Culture medium for colonization of intestinal flora and intestinal gas of middle-aged and elderly people and application of culture medium

Abstract

The invention discloses a culture medium for colonization of intestinal flora and intestinal gas of middle-aged and elderly people. The culture medium contains yeast extract, tryptone, mucoprotein, mycoprotein, worm protein, soybean protein, fructo-oligosaccharide, galactooligosaccharide, inulin, resistant dextrin, dietary fiber, sorghum, buckwheat, oat, coix seed, corn germ oil, olive oil, linseed oil, walnut oil, sialic acid, algal oil DHA, potassium chloride, sodium chloride, monopotassium phosphate, sodium sulfate and bile salt. A plant extract mixture, a vitamin mixed solution and the like. By means of the bionic colon reactor, the real intestinal environment of the middle-aged and elderly people in China is well simulated in vitro by referring to real in-vivo parameters of the middle-aged and elderly people, the intestinal flora ecology and intestinal gas distribution of the middle-aged and elderly people are reproduced, and a database of normal intestinal microorganisms and intestinal gas of the middle-aged and elderly people can be constructed; accurate diagnosis and personalized treatment of intestinal microecology of the middle-aged and elderly people are facilitated, and the health state of the middle-aged and elderly people can be better evaluated and preventive measures can be better formulated.

Description

Technical Field

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

[0002] The gut microbiome constitutes a crucial "microbial organ" that profoundly influences human metabolism, immune regulation, and nervous system function. The uniqueness of an individual's gut microbiome stems from a variety of factors, including but not limited to age, sex, geographic location, ethnic background, medication history, physiological state, lifestyle choices, and genetics. Despite these differences, healthy gut microbiota often exhibit certain structural commonalities, forming a relatively stable microbiome pattern.

[0003] In a healthy state, the intestinal microbiome maintains a delicate balance with the host (human body) and its environment, the so-called "homeostasis", which is crucial for maintaining the overall health of the body. The intestine is a complex system that not only contains a variety of microbial populations, but also involves a variety of gases produced by these microorganisms through metabolic activities, such as carbon dioxide, hydrogen, methane, hydrogen sulfide, and various trace gases. The production of these gases is a direct result of the interaction between biochemical processes in the intestine and the microbial community. Precise analysis of intestinal gas composition and its dynamic changes with dietary intervention provides us with a way to deeply understand the functional characteristics of the intestinal microbiome, its metabolites, and its potential impact on host health. Deepening the understanding of the complex mechanisms of gas production in the intestine is expected to provide a scientific basis for improving prevention strategies, diagnostic accuracy, treatment effects, and long-term monitoring capabilities for a variety of diseases.

[0004] For a specific population, the normal microbiome composition and intestinal gas profiles can serve as important benchmarks for measuring health status. By comparing an individual's microbiome composition and intestinal gas profile with standard reference data for that population, insights can be gained into whether the individual's health status meets standards and whether potential health risks exist. Therefore, establishing a standardized reference framework for microbiota and intestinal gas profiles for specific populations is crucial for achieving precision medicine and personalized treatment strategies. It facilitates more accurate assessments of health status and the development of effective prevention programs; it promotes understanding of interpopulational differences, guiding drug development; and it aids in evaluating the effectiveness of clinical interventions and determining 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. Furthermore, most microbiome colonization strategies are designed based on findings from studies in Westernized populations, while studies on microbiome colonization strategies for Chinese populations, particularly those targeting the middle-aged and elderly, are limited. The intestinal microbiota of middle-aged and elderly people undergoes significant changes with aging, and these changes may have important health implications. The intestinal microbiota of middle-aged and elderly people exhibits reduced diversity, fewer probiotics, an increase in opportunistic pathogens, and altered metabolic function. These characteristics of the intestinal microbiota reflect age-related physiological changes. These microbiota are primarily composed of members of the genera Bacteroides, Prevotella, Clostridium, Ruminococcus, Faecalibacterium, Bifidobacterium, Escherichia, Klebsiella, and Akkermansia. Furthermore, there are few reports domestically and internationally on intestinal gas production in middle-aged and elderly people. Summary of the Invention

[0005] Purpose of the invention: The present invention provides a culture medium for colonizing the intestinal flora and intestinal gas of middle-aged and elderly people, providing a basis for in vitro colonization of the intestinal flora and intestinal gas of middle-aged and elderly people.

