A device and method for fully automatically simulating human gastrointestinal digestion and fermentation

Abstract

The present invention discloses a device and method for fully automatic simulation of human gastrointestinal digestion and fermentation, the device comprises a device frame, a device electrical control box and an oral digestion tank, a stomach digestion tank, a duodenum digestion tank, a jejunum digestion tank, an ileum digestion tank, an ascending colon digestion tank, a transverse colon digestion tank and a descending colon digestion tank installed in the device frame for simulating human gastrointestinal digestion and fermentation and connected in sequence. This application will for the first time provide a detailed description of a fully automatic simulation device and method for all components of the human gastrointestinal tract, and for the first time provide a detailed simulation description of the structure and design of each part. The device and its implementation method can observe the digestion and fermentation of various foods, active or nutrient substances after entering the human body, and help explore the digestion and fermentation of various foods, active or nutrient substances after entering the human body.

Description

Technical Field

[0001] The present invention belongs to the fields of bionics, human physiology, microbiology, nutrition, organic chemistry and analytical testing technology, and more precisely, relates to a device and method for fully automatically simulating digestive fermentation in the human gastrointestinal tract. Background Art

[0002] The digestion and absorption of food by the human body is a very complex process. After food is ingested, it is converted into nutrients for the growth of the body. During the human digestion process, two main processes occur almost simultaneously: (1) the size of food particles is reduced during mechanical transport in the digestive tract; (2) the large molecules in the food are hydrolyzed into small molecules and then absorbed into the blood. The food crushing process mainly occurs in the mouth and stomach, while enzymatic hydrolysis and the absorption of nutrients and water mainly occur in the small intestine and large intestine.

[0003] At present, the digestive system has become the focus of various researches in various fields (such as nutrition, toxicology, physiology and microbiology). Unfortunately, studying this complex multi-stage process of human digestion is economically and technically difficult, and is also subject to ethical constraints when potential toxic and harmful substances are involved in the research process. Therefore, there is an urgent need for a flexible, accurate and reproducible in vitro model to simulate the physiological processes of human digestion and absorption.

[0004] In the past, scientists from various countries have been trying to make gastrointestinal models, but most in vitro models are static, contain only a few simulation parameters, and only target specific organs. For example, the gastrointestinal digestive system digestive juice developed by Dutch scientists AGOomen et al. in 2002 only contains saliva, gastric juice, pancreatic juice and bile. However, in the process of simulating the complex physiological, biochemical and physicochemical properties of the digestive tract, it is very important to simulate the time, pH, and enzymatic conditions of each meal or the substance to be studied during the digestive process. Therefore, some dynamic multi-digestive organ models have been developed and applied to research, such as the human gastric simulator (HGS) developed by American scientists Kong F and Singh PR in 2010 to simulate the digestion of food in the stomach. The simulated human intestinal micro-ecosystem (SHIME) reactor was developed and developed by Belgian scientists K. Molly, M. Vande Woestyne, and W. Verstraete in 1993.

[0005] The establishment and method research of human gastrointestinal bionic system started earlier abroad and received more attention, while domestic research is relatively lagging behind. Currently, only a few institutions are conducting research on devices and methods. For example, utility model patent authorization number CN201634678U discloses a small fermentation device that simulates the intestinal environment. The device can be used for laboratory aerobic, anaerobic, gas-producing, non-gas-producing and other micro-fermentations, and can accurately collect and measure the volume of gas produced by fermentation. Invention patent authorization number CN102533543B discloses a device that can simulate the human intestinal environment for intestinal microbial culture. The device can provide and maintain a relatively constant temperature and humidity, and can have a certain oscillation to achieve the effect of simulating the intestinal environment. Utility model patent authorization number CN202658158U discloses an intelligent biological gastrointestinal digestive system simulation control device, which can be controlled by a computer to simulate the gastrointestinal digestive system. Invention patent authorization number CN101482460B discloses a unit animal bionic digestive system and a method for simulating unit animal digestion based on the system. The system and method realize the process of simulating animal gastrointestinal digestion and absorption of feed under in vitro conditions. Invention patent authorization number CN101665758B discloses a simulation test method for reproducing the human gastrointestinal tract and microecological system under in vitro conditions. This method is based on the human intestinal microecological system simulation device introduced by Ghent University in Belgium, and is technically improved according to the living habits of Oriental people to achieve the reproduction of the human gastrointestinal system under in vitro conditions.

[0006] These patent documents have greatly enriched the devices and methods of use suitable for digestive fermentation, but so far, these patents have not provided detailed descriptions on simulating specific oral digestion; secondly, these patents have not provided detailed simulations and descriptions of the gastric emptying process in the simulation of the stomach; at the same time, these patents have not provided detailed descriptions on the parameters for controlling the small intestine simulation system and the large intestine fermentation system; some of these patents are based only on animal-related parameters and have little relevance to humans.

[0007] This patent application will, for the first time, simulate the digestion and glycolysis of various parts completely according to the human body's digestion and glycolysis process and physiological environment, and provide detailed descriptions of each digestion and glycolysis part. At the same time, the process of each digestion and glycolysis part is fully automatically and dynamically simulated, which is more in line with the real human physiological environment. Summary of the invention

[0008] In view of the above problems, the present invention provides a device and method for fully automatically simulating digestion and fermentation of the human gastrointestinal tract. The present invention will for the first time provide a detailed description of the fully automatic simulation device and method for all components of the human gastrointestinal tract, and for the first time provide a detailed simulation description of the structure and design of each part. The device and implementation method can observe the digestion and fermentation of various foods, active or nutrient substances after entering the human body, and help explore the digestion and fermentation of various foods, active or nutrient substances after entering the human body.

[0009] The technical solution adopted by the present invention to solve the technical problem is:

[0010] A fully automatic device for simulating digestion and fermentation of the human gastrointestinal tract, the device comprising a device frame, a device electrical control box, and an oral digestion tank, a stomach digestion tank, a duodenum digestion tank, a jejunum digestion tank, an ileum digestion tank, an ascending colon digestion tank, a transverse colon digestion tank, and a descending colon digestion tank installed in the device frame and used for simulating digestion and fermentation of the human gastrointestinal tract, and the digestion tanks are connected in sequence, wherein each digestion tank is connected by a transfer pipeline, and the jejunum digestion tank, the ileum digestion tank, the ascending colon digestion tank, and the transverse colon digestion tank are connected with a membrane absorption device;

[0011] Each digestion tank comprises a tank cover, a tank cover flange, a tank inner layer, a cleaning ball arranged in the tank inner layer, a liquid transfer interface and a drain valve arranged at the lower end of the tank inner layer, and a tank outer layer jacket, and the tank cover is provided with a digestion liquid port, a digestion sample port, a drain port, a sampling port, an inoculation port, an acid liquid port, an alkali liquid port, an air inlet, an air outlet, and a cleaning ball interface;

[0012] The membrane absorption device comprises a fixture fixing base and a front fixture and a rear fixture installed thereon, a flat membrane is arranged between the front fixture and the rear fixture, a digestion liquid / fermentation liquid inlet is arranged at the lower end of the front fixture, and a concentrated liquid reflux port is arranged at the upper end, the digestion liquid / fermentation liquid inlet and the concentrated liquid reflux port are both connected to the corresponding digestion tank through a transfer pipeline, and the flat membrane is connected to a dialysis bag through a membrane absorption pipeline;

[0013] The sampling port of each digestion tank is connected to a sampling pipeline, a sampling needle is arranged at the end of the sampling pipeline, and the sampling needle is installed above the automatic sampling equipment.

[0014] As an improvement of the above technical solution, the automatic sampling equipment includes a sampling bottle rotating table, a plurality of sampling bottles are installed on the upper end surface of the sampling bottle rotating table, a sampling needle slider and a slide rail are arranged directly above the sampling bottle rotating table, and the sampling needle is electrically or manually moved horizontally and vertically with the cooperation of the sampling needle slider and the slide rail to be inserted into the sampling bottle for quantitative sampling, an ethanol needle slider is installed on the slide rail, and an ethanol needle is installed at the front end of the ethanol needle slider.

[0015] As an improvement of the above technical solution, the digester tank cover is also provided with a dissolved oxygen electrode interface, a pH electrode interface and a tank temperature electrode interface.

[0016] As an improvement of the above technical solution, the outer jacket of the digester tank body is connected with a tank jacket water inlet and a tank jacket water outlet.

[0017] As an improvement of the above technical solution, the front clamp and the rear clamp of the membrane absorption device are fixed by membrane fastening screws.

[0018] As an improvement of the above technical solution, the dialysis bag of the membrane absorption device is connected to the corresponding digestion tank through a transfer pipeline.

[0019] As an improvement of the above technical solution, the transfer pipeline, membrane absorption pipeline and sampling pipeline are all equipped with peristaltic pumps.

