Culture medium for colonization of intestinal flora and intestinal gas of people with chronic gastritis and application of culture medium

Abstract

The invention discloses a culture medium for colonization of intestinal flora and intestinal gas of people with chronic gastritis, and belongs to the technical field of biology. The culture medium contains yeast extract, tryptone, mucoprotein, mycoprotein, worm protein, soybean protein, casein, whey protein, vitellin, fructo-oligosaccharide, galactooligosaccharide, wheat flour, inulin, resistant dextrin, dietary fiber, sialic acid, algal oil DHA, calcium chloride, potassium chloride, sodium chloride, monopotassium phosphate and the like. By means of the bionic colon reactor, by referring to real in-vivo parameters of people with chronic gastritis, the real intestinal environment of people with chronic gastritis in China is well simulated in vitro, intestinal flora ecology and intestinal gas distribution of people with chronic gastritis reappear, and a normal intestinal microorganism and intestinal gas database of people with chronic gastritis can be constructed. The method is beneficial to accurate diagnosis and personalized treatment of intestinal microecology of people with chronic gastritis, and can better evaluate the health state of people with chronic gastritis and formulate preventive measures.

Description

Technical Field

[0001] The invention relates to a culture medium for colonizing intestinal flora and intestinal gas in people with chronic gastritis and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] The gut microbiome refers to the microbial community itself, the relative abundance of individual species, and their functions. The intestine is rich in diverse microbial communities that extract energy from food digestion to promote the development and maturation of the intestinal and systemic immune systems, forming a symbiotic relationship with the human body and jointly maintaining immune homeostasis and balance in the gut. Each person possesses a unique gut microbiome, which varies from population to population, primarily influenced by factors such as age, gender, geographic location, medications, physiological status, lifestyle, and genetic background. However, normal microbial communities tend to share similar microbiome compositions. In a healthy state, the gut microbiome exhibits strong resilience and ability to recover from external disturbances. The human gut microbiome is a complex ecological system. Imbalances can lead to changes in gut microbial function and a significant decrease in microbial diversity, which in turn can affect metabolic health and contribute to the development of disease.

[0003] Furthermore, the inner workings of the intestine are complex, encompassing not only a variety of microorganisms but also the gases they produce, such as carbon dioxide, hydrogen, methane, hydrogen sulfide, and various trace gases, all of which are produced by chemical interactions within the intestine and by the microbiome. Analyzing these intestinal gases and their responses to dietary changes can reveal the products and functions of the gut microbiome and their impact on human health. This can provide a better understanding of the complex interactions that produce gases in the intestine, thereby improving our ability to prevent, diagnose, treat, and monitor many diseases.

[0004] Reference microbiota and intestinal gas profiles of a specific population can serve as important indicators for assessing that population's health status. By measuring an individual's microbiome composition and intestinal gas profile and comparing it with a reference sample from the same population, we can understand their health status and potential health risks. Therefore, establishing a reference microbiome and intestinal gas profile for a specific population can help provide accurate diagnosis and personalized treatment; better assess health status and develop preventive strategies; gain a deeper understanding of population differences and guide drug development; assist in assessing the effectiveness of clinical interventions; and determine disease susceptibility in specific populations. For example, for infants and young children, maintaining a healthy intestinal microbiome is crucial for immune system development and overall health. For the elderly, maintaining a balanced intestinal microbiome through dietary adjustments and probiotic supplementation can prevent the development of intestinal diseases and other chronic conditions. However, achieving a perfect in vitro colonization of the intestinal microbiome and studying its mechanisms and mechanisms remains a major challenge in microbiome research. Most microbiome colonization strategies are based on findings from studies in Westernized populations, while studies specifically targeting Chinese populations, particularly those with chronic gastritis, are limited. The microbial communities of people with chronic gastritis abroad have been observed, with Helicobacter pylori infection being the core trigger. This alters the body's immune response and metabolism, destroys the gastric mucosal barrier, and triggers inflammation, resulting in a relatively fragile intestinal flora with poor diversity. Furthermore, a variety of factors, including medication and chemical stimulation, immune and genetic factors, and irregular lifestyles and diets, can affect the colonization and succession of intestinal flora. These are primarily composed of members of the genera Helicobacter pylori, Escherichia, Coprococcus, Bacteroides, Clostridium, Actinomyces, and Bifidobacterium. Furthermore, there are few reports both domestically and internationally on the intestinal gas produced by people with chronic gastritis. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a culture medium for colonizing intestinal flora and intestinal gas in people with chronic gastritis, so as to solve the problem that there is little research on intestinal colonization strategies in people with chronic gastritis in the prior art.

[0006] The technical problem that the present invention also aims to solve is to provide a method for in vitro colonization of intestinal flora and intestinal gas in people with chronic gastritis, so as to provide a method for simulating the real intestinal environment of people with chronic gastritis, reproducing the intestinal flora ecology and intestinal gas distribution of people with chronic gastritis, and providing data support for the study of intestinal flora in people with chronic gastritis.

