A method for preparing fecal bacteria for fecal microbiota transplantation

Abstract

The present application relates to a kind of preparation method of fecal bacteria for intestinal flora transplantation.The present application includes the following steps: (1) preparation of intestinal flora reticular carrier; (2) preparation of intestinal flora suspension; (3) preparation of bionic intestine; (4) in vitro continuous culture of intestinal flora; (5) collection after intestinal flora culture.The present application provides a preparation method of fecal bacteria for intestinal flora transplantation, which realizes in vitro culture of intestinal flora by simulating the living environment of intestinal flora in human intestine, ensures the stability of flora quality and yield, eliminates the time limit of intestinal flora transplantation treatment, and greatly improves the timeliness of clinical application.The intestinal flora prepared by the present application has large yield and high activity.Furthermore, the intestinal flora of the present application has obvious improvement in relieving weight loss of inflammatory bowel disease patients, relieving food utilization coefficient decline of inflammatory bowel disease patients, relieving red blood cell quantity decline of inflammatory bowel disease patients, reducing white blood cell quantity of inflammatory bowel disease patients and improving intestinal beneficial bacteria content.

Description

Technical Field

[0001] This invention relates to a method for preparing fecal microbiota for intestinal microbiota transplantation, belonging to the field of intestinal microbiota transplantation technology. Background Technology

[0002] Intestinal microbiota transplantation, also known as fecal microbiota transplantation (FMT), is a microecological treatment method that isolates functional microbiota from the feces of a healthy donor and transplants them into the digestive tract of a patient, thereby reconstructing the patient's intestinal microbiota. Currently, this method has been used to treat a variety of gastrointestinal and metabolic diseases, such as chronic functional constipation, refractory Clostridium difficile infection, and inflammatory bowel disease.

[0003] Tracing the origins of fecal microbiota (FMT), the earliest written record of its use in treating human diseases can be found in Ge Hong's *Zhouhou Beiji Fang* (also known as *Zhouhou Fang*), written during the Eastern Jin Dynasty (300-400 AD). The text describes using human feces to treat patients suffering from food poisoning, diarrhea, fever, and near death, stating that "drinking it brought them back to life." Li Shizhen's *Compendium of Materia Medica* (1596 edition) from the Ming Dynasty records more than twenty different treatments using human feces. Modern literature on FMT dates back to 1958, when Ben Eiseman, a surgeon at the University of Colorado School of Medicine, and his colleagues used fecal matter from healthy individuals to create a solution for enemas in four patients with severe pseudomembranous colitis who had not responded to conventional antibiotics and hormone therapy. The results successfully cured three critically ill patients, while the remaining patient died from a disease unrelated to intestinal infection.

[0004] In 2013, the first controlled clinical trial of fibrinogen-mediated transluminal thrombocytopenic purpura (FMT) for recurrent Clostridium difficile enteritis demonstrated that FMT was significantly more effective than vancomycin. FMT has shown unparalleled superiority over other treatment methods in both animal and clinical trials. It was named one of the top ten biomedical breakthroughs of 2013 by *Science* and *TIME*.

[0005] Currently, all clinical intestinal microbiota transplantation (IVT) treatments involve preparing standard bacterial solutions from fresh fecal samples taken from donors. This fresh fecal microbiota standard solution requires a timeframe of less than 6 hours from fecal collection to treatment. This time constraint, coupled with the difficulty in finding suitable donors and the high cost of donor screening, hinders the clinical application of IVT. Therefore, finding a rapid and highly active method for preparing fecal microbiota for IVT is crucial. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing fecal microbiota for intestinal microbiota transplantation.

[0007] The technical solution of the present invention is as follows:

[0008] A method for preparing fecal microbiota for intestinal flora transplantation includes the following steps:

[0009] (1) Preparation of intestinal microbiota network carrier: Plant fibers are woven into a network structure, and then the network structure is assembled into a cylindrical fiber network. The cylindrical fiber network is then soaked in a 2-5% mucin solution in a shaker for 15-20 minutes at a shaker speed of 10-20 rpm to obtain the intestinal microbiota network carrier.

[0010] (2) Preparation of intestinal flora suspension: Collect feces from normal healthy donors, then dilute the feces in physiological saline, shake it evenly using a homogenizer, and filter it with medical gauze to obtain intestinal flora suspension;

[0011] (3) Preparation of bionic intestine: Based on the precise human intestinal anatomy model of the Department of Gastroenterology, a tubular human intestinal mold is made, and then transparent silicone is injected into the mold. After solidification and molding, it is removed to obtain the bionic intestine.

[0012] (4) Continuous in vitro culture of gut microbiota:

[0013] The intestinal microbiota network carrier prepared in step (1) was placed at the end of the bionic intestine. The intestinal microbiota suspension prepared in step (2) was inoculated into the bionic intestine prepared in step (3). Then, sterile water, nutrients, amylase, protease, cellulase and lipase were added in sequence and anaerobic culture was carried out for 1 to 4 weeks. Sterile water, nutrients, amylase, protease, cellulase and lipase were added every 8 hours. Sterile water, nutrients, amylase, protease, cellulase and lipase were discharged every 24 hours.

[0014] (5) Collection of gut microbiota after culture:

[0015] The intestinal microbiota reticulum carrier was removed and soaked in physiological saline in a shaker 4 to 6 times, with each soaking time being 2 to 6 minutes and the shaker speed being 2 to 10 rpm. After soaking, it was stored in an anaerobic environment to obtain fecal microbiota for intestinal microbiota transplantation.

[0016] According to a preferred embodiment of the present invention, in step (1), the plant fiber is plant fiber from fruits, vegetables, beans, nuts, grains and fungi.

[0017] According to a preferred embodiment of the present invention, in step (1), the length of each side line of the mesh structure is consistent, ranging from 0.1 to 1 cm.

[0018] According to a preferred embodiment of the present invention, in step (1), the cylindrical fiber network has a diameter of 50–100 μm and a total length of 1.2–1.5 m. The morphology of this cylindrical fiber network is consistent with the biomimetic intestine subsequently prepared.

