Anti-digestion polysaccharide-based food colon simulated glycolysis and multi-dimensional online analysis system

Abstract

The invention discloses an anti-digestion polysaccharide-based food colon simulated glycolysis and multi-dimensional online analysis system, and belongs to the technical field of food. According to the method, glycolysis simulation of each colon part is completely carried out according to a human body digestion and glycolysis process and a physiological environment, and intestinal mucosa pellets are added to simulate a human body intestinal mucosa environment, so that the degradation process of partial microorganisms of the human body colon for resisting digestion of polysaccharide-based food is simulated under an in-vitro condition; and the glycolysis condition of the digested polysaccharide-based food entering the colon can be observed on line, and the device fits the real physiological environment of people.

Description

Technical Field

[0001] The invention relates to a colonic simulated fermentation and multi-dimensional online analysis system for digestion-resistant polysaccharide-based foods, belonging to the technical field of food. Background Art

[0002] The human digestive organs are usually divided into digestive glands and digestive tracts including the mouth, esophagus, stomach, small intestine and large intestine, which together constitute the human digestive system. Among them, the colon is an important part of the human digestive system and is the lower part of the digestive tract. The bacteria in the colon constitute a huge and complex ecosystem. The bacteria contain enzymes that can decompose or ferment cellulose and resistant sugars to produce short-chain fatty acids, carbon dioxide and methane. Since in vivo digestion experiments are limited by high experimental costs, long cycles, poor reproducibility of results, and animal experiments will cause great pain to the test animals, in vitro colon fermentation simulators are now often used to replace animal experiments to study the changes of resistant polysaccharides during digestion and fermentation.

[0003] Patent CN108364553A discloses that by real-time regulation of the speed of gastric emptying and intestinal transit, the real-time flow of food between different digestive tracts is achieved; the digestive state and environment of the human stomach-small intestine are simulated more realistically and comprehensively; Patent CN112029663A discloses an integrated in vitro simulated digestion 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. Through the synergistic effect of each system, the in vitro alternative model not only simulates the gastrointestinal digestion and absorption process in vitro, but also simulates the fermentation and decomposition process of the intestinal microbial community.

[0004] Most of the current bionic colon fermentation systems ignore the important component of the intestinal mucosa, which makes it difficult for the intestinal flora to colonize and has low stability, affecting the stability of the entire ecosystem in the fermenter, being unfavorable for the survival of bacteria with weak competitiveness, and easily causing pollution. There is a lack of simulation of the real intestinal environment; and most of them characterize the fermentation process of food through sampling and analysis, resulting in the samples taken out not necessarily being representative, causing the results of in vitro fermentation tests to be different from the actual fermentation process.

[0005] Therefore, developing a method based on intestinal mucosal spheres that is closer to the actual intestinal digestion situation in order to gain a deeper understanding of the digestion process of resistant polysaccharide-based foods has extremely high practical and economic value. Summary of the invention

[0006] To solve the above problems, the present invention prepares intestinal mucosal beads that simulate the human intestinal mucosal environment, and directly characterizes the fermentation products and gas component contents during the fermentation process in the fermentation system (in situ), thereby continuously monitoring the fermentation dynamics in a non-invasive manner.

[0007] The first object of the present invention is to provide a method for simulating the dynamic digestion of polysaccharide-based foods by the colon in vitro, wherein the intestinal mucosal pellets are added to a colon digestion simulation device, polysaccharide-based foods are added, and the digestion process and emptying process of the colon are simulated;

[0008] The preparation method of the mucosal sphere is as follows:

[0009] Agar is added into water to dissolve, and mucin is added to obtain a mucosal gel solution; the mucosal gel solution is solidified to obtain mucosal spheres.

