS); protein hydrolysis degree by 2,4,6-trinitrobenzene sulfonic acid (TNBSA) assay and native polyacrylamide gel electrophoresis; and bioactive compound concentration. Caco-2 cell viability was not affected up to 21 × 10 mg mL of riboflavin and 16 × 10 mg mL quercetin on β-Lg micro- and nanostructures. In the oral phase, β-Lg structures' particle size, PDI and surface charge values were not changed comparing to the initial β-Lg structures (i.e., before being subjected to in vitro GI digestion). During gastric digestion, β-Lg structures were resistant to proteolytic enzymes and to acid environment of the stomach - confirmed by TNBSA and native gel electrophoresis. In vitro digestion results indicated that β-Lg micro- and nanostructures protected both hydrophilic and hydrophobic compounds from gastric conditions and deliver them to target site (i.e., intestinal phase). In addition, β-Lg structures were capable to enhance riboflavin and quercetin bioaccessibility and bioavailability potential compared to bioactive compounds in their free form. This study indicated that β-Lg micro- and nanostructures were capable to enhance hydrophilic and hydrophobic compounds bioavailability potential and they can be used as oral delivery systems.
Higher microbial diversity in raw than in pasteurized milk Raclette-type cheese enhances peptide and metabolite diversity after in vitro digestion.
Egger Lotti,Ménard Olivia,Abbühl Lychou,Duerr Desirée,Stoffers Helena,Berthoud Hélène,Meola Marco,Badertscher René,Blaser Carola,Dupont Didier,Portmann Reto
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.
Monitoring protein hydrolysis by pepsin using pH-stat: In vitro gastric digestions in static and dynamic pH conditions.
Mat Damien J L,Cattenoz Thomas,Souchon Isabelle,Michon Camille,Le Feunteun Steven
This study intends to demonstrate that acid titration at low pH is very well adapted to the monitoring of pepsin activity. After a description of the underlying principles, this approach was used during in vitro gastric digestions of a model of complex food containing 15wt% of whey proteins, according to both static (2h at pH = 3, Infogest protocol) and dynamic pH conditions (from pH 6.3 down to 2 in 1h). Pepsin activity was quantitatively assessed in all experiments through the calculation of degrees of hydrolysis (DH). Final values of 3.7 and 3.0% were obtained in static and dynamic pH conditions, respectively, and validated using an independent method. Results also show that about 92% of the peptides were detected at pH = 3, and 100% for pH≤2.5. Overall, the proposed approach proved to be very worthy to study protein hydrolysis during in vitro gastric digestions.
A standardised semi-dynamic in vitro digestion method suitable for food - an international consensus.
Mulet-Cabero Ana-Isabel,Egger Lotti,Portmann Reto,Ménard Olivia,Marze Sébastien,Minekus Mans,Le Feunteun Steven,Sarkar Anwesha,Grundy Myriam M-L,Carrière Frédéric,Golding Matt,Dupont Didier,Recio Isidra,Brodkorb André,Mackie Alan
Food & function
The link between food and human health is increasingly a topic of interest. One avenue of study has been to assess food disintegration and interactions within the gastrointestinal tract. In vitro digestion models have been widely used to overcome the constrictions associated with in vivo methodology. The COST Action INFOGEST developed an international, harmonised protocol for static simulation of digestion in the upper gastrointestinal tract of adults. This protocol is widely used; however, it is restricted to providing end-point assessment without considering the possible structural changes. On the other hand, there are dynamic models that provide more physiologically relevant data but are expensive and difficult to access. There is a gap between these models. The method outlined in this article provides an intermediate model; it builds upon the harmonised static model and now includes crucial kinetic aspects associated with the gastric phase of digestion, including gradual acidification, fluid and enzyme secretion and emptying. This paper provides guidance and standardised recommendations of a physiologically relevant semi-dynamic in vitro simulation of upper gastrointestinal tract digestion, with particular focus on the gastric phase. Adaptations of this model have already been used to provide kinetic data on nutrient digestion and structural changes during the gastric phase that impact on nutrient absorption. Moreover, it provides a simple tool that can be used in a wide range of laboratories.
Can dynamic digestion systems mimic the physiological reality?
Dupont D,Alric M,Blanquet-Diot S,Bornhorst G,Cueva C,Deglaire A,Denis S,Ferrua M,Havenaar R,Lelieveld J,Mackie A R,Marzorati M,Menard O,Minekus M,Miralles B,Recio I,Van den Abbeele P
Critical reviews in food science and nutrition
During the last decade, there has been a growing interest in understanding the fate of food during digestion in the gastrointestinal tract in order to strengthen the possible effects of food on human health. Ideally, food digestion should be studied on humans but this is not always ethically and financially possible. Therefore simple static digestion models mimicking the gastrointestinal tract have been proposed as alternatives to experiments but these models are quite basic and hardly recreate the complexity of the digestive tract. In contrast, dynamic models that allow pH regulation, flow of the food and injection in real time of digestive enzymes in the different compartments of the gastrointestinal tract are more promising to accurately mimic the digestive process. Most of the systems developed so far have been compared for their performances to data obtained on animals and/or humans. The objective of this article is to review the validation towards data of some of the dynamic digestion systems currently available in order to determine what aspects of food digestion they are able to mimic. Eight dynamic digestion systems are presented as well as their validation towards data. Advantages and limits of each simulator is discussed. This is the result of a cooperative international effort made by some of the scientists involved in Infogest, an international network on food digestion.
