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.

Intestinal health functions of colonic microbial metabolites: a review.

This review tries to find a scientific answer on the following two questions: (1) to what extent do we understand the specific role of colonic microbial metabolites, especially short-chain fatty acids (SCFA), in maintaining the health status and prevention of diseases of the colon and the host; (2) to what extent can we influence or even control the formation of colonic microbial metabolites which are beneficial for the health status. The review focuses on the following topics: energy source, intestinal motility, defence barrier, oxidative stress with special attention for antiinflammatory and anti-carcinogen functions, and satiety. Also the risk of overproduction of SCFA is discussed. Reviewing the literature as present today, it can be concluded that physiological levels of SCFA are vital for the health and well-being of the host and that the presence of carbohydrates (dietary fibre, prebiotics) is essential to favour the metabolic activity in the direction of carbohydrate fermentation. For optimal motor activity of the ileum and colon, to regulate the physiological intestinal mobility, steadily fermentable dietary fibres or prebiotics are crucial. The formation of SCFA, especially propionate and butyrate, up to high physiological levels in the colon, much likely also contributes to the defence mechanisms of the intestinal wall. No final answer can be given yet about the role of SCFA in anti-inflammation and anti-carcinogenicity, but recently published research shows possible mechanisms in this field. The intake of prebiotics or specific dietary fibres promotes the formation of SCFA within the physiological range, and more or less specifically increases the levels of propionate and butyrate. In this way, they provide benefit to the host, especially the natural regulation of the digestive system.

Efficient degradation of gluten by a prolyl endoprotease in a gastrointestinal model: implications for coeliac disease.

BACKGROUND: Coeliac disease is caused by an immune response to gluten. As gluten proteins are proline rich they are resistant to enzymatic digestion in the gastrointestinal tract, a property that probably contributes to the immunogenic nature of gluten. AIMS: This study determined the efficiency of gluten degradation by a post-proline cutting enzyme, Aspergillus niger prolyl endoprotease (AN-PEP), in a dynamic system that closely mimics the human gastrointestinal tract (TIM system). METHODS: Two experiments were performed. In the first, a slice of bread was processed in the TIM system with and without co-administration of AN-PEP. In the second, a standard fast food menu was used. Samples of the digesting meals were taken from the stomach, duodenum, jejunum and ileum compartments at time zero until 4 hours after the start of the experiment. In these samples the levels of immunogenic peptides from gliadins and glutenins were assessed by monoclonal antibody-based competition assays, Western blot analysis and proliferation T-cell assays. RESULTS: AN-PEP accelerated the degradation of gluten in the stomach compartment to such an extent that hardly any gluten reached the duodenum compartment. CONCLUSION: AN-PEP is capable of accelerating the degradation of gluten in a gastrointestinal system that closely mimics in-vivo digestion. This implies that the co-administration of AN-PEP with a gluten-containing meal might eliminate gluten toxicity, thus offering patients the possibility of abandoning (occasionally) their strict gluten-free diet.

Research goals for folate and related B vitamin in Europe.

In the past decade, the understanding of folate bioavailability, metabolism and related health issues has increased, but several problems remain, including the difficulty of delivering the available knowledge to the populations at risk. Owing to the low compliance of taking folic acid supplements, for example, among women of child-bearing age who could lower the risk of having a baby with a neural tube defect, food-based strategies aimed at increasing the intake of folate and other B-group vitamins should be a priority for future research. These should include the development of a combined strategy of supplemental folate (possibly with vitamin B(12)), biofortification using engineered plant-derived foods and micro-organisms and food fortification for increasing folate intakes in the general population. Currently, the most effective population-based strategy to reduce NTDs remains folic acid fortification. However, the possible adverse effect of high intakes of folic acid on neurologic functioning among elderly persons with vitamin B(12) deficiency needs urgent investigation. The results of ongoing randomized controlled studies aimed at reducing the prevalence of hyperhomocysteinemia and related morbidity must be available before food-based total population approaches for treatment of hyperhomocysteinemia can be recommended. Further research is required on quantitative assessment of folate intake and bioavailability, along with a more thorough understanding of physiological, biochemical and genetic processes involved in folate absorption and metabolism.