[0006] Technical solution: The present invention provides a culture medium for colonizing intestinal flora and intestinal gas in middle-aged and elderly people. The content of each component in each 1L of the culture medium is as follows: 2-3g of yeast extract, 2-3g of tryptone, 1.5-2g of mucin, 1-2g of mycoprotein, 0.2-0.3g of insect protein, 1-1.5g of soy protein, 3-7g of oligofructose, 1-2.5g of oligogalactose, 0.5-2g of inulin, 1.5-2g of resistant dextrin, 1.5-2g of dietary fiber, 1-2g of sorghum, 1-2g of buckwheat, and 1-2g of oats. , the added amount of coix seed is 1-2g, the added amount of corn germ oil is 0.5-1.5g, the added amount of olive oil is 0.5-1.5g, the added amount of linseed oil is 0.5-1.5g, the added amount of walnut oil is 0.5-1.5g, the added amount of sialic acid is 0.5-1g, the added amount of algal oil DHA is 1-1.5g, the added amount of potassium chloride is 1-1.5g, the added amount of sodium chloride is 1-1.5g, the added amount of potassium dihydrogen phosphate is 1.5-2.5g, the added amount of sodium sulfate is 0.5-2g, the added amount of bile salt is 0.4-0.6g, the added amount of plant extract mixture is 0.5-1g, and the added amount of vitamin mixture is 0.5-1.5mL. All of the above reagents are analytical grade concentrations.

[0007] Preferably, the content of each component in each 1L of culture medium is as follows: 2.5g yeast extract, 2.5g tryptone, 1.75g mucin, 1.5g mycoprotein, 0.25g insect protein, 1.25g soy protein, 5g fructooligosaccharide, 1.75g galacto-oligosaccharide, 1.25g inulin, 1.75g resistant dextrin, 1.75g dietary fiber, 1.5g sorghum, 1.5g buckwheat, 1.5g oatmeal, 1.5g gluten ...2 The added amount is 1.5g, the added amount of coix seed is 1.5g, the added amount of corn germ oil is 1g, the added amount of olive oil is 1g, the added amount of linseed oil is 1g, the added amount of walnut oil is 1g, the added amount of sialic acid is 0.75g, the added amount of algal oil DHA is 1.25g, the added amount of potassium chloride is 1.25g, the added amount of sodium chloride is 1.25g, the added amount of potassium dihydrogen phosphate is 2g, the added amount of sodium sulfate is 1.25g, the added amount of bile salt is 0.5g, the added amount of plant extract mixture is 0.75g, and the added amount of vitamin mixture is 1mL.

[0008] Furthermore, the mucin comprises one or a mixture of porcine mucin, bovine mucin, and mussel mucin, preferably porcine mucin.

[0009] Furthermore, 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, and Cordyceps sinensis powder.

[0010] Furthermore, the insect protein includes proteins extracted from eggs, larvae, adults, pupae, moths, etc., such as bean worm powder and silkworm pupae.

[0011] Furthermore, the plant extract mixture includes puerarin, carotene, bioflavonoids, phytoandrogen, soy isoflavones, limonene, lycopene, catechins, anthocyanidins, resveratrol, etc.

[0012] Preferably, per 1 L of the vitamin mixture, the added amount of vitamin B1 is 0.4 g, the added amount of vitamin B2 is 0.4 g, the added amount of niacin is 4 g, the added amount of folic acid is 8 g, and the added amount of pantothenic acid is 12 g.

[0013] The present invention also provides a method for in vitro colonization of intestinal flora and intestinal gas in middle-aged and elderly people, the method comprising the following steps:

[0014] (1) Collect 6 g of fresh feces from middle-aged or elderly individuals, add them to 30 mL of PBS, and stir evenly. Then, filter the solids with gauze to obtain a fecal suspension.

[0015] (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 the middle-aged and elderly in the ascending colon, transverse colon and descending colon stages respectively;

[0016] 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.

