Method for simulating small intestine-colon dynamic digestion and glycolysis of polysaccharide-based food

Abstract

The invention discloses a method for simulating small intestine-colon dynamic digestion and glycolysis of polysaccharide-based food, and belongs to the technical field of food detection. The small intestine-colon simulated digestion and absorption system special for the polysaccharide-based food comprises a small intestine digestion simulation system, a sampling and emptying system, a large intestine glycolysis simulation system and a central intelligent control and display system (a pH control system, a temperature control system, a stirring rate control system, a gas path system and a flow rate control system). Aiming at the limitations that an existing human gastrointestinal tract digestion in-vitro simulation system cannot be continuous, is short in period and the like, the in-vivo real digestion and glycolysis physiological environment and process are simulated, intestinal peristalsis waves and digestion and glycolysis of polysaccharide-based food are simulated, automatic sampling can be achieved, generation of glucose in small intestines can be detected on line, and the in-vitro simulation system fits the real human physiological environment.

Description

Technical Field

[0001] The invention relates to a method for simulating the dynamic digestion and fermentation of polysaccharide-based food in small intestine and colon, belonging to the technical field of food. Background Art

[0002] Polysaccharide-based foods are foods that contain a large amount of polysaccharides, such as grains, beans, vegetables, fruits, seaweed and mushrooms. Compared with conventional foods, polysaccharide-based foods are rich in dietary fiber, vitamins and minerals, have a lower glycemic index, can stabilize blood sugar, promote intestinal health, and provide antioxidant and immunomodulatory effects. These characteristics give them significant advantages in improving digestion, lowering cholesterol, enhancing immunity and preventing chronic diseases, helping to meet nutritional needs and promote overall health.

[0003] The in vitro simulated digestion model is an experimental technique used to study the physical and chemical changes of food in the digestive tract. By simulating the human digestive process in vitro, scientists can study the digestion and absorption mechanism of food components, providing important theoretical basis and experimental data for fields such as food science, nutrition and pharmacology.

[0004] There are many methods for simulating digestive models in vitro, such as: Single-phase simulation method: This method simulates a part of the digestive system, such as simulating only the effect of gastric juice or small intestinal juice on food. The food sample is mixed with simulated digestive juice (such as gastric juice or small intestinal juice) and reacted at a certain temperature and time. The advantage of the single-phase simulation method is that it is simple to operate and can focus on studying a specific digestive stage. Multiphase simulation method: The multiphase simulation method combines different digestive stages such as the mouth, stomach and small intestine, and gradually simulates the changes of food in different digestive environments. This method usually includes three main steps: oral stage: simulate the secretion of saliva and mechanical chewing; gastric stage: simulate the secretion of gastric juice and the mechanical stirring of the stomach; small intestine stage: simulate the secretion of bile and pancreatic juice and the stirring of the small intestine; dynamic simulation system: The dynamic simulation system is further refined to simulate the dynamic changes in the digestive process, such as the gradual secretion of digestive juice, gastrointestinal peristalsis and the transfer of chyme. Such systems often use specially designed devices, such as in vitro gastrointestinal models (TIMs) or dynamic gastrointestinal models (DGMs), which can reproduce the digestive process more realistically.

[0005] Patent CN118294600A can simulate the digestive environment in the human gastrointestinal tract by adding a sample containing amylose to the bionic digestive system model and performing peristaltic compression to simulate the digestion and emptying process of amylose in the digestive system. Patent CN112029663A integrates 12 systems into an integrated in vitro simulation digestion model through an integrated human gastrointestinal digestion and absorption in vitro substitute model, including a gastric digestion simulation system, a small intestinal digestion simulation system, a small intestinal absorption simulation system, a large intestinal decomposition simulation system, a central intelligent control system, a pH control system, a temperature control system, a stirring rate control system, a conveying system, a central purification system, an automatic cleaning and disinfection system, and an anaerobic system, to achieve in vitro simulation of the gastrointestinal digestion and absorption process. Patent CN114657230A discloses the preparation of a standardized fecal inoculum to simulate gastro-small intestinal digestion.

[0006] However, there are many problems with the above-mentioned in vitro simulated digestion model. For example, the sampling method of in vitro simulated digestion experiments is mostly manual, which is prone to sample duplication and poor sample uniformity due to subjective factors, directly affecting the accuracy of the test results, and the samples are easily contaminated; at the same time, the current in vitro simulated digestion and fermentation experiments are either carried out separately in the small intestine and colon, and the digestion products of the small intestine are manually transferred into the colon digestive system, or dynamically enter the colon fermentation system at a constant speed, while the real intestinal peristalsis in the body is carried out in segments, and each peristaltic wave pushes the chyme a short distance and then disappears. The current in vitro simulated digestion and fermentation experiments cannot fully feedback and express the real in vivo reactions.

