In vitro human digestion models for food applications

In vitro digestion models are widely used to study the structural changes, digestibility, and release of food components under simulated gastrointestinal conditions. However, the results of in vitro digestion models are often different to those found using in vivo models because of the difficulties in accurately simulating the highly complex physicochemical and physiological events occurring in animal and human digestive tracts. This paper provides an overview of current trends in the development and utilisation of in vitro digestion models for foods, as well as information that can be used to develop improved digestion models. Our survey of in vitro digestion models found that the most predominant food samples tested were plants, meats, fish, dairy, and emulsion-based foods. The most frequently used biological molecules included in the digestion models were digestive enzymes (pancreatin, pepsin, trypsin, chymotrypsin, peptidase, α-amylase, and lipase), bile salts, and mucin. In all the in vitro digestion models surveyed, the digestion temperature was 37 °C although varying types and concentrations of enzymes were utilised. With regard to digestion times, 2 h (the stomach, small intestine, and large intestine each) was predominantly employed. This survey enhances the understanding of in vitro digestion models and provides indications for the development of improved in vitro digestion models for foods or pharmaceuticals.     

Structural breakdown of starch-based foods during gastric digestion and its link to glycemic response: In vivo and in vitro considerations

The digestion of starch-based foods in the small intestine as well as factors affecting their digestibility have been previously investigated and reviewed in detail. Starch digestibility has been studied both in vivo and in vitro, with increasing interest in the use of in vitro models. Although previous in vivo studies have indicated the effect of mastication and gastric digestion on the digestibility of solid starch-based foods, the physical breakdown of starch-based foods prior to small intestinal digestion is often less considered. Moreover, gastric digestion has received little attention in the attempt to understand the digestion of solid starch-based foods in the digestive tract. In this review, the physical breakdown of starch-based foods in the mouth and stomach, the quantification of these breakdown processes, and their links to physiological outcomes, such as gastric emptying and glycemic response, are discussed. In addition, the physical breakdown aspects related to gastric digestion that need to be considered when developing in vitro–in vivo correlation in starch digestion studies are discussed. The discussion demonstrates that physical breakdown prior to small intestinal digestion, especially during gastric digestion, should not be neglected in understanding the digestion of solid starch-based foods.     

Optimization of a gastrointestinal model applicable to the evaluation of bioaccessibility in fish feeds

BACKGROUND: Although several types of in vitro digestibility assays have been applied to nutritional evaluation of feeds for aquatic organisms, all of them are based on the use of closed reactors and do not simulate the gastric phase of the digestion. Our objective was to evaluate the suitability of a gastrointestinal model based on the use of a digestion cell provided by a semi‐permeable reaction chamber, which allows continuous removal of digestion products as they are produced. We tested the effects of some factors, like the inclusion of a gastric phase, reaction temperature or bile salts on the hydrolysis of feed proteins by fish enzymes. RESULTS: We found that the most suitable operational conditions to simulate the digestion process must include a short acid pre‐digestion as well as the use of bile salts in the reaction mixture. Acid pre‐digestion resulted in a significant increase in the liberation of amino acids which represented more than twice that measured when using a single phase. The addition of two bile salts (45 µmol L^?1 sodium taurocholate + chenodesoxycolate) resulted in almost a threefold increase in the hydrolysis of feed protein. The use of the described open system also allows the evaluation of carbohydrate hydrolysis as well as determination of residual undigested matter, in a similar manner to that carried out in ruminants with the DAISY system. CONCLUSION: Results suggest the system can be a very suitable model for evaluation of bioaccessibility in fish feeds. Copyright © 2009 Society of Chemical Industry     

Effects of simulated gastric and intestinal digestion on chitooligosaccharides in two in vitro models

Chitooligosaccharides (COS) with different degree of polymerisation (DP) have different physiological activities such as anti-tumour and anti-hyperlipidemia activities. However, the digestive process might lead to the change of DP of COS as well as cause the change of physiological activities in vivo. In this study, two in vitro digestion models (static and dynamic) were used to investigate digestion behaviours of COS and the influencing factors during digestion with or without food matrix. The results showed that COS with DP 2–5 were indigestible and COS with DP 6–10 degraded with time during gastric and intestinal digestion. Pepsin, pancreatin and lipase were the main factors leading to degradation. Food matrix could protect COS with DP 6–10 from degradation during gastric digestion. However, the degradation of COS with DP 6–10 accelerated and the degradation of COS with DP 5 occurred during intestinal digestion with food matrix due to pancreatin, lipase and bile salt.     