[0010] In one embodiment, the colon digestion simulation device includes an interconnected large intestine fermentation simulation system, a central intelligent control and display system, an exhaust gas detection system, and an online detection system for bacterial count and short-chain fatty acids;

[0011] Among them, the large intestine fermentation simulation system includes: culture medium feeding bottle, pH control feeding bottle, ascending colon fermentation tank, transverse colon fermentation tank, descending colon fermentation tank, and anaerobic cylinder;

[0012] The culture medium feeding bottle includes a first culture medium feeding bottle, a second culture medium feeding bottle and a third culture medium feeding bottle; the pH control feeding bottle includes a first pH control feeding bottle, a second pH control feeding bottle and a third pH control feeding bottle

[0013] The central intelligent control and display system includes the overall control and display screen, pH control system, temperature control system, stirring rate control system, gas circuit system, and flow rate control system;

[0014] The tail gas detection system includes a tail gas detector and a product collection bottle;

[0015] The online detection system for bacterial count and short-chain fatty acids includes a bacterial count and short-chain fatty acid detector; the bacterial count and short-chain fatty acid detector includes a first bacterial count and short-chain fatty acid detector, a second bacterial count and short-chain fatty acid detector and a third bacterial count and short-chain fatty acid detector.

[0016] 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.

[0017] 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.

[0018] In one embodiment, 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;

[0019] 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;

[0020] 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;

[0021] The tail gas detector is connected to the descending colon fermentation tank and the product collection bottle through a silicone tube; the tail gas detector is connected to the main control and display screen through a communication line;

[0022] The bacteria quantity and short-chain fatty acid detector is located at the bottom of the ascending colon fermentation tank, the bacteria quantity and short-chain fatty acid detector is located at the bottom of the transverse colon fermentation tank, and the bacteria quantity and short-chain fatty acid detector is located at the bottom of the descending colon fermentation tank; the bacteria quantity and short-chain fatty acid detector is connected to the main control and display screen through a communication line.

[0023] In one embodiment, the usage ratio of agar, mucin and water is 1-3 g: 0.3-0.6 g: 50 mL.

[0024] In one embodiment, the pH of the mucosal gel solution is adjusted to 7.5-8.5.

[0025] In one embodiment, the intestinal mucosal spheres have a diameter of 0.3 to 0.7 cm.

[0026] In one embodiment, the intestinal mucosal spheres are located in the ascending colon fermentation tank, the transverse colon fermentation tank, and the descending colon fermentation tank of the colon digestion simulation device, and the number is 3 to 7.

[0027] The second object of the present invention is to provide a colon simulation fermentation and analysis system for polysaccharide-based foods, wherein the colon simulation fermentation and analysis system contains intestinal mucosal spheres;

[0028] The colon simulation fermentation and analysis system includes an interconnected large intestine fermentation simulation system, a central intelligent control and display system, an exhaust gas detection system, and an online detection system for bacterial count and short-chain fatty acids; wherein the large intestine fermentation simulation system includes: 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 cylinder;

[0029] The culture medium feeding bottle includes a first culture medium feeding bottle, a second culture medium feeding bottle and a third culture medium feeding bottle; the pH control feeding bottle includes a first pH control feeding bottle, a second pH control feeding bottle and a third pH control feeding bottle

[0030] The central intelligent control and display system includes the overall control and display screen, pH control system, temperature control system, stirring rate control system, gas circuit system, and flow rate control system;

[0031] The tail gas detection system includes a tail gas detector and a product collection bottle;

[0032] The online detection system for bacterial count and short-chain fatty acids includes a bacterial count and short-chain fatty acid detector; the bacterial count and short-chain fatty acid detector includes a first bacterial count and short-chain fatty acid detector, a second bacterial count and short-chain fatty acid detector and a third bacterial count and short-chain fatty acid detector.

[0033] In one embodiment, the method for preparing the mucosal spheres is as follows: adding agar to water, dissolving it, adding mucin to obtain a mucosal gel solution; solidifying the mucosal gel solution to obtain mucosal spheres;

[0034] Optionally, the dosage ratio of agar, mucin and water is 1-3g:0.3-0.6g:50mL; the diameter of the intestinal mucosal spheres is 0.3-0.7cm; the intestinal mucosal spheres are located in the ascending colon fermentation tank, transverse colon fermentation tank and descending colon fermentation tank of the colon digestion simulation device, and the number is 3-7.

[0035] The third object of the present invention is to provide an application of any of the above methods or any of the above systems in the field of food, medicine or feed, wherein the application includes: detecting the digestion and absorption effects of food, medicine or feed.

