A system for anti-digestive polysaccharide-based food colon simulation fermentation and multi-dimensional online analysis

Abstract

The application discloses an anti-digestive polysaccharide-based food colon simulation fermentation and multi-dimensional online analysis system and belongs to the technical field of food. The fermentation simulation of each colon part is completely carried out according to the human digestive fermentation process and physiological environment, and intestinal mucosa globules are added to simulate the human intestinal mucosa environment, so that the degradation process of the anti-digestive polysaccharide-based food by part microorganisms in the human colon under the ex vivo condition is simulated, and the fermentation condition of the anti-digestive polysaccharide-based food after entering the colon can be observed online, which is in line with the real human physiological environment.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of anti-digestive polysaccharide-based food colon simulation fermentation and multidimensional online analysis system, belong to food technology field. BACKGROUND

[0002] Human digestive organs are usually divided into digestive glands and digestive tracts including mouth, esophagus, stomach, small intestine and large intestine, which together form the human digestive system, wherein the colon is an important part of the human digestive system and is the lower segment of the digestive tract. The bacteria in the colon form a large and complex ecosystem, and the bacteria contain enzymes that can break down or ferment cellulose and indigestible sugars, producing short-chain fatty acids, carbon dioxide and methane, etc. Due to the limitations of in vivo digestion experiments, such as high experimental cost, long cycle, poor repeatability of results, and great pain to test animals, in vitro colon fermentation simulators are now used to replace animal experiments to study the changes of anti-digestive polysaccharides during digestion and fermentation.

[0003] Patent CN108364553A discloses that by real-time regulation of the speed of gastric emptying and intestinal transport, the real-time flow of food between different digestive tracts is realized; a more realistic and comprehensive simulation of the digestive state and environment of the human stomach and small intestine is achieved; patent CN112029663A discloses an integrated in vitro simulation 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, this in vitro replacement model not only simulates the digestion and absorption process of the gastrointestinal tract, but also simulates the fermentation and decomposition process of the intestinal microbiota.

[0004] Most current biomimetic colon fermentation systems ignore the important component of intestinal mucosa, leading to difficulty in colonization of intestinal flora, low stability, affecting the stability of the entire fermentation system, not conducive to the survival of bacteria with weak competitiveness, and easily causing pollution, lacking simulation of the real intestinal environment; and most of them characterize the fermentation process of food by sampling analysis, resulting in the samples taken may not be representative, causing differences between the in vitro fermentation test results and the real fermentation process.

[0005] Therefore, based on the intestinal mucosa globule, a method closer to the real intestinal digestion situation is developed to better understand the digestion process of anti-digestive polysaccharide-based food, which has very high practical and economic value. SUMMARY

[0006] To solve the above problems, the present application simulates the intestinal mucosa environment of human body by preparing intestinal mucosa beads, and directly in-situ characterizes the content of fermentation products and gas components in the fermentation process in the fermentation system, so as to continuously monitor the fermentation kinetics in a non-invasive manner.

[0007] The first object of the present application is to provide a method for simulating the dynamic digestion of polysaccharide-based food in the colon in vitro, which comprises adding intestinal mucosa beads to a colon digestion simulation device, adding polysaccharide-based food, and simulating the digestion and emptying processes of the colon.

[0008] The preparation method of the mucosa beads is as follows:

[0009] The agar is added to water, dissolved, and mucin is added to obtain a mucosa gel solution; and the mucosa gel solution is coagulated to obtain mucosa beads.

[0010] In an embodiment, the colon digestion simulation device comprises a large intestine fermentation simulation system, a central intelligent control and display system, a tail gas detection system, a bacteria amount and short-chain fatty acid online detection system, which are connected to each other.

[0011] The large intestine fermentation simulation system comprises a culture medium feed bottle, a pH control feed bottle, an ascending colon fermentation tank, a transverse colon fermentation tank, a descending colon fermentation tank, and an anaerobic gas bottle.

[0012] The culture medium feed bottle comprises a first culture medium feed bottle, a second culture medium feed bottle, and a third culture medium feed bottle; and the pH control feed bottle comprises a first pH control feed bottle, a second pH control feed bottle, and a third pH control feed bottle.