[0007] Technical solution: To solve the above technical problems, the present invention provides the following technical solutions:

[0008] A culture medium for colonizing intestinal flora and intestinal gas in people with chronic gastritis. The contents of each component in 1L of the culture medium are as follows: 3-4g yeast extract, 2-4g tryptone, 0.5-1.5g mucin, 1-2g mycoprotein, 0.5-1g insect protein, 2.5-3.5g soy protein, 2-3g casein, 1-2g whey protein, 1.5-2.5g phosvitin, 2-4g fructooligosaccharide, 2-4g galacto-oligosaccharide, 3-5g wheat flour, 1-2g inulin, 1-2g resistant dextrin, 2-4g dietary fiber, 0.5-1.5g sialic acid, and algae oil DHA. 1-2g, calcium chloride 0.5-1g, potassium chloride 0.25-0.75g, sodium chloride 1-2g, potassium dihydrogen phosphate 1-1.5g, sodium sulfate 0.5-1.5g, bile salts 0.2-0.4g, plant extract mixture 0.5-1g, vitamin mixture 4-6mL. All the above reagents are analytical grade.

[0009] Preferably, the content of each component in each 1L of culture medium is as follows: 3.5g yeast extract, 3g tryptone, 1g mucin, 1.5g mycoprotein, 0.75g insect protein, 3g soy protein, 2.5g casein, 1.5g whey protein, 2g phosvitin, 3g fructooligosaccharide, 3g galacto-oligosaccharide, 4g wheat flour, 1.5g inulin, 1.5g resistant dextrin, 3g dietary fiber, 1g sialic acid, 1.5g algal oil DHA, 0.75g calcium chloride, 0.5g potassium chloride, 1.5g sodium chloride, 1.25g potassium dihydrogen phosphate, 1g sodium sulfate, 0.3g bile salt, 0.75g plant extract mixture, and 5mL vitamin mixture.

[0010] The mucin includes porcine mucin, bovine mucin and mussel mucin, preferably bovine mucin.

[0011] The mycoprotein includes shiitake mushroom powder, ganoderma lucidum powder, wood ear powder, boletus powder, bamboo fungus powder, Agaricus blazei powder, Hericium erinaceus powder, Pleurotus eryngii powder, Cordyceps sinensis powder, preferably ganoderma lucidum powder.

[0012] The insect protein includes proteins extracted from eggs, larvae, adults, pupae, moths, etc., such as bean worm powder and silkworm pupae, preferably silkworm pupae powder.

[0013] The plant extract mixture includes puerarin, carotene, bioflavonoids, phytoandrogen, soy isoflavones, limonene, lycopene, catechin, anthocyanidin, and resveratrol, preferably carotene and bioflavonoids.

[0014] The formula of the vitamin mixture is as follows: vitamin B1 0.2g / L, vitamin B2 0.2g / L, niacin 2g / L, folic acid 6g / L, vitamin B12 0.8g / L, vitamin C 5g / L, vitamin E 2g / L, vitamin D 0.2g / L, vitamin A 0.2g / L.

[0015] The present invention also provides a method for in vitro colonization of intestinal flora and intestinal gas in people with chronic gastritis, the method comprising the following steps:

[0016] (1) 6 g of fresh feces from a patient with chronic gastritis was collected and placed in 30 mL of PBS, stirred evenly, and then the solid was filtered with gauze to obtain a fecal suspension;

[0017] (2) injecting the fecal suspension obtained in step (1) into the ascending colon reactor, transverse colon reactor, and descending colon reactor of the above-mentioned culture medium, and culturing them under anaerobic conditions for 24 hours, thereby obtaining the intestinal flora and intestinal gas of chronic gastritis patients at the ascending colon, transverse colon, and descending colon stages, respectively;

[0018] In step (1), fresh feces were collected by a feces collector, and then each gram of feces was suspended in 5 mL of PBS. The time from feces collection to injection into the reactor was controlled within 10 minutes.

[0019] In step (2), the dynamic culture is carried out at 37°C under anaerobic conditions for 24 hours, and the simulated peristalsis frequency of each reactor is 1 to 2 times per minute in the ascending colon, 1 to 2 times per minute in the transverse colon, and 2 to 3 times per minute in the descending colon; the stable pH of the ascending colon reactor is 5.75 to 6.25, the stable pH of the transverse colon reactor is 6.35 to 6.65, and the stable pH of the descending colon reactor is 6.75 to 7.34. The rate of transfer of the contents of the ascending colon into the transverse colon is 1 to 2 ml / min, and the rate of transfer of the contents of the transverse colon into the descending colon is 2 to 3 ml / min. The ratio of the fecal suspension to the culture medium in each reactor is 5 mL:100 mL.

[0020] In step (2), the stable pH of each reactor is adjusted up and down by 0.5 mol of hydrochloric acid and 0.5 mol of sodium hydroxide.

