Culture medium for colonization of intestinal flora and intestinal gas of obesity people and application of culture medium

Abstract

The invention discloses a culture medium for colonization of intestinal flora and intestinal gas of obesity people, and belongs to the technical field of biology. The culture medium contains yeast extract, tryptone, mucoprotein, mycoprotein, worm protein, soybean protein, fructo-oligosaccharide, galactooligosaccharide, seed polysaccharide, sorghum, buckwheat, oat, coix seed powder, wheat flour, corn flour, soybean flour, inulin and the like. By means of the bionic colon reactor, the real intestinal environment of obesity people in China is well simulated in vitro by referring to real in-vivo parameters of obesity people, the intestinal flora ecology and intestinal gas distribution are reproduced, and a normal intestinal microorganism and intestinal gas database of obesity people can be constructed. The method is helpful for accurate diagnosis and personalized treatment of intestinal microecology of obesity people, and better evaluates health status of obesity people and formulates preventive measures.

Description

Technical Field

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

[0002] The intestinal microbiota can play an important role in carbohydrate metabolism, neuroendocrine regulation, and immune regulation. Although the intestinal microbiota is a relatively stable state, it is not static. Its composition and structure vary greatly between individuals, and multiple factors affect the diversity of the intestinal microbiota. Factors that cause differences in intestinal microbiota between different groups mainly include age, gender, geographic location, race, drugs, physiological state, lifestyle, diet and genetic background, but their normal microbial community composition has specific characteristics and often has similar microbiome composition. The intestinal microbiota of a healthy human is a complex ecosystem with high stability and a certain resistance and recovery ability to external disturbances. It usually does not cause homeostasis imbalance and lead to the occurrence of disease.

[0003] Moreover, 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 volatile 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. A better understanding of the complex interactions that produce gases in the intestine will improve our ability to prevent, diagnose, treat, and monitor many diseases.

[0004] Normal microorganisms and intestinal gas references for a specific population can serve as an important indicator for assessing the health status of that population. By detecting the composition of an individual's microbial community and intestinal gas distribution and comparing it with the normal reference of the same population, we can understand whether the individual's health is good and whether there are potential health risks. Therefore, establishing normal microorganisms and intestinal gas references for a specific population can help with accurate diagnosis and personalized treatment; better assess health status and formulate prevention strategies; gain a deeper understanding of population differences and guide drug development; assist in judging the effectiveness of clinical interventions and determine the disease susceptibility of a specific population. However, the perfect in vitro colonization of the intestinal microbiome to study its mechanisms and mechanisms has always been an important challenge in microbiome science. In addition, most microbiome colonization strategies are designed based on the research results of Westernized populations, and there are few studies on microbiome colonization strategies for domestic populations, especially intestinal colonization in obese populations.

[0005] The microbial communities of obese people abroad have been observed. A high-calorie, high-fat, and high-sugar diet promotes the growth of harmful bacteria and inhibits the reproduction of beneficial bacteria, which changes the body's immune response and metabolism, resulting in a relatively fragile intestinal flora with poor diversity. In addition, a variety of factors such as genetic factors, drug factors, psychological factors, endocrine factors, and a lifestyle lacking exercise can affect the colonization and succession of intestinal flora. They are mainly composed of members of the genera Faecalibacterium, Dialister, Clostridium, Ruminococcus, and Bacteroides. In addition, there are few reports both at home and abroad on the intestinal gas produced by obese people. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a culture medium for colonizing the intestinal flora and intestinal gas of obese people, so as to solve the problem that there is little research on the intestinal colonization strategy of obese people in the prior art.

[0007] 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 obese people, so as to provide a method that simulates the real intestinal environment of obese people, reproduces the intestinal flora ecology and intestinal gas distribution of obese people, and provides data support for the study of intestinal flora in obese people.