[0013] The central intelligent control and display system comprises a total control and display screen, a pH control system, a temperature control system, a stirring rate control system, a gas path system, and a flow rate control system.

[0014] The tail gas detection system comprises a tail gas detector and a product collection bottle.

[0015] The bacteria amount and short-chain fatty acid online detection system comprises a bacteria amount and short-chain fatty acid detector; and the bacteria amount and short-chain fatty acid detector comprises a first bacteria amount and short-chain fatty acid detector, a second bacteria amount and short-chain fatty acid detector, and a third bacteria amount and short-chain fatty acid detector.

[0016] In an embodiment, the polysaccharide-based food in the present application includes, but is not limited to, rice, noodles, biscuits, and bread, and can also be different types of starch.

[0017] In an embodiment, the different types of starch in the polysaccharide-based food include cassava starch, corn starch, sweet potato starch, potato starch, slow-digesting starch, resistant starch, acetylated starch, etc.

[0018] In an embodiment, in the large intestine fermentation simulation system, the first culture medium feeding bottle, the first pH control feeding bottle of large intestine and the ascending colon fermentation tank are connected by silica gel pipes; the second culture medium feeding bottle, the second pH control feeding bottle and the transverse colon fermentation tank are connected by silica gel pipes; the third culture medium feeding bottle, the third pH control feeding bottle and the descending colon fermentation tank are connected by silica gel pipes.

[0019] The first culture medium feeding bottle, the second culture medium feeding bottle and the third culture medium feeding bottle are connected with the total control and display screen by communication lines, and the first pH control feeding bottle, the second pH control feeding bottle and the third pH control feeding bottle are connected with the total control and display screen by communication lines.

[0020] The anaerobic gas cylinder is connected with the ascending colon fermentation tank, the transverse colon fermentation tank and the descending colon fermentation tank by silica gel pipes, and is connected with the total control and display screen by communication lines.

[0021] The tail gas detector is connected with the descending colon fermentation tank and the product collection bottle by silica gel pipes, and is connected with the total control and display screen by communication lines.

[0022] The bacteria amount and short-chain fatty acid detector is located at the bottom of the ascending colon fermentation tank, the transverse colon fermentation tank and the descending colon fermentation tank, and is connected with the total control and display screen by communication lines.

[0023] In an embodiment, the agar, mucin and water are used in a ratio of 1-3 g: 0.3-0.6 g: 50 mL.

[0024] In an embodiment, the mucosa gel solution is adjusted to a pH of 7.5-8.5.

[0025] In an embodiment, the intestinal mucosa beads have a diameter of 0.3-0.7 cm.

[0026] In an embodiment, the intestinal mucosa beads 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 3-7.

[0027] A second object of the present application is to provide a colon simulation fermentation and analysis system for polysaccharide-based food, which contains intestinal mucosa beads.

[0028] The colon simulation fermentation and analysis system comprises a large intestine fermentation simulation system, a central intelligent control and display system, a tail gas detection system and a bacteria amount and short-chain fatty acid online detection system which are connected with each other, wherein 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 gas cylinder.

[0029] The medium feeding bottles include a first medium feeding bottle, a second medium feeding bottle and a third medium feeding bottle; the pH control feeding bottles include 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 a total control and display screen, a pH control system, a temperature control system, a stirring rate control system, a gas path system and a flow rate control system

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

[0032] The bacteria amount and short-chain fatty acid online detection system includes a bacteria amount and short-chain fatty acid detector; the bacteria amount and short-chain fatty acid detector includes a first bacteria amount and short-chain fatty acid detector, a second bacteria amount and short-chain fatty acid detector and a third bacteria amount and short-chain fatty acid detector.

[0033] In an embodiment, the preparation method of the mucosa small ball is: adding agar into water, dissolving, adding mucin to obtain a mucosa gel solution; and solidifying the mucosa gel solution to obtain the mucosa small ball.

[0034] Optionally, the use amount ratio of agar, mucin and water is 1-3 g: 0.3-0.6 g: 50 mL; the diameter of the intestinal mucosa small ball is 0.3-0.7 cm; and the intestinal mucosa small ball is 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 3-7.