[0019] According to a preferred embodiment of the present invention, in step (2), the mass-to-volume ratio of feces to saline solution is 1:(3-30), with units of g / mL.

[0020] According to a preferred embodiment of the present invention, in step (3), the tubular human intestinal mold is composed of a core mold and a sleeve forming an internal cavity, into which transparent silicone is injected.

[0021] According to a preferred embodiment of the present invention, in step (3), the bionic intestine has a thickness of 1-4 mm, a length of 9-10 m, a diameter of 1-2 cm for the first 4-6 m, and a diameter of 2-3 cm for the remainder.

[0022] According to a preferred embodiment of the present invention, in step (4), the volume ratio of the intestinal flora suspension to sterile water is 1:(0.5-1).

[0023] According to a preferred embodiment of the present invention, in step (4), the nutrient agent is composed of the following raw materials in parts by weight:

[0024] Hydrolyzed whey protein 2-9 parts, whey protein concentrate 2-10 parts, soy protein isolate 2-7 parts, other proteins 2-7 parts, amino acid premix 1.35-11.5 parts, nucleotide premix 1-5 parts, maltodextrin 30-53 parts, fructooligosaccharides 1-5 parts, galactooligosaccharides 1-5 parts, polydextrose 1-4 parts, vegetable oil powder 7-20 parts, mineral premix 4-18 parts, vitamin premix 0.23-6.75 parts;

[0025] The hydrolyzed whey protein and whey protein concentrate mentioned are lactose-free whey proteins;

[0026] The other proteins are black bean protein, corn protein, wheat protein, pea protein, collagen, albumin, or sea cucumber protein;

[0027] The amino acid premix, per 1g, comprises: 50-75mg L-glutamine, 53-72mg threonine, 65-87mg serine, 20-41mg proline, 2-10mg cysteine, 0.001-0.006mg taurine, with the remainder being maltodextrin.

[0028] The nucleotide premix comprises, per 1g: 15-35mg of nucleotides, with the remainder being maltodextrin; the nucleotides are one or a mixture of two or more of the following: 5'-cytidine monophosphate (5'-CMP), 5'-uridine monophosphate (5'-UMP), 5'-adenosine monophosphate (5'-AMP), disodium 5'-inosinate, disodium 5'-guanylate, disodium 5'-uridine, or disodium 5'-cytidine.

[0029] The mineral premix, per 1g, comprises: 128-176mg sodium citrate monohydrate, 88-128mg potassium chloride, 0.1-0.8mg copper gluconate, 152-168mg magnesium gluconate, 1.5-4mg ferrous fumarate, 2-6mg zinc gluconate, 0.5-3mg manganese gluconate, 97-135mg dicalcium phosphate, 0.004-0.009mg potassium iodide, 0.002-0.009mg sodium selenite, 50-80μg chromium chloride, 70-100μg ammonium molybdate, 0.3-0.7mg potassium fluoride, with the balance being maltodextrin.

[0030] The vitamin premix, per 1g, comprises: 0.1–1.5 IU of vitamin A acetate oil, 0.01–0.1 IU of vitamin D3, 0.1–1 mg of vitamin E acetate, 0.01–0.1 mg of vitamin K1, 0.008–0.15 mg of thiamine hydrochloride (B1), 0.007–0.12 mg of riboflavin (B2), 0.02–0.18 mg of pyridoxine hydrochloride (B6), 0.01–0.14 mg of cyanocobalamin (B12), 0.1–1 mg of nicotinamide, 0.001–0.01 mg of folic acid, 0.02–0.21 mg of D-calcium pantothenate, 1–3 mg of L-ascorbic acid, 0.1–1.4 mg of biotin, 0.01–0.12 mg of inositol, 0.02–0.18 mg of L-carnitine tartrate, with the balance being maltodextrin.

[0031] According to a preferred embodiment of the present invention, in step (4), the amount of amylase added is 35-135 U / L, the amount of protease added is 6-20 U / L, the amount of cellulase added is 7-15 U / L, and the amount of lipase added is 18-30 U / L.

[0032] According to a preferred embodiment of the present invention, in step (4), the pH value of the bionic intestine during the anaerobic culture process is 6.5 to 8.5, the outer side of the bionic intestine is sterile water at a temperature of 36.9 to 37.9°C, the gas content in the bionic intestine is 90 to 110 mL, and the pressure is monitored by a pressure sensor with a pressure range of 2 to 4 mm Hg; when the pressure is too high, the excess gas is discharged to maintain the gas content in the bionic intestine at 90 to 110 mL.

[0033] According to a preferred embodiment of the present invention, in step (4), during the anaerobic culture process, the biomimetic intestine moves in a combination of segmented movement and peristalsis. The segmented movement occurs at a rate of 3 to 5 times per minute, and the peristaltic propulsion speed is 0.7 to 0.9 cm / min. The purpose of the segmented movement is to compress the biomimetic intestine, and the purpose of the peristalsis is to push the substances in the biomimetic intestine from top to bottom. Both segmented movement and peristalsis simulate the movement pattern of the intestine inside the human body.

[0034] The beneficial effects of this invention are as follows:

[0035] 1. The fecal microbiota preparation method for intestinal microbiota transplantation provided by this invention achieves in vitro culture of intestinal microbiota by simulating the living environment of microbiota in the human gut, ensuring the stability of microbiota quality and yield, removing the time limit of intestinal microbiota transplantation treatment, and providing intestinal microbiota in a timely manner according to the needs of patients, greatly improving the timeliness of clinical application.

[0036] 2. The intestinal flora prepared by this invention has a high yield and high activity. Compared with traditional fecal microbiota, the intestinal flora of this invention has significant improvements in alleviating weight loss, food utilization rate, red blood cell count, and white blood cell count in patients with inflammatory bowel disease, and in increasing the content of beneficial bacteria in the intestine.

[0037] 3. This invention uses plant fiber materials as a mesh carrier, and then attaches mucin to the mesh carrier. During the culture process, the intestinal flora attaches to the mesh carrier, making the intestinal flora easy to isolate and colonize. In clinical application, it is more convenient than the traditional enema method, and it is easier to colonize in the body after transplantation.