[0007] Therefore, there is an urgent need for a model that can simulate the dynamic digestion and fermentation of resistant polysaccharide-based foods in the small intestine and colon, so as to gain a deeper understanding of the digestion process of resistant polysaccharide-based foods. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a method for simulating the dynamic digestion and glycolysis of resistant polysaccharide foods in the small intestine and colon, in which a robotic arm and a centrifugal system are placed, which can automatically oscillate and take samples at regular intervals and pump the small intestinal digestion products (the undigested precipitation part in the digestion process and 20% of the small intestinal digestive fluid) into the colon digestion device, which is more in line with the actual digestion conditions of the human body.

[0009] The first object of the present invention is to provide a method for simulating the dynamic digestion and fermentation of polysaccharide-based food in the small intestine and colon, wherein the method simulates the digestion and emptying process of the small intestine and colon by adding polysaccharide-based food into a dynamic digestion and fermentation device;

[0010] The dynamic digestion and fermentation device includes a small intestine digestion simulation system, a sampling and emptying system, a large intestine fermentation simulation system, a central intelligent control system and a display system that are interconnected;

[0011] The small intestine digestion simulation system comprises: a small intestine simulation tank, a small intestine pH control feeding bottle, and a mechanical arm oscillation device;

[0012] The sampling and detection system comprises: a sampling, detection and emptying device; wherein the sampling, detection and emptying device comprises an automatic sampling device, a peristaltic pump, a centrifugal device and a glucose detection device;

[0013] The large intestine fermentation simulation system comprises: a culture medium feeding bottle, a pH control feeding bottle, an ascending colon fermentation tank, a transverse colon fermentation tank, a descending colon fermentation tank, and an anaerobic bottle; wherein the culture medium feeding bottle comprises a first culture medium feeding bottle, a second culture medium feeding bottle, and a third culture medium feeding bottle; the pH control feeding bottle comprises a first pH control feeding bottle, a second pH control feeding bottle, and a third pH control feeding bottle;

[0014] The central intelligent control system includes: a general control and display screen, a pH control system, a temperature control system, a stirring rate control system, a gas circuit system, and a flow rate control system, which are used to control pH, temperature, stirring rate, gas transmission, and digestion liquid transmission.

[0015] In one embodiment, the polysaccharide-based food of the present invention includes but is not limited to rice, noodles, biscuits, bread, and may also be different types of starch.

[0016] In one embodiment, the different types of starch in the polysaccharide-based food include tapioca starch, corn starch, sweet potato starch, potato starch, slowly digestible starch, resistant starch, acetylated starch, and the like.

[0017] In one embodiment, in the small intestine digestion simulation system, the mechanical arm oscillating device and the small intestine simulation tank are connected via a transmission device; the mechanical arm oscillating device and the general control and display screen are connected via a communication line;

[0018] The small intestine pH feeding bottle and the small intestine simulation tank are connected through a silicone tube; the small intestine pH feeding bottle and the main control and display screen are connected through a communication line;

[0019] In the large intestine fermentation simulation system, the first culture medium feeding bottle, the large intestine first pH control feeding bottle and the ascending colon fermentation tank are connected by a silicone tube; the second culture medium feeding bottle, the second pH control feeding bottle and the transverse colon fermentation tank are connected by a silicone tube; the third culture medium feeding bottle, the third pH control feeding bottle and the descending colon fermentation tank are connected by a silicone tube;

[0020] The first culture medium feeding bottle, the second culture medium feeding bottle and the third culture medium feeding bottle are connected to the general control and display screen through a communication line, and the first pH control feeding bottle, the second pH control feeding bottle and the third pH control feeding bottle are connected to the general control and display screen through a communication line;

[0021] The anaerobic cylinder is connected to the ascending colon fermentation tank, the transverse colon fermentation tank, and the descending colon fermentation tank through a silicone tube; the anaerobic cylinder is connected to the main control and display screen through a communication line;

[0022] The sampling, detection and emptying detection device is connected with the small intestine simulation tank, the ascending colon fermentation tank, the transverse colon fermentation tank and the descending colon fermentation tank through a silicone tube; the sampling, detection and emptying detection device is connected with the general control and display screen through a communication line.