Can dynamic in vitro digestion systems mimic the physiological reality?

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 in vivo on humans but this is not always ethically and financially possible. Therefore simple static in vitro digestion models mimicking the gastrointestinal tract have been proposed as alternatives to in vivo 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 in vivo data obtained on animals and/or humans. The objective of this article is to review the validation towards in vivo 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 in vivo 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     

Influence of initial emulsifier type on microstructural changes occurring in emulsified lipids during in vitro digestion

The purpose of this study was to examine the impact of emulsifier type on the micro-structural changes that occur to emulsified lipids as they pass through a model gastrointestinal system. Lipid droplets initially coated by different kinds of emulsifiers (lecithin, Tween 20, whey protein isolate and sodium caseinate) were prepared using a high speed blender. The emulsified lipids were then passed through an in vitro digestion model that simulated the composition (pH, minerals, surface active components, and enzymes) of mouth, stomach and small intestine juices. The change in structure and properties of the lipid droplets were monitored by laser scanning confocal fluorescence microscopy, conventional optical microscopy, light scattering, and micro-electrophoresis. In general, there was a decrease in mean droplet diameter (d₃₂) as the droplets moved from mouth to stomach to small intestine. The electrical charge on the droplets stabilized by lecithin, Tween 20 and sodium caseinate were negative throughout the model GI system, while those stabilized by whey protein were positive in the stomach. This suggests that at least some of this globular protein remained attached to the droplet surfaces. The data was interpreted in terms of the competitive adsorption of phospholipids/bile salts with the adsorbed emulsifiers, as well as the enzymatic digestion of proteins and lipids. These results enhance our understanding of the physicochemical and structural changes that may occur to emulsified lipids within the gastrointestinal tract, which may have important consequences for the design of functional foods that alter lipid bioavailability. Nevertheless, there were appreciable differences between the behavior of emulsions within the in vitro model used in this study and literature reports of their behavior within in vivo studies, which highlights the need for more realistic in vitro digestion models.     

In vitro binding of bile salts by lentil flours, lentil protein concentrates and lentil protein hydrolysates

The binding capacity of bile salts by lentil flours produced from two varieties, Blaze and Laird and their protein concentrates and hydrolysates were studied. Sodium cholate, sodium deoxycholate, sodium taurocholate, sodium glycocholate and sodium chenodeoxycholate were tested individually, and their binding interactions with the lentil products were analyzed using the Trinity Biotech Bile Acids Kit 450-10 and compared to cholestyramine. All tested samples bound the bile salts investigated, and the amount of bile salts bound (> 70%) was sometimes greater than that bound by cholestyramine. Overall, there were no major differences in the bile salt binding capacities of similar samples prepared from the two varieties of lentil. In vitro digestion of the lentil proteins by pepsin/trypsin/??-chymotrypsin, alcalase/flavourzyme and papain significantly reduced the bile salt binding capacity compared to the undigested samples except in the case of sodium deoxycholate where no significant differences in bile salt binding were observed before and after hydrolysis. Binding of bile salts has been linked to cholesterol reduction, thus, the ability of the lentil products to bind bile salts is of interest as it may suggest that lentils could potentially have cholesterol-reducing properties.     

Effect of gastric pH and bile acids on the survival of Listeria monocytogenes and Salmonella Typhimurium during simulated gastrointestinal digestion

The human gastrointestinal tract (GIT) harbors numerous defensive mechanisms to impede pathogen colonization, including gastric acidity and bile acids in the small intestine. This study aims to elucidate the survival of Salmonella Typhimurium and Listeria monocytogenes under different levels of gastric pH (2.0–3.5) and bile acid concentrations (2.5 mM – 10.0 mM), during an in vitro digestion process following the ingestion of a contaminated food model system. The results showed that S. Typhimurium  was more susceptible to gastric acidity than L. monocytogenes, yet more bile acid tolerant. Interestingly, the bile acids bactericidal effect towards L. monocytogenes was higher if the previous exposure to the gastric acidity was harsher. Our findings suggest that, despite the different microbial behavior of the two species in the simulated GIT, the effects of gastric acidity and bile acids alone could not prevent their survival in the intestine.Industrial relevanceS. Τyphimurium and L. monocytogenes represent biological threats that are of major concern for the food industry due to their ubiquitous nature and their ability to trigger foodborne illnesses. Their ability to survive the gastrointestinal passage is crucial for their subsequent intestinal colonization that can lead to host infection. Therefore, obtaining a deeper insight into the factors affecting pathogen survival in the gut is vital for the establishment of more efficient strategies to prevent foodborne diseases. Given the limitations and ethical constraints of conducting studies like this in vivo, the applied in vitro digestion model system along with the mathematical modelling can provide a valuable alternative that can be readily utilized in the food and pharmaceutical industry as a platform for similar digestion studies.     