[0036] The fourth object of the present invention is to provide a method for improving the credibility of in vitro simulation of colonic dynamic digestion of polysaccharide-based foods, by adding intestinal mucosal pellets to a colonic digestion simulation device;

[0037] The preparation method of the mucosal sphere is as follows: adding agar into water, dissolving it, adding mucin to obtain a mucosal gel solution; solidifying the mucosal gel solution to obtain the mucosal sphere;

[0038] Optionally, the dosage ratio of agar, mucin and water is 1-3g:0.3-0.6g:50mL; the diameter of the intestinal mucosal spheres is 0.3-0.7cm; the intestinal mucosal spheres are located in the ascending colon fermentation tank, transverse colon fermentation tank and descending colon fermentation tank of the colon digestion simulation device, and the number is 3-7.

[0039] In one embodiment, the credibility is that the flora structure and short-chain fatty acid ratio are closer to the flora and short-chain fatty acid structure of the real intestine.

[0040] In one embodiment, the colon digestion simulation device includes an interconnected large intestine fermentation simulation system, a central intelligent control and display system, an exhaust gas detection system, and an online detection system for bacterial count and short-chain fatty acids;

[0041] Among them, the large intestine fermentation simulation system includes: culture medium feeding bottle, pH control feeding bottle, ascending colon fermentation tank, transverse colon fermentation tank, descending colon fermentation tank, and anaerobic cylinder;

[0042] The culture medium feeding bottle includes a first culture medium feeding bottle, a second culture medium feeding bottle and a third culture medium feeding bottle; the pH control feeding bottle includes a first pH control feeding bottle, a second pH control feeding bottle and a third pH control feeding bottle

[0043] The central intelligent control and display system includes the overall control and display screen, pH control system, temperature control system, stirring rate control system, gas circuit system, and flow rate control system;

[0044] The tail gas detection system includes a tail gas detector and a product collection bottle;

[0045] The online detection system for bacterial count and short-chain fatty acids includes a bacterial count and short-chain fatty acid detector; the bacterial count and short-chain fatty acid detector includes a first bacterial count and short-chain fatty acid detector, a second bacterial count and short-chain fatty acid detector and a third bacterial count and short-chain fatty acid detector.

[0046] In one embodiment, 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;

[0047] 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;

[0048] 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;

[0049] The tail gas detector is connected to the descending colon fermentation tank and the product collection bottle through a silicone tube; the tail gas detector is connected to the main control and display screen through a communication line;

[0050] The bacteria quantity and short-chain fatty acid detector is located at the bottom of the ascending colon fermentation tank, the bacteria quantity and short-chain fatty acid detector is located at the bottom of the transverse colon fermentation tank, and the bacteria quantity and short-chain fatty acid detector is located at the bottom of the descending colon fermentation tank; the bacteria quantity and short-chain fatty acid detector is connected to the main control and display screen through a communication line.

[0051] In one embodiment, the preparation method of the intestinal mucosal spheres added to the large intestine glycolysis simulation system is as follows: weigh 2 to 3 g of agar and heat it in 50 to 80 mL of distilled water to dissolve it. After the solution becomes clear and transparent, cool it to 55 to 65° C. and add 0.4 to 0.6 g of mucin. After it is fully dissolved, adjust the pH of the solution to 7.5 to 8.5; add the mucosal gel solution to the mold to solidify it into small balls, and sterilize it with ultraviolet light in a clean bench for 20 to 50 minutes.

[0052] In one embodiment, the central intelligent control system includes a touch screen intelligent controller, which is connected to the remaining systems through communication lines; the pH control system includes five pH electrodes, pH controllers, acid bottles, alkali bottles, acid peristaltic pumps and alkali peristaltic pumps;

[0053] The pH electrode is connected to the pH controller via a communication line; the pH controller is connected to the 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 acid and alkali; the acid bottle and alkali bottle are respectively connected to the acid and alkali inlets of the large intestine decomposition simulation system via silicone tubes;

[0054] 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;

[0055] The stirring rate control system includes five magnetic stirring devices, which are connected to the central intelligent control system through a communication line.

[0056] In one embodiment, the steps for constructing the intestinal microbial ecology of the large intestine glycolysis simulation system are as follows: collect feces from 3 to 5 adults who have not taken antibiotics for 6 months, sample 50g of feces, mix and add a phosphate buffer solution with a pH of 6.5 to 7.5, and add 0.5 to 2g of sodium thioacetate as a reducing agent, use a homogenizer to homogenize the sample, and after homogenization, centrifuge the sample at 3000rpm for 5 to 10min. After centrifugation, inoculate 50 to 80mL, 50 to 80mL, and 50 to 80mL of intestinal microbial culture liquid in three cylindrical glass reactors in the large intestine decomposition simulation system, respectively, and then add 500 to 800mL, 500 to 800mL and 500 to 800mL of SHIME culture medium to the three reactors, respectively, stir at 30 to 50rpm at 37°C for 12 to 24h, and pass anaerobic gas (5% hydrogen, 10% carbon dioxide, 85% nitrogen) for 10 to 30min every 8h. 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.