[0038] 4. The fecal microbiota preparation method for intestinal microbiota transplantation provided by this invention only requires the microbiota donor to provide fecal microbiota a few times or even once, overcoming the problems of long-term dietary restrictions for microbiota donors and repeated health examinations before donation, thus reducing the cost of intestinal microbiota transplantation. Furthermore, each process of this invention is simulated in vitro, reducing discomfort for both the operator and the recipient and expanding its application scope. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to these specific embodiments.

[0040] Example 1

[0041] A method for preparing fecal microbiota for intestinal flora transplantation includes the following steps:

[0042] (1) Preparation of intestinal microbiota network carrier: Plant fibers are woven into a network structure, and then the network structure is assembled into a cylindrical fiber network. The cylindrical fiber network is then soaked in a 3% mucin solution in a shaker for 18 minutes at a shaking speed of 15 rpm to obtain the intestinal microbiota network carrier.

[0043] The length of each side of the mesh structure is the same, 0.5 cm; the diameter of the cylindrical fiber mesh is 80 μm and the total length is 1.35 m. The morphology of this cylindrical fiber mesh is consistent with the bionic intestine prepared later.

[0044] (2) Preparation of intestinal flora suspension: Collect feces from normal healthy people, then dilute the feces in physiological saline at a mass-volume ratio of 1:20, shake it evenly using a homogenizer, and filter it with medical gauze to obtain intestinal flora suspension;

[0045] (3) Preparation of bionic intestine: Based on the precise human intestinal anatomy model of the Department of Gastroenterology, a tubular human intestinal mold is made. The tubular human intestinal mold consists of a core mold and a sleeve forming an internal cavity. Then, transparent silicone is injected into the internal cavity of the mold, solidified and removed to obtain a bionic intestine.

[0046] The biomimetic intestine is 2mm thick and 9.5m long, with the first 5m having a diameter of 1.5cm and the remaining 4.5m having a diameter of 2.5cm.

[0047] (4) Continuous in vitro culture of gut microbiota:

[0048] The intestinal microbiota network carrier prepared in step (1) was placed at the end of the bionic intestine. The intestinal microbiota suspension prepared in step (2) was inoculated into the bionic intestine prepared in step (3). Then, sterile water, nutrients, amylase, protease, cellulase and lipase were added in sequence and anaerobic culture was carried out for 2 weeks. Sterile water, nutrients, amylase, protease, cellulase and lipase were added every 8 hours. Sterile water, nutrients, amylase, protease, cellulase and lipase were discharged every 24 hours.

[0049] The volume ratio of the intestinal flora suspension to sterile water is 1:0.75; the amount of amylase added is 85 U / L, the amount of protease added is 15 U / L, the amount of cellulase added is 10 U / L, and the amount of lipase added is 25 U / L.

[0050] (5) Collection of gut microbiota after culture:

[0051] The intestinal microbiota reticulum carrier was removed and soaked in physiological saline in a shaker 5 times, each time for 5 minutes, with a shaker speed of 5 rpm. After soaking, it was stored in an anaerobic environment to obtain fecal microbiota for intestinal microbiota transplantation.

[0052] In step (4), the nutrient is composed of the following parts by weight of raw materials: 5 parts hydrolyzed whey protein, 6 parts concentrated whey protein, 5 parts soy protein isolate, 5 parts other proteins, 8 parts amino acid premix, 3 parts nucleotide premix, 40 parts maltodextrin, 3 parts fructooligosaccharides, 3 parts galactooligosaccharides, 3 parts polydextrose, 10 parts vegetable oil powder, 12 parts mineral premix, and 3.5 parts vitamin premix.

[0053] The other proteins mentioned are a mixture of pea protein, black bean protein, and wheat protein in equal weight ratios;

[0054] The amino acid premix, per 1g, comprises the following raw materials: L-glutamine 60.124mg, threonine 62.751mg, serine 72.142mg, proline 25.457mg, cysteine ​​5.412mg, taurine 0.003mg, with the balance being maltodextrin; the nucleotide premix, per 1g, comprises nucleotides 24.514mg, with the balance being maltodextrin; the nucleotides are a mixture of 5'-cytidine monophosphate (5'-CMP), 5'-uridine monophosphate (5'-UMP), 5'-adenosine monophosphate (5'-AMP), and disodium 5'-inosinate in equal weight proportions; the mineral premix, per 1g... The raw material composition is as follows: sodium citrate monohydrate 142.251 mg, potassium chloride 105.542 mg, copper gluconate 0.612 mg, magnesium gluconate 158.024 mg, ferrous fumarate 2.189 mg, zinc gluconate 4.583 mg, manganese gluconate 1.521 mg, calcium hydrogen phosphate 120.421 mg, potassium iodide 0.006 mg, sodium selenite 0.005 mg, chromium chloride 71.30 μg, ammonium molybdate 85.42 μg, potassium fluoride 0.523 mg, with the balance being maltodextrin; the vitamin premix, per 1 g, has the following raw material composition: vitamin A acetate oil 0.741 IU, vitamin D3... 0.062 IU, Vitamin E acetate 0.584 mg, Vitamin K1 0.068 mg, Thiamine hydrochloride (B1) 0.061 mg, Riboflavin (B2) 0.053 mg, Pyridoxine hydrochloride (B6) 0.078 mg, Cyanocobalamin (B12) 0.07 mg, Nicotinamide 0.557 mg, Folic acid 0.006 mg, D-calcium pantothenate 0.135 mg, L-ascorbic acid 1.687 mg, Biotin 0.738 mg, Inositol 0.074 mg, L-carnitine tartrate 0.084 mg, with the balance being maltodextrin.

[0055] During the anaerobic culture process, the pH value of the bionic intestine is 7, the outer side of the bionic intestine is sterile water at a temperature of 37.3℃, the gas content in the bionic intestine is 100mL, and the pressure is monitored by a pressure sensor, with the pressure range being 2-4mm Hg; when the pressure is too high, the excess gas is discharged to maintain the gas content in the bionic intestine at 100mL.