[0023] In one embodiment, the digestion and emptying process of the simulated small intestine is:

[0024] Add polysaccharide-based foods into the small intestine digestion simulation system for digestion, and use a robotic arm to shake the reaction tank;

[0025] Using a sampling and detection system, taking out digestive fluid from the small intestine simulation tank (i.e., the emptying volume of digestive fluid in the simulated small intestine tank) according to the emptying equation, centrifuging to obtain a supernatant and a precipitate, and transferring the supernatant and the precipitate to a large intestine fermentation simulation system;

[0026] The emptying equation is as follows:

[0027]

[0028] Wherein, t represents the digestion time (min) of the small intestinal digestion simulation system, that is, the time after the start of small intestinal digestion.

[0029] In one embodiment, the digestion conditions in the small intestine simulation tank are 36-38° C., pH 6.8-7.2, and 300-400 rpm.

[0030] In one embodiment, the transfer ratio of the supernatant is 18-22% v / v.

[0031] In one embodiment, a small intestinal digestion simulation system simulates the small intestinal digestion process of polysaccharide-based foods in the human body, with stirring at 200-400 rpm at 37°C and a pH controlled at 6.5-7.0. The reaction tank is shaken and evenly mixed by a robotic arm at regular intervals according to the emptying equation using a sampling, detection and emptying system, and then the digestive fluid is taken out of the digestion system and pumped into a centrifugal system for centrifugation. The supernatant is immediately tested using an online glucose detection system to obtain the glucose release amount of the polysaccharide-based food to be tested at each time point under the in vitro simulated digestion reaction, and the undigested precipitate and part of the supernatant are pumped into the simulated colon device by a peristaltic pump for further fermentation.

[0032] In one embodiment, when the digestion time of the small intestine digestion simulation system reaches 10 min, the amount of digestive fluid discharged from the simulated small intestine tank is 1 mL.

[0033] In one embodiment, the human intestinal microbial ecology is constructed by inoculating fecal flora from healthy people.

[0034] In one embodiment, the method for constructing a large intestine fermentation simulation system is as follows:

[0035] Add the stool sample to phosphate buffer solution, add sodium thioacetate, homogenize, and centrifuge to obtain a microbial solution, add the microbial solution to an ascending colon fermentation tank, a transverse colon fermentation tank, and a descending colon fermentation tank, respectively, and then add culture medium for cultivation; after the microbial solution is stabilized, control the pH values ​​of the ascending colon fermentation tank, the transverse colon fermentation tank, and the descending colon fermentation tank to 5.6-6.0, 6.0-6.4, and 6.4-6.9, respectively.

[0036] In one embodiment, the culture conditions of the microbial culture liquid are 36-38° C., stirring at 30-40 rpm for 12-24 hours, and passing anaerobic gas (5% hydrogen, 10% carbon dioxide, 85% nitrogen) for 10-15 minutes every 6-8 hours.

[0037] In one embodiment, the volume ratio of the microbial liquid in the ascending colon fermenter, the transverse colon fermenter, and the descending colon fermenter is 4.5-5.5:7.5-8.5:4.5-5.5.

[0038] In one embodiment, the volume ratio of the microbial liquid to the culture medium in the ascending colon fermenter, the transverse colon fermenter, and the descending colon fermenter is 0.8-1.2:10.

[0039] In one embodiment, the steps for constructing the intestinal microbial ecology of the large intestine fermentation simulation system are as follows:

[0040] Feces were collected from 3 to 5 adults who had not taken antibiotics for 6 months. 50 g of feces were sampled and mixed and added to a phosphate buffer solution with a pH of 6.5 to 7.5, and 0.5 to 2 g of sodium thioacetate was added as a reducing agent. The sample was homogenized using a homogenizer. After homogenization, the sample was centrifuged at 3000 rpm for 5 to 10 min. After centrifugation, 50 to 80 mL, 50 to 80 mL, and 50 to 80 mL of intestinal microbial culture liquid were inoculated into three cylindrical glass reactors in the large intestine decomposition simulation system, respectively. Then, 500 to 800 mL, 500 to 800 mL, and 500 to 800 mL of SHIME culture medium were added to the three reactors, respectively. The mixture was stirred at 30 to 50 rpm at 37°C for 12 to 24 h, and anaerobic gas (5% hydrogen, 10% carbon dioxide, 85% nitrogen) was passed for 10 to 30 min every 8 h. After stabilization, the pH values ​​of the three reactors were controlled at 5.6-6.0, 6.0-6.4 and 6.4-6.9, respectively, by adding 0.05-0.1 mol / L HCl or 0.05-0.1 mol / L NaOH.