[0057] In one embodiment, 24 hours after the first inoculation, in order to maintain the normal growth of microorganisms, nutrients are replenished and 200-400 ml of culture medium is discharged daily to maintain the constant volume, and 3-5 new mucosal pellets are used to replace the mucosal pellets in the tank to simulate mucosal regeneration.

[0058] In one embodiment, the fecal intestinal flora inoculation is followed by continuous fermentation for 7 days and becomes stable.

[0059] Beneficial effects of the present invention

[0060] The present invention provides a colon simulation fermentation and multi-dimensional online analysis system for digestion-resistant polysaccharide-based foods, which can simulate the fermentation activity of the colon through in vitro experiments, measure the fermentation gas and fermentation products of the food to be tested, and regulate and monitor the degradation kinetics of the digestion-resistant polysaccharide-based foods in real time.

[0061] Specific:

[0062] (1) Introducing intestinal mucosal microspheres to simulate the human intestinal mucosal environment can more carefully and accurately simulate the composition of human intestinal microorganisms in vitro, making the in vitro intestinal simulation fermentation system closer to the real intestine and improving the simulation effect and reliability of the experiment.

[0063] (2) Compared with the prior art, the present invention performs online characterization of the fermentation product content during the fermentation process in the fermentation system (in situ), and continuously monitors the fermentation dynamics in a non-invasive manner. The fermentation rules of intestinal flora-resistant food are determined by diversified gas detection. In situ detection of short-chain fatty acids (acetic acid, propionic acid, butyric acid, etc.) during the fermentation process is achieved by infrared devices, which can quickly and accurately identify changes in intestinal bacteria and their metabolites, and avoid errors caused by a small number of sampling points. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Schematic diagram of the fermentation system for simulating colon environment of the present invention; wherein 101 is an ascending colon fermentation tank, 102 is a transverse colon fermentation tank, 103 is a descending colon fermentation tank, 201-203 are culture medium feeding bottles, 301-303 are pH control feeding bottles, 401 is a product collection bottle, 501 is an exhaust gas detector, 601 is an anaerobic cylinder, 703 is a general control and display screen, and 801-803 are bacterial quantity and short-chain fatty acid detectors;

[0065] Figure 2 is a bacterial abundance diagram of the embodiment and the comparative example after continuous fermentation for 7 days after inoculation and stabilization; wherein, "AC" represents the ascending colon, "DC" represents the descending colon, "TC" represents the transverse colon, "Initial" represents the original inoculated bacterial community, "A" represents the bacterial community of embodiment 1, and "B, C, D, E" represent the bacterial community abundance of comparative example 1 (1), comparative example 1 (2), comparative example 2, and comparative example 3, respectively;

[0066] Figure 3 is the PCA diagram of the bacterial flora after continuous fermentation for 7 days after inoculation in the embodiment and the comparative example and tending to be stable;

[0067] Figure 4 24h after adding the sample to the fermentation broth; DETAILED DESCRIPTION

[0068] 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.

[0069] Device:

[0070] Colonic simulated fermentation and multi-dimensional online analysis system for digestion-resistant polysaccharide-based foods Figure 1 As shown, it includes interconnected large intestine fermentation simulation system, central intelligent control and display system (pH control system, temperature control system, stirring rate control system, gas path system, flow rate control system), tail gas detection system, bacterial quantity and short-chain fatty acid online detection system;

[0071] The large intestine fermentation simulation system includes culture medium feeding bottles 201-203, pH control feeding bottles 301-303, ascending colon fermentation tank 101, transverse colon fermentation tank 102, descending colon fermentation tank 103, and anaerobic cylinder 601;

[0072] 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, and the pH control feeding bottles include a first pH control feeding bottle 302, a second pH control feeding bottle 303 and a third pH control feeding bottle 304;

[0073] The central intelligent control and display system includes a general control and display screen 703, a pH control system, a temperature control system, a stirring rate control system, a gas path system, and a flow rate control system.