Antimutagenic activity of green tea and black tea extracts studied in a dynamic in vitro gastrointestinal model.

An in vitro gastrointestinal model, which simulates the conditions in the human digestive tract, was used to determine potential antimutagenic activity of extracts of black tea and green tea. In this paper, results are presented on the availability for absorption of potential antimutagenic compounds present in tea and on the influence of the food matrix on this activity. Between 60 and 180min after the tea was introduced into the model, antimutagenic activity was recovered from the jejunal compartment by means of dialysis: the dialysate appeared to inhibit the mutagenicity of the food mutagen MeIQx in the direct plate assay with Salmonella typhimurium (Ames test). The maximum inhibition was measured at 2h after the start of the experiment and was comparable for black tea and green tea extract. To determine the influence of food matrices on the antimutagenic activity of tea, the model was loaded with black tea together with milk or a homogenized standard breakfast. The maximum inhibition observed with black tea was reduced by 22, 42 and 78% in the presence of whole milk, semi-skimmed milk, and skimmed milk, respectively. Whole milk and skimmed milk abolished the antimutagenic activity of green tea by more than 90%; for semi-skimmed milk the inhibition was more than 60%. When a homogenized breakfast was added into the model together with the black tea extract, the antimutagenic activity was completely eliminated. When tea and MeIQx were added together into the digestion model, MeIQx mutagenicity was efficiently inhibited, with green tea showing a slightly stronger antimutagenic activity than black tea. In this case, the addition of milk had only a small inhibiting effect on the antimutagenicity. Antioxidant capacity and the concentration of catechins were also measured in the jejunal dialysates. The reduction in antimutagenic activity corresponded with reduction in antioxidant capacity and with a decrease of concentration of three catechins, viz. catechin, epigallocatechin gallate and epigallocatechin. The in vitro gastrointestinal model appears to be a useful tool to study the antimutagenicity of food components.

Application of a dynamic in vitro gastrointestinal tract model to study the availability of food mutagens, using heterocyclic aromatic amines as model compounds.

The TNO gastro-Intestinal tract Model (TIM) is a dynamic computer-controlled in vitro system that mimics the human physiological conditions in the stomach and small intestine. In the current TIM physiological parameters such as pH, temperature, peristaltic movements, secretion of digestion enzymes, bile and pancreatic juices, and absorption of digested products-by removal through dialysis-was simulated. Heterocyclic aromatic amines (HAA; viz. IQ, MeIQ, MeIQx and PhIP) were used as model compounds for food mutagens, and the passage through TIM was investigated for each of these compounds separately. Subsequently, the influence of a matrix and different rates of passage on the availability for absorption and distribution were studied in experiments with prepared meat, supplemented with MeIQx. Samples taken at various time points from the jejunal and ileal dialysates and from the lumen at the end of the small intestine (ileal delivery) were tested for the presence of mutagenic activity in the Ames test with Salmonella typhimurium strain TA98 as indicator, in the presence of mammalian metabolic activation (rat S9 mix). The results show that, comparable with the human in vivo situation, all four HAA are quickly removed (approx. 50% in 2 hr; approx. 95% in 6 hr) and mainly recovered from the lumen into the jejunal and ileal dialysates (94% of recovery). Only 5+/-1.5% is recovered in the chyme at the end of the small intestine. When MeIQx was added to meat, its availability for absorption was slower, although the influence of the gastrointestinal passage time on the availability of MeIQx was more pronounced than this matrix effect. More MeIQx was found in the jejunal dialysate (23%; P<0.01) and less in the ileal delivery (8%; P<0.01) when simulating the gastrointestinal passage of solid meals was compared to simulating that of liquid meals. The present experiments demonstrate that TIM can be applied to study in vitro the availability of heterocyclic aromatic amines in the gastrointestinal tract. More generally, these studies indicate that TIM shows promise as a useful tool for various research purposes dealing with the availability for absorption of mutagenic as well as antimutagenic components in food.

Safe biotechnology 9: values in risk assessment for the environmental application of microorganisms. The Safety in Biotechnology Working Party of the European Federation of Biotechnology.