[0008] Technical solution: To solve the above technical problems, the present invention provides the following technical solutions:

[0009] A culture medium for colonizing intestinal flora and intestinal gas in obese people, wherein the contents of each component in each 1L of the culture medium are as follows: 2-4g of yeast extract, 2-3g of tryptone, 0.5-1.5g of mucin, 1-2g of mycoprotein, 0.3-0.5g of insect protein, 0.5-1.5g of soy protein, 1-3g of fructooligosaccharide, 1-3g of galacto-oligosaccharide, 3-7g of seed polysaccharide, 1-3g of sorghum, 1-3g of buckwheat, 1-3g of oats, 1-3g of coix seed flour, 1-3g of wheat flour, and 1-3g of corn flour. The following ingredients are added: 1-3g of sodium chloride, 1-3g of soybean flour, 1-2g of inulin, 1-2g of resistant dextrin, 0.25-0.75g of dietary fiber, 0.5-1.5g of sialic acid, 2-4g of algal oil DHA, 0.25-0.75g of calcium chloride, 0.25-0.75g of potassium chloride, 1-2g of sodium chloride, 1-1.5g of potassium dihydrogen phosphate, 0.5-1.5g of sodium sulfate, 0.2-0.4g of bile salts, 0.4-0.8g of plant extract mixture, and 1-3mL of vitamin mixture. All the above reagents are analytical grade.

[0010] In one embodiment of the present invention, the content of each component in each 1L of culture medium is as follows: 3g of yeast extract, 2.5g of tryptone, 1g of mucin, 1.5g of mycoprotein, 0.4g of insect protein, 1g of soy protein, 2g of oligofructose, 2g of oligogalactose, 5g of seed polysaccharide, 2g of sorghum, 2g of buckwheat, 2g of oats, 2g of coix seed flour, 2g of wheat flour, 2g of corn flour, g, 2g of soybean powder, 1.5g of inulin, 1.5g of resistant dextrin, 0.5g of dietary fiber, 1g of sialic acid, 3g of algal oil DHA, 0.5g of calcium chloride, 0.5g of potassium chloride, 1.5g of sodium chloride, 1.25g of potassium dihydrogen phosphate, 0.75g of sodium sulfate, 0.3g of bile salts, 0.6g of plant extract mixture, and 2mL of vitamin mixture.

[0011] The mucin includes porcine mucin, bovine mucin and mussel mucin, preferably porcine mucin.

[0012] 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 shiitake mushroom powder.

[0013] 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.

[0014] The seed polysaccharides include pumpkin seed polysaccharides, watermelon seed polysaccharides, blackberry seed polysaccharides, chia seed polysaccharides, and grape seed polysaccharides, preferably grape seed polysaccharides.

[0015] The plant extract mixture includes puerarin, carotene, bioflavonoids, phytoandrogens, soy isoflavones, limonene, lycopene, catechins, anthocyanidins, and resveratrol, preferably puerarin, carotene, and anthocyanidins.

[0016] The vitamin mixture contains 0.3g of vitamin B1, 0.3g of vitamin B2, 4g of niacin, 8g of folic acid, 10g of pantothenic acid, and 10g of vitamin B6 per 1L. 12 The amount added is 0.3g.

[0017] The present invention also provides a method for in vitro colonization of intestinal flora and intestinal gas in obese people, the method comprising the following steps:

[0018] (1) 6 g of fresh feces from obese people were collected and placed in 30 mL of PBS, stirred evenly, and then the solid was filtered with gauze to obtain a fecal suspension;

[0019] (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 obese people in the ascending colon, transverse colon, and descending colon stages, respectively;

[0020] In step (1), fresh feces are collected by a feces collector, and then each gram of feces is suspended in 5 mL of PBS. The time from feces collection to injection into the reactor is controlled within 10 minutes.

[0021] 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 3 to 5 times per minute for the ascending colon, 1 to 3 times per minute for the transverse colon, and 2 to 4 times per minute for the descending colon; the stable pH of the ascending colon reactor is 5.7 to 6.3, the stable pH of the transverse colon reactor is 6.3 to 6.6, and the stable pH of the descending colon reactor is 6.7 to 7.3. The rate of transfer of the contents of the ascending colon into the transverse colon is 2 to 4 ml / min, and the rate of transfer of the contents of the transverse colon into the descending colon is 4 to 6 ml / min. The ratio of the fecal suspension to the culture medium in each reactor is 5 mL:100 mL.