[0021] In step (2), if the intestinal flora of chronic gastritis patients needs to be kept in a steady state for a long time, fresh culture medium can be injected every 8 hours for stable growth, and the fresh culture medium accounts for 10% of the total culture volume.

[0022] The headspace gas of the reactor was composed of 100% nitrogen to ensure an anaerobic environment. As the culture time increased, colonization samples and intestinal gas were regularly taken from the reactor for measurement.

[0023] Beneficial effects:

[0024] The bionic colon reactor, based on gastrointestinal physiology, biotechnology, and fermentation engineering, effectively simulates the actual intestinal environment of chronic gastritis patients in China in vitro by referencing real-world parameters found in humans. The method utilizes intestinal flora culture media from chronic gastritis patients combined with the bionic colon reactor to establish a colonization strategy for intestinal microorganisms and intestinal gas in chronic gastritis patients. This method can construct a database of normal intestinal microorganisms and intestinal gas in chronic gastritis patients, facilitating accurate diagnosis and personalized treatment of the intestinal microbiome in chronic gastritis patients, and better assessing their health status and developing preventive measures. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Comparison of species richness of intestinal flora and intestinal gas cultured in common culture medium and culture medium of chronic gastritis patients;

[0026] Figure 2 : Comparison of gas composition after culture of intestinal flora and intestinal gas culture medium in chronic gastritis patients. DETAILED DESCRIPTION

[0027] The culture medium components involved in the following examples are described as follows:

[0028] The yeast extract, tryptone, mucin, mycoprotein, insect protein, soy protein, casein, whey protein, yolk phosvitin, oligofructose, oligogalactose, wheat flour, inulin, resistant dextrin, dietary fiber, sialic acid, algae oil DHA, calcium chloride, potassium chloride, sodium chloride, potassium dihydrogen phosphate, sodium sulfate, bile salts, plant extract mixture, and vitamin mixture are all of analytical grade concentration.

[0029] The mucin includes porcine mucin, bovine mucin, and mussel mucin, preferably bovine mucin.

[0030] The mycoprotein includes shiitake mushroom powder, ganoderma lucidum powder, wood ear powder, boletus powder, bamboo fungus powder, Agaricus blazei powder, Hericium erinaceus powder, Pleurotus eryngii powder, Cordyceps sinensis powder, preferably ganoderma lucidum powder.

[0031] The insect protein includes proteins extracted from eggs, larvae, adults, pupae, moths, etc., such as bean worm powder and silkworm pupae, preferably silkworm pupae powder.

[0032] The plant extract mixture comprises puerarin, carotene, bioflavonoids, phytoandrogen, soy isoflavones, limonene, lycopene, catechin, anthocyanidin, and resveratrol, preferably carotene and bioflavonoids.

[0033] The formula of the vitamin mixture is as follows: vitamin B1 0.2g / L, vitamin B2 0.2g / L, niacin 2g / L, folic acid 6g / L, vitamin B12 0.8g / L, vitamin C 5g / L, vitamin E 2g / L, vitamin D 0.2g / L, and vitamin A 0.2g / L.

[0034] When the culture medium is a solid culture medium, the added amount of agar powder is 1-1.5%.

[0035] Example 1: In vitro spatiotemporal dynamic colonization and cultivation of intestinal flora and intestinal gas in patients with chronic gastritis

[0036] The specific steps are as follows:

[0037] (1) Preparation of fecal suspension

[0038] Fresh stool samples were collected from five individuals with chronic gastritis. None of the individuals had taken antibiotics or other medications that could interfere with the intestinal microbiome in the past three months, nor had any gastrointestinal diseases. Fresh stool was collected using a stool collector. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0039] (2) Preparation of culture medium

[0040] Prepare the culture medium according to the formula in Table 1:

[0041] Table 1 In vitro colonization culture medium of intestinal flora and intestinal gas in people with chronic gastritis

[0042]

[0043]

[0044] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0045] 200 mL of culture medium was added to the ascending colon reactor, the transverse colon reactor, and the descending colon reactor, followed by 10 mL of fecal suspension. The cells were incubated dynamically at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in each reactor was 1-2 beats per minute for the ascending colon, 1-2 beats per minute for the transverse colon, and 2-3 beats per minute for the descending colon. The stable pH of the ascending colon reactor was 5.75-6.25, the stable pH of the transverse colon reactor was 6.35-6.65, and the stable pH of the descending colon reactor was 6.75-7.34. The transfer rate of contents from the ascending colon to the transverse colon was 1-2 ml / min, and the transfer rate of contents from the transverse colon to the descending colon was 2-3 ml / min. The stable pH of each reactor was adjusted upward and downward using 0.5 M hydrochloric acid and 0.5 M sodium hydroxide. For intestinal flora from people with chronic gastritis that maintain a steady state for a long time, fresh 10% culture medium can be added every 8 hours to maintain stable growth.