Risk assessment for the deliberate release of microorganisms into the environment is traditionally carried out on a case-by-case basis. In a similar approach to that used when assessing human pathogenicity, we propose an alternative approach by introducing risk classes to facilitate or complement this type of risk assessment. These consider several sets of scenarios that address the different values that need to be protected. Examples of this approach include risk-class definitions for soil fertility and biodiversity.

A computer-controlled system to simulate conditions of the large intestine with peristaltic mixing, water absorption and absorption of fermentation products.

This paper introduces a new type of system to simulate conditions in the large intestine. This system combines removal of metabolites and water with peristaltic mixing to obtain and handle physiological concentrations of microorganisms, dry matter and microbial metabolites. The system has been designed to be complementary to the dynamic multi-compartmental system that simulates conditions in the stomach and small intestine described by Minekus et al. [Minekus M, Marteau P, Havenaar R, Huis in't Veld JHJ (1995) ATLA 23:197-209]. High densities of microorganisms, comparable to those found in the colon in vivo, were achieved by absorption of water and dialysis of metabolites through hollow-fibre membranes inside the reactor compartments. The dense chyme was mixed and transported by peristaltic movements. The potential of the system as a tool to study fermentation was demonstrated in experiments with pectin, fructo-oligosaccharide, lactulose and lactitol as substrates. Parameters such as total acid production and short-chain fatty acid (SCFA) patterns were determined with time to characterize the fermentation. The stability of the microflora in the system was tested after inoculation with fresh fecal samples and after inoculation with a microflora that was maintained in a fermenter. Both approaches resulted in total anaerobic bacterial counts higher than 10(10) colony-forming units/ml with physiological levels of Bifidobacterium, Lactobacillus, Enterobacteriaceae and Clostridium. The dry matter content was approximately 10%, while the total SCFA concentration was maintained at physiological concentrations with similar molar ratios for acetic acid, propionic acid and butyric acid as measured in vivo.

Description of a dynamic in vitro model of the dog gastrointestinal tract and an evaluation of various transit times for protein and calcium.

In order to manufacture complete and balanced dog diets, it is important to know the nutrient requirements of dogs and the availability of these nutrients from food. As pet food manufacturers are restricted in their options for (invasive) animal studies, due to ethical constraints, it is important to have alternative methods for researching the effects of various dog diets. To simulate the gastrointestinal tract of the dog, the dynamic gastrointestinal tract model developed by Minekus et al. was further developed and modified in this study. The model consists of four compartments which simulate the stomach and small intestine (duodenum, jejunum and ileum). Each compartment is made of glass, with a flexible inner wall. This wall can be compressed by increasing the pressure of the surrounding water, mimicking the peristaltic movements and mixing seen in vivo. The model is computer-controlled to simulate physiological parameters such as pH, transit time and secretion of digestive juices, as derived from the literature. Gastric meal delivery and the effects of intestinal transit time on protein digestibility and availability for absorption of calcium from dog food were studied to evaluate the model. The gastric meal delivery of dry dog food was identical to a preset curve, which was based on in vivo data from healthy dogs. The emptying time for canned dog food was somewhat slower than the preset values, probably due to the viscosity of the meal. The differences between the preset values and the measured delivery were not significant. The digestibility of protein and the availability of calcium for absorption increased with a longer transit time. A significant difference was found between medium and slow transit times for the nitrogen content in the ileal delivery effluent and the jejunal dialysates (p < 0.05). The same trend was seen for calcium (not significant). The overall conclusion is that the model is a useful tool for mimicking the gastrointestinal tract of dogs. Parameters such as pH, transit time and enzyme activity can be mimicked and can be kept within a physiological range.

The effect of consumption of milk fermented by Lactobacillus casei strain Shirota on the intestinal microflora and immune parameters in humans.