[0022] 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.

[0023] In step (2), if it is necessary to maintain a steady state of intestinal flora in obese people for a long time, fresh 10% culture medium can be injected every 8 hours for stable growth.

[0024] 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.

[0025] Beneficial effects:

[0026] This biomimetic colon bioreactor, based on gastrointestinal physiology, biotechnology, and fermentation engineering, simulates the actual intestinal environment of obese individuals in China in vitro by referencing real-world parameters found in obese individuals. It also recreates their intestinal flora ecology and intestinal gas distribution. This method utilizes intestinal flora culture media from obese individuals in combination with the biomimetic colon bioreactor to establish a strategy for colonizing intestinal microorganisms and intestinal gas in obese individuals. This method can construct a database of normal intestinal microorganisms and intestinal gas in obese individuals, facilitating accurate diagnosis and personalized treatment of intestinal microbiota in obese individuals, and enabling better assessment of their health status and development of preventive measures. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Comparison of species richness of intestinal flora and intestinal gas colonization culture medium in obese subjects.

[0028] Figure 2 : Comparison of gas composition after culture in normal culture medium and culture medium colonized with intestinal flora and intestinal gas in obese people. DETAILED DESCRIPTION

[0029] The culture medium components involved in the following examples are described as follows:

[0030] The yeast extract, tryptone, mucin, mycoprotein, insect protein, soy protein, fructooligosaccharide, galacto-oligosaccharide, seed polysaccharide, sorghum, buckwheat, oat, coix seed flour, wheat flour, corn flour, soybean flour, inulin, resistant dextrin, dietary fiber, sialic acid, algal oil DHA, calcium chloride, potassium chloride, sodium chloride, potassium dihydrogen phosphate, sodium sulfate, bile salt, plant extract mixture, and vitamin mixture are all of analytical grade concentration.

[0031] The mucin includes porcine mucin, bovine mucin, and mussel mucin, preferably porcine mucin.

[0032] 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 shiitake mushroom powder.

[0033] 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.

[0034] The seed polysaccharide comprises pumpkin seed polysaccharide, watermelon seed polysaccharide, blackberry seed polysaccharide, chia seed polysaccharide, and grape seed polysaccharide, preferably grape seed polysaccharide.

[0035] The plant extract mixture comprises puerarin, carotene, bioflavonoids, phytoandrogen, soy isoflavones, limonene, lycopene, catechin, anthocyanidin, and resveratrol, preferably puerarin, carotene, and anthocyanidin.

[0036] The vitamin mixture contains 0.3 g of vitamin B1, 0.3 g of vitamin B2, 4 g of niacin, 8 g of folic acid, 10 g of pantothenic acid, and 0.3 g of vitamin B12 per 1 L.

[0037] When the culture medium is a solid culture medium, the added amount of agar powder is 1-1.5%.

[0038] Example 1: In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas in obese people

[0039] The specific steps are as follows:

[0040] (1) Preparation of fecal suspension

[0041] Fresh stool samples were collected from five obese individuals. The obese individuals had not 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.

[0042] (2) Preparation of culture medium

[0043] Prepare the culture medium according to the formula in Table 1:

[0044] Table 1 In vitro colonization culture medium of intestinal flora and intestinal gas in obese people

[0045]

[0046]

[0047] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0048] 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 then dynamically incubated at 37°C under anaerobic conditions for 24 hours. The simulated peristaltic frequency in each reactor was 3-5 beats per minute in the ascending colon, 1-3 beats per minute in the transverse colon, and 2-4 beats per minute in the descending colon. The stable pH in the ascending colon reactor was 5.7-6.3, the stable pH in the transverse colon reactor was 6.3-6.6, and the stable pH in the descending colon reactor was 6.7-7.3. The transfer rate of contents from the ascending colon into the transverse colon was 2-4 mL / min, and the transfer rate of contents from the transverse colon into the descending colon was 4-6 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 microbiota from obese individuals that maintain a steady state for a long time, fresh 10% culture medium can be added every 8 hours to maintain stable growth.