[0035] A third object of the present application is to provide the use of any of the above-mentioned methods or any of the above-mentioned systems in the field of food, medicine or feed, which use comprises: detecting the digestion and absorption effect of food, medicine or feed.

[0036] A fourth object of the present application is to provide a method for improving the reliability of in-vitro simulation of colon dynamic digestion of polysaccharide-based food, which adds intestinal mucosa small balls in a colon digestion simulation device.

[0037] The preparation method of the mucosa small ball is: adding agar into water, dissolving, adding mucin to obtain a mucosa gel solution; and solidifying the mucosa gel solution to obtain the mucosa small ball.

[0038] Optionally, the use amount ratio of agar, mucin and water is 1-3 g: 0.3-0.6 g: 50 mL; the diameter of the intestinal mucosa small ball is 0.3-0.7 cm; and the intestinal mucosa small ball is 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 3-7.

[0039] In an embodiment, the credibility is that the flora structure, the proportion of short-chain fatty acids is closer to the flora of the real intestine, the structure of short-chain fatty acids.

[0040] In an embodiment, the colon digestion simulation device comprises a large intestine fermentation simulation system, a central intelligent control and display system, a tail gas detection system, a bacteria amount and short-chain fatty acid online detection system connected with each other;

[0041] 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 gas bottle.

[0042] 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.

[0043] The central intelligent control and display system comprises a total control and display screen, a pH control system, a temperature control system, a stirring rate control system, a gas path system, and a flow rate control system.

[0044] The tail gas detection system comprises a tail gas detector and a product collection bottle.

[0045] The bacteria amount and short-chain fatty acid online detection system comprises a bacteria amount and short-chain fatty acid detector; the bacteria amount and short-chain fatty acid detector comprises a first bacteria amount and short-chain fatty acid detector, a second bacteria amount and short-chain fatty acid detector, and a third bacteria amount and short-chain fatty acid detector.

[0046] In an 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 silica gel pipe; the second culture medium feeding bottle, the second pH control feeding bottle, and the transverse colon fermentation tank are connected by a silica gel pipe; the third culture medium feeding bottle, the third pH control feeding bottle, and the descending colon fermentation tank are connected by a silica gel pipe.

[0047] The first culture medium feeding bottle, the second culture medium feeding bottle, and the third culture medium feeding bottle are connected with the total control and display screen by a communication line; the first pH control feeding bottle, the second pH control feeding bottle, and the third pH control feeding bottle are connected with the total control and display screen by a communication line.

[0048] The anaerobic gas bottle is connected with the ascending colon fermentation tank, the transverse colon fermentation tank, and the descending colon fermentation tank by a silica gel pipe; the anaerobic gas bottle is connected with the total control and display screen by a communication line.

[0049] The tail gas detector is connected with the descending colon fermentation tank and the product collection bottle by a silica gel pipe; the tail gas detector is connected with the total control and display screen by a communication line.

[0050] The bacteria amount and short-chain fatty acid detector is located at the bottom of the ascending colon fermentation tank, the bacteria amount and short-chain fatty acid detector is located at the bottom of the horizontal colon fermentation tank, and the bacteria amount and short-chain fatty acid detector is located at the bottom of the descending colon fermentation tank; the bacteria amount and short-chain fatty acid detector and the total control and display screen are connected through communication lines.

[0051] In an embodiment, the preparation method of the intestinal mucosa pellets added in the large intestine fermentation simulation system is as follows: 2-3 g agar is weighed and dissolved in 50-80 mL distilled water, and after the solution is clear and transparent, it is cooled to 55-65 ℃, 0.4-0.6 g mucin is added, and the solution is adjusted to pH 7.5-8.5 after being fully dissolved; the mucosa gel solution is added to a mold to solidify into pellets, and ultraviolet sterilization is performed for 20-50 minutes in a clean bench.