OBJECTIVE: To determine the effect of consumption of milk fermented by Lactobacillus casei strain Shirota (L. casei Shirota) on the composition and metabolic activities of the intestinal microflora, and immune parameters in humans. SUBJECTS: Twenty healthy male subjects aged 40-65 years were selected. DESIGN: A placebo-controlled trial was performed in which 10 subjects were randomly assigned to a control and 10 to a treatment group. During the first and last two weeks of the 8-week study the subjects received a strictly controlled diet without fermented products. The same controlled diet was given during the intermediate 4-week test period but then the treatment group received three times daily 100 ml of fermented milk containing 10(9) CFU L. casei Shirota/ml, whereas the same amount of unfermented milk was given to the subjects in the control group. RESULTS: In comparison to the control group, the consumption of L. casei Shirota-fermented milk resulted in an increase of the Lactobacillus count in the faeces in which the administered L. casei Shirota was predominant at the level of 10(7) CFU/g wet faeces. This was associated with a significant increase in Bifidobacterium counts (P < 0.05). Some shifts in the other bacterial species were found, such as a decreased number of Clostridium; however the differences were not statistically different between the treatment and the control groups. The beta-glucuronidase and beta-glucosidase activities per 10(10) bacteria decreased significantly (P < 0.05) at the second week of the 4-week test period with the consumption of L. casei Shirota-fermented milk. Furthermore, the consumption of the fermented milk product resulted in a slight but significant increase in the moisture content of the faecal samples (P < 0.05). No treatment effects were observed for any of the immune parameters measured (including natural killer (NK) cell activity, phagocytosis and cytokine production). CONCLUSIONS: The results suggest that consumption of L. casei Shirota-fermented milk is able to modulate the composition and metabolic activity of the intestinal flora and indicate that L. casei Shirota-fermented milk does not influence the immune system of healthy immunocompetent males.

A review of the physiology of the canine digestive tract related to the development of in vitro systems.

Food and nutrition studies in animals and human beings often meet with technical difficulties and sometimes with ethical questions. An alternative to research in living animals is the dynamic multicompartmental in vitro model for the gastrointestinal tract described by Minekus et al. (1995) and Havenaar & Minekus (1996). The dynamic conditions that are simulated in this model are peristaltic movements, transit times, pH responses, secretion of enzymes and electrolytes and absorption of nutrients and water. To obtain data for an in vitro model of the dog gastrointestinal tract, the literature was surveyed for physiological responses to different types of dog food. These included: values of enzyme activities, electrolyte concentrations, gastric emptying and intestinal transit times, pH values, secretion and composition of bile and absorption rates in different parts of the dog gastrointestinal tract. The review focuses on research carried out on healthy, adult dogs of 10-20 kg and on parameters related to the oral cavity, stomach and small intestine. This literature research gives sufficient data on the physiology of the canine digestive tract for the development of an in vitro dynamic model that adequately simulates the functions of the stomach and small intestine of the dog.

Safe biotechnology. 8. Transport of infectious and biological materials. Working Party "Safety in Biotechnology" of the European Federation of Biotechnology.

The transport of infectious and biological material is regulated by a number of international organizations. This mini-review has been compiled to increase awareness within the scientific community of problems caused by differences in terminology (such as infectious materials/substances, biological products, diagnostic specimens, genetically modified microorganisms) and certain technical aspects of the main international guidelines, and to assist policy makers in the creation of harmonized guidelines. A list of relevant Internet resources has been compiled.

Survival of lactic acid bacteria in a dynamic model of the stomach and small intestine: validation and the effects of bile.

This study was conducted to validate a dynamic model of the stomach and small intestine to quantify the survival of lactic acid bacteria and to assess the influence of gastrointestinal secretions. The survival of a single strain of each of the following species, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus bulgaricus, and Streptococcus thermophilus, was measured under physiological conditions (e.g., peristalsis, changes in pH, and changes in concentrations of enzymes and bile) and were compared with data obtained from humans. No significant differences were found between the in vitro and in vivo data, indicating that the model has a predictive value for the survival of these bacteria in humans. The survival of these strains of lactic acid bacteria in the gastrointestinal model was investigated under two different conditions in the small intestine: simulation of physiological secretion of bile and low bile secretion. Reductions in viability were significantly different between the bacterial species. The dose-response effect of bile on the survival of the tested bacteria was significant, demonstrating the bactericidal effect of bile salts. This study demonstrates the differences among bacterial species in their sensitivity to gastric and intestinal secretions.

Safe biotechnology. 7. Classification of microorganisms on the basis of hazard. Working Party "Safety in Biotechnology" of the European Federation Biotechnology.