[0049] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0050] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0051] The results showed that: in terms of intestinal flora structure, the culture medium of the present invention can significantly increase the colonization abundance of Faecalibacterium, Dialister, Clostridium, Ruminococcus, and Bacteroides compared to 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 obese people. 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 obese people. Specific parameters such as Figure 1 , as shown in Table 10 and Table 11.

[0052] Example 2: In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas in obese people

[0053] The specific steps are as follows:

[0054] (1) Preparation of fecal suspension

[0055] Fresh stool samples were collected from five obese individuals. The obese individuals had not 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.

[0056] (2) Preparation of culture medium

[0057] Prepare the culture medium according to the formula in Table 2:

[0058] Table 2 In vitro colonization culture medium of intestinal flora and intestinal gas in obese people

[0059]

[0060]

[0061] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0062] 200 mL of culture medium was added to the ascending colon reactor, the transverse colon reactor, and the descending colon reactor, each of which was injected with 10 mL of fecal suspension. The cells were then dynamically cultured at 37°C under anaerobic conditions for 24 hours. The simulated peristaltic frequency in each reactor was 3-5 beats per minute for the ascending colon, 1-3 beats per minute for the transverse colon, and 2-4 beats per minute for the descending colon. The stable pH of the ascending colon reactor was 5.7-6.3, the stable pH of the transverse colon reactor was 6.3-6.6, and the stable pH of the descending colon reactor was 6.7-7.3. The transfer rate of contents from the ascending colon to the transverse colon was 2-4 ml / min, and the transfer rate of contents from the transverse colon to the descending colon was 4-6 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 obese individuals that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0063] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0064] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0065] The results showed that: in terms of intestinal flora structure, the culture medium of the present invention can significantly increase the colonization abundance of Faecalibacterium, Dialister, Clostridium, Ruminococcus, and Bacteroides compared to 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 obese people. 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 obese people. Specific parameters such as Figure 1 , as shown in Table 10 and Table 11.

[0066] Example 3: In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas in obese people

[0067] The specific steps are as follows:

[0068] (1) Preparation of fecal suspension

[0069] Fresh stool samples were collected from five obese individuals. The obese individuals had not taken antibiotics or other medications that could disrupt the intestinal microbiome for 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.

[0070] (2) Preparation of culture medium

[0071] Prepare the culture medium according to the formula in Table 3:

[0072] Table 3 In vitro colonization culture medium of intestinal flora and intestinal gas in obese people

[0073]

[0074]

[0075] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0076] 200 mL of culture medium was added to the ascending colon reactor, the transverse colon reactor, and the descending colon reactor, and 10 mL of fecal suspension was injected into each reactor. The cells were dynamically cultured under anaerobic conditions at 37°C for 24 hours. The simulated peristalsis frequency in each reactor was 3-5 times per minute for the ascending colon, 1-3 times per minute for the transverse colon, and 2-4 times per minute for the descending colon. The stable pH in the ascending colon reactor was 5.7-6.3, the stable pH in the transverse colon reactor was 6.3-6.6, and the stable pH in the descending colon reactor was 6.7-7.3. The rate of transfer of contents from the ascending colon to the transverse colon was 2-4 ml / min, and the rate of transfer of contents from the transverse colon to the descending colon was 4-6 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 obese individuals that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0077] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0078] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0079] The results showed that: in terms of intestinal flora structure, the culture medium of the present invention can significantly increase the colonization abundance of Faecalibacterium, Dialister, Clostridium, Ruminococcus, and Bacteroides compared to 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 obese people. 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 obese people. Specific parameters such as Figure 1 , as shown in Table 10 and Table 11.

[0080] Comparative Example 1: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in obese people (the amount of culture medium components added is lower than the minimum value)

[0081] The specific steps are as follows:

[0082] (1) Preparation of fecal suspension

[0083] Fresh stool samples were collected from five obese individuals. The obese individuals had not 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.