[0056] During the anaerobic culture process, the biomimetic intestine moves in a combination of segmentation and peristalsis. The segmentation rate is 4 times per minute, and the peristalsis propulsion speed is 0.8 cm / min. The purpose of the segmentation is to compress the biomimetic intestine, and the purpose of the peristalsis is to push the substances in the biomimetic intestine from top to bottom. Both segmentation and peristalsis simulate the movement pattern of the intestine inside the human body.

[0057] Example 2

[0058] A method for preparing fecal microbiota for intestinal flora transplantation includes the following steps:

[0059] (1) Preparation of intestinal microbiota network carrier: Plant fibers are woven into a network structure, and then the network structure is assembled into a cylindrical fiber network. The cylindrical fiber network is then soaked in a 2% mucin solution in a shaker for 20 minutes at a shaking speed of 10 rpm to obtain the intestinal microbiota network carrier.

[0060] The length of each side of the mesh structure is the same, 0.1 cm; the diameter of the cylindrical fiber mesh is 50 μm and the total length is 1.2 m. The morphology of this cylindrical fiber mesh is consistent with the bionic intestine subsequently prepared.

[0061] (2) Preparation of intestinal flora suspension: Collect feces from normal healthy people, then dilute the feces in physiological saline at a mass-volume ratio of 1:30, shake it evenly using a homogenizer, and filter it with medical gauze to obtain intestinal flora suspension;

[0062] (3) Preparation of bionic intestine: Based on the precise human intestinal anatomy model of the Department of Gastroenterology, a tubular human intestinal mold is made. The tubular human intestinal mold consists of a core mold and a sleeve forming an internal cavity. Then, transparent silicone is injected into the internal cavity of the mold, solidified and removed to obtain a bionic intestine.

[0063] The bionic intestine is 1 mm thick and 10 m long. The diameter of the first 4 m is 1 cm, and the diameter of the remaining 6 m is 2 cm. There are bends every 3 cm.

[0064] (4) Continuous in vitro culture of gut microbiota:

[0065] The intestinal microbiota network carrier prepared in step (1) was placed at the end of the bionic intestine. The intestinal microbiota suspension prepared in step (2) was inoculated into the bionic intestine prepared in step (3). Then, sterile water, nutrients, amylase, protease, cellulase and lipase were added in sequence and anaerobic culture was carried out for 1 week. Sterile water, nutrients, amylase, protease, cellulase and lipase were added every 8 hours. Sterile water, nutrients, amylase, protease, cellulase and lipase were discharged every 24 hours.

[0066] The volume ratio of the intestinal flora suspension to sterile water is 1:0.5; the amount of amylase added is 35 U / L, the amount of protease added is 6 U / L, the amount of cellulase added is 7 U / L, and the amount of lipase added is 18 U / L.

[0067] (5) Collection of gut microbiota after culture:

[0068] The intestinal microbiota reticulum carrier was removed and soaked in physiological saline in a shaker 4 times, each time for 2 minutes and at a shaker speed of 2 rpm. After soaking, it was stored in an anaerobic environment to obtain fecal microbiota for intestinal microbiota transplantation.

[0069] In step (4), the nutrient is composed of the following raw materials in parts by weight: 2 parts hydrolyzed whey protein, 2 parts concentrated whey protein, 2 parts soy protein isolate, 2 parts other proteins, 1.35 parts amino acid premix, 1 part nucleotide premix, 30 parts maltodextrin, 1 part fructooligosaccharide, 1 part galactooligosaccharide, 1 part polydextrose, 7 parts vegetable oil powder, 4 parts mineral premix, and 0.23 parts vitamin premix.

[0070] The hydrolyzed whey protein and whey protein concentrate mentioned are lactose-free whey proteins;

[0071] The other proteins mentioned are a mixture of corn protein, black bean protein, and collagen in equal weight proportions.

[0072] The amino acid premix, per 1g, has the following raw material composition: L-glutamine 54.124mg, threonine 55.751mg, serine 66.142mg, proline 30.457mg, cysteine ​​3.412mg, taurine 0.002mg, with the remainder being maltodextrin.

[0073] The nucleotide premix, per 1g, contains 18.514mg of nucleotides, with the remainder being maltodextrin; the nucleotides are a mixture of 5'-adenosine monophosphate (5'-AMP), disodium 5'-inosinate, disodium 5'-guanylate, disodium 5'-uridine, and disodium 5'-cytidine in equal weight ratios; the mineral premix, per 1g, comprises the following raw materials: 131.251mg sodium citrate monohydrate, 90.542mg potassium chloride, 0.312mg copper gluconate, and magnesium gluconate. The premixed vitamins contain 153.924 mg of ferrous fumarate, 1.689 mg of zinc gluconate, 2.983 mg of manganese gluconate, 0.921 mg of dicalcium phosphate, 115.296 mg of potassium iodide, 0.005 mg of sodium selenite, 0.003 mg of chromium chloride, 55.30 μg of ammonium molybdate, 75.42 μg of potassium fluoride, and the balance is maltodextrin. The vitamin premix, per 1 g, consists of the following ingredients: 0.241 IU of vitamin A acetate oil, vitamin D3... 0.022 IU, Vitamin E acetate 0.184 mg, Vitamin K1 0.028 mg, Thiamine hydrochloride (B1) 0.021 mg, Riboflavin (B2) 0.023 mg, Pyridoxine hydrochloride (B6) 0.038 mg, Cyanocobalamin (B12) 0.03 mg, Nicotinamide 0.257 mg, Folic acid 0.003 mg, D-calcium pantothenate 0.095 mg, L-ascorbic acid 1.287 mg, Biotin 0.538 mg, Inositol 0.034 mg, L-carnitine tartrate 0.064 mg, with the balance being maltodextrin.

[0074] During the anaerobic culture process, the pH value of the bionic intestine is 6.5, the outer side of the bionic intestine is sterile water at a temperature of 37.3℃, and the gas content in the bionic intestine is 100mL, which is monitored by a pressure sensor, with the pressure range being 2-4mm Hg; when the pressure is too high, the excess gas is discharged to maintain the gas content in the bionic intestine at 100mL.

[0075] During the anaerobic culture process, the biomimetic intestine moves in a combination of segmental movement and peristalsis, with segmental movement occurring 3 times per minute and peristalsis propulsion speed of 0.9 cm / min.