The current systems for classifying human pathogens on the basis of hazard are well developed and their basic criteria are in general agreement one with another. Of more importance, the safety practices based on these classifications have generally been successful. They have enabled extensive research activities, medical practice and industrial production to be conducted on an ever-increasing scale, involving dangerous microorganisms (e.g. in vaccine production and treatment of infected patients) with a very low incidence of adverse effects on the workers involved and the general public. Although the EU has adopted a harmonised list of agents in groups 1-4 there is as yet no complete agreement among member states and individual microbiologists. The purpose of this paper is to present a historical survey and to discuss the current processes for identifying and classifying the hazards posed by the use of microorganisms in research and technology. This is essential in the design of appropriate methods of counteracting potential risks.

The effectiveness of Ivomec and Neguvon in the control of murine mites.

The effectiveness of different methods of external applications of Neguvon and Ivomec for treating infestations with Myobia musculi and Myocoptes spp. in conventional colonies of laboratory mice was investigated. Ivomec appeared to be effective over a longer period of time than Neguvon. There was no difference in the results obtained with different methods of application, but spraying seems to be more practical for large colonies of mice than individual dipping.

The use of repeated treatment with Ivomec and Neguvon spray in the control of murine fur mites and oxyurid worms.

In a previous study, a single external treatment with Ivomec appeared to be more effective than Neguvon treatment. In this study the antiendoparasitic qualities of external application of Ivomec were investigated, together with the effectiveness of a combined Neguvon and Ivomec treatment. After 3 treatments, all mice were mite- and worm-free: they remained free of ectoparasites until 18 weeks after the last treatment; eggs of endoparasites reappeared 9 weeks after the last treatment.

Assessment of discomfort in gallstone-bearing mice: a practical example of the problems encountered in an attempt to recognize discomfort in laboratory animals.

In order to obtain practical experience on the recognition, assessment and evaluation of discomfort in laboratory animals, the degree of discomfort was studied in gallstone-free and gallstone-bearing mice. Out of nine parameters to which scores were assigned per individual mouse, only the response to palpation of the right hypochondrium was found to score significantly higher in gallstone-bearing mice. That is, the incidence of squeaking and the magnitude of muscular contractions were significantly higher in these animals compared with the gallstone-free mice. The stance of the gallstone-bearing mice also tended to be abnormal, although the difference between gallstone-free and gallstone-bearing animals did not reach statistical significance. Although this study does not prove unequivocally that the induction of gallstones per se causes discomfort or pain in mice, we tentatively conclude that it does. We feel that this should be taken into account in any projected work in which gallstone induction in animals may occur. There was considerable between-assessor variation in the assignment of scores to the variables used to assess discomfort, including the response to palpation. It is concluded that the selection of parameters and the experience and/or attitude of the assessor are critically important when the magnitude of discomfort, if any, is assessed in experimental animals.

The anti-cariogenic potential of xylitol in comparison with sodium fluoride in rat caries experiments.

Previous rat caries experiments in our laboratory demonstrated an anti-cariogenic and most probably a remineralizing effect of xylitol. The purpose of the present study was to compare the caries-inhibiting property of xylitol and fluoride in drinking water. The study consisted of two experiments with Osborne-Mendel rats, superinfected with S. mutans. In the first experiment, the rats were fed ad libitum a cariogenic diet containing 20% sucrose and 5% glucose (diet SSP 20/5) and tap water containing 0%, 2%, or 4% xylitol. An additional control group was fed diet SSP 20/5 supplemented with 5% xylitol. In the second experiment, the rats were also fed diet SSP 20/5, but received tap water, demineralized water (both containing less than 0.1 ppm F-), or tap water containing 10 and 40 ppm fluoride. Some animals in the 4% xylitol drinking water group showed a mild osmotic diarrhea for the first four days and a retarded increase in body weight of limited duration. S. mutans colonized to a similar level in all groups of both experiments. Fluoride as well as xylitol in the drinking water reduced the fissure caries scores significantly. However, 10 and 40 ppm fluoride had a more pronounced inhibiting effect on the caries experience than did 4% xylitol in the drinking water or 5% xylitol in the diet.