[0052] In an embodiment, the central intelligent control system includes a touch screen intelligent controller connected with the rest of the systems through communication lines; the pH control system includes five pH electrodes, five pH controllers, five acid liquid bottles, five alkali liquid bottles, five acid liquid peristaltic pumps and five alkali liquid peristaltic pumps;

[0053] The pH electrode is connected with the pH controller through communication lines; the pH controller is connected with the central intelligent controller through communication lines to transmit the pH values of each simulation system to the central intelligent controller in real time, and to control the acid liquid and alkali liquid peristaltic pumps to open the transfer of acid liquid and alkali liquid; the acid liquid bottle and the alkali liquid bottle are connected with the acid liquid inlet and the alkali liquid inlet of the large intestine decomposition simulation system through silica gel tubes, respectively;

[0054] The temperature control system is connected with the central intelligent control system through communication lines, and includes a movable upper cover, a ring-shaped water bath dish, a water bath pump, a plurality of semiconductor temperature control systems and a temperature sensor; the plurality of temperature control systems are connected with the temperature sensor through communication lines, the real-time temperature of the water bath is fed back to the central intelligent control system through communication lines by the temperature sensor to form a record, and the opening or closing of the heater is controlled;

[0055] The stirring rate control system includes five magnetic stirring devices connected with the central intelligent control system through communication lines.

[0056] In an embodiment, the steps of constructing the large intestine fermentation simulation system to simulate the intestinal microbial ecology are as follows: 3-5 adults who have not taken antibiotics for 6 months are selected, 50g of the collected feces is sampled, mixed, and added to a phosphate buffer solution with a pH of 6.5-7.5, 0.5-2g of sodium thioacetate is added as a reducing agent, the sample is homogenized using a homogenizer, after homogenization, the sample is centrifuged at a speed of 3000 rpm for 5-10 min, after centrifugation, 50-80mL, 50-80mL, and 50-80mL of intestinal microbial bacterial solution are inoculated into three cylindrical glass reactors in the large intestine decomposition simulation system, respectively, then 500-800mL, 500-800mL, and 500-800mL of SHIME medium are added to the three reactors, respectively, 37°C, 30-50rpm stirring for 12-24h, and anaerobic gas (hydrogen 5%, carbon dioxide 10%, nitrogen 85%) is passed through every 8h for 10-30min. After stabilization, the pH values of the three reactors are controlled at 5.6-6.0, 6.0-6.4, and 6.4-6.9, respectively, by adding 0.05-0.1mol / L HCl or 0.05-0.1mol / L NaOH.

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

[0058] In an embodiment, after the fecal intestinal flora is inoculated, the fermentation is continuously carried out for 7d to tend to be stable.

[0059] Advantages of the present application

[0060] The present application provides a colon simulation fermentation and multi-dimensional online analysis system for anti-digestive polysaccharide-based food, which can simulate the fermentation activity of the colon in vitro, measure the fermentation gas and fermentation products of the food to be tested, and real-time adjust and monitor the degradation kinetics of the anti-digestive polysaccharide-based food.

[0061] Specifically:

[0062] (1) The intestinal mucosa balls are introduced to simulate the intestinal mucosa environment of the human body, which can more accurately and conveniently simulate the composition of the intestinal microorganisms of the human body in vitro, make the in vitro intestinal simulation fermentation system closer to the real intestine, and improve the simulation effect and the reliability of the experiment.

[0063] (2) Compared with the prior art, the present application on-line characterizes the content of fermentation products in the fermentation system (in situ) during the fermentation process, continuously monitors the fermentation kinetics in a non-invasive manner. The fermentation rule of the intestinal flora-anti-digestion food is judged by multi-element gas detection. The in-situ detection of short-chain fatty acids (acetic acid, propionic acid, butyric acid, etc.) in the fermentation process is realized by an infrared device, which quickly and accurately determines the changes of intestinal bacteria and their metabolites, and avoids errors caused by few sampling points. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1 is a schematic diagram of the fermentation system simulating the colon environment of the present application; wherein 101 is the ascending colon fermentation tank, 102 is the transverse colon fermentation tank, 103 is the 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 a tail gas detector, 601 is an anaerobic gas bottle, 703 is a total control and display screen, 801-803 are bacteria amount and short-chain fatty acid detectors;