[0084] (2) Preparation of culture medium

[0085] Prepare the culture medium according to the formula in Table 4:

[0086] Table 4 In vitro colonization culture medium of intestinal flora and intestinal gas in obese people

[0087]

[0088] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0089] 200 mL of culture medium was added to the ascending colon reactor, the transverse colon reactor, and the descending colon reactor, and 10 mL of fecal suspension was injected into each reactor. The cells were then dynamically cultured at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in each reactor was 3-5 beats per minute in the ascending colon, 1-3 beats per minute in the transverse colon, and 2-4 beats per minute in the descending colon. The stable pH in the ascending colon reactor was 5.7-6.3, the stable pH in the transverse colon reactor was 6.3-6.6, and the stable pH in the descending colon reactor was 6.7-7.3. The rate of transfer of contents from the ascending colon to the transverse colon was 2-4 ml / min, and the rate of transfer of contents from the transverse colon to the descending colon was 4-6 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 obese individuals that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0090] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0091] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0092] The results showed that in terms of intestinal flora structure, compared with the intestinal environment of real obese people, the colonization abundance of Faecalibacterium, Dialister, Clostridium, Ruminococcus, and Bacteroides in the fermentation broth was reduced. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide, and volatile gases (VOCs) were the main components, but the gas production was different from the real intestinal gas of obese people. Specific parameters such as Figure 1 , as shown in Table 10 and Table 11.

[0093] Comparative Example 2: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in obese people (the amount of culture medium components added is higher than the maximum value)

[0094] The specific steps are as follows:

[0095] (1) Preparation of fecal suspension

[0096] Fresh stool samples were collected from five obese individuals. The obese individuals had not taken antibiotics or other medications that could disrupt the intestinal microbiome for 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.

[0097] (2) Preparation of culture medium

[0098] The culture medium was prepared according to the formula in Table 5:

[0099] Table 5 In vitro colonization culture medium of intestinal flora and intestinal gas in obese people

[0100]

[0101]

[0102] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0103] 200 mL of culture medium was added to the ascending colon reactor, the transverse colon reactor, and the descending colon reactor, and 10 mL of fecal suspension was injected into each reactor. The cells were then dynamically cultured at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in each reactor was 3-5 beats per minute in the ascending colon, 1-3 beats per minute in the transverse colon, and 2-4 beats per minute in the descending colon. The stable pH in the ascending colon reactor was 5.7-6.3, the stable pH in the transverse colon reactor was 6.3-6.6, and the stable pH in the descending colon reactor was 6.7-7.3. The rate of transfer of contents from the ascending colon to the transverse colon was 2-4 ml / min, and the rate of transfer of contents from the transverse colon to the descending colon was 4-6 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 obese individuals that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0104] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0105] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0106] The results showed that in terms of intestinal flora structure, compared with the intestinal environment of real obese people, the colonization abundance of Faecalibacterium, Dialister, Clostridium, Ruminococcus, and Bacteroides 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 10 and Table 11.

[0107] Comparative Example 3: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in obese people (without adding porcine mucin)

[0108] The specific steps are as follows:

[0109] (1) Preparation of fecal suspension

[0110] Fresh stool samples were collected from five obese individuals. The obese individuals had not taken antibiotics or other medications that could disrupt the intestinal microbiome for 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.

[0111] (2) Preparation of culture medium

[0112] The culture medium was prepared according to the formula in Table 6:

[0113] Table 6 In vitro colonization culture medium of intestinal flora and intestinal gas in obese people

[0114]

[0115]

[0116] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0117] 200 mL of culture medium was added to the ascending colon reactor, the transverse colon reactor, and the descending colon reactor, and 10 mL of fecal suspension was injected into each reactor. The cells were then dynamically cultured at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in each reactor was 3-5 beats per minute in the ascending colon, 1-3 beats per minute in the transverse colon, and 2-4 beats per minute in the descending colon. The stable pH in the ascending colon reactor was 5.7-6.3, the stable pH in the transverse colon reactor was 6.3-6.6, and the stable pH in the descending colon reactor was 6.7-7.3. The rate of transfer of contents from the ascending colon to the transverse colon was 2-4 ml / min, and the rate of transfer of contents from the transverse colon to the descending colon was 4-6 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 obese individuals that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0118] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0119] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0120] The results showed that in terms of intestinal flora structure, the abundance of Faecalibacterium, Dialister, and Clostridium in the fermentation broth was significantly reduced compared with the intestinal environment of real obese people. 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 obese people. Specific parameters such as Figure 1 , as shown in Table 10 and Table 11.