[0041] In one embodiment, the central intelligent control system includes a master control and display screen, a pH control system, a temperature control system, a stirring rate control system, a gas circuit system, and a flow rate control system, and the master control and display screen are connected to the remaining systems via communication lines;

[0042] A pH control system, comprising five pH electrodes, a pH controller, an acid bottle, an alkali bottle, an acid peristaltic pump and an alkali peristaltic pump; the pH electrode is connected to the pH controller via a communication line; the pH controller is connected to a central intelligent controller via a communication line to transmit the pH value of each simulation system to the central intelligent controller in real time, and control the acid and alkali peristaltic pumps to start the transfer of the acid and alkali; the acid bottle and the alkali bottle are respectively connected to the acid and alkali inlets of the small intestine digestion simulation system and the large intestine decomposition simulation system via silicone tubes;

[0043] The temperature control system is connected to the central intelligent control system through a communication line, and includes a movable upper cover, an annular water bath, a water bath pump, multiple semiconductor temperature control systems and a temperature sensor; the multiple temperature control systems are connected to the temperature sensor through a communication line, and the real-time temperature of the water bath is fed back to the central intelligent control system through the communication line by the temperature sensor to form a record, and the heater is controlled to be turned on or off;

[0044] A stirring rate control system, including five magnetic stirring devices, which are connected to the central intelligent control system via a communication line;

[0045] The gas system consists of an anaerobic cylinder, a bacterial filter, a one-way valve, pipes and interfaces;

[0046] The flow rate control system includes a number of peristaltic pumps and pipelines.

[0047] The second object of the present invention is to provide application of any of the above methods in the field of food, medicine or feed.

[0048] In one embodiment, the application includes: detecting the digestion and absorption effects of food, medicine or feed.

[0049] Beneficial effects of the present invention

[0050] The present invention provides a method for simulating the dynamic digestion and fermentation of polysaccharide-based foods in the small intestine and colon. The method can simulate the enzymatic digestion reaction activity of the small intestine and the fermentation activity of the colon through in vitro experiments, measure the rate at which glucose is released by the carbohydrate food to be tested, and regulate and monitor the gastrointestinal degradation kinetics of foods mainly based on carbohydrates during the digestion process in real time.

[0051] Specifically:

[0052] (1) The device for simulating the dynamic digestion and glycolysis of resistant polysaccharide-based foods in the small intestine and colon provided by the present invention is equipped with a mechanical arm and a centrifugal system, which can automatically oscillate and centrifuge at each time point and pump the small intestine digestion products, the undigested precipitate part and 20% of the small intestine digestive fluid into the colon glycolysis device based on the emptying equation during the digestion process; the simulation method of the present invention has a short-chain fatty acid composition that is closer to the real human body and is more in line with the real digestive environment than the existing method of directly pumping into the colon glycolysis system.

[0053] (2) Compared with the prior art, the present invention can automatically take samples and online detect the production of glucose in an in vitro simulated small intestinal digestive system, overcoming the problems of poor sample uniformity caused by subjective factors in traditional manual sampling and measurement methods. The model has good stability and repeatability, meeting the requirements of standardized scientific research and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Schematic diagram of the device for simulating small intestine-colon dynamic digestion and fermentation of the present invention; wherein 101 is a small intestine simulation tank, 201-203 are feeding bottles, 301-304 are pH control feeding bottles, 401 is an ascending colon fermentation tank, 402 is a transverse colon fermentation tank, 403 is a descending colon fermentation tank, 501 is a sampling, detection and emptying device, 601 is an anaerobic cylinder, 701 is a general control and display screen, and 801 is a mechanical arm oscillation device;

[0055] Figure 2 is a graph showing the glucose content in the small intestine supernatant during digestion in the embodiments and comparative examples;

[0056] Figure 3 It is a diagram showing the distribution results of the short-chain fatty acids of the embodiments and comparative examples. DETAILED DESCRIPTION

[0057] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0058] Device:

[0059] Dynamic digestion and fermentation device Figure 1 As shown, it includes interconnected small intestine digestion simulation system, sampling and emptying system, large intestine fermentation simulation system, central intelligent control and display system;

[0060] The small intestine digestion simulation system includes a small intestine simulation tank 101, a small intestine pH control feeding bottle 301, and a mechanical arm oscillating device 801;

[0061] The sampling and detection system includes: sampling, detection and device 501, the sampling, detection and device 501 includes an automatic sampling device, a peristaltic pump, a centrifugal device and a glucose detection device;

[0062] The large intestine fermentation simulation system includes culture medium feeding bottles 201-203, large intestine pH control feeding bottles 302-304, ascending colon fermentation tank 401, transverse colon fermentation tank 402, descending colon fermentation tank 403, and anaerobic cylinder 601;

[0063] Among them, the feeding bottles include a first culture medium feeding bottle 201, a second culture medium feeding bottle 202 and a third culture medium feeding bottle 203; the large intestine pH control feeding bottle includes a first pH control feeding bottle 302, a second pH control feeding bottle 303 and a third pH control feeding bottle 304;

[0064] The central intelligent control and display system includes a general control and display screen 701, a pH control system, a temperature control system, a stirring rate control system, a gas path system, and a flow rate control system.