An in vitro method to simulate phenolic compound release from the food matrix in the gastrointestinal tract

A method to simulate in vitro the physiological conditions in the stomach and small intestine has been developed as a modification of a method already described by Miller et al. [11]. This method enables the study of phenolic compound release from the food matrix and their transformation during digestion, and has been applied to orange juice, strawberries, and strawberry jam. The dialysis rates obtained for the different phenolics in the new method are much better than those found in the original one. Some food constituents, as is the case of sugars in jams, can affect the dialysis rates in vitro, showing that the results obtained for dialysis should be taken with caution in these products. These assays have shown that strawberry anthocyanins are largely degraded (transformed) during the digestion and that free ellagic acid is released from ellagitannins leading to a ten-fold increase of this compound during digestion.     

Stability of polyphenols in chokeberry (Aronia melanocarpa) subjected to in vitro gastric and pancreatic digestion

Berries and red fruits are dietary sources of polyphenols with reported health benefits. As part of the diet, polyphenols are ingested as complex mixtures immersed in a food matrix which is digested in the gut. Epithelial cells lining the gut are regularly exposed to these digested mixtures. To understand the effects of dietary polyphenols on human gut health it is essential to determine their stability and fate in the lumen. In this work, we investigated the effects of an in vitro gastric and pancreatic digestion on the stability and composition of the major polyphenols in chokeberry juice. Digestion was carried out with a mixture of pepsin-HCl for 2 h, followed by a 2 h incubation with pancreatin and bile salts at 37 °C with shaking, in the absence of light and under N₂. After digestion, the chokeberry samples were acidified, filtered and HPLC-DAD/HPLC–MS–MS analysed to determine the content of total soluble recovered phenolics. Gastric digestion had no substantial effect on any of the major phenolic compounds in chokeberry, namely anthocyanins, flavan-3-ols, flavonols and caffeic acid derivatives. However, these compounds were significantly altered during the pancreatic digestion and this effect was more marked for anthocyanins (approximately 43% was lost during the 2 h treatment with pancreatin). Flavonols and flavan-3-ols decreased by 26% and 19%, respectively. Neochlorogenic acid decreased by 28% whereas chlorogenic acid was increased by 24%. In vitro digestion of standard phenolic compounds, representing each of the groups of phenolics in chokeberry, confirmed some of the observed changes. Interactions with the digestive enzymes were not responsible for the observed losses which were mostly due to the chemical conditions during pancreatic digestion. Our results, in accordance with previously published results, show that dietary polyphenols are highly sensitive to the mild alkaline conditions in the small intestine and that a good proportion of these compounds can be transformed into other unknown and/or undetected structural forms with different chemical properties and, consequently, different bioaccessibility, bioavailability and biological activity.     

Bioaccessibility of carotenoids from plant and animal foods

The frequent consumption of carotenoid-rich foods has been associated with numerous health benefits, such as the supply of provitamin A. To exert these health benefits, carotenoids need to be efficiently liberated from the food matrix, micellized in the small intestine, taken up by the enterocytes and absorbed into the human blood stream. Enormous efforts have been made to better understand these processes. Because human studies are costly, labor-intense and time-consuming, the evaluation of carotenoid liberation and micellization at the laboratory scale using simulated in vitro digestion models has proven to be an important tool for obtaining preliminary results prior to conducting human studies. In particular, the liberation from the food matrix and the intestinal micellization can be mimicked by simulated digestion, yielding an estimate of the so-called bioaccessibility of a carotenoid. In the present review, we provide an overview of the carotenoid digestion process in vivo, the currently used in vitro digestion models and the outcomes of previous bioaccessibility studies, with a special focus on correlations with concomitantly conducted human studies. Furthermore, we advocate for the on-going requirement of better standardized digestion protocols and, in addition, we provide suggestions for the complementation of the acquired knowledge and current nutritional recommendations. © 2018 Society of Chemical Industry     

Influence of simulated upper intestinal parameters on the efficiency of beta carotene micellarisation using an in vitro model of digestion

This study was undertaken to better understand how variations in the parameters used to simulate the upper intestinal environment influence the transfer of a lipophilic molecule, beta carotene (BC), from the oily to the aqueous phase of digestate using a static model of digestion. Bile, pancreatin and pH were important and inter-related factors in determining the efficiency of BC transfer (P < 0.05). Less than 4% and 8% of the BC was transferred to the aqueous phase in the absence of bile and pancreatin, respectively. Generally, the proportion of BC transferred increased with bile and pancreatin concentrations and with pH. Under conditions which simulated the fed versus fasted states of digestion, significantly more BC was incorporated in the aqueous fraction (46.5% versus 18.8%). These results underscore the need to carefully consider and define the experimental parameters used for in vitro assays to study the digestion of carotenoids and other lipophilic molecules.     