[0046] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0047] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0048] The results showed that: in terms of intestinal flora structure, the culture medium of the present invention can significantly increase the colonization abundance of Helicobacter pylori, Escherichia, Coprococcus, Bacteroides, Clostridium, Actinomyces, and Bifidobacterium compared with the traditional culture medium (main ingredients: ox brain extract powder, ox heart extract powder, peptone, casein peptone, glucose, sodium chloride, disodium hydrogen phosphate, agar), which is similar to the real intestinal environment of people with chronic gastritis. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide and volatile gases (VOC) are the main components, which are similar to the real intestinal gas of people with chronic gastritis. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0049] Example 2: In vitro spatiotemporal dynamic colonization and cultivation of intestinal flora and intestinal gas in patients with chronic gastritis

[0050] The specific steps are as follows:

[0051] (1) Preparation of fecal suspension

[0052] Fresh stool samples were collected from five individuals with chronic gastritis. None of the individuals had taken antibiotics or other medications that could interfere with the intestinal microbiome in the past three months, nor had any gastrointestinal diseases. Fresh stool was collected using a stool collector. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0053] (2) Preparation of culture medium

[0054] Prepare the culture medium according to the formula in Table 2:

[0055] Table 2 In vitro colonization culture medium of intestinal flora and intestinal gas in patients with chronic gastritis

[0056]

[0057] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0058] 200 mL of culture medium was added to the ascending colon reactor, the transverse colon reactor, and the descending colon reactor, followed by 10 mL of fecal suspension, and dynamic incubation at 37°C for 24 hours under anaerobic conditions. The simulated peristaltic frequency in each reactor was 1-2 beats per minute for the ascending colon, 1-2 beats per minute for the transverse colon, and 2-3 beats per minute for the descending colon. The stable pH of the ascending colon reactor was 5.75-6.25, the transverse colon reactor was 6.35-6.65, and the descending colon reactor was 6.75-7.34. The transfer rate of contents from the ascending colon to the transverse colon was 1-2 ml / min, and the transfer rate of contents from the transverse colon to the descending colon was 2-3 ml / min. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For intestinal flora from people with chronic gastritis that maintain a steady state for a long time, fresh 10% culture medium can be added every 8 hours to maintain stable growth.

[0059] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0060] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0061] The results showed that: in terms of intestinal flora structure, the culture medium of the present invention can significantly increase the colonization abundance of Helicobacter pylori, Escherichia, Coprococcus, Bacteroides, Clostridium, Actinomyces, and Bifidobacterium compared with the traditional culture medium (main ingredients: ox brain extract powder, ox heart extract powder, peptone, casein peptone, glucose, sodium chloride, disodium hydrogen phosphate, agar), which is similar to the real intestinal environment of people with chronic gastritis. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide and volatile gases (VOC) are the main components, which are similar to the real intestinal gas of people with chronic gastritis. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0062] Example 3: In vitro spatiotemporal dynamic colonization and cultivation of intestinal flora and intestinal gas in patients with chronic gastritis

[0063] The specific steps are as follows:

[0064] (1) Preparation of fecal suspension

[0065] Fresh stool samples were collected from five individuals with chronic gastritis. None of the individuals had taken antibiotics or other medications that could interfere with the intestinal microbiome in the past three months, nor had any gastrointestinal diseases. Fresh stool was collected using a stool collector. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0066] (2) Preparation of culture medium

[0067] Prepare the culture medium according to the formula in Table 3:

[0068] Table 3 In vitro colonization culture medium of intestinal flora and intestinal gas in patients with chronic gastritis

[0069]

[0070] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0071] 200 mL of culture medium was added to the ascending colon reactor, the transverse colon reactor, and the descending colon reactor, followed by 10 mL of fecal suspension, and dynamic incubation at 37°C for 24 hours under anaerobic conditions. The simulated peristalsis frequency in each reactor was 1-2 beats per minute for the ascending colon, 1-2 beats per minute for the transverse colon, and 2-3 beats per minute for the descending colon. The stable pH of the ascending colon reactor was 5.75-6.25, the stable pH of the transverse colon reactor was 6.35-6.65, and the stable pH of the descending colon reactor was 6.75-7.34. The transfer rate of contents from the ascending colon to the transverse colon was 1-2 ml / min, and the transfer rate of contents from the transverse colon to the descending colon was 2-3 ml / min. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For intestinal flora from people with chronic gastritis that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0072] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0073] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0074] The results showed that: in terms of intestinal flora structure, the culture medium of the present invention can significantly increase the colonization abundance of Helicobacter pylori, Escherichia, Coprococcus, Bacteroides, Clostridium, Actinomyces, and Bifidobacterium compared with the traditional culture medium (main ingredients: ox brain extract powder, ox heart extract powder, peptone, casein peptone, glucose, sodium chloride, disodium hydrogen phosphate, agar), which is similar to the real intestinal environment of people with chronic gastritis. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide and volatile gases (VOC) are the main components, which are similar to the real intestinal gas of people with chronic gastritis. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0075] Comparative Example 1: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in patients with chronic gastritis (the amount of culture medium components added is lower than the minimum value)