[0065] In the small intestine digestion simulation system, the mechanical arm oscillating device 801 and the small intestine simulation tank 101 are connected through a transmission device; the mechanical arm oscillating device 801 and the general control and display screen 701 are connected through a communication line;

[0066] The small intestine pH feeding bottle 301 and the small intestine simulation tank 101 are connected through a silicone tube; the small intestine pH feeding bottle 301 and the main control and display screen 701 are connected through a communication line.

[0067] In the large intestine fermentation simulation system, the first culture medium feeding bottle 201, the large intestine first pH control feeding bottle 302 and the ascending colon fermentation tank 401 are connected by a silicone tube; the second culture medium feeding bottle 202, the second pH control feeding bottle 303 and the transverse colon fermentation tank 402 are connected by a silicone tube; the third culture medium feeding bottle 203, the third pH control feeding bottle 304 and the descending colon fermentation tank 403 are connected by a silicone tube;

[0068] The first culture medium feeding bottle 201, the second culture medium feeding bottle 202 and the third culture medium feeding bottle 203 are connected to the main control and display screen 701 through a communication line, and the first pH control feeding bottle 302, the second pH control feeding bottle 303 and the third pH control feeding bottle 304 are connected to the main control and display screen 701 through a communication line;

[0069] The anaerobic cylinder 601 is connected to the ascending colon fermentation tank 401, the transverse colon fermentation tank 402, and the descending colon fermentation tank 403 through a silicone tube; the anaerobic cylinder 601 is connected to the main control and display screen 701 through a communication line.

[0070] The sampling, detecting and emptying device 501 is connected to the small intestine simulation tank 101, the ascending colon fermentation tank 401, the transverse colon fermentation tank 402, and the descending colon fermentation tank 403 through silicone tubes; the sampling, detecting and emptying device 501 is connected to the main control and display screen 701 through a communication line.

[0071] raw material:

[0072] Corn starch was purchased from Hangzhou Prostar Starch Co., Ltd.;

[0073] The preparation process of acetylated starch is as follows: a certain amount of corn starch is dispersed in deionized water to prepare a 30% (w / w) starch suspension, which is placed on a heating plate and maintained at a temperature of 40°C. The pH of the suspension is adjusted to 8-9 with a 1 mol / L NaOH solution, and then 15% (based on starch dry basis, v / w, mL / g) acetic anhydride is added dropwise within 0.5 h, and the reaction is continued for 2 h. Finally, the pH of the system is adjusted to 6.5 with a 1 mol / L HCl solution to terminate the reaction. The obtained acylated starch is washed twice with deionized water and ethanol, and then dried in a 40°C oven, ground and passed through a 100-mesh sieve to obtain a sample;

[0074] Pancreatic enzyme was purchased from Sigma-Aldrich Company with an enzyme activity of 1000 U / mg;

[0075] Amyloglucosidase was purchased from Sigma-Aldrich with an enzyme activity of 260 U / mg;

[0076] SHIME medium formula: 1.0 g / L arabinogalactan, 2.0 g / L pectin, 3.0 g / L starch, 1.0 g / L xylan, 0.5 g / L cysteine, 0.4 g / L glucose, 3.0 g / L yeast extract, 4.0 g / L mucin and 1.0 g / L peptone.

[0077] Artificial intestinal fluid: 0.055mol / L KCl, 0.006mol / L KH2PO4, 0.684mol / L NaHCO3, 0.309mol / LNaCl, 0.003mol / L MgCl2, 0.126mol / L HCl, 0.21×10 -3 mol CaCl2·2H2O, 4mg / mL pancreatic enzyme.

[0078] Detection method:

[0079] 1. Determination of short-chain fatty acids (SCFAs)

[0080] Take 25 mg of the sample to be tested in a 2 mL EP tube, add pure water and vortex evenly, centrifuge at 4 ° C for 20 min (5000 r / min). Take 0.8 mL of the supernatant in a 2 mL EP tube, add 0.1 mL of H2SO4 solution (50%) and 0.8 mL of the extract, centrifuge for 15 min (4 ° C, 10000 r / min), and then stand at -20 ° C for 30 min. Finally, take the supernatant in a sample injection bottle and detect it by gas chromatography-mass spectrometry (GC-MS).