[0074] The tail gas detection system includes a tail gas detector 501 and a product collection bottle 401 .

[0075] The bacterial count and short-chain fatty acid online detection system includes bacterial count and short-chain fatty acid detectors 801-803;

[0076] Among them, the bacterial count and short-chain fatty acid detector includes a first bacterial count and short-chain fatty acid detector 801, a second bacterial count and short-chain fatty acid detector 802 and a third bacterial count and short-chain fatty acid detector 803.

[0077] In the large intestine fermentation simulation system, the first culture medium feeding bottle 201, the large intestine first pH control feeding bottle 301 and the ascending colon fermentation tank 101 are connected by a silicone tube; the second culture medium feeding bottle 202, the second pH control feeding bottle 302 and the transverse colon fermentation tank 102 are connected by a silicone tube; the third culture medium feeding bottle 203, the third pH control feeding bottle 303 and the descending colon fermentation tank 103 are connected by a silicone tube;

[0078] 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 703 via a communication line, and the first pH control feeding bottle 301, the second pH control feeding bottle 302 and the third pH control feeding bottle 303 are connected to the main control and display screen 703 via a communication line;

[0079] The anaerobic cylinder 601 is connected to the ascending colon fermentation tank 101, the transverse colon fermentation tank 102, and the descending colon fermentation tank 103 through a silicone tube; the anaerobic cylinder 601 is connected to the general control and display screen 703 through a communication line.

[0080] The tail gas detector 501 is connected to the descending colon fermentation tank 103 and the product collection bottle 401 through a silicone tube; the tail gas detector 501 is connected to the main control and display screen 703 through a communication line.

[0081] The bacterial count and short-chain fatty acid detector 801 is located at the bottom of the ascending colon fermentation tank 101, the bacterial count and short-chain fatty acid detector 802 is located at the bottom of the transverse colon fermentation tank 102, and the bacterial count and short-chain fatty acid detector 803 is located at the bottom of the descending colon fermentation tank 103; the bacterial count and short-chain fatty acid detectors 801-803 are connected to the main control and display screen 703 via a communication line.

[0082] raw material:

[0083] Corn starch was purchased from Hangzhou Prostar Starch Co., Ltd.;

[0084] The preparation process of acetylated starch is as follows:

[0085] A certain amount of corn starch was dispersed in deionized water to prepare a 30% (w / w) starch suspension, which was placed on a heating plate and maintained at 40°C. The pH of the suspension was adjusted to 8-9 with a 1 mol / L NaOH solution, and then 15% (v / w) acetic anhydride was added dropwise within 0.5 h, and the reaction was continued for 2 h. Finally, the pH of the system was adjusted to 6.5 with a 1 mol / L HCl solution to terminate the reaction. The acylated starch was washed twice with deionized water and ethanol, then dried in an oven at 40°C, ground and sieved through a 100-mesh sieve to obtain a sample;

[0086] SHIME medium: 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.

[0087] Detection method:

[0088] 1. Sample collection for in vivo control experiments

[0089] Healthy male 8-week-old C57BL / 6J mice were selected as in vivo controls. The mice were fasted for 20 hours before the experiment to allow the original contents in the gastrointestinal tract to be digested or emptied as much as possible. The starch samples were prepared into a 2.5g / mL suspension with deionized water, mixed, and then gavaged into the mice at the same dose (2mg / g body weight). Fresh feces of the mice were collected and immediately frozen in liquid nitrogen, and the fecal samples were stored at -80°C for later use.

[0090] 2. Determination of short-chain fatty acids (SCFAs) in mouse feces

[0091] 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 an injection bottle and detect it by gas chromatography-mass spectrometry (GC-MS). Chromatographic conditions: Chromatographic column: Agilent HP-FFAP capillary column (30m×250μm×0.25μm); temperature program: 80 ° C for 1 min, increase to 200 ° C at 10 ° C / min, maintain for 5 min, increase to 240 ° C at 40 ° C / min, maintain for 1 min; carrier gas (He) flow rate 3 mL / min, injection volume 1 μL; split ratio: 5:1. Mass spectrometry conditions: ionization voltage -70 eV; transfer line temperature 240 °C; ion source temperature 200 °C; quadrupole temperature 150 °C; mass scan range m / z 33-150.