[0076] Example 3

[0077] A method for preparing fecal microbiota for intestinal flora transplantation includes the following steps:

[0078] (1) Preparation of intestinal microbiota network carrier: Plant fibers are woven into a network structure, and then the network structure is assembled into a cylindrical fiber network. The cylindrical fiber network is then soaked in a 5% mucin solution in a shaker for 15 minutes at a shaking speed of 20 rpm to obtain the intestinal microbiota network carrier.

[0079] The length of each side of the mesh structure is the same, 1 cm; the diameter of the cylindrical fiber mesh is 100 μm and the total length is 1.5 m. The morphology of this cylindrical fiber mesh is consistent with the bionic intestine prepared later.

[0080] (2) Preparation of intestinal flora suspension: Collect feces from normal healthy people, then dilute the feces in physiological saline at a mass-volume ratio of 1:3, shake it evenly using a homogenizer, and filter it with medical gauze to obtain intestinal flora suspension;

[0081] (3) Preparation of bionic intestine: Based on the precise human intestinal anatomy model of the Department of Gastroenterology, a tubular human intestinal mold is made. The tubular human intestinal mold consists of a core mold and a sleeve forming an internal cavity. Then, transparent silicone is injected into the internal cavity of the mold, solidified and removed to obtain a bionic intestine.

[0082] The bionic intestine is 4mm thick and 9m long. The diameter of the first 6m is 2cm, and the diameter of the remaining 3m is 3cm. There are bends every 5cm.

[0083] (4) Continuous in vitro culture of gut microbiota:

[0084] The intestinal microbiota network carrier prepared in step (1) was placed at the end of the bionic intestine. The intestinal microbiota suspension prepared in step (2) was inoculated into the bionic intestine prepared in step (3). Then, sterile water, nutrients, amylase, protease, cellulase and lipase were added in sequence and anaerobic culture was carried out for 4 weeks. Sterile water, nutrients, amylase, protease, cellulase and lipase were added every 8 hours. Sterile water, nutrients, amylase, protease, cellulase and lipase were discharged every 24 hours.

[0085] The hydrolyzed whey protein and whey protein concentrate mentioned are lactose-free whey proteins.

[0086] The other proteins mentioned are a mixture of pea protein, black bean protein, wheat protein, albumin, etc. in equal weight proportions;

[0087] The amino acid premix, per 1g, comprises the following raw materials: L-glutamine 65.124mg, threonine 64.751mg, serine 79.142mg, proline 33.457mg, cysteine ​​6.412mg, taurine 0.005mg, with the balance being maltodextrin; the nucleotide premix, per 1g, comprises: nucleotides 34.514mg, with the balance being maltodextrin; the nucleotides are a mixture of 5'-uridine monophosphate (5'-UMP), 5'-adenosine monophosphate (5'-AMP), disodium 5'-inosinate, and disodium 5'-guanylate in equal weight proportions; the mineral premix, per 1g, comprises the following raw materials... The composition is as follows: sodium citrate monohydrate 172.251 mg, potassium chloride 118.542 mg, copper gluconate 0.752 mg, magnesium gluconate 165.024 mg, ferrous fumarate 3.389 mg, zinc gluconate 5.583 mg, manganese gluconate 2.521 mg, calcium hydrogen phosphate 125.435 mg, potassium iodide 0.008 mg, sodium selenite 0.008 mg, chromium chloride 78.30 μg, ammonium molybdate 96.42 μg, potassium fluoride 0.623 mg, with the balance being maltodextrin; the vitamin premix, per 1 g, has the following raw material composition: vitamin A acetate oil 1.341 IU, vitamin D3... 0.092 IU, Vitamin E acetate 0.884 mg, Vitamin K1 0.088 mg, Thiamine hydrochloride (B1) 0.12 mg, Riboflavin (B2) 0.099 mg, Pyridoxine hydrochloride (B6) 0.128 mg, Cyanocobalamin (B12) 0.11 mg, Nicotinamide 0.857 mg, Folic acid 0.009 mg, D-calcium pantothenate 0.185 mg, L-ascorbic acid 2.687 mg, Biotin 1.238 mg, Inositol 0.099 mg, L-carnitine tartrate 0.114 mg, with the balance being maltodextrin.

[0088] The volume ratio of the intestinal flora suspension to sterile water is 1:1; the amount of amylase added is 135 U / L, the amount of protease added is 20 U / L, the amount of cellulase added is 15 U / L, and the amount of lipase added is 30 U / L.

[0089] (5) Collection of gut microbiota after culture:

[0090] The intestinal microbiota reticulum carrier was removed and soaked in physiological saline on a shaker 6 times, each time for 6 minutes, at a shaker speed of 10 rpm. After soaking, it was stored in an anaerobic environment to obtain fecal microbiota for intestinal microbiota transplantation.

[0091] In step (4), the nutrient is composed of the following raw materials in parts by weight: 9 parts hydrolyzed whey protein, 10 parts concentrated whey protein, 7 parts soy protein isolate, 7 parts other proteins, 11.5 parts amino acid premix, 5 parts nucleotide premix, 53 parts maltodextrin, 5 parts fructooligosaccharides, 5 parts galactooligosaccharides, 4 parts polydextrose, 20 parts vegetable oil powder, 18 parts mineral premix, and 6.75 parts vitamin premix.

[0092] During the anaerobic culture process, the pH value of the bionic intestine is 8.5, the outer side of the bionic intestine is sterile water at a temperature of 37.3℃, the gas content in the bionic intestine is 100mL, and the pressure is monitored by a pressure sensor, with the pressure range being 2-4mm Hg; when the pressure is too high, the excess gas is discharged to maintain the gas content in the bionic intestine at 100mL.

[0093] During the anaerobic culture process, the biomimetic intestine moves in a combination of segmental movement and peristalsis, with segmental movement occurring 5 times per minute and peristalsis propulsion speed of 0.7 cm / min.