[0081] Chromatographic conditions: Chromatographic column: Agilent HP-FFAP capillary column (30m×250μm×0.25μm); Temperature program: 80℃ for 1min, increase to 200℃ at 10℃ / min, maintain for 5min, increase to 240℃ at 40℃ / min, maintain for 1min; Carrier gas (He) flow rate 3mL / min, injection volume 1μL; Split ratio: 5:1. Mass spectrometry conditions: ionization voltage -70eV; transfer line temperature 240℃; ion source temperature 200℃; quadrupole temperature 150℃; mass scan range m / z 33~150.

[0082] 2. Sample collection for in vivo control experiments

[0083] Before the experiment, mice were fasted but not watered for 20 hours to allow the original contents in the gastrointestinal tract to be digested or emptied as much as possible. Starch was prepared into a certain concentration suspension (2.5g / mL), mixed, and the required dose (0.4mL) was drawn through a syringe for gavage to mice. Fresh feces of mice were collected and immediately frozen in liquid nitrogen, and fecal samples were stored at -80°C for later use.

[0084] Example 1: A method for simulating the dynamic digestion and fermentation of digestion-resistant polysaccharide-based foods in the small intestine and colon

[0085] A method for simulating the dynamic digestion and fermentation of digestion-resistant polysaccharide-based foods in the small intestine and colon, by Figure 1 The fermentation dynamic digestion and glycolysis device shown simulates the enzymatic digestion reaction activity of the human small intestine and the fermentation activity of the colon in vitro experiments.

[0086] The method comprises the following steps: measuring the glucose release rate of the food to be tested and the types and contents of the fermentation products by using a fermentation dynamic digestion and fermentation device;

[0087] (1) The intestinal microbial ecology was constructed by inoculating feces of healthy people and performing anaerobic culture in SHIME medium for 24 hours. Feces were collected from 3 to 5 adults who had not taken antibiotics for 6 months. 50 g of feces were sampled and mixed with a phosphate buffer solution with a pH of 7 at a ratio of 1:10 (g:mL), and 1 g of sodium thioacetate was added as a reducing agent. The sample was homogenized using a homogenizer. After homogenization, the sample was centrifuged at 3000 rpm for 5 minutes. After centrifugation, the ascending colon fermentation tank (401), transverse colon fermentation tank (402), and descending colon fermentation tank in the large intestine fermentation simulation system were respectively used. (403) Inoculate 50mL, 80mL, and 50mL of intestinal microbial liquid into the reactor, then add 500mL, 800mL, and 500mL of SHIME medium into the three reactors respectively, stir at 30rpm at 37°C for 24h, and pass anaerobic gas (hydrogen 5%, carbon dioxide 10%, nitrogen 85%) for 10min every 8h; control the pH values ​​of the three reactors within the range of 5.6-6.0, 6.0-6.4, and 6.4-6.9, respectively (different pH values ​​within the range will not affect the results), and continue fermentation for 24h after inoculation to stabilize the flora environment;

[0088] (2) Add 260 mL of artificial intestinal fluid to the simulated small intestine tank (101), and add 10 g of acetylated starch for digestion, stir at 37° C. and 300 rpm, control the pH to 6.8, and use the sampling, detection and emptying device (501) to sample according to the emptying equation:

[0089]

[0090] Here, t represents the time (min) from the start of small intestinal digestion.

[0091] After the reaction tank is shaken 10 times by a mechanical arm every 10 minutes, a sample is taken from the simulated small intestine tank (101) (the sampling volume is the simulated small intestine tank digestion fluid emptying volume calculated by the emptying equation), the digestion fluid is taken out and pumped into the sampling and detection device (501) for detection (centrifugation to obtain a precipitate and a supernatant, and the glucose content in the supernatant is detected), and the glucose release amount of the polysaccharide-based food to be tested at each time point under the in vitro simulated digestion reaction is obtained. The results are as follows: Figure 2 As shown;

[0092] The undigested precipitate and 20% v / v supernatant of the sample obtained from the simulated small intestine tank (101) are pumped into the colon fermentation tank for further fermentation, wherein the digestive fluid in the ascending colon fermentation tank (401), the transverse colon fermentation tank (402) and the descending colon fermentation tank (403) is slowly and continuously emptied at a rate of 12.5 mL / h (i.e., 12.5 mL is transferred from the ascending colon fermentation tank to the transverse colon fermentation tank per hour; 12.5 mL is transferred from the transverse colon fermentation tank to the descending colon fermentation tank per hour). After 24 hours of fermentation and cultivation, the simulated digestion is completed, and the digestive fluid in the descending colon fermentation tank is stored in a -80°C refrigerator for subsequent testing.