Dynamic gastric digestion of a commercial whey protein concentrate†.
Miralles Beatriz,Del Barrio Roberto,Cueva Carolina,Recio Isidra,Amigo Lourdes
Journal of the science of food and agriculture
BACKGROUND:A dynamic gastrointestinal simulator, simgi , has been applied to assess the gastric digestion of a whey protein concentrate. Samples collected from the outlet of the stomach have been compared to those resulting from the static digestion protocol INFOGEST developed on the basis of physiologically inferred conditions. RESULTS:Progress of digestion was followed by SDS-PAGE and LC-MS/MS. By SDS-PAGE, serum albumin and α-lactalbumin were no longer detectable at 30 and 60 min, respectively. On the contrary, β-lactoglobulin was visible up to 120 min, although in decreasing concentrations in the dynamic model due to the gastric emptying and the addition of gastric fluids. Moreover, β-lactoglobulin was partly hydrolysed by pepsin probably due to the presence of heat-denatured forms and the peptides released using both digestion models were similar. Under dynamic conditions, a stepwise increase in number of peptides over time was observed, while the static protocol generated a high number of peptides from the beginning of digestion. CONCLUSION:Whey protein digestion products using a dynamic stomach are consistent with those generated with the static protocol but the kinetic behaviour of the peptide profile emphasises the effect of the sequential pepsin addition, peristaltic shaking, and gastric emptying on protein digestibility. © 2017 Society of Chemical Industry.
Digestion of milk proteins: Comparing static and dynamic in vitro digestion systems with in vivo data.
Egger Lotti,Ménard Olivia,Baumann Christian,Duerr Desirée,Schlegel Patrick,Stoll Peter,Vergères Guy,Dupont Didier,Portmann Reto
Food research international (Ottawa, Ont.)
In the frame of the COST action INFOGEST, a static in vitro digestion protocol has been elaborated aiming at the improvement of data comparability by harmonizing the experimental conditions. The success in harmonization was confirmed with inter-laboratory trials using skim milk powder as a standardized model food. Moreover, the physiological relevance of the gastric and intestinal endpoints of the static digestion protocol was demonstrated in a pig in vivo trial, with the same skim milk powder and samples collected from different sections of the digestive tract, as well as in a human study with from jejunal effluents. In vivo, digestion is a dynamic process influenced by peristalsis and by the gradual secretion of enzymes and juices and the dwell time of the food. To mimic these physiological mechanisms, dynamic in vitro digestion protocols are widely used. Until now, the differences of protein hydrolysis taking place during dynamic and static in vitro digestion have not been investigated. In this study, the gradual hydrolysis of the main milk proteins present in skim milk powder was digested with the dynamic DIDGI®-system using adult digestion protocol and the static harmonized INFOGEST method. Protein hydrolysis was analyzed by gel electrophoresis, peptide patterns were measured with mass spectrometry, and free amino acids with high pressure liquid chromatography. The peptide patterns at the gastric and intestinal endpoints of in vitro digestion showed a good approximation to the in vivo results from pigs. Moreover, gradual peptide generation was comparable in both in vitro digestion conditions. However, the dynamic protocol reflected the physiological situation better at the level of free amino acid release. Nonetheless, in both in vitro digestion protocols, absorption of free amino acids is not simulated, and they are therefore limited in reflecting the in vivo situation at this level.
Physiological comparability of the harmonized INFOGEST in vitro digestion method to in vivo pig digestion.
Egger Lotti,Schlegel Patrick,Baumann Christian,Stoffers Helena,Guggisberg Dominik,Brügger Cédric,Dürr Desirée,Stoll Peter,Vergères Guy,Portmann Reto
Food research international (Ottawa, Ont.)