[0121] Comparative Example 4: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in obese people (without sodium sulfate addition)

[0122] The specific steps are as follows:

[0123] (1) Preparation of fecal suspension

[0124] Fresh stool samples were collected from five obese individuals. The obese individuals had not taken antibiotics or other medications that could disrupt the intestinal microbiome for 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.

[0125] (2) Preparation of culture medium

[0126] The culture medium was prepared according to the formula in Table 7:

[0127] Table 7 In vitro colonization culture medium of intestinal flora and intestinal gas in obese people

[0128]

[0129] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0130] 200 mL of culture medium was added to the ascending colon reactor, the transverse colon reactor, and the descending colon reactor, and 10 mL of fecal suspension was injected into each reactor. The cells were then dynamically cultured at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in each reactor was 3-5 beats per minute in the ascending colon, 1-3 beats per minute in the transverse colon, and 2-4 beats per minute in the descending colon. The stable pH in the ascending colon reactor was 5.7-6.3, the stable pH in the transverse colon reactor was 6.3-6.6, and the stable pH in the descending colon reactor was 6.7-7.3. The rate of transfer of contents from the ascending colon to the transverse colon was 2-4 ml / min, and the rate of transfer of contents from the transverse colon to the descending colon was 4-6 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 obese individuals that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0131] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0132] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0133] The results showed that in terms of intestinal flora structure, compared with the intestinal environment of real obese people, the colonization abundance of Faecalibacterium, Dialister, Clostridium, Ruminococcus, and Bacteroides 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 10 and Table 11.

[0134] Comparative Example 5: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in obese people (without adding grape seed polysaccharide)

[0135] The specific steps are as follows:

[0136] (1) Preparation of fecal suspension

[0137] Fresh stool samples were collected from five obese individuals. The obese individuals had not taken antibiotics or other medications that could disrupt the intestinal microbiome for 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.

[0138] (2) Preparation of culture medium

[0139] Prepare the culture medium according to the formula in Table 8:

[0140] Table 8 In vitro colonization culture medium of intestinal flora and intestinal gas in obese people

[0141]

[0142]

[0143] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0144] 200 mL of culture medium was added to the ascending colon reactor, the transverse colon reactor, and the descending colon reactor, and 10 mL of fecal suspension was injected into each reactor. The cells were then dynamically cultured at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in each reactor was 3-5 beats per minute in the ascending colon, 1-3 beats per minute in the transverse colon, and 2-4 beats per minute in the descending colon. The stable pH in the ascending colon reactor was 5.7-6.3, the stable pH in the transverse colon reactor was 6.3-6.6, and the stable pH in the descending colon reactor was 6.7-7.3. The rate of transfer of contents from the ascending colon to the transverse colon was 2-4 ml / min, and the rate of transfer of contents from the transverse colon to the descending colon was 4-6 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 obese individuals that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0145] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0146] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0147] The results showed that in terms of intestinal flora structure, the abundance of Faecalibacterium, Dialister, and Clostridium in the fermentation broth was significantly reduced compared with the intestinal environment of real obese people. 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 obese people. Specific parameters such as Figure 1 , as shown in Table 10 and Table 11.

[0148] Comparative Example 6: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in obese people (without addition of sorghum, buckwheat, and oats)

[0149] The specific steps are as follows:

[0150] (1) Preparation of fecal suspension

[0151] Fresh stool samples were collected from five obese individuals. The obese individuals had not taken antibiotics or other medications that could disrupt the intestinal microbiome for 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.