[0093] Comparative Example 1: Changing the transfer ratio of the supernatant of the small intestinal digestive juice

[0094] (1) Based on Example 1, the transfer ratio of the supernatant of the small intestinal digestive fluid in step (2) was changed to 10% v / v, and the other steps remained consistent with the example.

[0095] (2) Based on Example 1, the transfer ratio of the supernatant of the small intestinal digestive fluid in step (2) was changed to 30% v / v, and the other steps remained consistent with the example.

[0096] Comparative Example 2: Changing the emptying mode

[0097] On the basis of Example 1, the emptying mode of the simulated small intestine tank (101) in step (2) is changed to constant speed emptying, that is, 1 ml of digestive fluid is taken out from the digestion system every 10 minutes and pumped into the centrifugal system for centrifugation, and the supernatant is immediately tested using the glucose online detection system, and the undigested precipitate and 20% v / v of the supernatant in the simulated small intestine tank (101) are pumped into the simulated colon device for further fermentation through a peristaltic pump, and the other steps remain consistent with the example.

[0098] Comparative Example 3: Classic static simulated digestion

[0099] According to the standard method reported in the literature (A standardised semi-dynamic in vitro digestion method suitable for food - an international consensus [J]. Food & Function, 2020, Vol. 11 (2): 1702-1720), the digestive environment of healthy adults was simulated. The specific method is as follows:

[0100] The mixed enzyme preparation method is as follows: 4.5 g of pancreatic enzyme is dissolved in 40 mL of deionized water, vortexed for 10 min to make it evenly dispersed, centrifuged at 5000×g for 10 min, and 27 mL of supernatant is mixed with 3.2 mL of amyloglucosidase.

[0101] First, weigh 200 mg of sample into a centrifuge tube, add 4 mL of deionized water, 4 mL of sodium acetate buffer (pH 5.2) and 10 glass balls in sequence, and mix them evenly. Then, place the mixture in a 37°C water bath shaker and vibrate for 30 min (170 r / min).

[0102] After that, 2 mL of the mixed enzyme was added to the mixed system. At 0, 10, 20, 30, 45, 60, 90, 120, 150 and 180 min, 0.1 mL of the enzymatic solution was placed in 0.9 mL of ethanol solution (90%) and centrifuged (10000×g, 5 min). The supernatant was taken and the glucose content in the supernatant was determined using a GOPOD kit.

[0103] After 180 min of simulated digestion in the small intestine, all the digestion products of the small intestine were transferred to the large intestine fermentation tank for fermentation. The fermentation process was consistent with that in the embodiment.

[0104] Example 2: Simulation effect detection

[0105] Example 1 and Comparative Examples 1 to 3 After 24 hours of fermentation, the fermentation broth was taken to detect the content of short-chain fatty acids.

[0106] The results of short-chain fatty acid content in fermentation broth are as follows Figure 3 As shown in Table 1. According to the literature "Gastrointestinal effects of resistant starch, soluble maize fibre and pullulan in healthy adults. [J]. The British journal of nutrition, 2013, Vol. 110 (6): 1068-1074", after 20 healthy subjects with normal BMI ingested resistant starch, the proportion of SCFAs in feces was distributed as follows: acetic acid 41.3%, propionic acid 25.6%, butyric acid 33.1%.

[0107] The results show that the simulation method of Example 1 is closer to the actual small intestinal digestion conditions in vivo and is significantly better than Comparative Examples 1 to 3.

[0108] Table 1 Distribution of short-chain fatty acids (%)

[0109] Acetic acid Propionic acid Butyric acid Example 1 40.9±0.85 20.6±0.78 38.5±0.69 Comparative Example 1 (1) 76.06±1.02 12.47±0.85 11.47±0.78 Comparative Example 1(2) 67.21±1.02 21.02±0.85 11.78±0.78 Comparative Example 2 62.38±1.02 30.03±0.85 7.59±0.78 Comparative Example 3 87.22±0.78 6.43±0.65 6.35±0.56 Mouse feces 47.2±3.45 17.2±2.46 35.6±2.54