[0094] Comparative Example

[0095] Preparation of mouse intestinal flora solution: Fresh mouse feces were placed in a nitrogen bioengineering chamber, weighed 2g, and placed in a stirrer. 10mL of sterile 0.85% NaCl solution was added for the first homogenization treatment. The fecal slurry was filtered through a 5mm stainless steel filter to remove large particles. Then, 10mL of sterile 0.85% NaCl solution was added and homogenized a second time to obtain fecal microbiota slurry. The fecal microbiota slurry was then filtered through stainless steel filters of 2.0mm, 1.0mm, 0.5mm, and 0.25mm to remove unabsorbed food residue and small particles. The mixture was centrifuged at 4000 rpm for 10min, and resuspended in 4ml of sterile 0.85% NaCl to obtain intestinal flora solution (conventional fecal microbiota).

[0096] Experimental Example

[0097] 1. Effects on mouse body weight

[0098] Grouping: SPF-grade female Balb / c mice, weighing 18–22 g, were randomly divided into four groups: inflammatory bowel disease model group, experimental group 1, experimental group 2, experimental group 3, and experimental group 4, with 15 inflammatory bowel disease model mice in each group. Experimental groups 1, 2, and 3 were treated with the methods described in Examples 1, 2, and 3, respectively, while experimental group 4 was treated with traditional intestinal flora solution.

[0099] Inflammatory bowel disease model: Mice with inflammatory bowel disease were established by gavage with a mixture of 0.1 ml of trinitrobenzenesulfonic acid and ethanol. The concentration of trinitrobenzenesulfonic acid in the mixture was 20 g / L, and the concentration of ethanol was 50%.

[0100] Preparation of mice before intestinal flora transplantation: Before intestinal flora transplantation, mice were fasted for 4 hours and anesthetized by intraperitoneal injection of 10% chloral hydrate (0.03ml / 10g~0.04ml / 10g). After about 5 minutes, the mice were completely anesthetized and fixed in a supine position.

[0101] Intervention method for experimental groups 1-3: 4cm of mesh carriers containing intestinal flora were prepared according to Examples 1-3, and then the mesh carriers containing intestinal flora were placed in the distal colon of mice in experimental groups 1-3 using a mouse colonoscope.

[0102] Intervention method for experimental group 4: 4 mL of the intestinal flora solution (traditional fecal bacteria) prepared in comparative example 1 was injected into the distal colon of mice in experimental group 4 using a mouse colonoscope.

[0103] Feeding method: Mice were administered the following feed solutions via gavage: 0.6g (dry weight powder of mouse feed) / 10g (body weight of mouse). The gavage solution was 0.2ml of physiological saline. Mice were housed individually. The final change in body weight of each group was calculated. The results are shown in Table 1.

[0104] Table 1. Effects of gut microbiota transplantation on body weight in mice with inflammatory bowel disease.

[0105]

[0106] Note: a: P < 0.05 vs. after inflammatory bowel disease modeling in each group; b: P < 0.05 vs. after 2 weeks of intervention in each group; c, d: P < 0.05 vs. experimental groups 1, 2, and 3 of the inflammatory bowel disease model group at different intervention stages.

[0107] As shown in Table 1, compared with the modeling of inflammatory bowel disease, after intervention for 2 and 4 weeks using the fecal microbiota prepared in Examples 1-3 of this invention for intestinal microbiota transplantation and the traditional fecal microbiota prepared in Comparative Example 1, the weight loss of inflammatory bowel disease model mice was significantly alleviated (P < 0.05), and the weight at 4 weeks of intervention was higher than that at 2 weeks of intervention (P < 0.05). However, compared with the fecal microbiota prepared in Examples 1-3 of this invention, the traditional fecal microbiota prepared in Comparative Example 1 showed a significantly lower degree of weight loss alleviation in experimental group 4 than in experimental groups 1-3 (P < 0.05), indicating that the fecal microbiota prepared in this invention for intestinal microbiota transplantation has a large yield and high activity, and is significantly superior to traditional fecal microbiota in alleviating weight loss in patients with inflammatory bowel disease.

[0108] 2. Effects on food utilization in mice

[0109] The modeling, experimental grouping, and intervention measures were consistent with those in Method 1.

[0110] During the intervention period, mice with the inflammatory bowel disease model were housed individually, and their food intake and weight changes were measured. The food utilization coefficient was calculated, and the results are shown in Table 2.

[0111] Wherein: Food utilization coefficient = weight gain (g) ÷ food weight (g) × 100%.

[0112] Table 2. Effects of gut microbiota transplantation on food utilization coefficient in inflammatory bowel disease model mice.

[0113]

[0114] Note: a: P < 0.05 vs. after inflammatory bowel disease modeling in each group; b: P < 0.05 vs. after 2 weeks of intervention in each group; c, d: P < 0.05 vs. experimental groups 1, 2, and 3 of the inflammatory bowel disease model group at different intervention stages.

[0115] As shown in Table 2, compared with the modeling of inflammatory bowel disease, after intervention for 2 and 4 weeks using fecal microbiota prepared in Examples 1-3 of this invention for intestinal microbiota transplantation and conventional fecal microbiota prepared in Comparative Example 1, the decrease in food utilization coefficient of inflammatory bowel disease model mice was significantly alleviated (P < 0.05), and the food utilization coefficient after 4 weeks of intervention was higher than that after 2 weeks of intervention (P < 0.05). However, compared with the fecal microbiota prepared in Examples 1-3 of this invention for intestinal microbiota transplantation, the decrease in food utilization coefficient of experimental group 4 was significantly greater than that of experimental groups 1-3 (P < 0.05), indicating that the fecal microbiota prepared by this invention for intestinal microbiota transplantation has a large yield and high activity, and is significantly superior to conventional fecal microbiota in alleviating the decrease in food utilization coefficient of patients with inflammatory bowel disease.

[0116] 3. Effects on mouse blood cells

[0117] The modeling, experimental grouping, and intervention measures were consistent with those in Method 1.

[0118] Before intestinal flora transplantation, blood was collected from each group of inflammatory bowel disease model mice to detect the number of blood cells. Two and four weeks after intestinal flora transplantation, blood was collected from each inflammatory bowel disease model mouse to detect the number of blood cells. The changes in the number of blood cells were calculated, and the results are shown in Tables 3 and 4.