[0065] Figure 2 is a bacteria phylum abundance diagram after 7 days of continuous fermentation after inoculation of the examples and comparative examples and tends to be stable; wherein "AC" represents the ascending colon, "DC" represents the descending colon, "TC" represents the transverse colon, "Initial" is the original flora inoculated, "A" is the flora of Example 1, "B, C, D, E" are the flora abundance of Comparative Example 1(1), Comparative Example 1(2), Comparative Example 2, Comparative Example 3, respectively;

[0066] Figure 3 is a bacteria flora PCA diagram after 7 days of continuous fermentation after inoculation of the examples and comparative examples and tends to be stable;

[0067] Figure 4 is a short-chain fatty acid proportion distribution result diagram in the fermentation broth after 24 hours of adding the sample of the examples and comparative examples; DETAILED DESCRIPTION

[0068] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are for better explanation of the present application and are not used to limit the present application.

[0069] Device:

[0070] The anti-digestion polysaccharide-based food colon simulation fermentation and multi-dimensional on-line analysis system is as shown in Figure 1 , which comprises a large intestine fermentation simulation system, a central intelligent control and display system (pH control system, temperature control system, stirring rate control system, gas path system, flow rate control system), a tail gas detection system, and a bacteria amount and short-chain fatty acid on-line detection system.

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

[0072] The feeding bottles comprise a first culture medium feeding bottle 201, a second culture medium feeding bottle 202, and a third culture medium feeding bottle 203; the pH control feeding bottles comprise a first pH control feeding bottle 301, a second pH control feeding bottle 302, and a third pH control feeding bottle 303.

[0073] The central intelligent control and display system comprises a total 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 comprises a tail gas detector 501 and a product collection bottle 401.

[0075] The bacterial amount and short-chain fatty acid online detection system comprises bacterial amount and short-chain fatty acid detectors 801-803.

[0076] The bacterial amount and short-chain fatty acid detectors comprise a first bacterial amount and short-chain fatty acid detector 801, a second bacterial amount and short-chain fatty acid detector 802, and a third bacterial amount and short-chain fatty acid detector 803.

[0077] In the large intestine fermentation simulation system, the first culture medium feeding bottle 201, the first pH control feeding bottle 301 of the large intestine, and the caecum fermentation tank 101 are connected by silica gel pipes; the second culture medium feeding bottle 202, the second pH control feeding bottle 302, and the transverse colon fermentation tank 102 are connected by silica gel pipes; and the third culture medium feeding bottle 203, the third pH control feeding bottle 303, and the descending colon fermentation tank 103 are connected by silica gel pipes.

[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 total control and display screen 703 by communication lines; 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 total control and display screen 703 by communication lines.

[0079] The anaerobic gas cylinder 601 is connected to the caecum fermentation tank 101, the transverse colon fermentation tank 102, and the descending colon fermentation tank 103 by silica gel pipes; and the anaerobic gas cylinder 601 is connected to the total control and display screen 703 by communication lines.

[0080] The tail gas detector 501 is connected to the descending colon fermentation tank 103 and the product collection bottle 401 by silica gel pipes; and the tail gas detector 501 is connected to the total control and display screen 703 by communication lines.

[0081] The bacteria amount and short-chain fatty acid detector 801 is located at the bottom of the ascending colon fermentation tank 101, the bacteria amount and short-chain fatty acid detector 802 is located at the bottom of the transverse colon fermentation tank 102, and the bacteria amount and short-chain fatty acid detector 803 is located at the bottom of the descending colon fermentation tank 103; the bacteria amount and short-chain fatty acid detectors 801-803 and the total control and display screen 703 are connected through communication lines.

[0082] Raw materials:

[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 kept at a temperature of 40°C. The pH of the suspension was adjusted to 8-9 with 1 mol / L NaOH solution, 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 1 mol / L HCl solution to terminate the reaction. The obtained acetylated starch was washed with deionized water and ethanol for 2 times respectively, then dried in an oven at 40°C, ground and passed through a 100 mesh sieve to obtain the sample;

[0086] The SHIME culture 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. In vivo control sample collection

[0089] Healthy male 8-week-old C57BL / 6J mice were selected for in vivo control. Before the experiment, the mice were fasted for 20 h without water, and the contents in the original gastrointestinal tract were as much as possible to be digested or emptied. The starch sample was prepared into a 2.5 g / mL suspension with deionized water, mixed uniformly, and then the mice were gavaged with the same dose (2 mg / 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 standby.