[0076] The specific steps are as follows:

[0077] (1) Preparation of fecal suspension

[0078] Fresh stool samples were collected from five individuals with chronic gastritis. None of the individuals had taken antibiotics or other medications that could interfere with the intestinal microbiome in the past three months, nor had any gastrointestinal diseases. Fresh stool was collected using a stool collector. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0079] (2) Preparation of culture medium

[0080] Prepare the culture medium according to the formula in Table 4:

[0081] Table 4 In vitro colonization culture medium of intestinal flora and intestinal gas in patients with chronic gastritis

[0082]

[0083]

[0084] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0085] Add 200 mL of culture medium to the ascending colon reactor, transverse colon reactor, and descending colon reactor, and inject 10 mL of fecal suspension into each reactor. Dynamic culture was carried out at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in each reactor was 1-2 times per minute in the ascending colon, 1-2 times per minute in the transverse colon, and 2-3 times per minute in the descending colon. The stable pH of the ascending colon reactor was 5.75-6.25, the stable pH of the transverse colon reactor was 6.35-6.65, and the stable pH of the descending colon reactor was 6.75-7.34. The transfer rate of contents from the ascending colon to the transverse colon was 1-2 ml / min, and the transfer rate of contents from the transverse colon to the descending colon was 2-3 ml / min. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For intestinal flora from people with chronic gastritis that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0086] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0087] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0088] The results showed that in terms of intestinal flora structure, compared with the intestinal environment of real chronic gastritis people, the colonization abundance of Helicobacter pylori, Escherichia, Coprococcus, Bacteroides, Clostridium, Actinomyces, and Bifidobacterium in the fermentation broth was reduced. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide and volatile gases (VOC) were the main components, but the gas production was different from the real intestinal gas of chronic gastritis people. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0089] Comparative Example 2: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in patients with chronic gastritis (the amount of culture medium components added is higher than the maximum value)

[0090] The specific steps are as follows:

[0091] (1) Preparation of fecal suspension

[0092] Fresh stool samples were collected from five individuals with chronic gastritis. None of the individuals had taken antibiotics or other medications that could interfere with the intestinal microbiome in the past three months, nor had any gastrointestinal diseases. Fresh stool was collected using a stool collector. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0093] (2) Preparation of culture medium

[0094] The culture medium was prepared according to the formula in Table 5:

[0095] Table 5 In vitro colonization culture medium of intestinal flora and intestinal gas in patients with chronic gastritis

[0096]

[0097] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0098] Add 200 mL of culture medium to the ascending colon reactor, transverse colon reactor, and descending colon reactor, and inject 10 mL of fecal suspension into each reactor. Dynamic culture was carried out at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in each reactor was 1-2 times per minute in the ascending colon, 1-2 times per minute in the transverse colon, and 2-3 times per minute in the descending colon. The stable pH of the ascending colon reactor was 5.75-6.25, the stable pH of the transverse colon reactor was 6.35-6.65, and the stable pH of the descending colon reactor was 6.75-7.34. The transfer rate of contents from the ascending colon to the transverse colon was 1-2 ml / min, and the transfer rate of contents from the transverse colon to the descending colon was 2-3 ml / min. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For intestinal flora from people with chronic gastritis that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0099] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0100] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0101] The results showed that in terms of intestinal flora structure, compared with the intestinal environment of real chronic gastritis people, the colonization abundance of Helicobacter pylori, Escherichia, Coprococcus, Bacteroides, Clostridium, Actinomyces, and Bifidobacterium in the fermentation broth was reduced. In terms of intestinal gas, carbon dioxide decreased and volatile gases (VOC) increased. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0102] Comparative Example 3: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in people with chronic gastritis (without adding bovine mucin)

[0103] The specific steps are as follows:

[0104] (1) Preparation of fecal suspension

[0105] Fresh stool samples were collected from five individuals with chronic gastritis. None of the individuals had taken antibiotics or other medications that could interfere with the intestinal microbiome in the past three months, nor had any gastrointestinal diseases. Fresh stool was collected using a stool collector. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0106] (2) Preparation of culture medium

[0107] The culture medium was prepared according to the formula in Table 6:

[0108] Table 6 In vitro colonization culture medium of intestinal flora and intestinal gas in patients with chronic gastritis

[0109]

[0110] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0111] Add 200 mL of culture medium to the ascending colon reactor, transverse colon reactor, and descending colon reactor, and inject 10 mL of fecal suspension into each reactor. Dynamic culture was carried out at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in each reactor was 1-2 times per minute in the ascending colon, 1-2 times per minute in the transverse colon, and 2-3 times per minute in the descending colon. The stable pH of the ascending colon reactor was 5.75-6.25, the stable pH of the transverse colon reactor was 6.35-6.65, and the stable pH of the descending colon reactor was 6.75-7.34. The transfer rate of contents from the ascending colon to the transverse colon was 1-2 ml / min, and the transfer rate of contents from the transverse colon to the descending colon was 2-3 ml / min. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For intestinal flora from people with chronic gastritis that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0112] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0113] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0114] The results showed that in terms of intestinal flora structure, compared with the intestinal environment of real chronic gastritis people, the colonization abundance of Helicobacter pylori, Escherichia, Coprococcus, Clostridium, and Actinomyces in the fermentation broth was significantly reduced. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide, and volatile gases (VOCs) were the main components, which were no different from the real intestinal gas of chronic gastritis people. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0115] Comparative Example 4: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in people with chronic gastritis (without sodium sulfate addition)