[0119] The white blood cell and red blood cell counts were determined according to the methods described in the "Clinical Laboratory Manual".

[0120] Table 3. Effects of gut microbiota transplantation on erythrocyte count in inflammatory bowel disease model mice.

[0121]

[0122]

[0123] Note: a: P < 0.05 vs. after inflammatory bowel disease modeling in each group; b: P < 0.05 vs. after 2 weeks of intervention in each group; c, d: P < 0.05 vs. experimental groups 1, 2, and 3 of the inflammatory bowel disease model group at different intervention stages.

[0124] As shown in Table 3, compared with the model of inflammatory bowel disease, after intervention for 2 and 4 weeks using the fecal microbiota prepared in Examples 1-3 of this invention for intestinal microbiota transplantation and the traditional fecal microbiota prepared in Comparative Example 1, the decrease in red blood cell count in inflammatory bowel disease model mice was significantly alleviated (P < 0.05), and the red blood cell count after 4 weeks of intervention was higher than that after 2 weeks of intervention (P < 0.05). However, compared with the fecal microbiota prepared in Examples 1-3 of this invention, the traditional fecal microbiota prepared in Comparative Example 1 showed a significantly greater decrease in red blood cell count in experimental group 4 than in experimental groups 1-3 (P < 0.05), indicating that the fecal microbiota prepared in this invention for intestinal microbiota transplantation has a high yield and high activity, and is significantly superior to traditional fecal microbiota in alleviating the decrease in red blood cell count in patients with inflammatory bowel disease.

[0125] Table 4. Effects of gut microbiota transplantation on white blood cell count in inflammatory bowel disease model mice.

[0126]

[0127] Note: a: P < 0.05 vs. after inflammatory bowel disease modeling in each group; b: P < 0.05 vs. after 2 weeks of intervention in each group; c, d: P < 0.05 vs. experimental groups 1, 2, and 3 of the inflammatory bowel disease model group at different intervention stages.

[0128] As shown in Table 4, compared with the modeling of inflammatory bowel disease, the increase in white blood cell count in inflammatory bowel disease model mice was significantly alleviated after 2 and 4 weeks of intervention using the fecal microbiota prepared in Examples 1-3 of the present invention and the traditional fecal microbiota prepared in Comparative Example 1 (P < 0.05), and the white blood cell count after 4 weeks of intervention was lower than that after 2 weeks of intervention (P < 0.05). However, compared with the fecal microbiota prepared in Examples 1-3 of the present invention, the increase in white blood cell count in experimental group 4 was significantly higher than that in experimental groups 1-3 (P < 0.05), indicating that the fecal microbiota prepared in the present invention has a large yield and high activity, and is significantly superior to traditional fecal microbiota in reducing the white blood cell count in patients with inflammatory bowel disease.

[0129] 4. Effects on the gut microbiota of mice

[0130] The modeling was consistent with Method 1. After successful modeling, mice were randomly divided into four groups: Experimental Group 1, Experimental Group 2, Experimental Group 3, and Experimental Group 4, with 45 inflammatory bowel disease (IBD) model mice in each group. Before intestinal microbiota transplantation, 15 IBD model mice from each group were sacrificed, and their cecal contents were cultured and counted for Bifidobacteria. During intestinal microbiota transplantation, mice were housed individually. At 2 and 4 weeks after intestinal microbiota transplantation, 15 IBD model mice from each group were sacrificed, and their cecal contents were cultured and counted for Bifidobacteria. The changes in the number of Bifidobacteria in the mouse intestines before and after intestinal microbiota transplantation (at weeks 2 and 4) were calculated, and the results are shown in Table 5.

[0131] The method for counting Bifidobacteria is as follows: after weighing the sample, serially dilute it 10 times. Take 5 μL of the diluted solution at a certain dilution and spread it on a pre-prepared culture medium (400 ml tomato juice, 15 g polyvalent peptone, 6 g yeast extract, 20 g glucose, 0.5 g starch, 80 ml Tween, 20 g agar powder, heated to dissolve, adjust the pH to 6.8-7.0, sterilize at 115℃ for 20 min, cool to 55-60℃, and add neomycin sulfate to make the final concentration 100 mg / L). Set up 3 parallel groups for each dilution, and anaerobic culture at 37℃ for 72 h using the anaerobic tank method before counting.

[0132] Table 5. Effects of gut microbiota transplantation on the number of Bifidobacteria in the gut of mice with inflammatory bowel disease.

[0133]

[0134] Note: The data in the table are the common logarithms of the number of bacteria per gram of feces; a: P < 0.05 vs after inflammatory bowel disease modeling in each group; b: P < 0.05 vs after 2 weeks of intervention in each group; c, d: P < 0.05 vs inflammatory bowel disease model groups 1, 2, and 3 at different intervention periods.

[0135] As shown in Table 5, compared with the modeling of inflammatory bowel disease, after intervention for 2 and 4 weeks using the fecal microbiota prepared in Examples 1-3 of this invention for intestinal microbiota transplantation and the traditional fecal microbiota prepared in Comparative Example 1, the decrease in the number of Bifidobacteria in the inflammatory bowel disease model mice was significantly alleviated (P < 0.05), and the content of Bifidobacteria after 4 weeks of intervention was higher than that after 2 weeks of intervention (P < 0.05). However, compared with the fecal microbiota prepared in Examples 1-3 of this invention for intestinal microbiota transplantation, the content of Bifidobacteria in experimental group 4 was significantly lower than that in experimental groups 1-3 (P < 0.05), indicating that the fecal microbiota prepared by this invention for intestinal microbiota transplantation has a large yield and high activity, and is significantly better than traditional fecal microbiota in increasing the content of beneficial bacteria in patients with inflammatory bowel disease, thereby improving their immunity.