[0020] The present invention also provides a method for fully automatically simulating digestion and glycolysis of the human gastrointestinal tract, which is applied to any of the above-mentioned devices for fully automatically simulating digestion and glycolysis of the human gastrointestinal tract, and the method comprises the following steps:

[0021] Step 1: Connect each digestion tank, supporting pipeline, supply pipeline and detection electrode;

[0022] Step 2: Disinfect empty cans

[0023] Turn on the magnetic stirring of each digestion tank, enter high-pressure pure water for cleaning, and the residue is discharged from the drain valve at the bottom of each digestion tank. At the same time, each transfer peristaltic pump pipeline and sampling pipeline are rinsed with pure water for 3-10 minutes. After the end, steam is introduced to sterilize each digestion tank at high temperature for 25-30 minutes. After the end, nitrogen is introduced, and then 80-100mL ethanol is introduced to sterilize each transfer peristaltic pump pipeline and sampling pipeline. After the end, 200-300mL pure water is introduced to clean each digestion tank and each transfer peristaltic pump pipeline and sampling pipeline. After the end, high-temperature nitrogen is introduced to dry each digestion tank. After the end, normal temperature nitrogen is introduced with a flow rate of 1-10mL / min to ensure positive pressure and anaerobic environment of each digestion tank;

[0024] Step 3: Simulate digestion

[0025] The simulated digestion was performed in the following order:

[0026] Oral digestion → stomach digestion → duodenum digestion → jejunum digestion → ileum digestion;

[0027] Oral digestion: Connect the sterilized replenishing fluids to the oral digestion tank, turn on the magnetic stirring, and start the circulating temperature control. Before digestion, pump 15-20mL of oral digestion fluid, then use the transfer peristaltic pump to pump the digestion sample into the oral digestion tank, pump in 80-100mL of digestion sample, flow rate 80-100mL / min, then pump in 70-80mL of oral digestion fluid, flow rate 80-100mL / min, 0.3-0.5mL of CaCl2 solution, flow rate 0.8-1.0mL / min, pH = 6.8-7.0, temperature 37°C, digestion 3 -5min, sampling time points: 0min, 3-5min, magnetic stirring is always running; when oral digestion is finished and the peristaltic pump for transferring oral digestive fluid stops working, gastric digestion is started, and pre-cleaning is automatically performed at the same time: first, the oral digestion tank drain valve is automatically opened, and the tank body is cleaned through the shower head above the oral digestion tank body. After cleaning for 3-10min, the oral digestion tank drain valve is closed, and then 80%-90% of the tank volume is immersed in pure water, and the oral digestion tank sampling pipeline is started for pre-cleaning, and the cleaning is performed for 1-2min, and the transfer pipeline is waited for cleaning, and the automatic cleaning and sterilization operation is performed;

[0028] Gastric digestion: Connect the sterilized replenishing fluids to the gastric digestion tank, turn on the magnetic stirring, pump 50-60mL of gastric digestion fluid into the gastric digestion tank before digestion, transfer 180-200mL of oral digestion sample to the gastric digestion tank, and after the transfer, pump 130-150mL of gastric digestion fluid into the tank through the peristaltic pump, with a flow rate of 80-100mL / min, 0.3-0.5mL of CaCl2 solution, a flow rate of 0.8-1.0mL / min, and pH = 0.0005t 2 -0.079t+4.47, digestion 120-150min, sampling time points 0, 10, 20, 30, 60, 120-150min, magnetic stirring runs three times per minute, each time lasting 10-15s; when gastric digestion is completed and the gastric digestion fluid transfer peristaltic pump stops working, duodenal digestion is turned on, and pre-cleaning is automatically performed at the same time: first, the gastric digestion tank drain valve is automatically opened, and the transfer peristaltic pump between the oral digestion tank and the gastric digestion tank is turned on, and the pure water in the oral digestion tank is quickly transferred for 1-2min, and the pre-cleaning of the transfer pipeline between the oral digestion tank and the gastric digestion tank is completed, and then the tank body is cleaned through the shower above the gastric digestion tank body. After cleaning for 3-10min, the gastric digestion tank drain valve is closed, and then 80%-90% of the tank volume is immersed in pure water, and the pre-cleaning of the gastric digestion tank sampling pipeline is turned on, and the cleaning is carried out for 1-2min, and the transfer pipeline is waited for cleaning, and the automatic cleaning and sterilization operation is performed;

[0029] Duodenal digestion: connect the disinfected replenishing fluids to the duodenal digestion tank, turn on the magnetic stirring, transfer the peristaltic pump to transfer 350-400mL of gastric digestion sample to the duodenal digestion tank. After the transfer is completed, pump in 380-400mL of intestinal electrolytes through the peristaltic pump, with a flow rate of 80-100mL / min, 0.6-0.8mlCaCl2 solution, a flow rate of 0.8-1.0mL / min, pH=6.1-6.2, digest for 45-50min, and sampling time points of 0, 10, 20, 45-50min. The magnetic stirring is always running; when the duodenal digestion is completed and the peristaltic pump for duodenal digestion fluid transfer is stopped When the machine stops working, the jejunum digestion is turned on and pre-cleaning is automatically performed at the same time: first, the drain valve of the duodenal digestion tank is automatically opened, and the transfer peristaltic pump between the stomach digestion tank and the duodenal digestion tank is turned on to quickly transfer the pure water in the stomach digestion tank for 1-2 minutes, and complete the pre-cleaning of the transfer pipeline between the stomach digestion tank and the duodenal digestion tank. Then, the tank body is cleaned through the shower above the duodenal digestion tank body. After cleaning for 3-10 minutes, the drain valve of the duodenal digestion tank is closed, and then 80%-90% of the tank volume is immersed in pure water. The pre-cleaning of the sampling pipeline of the duodenal digestion tank is turned on for 1-2 minutes, and the transfer pipeline is waited for cleaning, and the automatic cleaning and sterilization operation is performed;

[0030] Jejunal digestion: connect the disinfected replenishing fluids to the jejunal digestion tank, turn on the magnetic stirring, and transfer all the duodenal digestion samples to the jejunal digestion tank through the transfer peristaltic pump, with a total volume of 700-800mL, pH=6.4-6.5, digestion time 45-50min, sampling time points 0, 10, 20, 45-50min, and magnetic stirring running all the time. After sampling, the peristaltic pump is used to complete the absorption, with a flow rate of 50-100mL / min. During the absorption process, the absorbed water molecules are pumped back into the jejunal digestion tank for cyclic absorption, and small molecules are absorbed as much as possible. The absorption lasts for 15min-30min until 55%-60% of the total volume is absorbed, and the remaining volume is 300-320mL; wait until the jejunal digestion is completed and the jejunal digestion fluid is transferred When the peristaltic pump stops working, the ileum digestion is turned on and pre-cleaning is automatically performed at the same time: first, the jejunum digestion tank drain valve is automatically opened, and the transfer peristaltic pump between the duodenum digestion tank and the jejunum digestion tank is turned on to quickly transfer the pure water in the duodenum digestion tank for 1-2 minutes, and complete the pre-cleaning of the transfer pipeline between the duodenum digestion tank and the jejunum digestion tank. Then, the tank body is cleaned through the shower above the jejunum digestion tank body. After cleaning for 3-10 minutes, the jejunum digestion tank drain valve is closed, and then 80%-90% of the tank volume is immersed in pure water. The pre-cleaning of the jejunum digestion tank sampling pipeline is turned on for 1-2 minutes, and then the cleaning of the jejunum digestion tank membrane absorption pipeline is turned on. The cycle cleaning is 3-5 minutes, and the transfer pipeline is waited for cleaning, and the automatic cleaning and sterilization operation is performed;

[0031] Ileal digestion: connect the disinfected replenishing fluids to the ileal digestion tank, turn on the magnetic stirring, and transfer the remaining digestion sample in the jejunal digestion tank to the ileal digestion tank through the transfer peristaltic pump, with a flow rate of 80-100mL / min, pH=7.4-7.5, digestion time of 90-100min, sampling time points of 0, 10, 20, 30, 60, 90-100min, and magnetic stirring running all the time. After sampling, the peristaltic pump is used to complete the absorption. During the absorption process, the absorbed water molecules are pumped back into the ileal digestion tank for cyclic absorption, and small molecules are absorbed as much as possible for 15-30min, until 55%-60% of the total volume is absorbed, and the remaining volume is 120-130mL; after the ileal digestion is completed and the ileal digestion fluid is transferred to the peristaltic pump, the water molecules are pumped back into the ileal digestion tank for cyclic absorption, and small molecules are absorbed as much as possible for 15-30min. When the pump stops working, the ascending colon digestion is turned on and pre-cleaning is automatically performed at the same time: first, the sewage valve of the ileum digestion tank is automatically opened, and the transfer peristaltic pump between the jejunum digestion tank and the ileum digestion tank is turned on to quickly transfer the pure water in the jejunum digestion tank for 1-2 minutes, and complete the pre-cleaning of the transfer pipeline between the jejunum digestion tank and the ileum digestion tank. Then, the tank body is cleaned through the shower above the ileum digestion tank body. After cleaning for 3-10 minutes, the sewage valve of the ileum digestion tank is closed, and then 80%-90% of the tank volume is immersed in pure water. The pre-cleaning of the sampling pipeline of the ileum digestion tank is turned on for 1-2 minutes, and then the cleaning of the membrane absorption pipeline of the ileum digestion tank is turned on. The cycle cleaning is 3-5 minutes, and the transfer pipeline is waited for cleaning, and the automatic cleaning and sterilization operation is performed;

[0032] Step 4: Simulate fermentation

[0033] The simulated fermentation was carried out in the following order:

[0034] Ascending colon fermentation → transverse colon fermentation → descending colon fermentation;