[0116] The specific steps are as follows:

[0117] (1) Preparation of fecal suspension

[0118] Fresh stool samples were collected from five individuals with chronic gastritis. None of the individuals had taken antibiotics or other medications that could interfere with the intestinal microbiome in the past three months, nor had any gastrointestinal diseases. Fresh stool was collected using a stool collector. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0119] (2) Preparation of culture medium

[0120] The culture medium was prepared according to the formula in Table 7:

[0121] Table 7 In vitro colonization culture medium of intestinal flora and intestinal gas in patients with chronic gastritis

[0122]

[0123]

[0124] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0125] Add 200 mL of culture medium to the ascending colon reactor, transverse colon reactor, and descending colon reactor, and inject 10 mL of fecal suspension into each reactor. Dynamic culture was carried out at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in each reactor was 1-2 times per minute in the ascending colon, 1-2 times per minute in the transverse colon, and 2-3 times per minute in the descending colon. The stable pH of the ascending colon reactor was 5.75-6.25, the stable pH of the transverse colon reactor was 6.35-6.65, and the stable pH of the descending colon reactor was 6.75-7.34. The transfer rate of contents from the ascending colon to the transverse colon was 1-2 ml / min, and the transfer rate of contents from the transverse colon to the descending colon was 2-3 ml / min. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For intestinal flora from people with chronic gastritis that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0126] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0127] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0128] The results showed that in terms of intestinal flora structure, compared with the intestinal environment of real chronic gastritis people, the colonization abundance of Helicobacter pylori, Escherichia, Coprococcus, Bacteroides, Clostridium, Actinomyces, and Bifidobacterium in the fermentation broth was reduced. In terms of intestinal gas, no hydrogen sulfide was produced. This is because the culture medium lacks sulfur and the intestinal flora cannot use sulfur metabolism to produce hydrogen sulfide. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0129] Comparative Example 5: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in people with chronic gastritis (without adding casein and whey protein)

[0130] The specific steps are as follows:

[0131] (1) Preparation of fecal suspension

[0132] Fresh stool samples were collected from five individuals with chronic gastritis. None of the individuals had taken antibiotics or other medications that could interfere with the intestinal microbiome in the past three months, nor had any gastrointestinal diseases. Fresh stool was collected using a stool collector. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0133] (2) Preparation of culture medium

[0134] Prepare the culture medium according to the formula in Table 8:

[0135] Table 8 In vitro colonization culture medium of intestinal flora and intestinal gas in patients with chronic gastritis

[0136]

[0137] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0138] Add 200 mL of culture medium to the ascending colon reactor, transverse colon reactor, and descending colon reactor, and inject 10 mL of fecal suspension into each reactor. Dynamic culture was carried out at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in each reactor was 1-2 times per minute in the ascending colon, 1-2 times per minute in the transverse colon, and 2-3 times per minute in the descending colon. The stable pH of the ascending colon reactor was 5.75-6.25, the stable pH of the transverse colon reactor was 6.35-6.65, and the stable pH of the descending colon reactor was 6.75-7.34. The transfer rate of contents from the ascending colon to the transverse colon was 1-2 ml / min, and the transfer rate of contents from the transverse colon to the descending colon was 2-3 ml / min. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For intestinal flora from people with chronic gastritis that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0139] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0140] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0141] The results showed that in terms of intestinal flora structure, compared with the intestinal environment of real chronic gastritis people, the colonization abundance of Helicobacter pylori, Escherichia, Coprococcus, Clostridium, and Actinomyces in the fermentation broth was significantly reduced. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide, and volatile gases (VOCs) were the main components, which were no different from the real intestinal gas of chronic gastritis people. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0142] Comparative Example 6: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in people with chronic gastritis (without addition of yolk phosvitin)

[0143] The specific steps are as follows:

[0144] (1) Preparation of fecal suspension

[0145] Fresh stool samples were collected from five individuals with chronic gastritis. None of the individuals had taken antibiotics or other medications that could interfere with the intestinal microbiome in the past three months, nor had any gastrointestinal diseases. Fresh stool was collected using a stool collector. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0146] (2) Preparation of culture medium

[0147] The culture medium was prepared according to the formula in Table 9:

[0148] Table 9 In vitro colonization culture medium of intestinal flora and intestinal gas in patients with chronic gastritis

[0149]

[0150]