罗非鱼源肠道益生菌的分离鉴定及其体外益生特性分析

为了筛选出优良的鱼源益生菌，作者以吉富罗非鱼(Oreochromis niloticus)为实验材料，进行肠道芽孢杆菌和乳酸菌的分离、鉴定，并对分离得到的菌株进行抑菌活性、耐酸性、耐胆盐、人工胃肠液耐受、溶血活性和抗生素敏感性等体外益生特性分析。结果表明，从罗非鱼肠道中共分离得到109株菌，其中包括79株乳酸菌和30株芽孢杆菌，经过一系列的实验最终筛选出3株乳酸乳球菌（Lactococcus lactis）。生物学特性研究表明，3株菌均无溶血活性，对抗生素具有一定的敏感性，对强酸、胆盐和人工胃肠液具有一定的耐受性，对水产病原菌具有不同的抑菌活性。筛选得到的3株菌丰富了鱼源益生菌资源，它们均可作为优良的鱼源益生菌候选菌株。其中，菌株R1对强酸和人工胃肠液耐受性最强，菌株R29耐胆盐能力最强，可进行进一步的益生特性研究，挖掘其作为食品源益生菌的益生效果。     

海藻糖基麦芽五糖及其微球的体外消化和酵解

为研究包埋前后的海藻糖基麦芽五糖(N-G7)在模拟消化液及体外酵解过程中的变化,采用口腔、胃部及小肠的体外消化模型分析消化前后溶液中N-G7的质量和N-G7微球(ALGCa)中的糖组成变化,应用体外发酵技术评价N-G7微球的发酵行为,检测不同时间点的pH值、菌体浓度(OD₆₀₀)、产气量、短链脂肪酸(SCFA)浓度等指标的变化,并探究其对人体肠道微生物构成的影响。结果表明：N-G7在口腔消化2 min后在强酸性的胃部环境中几乎不被水解,在小肠中N-G7的水解率达到了82.70%。采用不同质量分数(1%、2%、3%)的海藻酸钙对其进行包埋,形成的微球保留80%以上的N-G7到达结肠。在体外酵解中,各实验组的pH随时间延长逐渐降低,OD600随时间延长逐渐增加。质量分数2%的ALG-Ca组产气量和碳源消耗率在0～6 h内达到最低,而后逐渐增加,证明包埋后的N-G7发酵体系属于缓慢发酵。与对照相比,质量分数2%的ALG-Ca组产生大量SCFA,肠道菌群组成发生变化,更有利于益生菌的生长。     

Dietary interference on the oxidation and hydrolysis of liposomes during in vitro digestion

In this study, the influence of dietary compounds, including antioxidants (ethylenediaminetetraacid, ascorbic acid, catechin), chitosan and lactoferrin, on the stability of liposomes during in vitro adult and infant digestion was investigated in terms of formation of thiobarbituric acid-reactive substances (TBARS) and free fatty acid release. Liposomes were more sensitive to degradation in adults than in infants, suggesting lower pH (1.5) and higher concentration of pancreatic enzymes (3.2 mg mL⁻¹) and bile salts (5 mg mL⁻¹) that indicated greater damage to the structure of liposomes. Compared to ethylenediaminetetraacid and ascorbic acid, catechin presented the most apparent protective effect against liposomal oxidation by scavenging free radicals. Chitosan promoted the formation of TBARS, while lactoferrin facilitated the hydrolysis rate of liposomes under both adult and infant conditions. This study provided an insight into the development of healthcare products and functional foods related to liposomes for special populations.     

Advances in the in vitro digestion and fermentation of polysaccharides

In vitro digestion models are widely used to study the structural changes, digestion and release of food components under simulated gastrointestinal conditions. As compared to the in vivo digestion tests, the in vitro digestion reflects the digestion and utilisation of food after ingestion and has the advantages of being time consuming, inexpensive, reproducible and free from moral and ethical restrictions. This study reviewed the current research studies on the in vitro simulated digestion of polysaccharides conducted in the last 5 years and focused on the oral, gastric and intestinal digestion models, with the aim of providing a basis for the further testing of changes in the content, structure and active ingredients of polysaccharides before and after digestion.     