[0152] (2) Preparation of culture medium

[0153] The culture medium was prepared according to the formula in Table 9:

[0154] Table 9 In vitro colonization culture medium of intestinal flora and intestinal gas in obese people

[0155]

[0156]

[0157] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0158] 200 mL of culture medium was added to the ascending colon reactor, the transverse colon reactor, and the descending colon reactor, and 10 mL of fecal suspension was injected into each reactor. The cells were then dynamically cultured at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in each reactor was 3-5 beats per minute in the ascending colon, 1-3 beats per minute in the transverse colon, and 2-4 beats per minute in the descending colon. The stable pH in the ascending colon reactor was 5.7-6.3, the stable pH in the transverse colon reactor was 6.3-6.6, and the stable pH in the descending colon reactor was 6.7-7.3. The rate of transfer of contents from the ascending colon to the transverse colon was 2-4 ml / min, and the rate of transfer of contents from the transverse colon to the descending colon was 4-6 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 obese individuals that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0159] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0160] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0161] The results showed that in terms of intestinal flora structure, the abundance of Faecalibacterium, Dialister, and Clostridium in the fermentation broth was significantly reduced compared with the intestinal environment of real obese people. 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 obese people. Specific parameters such as Figure 1 , as shown in Table 10 and Table 11.

[0162] Table 10 Comparison of species richness of intestinal flora and intestinal gas cultured in normal culture medium and in obese people

[0163]

[0164]

[0165] Table 11 Comparison of gas composition after culture of intestinal flora and intestinal gas culture medium in normal culture medium and obese people

[0166]

[0167] 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 obese people, characterized in that: The content of each component in 1L of culture medium is as follows: Yeast extract 2-4g, tryptone 2-3g, mucin 0.5-1.5g, mycoprotein 1-2g, insect protein 0.3-0.5g, soy protein 0.5-1.5g, oligofructose 1-3g, oligogalactose 1-3g, seed polysaccharide 3-7g, sorghum 1-3g, buckwheat 1-3g, oats 1-3g, coix seed flour 1-3g, wheat flour 1-3g, corn flour 1-3g, soybean flour 1-3g, inulin 1-2g, resistant dextrin 1-2g, dietary fiber 0.25-0.75g, sialic acid 0.5-1.5g, algal oil DHA 2-4g, calcium chloride 0.25-0.75g, 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 0.4-0.8g, vitamin mixture 1-3mL.

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

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

4. The culture medium for colonizing intestinal flora and intestinal gas in obese people according to claim 1 is characterized in that The seed polysaccharide includes one of pumpkin seed polysaccharide, watermelon seed polysaccharide, blackberry seed polysaccharide, chia seed polysaccharide, grape seed polysaccharide or a mixture thereof or a mixture of several thereof.

5. The culture medium for colonizing intestinal flora and intestinal gas in obese people according to claim 1, characterized in that The formula of the vitamin mixture is as follows: vitamin B1 0.3g / L, vitamin B2 0.3g / L, niacin 4g / L, folic acid 8g / L, pantothenic acid 10g / L, vitamin B 12 0.3g / L.

6. A method for in vitro colonization of intestinal flora and intestinal gas in obese people, characterized in that: The following steps are involved: (1) Obese human feces 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 obese people, and cultured under anaerobic conditions for 24 hours to obtain the intestinal flora and intestinal gas of obese people in the ascending colon, transverse colon, and descending colon stages, respectively.

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

8. The method for in vitro colonization of intestinal flora and intestinal gas in obese people 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 3 to 5 times per minute for the ascending colon, 1 to 3 times per minute for the transverse colon, and 2 to 4 times per minute for the descending colon; the stable pH of the ascending colon reactor is 5.7 to 6.3, the stable pH of the transverse colon reactor is 6.3 to 6.6, and the stable pH of the descending colon reactor is 6.7 to 7.3; the rate of transfer of the contents of the ascending colon into the transverse colon is 2 to 4 ml / min, and the rate of transfer of the contents of the transverse colon into the descending colon is 4 to 6 ml / min; and 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 obese people 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 obese people according to claim 6 is characterized in that: In step (2), fresh 10% culture medium was injected every 8 h.

Images