[0035] Ascending colon fermentation: connect the disinfected feed fluids to the ascending colon digestion tank, turn on the magnetic stirring, transfer the peristaltic pump to transfer all the ileal digestion fluid to the ascending colon digestion tank, add 120-130mL of ascending colon culture fluid through the peristaltic pump, the flow rate is 80-100mL / min, pH=5.5-6.0, fermentation time is 6-8h, sampling time points are 0, 3, 6h or 0, 4, 8h, magnetic stirring is always running, after sampling is completed, the peristaltic pump is used to complete the absorption, during the absorption process, the absorbed water molecules are pumped back to the ascending colon digestion tank, circulated and absorbed, small molecules are absorbed as much as possible, and the absorption is 15-30min, until 55%-60% of the total volume is absorbed, and the remaining volume is 90-100mL; wait until the ascending colon fermentation is completed and the ascending colon fermentation fluid is transferred The peristaltic pump stops working, the transverse colon fermentation starts, and pre-cleaning is automatically performed at the same time: first, the sewage valve of the ascending colon digestion tank is automatically opened, and the transfer peristaltic pump between the ileum digestion tank and the ascending colon digestion tank is opened to quickly transfer the pure water in the ileum digestion tank for 1-2 minutes, and complete the pre-cleaning of the transfer pipeline between the ileum digestion tank and the ascending colon digestion tank. Then, the tank body is cleaned through the shower above the ascending colon digestion tank body. After cleaning for 3-10 minutes, the sewage valve of the ascending colon digestion tank is closed, and then 80%-90% of the tank volume is immersed in pure water. The pre-cleaning of the sampling pipeline of the ascending colon digestion tank is started, and the cleaning is started for 1-2 minutes. Then, the cleaning of the membrane absorption pipeline of the ascending colon digestion tank is started, and the circulation cleaning is carried out for 3-5 minutes. Wait for the cleaning of the transfer pipeline, and the automatic cleaning and sterilization operation is performed;

[0036] Transverse colon fermentation: connect the disinfected feed fluids to the transverse colon digestion tank, turn on the magnetic stirring, transfer the peristaltic pump to transfer all the ascending colon fermentation liquid to the transverse colon digestion tank, add 90-100mL of ascending colon culture liquid through the peristaltic pump, the flow rate is 80-100mL / min, pH=6.0-6.4, fermentation time is 12-16h, sampling time points are 0, 4, 8, 12, 16h, magnetic stirring is always running, after sampling is completed, the peristaltic pump is used to complete the absorption, and the absorbed water molecules are pumped back into the transverse colon digestion tank during the absorption process, and the small molecules are absorbed as much as possible for 15-30min, until 55%-60% of the total volume is absorbed, and the remaining volume is 60-70mL; wait until the transverse colon fermentation is completed and the transverse colon fermentation liquid is transferred The peristaltic pump stops working, the descending colon fermentation starts, and pre-cleaning is automatically performed at the same time: first, the drain valve of the transverse colon digestion tank is automatically opened, and the transfer peristaltic pump between the ascending colon digestion tank and the transverse colon digestion tank is opened to quickly transfer the pure water in the ascending colon digestion tank for 1-2 minutes, and complete the pre-cleaning of the transfer pipeline between the ascending colon digestion tank and the transverse colon digestion tank. Then, the tank body is cleaned through the shower head above the transverse colon digestion tank body. After cleaning for 3-10 minutes, the drain valve of the transverse colon digestion tank is closed, and then 90% of the tank volume is immersed in pure water. The pre-cleaning of the sampling pipeline of the transverse colon digestion tank is started, and the cleaning is started for 1-2 minutes. Then, the cleaning of the membrane absorption pipeline of the transverse colon digestion tank is started, and the cycle cleaning is carried out for 3-5 minutes. Wait for the cleaning of the transfer pipeline, and the automatic cleaning and sterilization operation is performed;

[0037] Descending colon fermentation: connect the disinfected feed liquids to the descending colon digestion tank, turn on the magnetic stirring, transfer the peristaltic pump to transfer all the fermentation liquid of the transverse colon to the descending colon, add 60-70mL of ascending colon culture liquid through the peristaltic pump, the flow rate is 80-100mL / min, pH=6.5-6.9, fermentation time is 6-8h, sampling time points are 0, 3, 6 or 0, 4, 8h, magnetic stirring is always running, after sampling is completed, transfer it to the waste liquid bucket through the transfer peristaltic pump; when the fermentation of the descending colon is completed and the peristaltic pump for transferring the fermentation liquid of the descending colon stops working, automatically perform pre-cleaning: first, automatically Open the drain valve of the descending colon digester, start the transfer peristaltic pump between the transverse colon digester and the descending colon digester, quickly transfer the pure water in the ascending colon digester for 1-2 minutes, complete the pre-cleaning of the transfer pipeline between the transverse colon digester and the descending colon digester, and then clean the tank body through the shower head above the descending colon digester. After cleaning for 3-10 minutes, close the drain valve of the descending colon digester, and then soak it in pure water that accounts for 80%-90% of the tank volume. At the same time, start the pre-cleaning of the descending colon sampling pipeline, transfer pipeline, and waste pipeline. Clean for 1-2 minutes and wait for automatic cleaning and sterilization operation.

[0038] As an improvement of the above technical solution, the method further comprises the following steps:

[0039] Step 5: Automatic cleaning and sterilization

[0040] After all digestion and fermentation are completed, the digestion tanks, transfer pipelines, membrane absorption pipelines, and sampling pipelines are waiting to be formally cleaned and sterilized together:

[0041] The bottom drain valves of the digestion tanks for the oral cavity, stomach, duodenum, jejunum, ileum, ascending colon, transverse colon, and descending colon are automatically opened to discharge the wastewater in the tanks. Each digestion tank is cleaned again with high-pressure pure water, and the residue is discharged from the bottom drain valve of the tank. At the same time, the transfer peristaltic pump pipeline enters the tank side for flushing with pure water. After 3-10 minutes of cleaning, the tank without membrane absorption device is in a pending state, and the tank with membrane absorption device closes the bottom drain valve for membrane regeneration and cleaning;

[0042] Membrane regeneration and cleaning steps: pump 400-500mL of pure water into the corresponding tank, clean for 5-10min, discharge the waste water, pump in 400-500mL of membrane cleaning agent, clean for 30-40min, pump in 400-500mL of pure water, clean for 5-10min, discharge the waste water, then the corresponding tank enters steam high-temperature disinfection, and then pass air, close the bottom drain valve, and then enter 250-300mL of pure water into each corresponding tank, turn on all transfer peristaltic pumps to clean the transfer pipeline, and clean the sampling pipeline at the same time, discharge the waste water after cleaning for 3-5min, and then add 250-300mL of ethanol to each corresponding tank to disinfect the membrane absorption device, transfer pipeline, and sampling pipeline. After the end, add 400-500mL of pure water to clean the tank body and membrane absorption device, transfer pipeline, and sampling pipeline. After the end, pass high-temperature nitrogen to dry the tank body and each pipeline, and then pass normal temperature nitrogen after the end.

[0043] Other things that need special explanation:

[0044] (1) Each step of the digestion / fermentation process has automatic sampling:

[0045] Sampling system control method: the sampling bottle rotating table can rotate automatically, the samples are stored at a low temperature of about 0-2℃, at least 80-100 sampling bottles are kept in reserve, and each sampling bottle has a corresponding number. The program will distinguish which digester the sample belongs to by color to prevent confusion; the sampling system is equipped with a separate control system, and establishes industrial Ethernet communication with the main program of the electrical control box of the digestion and glycolysis system. The corresponding digester sampling time is set through the control system. When the control program detects the sampling command, it automatically samples and adds ethanol to inactivate the enzyme for storage.

[0046] Disinfection steps for sampling needles and sampling pipelines: To ensure that there is no residue in the sampling (which may easily lead to mixed contamination), the sampling pipeline close to the sampling needle must be cleaned and disinfected before and after sampling, and heated nitrogen must be passed through to blow dry. After each sampling, the digestion sample remaining in the pipeline close to the digestion (fermentation) tank side of the sampling pipeline is pushed back into the digestion tank by nitrogen to continue digestion (or fermentation), thereby making the sampling more representative.

[0047] After sampling, the simulated digestion and fermentation process is repeated three times, and the digestion and fermentation of the substance to be tested are detected using corresponding detection methods, such as liquid phase and gas phase.

[0048] (2) In each step, the transfer peristaltic pump extends the transfer time by 1-2 minutes when transferring the digestion sample, so as to transfer the digestion sample from the previous part completely and reduce the residue in the transfer pipeline.

[0049] (3) In each step, the replenishing liquid is equipped with a 37°C water bath heating device; at least two sets of glass tanks containing digestion samples, replenishing liquid, simulated absorption liquid, etc. and corresponding rubber pipes are provided.

[0050] (4) All tanks need to be equipped with an annular hollow ring attached to the wall to better simulate the secretion of digestive juices and acid and alkali solutions; the stomach needs to be equipped with a 1-2 mm sieve to simulate the stomach screening function.

[0051] The beneficial effects brought by the present invention are:

[0052] The core of the fully automatic simulation of human gastrointestinal digestion and fermentation device of the present invention is that the device and its implementation method provide a detailed description of the fully automatic simulation of all components of the human gastrointestinal tract for the first time, and also provide a detailed simulation description of the design of the structure of each part and its corresponding function for the first time.