[0092] Example 1: A method for simulating colonic fermentation and multi-dimensional online analysis of digestion-resistant polysaccharide-based foods

[0093] A method for colonic simulated fermentation and multi-dimensional online analysis of digestion-resistant polysaccharide-based foods, 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.

[0094] The in vitro test simulates the enzymatic digestion reaction activity of the human small intestine and the fermentation activity of the colon through a fermentation dynamic digestion and fermentation device, and measures the glucose release rate of the carbohydrate food to be tested and the types and contents of the fermentation products, including the following steps:

[0095] (1) Preparation of intestinal mucosal spheres

[0096] Mucin is the main structural and functional component of the mucus covering the surface of the intestinal epithelium. Agar is selected as the coagulant of the mucosal sphere. The preparation method of the intestinal mucosal sphere is as follows:

[0097] Weigh 2 g of agar and dissolve it in 50 mL of distilled water, heat it to 100 °C and dissolve it. After the solution becomes clear and transparent, cool it to 60 °C and add 0.5 g of mucin. After it is fully dissolved, adjust the pH of the solution to 8 to obtain a mucosal gel solution. Add the mucosal gel solution into a mold to solidify it into a small ball with a diameter of 0.5 cm, and sterilize it with ultraviolet light in a clean bench for 20 minutes.

[0098] (2) Simulating the intestinal environment

[0099] The intestinal microbial ecology was constructed by inoculating feces from 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 phosphate buffer solution with a pH of 7 at a ratio of 1:10 (g:mL). 1 g of sodium thioacetate was added as a reducing agent, and the sample was homogenized using a homogenizer.

[0100] After homogenization, the sample was centrifuged at 3000 rpm for 5 minutes. After centrifugation, 50 mL, 80 mL, and 50 mL of intestinal microbial culture were inoculated into the ascending colon fermentation tank (101), the transverse colon fermentation tank (102), and the descending colon fermentation tank (103) in the large intestine fermentation simulation system, respectively. Then, 5 mucosal spheres were added to the three reactors, and then 500 mL, 800 mL, and 500 mL of SHIME culture medium were added, respectively. The mixture was stirred at 30 rpm for 24 hours at 37°C, and anaerobic gas (5% hydrogen, 10% carbon dioxide, 85% nitrogen) was passed for 10 minutes every 8 hours. After stabilization, the pH values ​​of the three reactors were controlled within the ranges of 5.6-6.0, 6.0-6.4, and 6.4-6.9, respectively (different pH values ​​within the ranges would not affect the results). The magnetic stirring device was turned on at a speed of 30 rpm to simulate fermentation at 37°C.

[0101] After the first inoculation and cultivation for 24 hours, in order to maintain the normal growth of microorganisms, nutrients were added and 300 mL of culture medium was discharged daily to maintain the constant volume, and the mucosal pellets in the tank were replaced with 3 new mucosal pellets to simulate mucosal regeneration. After the inoculation of fecal intestinal flora, continuous fermentation for 7 days tended to be stable, and part of the fermentation liquid was stored in a -80℃ refrigerator for flora determination.

[0102] (3) Detection of sample digestion effect

[0103] Take 12.5mL of the test substance (resistant starch suspension, 10% g / 100mL, based on starch dry basis) and add it to the ascending colon fermentation tank as a nutrient supplement. Transfer 100mL to the next fermentation tank at a rate of 20mL / min every 8 hours. Transfer in sequence and ferment for 24 hours. The ascending colon simulation tank, transverse colon simulation tank, and descending colon simulation tank are adjusted with hydrochloric acid (0.05mol / L) and sodium hydroxide (0.1mol / L). The pH is maintained between 5.6 and 6.0, 6.0 and 6.4, and 6.5 and 6.9, respectively (different pH values ​​within the range will not affect the results), to obtain the fermented test substance.

[0104] Comparative Example 1: Changing the Ratio of Intestinal Mucosal Balls

[0105] (1) On the basis of Example 1, the ratio of the intestinal mucosal spheres in step (1) was changed to 1 g agar and 1 g mucin, and the other steps were kept consistent with Example 1 to obtain the fermented test substance.

[0106] (2) Based on Example 1, the ratio of the intestinal mucosal spheres in step (1) was changed to 4 g agar and 0.25 g mucin, and the other steps were kept consistent with Example 1 to obtain the fermented test substance.