[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 supernatant in a 2 mL EP tube, add 0.1 mL of H2SO4 solution (50%) and 0.8 mL of extract, centrifuge for 15 min (4°C, 10000 r / min) and stand in -20°C for 30 min. Finally, take the supernatant into a sample bottle and detect by gas chromatography-mass spectrometry (GC-MS). Chromatographic conditions: chromatographic column: Agilent HP-FFAP capillary column (30 m x 250 μm x 0.25 μm); temperature program: 80°C for 1 min, 10°C / min to 200°C, hold for 5 min, 40°C / min to 240°C, hold 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 colon simulation fermentation and multi-dimensional online analysis of anti-digestive polysaccharide-based food

[0093] A method for colon simulation fermentation and multi-dimensional online analysis of anti-digestive polysaccharide-based food, through a fermentation dynamic digestion and fermentation device as shown in Figure 1 The in vitro test simulates the enzyme digestion reaction activity of the small intestine and the fermentation activity of the colon of the human body.

[0094] Through the fermentation dynamic digestion and fermentation device, the in vitro test simulates the enzyme digestion reaction activity of the small intestine and the fermentation activity of the colon of the human body, and measures the glucose release rate and fermentation product types and content of the carbohydrate food to be tested, including the following steps:

[0095] (1) Preparation of intestinal mucosa globule

[0096] Mucin is the main structural and functional component in the mucus covering the intestinal epithelial surface, and agar is selected as the coagulant of the mucosa globule. The preparation method of the intestinal mucosa globule is as follows:

[0097] Weigh 2 g of agar in 50 mL of distilled water and heat to 100°C to dissolve. After the solution is clear and transparent, cool it to 60°C, add 0.5 g of mucin, dissolve thoroughly, adjust the pH of the solution to 8, and obtain the mucosa gel solution. Pour the mucosa gel solution into the mold to coagulate into globules with a diameter of 0.5 cm, and ultraviolet sterilize in a clean bench for 20 minutes.

[0098] (2) Simulation of intestinal environment

[0099] The intestinal microecology was constructed by inoculating the feces of healthy people in the SHIME medium for 24h anaerobic culture. The feces of 3-5 adults who had not taken antibiotics for 6 months were collected. 50g of the feces was sampled and mixed with 1:10 (g:mL) phosphate buffer solution at pH 7, and 1g of sodium thioacetate was added as a reducing agent. The sample was homogenized using a homogenizer;

[0100] After homogenization, the sample was centrifuged at 3000rpm for 5min. After centrifugation, 50mL, 80mL and 50mL of intestinal microorganism liquid were inoculated into the ascending colon fermentation tank (101), transverse colon fermentation tank (102) and descending colon fermentation tank (103) in the colon fermentation simulation system, respectively. Then 5 mucosa balls were added into each reactor, and 500mL, 800mL and 500mL of SHIME medium were added, respectively. The three reactors were stirred at 37℃ and 30rpm for 24h, and anaerobic gas (hydrogen 5%, carbon dioxide 10%, nitrogen 85%) was passed through each reactor for 10min every 8h. After stabilization, the pH values of the three reactors were controlled in the ranges 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). The magnetic stirring device was turned on at a speed of 30rpm, and the simulation fermentation was carried out at 37℃.

[0101] After the first inoculation and culture for 24h, in order to maintain the normal growth of microorganisms, the nutrients were replenished daily and 300mL of culture medium was discharged to maintain the volume unchanged. Three new mucosa balls were used to replace the mucosa balls in the tank to simulate mucosa regeneration. After inoculation of the fecal intestinal flora, continuous fermentation was carried out for 7d to reach stability. Part of the fermentation liquid was stored in a-80℃ refrigerator for bacterial community determination.