[0053] The fully automatic simulation device and method for human gastrointestinal digestion and fermentation can observe the digestion and fermentation of various foods, active or nutritional substances after entering the human body, and help explore the digestion and fermentation of various foods, active or nutritional substances after entering the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0055] Attached Figure 1 This is a system flow chart of the fully automatic simulation of human gastrointestinal digestion and fermentation device of the present invention;

[0056] Attached Figure 2 It is a side view of the digestion tank structure in the present invention;

[0057] Attached Figure 3It is a top view of the digestion tank structure in the present invention;

[0058] Attached Figure 4 It is a three-dimensional diagram of the digestion tank structure in the present invention;

[0059] Attached Figure 5 It is a simplified structural diagram of the membrane absorption device in the present invention;

[0060] Attached Figure 6 It is a schematic diagram of the structure and the membrane absorption principle of the present invention taking the jejunum digestion tank to the ileum digestion tank as an example;

[0061] Attached Figure 7 It is a schematic diagram of the structure of the automatic sampling device in the present invention.

[0062] The label names corresponding to the serial numbers in the figure are as follows:

[0063] 101-tank inner layer, 102-cleaning ball, 103-tank outer jacket, 104-pipetting interface, 105-drain valve, 106-digestion liquid port, 107-digestion sample port, 108-drainage port, 109-sampling port, 110-inoculation port, 111-acid liquid port, 112-alkali liquid port, 113-air inlet, 114-air outlet, 115-cleaning ball interface, 116-dissolved oxygen electrode interface, 117-pH electrode interface, 118-tank temperature electrode interface, 119-tank jacket water inlet, 120-tank jacket water outlet;

[0064] 201-fixture fixing base, 202-front fixture, 203-flat membrane, 204-rear fixture, 205-digestion solution / fermentation solution inlet, 206-concentrate reflux port, 207-small molecule permeation port, 208-membrane fastening screws;

[0065] 301 - sampling bottle rotating table, 302 - sampling needle slider, 303 - slide rail, 304 - ethanol needle slider. DETAILED DESCRIPTION

[0066] Example 1

[0067] This embodiment will introduce the device for fully automatically simulating human gastrointestinal digestion and glycolysis of the present application in conjunction with the accompanying drawings.

[0068] A fully automatic device for simulating digestion and fermentation of the human gastrointestinal tract, the device comprises a device frame, an electrical control box, and a digestion tank for the mouth, stomach, duodenum, jejunum, ileum, ascending colon, transverse colon, and descending colon installed in the device frame for simulating digestion and fermentation of the human gastrointestinal tract. Figure 1The digestion tanks are connected in sequence through transfer pipelines, and the jejunum digestion tank, ileum digestion tank, ascending colon digestion tank, and transverse colon digestion tank are connected with a membrane absorption device. The sampling port 109 of each digestion tank is connected with a sampling pipeline, and a sampling needle is provided at the end of the sampling pipeline, and the sampling needle is installed above the automatic sampling device.

[0069] See attached Figures 2 to 4 Each of the above-mentioned digestion tanks includes a tank cover, a tank cover flange, a tank inner layer 101, a cleaning ball 102 arranged in the tank inner layer 101, a pipetting interface 104 and a drain valve 105 arranged at the lower end of the tank inner layer 101, and a tank outer layer jacket 103. The tank cover is provided with a digestion liquid port 106, a digestion sample port 107, a drain port 108, a sampling port 109, an inoculation port 110, an acid liquid port 111, an alkali liquid port 112, an air inlet 113, an air outlet 114 and a cleaning ball interface 115.

[0070] At the same time, the digestion tank cover is also provided with a dissolved oxygen electrode interface 116, a pH electrode interface 117 and a tank temperature electrode interface 118. The outer jacket 103 of the digestion tank body is connected with a tank jacket water inlet 119 and a tank jacket water outlet 120.

[0071] See attached Figure 5 The membrane absorption device includes a clamp fixing base 201 and a front clamp 202 and a rear clamp 204 installed thereon, the front clamp 202 and the rear clamp 204 are fixed by a membrane fastening screw 208, a flat membrane 203 is arranged between the front clamp 202 and the rear clamp 204, a digestion liquid / fermentation liquid inlet 205 is arranged at the lower end of the front clamp 202, and a concentrated liquid reflux port 206 is arranged at the upper end, the digestion liquid / fermentation liquid inlet 205 is connected to the digestion tank sampling port 109 through a transfer pipeline, the concentrated liquid reflux port 206 is connected to the digestion tank discharge port 108 through a transfer pipeline, the flat membrane 203 is connected to the dialysis bag through the small molecule permeation port 207 and the membrane absorption pipeline, the dialysis bag adds a small amount of physiological saline to the absorbed water molecules through the transfer pipeline and refluxes to the corresponding digestion tank (the corresponding digestion tanks of the ascending, transverse and descending colon can optionally be injected with nutrient solution), and docks with the inoculation port 110 of the digestion tank.

[0072] See attached Figure 6, taking the jejunal digestion tank to the ileal digestion tank as an example, the tank connection structure and membrane absorption principle are illustrated. After the digestion (fermentation) of the jejunal digestion tank is completed and the sampling is completed, the transfer peristaltic pump is started to transfer the digestion sample to the membrane absorption device, and the absorption is completed under the action of the peristaltic pump (flow rate 50-100mL / min), and small molecules are absorbed as much as possible (≦5.8-6.0KDa). The small molecules enter the dialysis bag, and then the absorbed water molecules are returned to the digestion tank. The concentrated liquid is returned to the jejunal tank under the pressure of the peristaltic pump, and the absorption is circulated for 15min-30min, and 55%-60% of the total volume is absorbed. When the fermentation liquid is separated to the process requirements, the membrane absorption program is ended, the transfer peristaltic pump is started, and the digestion sample is sent to the ileal digestion tank to continue the next step of digestion.

[0073] See attached Figure 7 The automatic sampling device includes a sampling bottle rotating table 301. Several sampling bottles are installed on the upper end surface of the sampling bottle rotating table 301. The samples are stored at a low temperature of about 0-2°C. At least 80-100 sampling bottles are spare, and each sampling bottle has a corresponding number. The program will distinguish which digestion tank the sample belongs to by color to prevent confusion. A sampling needle slider 302 and a slide rail 303 are arranged directly above the sampling bottle rotating table 301. The sampling needle moves horizontally and vertically electrically or manually under the cooperation of the sampling needle slider 302 and the slide rail 303. After inserting the sampling bottle downward, quantitative sampling is performed. The sampling needle is connected to the sampling port 109 of the digestion tank through a sampling pipeline.

[0074] At the same time, an ethanol needle slider 304 is installed on the slide rail 303, and an ethanol needle is installed at the front end of the ethanol needle slider 304. After the sampling is completed, ethanol will be added to the sample to inactivate the enzyme and preserve it.

[0075] The above-mentioned sampling system is equipped with a separate control system, and establishes industrial Ethernet communication with the main program of the electrical control box of the digestion and fermentation system. The corresponding digestion tank sampling time is set through the control system, and automatic sampling is performed when the control program detects the sampling command.

[0076] After sampling, the simulated digestion and fermentation process of the present application is repeated three times, and corresponding detection methods, such as liquid phase and gas phase detection, are used to detect the digestion and fermentation of the substance to be tested.

[0077] The PLC electrical control system involved in this embodiment partially applies existing technologies, and this application does not involve any improvements thereto; in order to realize the functions of each unit, such as: the control of the sampling bottle rotating table, the electric coordination between the slider and the slide rail, etc., which are not described in detail in this embodiment, existing technologies can be applied.

[0078] Example 2

[0079] This embodiment will introduce in detail a method of fully automatic simulation of human gastrointestinal digestion and glycolysis of the present application.

[0080] The method is applied to the device for fully automatically simulating human gastrointestinal digestion and fermentation in Example 1, and the method specifically comprises the following steps:

[0081] Step 1: Connect each digestion tank, supporting pipeline, supply pipeline and detection electrode;

[0082] Step 2: Disinfect empty cans

[0083] Turn on the magnetic stirring of each digestion tank, enter high-pressure pure water for cleaning, and the residue is discharged from the bottom drain valve 105 of each digestion tank. At the same time, each transfer peristaltic pump pipeline and sampling pipeline are rinsed with pure water for 3-10 minutes. After the end, steam is introduced to perform high-temperature disinfection on each digestion tank for 25-30 minutes. After the end, nitrogen is introduced, and then 80-100mL ethanol is introduced to each transfer peristaltic pump pipeline and sampling pipeline for disinfection. After the end, 200-300mL pure water is introduced to clean each digestion tank and each transfer peristaltic pump pipeline and sampling pipeline. After the end, high-temperature nitrogen is introduced to dry each digestion tank. After the end, normal temperature nitrogen is introduced with a flow rate of 1-10mL / min to ensure positive pressure and anaerobic environment of each digestion tank;

[0084] Step 3: Simulate digestion

[0085] The simulated digestion was performed in the following order:

[0086] Oral digestion → stomach digestion → duodenum digestion → jejunum digestion → ileum digestion;