Claims

1. A method for preparing fecal microbiota for intestinal flora transplantation, characterized in that, Includes the following steps: (1) Preparation of intestinal microbiota network carrier: Plant fibers are woven into a network structure, and then the network structure is assembled into a cylindrical fiber network. The cylindrical fiber network is then soaked in a 2-5% mucin solution in a shaker for 15-20 minutes at a shaker speed of 10-20 rpm to obtain the intestinal microbiota network carrier. The mesh structure has a uniform length of 0.1-1 cm for each side; the cylindrical fiber mesh has a diameter of 50-100 μm and a total length of 1.2-1.5 m. (2) Preparation of intestinal flora suspension: Collect feces from normal healthy donors, then dilute the feces in physiological saline, shake it evenly using a homogenizer, and filter it with medical gauze to obtain intestinal flora suspension; (3) Preparation of bionic intestine: Based on the precise human intestinal anatomy model of the Department of Gastroenterology, a tubular human intestinal mold is made, and then transparent silicone is injected into the mold. After solidification, it is removed to obtain the bionic intestine. (4) Continuous in vitro culture of gut microbiota: The intestinal microbiota network carrier prepared in step (1) was placed at the end of the bionic intestine. The intestinal microbiota suspension prepared in step (2) was inoculated into the bionic intestine prepared in step (3). Then, sterile water, nutrients, amylase, protease, cellulase and lipase were added in sequence and anaerobic culture was carried out for 1 to 4 weeks. Sterile water, nutrients, amylase, protease, cellulase and lipase were added every 8 hours. Sterile water, nutrients, amylase, protease, cellulase and lipase were discharged every 24 hours. During the anaerobic culture process, the pH value of the bionic intestine is 6.5-8.5, the outer side of the bionic intestine is sterile water at a temperature of 36.9-37.9℃, the gas content inside the bionic intestine is 90-110mL, and the pressure is monitored by a pressure sensor, with the pressure range being 2-4mmHg; when the pressure is too high, excess gas is expelled to maintain the gas content inside the bionic intestine at 90-110mL; the bionic intestine moves in a combination of segmental movement and peristalsis, with segmental movement occurring 3-5 times / min and peristaltic propulsion speed of 0.7-0.9 cm / min; The nutrient solution is composed of the following raw materials in parts by weight: Hydrolyzed whey protein 2-9 parts, whey protein concentrate 2-10 parts, soy protein isolate 2-7 parts, other proteins 2-7 parts, amino acid premix 1.35-11.5 parts, nucleotide premix 1-5 parts, maltodextrin 30-53 parts, fructooligosaccharides 1-5 parts, galactooligosaccharides 1-5 parts, polydextrose 1-4 parts, vegetable oil powder 7-20 parts, mineral premix 4-18 parts, vitamin premix 0.23-6.75 parts; The hydrolyzed whey protein and whey protein concentrate mentioned are lactose-free whey proteins; The other proteins are black bean protein, corn protein, wheat protein, pea protein, collagen, albumin, or sea cucumber protein; The amino acid premix, per 1g, comprises: 50-75mg L-glutamine, 53-72mg threonine, 65-87mg serine, 20-41mg proline, 2-10mg cysteine, 0.001-0.006mg taurine, with the remainder being maltodextrin. The nucleotide premix comprises, per 1g: 15-35mg of nucleotides, with the remainder being maltodextrin; the nucleotides are one or a mixture of two or more of the following: 5'-cytidine monophosphate (5'-CMP), 5'-uridine monophosphate (5'-UMP), 5'-adenosine monophosphate (5'-AMP), disodium 5'-inosinate, disodium 5'-guanylate, disodium 5'-uridine, or disodium 5'-cytidine. The mineral premix, per 1g, comprises: 128-176mg sodium citrate monohydrate, 88-128mg potassium chloride, 0.1-0.8mg copper gluconate, 152-168mg magnesium gluconate, 1.5-4mg ferrous fumarate, 2-6mg zinc gluconate, 0.5-3mg manganese gluconate, 97-135mg dicalcium phosphate, 0.004-0.009mg potassium iodide, 0.002-0.009mg sodium selenite, 50-80μg chromium chloride, 70-100μg ammonium molybdate, 0.3-0.7mg potassium fluoride, with the balance being maltodextrin. The vitamin premix, per 1g, comprises: 0.1-1.5 IU of vitamin A acetate oil, 0.01-0.1 IU of vitamin D3, 0.1-1 mg of vitamin E acetate, 0.01-0.1 mg of vitamin K1, 0.008-0.15 mg of thiamine hydrochloride (B1), 0.007-0.12 mg of riboflavin (B2), 0.02-0.18 mg of pyridoxine hydrochloride (B6), and 0.01-0.14 mg of cyanocobalamin (B12). The formula contains: nicotinamide 0.1-1 mg, folic acid 0.001-0.01 mg, D-calcium pantothenate 0.02-0.21 mg, L-ascorbic acid 1-3 mg, biotin 0.1-1.4 mg, inositol 0.01-0.12 mg, L-carnitine tartrate 0.02-0.18 mg, with the remainder being maltodextrin. The amount of amylase added is 35~135U / L, the amount of protease added is 6~20U / L, the amount of cellulase added is 7~15U / L, and the amount of lipase added is 18~30U / L. (5) Collection of gut microbiota after culture: The intestinal microbiota reticulum carrier was removed and soaked in physiological saline on a shaker 4-6 times, with each soaking time being 2-6 minutes and the shaker speed being 2-10 rpm. After soaking, it was stored in an anaerobic environment to obtain fecal microbiota for intestinal microbiota transplantation.

2. The method for preparing intestinal flora transplantation as described in claim 1, characterized in that, In step (1), the plant fiber is the plant fiber of fruits, vegetables, beans, nuts, grains and fungi.

3. The method for preparing intestinal flora transplantation as described in claim 1, characterized in that, In step (2), the mass-to-volume ratio of feces to saline solution is 1:(3~30), with units of g / mL.

4. The method for preparing intestinal flora transplantation as described in claim 1, characterized in that, In step (3), the tubular human intestinal mold consists of a core mold and a sleeve forming an internal cavity, into which transparent silicone is injected; the bionic intestine has a thickness of 1~4mm, a length of 9~10m, a diameter of 1~2cm for the first 4~6m, and a diameter of 2~3cm for the remainder.

5. The method for preparing intestinal flora transplantation as described in claim 1, characterized in that, In step (4), the volume ratio of the intestinal flora suspension to sterile water is 1:(0.5~1).

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