Digestive system simulator apparatus for high-yield screening

Abstract

The invention concerns a digestive system simulator apparatus for high-yield screening comprising an in vitro apparatus for individualized batch simulation of the digestive system, more precisely the colon, using dozens of independent, parallel, hermetically sealed reactors, partially submerged in a water bath to allow temperature and agitation control, with a flow of nitrogen in each reactor to provide an anaerobic atmosphere, as the microorganisms in the colon are restricted or facultative anaerobes. In this way, the present batch apparatus can, for example, allow several active ingredients in different doses and combinations thereof, as well as several comparators, to be tested simultaneously, one in each reactor, whereas dynamic colonic fermenters only allow one active ingredient or one comparator in a single dose at a time.

Description

“DIGESTIVE SYSTEM SIMULATOR APPARATUS FOR HIGH-THROUGH SCREENING”

[0001] The present invention is in the field of biology, more specifically, in the field of in vitro simulating devices of the digestive system. The development of new devices simulating the digestive system also allows for the improved study of different intestinal microbiotas, whether healthy or pathological, the evaluation of different treatments on the intestinal microbiota aiming at its modulation and, finally, its effects on the human organism.

[0002] Digestion is a complex process of chemical and physical transformations carried out by the body with the intention of obtaining smaller, soluble and absorbable compounds from food. After digestion, the absorption of such compounds begins and, finally, after digestion and absorption, all elements that were not digested or absorbed are eliminated from the body through defecation.

[0003] Therefore, it is a process that begins in the mouth, with chemical and physical processes through chewing and the release of salivary enzymes for the preliminary degradation of macromolecules. After this stage, the food is carried by peristaltic movements to the stomach. There, other specific enzymes, such as proteases, act in the digestion of proteins. After passing through the stomach, the still undigested macromolecules reach the small intestine, where they are acted upon by pancreatic enzymes, such as lipases (fat digestion), amylases (carbohydrate digestion) and again proteases (protein digestion), in addition to the bile secreted by the gallbladder, to provide absorption of small nutrients in the small intestine itself.

[0004] The intestine is initially composed of the small intestine, in which, as previously mentioned, a large part of the nutrients will be absorbed, carried into the bloodstream and conducted to all the cells of the body. What is not absorbed by the small intestine (water, macromolecules not fully digested, hydrophilic molecules, which are not absorbable, insoluble fibers, etc.) will go to the large intestine, the largest part of which is made up of the colon. Due to its anaerobic nature, the colon is home to the intestinal microbiota, which is responsible for the main fermentation processes, allowing the digestion of complex carbohydrates and some proteins. The colon is also where water and mineral salts are absorbed and feces are formed.

[0005] The intestinal microbiota contains trillions of microorganisms, which corresponds to millions of genes, almost a hundred times the amount of human genes. The intestinal microbiota is composed of several viruses, bacteria, fungi and archaea that can have profound effects on controlling the proliferation of pathogenic bacteria present in the gastrointestinal tract, the production and absorption of vitamins and nutrients, and the regulation of the gastrointestinal, immune, endocrine and nervous systems. Although it is already known that more than 99% of the species in the intestine are anaerobic, in-depth studies on the subject are still necessary, since not all species present can be cultivated in conventional culture media.

[0006] Recent discoveries have increasingly demonstrated the great relevance and impact of the composition of the intestinal microbiota on the state of health and general well-being of human beings, suggesting that there is a mutualistic and symbiotic relationship, of cooperation, with its host. At the same time, it has been reported that a growing number of pathologies, such as those affecting the gastrointestinal, immune, endocrine and nervous systems, have relevant contributions or even origin from dysbiosis (imbalance) of the intestinal microbiota.

[0007] Therefore, more in-depth studies are needed on how the physical-chemical processes of digestion in the human body and their alterations can lead to dysbiosis, as the imbalance of the intestinal microbiota affects a series of physiological processes, resulting in the emergence and / or worsening of intestinal and extra-intestinal pathological conditions. To this end, the physical-chemical variables of temperature and pH, the action of different enzymes in the digestion of macromolecules in the different compartments of the gastrointestinal tract and also the impact of different profiles of intestinal microbiota and metabolites produced on the human body can be considered. It is also necessary to analyze how different foods, ingredients, nutrients, microorganisms, biomolecules, organic molecules, natural products, metabolites, etc., collectively categorized as prebiotic, probiotic, postbiotic or symbiotic actives, can modulate the composition and diversity of the intestinal microbiota and act in the prevention, attenuation and even reversal of certain diseases.