[0087] Oral digestion: Connect the disinfected replenishing fluids to the oral digestion tank, turn on the magnetic stirring, and start the circulating temperature control. Before digestion, pump 15-20mL of oral digestion fluid, then use the transfer peristaltic pump to pump the digestion sample into the oral digestion tank, pump in 80-100mL of digestion sample, flow rate 80-100mL / min, then pump in 70-80mL of oral digestion fluid, flow rate 80-100mL / min, 0.3-0.5mL of CaCl2 solution, flow rate 0.8-1.0mL / min, pH = 6.8-7.0, temperature 37°C, digestion 3-5m in, sampling time points: 0min, 3-5min, magnetic stirring is always running; when oral digestion is finished and the peristaltic pump for transferring oral digestive fluid stops working, gastric digestion is started, and pre-cleaning is automatically performed at the same time: first, the oral digestion tank drain valve 105 is automatically opened, and the tank body is cleaned through the shower head above the oral digestion tank body. After cleaning for 3-10min, the oral digestion tank drain valve 105 is closed, and then the tank volume is immersed in 80%-90% pure water, and the oral digestion tank sampling pipeline is started for pre-cleaning, and the cleaning is performed for 1-2min, and the transfer pipeline is waited for cleaning, and the automatic cleaning and sterilization operation is performed;

[0088] Gastric digestion: Connect the sterilized replenishing fluids to the gastric digestion tank, turn on the magnetic stirring, pump 50-60mL of gastric digestion fluid into the gastric digestion tank before digestion, transfer 180-200mL of oral digestion sample to the gastric digestion tank, and after the transfer, pump 130-150mL of gastric digestion fluid into the tank through the peristaltic pump, with a flow rate of 80-100mL / min, 0.3-0.5mL of CaCl2 solution, a flow rate of 0.8-1.0mL / min, and pH = 0.0005t 2 -0.079t+4.47, digestion 120-150min, sampling time points 0, 10, 20, 30, 60, 120-150min, magnetic stirring runs three times per minute, each time lasting 10-15s; when gastric digestion is completed and the gastric digestion fluid transfer peristaltic pump stops working, duodenal digestion is turned on, and pre-cleaning is automatically performed at the same time: first, the gastric digestion tank drain valve 105 is automatically opened, and the transfer peristaltic pump between the oral digestion tank and the gastric digestion tank is turned on, and the pure water in the oral digestion tank is quickly transferred for 1-2min, and the pre-cleaning of the transfer pipeline between the oral digestion tank and the gastric digestion tank is completed, and then the tank body is cleaned through the shower above the gastric digestion tank body. After cleaning for 3-10min, the gastric digestion tank drain valve 105 is closed, and then 80%-90% of the tank volume is immersed in pure water, and the pre-cleaning of the gastric digestion tank sampling pipeline is turned on, and the cleaning is carried out for 1-2min, and the transfer pipeline is waited for cleaning, and the automatic cleaning and sterilization operation is performed;

[0089] Duodenal digestion: connect the disinfected replenishing fluids to the duodenal digestion tank, turn on the magnetic stirring, transfer the peristaltic pump to transfer 350-400mL of gastric digestion sample to the duodenal digestion tank. After the transfer is completed, pump in 380-400mL of intestinal electrolytes through the peristaltic pump, with a flow rate of 80-100mL / min, 0.6-0.8mlCaCl2 solution, a flow rate of 0.8-1.0mL / min, pH=6.1-6.2, digest for 45-50min, and sampling time points of 0, 10, 20, 45-50min. The magnetic stirring is always running; stop the peristaltic pump after the duodenal digestion is completed and the duodenal digestion fluid transfer is completed. When the jejunum digestion is started, the jejunum digestion is automatically pre-cleaned at the same time: first, the duodenal digestion tank drain valve 105 is automatically opened, and the transfer peristaltic pump between the stomach digestion tank and the duodenal digestion tank is turned on to quickly transfer the pure water in the stomach digestion tank for 1-2 minutes, and the pre-cleaning of the transfer pipeline between the stomach digestion tank and the duodenal digestion tank is completed. Then, the tank body is cleaned through the shower head above the duodenal digestion tank body. After cleaning for 3-10 minutes, the duodenal digestion tank drain valve 105 is closed, and then 80%-90% of the tank volume is immersed in pure water, and the pre-cleaning of the duodenal digestion tank sampling pipeline is turned on for cleaning for 1-2 minutes, and the transfer pipeline is waited for cleaning, and the automatic cleaning and sterilization operation is performed;

[0090] Jejunal digestion: connect the disinfected replenishing fluids to the jejunal digestion tank, turn on the magnetic stirring, and transfer all the duodenal digestion samples to the jejunal digestion tank through the transfer peristaltic pump, with a total volume of 700-800mL, pH=6.4-6.5, digestion time 45-50min, sampling time points 0, 10, 20, 45-50min, and magnetic stirring running all the time. After sampling, the peristaltic pump is used to complete the absorption, with a flow rate of 50-100mL / min. During the absorption process, the absorbed water molecules are pumped back into the jejunal digestion tank for cyclic absorption, and small molecules are absorbed as much as possible. The absorption takes 15min-30min until 55%-60% of the total volume is absorbed, and the remaining volume is 300-320mL; wait until the jejunal digestion is completed and the jejunal digestion fluid is transferred to the peristaltic pump When the work is stopped, the ileum digestion is turned on and pre-cleaning is automatically performed at the same time: first, the jejunum digestion tank drain valve 105 is automatically opened, and the transfer peristaltic pump between the duodenum digestion tank and the jejunum digestion tank is turned on to quickly transfer the pure water in the duodenum digestion tank for 1-2 minutes, and the pre-cleaning of the transfer pipeline between the duodenum digestion tank and the jejunum digestion tank is completed. Then, the tank body is cleaned through the shower above the jejunum digestion tank body. After cleaning for 3-10 minutes, the jejunum digestion tank drain valve 105 is closed, and then 80%-90% of the tank volume is immersed in pure water, and the pre-cleaning of the jejunum digestion tank sampling pipeline is turned on for 1-2 minutes. Then, the cleaning of the jejunum digestion tank membrane absorption pipeline is turned on, and the cycle cleaning is 3-5 minutes. Wait for the cleaning of the transfer pipeline, and the automatic cleaning and sterilization operation is performed;

[0091] Ileal digestion: Connect the disinfected replenishing fluids to the ileal digestion tank, turn on the magnetic stirring, and transfer the remaining digestion sample in the jejunal digestion tank to the ileal digestion tank through the transfer peristaltic pump, with a flow rate of 80-100mL / min, pH=7.4-7.5, digestion time of 90-100min, sampling time points of 0, 10, 20, 30, 60, 90-100min, and magnetic stirring running all the time. After sampling, the peristaltic pump is used to complete the absorption. During the absorption process, the absorbed water molecules are pumped back into the ileal digestion tank for cyclic absorption, and small molecules are absorbed as much as possible. The absorption lasts for 15-30min until 55%-60% of the total volume is absorbed, and the remaining volume is 120-130mL. When the ileal digestion is completed and the peristaltic pump for ileal digestion fluid transfer is stopped, When the work is stopped, the ascending colon digestion is turned on and pre-cleaning is automatically performed at the same time: first, the ileum digestion tank drain valve 105 is automatically opened, and the transfer peristaltic pump between the jejunum digestion tank and the ileum digestion tank is turned on to quickly transfer the pure water in the jejunum digestion tank for 1-2 minutes, and the pre-cleaning of the transfer pipeline between the jejunum digestion tank and the ileum digestion tank is completed. Then, the tank body is cleaned through the shower above the ileum digestion tank body. After cleaning for 3-10 minutes, the ileum digestion tank drain valve 105 is closed, and then 80%-90% of the tank volume is immersed in pure water. The pre-cleaning of the ileum digestion tank sampling pipeline is turned on for 1-2 minutes, and then the cleaning of the ileum digestion tank membrane absorption pipeline is turned on. The cycle cleaning is 3-5 minutes, and the cleaning of the transfer pipeline is waited for. The automatic cleaning and sterilization operation is performed;

[0092] Step 4: Simulate fermentation

[0093] The simulated fermentation was carried out in the following order:

[0094] Ascending colon fermentation → transverse colon fermentation → descending colon fermentation;

[0095] Ascending colon fermentation: connect the disinfected feed fluids to the ascending colon digestion tank, turn on the magnetic stirring, transfer the peristaltic pump to transfer all the ileal digestion fluid to the ascending colon digestion tank, add 120-130mL of ascending colon culture fluid through the peristaltic pump, the flow rate is 80-100mL / min, pH=5.5-6.0, fermentation time is 6-8h, sampling time points are 0, 3, 6h or 0, 4, 8h, magnetic stirring is always running, after sampling is completed, the absorption is completed by the peristaltic pump, and the absorbed water molecules are pumped back to the ascending colon digestion tank during the absorption process, and the small molecules are absorbed as much as possible for 15-30min, until 55%-60% of the total volume is absorbed, and the remaining volume is 90-100mL; wait until the ascending colon fermentation is completed and the ascending colon fermentation fluid is transferred to the peristaltic pump Stop working, start the fermentation of transverse colon, and automatically perform pre-cleaning at the same time: first, automatically open the sewage valve 105 of the ascending colon digestion tank, start the transfer peristaltic pump between the ileum digestion tank and the ascending colon digestion tank, quickly transfer the pure water in the ileum digestion tank for 1-2 minutes, complete the pre-cleaning of the transfer pipeline between the ileum digestion tank and the ascending colon digestion tank, and then clean the tank body through the shower head above the ascending colon digestion tank body. After cleaning for 3-10 minutes, close the sewage valve 105 of the ascending colon digestion tank, and then enter the tank volume again. Soak in pure water of 80%-90% of the tank volume, start the pre-cleaning of the sampling pipeline of the ascending colon digestion tank, clean for 1-2 minutes, then start the cleaning of the membrane absorption pipeline of the ascending colon digestion tank, circulate the cleaning for 3-5 minutes, wait for the cleaning of the transfer pipeline, and automatically clean and sterilize the operation;