[0102] (3) Detection of sample digestion effect

[0103] 12.5mL of the test substance (resistant starch suspension, 10% g / 100mL, based on starch dry basis) was added to the ascending colon fermentation tank as nutrient replenishment. Every 8h, 100mL was transferred to the next fermentation tank at a rate of 20mL / min. After 24h of fermentation, the ascending colon simulation tank, transverse colon simulation tank and descending colon simulation tank were adjusted by hydrochloric acid (0.05mol / L) and sodium hydroxide (0.1mol / L), and the pH values were maintained at 5.6-6.0, 6.0-6.4 and 6.5-6.9, respectively (different pH values within the range will not affect the results). The fermented test substance was obtained.

[0104] Comparative Example 1: Change the ratio of intestinal mucosa balls

[0105] (1) Based on Example 1, the ratio of intestinal mucosa globule in step (1) was changed to 1 g agar, 1 g mucin, and other steps were kept the same as Example 1 to obtain the fermented sample.

[0106] (2) Based on Example 1, the ratio of intestinal mucosa globule in step (1) was changed to 4 g agar, 0.25 g mucin, and other steps were kept the same as Example 1 to obtain the fermented sample.

[0107] Comparative Example 2: Change the particle size of intestinal mucosa globule

[0108] Based on Example 1, the diameter of intestinal mucosa globule in step (1) was changed to 1 cm, and other steps were kept the same as Example 1 to obtain the fermented sample.

[0109] Comparative Example 3: No addition of mucosa globule

[0110] Based on Example 1, no intestinal mucosa globule was added in step (2), and other steps were kept the same as Example 1 to obtain the fermented sample.

[0111] Example 2: Detection of fermentation effect

[0112] The fermented samples obtained from Example 1 and Comparative Examples 1-3 were taken to detect the composition of microbial flora and short-chain fatty acids, with the proportion of short-chain fatty acids in fresh mouse feces as a control.

[0113] The composition of microbial flora and PCA results are shown in Figure 2 , Figure 3 Principal component analysis (PCA, based on Bray-Curtis distance algorithm) is a method of reducing the dimensionality of multi-dimensional data, which is used to evaluate Beta diversity. If the microbial composition is more similar, the distance in the figure is closer.

[0114] As shown in Figure 3 , compared with Comparative Examples 1-3, Example 1 is closest to Initial, while Comparative Examples (especially Comparative Examples 2 and 3) are far from Initial, indicating that the composition of microbial flora in the fermented sample under the simulation method of Example is closer to the original microbial flora, and this simulation method is closer to the intestinal fermentation environment. Therefore, changing the raw material ratio, diameter or number of intestinal mucosa globule will affect the intestinal fermentation environment.

[0115] The fermentation liquid was taken after 24 h of fermentation culture after adding the sample in Example 1 and Comparative Examples 1-3, and the composition of short-chain fatty acids was detected, and the results are shown in Figure 4And shown in Table 1, compared with the in vivo results, the acetic acid proportion of the comparative examples is different degrees of high, the butyric acid content is low, especially the comparative example 3 is very different from the in vivo results, and the simulation method of the examples is closer to the in vivo fermentation results, which is significantly better than the comparative examples.

[0116] Table 1 Short-chain fatty acid proportion distribution (%)

[0117]

[0118] Although the present application has been disclosed in the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for in vitro simulation of dynamic digestion of a polysaccharide-based food product in the colon, characterized in that, The method adds the intestinal mucosa globule into a colon digestion simulation device, adds polysaccharide-based food, simulates the digestion process and emptying process of the colon; The preparation method of the mucosa globule is: Agar is added into water, dissolved, mucin is added, the dosage ratio of agar, mucin and water is 2 g:0.5 g:50 mL, mucosa gel solution is obtained, and pH is adjusted to 8; the mucosa gel solution is coagulated to obtain mucosa globules with a diameter of 0.5 cm; The mucosa globules are located in ascending colon fermentation tanks, transverse colon fermentation tanks and descending colon fermentation tanks of the colon digestion simulation device, and the number of the mucosa globules in the ascending colon fermentation tanks, the transverse colon fermentation tanks and the descending colon fermentation tanks is 5 respectively.