[0151] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0152] Add 200 mL of culture medium to the ascending colon reactor, transverse colon reactor, and descending colon reactor, and inject 10 mL of fecal suspension into each reactor. Dynamic culture was carried out at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in each reactor was 1-2 times per minute in the ascending colon, 1-2 times per minute in the transverse colon, and 2-3 times per minute in the descending colon. The stable pH of the ascending colon reactor was 5.75-6.25, the stable pH of the transverse colon reactor was 6.35-6.65, and the stable pH of the descending colon reactor was 6.75-7.34. The transfer rate of contents from the ascending colon to the transverse colon was 1-2 ml / min, and the transfer rate of contents from the transverse colon to the descending colon was 2-3 ml / min. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For intestinal flora from people with chronic gastritis that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0153] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0154] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0155] The results showed that in terms of intestinal flora structure, compared with the intestinal environment of real chronic gastritis people, the colonization abundance of Helicobacter pylori, Escherichia, Coprococcus, Clostridium, and Actinomyces in the fermentation broth was significantly reduced. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide, and volatile gases (VOCs) were the main components, which were no different from the real intestinal gas of chronic gastritis people. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0156] Comparative Example 7: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in people with chronic gastritis (without adding silkworm pupa powder)

[0157] The specific steps are as follows:

[0158] (1) Preparation of fecal suspension

[0159] Fresh stool samples were collected from five individuals with chronic gastritis. None of the individuals had taken antibiotics or other medications that could interfere with the intestinal microbiome in the past three months, nor had any gastrointestinal diseases. Fresh stool was collected using a stool collector. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0160] (2) Preparation of culture medium

[0161] The culture medium was prepared according to the formula in Table 12:

[0162] Table 10 In vitro colonization culture medium of intestinal flora and intestinal gas in patients with chronic gastritis

[0163]

[0164]

[0165] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0166] Add 200 mL of culture medium to the ascending colon reactor, transverse colon reactor, and descending colon reactor, and inject 10 mL of fecal suspension into each reactor. Dynamic culture was carried out at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in each reactor was 1-2 times per minute in the ascending colon, 1-2 times per minute in the transverse colon, and 2-3 times per minute in the descending colon. The stable pH of the ascending colon reactor was 5.75-6.25, the stable pH of the transverse colon reactor was 6.35-6.65, and the stable pH of the descending colon reactor was 6.75-7.34. The transfer rate of contents from the ascending colon to the transverse colon was 1-2 ml / min, and the transfer rate of contents from the transverse colon to the descending colon was 2-3 ml / min. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For intestinal flora from people with chronic gastritis that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0167] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0168] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0169] The results showed that in terms of intestinal flora structure, compared with the intestinal environment of real chronic gastritis people, the colonization abundance of Helicobacter pylori, Escherichia, Coprococcus, Clostridium, and Actinomyces in the fermentation broth was significantly reduced. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide, and volatile gases (VOCs) were the main components, which were no different from the real intestinal gas of chronic gastritis people. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0170] Comparative Example 8: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in people with chronic gastritis (without the addition of carotene and bioflavonoids)

[0171] The specific steps are as follows:

[0172] (1) Preparation of fecal suspension

[0173] Fresh stool samples were collected from five individuals with chronic gastritis. None of the individuals had taken antibiotics or other medications that could interfere with the intestinal microbiome in the past three months, nor had any gastrointestinal diseases. Fresh stool was collected using a stool collector. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0174] (2) Preparation of culture medium

[0175] The culture medium was prepared according to the formula in Table 11:

[0176] Table 11 In vitro colonization culture medium of intestinal flora and intestinal gas in patients with chronic gastritis

[0177]

[0178] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0179] Add 200 mL of culture medium to the ascending colon reactor, transverse colon reactor, and descending colon reactor, and inject 10 mL of fecal suspension into each reactor. Dynamic culture was carried out at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in each reactor was 1-2 times per minute in the ascending colon, 1-2 times per minute in the transverse colon, and 2-3 times per minute in the descending colon. The stable pH of the ascending colon reactor was 5.75-6.25, the stable pH of the transverse colon reactor was 6.35-6.65, and the stable pH of the descending colon reactor was 6.75-7.34. The transfer rate of contents from the ascending colon to the transverse colon was 1-2 ml / min, and the transfer rate of contents from the transverse colon to the descending colon was 2-3 ml / min. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For intestinal flora from people with chronic gastritis that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0180] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0181] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0182] The results showed that in terms of intestinal flora structure, compared with the intestinal environment of real chronic gastritis people, the colonization abundance of Helicobacter pylori, Escherichia, Coprococcus, Clostridium, and Actinomyces in the fermentation broth was significantly reduced. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide, and volatile gases (VOCs) were the main components, which were no different from the real intestinal gas of chronic gastritis people. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0183] Table 12 Comparison of species richness of intestinal flora and intestinal gas culture medium in patients with chronic gastritis

[0184]