[0096] Transverse colon fermentation: connect the disinfected feed fluids to the transverse colon digestion tank, turn on the magnetic stirring, transfer the peristaltic pump to transfer all the ascending colon fermentation liquid to the transverse colon digestion tank, add 90-100mL of ascending colon culture liquid through the peristaltic pump, the flow rate is 80-100mL / min, pH=6.0-6.4, fermentation time is 12-16h, sampling time points are 0, 4, 8, 12, 16h, magnetic stirring is always running, after sampling is completed, the peristaltic pump is used to complete the absorption, during the absorption process, the absorbed water molecules are pumped back into the transverse colon digestion tank, circulated and absorbed, small molecules are absorbed as much as possible, and the absorption is 15-30min, until 55%-60% of the total volume is absorbed, and the remaining volume is 60-70mL; wait until the transverse colon fermentation is completed and the transverse colon fermentation liquid is transferred to the peristaltic pump Stop working, start the fermentation of the descending colon, and automatically perform pre-cleaning at the same time: first, automatically open the drain valve 105 of the transverse colon digestion tank, start the transfer peristaltic pump between the ascending colon digestion tank and the transverse colon digestion tank, quickly transfer the pure water in the ascending colon digestion tank for 1-2 minutes, complete the pre-cleaning of the transfer pipeline between the ascending colon digestion tank and the transverse colon digestion tank, and then clean the tank body through the shower head above the transverse colon digestion tank body. After cleaning for 3-10 minutes, close the drain valve 105 of the transverse colon digestion tank, and then soak in pure water that accounts for 90% of the tank volume, start the pre-cleaning of the sampling pipeline of the transverse colon digestion tank, clean for 1-2 minutes, then start the cleaning of the membrane absorption pipeline of the transverse colon digestion tank, circulate the cleaning for 3-5 minutes, wait for the cleaning of the transfer pipeline, and automatically clean and sterilize the operation;

[0097] Descending colon fermentation: connect the disinfected feed liquids to the descending colon digestion tank, turn on the magnetic stirring, transfer the peristaltic pump to transfer all the fermentation liquid of the transverse colon to the descending colon, add 60-70mL of ascending colon culture liquid through the peristaltic pump, the flow rate is 80-100mL / min, pH=6.5-6.9, fermentation time is 6-8h, sampling time points are 0, 3, 6 or 0, 4, 8h, magnetic stirring is always running, after sampling is completed, transfer it to the waste liquid bucket through the transfer peristaltic pump; when the fermentation of the descending colon is completed and the peristaltic pump for transferring the fermentation liquid of the descending colon stops working, automatically perform pre-cleaning: first automatically turn on the descending colon fermentation liquid transfer pump, and then turn on the magnetic stirring pump to transfer the fermentation liquid of the descending colon to the waste liquid tank. The colon digester tank drain valve 105 is turned on, and the transfer peristaltic pump between the transverse colon digester tank and the descending colon digester tank is turned on to quickly transfer the pure water in the ascending colon digester tank for 1-2 minutes, and the transfer pipeline between the transverse colon digester tank and the descending colon digester tank is pre-cleaned. The tank body is then cleaned through the shower head above the descending colon digester tank body. After cleaning for 3-10 minutes, the descending colon digester tank drain valve 105 is closed, and then 80%-90% of the tank volume is immersed in pure water. At the same time, the descending colon sampling pipeline, transfer pipeline, and waste discharge pipeline are pre-cleaned for 1-2 minutes, and the automatic cleaning and sterilization operation is waited for.

[0098] The above method further comprises the following steps:

[0099] Step 5: Automatic cleaning and sterilization

[0100] After all digestion and fermentation are completed, the digestion tanks, transfer pipelines, membrane absorption pipelines, and sampling pipelines are waiting to be formally cleaned and sterilized together:

[0101] The oral cavity, stomach, duodenum, jejunum, ileum, ascending colon, transverse colon, and descending colon digestion tanks automatically open the bottom drain valve 105 to discharge the waste water in the tank, and each digestion tank enters the high-pressure pure water for cleaning again. The residue is discharged from the tank bottom drain valve 105, and at the same time, the transfer peristaltic pump pipeline enters the tank side for flushing. After 3-10 minutes of cleaning, the tank without membrane absorption device is in a pending state, and the tank with membrane absorption device closes the bottom drain valve 105 for membrane regeneration and cleaning;

[0102] Membrane regeneration and cleaning steps: 400-500mL pure water is pumped into the corresponding tank, cleaned for 5-10min, and the waste water is discharged. Then 400-500mL membrane cleaning agent is pumped in for cleaning for 30-40min, and 400-500mL pure water is pumped in for cleaning for 5-10min, and the waste water is discharged. Then the corresponding tank enters steam high-temperature disinfection. After completion, air is introduced, and the bottom drain valve 105 is closed. 250-300mL of pure water is introduced into each corresponding tank again. All transfer peristaltic pumps are turned on to clean the transfer pipeline. At the same time, the sampling pipeline is cleaned. After cleaning for 3-5min, the waste water is discharged, and 250-300mL of ethanol is added to each corresponding tank to disinfect the membrane absorption device, transfer pipeline, and sampling pipeline. After completion, 400-500mL pure water is added to clean the tank body and the membrane absorption device, transfer pipeline, and sampling pipeline. After completion, high-temperature nitrogen is introduced to dry the tank body and each pipeline. After completion, normal temperature nitrogen is introduced.

[0103] Other things that need special explanation:

[0104] (1) Each step of the digestion / fermentation process has automatic sampling:

[0105] Sampling system control method: the sampling bottle rotating table 301 can rotate automatically, and the samples are stored at a low temperature of about 0-2℃. There are at least 80-100 sampling bottles in reserve, and each sampling bottle has a corresponding number. The program will distinguish which digester the sample belongs to by color to prevent confusion; the sampling system is equipped with a separate control system, and establishes industrial Ethernet communication with the main program of the electrical control box of the digestion and glycolysis system. The corresponding digester sampling time is set through the control system. When the control program detects the sampling command, it automatically samples and adds ethanol to inactivate the enzyme for storage.

[0106] Disinfection steps for sampling needles and sampling pipelines: To ensure that there is no residue in the sampling (which may easily lead to mixed contamination), the sampling pipeline close to the sampling needle must be cleaned and disinfected before and after sampling, and heated nitrogen must be passed through to blow dry. After each sampling, the digestion sample remaining in the pipeline close to the digestion (fermentation) tank side of the sampling pipeline is pushed back into the digestion tank by nitrogen to continue digestion (or fermentation), thereby making the sampling more representative.

[0107] After sampling, the simulated digestion and fermentation process is repeated three times, and the digestion and fermentation of the substance to be tested are detected using corresponding detection methods, such as liquid phase and gas phase.

[0108] (2) In each step, the transfer peristaltic pump extends the transfer time by 1-2 minutes when transferring the digestion sample, so as to transfer the digestion sample from the previous part completely and reduce the residue in the transfer pipeline.

[0109] (3) In each step, the replenishing liquid is equipped with a 37°C water bath heating device; at least two sets of glass tanks containing digestion samples, replenishing liquid, simulated absorption liquid, etc. and corresponding rubber pipes are provided.

[0110] (4) All tanks need to be equipped with an annular hollow ring attached to the wall to better simulate the secretion of digestive juices and acid and alkali solutions; the stomach needs to be equipped with a 1-2 mm sieve to simulate the stomach screening function.

[0111] The above is a detailed description of the specific embodiments of the present invention, but the content is only a preferred embodiment of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

[0112] It should also be understood that although this specification is described according to embodiments, not every embodiment contains only one independent technical solution. This narrative method of this specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation methods that those skilled in the art can understand.

Claims

1. A fully automatic device for simulating digestion and fermentation of the human gastrointestinal tract, characterized in that: The device comprises an equipment frame, an equipment electrical control box, and an oral digestion tank, a stomach digestion tank, a duodenum digestion tank, a jejunum digestion tank, an ileum digestion tank, an ascending colon digestion tank, a transverse colon digestion tank, and a descending colon digestion tank installed in the equipment frame and used to simulate human gastrointestinal digestion and fermentation, and are sequentially connected, wherein each digestion tank is connected through a transfer pipeline, and the jejunum digestion tank, the ileum digestion tank, the ascending colon digestion tank, and the transverse colon digestion tank are connected with a membrane absorption device; Each digestion tank comprises a tank cover, a tank cover flange, a tank inner layer, a cleaning ball arranged in the tank inner layer, a liquid transfer interface and a drain valve arranged at the lower end of the tank inner layer, and a tank outer layer jacket; the tank cover is provided with a digestion liquid port, a digestion sample port, a drain port, a sampling port, an inoculation port, an acid liquid port, an alkali liquid port, an air inlet, an air outlet, and a cleaning ball interface; the digestion tank cover is also provided with a dissolved oxygen electrode interface, a pH electrode interface, and a tank temperature electrode interface; The membrane absorption device comprises a fixture fixing base and a front fixture and a rear fixture installed thereon, a flat membrane is arranged between the front fixture and the rear fixture, a digestion liquid / fermentation liquid inlet is arranged at the lower end of the front fixture, and a concentrated liquid reflux port is arranged at the upper end, the digestion liquid / fermentation liquid inlet and the concentrated liquid reflux port are both connected to the corresponding digestion tank through a transfer pipeline, and the flat membrane is connected to a dialysis bag through a small molecule permeation port and a membrane absorption pipeline; The sampling port of each digestion tank is connected to a sampling pipeline, a sampling needle is arranged at the end of the sampling pipeline, and the sampling needle is installed above the automatic sampling equipment.