2. The method of claim 1, wherein, The colon digestion simulation device comprises a large intestine fermentation simulation system, a central intelligent control and display system, a tail gas detection system, a bacteria amount and short-chain fatty acid online detection system and the like which are connected with each other; 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 gas bottle; 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 and display system comprises a total control and display screen, a pH control system, a temperature control system, a stirring rate control system, a gas path system and a flow rate control system; The tail gas detection system comprises a tail gas detector and a product collection bottle; The bacteria amount and short-chain fatty acid online detection system comprises a bacteria amount and short-chain fatty acid detector; the bacteria amount and short-chain fatty acid detector comprises a first bacteria amount and short-chain fatty acid detector, a second bacteria amount and short-chain fatty acid detector and a third bacteria amount and short-chain fatty acid detector.

3. The method of claim 2, wherein, In the large intestine fermentation simulation system, the first culture medium feeding bottle, the first pH control feeding bottle of the large intestine and the ascending colon fermentation tank are connected through silica gel pipes; the second culture medium feeding bottle, the second pH control feeding bottle and the transverse colon fermentation tank are connected through silica gel pipes; the third culture medium feeding bottle, the third pH control feeding bottle and the descending colon fermentation tank are connected through silica gel pipes; The first culture medium feeding bottle, the second culture medium feeding bottle and the third culture medium feeding bottle are connected with the total control and display screen through communication lines; the first pH control feeding bottle, the second pH control feeding bottle and the third pH control feeding bottle are connected with the total control and display screen through communication lines; The anaerobic gas bottle is connected with the ascending colon fermentation tank, the transverse colon fermentation tank and the descending colon fermentation tank through silica gel pipes; the anaerobic gas bottle is connected with the total control and display screen through communication lines; The tail gas detector is connected with the descending colon fermentation tank and the product collection bottle through silica gel pipes; the tail gas detector is connected with the total control and display screen through communication lines; The bacteria amount and short-chain fatty acid detector is located at the bottom of the ascending colon fermentation tank, the transverse colon fermentation tank and the descending colon fermentation tank; the bacteria amount and short-chain fatty acid detector is connected with the total control and display screen through communication lines.

4. A system for colonic simulation of the fermentation of a polysaccharide-based food product and analysis, characterised in that, The colon simulation fermentation and analysis system contains intestinal mucosa globules. The preparation method of the mucosa beads is: adding agar into water, dissolving, adding mucin to obtain a mucosa gel solution; solidifying the mucosa gel solution to obtain the mucosa beads; The intestinal mucosa beads 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 intestinal mucosa beads is 5. The colon simulation fermentation and analysis system comprises a large intestine fermentation simulation system, a central intelligent control and display system, a tail gas detection system, a bacteria amount and short-chain fatty acid online detection system which are connected with each other; wherein 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 gas bottle. 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 and display system comprises a total control and display screen, a pH control system, a temperature control system, a stirring rate control system, a gas path system and a flow rate control system. The tail gas detection system comprises a tail gas detector and a product collection bottle. The bacteria amount and short-chain fatty acid online detection system comprises a bacteria amount and short-chain fatty acid detector; the bacteria amount and short-chain fatty acid detector comprises a first bacteria amount and short-chain fatty acid detector, a second bacteria amount and short-chain fatty acid detector and a third bacteria amount and short-chain fatty acid detector.

5. Use of the method according to any one of claims 1 to 3 or the colon simulation fermentation and analysis system according to claim 4 in the field of food, pharmaceutical or feed products, characterized in that, The application comprises detecting the digestion and absorption effect of food, medicine or feed.

6. A method of improving the in vitro simulated colonic dynamic digestion polysaccharide-based food reliability, characterized in that, Intestinal mucosa beads are added into the colon digestion simulation device; The preparation method of the mucosa beads is: adding agar into water, dissolving, adding mucin to obtain a mucosa gel solution; solidifying the mucosa gel solution to obtain the mucosa beads; The intestinal mucosa beads 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 intestinal mucosa beads is 5.

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