[0110] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for simulating the dynamic digestion and fermentation of polysaccharide-based foods in the small intestine and colon, characterized in that: The method simulates the digestion and emptying process of the small intestine and colon by adding polysaccharide-based food into a dynamic digestion and fermentation device; The dynamic digestion and fermentation device includes an interconnected small intestine digestion simulation system, a sampling and emptying system, a large intestine fermentation simulation system, a central intelligent control system and a display system; It includes an automatic sampling device, a peristaltic pump, a centrifugal device and a glucose detection device; The large intestine fermentation simulation system comprises: a culture medium feeding bottle, a pH control feeding bottle, an ascending colon fermentation tank, a transverse colon fermentation tank, a descending colon fermentation tank, and an anaerobic bottle; wherein the culture medium feeding bottle comprises a first culture medium feeding bottle, a second culture medium feeding bottle, and a third culture medium feeding bottle; the pH control feeding bottle comprises a first pH control feeding bottle, a second pH control feeding bottle, and a third pH control feeding bottle; The central intelligent control system includes: a general control and display screen, a pH control system, a temperature control system, a stirring rate control system, a gas circuit system, and a flow rate control system.

2. The method according to claim 1, characterized in that In the small intestine digestion simulation system, the mechanical arm oscillating device and the small intestine simulation tank are connected through a transmission device; the mechanical arm oscillating device and the general control and display screen are connected through a communication line; The small intestine pH feeding bottle and the small intestine simulation tank are connected through a silicone tube; the small intestine pH feeding bottle and the main control and display screen are connected through a communication line; In the large intestine fermentation simulation system, the first culture medium feeding bottle, the large intestine first pH control feeding bottle and the ascending colon fermentation tank are connected by a silicone tube; the second culture medium feeding bottle, the second pH control feeding bottle and the transverse colon fermentation tank are connected by a silicone tube; the third culture medium feeding bottle, the third pH control feeding bottle and the descending colon fermentation tank are connected by a silicone tube; The first culture medium feeding bottle, the second culture medium feeding bottle and the third culture medium feeding bottle are connected to the general control and display screen through a communication line, and the first pH control feeding bottle, the second pH control feeding bottle and the third pH control feeding bottle are connected to the general control and display screen through a communication line; The anaerobic cylinder is connected to the ascending colon fermentation tank, the transverse colon fermentation tank, and the descending colon fermentation tank through a silicone tube; the anaerobic cylinder is connected to the main control and display screen through a communication line; The sampling, detecting and emptying device is connected with the small intestine simulation tank, the ascending colon fermentation tank, the transverse colon fermentation tank and the descending colon fermentation tank through a silicone tube; the sampling, detecting and emptying device is connected with the general control and display screen through a communication line.

3. The method according to claim 2, characterized in that The digestion conditions in the small intestine simulation tank were 36-38°C, pH 6.8-7.2, and 300-400 rpm.

4. The method according to claim 1, characterized in that The digestion and emptying process of the simulated small intestine is: Add the polysaccharide-based food into the small intestine digestion simulation system for digestion, and use a robotic arm to shake the small intestine simulation tank; The digestive fluid is taken out from the small intestine simulation tank using a sampling and emptying system according to the emptying equation, and the supernatant and precipitate are obtained by centrifugation, and the supernatant and precipitate are transferred to the large intestine fermentation simulation system; The emptying equation is as follows: Wherein, t represents the digestion time (min) of the small intestinal digestion simulation system.

5. The method according to claim 4, characterized in that The transfer ratio of the supernatant was 18-22% v / v.

6. The method according to claim 1, characterized in that The construction method of the large intestine fermentation simulation system is as follows: Add the stool sample to phosphate buffer solution, add sodium thioacetate, homogenize, and centrifuge to obtain a microbial solution, add the microbial solution to an ascending colon fermentation tank, a transverse colon fermentation tank, and a descending colon fermentation tank, respectively, and then add culture medium for cultivation; after the microbial solution is stabilized, control the pH values ​​of the ascending colon fermentation tank, the transverse colon fermentation tank, and the descending colon fermentation tank to 5.6-6.0, 6.0-6.4, and 6.4-6.9, respectively.

7. The method according to claim 6, characterized in that The culture conditions of the microbial culture liquid are 36-38°C, stirring at 30 rpm for 24 hours, and passing anaerobic gas for 10-15 minutes every 6-8 hours.

8. The method according to claim 6, characterized in that The volume ratio of the microbial culture liquid in the ascending colon fermentation tank, the transverse colon fermentation tank and the descending colon fermentation tank is 4.5-5.5:7.5-8.5:4.5-5.

5.

9. The method according to claim 6, characterized in that The volume ratio of the microbial liquid to the culture medium in the ascending colon fermentation tank, the transverse colon fermentation tank and the descending colon fermentation tank is 0.8-1.2:

10.

10. Use of the method according to any one of claims 1 to 9 in the field of food, medicine or feed, characterized in that: The application includes: detecting the digestion and absorption effects of food, medicine or feed.

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