Recently, a static in vitro digestion (IVD) protocol was published by Minekus and coworkers (Minekus et al., 2014) within the COST INFOGEST network. The protocol, concentrating on physiological enzyme activities had the main goal to improve the comparability of experimental data between labs. The protocol was validated in several inter-laboratory studies using skim milk powder (SMP) and indeed demonstrated improved harmonization compared with previous experiments with individual IVD protocols (Egger et al., 2016). Although the enzyme activities and salt concentrations of the harmonized protocol are based on available human in vivo data, confirmation of the protocol's physiological relevance has been lacking until now. The main goal of the study was therefore to compare the harmonized IVD protocol with data from in vivo digestion. Towards this aim, an in vivo pig experiment with the same SMP as used for the validation of the IVD protocol was performed followed by a comparison of protein hydrolysis between in vivo and in vitro results. Protein hydrolysis at different levels was analyzed with gel electrophoresis, mass spectrometry, high performance liquid chromatography, and spectrophotometric o-phthaldialdehyde determination of free amino acids. Principle component analysis was used for graphical data comparison. Milk proteins detected after gastric IVD corresponded to gastric and duodenal in vivo samples and intestinal IVD samples corresponded to distal jejunal in vivo samples. Peptides identified after the gastric phase of IVD, correlated with in vivo gastric samples (r=0.8) and intestinal IVD peptides correlated best with in vivo samples collected from the median jejunum (r=0.57). Free amino acids were in both systems mainly released during the intestinal phase of digestion. Protein hydrolysis in the harmonized IVD was similar to in vivo protein hydrolysis in pigs at the gastric and intestinal endpoints. Therefore, the harmonized static in vitro protocol is suited to study protein hydrolysis at these endpoints.
In Vitro Assessment of the Impact of Industrial Processes on the Gastrointestinal Digestion of Milk Protein Matrices Using the INFOGEST Protocol.
Atallah Nathalie,Deracinois Barbara,Boulier Audrey,Baniel Alain,Jouan-Rimbaud Bouveresse Delphine,Ravallec Rozenn,Flahaut Christophe,Cudennec Benoit
Foods (Basel, Switzerland)
The goal of this study was to determine the impact of industrial processes on the digestion of six milk protein matrices using the harmonized INFOGEST in vitro static digestion protocol. First, this method was optimized to simple protein matrices using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) to compare the intestinal protein hydrolysis obtained with increasing quantities of pancreatin. Similar results were achieved with the originally required pancreatin amount (trypsin activity of 100 U.mL) and with a quantity of pancreatin equivalent to a trypsin activity of 27.3 U.mL, which was thus used to perform the in vitro digestion of the milk matrices. Molecular weight profiles, peptide heterogeneity from LC-MS/MS data, calcium, free amino acid, and peptide concentrations were determined in the gastric and intestinal phases to compare the milk protein digests. Results showed that the industrial process affected not only the protein distribution of the matrices but also most likely the protein structures. Indeed, differences arose in terms of peptide populations generated when the caseins were reticulated or when their calcium concentrations were reduced.
INFOGEST static in vitro simulation of gastrointestinal food digestion.
Brodkorb André,Egger Lotti,Alminger Marie,Alvito Paula,Assunção Ricardo,Ballance Simon,Bohn Torsten,Bourlieu-Lacanal Claire,Boutrou Rachel,Carrière Frédéric,Clemente Alfonso,Corredig Milena,Dupont Didier,Dufour Claire,Edwards Cathrina,Golding Matt,Karakaya Sibel,Kirkhus Bente,Le Feunteun Steven,Lesmes Uri,Macierzanka Adam,Mackie Alan R,Martins Carla,Marze Sébastien,McClements David Julian,Ménard Olivia,Minekus Mans,Portmann Reto,Santos Cláudia N,Souchon Isabelle,Singh R Paul,Vegarud Gerd E,Wickham Martin S J,Weitschies Werner,Recio Isidra
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.
Protein digestion of different protein sources using the INFOGEST static digestion model.
Sousa Raquel,Portmann Reto,Dubois Sébastien,Recio Isidra,Egger Lotti
Food research international (Ottawa, Ont.)
In vitro digestion systems are valuable tools for understanding and monitoring the complex behavior of food degradation during digestion, thus proving to be good candidates for replacing in vivo assays. The aim of the present work was to study protein hydrolysis in a selection of different protein sources using the harmonized INFOGEST static protocol: three isolated proteins (collagen, zein, and whey protein) and five foods (sorghum flour, wheat bran cereals, peanuts, black beans, and pigeon peas). The proteins of all the substrates were analyzed by SDS-PAGE and HPLC-MS/MS. Individual amino acid composition was analyzed by high-performance liquid chromatography (HPLC). EAA/NEAA (essential amino acids/ nonessential amino acids) ratios in the substrates from low to high were as follows: wheat bran cereals, peanuts, collagen, zein, whey protein, sorghum, pigeon peas, and black beans. The results revealed sorghum, whey protein, and zein as good sources of BCAA. In all substrates, no intact protein from the substrates was visually detected by SDS-PAGE after the intestinal phase of in vitro digestion with the INFOGEST protocol. However, digestion-resistant peptides were detected in all substrates after the intestinal digestion phase. Protein hydrolysis was high in whey protein isolate and pigeon pea and low for wheat bran cereals and bovine collagen.