[0008] Due to the difficulty and complexity of carrying out such studies in animal models or in humans, over the years some in vitro simulating devices of the gastrointestinal system have been developed that could aid in this understanding, some of which are described in the state of the art.

[0009] For example, the technology called SHIME (Simulator of Human Intestinal Microbial Ecosystem) comprises a simulator of the gastrointestinal tract that contains 5 reactors connected to each other in a fixed apparatus of large size for simulating the stomach, small intestine and colon. Other technologies, such as SIMGI (Simulator of the Gastrointestinal Tract), focus only on simulating digestion in the stomach. PolyFermS, on the other hand, immobilizes fecal material, the source of intestinal microbiota, in a central colonization reactor, whose contents are distributed to other reactors. Another example of a gastrointestinal tract simulation apparatus is TIM-2, which has a vertical simulator of only the colon, which is capable of simulating small volumes and providing a more limited study on the effect of different active ingredients on the intestinal microbiota.Therefore, current in vitro simulation models of the gastrointestinal system comprise reactors or compartments that are connected to each other and can be powered by different sources to allow them to function, but which have a rigid design that is not very adaptable and practical. Additionally, the vast majority of them, as in the case of dynamic colonic fermenters, require a stabilization period of at least 2 weeks for the inoculated intestinal microbiota to adapt to in vitro conditions, a control period of at least 1 week and, finally, 7 to 14 days of treatment with the active ingredient of interest.

[0010] Therefore, some improvements in these devices are necessary, due to the long time, high quantities of consumables / reagents and high cost. operational, so that this technology is useful in screening different active ingredients, doses and their combinations, in evaluating intestinal microbiotas with different profiles and in optimizing culture media, protocols and experimental conditions.

[0011] Thus, the present invention solves this issue, providing an in vitro apparatus for batch simulation of the digestive system, more precisely the colon, in an individualized manner, through the use of dozens of independent, parallel reactors, partially submerged in a water bath to allow temperature and agitation control, hermetically sealed, with nitrogen flow in each reactor to provide an anaerobic atmosphere, since the microorganisms in the colon are restricted or facultative anaerobes.

[0012] Thus, the present batch apparatus can, for example, allow several actives in different doses and their combinations, in addition to several comparators, to be tested simultaneously, one in each reactor, while dynamic colonic fermenters allow only one active or one comparator in a single dose at a time.

[0013] Another advantage of this batch apparatus is that, since there are several individualized reactors, it is possible not only to include experimental replicates, but also to evaluate the action of an active ingredient on different intestinal microbiotas in parallel and simultaneously. In other words, this design allows the microbiotas of different donors to be studied in their individualized form, allowing a better understanding of the variability of effects between individuals. In dynamic colonic fermenters, a mixture of different intestinal microbiotas is often used.

[0014] Additionally, since there is no stabilization period for the intestinal microbiota inoculated in the reactors, the present invention allows testing microbiotas with profiles closer to those of the donors. In other words, this makes it possible to more clearly evaluate the effects of the active ingredients on certain microbial signatures specific to a disease or condition.

[0015] The batch apparatus allows screening in a short period of time, between 6 and 48 hours, and consumes much less reagents / consumables, due to both the shorter experimental time and the smaller volume in relation to dynamic colonic fermenters.

[0016] The batch apparatus, due to its fast response time, provides efficient optimization of culture media, protocols and experimental conditions.

[0017] In the case of culture media, the batch apparatus allows the use of media with low nutrient content to help highlight the action of the active substance under investigation and quickly identify the resulting metabolites and microorganisms involved, facilitating the interpretation of results. This can provide clues on how to direct microbial metabolism towards a specific objective (e.g., production of a beneficial metabolite or favoring the growth of a beneficial bacteria), promoting alternatives for modulating the intestinal microbiota. Dynamic colonic fermenters, on the other hand, due to the nature of this technology, require nutrient-rich media in long-term studies to maintain the intestinal microbiota in good condition during the experiment. This makes it difficult to separate the effect on the intestinal microbiota coming purely from the active substance under investigation.