[0107] Comparative Example 2: Changing the particle size of intestinal mucosal spheres

[0108] On the basis of the example, the diameter of the intestinal mucosal spheres in step (1) was changed to 1 cm, and the other steps were kept consistent with example 1 to obtain the fermented test substance.

[0109] Comparative Example 3: No mucosal beads added

[0110] On the basis of Example 1, the intestinal mucosal spheres were not added in step (2), and the other steps were kept consistent with Example 1 to obtain the fermented test substance.

[0111] Example 2: Fermentation effect detection

[0112] The fermented test objects obtained in Example 1 and Comparative Examples 1 to 3 were taken to detect the bacterial flora composition and short-chain fatty acid composition, and the proportion of short-chain fatty acids in fresh mouse feces was used as a control.

[0113] The bacterial community composition and PCA results are shown in Figure 2 , Figure 3 As shown. Principal component analysis (PCA, based on the Bray-Curtis distance algorithm) is a method of dimensionality reduction analysis of multidimensional data, which is used to evaluate Beta diversity. If the microbial composition is more similar, the closer their distance in the graph is.

[0114] Depend on Figure 3 It can be seen that compared with comparative examples 1 to 3, Example 1 is closest to Initial, while the comparative examples (especially comparative examples 2 and 3) are farther from Initial, indicating that the bacterial composition of the test object after fermentation in the simulation method of the embodiment is closer to the original bacterial composition, and this simulation method is closer to the intestinal fermentation environment. It can be seen that changing the raw material ratio, diameter or number of the intestinal mucosal spheres will affect the intestinal fermentation environment.

[0115] After adding samples to Example 1 and Comparative Examples 1 to 3, the fermentation broth was taken after 24 hours of fermentation and the composition of short-chain fatty acids was detected. The results are as follows Figure 4As shown in Table 1, compared with the in vivo results, the acetic acid ratios of the comparative examples were all higher to varying degrees, and the butyric acid content was lower, especially the comparative example 3 which was far from the in vivo results, while the simulation method of the embodiment was closer to the in vivo fermentation results and was significantly better than the comparative example.

[0116] Table 1 Distribution of short-chain fatty acids (%)

[0117] Acetic acid Propionic acid Propionic acid Example 1 39.91±0.85f <![CDATA[37.49±0.69 a ]]> <![CDATA[22.60±0.58 b ]]> Comparative Example 1 (1) <![CDATA[57.57±1.36 c ]]> <![CDATA[29.05±0.72 c ]]> <![CDATA[13.38±0.52 d ]]> Comparative Example 1(2) <![CDATA[52.71±0.87 d ]]> <![CDATA[32.27±0.64 b ]]> <![CDATA[15.02±1.07 d ]]> Comparative Example 2 <![CDATA[66.06±1.02 b ]]> <![CDATA[13.87±1.27 e ]]> <![CDATA[20.07±0.93 c ]]> Comparative Example 3 <![CDATA[83.43±0.78 a ]]> <![CDATA[9.32±0.65 f ]]> <![CDATA[7.25±0.56 e ]]> Mouse feces <![CDATA[47.2±3.45 e ]]> <![CDATA[17.2±2.54 d ]]> <![CDATA[35.6±2.46 a ]]>

[0118] 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 of polysaccharide-based foods by colon in vitro, characterized in that: The method includes adding intestinal mucosal pellets into a colon digestion simulation device, adding polysaccharide-based food, and simulating the digestion and emptying process of the colon; The preparation method of the mucosal sphere is as follows: Agar is added to water, dissolved, and mucin is added to obtain a mucosal gel solution; The mucosal gel solution is solidified to obtain mucosal pellets.

2. The method according to claim 1, characterized in that The colon digestion simulation device includes an interconnected large intestine fermentation simulation system, a central intelligent control and display system, an exhaust gas detection system, and an online detection system for bacterial count and short-chain fatty acids; Among them, the large intestine fermentation simulation system includes: culture medium feeding bottle, pH control feeding bottle, ascending colon fermentation tank, transverse colon fermentation tank, descending colon fermentation tank, anaerobic cylinder; The culture medium feeding bottle includes a first culture medium feeding bottle, a second culture medium feeding bottle and a third culture medium feeding bottle; the pH control feeding bottle includes a first pH control feeding bottle, a second pH control feeding bottle and a third pH control feeding bottle The central intelligent control and display system includes the overall control and display screen, pH control system, temperature control system, stirring rate control system, gas circuit system, and flow rate control system; The tail gas detection system includes a tail gas detector and a product collection bottle; The online detection system for bacterial count and short-chain fatty acids includes a bacterial count and short-chain fatty acid detector; the bacterial count and short-chain fatty acid detector includes a first bacterial count and short-chain fatty acid detector, a second bacterial count and short-chain fatty acid detector and a third bacterial count and short-chain fatty acid detector.