2. The device for fully automatic simulation of human gastrointestinal digestion and fermentation according to claim 1, characterized in that: The automatic sampling equipment includes a sampling bottle rotating table, a plurality of sampling bottles are installed on the upper end surface of the sampling bottle rotating table, a sampling needle slider and a slide rail are arranged directly above the sampling bottle rotating table, the sampling needle is electrically or manually moved horizontally and vertically in cooperation with the sampling needle slider and the slide rail to be inserted into the sampling bottle for quantitative sampling, an ethanol needle slider is installed on the slide rail, and an ethanol needle is installed at the front end of the ethanol needle slider.

3. The device for fully automatic simulation of human gastrointestinal digestion and fermentation according to claim 1, characterized in that: The outer jacket of the digester tank body is connected with a tank jacket water inlet and a tank jacket water outlet.

4. The device for fully automatic simulation of human gastrointestinal digestion and fermentation according to claim 1, characterized in that: The front clamp and the rear clamp of the membrane absorption device are fixed by membrane fastening screws.

5. The device for fully automatic simulation of human gastrointestinal digestion and fermentation according to claim 4, characterized in that: The dialysis bag of the membrane absorption device is connected to the corresponding digestion tank through a transfer pipeline.

6. The device for fully automatic simulation of human gastrointestinal digestion and fermentation according to claim 1, characterized in that: The transfer pipeline, membrane absorption pipeline and sampling pipeline are all equipped with peristaltic pumps.