In vitro model to correlate viscosity and bile acid-binding capacity of digested water-soluble and insoluble dietary fibres

Binding and excretion of bile acids in the small intestine by water-soluble and insoluble dietary fibres is one of the main mechanisms for their cholesterol-lowering effects. A model for the determination of the bile acid-binding capacity of dietary fibres was developed. The experimental set-up allowed to correlate the bile acid-binding capacities of different fibres with their viscosities after in vitro digestion. For cellulose, native oat fibre and psyllium fibres clear correlations between viscosity and bile acid-binding capacity could be observed, whereas for water-insoluble lupin fibre such a correlation did not exist. Heat-damaged oat fibre also showed bile acid-binding despite of significantly decreased viscosity. The data demonstrated that bile acid-binding of digested dietary fibres may not be solely based on their viscosity but may be influenced by additional binding forces.     

Food lipid oxidation under gastrointestinal digestion conditions: A review

Abstract

Unravelling the relationship between food and health requires a more in-depth knowledge of the various changes occurring in the gastrointestinal tract during digestion and which may ultimately affect the nutritional quality and safety of ingested food lipids before absorption into the bloodstream. In this context, this review deals with the oxidation process of food lipids under digestive conditions and the studies carried out on this topic using different digestion models: in vitro, in vivo or ex vivo, static or dynamic, and including one, two and/or three digestive phases (oral, gastric and duodenal). These studies have contributed to clarifying the occurrence and extent of lipid degradation under such a particular environment, many of them also highlighting the factors affecting the advance or delay of the oxidation of dietary lipids during digestion, like: food lipid content, unsaturation degree and initial oxidative status; the presence in the food bolus of compounds showing antioxidant activity (polyphenols, tocopherols…) either added or naturally present; the presence in the food bolus of proteins (including iron or not); food technological or culinary processings (salting, smoking, cooking…), among others. Likewise, the methodologies employed to study lipid oxidation under digestive conditions are also summarized and future research perspectives are discussed.     

The behavior of dietary fiber in the gastrointestinal tract determines its physiological effect

A diet rich in dietary fiber (DF) is considered healthy and recommended dietary intake of DF is established all over the world. The physiological effect of DF is mostly related to its behavior during digestion. In this review, the behavior of DF in the human digestive tract is discussed and linked to its physiological effect with special attention to four aspects of such behavior: (i) the modulation of bioavailability by the plant cell walls, (ii) the effect of DF on the rheological and colloidal state of digesta, (iii) the binding of DF with phenolic compounds, bile salts, mineral ions, and digestive enzymes, and (iv) DF fermentation in the large intestine and the corresponding effect on microbiota composition. It is stressed that the detailed chemical characterization of DF is crucial to explain its effect on health and that DF behavior in the digestive tract can be modulated by interactions with other food and meal components so that information of the bare content in DF of food is not sufficient to predict its physiological effect.     

Interfacial design of protein-stabilized emulsions for optimal delivery of nutrients

Proteins are often used as ingredients in food emulsions, as their amphiphilic structures provide electrostatic and steric stabilization. Significant attention has recently been directed at understanding how the composition and structure of oil-water interfaces change during digestion and how these can be manipulated to enhance the delivery of nutrients contained within the oil droplets. These efforts have necessitated the development of more sophisticated in vitro digestion models of greater physiological relevance and increased efforts in research to identify the role of the various digestive parameters on interfacial dynamics. The changes occurring at the oil-water interface will affect the adsorption of gastro-intestinal lipases and, ultimately, affect lipid digestion. The composition of a protein-stabilized oil droplet changes continuously during digestion, because of proteolysis and the formation of peptides with different affinities for the interface. In addition, natural bio-surfactants such as phospholipids and bile salts, other surface- active molecules present in foods, and the products of lipolysis (i.e. mono and diglycerides, lysophospholipids), all compete for access to the interface, and contribute to the dynamic changes occurring on the surface of the oil droplets. A better understanding of how to tailor the composition of oil droplet surfaces in food emulsions will aid in optimizing lipid digestion and, as a result, delivery of lipophilic nutrients. This review focuses on the physico-chemical changes occurring in protein-stabilized oil-in-water emulsions during gastric and small intestine digestion, and on how interfacial engineering could lead to differences in fatty acid release and the potential bioavailability of lipophilic molecules.