[0018] While dynamic colonic fermenters require a large space to accommodate the reactors that simulate the different portions of the colon, the batch apparatus is much more compact.

[0019] Regarding costs, the present batch apparatus is 10-20 times cheaper and 3-4 times faster to build than dynamic colonic fermenters. Experimental costs are also much lower, not only because of the shorter experimental time and smaller scale, requiring fewer reagents / consumables, but also because they do not require other integrated equipment, such as volume, residence time and pH control systems, and peristaltic pumps to move the culture and active medium from one reactor to another.

[0020] The batch apparatus also does not require specialized maintenance, making it cheaper and quicker to repair in the event of problems.

[0021] The batch apparatus does not require large amounts of osmosis or distilled water to control the temperature during the experiment, and is therefore more sustainable.

[0022] The batch apparatus has an anaerobic system, agitation system, temperature control and fermentation reactors on a single integrated platform, allowing easy transportation and installation in different locations in research laboratories.

[0023] This batch apparatus has lids designed to allow nitrogen to enter, sample collection and gas to exit to relieve the internal pressure generated during the fermentation process, making it very safe to use.

[0024] All reactors in this batch apparatus and their respective components are autoclavable, reducing the risk of external contamination.

[0025] In our search for patent documents that were potentially related to the said invention, some documents were found, such as CN 209468445, CN 203923186, LU 501780, CN 108251299 and CN 1 15433660.

[0026] Although they all concern different formats and configurations of fermentation simulator apparatus, none of the above documents or models was able to anticipate all the different aspects of the present invention.

[0027] The present invention, therefore, proves to be an expanded option in relation to the state of the art for simulating the gastrointestinal tract, since it requires less time, smaller quantities of consumables / reagents and lower costs for construction, maintenance and operation during experiments. The technology becomes especially useful in screening different active ingredients, doses and their combinations, in screening different comparators, in obtaining experimental replicates, in the individualized evaluation of intestinal microbiotas with different profiles and in optimizing culture media, protocols and experimental conditions.

[0028] The present invention emerges as a complementary technology to dynamic colonic fermenters in the evaluation of active ingredients in the modulation of the intestinal microbiota, but it is essential to be applied before using the latter.

[0029] Thus, the present invention comprises an in vitro batch intestinal fermentation simulator apparatus (25) comprising a mobile caster (24) with at least one cabinet (16) with: - at least one tank (8) containing water for a water bath (22) and an internal platform (15) for fitting into fasteners (23) of at least one intestinal fermentation reactor flask (19), said reactor arranged individually for simulating colon fermentation reactions in said tank (8); - device for On / Off (1 1 ), devices for pressure control (1 ), devices for input (2), output (4) and control of the flow (3) of nitrogen, devices for induction (17), control (12) and visualization (10) of temperature, devices for induction (14), control (13) and visualization (9) of agitation; - devices for controlling the safety (5 and 7) of the inputs and / or outputs (6) of the reactor flask (19); - where each reactor flask (19) comprises a lid (18) containing an inlet and / or outlet compartment (6) and at least one internal (20) and external (21) connection.

[0030] This report makes reference to the attached drawings, indicated as follows:

[0031] Figure 1 illustrates the top side view of the following elements of the intestinal batch fermentation simulator apparatus: (1) Pressure gauges, (2) Nitrogen inlet, (3) Flowmeter (to control nitrogen inlet), (4) Gas outlet, (8) Stainless steel tank, (9) Agitation display, (10) Temperature display, (1 1) On / Off, (12) Temperature control, (13) Agitation control, (16) Carbon steel cabinet, (24) Caster system for easy movement, (25) Digestive system simulator apparatus for high-throughput screening (Mini-xGIb / om / cs), equipment in an integrated and compact format.

[0032] Figure 2 illustrates the exploded side top view of the following elements of the intestinal batch fermentation simulator apparatus: (5) Safety valve for excess gas release; (6) Outlet for sampling or nitrogen inlet; (7) Safety clamp; (8) Stainless steel tank; (15) Internal platform with gallery with capacity for 20 100ml reactor flasks; (19) Reactor flask (100ml); (23) Reactor flask fasteners.