3. The method according to claim 2, characterized in that 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 tail gas detector is connected to the descending colon fermentation tank and the product collection bottle through a silicone tube; the tail gas detector is connected to the main control and display screen through a communication line; The bacteria quantity and short-chain fatty acid detector is located at the bottom of the ascending colon fermentation tank, the bacteria quantity and short-chain fatty acid detector is located at the bottom of the transverse colon fermentation tank, and the bacteria quantity and short-chain fatty acid detector is located at the bottom of the descending colon fermentation tank; the bacteria quantity and short-chain fatty acid detector is connected to the main control and display screen through a communication line.

4. The method according to claim 1, characterized in that: The dosage ratio of agar, mucin and water is 1-3 g: 0.3-0.6 g: 50 mL.

5. The method according to claim 1, characterized in that The diameter of the intestinal mucosal sphere is 0.3-0.7 cm.

6. The method according to claim 1, characterized in that The intestinal mucosal spheres are located in the ascending colon fermentation tank, the transverse colon fermentation tank and the descending colon fermentation tank of the colon digestion simulation device, and the number of the spheres is 3 to 7.

7. A colonic simulated fermentation and analysis system for polysaccharide-based foods, characterized in that: The colon simulation fermentation and analysis system contains intestinal mucosal spheres; The colon simulation fermentation and analysis system includes an interconnected large intestine fermentation simulation system, a central intelligent control and display system, an exhaust gas detection system, and an online detection system for bacterial count and short-chain fatty acids; wherein the large intestine fermentation simulation system includes: 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 cylinder; The culture medium feeding bottle includes a first culture medium feeding bottle, a second culture medium feeding bottle and a third culture medium feeding bottle; the pH control feeding bottle includes a first pH control feeding bottle, a second pH control feeding bottle and a third pH control feeding bottle The central intelligent control and display system includes the overall control and display screen, pH control system, temperature control system, stirring rate control system, gas circuit system, and flow rate control system; The tail gas detection system includes a tail gas detector and a product collection bottle; The online detection system for bacterial count and short-chain fatty acids includes a bacterial count and short-chain fatty acid detector; the bacterial count and short-chain fatty acid detector includes a first bacterial count and short-chain fatty acid detector, a second bacterial count and short-chain fatty acid detector and a third bacterial count and short-chain fatty acid detector.

8. The colon simulation fermentation and analysis system according to claim 7, characterized in that: The preparation method of the mucosal sphere is as follows: adding agar into water, dissolving it, adding mucin to obtain a mucosal gel solution; solidifying the mucosal gel solution to obtain the mucosal sphere; Optionally, the dosage ratio of agar, mucin and water is 1-3g:0.3-0.6g:50mL; the diameter of the intestinal mucosal spheres is 0.3-0.7cm; the intestinal mucosal spheres are located in the ascending colon fermentation tank, transverse colon fermentation tank and descending colon fermentation tank of the colon digestion simulation device, and the number is 3-7.

9. Use of the method according to any one of claims 1 to 6 or any one of claims 7 to 8 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.

10. A method for improving the credibility of in vitro simulated colon dynamic digestion of polysaccharide-based foods, characterized in that: Add intestinal mucosal pellets to the colon digestion simulation device; The preparation method of the mucosal sphere is as follows: adding agar into water, dissolving it, adding mucin to obtain a mucosal gel solution; solidifying the mucosal gel solution to obtain the mucosal sphere; Optionally, the dosage ratio of agar, mucin and water is 1-3g:0.3-0.6g:50mL; the diameter of the intestinal mucosal spheres is 0.3-0.7cm; the intestinal mucosal spheres are located in the ascending colon fermentation tank, transverse colon fermentation tank and descending colon fermentation tank of the colon digestion simulation device, and the number is 3-7.

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