[0185] Table 13 Comparison of gas composition after culture of intestinal flora and intestinal gas culture medium in patients with chronic gastritis

[0186]

[0187] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A culture medium for colonizing intestinal flora and intestinal gas in people with chronic gastritis, characterized in that: The content of each component in 1L of culture medium is as follows: Yeast extract 3-4g, tryptone 2-4g, mucin 0.5-1.5g, mycoprotein 1-2g, insect protein 0.5-1g, soy protein 2.5-3.5g, casein 2-3g, whey protein 1-2g, yolk phosvitin 1.5-2.5g, fructooligosaccharides 2-4g, galacto-oligosaccharides 2-4g, wheat flour 3-5g, inulin 1-2g, resistant dextrin 1-2g, dietary fiber 2-4g, sialic acid 0.5-1.5g, algal oil DHA 1-2g, calcium chloride 0.5-1g, potassium chloride 0.25-0.75g, sodium chloride 1-2g, potassium dihydrogen phosphate 1-1.5g, sodium sulfate 0.5-1.5g, bile salts 0.2-0.4g, plant extract mixture 0.5-1g, vitamin mixture 4-6mL.

2. The culture medium for colonizing intestinal flora and intestinal gas in chronic gastritis patients according to claim 1, characterized in that The mucin comprises one or a mixture of porcine mucin, bovine mucin and mussel mucin; The mycoprotein comprises shiitake mushroom powder, ganoderma lucidum powder, wood ear powder, boletus powder, bamboo fungus powder, Agaricus blazei powder, Hericium erinaceus powder, Pleurotus eryngii powder, Cordyceps sinensis powder or a mixture thereof.

3. The culture medium for colonizing intestinal flora and intestinal gas in chronic gastritis patients according to claim 1, characterized in that The insect protein includes proteins extracted from eggs, larvae, adults, pupae, and moths; The plant extract mixture comprises one or a mixture of several of puerarin, carotene, bioflavonoids, phytoandrogen, soy isoflavones, limonene, lycopene, catechins and anthocyanidins.

4. The culture medium for colonizing intestinal flora and intestinal gas in chronic gastritis patients according to claim 1, characterized in that The formula of the vitamin mixture is as follows: vitamin B1 0.2g / L, vitamin B2 0.2g / L, niacin 2g / L, folic acid 6g / L, vitamin B 12 0.8g / L, vitamin C 5g / L, vitamin E 2g / L, vitamin D 0.2g / L, vitamin A 0.2g / L.

5. Use of the culture medium for colonizing intestinal flora and intestinal gas in chronic gastritis patients according to any one of claims 1 to 4 in the study of intestinal flora in chronic gastritis patients.

6. A method for in vitro colonization of intestinal flora and intestinal gas in chronic gastritis patients, characterized in that: The following steps are involved: (1) Feces from a patient with chronic gastritis were collected, placed in PBS and stirred evenly, and then the solids were filtered with gauze to obtain a fecal suspension; (2) The fecal suspension obtained in step (1) is injected into an ascending colon reactor, a transverse colon reactor, and a descending colon reactor containing a culture medium for colonizing intestinal flora and intestinal gas in people with chronic gastritis, and cultured under anaerobic conditions for 24 hours to obtain the intestinal flora and intestinal gas of people with chronic gastritis at the ascending colon, transverse colon, and descending colon stages, respectively.

7. The method for in vitro colonization of intestinal flora and intestinal gas in chronic gastritis patients according to claim 6, characterized in that: In step (1), feces from people with chronic gastritis were collected using a feces collector, and each gram of feces was suspended in 5 mL of PBS. The time from feces collection to injection into the reactor was controlled within 10 minutes.

8. The method for in vitro colonization of intestinal flora and intestinal gas in chronic gastritis patients according to claim 6, characterized in that: In step (2), the anaerobic conditions are used for colonization culture, and the culture temperature is 37°C; the simulated peristalsis frequency of each reactor is 1 to 2 times per minute for the ascending colon, 1 to 2 times per minute for the transverse colon, and 2 to 3 times per minute for the descending colon; the stable pH of the ascending colon reactor is 5.75 to 6.25, the stable pH of the transverse colon reactor is 6.35 to 6.65, and the stable pH of the descending colon reactor is 6.75 to 7.

34. The rate of transfer of the contents of the ascending colon into the transverse colon is 1 to 2 ml / min, and the rate of transfer of the contents of the transverse colon into the descending colon is 2 to 3 ml / min. The ratio of the fecal suspension to the culture medium in each reactor is 5 mL:100 mL.

9. The method for in vitro colonization of intestinal flora and intestinal gas in chronic gastritis patients according to claim 6 is characterized in that: In step (2), the pH of each reactor is controlled by 0.5 mol of hydrochloric acid and 0.5 mol of sodium hydroxide.

10. The method for in vitro colonization of intestinal flora and intestinal gas in chronic gastritis patients according to claim 6 is characterized in that: In step (2), fresh culture medium was injected every 8 h.

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