7. A method for fully automatically simulating digestion and fermentation of the human gastrointestinal tract, characterized in that: The method is applied to the device for fully automatically simulating human gastrointestinal digestion and fermentation as claimed in any one of claims 1 to 6, and the method comprises the following steps: Step 1: Connect each digestion tank, supporting pipeline, supply pipeline and detection electrode; Step 2: Disinfect empty cans Turn on the magnetic stirring of each digestion tank, enter high-pressure pure water for cleaning, and the residue is discharged from the drain valve at the bottom of each digestion tank. At the same time, each transfer peristaltic pump pipeline and sampling pipeline are rinsed with pure water for 3-10 minutes. After the end, steam is introduced to sterilize each digestion tank at high temperature for 25-30 minutes. After the end, nitrogen is introduced, and then 80-100mL ethanol is introduced to sterilize each transfer peristaltic pump pipeline and sampling pipeline. After the end, 200-300mL pure water is introduced to clean each digestion tank and each transfer peristaltic pump pipeline and sampling pipeline. After the end, high-temperature nitrogen is introduced to dry each digestion tank. After the end, normal temperature nitrogen is introduced with a flow rate of 1-10mL / min to ensure positive pressure and anaerobic environment of each digestion tank; Step 3: Simulate digestion The simulated digestion was performed in the following order: Oral digestion → stomach digestion → duodenum digestion → jejunum digestion → ileum digestion; Oral digestion: Connect the sterilized replenishing fluids to the oral digestion tank, turn on the magnetic stirring, and start the circulating temperature control. Before digestion, pump 15-20mL of oral digestion fluid, then use the transfer peristaltic pump to pump the digestion sample into the oral digestion tank, pump in 80-100mL of digestion sample, flow rate 80-100mL / min, then pump in 70-80mL of oral digestion fluid, flow rate 80-100mL / min, 0.3-0.5mL of CaCl2 solution, flow rate 0.8-1.0mL / min, pH = 6.8-7.0, temperature 37°C, digestion 3 -5min, sampling time points: 0min, 3-5min, magnetic stirring is always running; when oral digestion is finished and the peristaltic pump for transferring oral digestive fluid stops working, gastric digestion is started, and pre-cleaning is automatically performed at the same time: first, the oral digestion tank drain valve is automatically opened, and the tank body is cleaned through the shower head above the oral digestion tank body. After cleaning for 3-10min, the oral digestion tank drain valve is closed, and then 80%-90% of the tank volume is immersed in pure water, and the oral digestion tank sampling pipeline is started for pre-cleaning, and the cleaning is performed for 1-2min, and the transfer pipeline is waited for cleaning, and the automatic cleaning and sterilization operation is performed; Gastric digestion: Connect the sterilized replenishing fluids to the gastric digestion tank, turn on the magnetic stirring, pump 50-60mL of gastric digestion fluid into the gastric digestion tank before digestion, transfer 180-200mL of oral digestion sample to the gastric digestion tank, and after the transfer, pump 130-150mL of gastric digestion fluid into the tank through the peristaltic pump, with a flow rate of 80-100mL / min, 0.3-0.5mL of CaCl2 solution, a flow rate of 0.8-1.0mL / min, and pH = 0.0005t 2 -0.079t+4.47, digestion 120-150min, sampling time points 0, 10, 20, 30, 60, 120-150min, magnetic stirring runs three times per minute, each time lasting 10-15s; when gastric digestion is completed and the gastric digestion fluid transfer peristaltic pump stops working, duodenal digestion is turned on, and pre-cleaning is automatically performed at the same time: first, the gastric digestion tank drain valve is automatically opened, and the transfer peristaltic pump between the oral digestion tank and the gastric digestion tank is turned on, and the pure water in the oral digestion tank is quickly transferred for 1-2min, and the pre-cleaning of the transfer pipeline between the oral digestion tank and the gastric digestion tank is completed, and then the tank body is cleaned through the shower above the gastric digestion tank body. After cleaning for 3-10min, the gastric digestion tank drain valve is closed, and then 80%-90% of the tank volume is immersed in pure water, and the pre-cleaning of the gastric digestion tank sampling pipeline is turned on, and the cleaning is carried out for 1-2min, and the transfer pipeline is waited for cleaning, and the automatic cleaning and sterilization operation is performed; Duodenal digestion: Connect the sterilized replenishing fluids to the duodenal digestion tank, turn on the magnetic stirring, and use the peristaltic pump to transfer 350-400 mL of gastric digestion sample to the duodenal digestion tank. After the transfer is completed, 380-400 mL of intestinal electrolytes are pumped in through the peristaltic pump at a flow rate of 80-100 mL / min and 0.6-0.8 ml CaCl2 solution, flow rate 0.8-1.0mL / min, pH=6.1-6.2, digestion 45-50min, sampling time points 0, 10, 20, 45-50min, magnetic stirring is always running; when the duodenal digestion is completed and the duodenal digestion liquid transfer peristaltic pump stops working, the jejunal digestion is started, and pre-cleaning is automatically performed at the same time: first, the duodenal digestion tank drain valve is automatically opened, and the transfer peristaltic pump between the stomach digestion tank and the duodenal digestion tank is opened, and the pure water in the stomach digestion tank is quickly transferred for 1-2min, and the transfer pipeline between the stomach digestion tank and the duodenal digestion tank is pre-cleaned, and then the tank body is cleaned through the shower above the duodenal digestion tank body. After cleaning for 3-10min, the duodenal digestion tank drain valve is closed, and then 80%-90% of the tank volume is immersed in pure water, and the pre-cleaning of the duodenal digestion tank sampling pipeline is started, and the cleaning is performed for 1-2min, and the transfer pipeline is waited for cleaning, and the automatic cleaning and sterilization operation is performed; Jejunal digestion: connect the disinfected replenishing fluids to the jejunal digestion tank, turn on the magnetic stirring, and transfer all the duodenal digestion samples to the jejunal digestion tank through the transfer peristaltic pump, with a total volume of 700-800mL, pH=6.4-6.5, digestion time 45-50min, sampling time points 0, 10, 20, 45-50min, and magnetic stirring running all the time. After sampling, the peristaltic pump is used to complete the absorption, with a flow rate of 50-100mL / min. During the absorption process, the absorbed water molecules are pumped back into the jejunal digestion tank for cyclic absorption, and small molecules are absorbed as much as possible. The absorption lasts for 15min-30min until 55%-60% of the total volume is absorbed, and the remaining volume is 300-320mL; wait until the jejunal digestion is completed and the jejunal digestion fluid is transferred When the peristaltic pump stops working, the ileum digestion is turned on and pre-cleaning is automatically performed at the same time: first, the jejunum digestion tank drain valve is automatically opened, and the transfer peristaltic pump between the duodenum digestion tank and the jejunum digestion tank is turned on to quickly transfer the pure water in the duodenum digestion tank for 1-2 minutes, and complete the pre-cleaning of the transfer pipeline between the duodenum digestion tank and the jejunum digestion tank. Then, the tank body is cleaned through the shower above the jejunum digestion tank body. After cleaning for 3-10 minutes, the jejunum digestion tank drain valve is closed, and then 80%-90% of the tank volume is immersed in pure water. The pre-cleaning of the jejunum digestion tank sampling pipeline is turned on for 1-2 minutes, and then the cleaning of the jejunum digestion tank membrane absorption pipeline is turned on. The cycle cleaning is 3-5 minutes, and the transfer pipeline is waited for cleaning, and the automatic cleaning and sterilization operation is performed; Ileal digestion: connect the disinfected replenishing fluids to the ileal digestion tank, turn on the magnetic stirring, and transfer the remaining digestion sample in the jejunal digestion tank to the ileal digestion tank through the transfer peristaltic pump, with a flow rate of 80-100mL / min, pH=7.4-7.5, digestion time of 90-100min, sampling time points of 0, 10, 20, 30, 60, 90-100min, and magnetic stirring running all the time. After sampling, the peristaltic pump is used to complete the absorption. During the absorption process, the absorbed water molecules are pumped back into the ileal digestion tank for cyclic absorption, and small molecules are absorbed as much as possible for 15-30min, until 55%-60% of the total volume is absorbed, and the remaining volume is 120-130mL; after the ileal digestion is completed and the ileal digestion fluid is transferred to the peristaltic pump, the water molecules are pumped back into the ileal digestion tank for cyclic absorption, and small molecules are absorbed as much as possible for 15-30min. When the pump stops working, the ascending colon digestion is turned on and pre-cleaning is automatically performed at the same time: first, the sewage valve of the ileum digestion tank is automatically opened, and the transfer peristaltic pump between the jejunum digestion tank and the ileum digestion tank is turned on to quickly transfer the pure water in the jejunum digestion tank for 1-2 minutes, and complete the pre-cleaning of the transfer pipeline between the jejunum digestion tank and the ileum digestion tank. Then, the tank body is cleaned through the shower above the ileum digestion tank body. After cleaning for 3-10 minutes, the sewage valve of the ileum digestion tank is closed, and then 80%-90% of the tank volume is immersed in pure water. The pre-cleaning of the sampling pipeline of the ileum digestion tank is turned on for 1-2 minutes, and then the cleaning of the membrane absorption pipeline of the ileum digestion tank is turned on. The cycle cleaning is 3-5 minutes, and the transfer pipeline is waited for cleaning, and the automatic cleaning and sterilization operation is performed; Step 4: Simulate fermentation The simulated fermentation was carried out in the following order: Ascending colon fermentation → transverse colon fermentation → descending colon fermentation; Ascending colon fermentation: connect the disinfected feed fluids to the ascending colon digestion tank, turn on the magnetic stirring, transfer the peristaltic pump to transfer all the ileal digestion fluid to the ascending colon digestion tank, add 120-130mL of ascending colon culture fluid through the peristaltic pump, the flow rate is 80-100mL / min, pH=5.5-6.0, fermentation time is 6-8h, sampling time points are 0, 3, 6h or 0, 4, 8h, magnetic stirring is always running, after sampling is completed, the peristaltic pump is used to complete the absorption, during the absorption process, the absorbed water molecules are pumped back to the ascending colon digestion tank, circulated and absorbed, small molecules are absorbed as much as possible, and the absorption is 15-30min, until 55%-60% of the total volume is absorbed, and the remaining volume is 90-100mL; wait until the ascending colon fermentation is completed and the ascending colon fermentation fluid is transferred The peristaltic pump stops working, the transverse colon fermentation starts, and pre-cleaning is automatically performed at the same time: first, the sewage valve of the ascending colon digestion tank is automatically opened, and the transfer peristaltic pump between the ileum digestion tank and the ascending colon digestion tank is opened to quickly transfer the pure water in the ileum digestion tank for 1-2 minutes, and complete the pre-cleaning of the transfer pipeline between the ileum digestion tank and the ascending colon digestion tank. Then, the tank body is cleaned through the shower above the ascending colon digestion tank body. After cleaning for 3-10 minutes, the sewage valve of the ascending colon digestion tank is closed, and then 80%-90% of the tank volume is immersed in pure water. The pre-cleaning of the sampling pipeline of the ascending colon digestion tank is started, and the cleaning is started for 1-2 minutes. Then, the cleaning of the membrane absorption pipeline of the ascending colon digestion tank is started, and the circulation cleaning is carried out for 3-5 minutes. Wait for the cleaning of the transfer pipeline, and the automatic cleaning and sterilization operation is performed; Transverse colon fermentation: connect the disinfected feed fluids to the transverse colon digestion tank, turn on the magnetic stirring, transfer the peristaltic pump to transfer all the ascending colon fermentation liquid to the transverse colon digestion tank, add 90-100mL of ascending colon culture liquid through the peristaltic pump, the flow rate is 80-100mL / min, pH=6.0-6.4, fermentation time is 12-16h, sampling time points are 0, 4, 8, 12, 16h, magnetic stirring is always running, after sampling is completed, the peristaltic pump is used to complete the absorption, and the absorbed water molecules are pumped back into the transverse colon digestion tank during the absorption process, and the small molecules are absorbed as much as possible for 15-30min, until 55%-60% of the total volume is absorbed, and the remaining volume is 60-70mL; wait until the transverse colon fermentation is completed and the transverse colon fermentation liquid is transferred The peristaltic pump stops working, the descending colon fermentation starts, and pre-cleaning is automatically performed at the same time: first, the drain valve of the transverse colon digestion tank is automatically opened, and the transfer peristaltic pump between the ascending colon digestion tank and the transverse colon digestion tank is opened to quickly transfer the pure water in the ascending colon digestion tank for 1-2 minutes, and complete the pre-cleaning of the transfer pipeline between the ascending colon digestion tank and the transverse colon digestion tank. Then, the tank body is cleaned through the shower head above the transverse colon digestion tank body. After cleaning for 3-10 minutes, the drain valve of the transverse colon digestion tank is closed, and then 90% of the tank volume is immersed in pure water. The pre-cleaning of the sampling pipeline of the transverse colon digestion tank is started, and the cleaning is started for 1-2 minutes. Then, the cleaning of the membrane absorption pipeline of the transverse colon digestion tank is started, and the cycle cleaning is carried out for 3-5 minutes. Wait for the cleaning of the transfer pipeline, and the automatic cleaning and sterilization operation is performed; Descending colon fermentation: connect the disinfected feed liquids to the descending colon digestion tank, turn on the magnetic stirring, transfer the peristaltic pump to transfer all the fermentation liquid of the transverse colon to the descending colon, add 60-70mL of ascending colon culture liquid through the peristaltic pump, the flow rate is 80-100mL / min, pH=6.5-6.9, fermentation time is 6-8h, sampling time points are 0, 3, 6 or 0, 4, 8h, magnetic stirring is always running, after sampling is completed, transfer it to the waste liquid bucket through the transfer peristaltic pump; when the fermentation of the descending colon is completed and the peristaltic pump for transferring the fermentation liquid of the descending colon stops working, automatically perform pre-cleaning: first, automatically Open the drain valve of the descending colon digester, start the transfer peristaltic pump between the transverse colon digester and the descending colon digester, quickly transfer the pure water in the ascending colon digester for 1-2 minutes, complete the pre-cleaning of the transfer pipeline between the transverse colon digester and the descending colon digester, and then clean the tank body through the shower head above the descending colon digester. After cleaning for 3-10 minutes, close the drain valve of the descending colon digester, and then soak it in pure water that accounts for 80%-90% of the tank volume. At the same time, start the pre-cleaning of the descending colon sampling pipeline, transfer pipeline, and waste pipeline. Clean for 1-2 minutes and wait for automatic cleaning and sterilization operation.

8. The method for fully automatic simulation of human gastrointestinal digestion and glycolysis according to claim 7, characterized in that: The method further comprises the following steps: Step 5: Automatic cleaning and sterilization After all digestion and fermentation are completed, the digestion tanks, transfer pipelines, membrane absorption pipelines, and sampling pipelines are waiting to be formally cleaned and sterilized together: The bottom drain valves of the digestion tanks for the oral cavity, stomach, duodenum, jejunum, ileum, ascending colon, transverse colon, and descending colon are automatically opened to discharge the wastewater in the tanks. Each digestion tank is cleaned again with high-pressure pure water, and the residue is discharged from the bottom drain valve of the tank. At the same time, the transfer peristaltic pump pipeline enters the tank side for flushing with pure water. After 3-10 minutes of cleaning, the tank without membrane absorption device is in a pending state, and the tank with membrane absorption device closes the bottom drain valve for membrane regeneration and cleaning; Membrane regeneration and cleaning steps: pump 400-500mL of pure water into the corresponding tank, clean for 5-10min, discharge the waste water, pump in 400-500mL of membrane cleaning agent, clean for 30-40min, pump in 400-500mL of pure water, clean for 5-10min, discharge the waste water, then the corresponding tank enters steam high-temperature disinfection, and then pass air, close the bottom drain valve, and then enter 250-300mL of pure water into each corresponding tank, turn on all transfer peristaltic pumps to clean the transfer pipeline, and clean the sampling pipeline at the same time, discharge the waste water after cleaning for 3-5min, and then add 250-300mL of ethanol to each corresponding tank to disinfect the membrane absorption device, transfer pipeline, and sampling pipeline. After the end, add 400-500mL of pure water to clean the tank body and membrane absorption device, transfer pipeline, and sampling pipeline. After the end, pass high-temperature nitrogen to dry the tank body and each pipeline, and then pass normal temperature nitrogen after the end.

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