[0033] Figure 3 illustrates the top view of the following elements of the intestinal batch fermentation simulator apparatus: (8) Stainless steel tank; (14) Reciprocating agitation device with an amplitude of 30 mm through bearings; (15) Internal platform with a gallery with capacity for 20 100 ml reactor flasks; (17) Thermometer; (23) Reactor flask holders.

[0034] Figure 4 illustrates the front view cut out in the left corner, highlighting the following element of the intestinal batch fermentation simulator apparatus: (22) Water bath for heating the reactor flasks.

[0035] Figure 5 illustrates the top front and bottom front views of the various elements that make up the reactor flask (100 ml): (5) Safety valve for excess gas release; (6) Outlet for sampling or nitrogen input; (7) Safety clip; (18) Screw cap; (19) Reactor flask (100ml_); (20) Internal connection; (21) External connection. Description of the invention and example of embodiment of the invention

[0036] Thus, the present invention comprises an in vitro batch intestinal fermentation simulator apparatus (25) comprising a mobile caster (24) with at least one cabinet (16) with: - at least one tank (8) containing water for a water bath (22) and an internal platform (15) for fitting into fasteners (23) of at least one intestinal fermentation reactor flask (19), said reactor arranged individually for simulating colon fermentation reactions in said tank (8); - device for On / Off (1 1 ), devices for pressure control (1 ), device for input (2), output (4) and control of the flow (3) of nitrogen, devices for induction (17), control (12) and visualization (10) of temperature, devices for induction (14), control (13) and visualization (9) of agitation; - devices for controlling the safety (5 and 7) of the inputs and / or outputs (6) of the reactor flask (19); - where each reactor flask (19) comprises a lid (18) containing an inlet and / or outlet compartment (6) and at least one internal (20) and external (21) connection, as illustrated and exemplified in figures 1, 2, 3, 4 and 5 of the present invention, which illustrate several views of the various elements of the intestinal batch fermentation simulator apparatus, indicating the following elements: (1) Pressure gauges; (2) Nitrogen inlet; (3) Flowmeter (to control the nitrogen inlet); (4) Gas outlet; (5) Safety valve for excess gas exhaust; (6) Outlet for sampling or nitrogen inlet; (7) Safety clamp; (8) Stainless steel tank; (9) Agitation display; (10) Temperature display; (11) On / Off; (12) Temperature control; (13) Agitation control; (14) Device for agitation with reciprocating movement with an amplitude of 30 mm through bearings and rollers;(15) Internal platform with gallery with capacity for 20 100 mL reactor flasks; (16) Carbon steel cabinet; (17) Thermometer; (18) Screw-on lid; (19) Reactor flask (100 mL); (20) Internal connection; (21) External connection; (22) Water bath for heating the reactor flasks, (23) Reactor flask fasteners; (24) Caster system for easy movement; (25) Digestive system simulator apparatus for high-throughput screening (Mini-xGIb / om / cs), equipment in integrated and compact format.;

Claims

CLAIM 1. APPARATUS FOR SIMULATING THE DIGESTIVE SYSTEM FOR HIGH-THROUGH SCREENING CHARACTERIZED BY COMPRISING an in vitro batch intestinal fermentation simulator apparatus (25) comprising a mobile caster (24) with at least one cabinet (16) with: - at least one tank (8) containing water for a water bath (22) and an internal platform (15) for fitting into fasteners (23) of at least one intestinal fermentation reactor flask (19), said reactor arranged individually for simulating colon fermentation reactions in said tank (8); - device for On / Off (1 1 ), devices for pressure control (1 ), devices for input (2), output (4) and control of the flow (3) of nitrogen, devices for induction (17), control (12) and visualization (10) of temperature, devices for induction (14), control (13) and visualization (9) of agitation; - devices for controlling the safety (5 and 7) of the inputs and / or outputs (6) of the reactor flask (19); - where each reactor flask (19) comprises a lid (18) containing an inlet and / or outlet compartment (6) and at least one internal (20) and external (21) connection.

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