[0017] In step (2), the dynamic culture is carried out at 37°C under anaerobic conditions for 24 hours, the simulated peristalsis frequency of the ascending colon reactor is 4-6 times per minute, the simulated peristalsis frequency of the transverse colon reactor is 2-3 times per minute, and the simulated peristalsis frequency of the descending colon reactor is 5-7 times per minute; the rate at which the contents of the ascending colon are transferred into the transverse colon is 2-4 ml / min, and the rate at which the contents of the transverse colon are transferred into the descending colon is 4-6 ml / min; the stable pH of the ascending colon reactor is 5.5-6.5, the stable pH of the transverse colon reactor is 6.0-7.0, and the stable pH of the descending colon reactor is 6.5-7.5, and the amount of fecal suspension added to each reactor is 5%.

[0018] 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.

[0019] In step (2), if it is necessary to maintain a steady state of intestinal flora in the elderly for a long time, fresh 10% culture medium can be injected every 8 hours for stable growth.

[0020] In one embodiment of the present invention, the gas in the reactor headspace is composed of 100% nitrogen to ensure an anaerobic environment. As the culture time increases, colonization samples and intestinal gas are regularly taken out of the reactor for measurement.

[0021] Beneficial effects:

[0022] This biomimetic colon bioreactor, based on gastrointestinal physiology, biotechnology, and fermentation engineering, simulates the actual intestinal environment of middle-aged and elderly individuals in China in vitro by referencing real-world in vivo parameters. It also recreates their intestinal flora ecology and intestinal gas distribution. This method utilizes a culture medium containing intestinal flora from middle-aged and elderly individuals combined with a biomimetic colon bioreactor to establish a colonization strategy for their intestinal microorganisms and intestinal gas. This method can construct a database of normal intestinal microorganisms and intestinal gas in middle-aged and elderly individuals, facilitating accurate diagnosis and personalized treatment of their intestinal microecology, better assessing their health status, and developing preventive measures. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Comparison of species richness of intestinal flora and intestinal gas colonization culture medium in middle-aged and elderly people.

[0024] Figure 2 : Comparison of gas composition after culture with ordinary culture medium and culture medium for colonization of intestinal flora and intestinal gas in middle-aged and elderly people. DETAILED DESCRIPTION

[0025] The culture medium components involved in the following examples are described as follows:

[0026] The yeast extract, tryptone, mucin, mycoprotein, insect protein, soy protein, fructooligosaccharide, galacto-oligosaccharide, inulin, resistant dextrin, dietary fiber, sorghum, buckwheat, oats, coix seed, corn germ oil, olive oil, linseed oil, walnut oil, sialic acid, algae oil DHA, potassium chloride, sodium chloride, potassium dihydrogen phosphate, sodium sulfate, bile salts, plant extract mixture, and vitamin mixture are all of analytical grade concentration.

[0027] The mucin includes porcine mucin, bovine mucin, mussel mucin and the like.

[0028] 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 and the like.

[0029] The insect protein includes proteins extracted from eggs, larvae, adults, pupae, moths, etc., such as bean worm powder, silkworm pupae, etc.

[0030] The plant extract mixture includes puerarin, carotene, bioflavonoids, phytoandrogen, soy isoflavones, limonene, lycopene, catechins, anthocyanidins, resveratrol, etc.

[0031] The vitamin mixture contains 0.4 g of vitamin B1, 0.4 g of vitamin B2, 4 g of niacin, 8 g of folic acid, and 12 g of pantothenic acid per 1 L. When the culture medium is a solid culture medium, the amount of agar powder added is 1.1-1.5%.

[0032] Example 1: In vitro spatiotemporal dynamic colonization and cultivation of intestinal flora and intestinal gas in middle-aged and elderly people

[0033] The specific steps are as follows:

[0034] (1) Preparation of fecal suspension

[0035] Fresh stool samples were collected from five middle-aged and elderly individuals aged 50 years. The subjects 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.

[0036] (2) Preparation of culture medium

[0037] Prepare the culture medium according to the formula in Table 1:

[0038] Table 1 In vitro colonization culture medium of intestinal flora and intestinal gas in middle-aged and elderly people

[0039]

[0040]

[0041] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0042] 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 incubated at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in the ascending colon reactor was 4-6 times per minute, the simulated peristalsis frequency in the transverse colon reactor was 2-3 times per minute, and the simulated peristalsis frequency in the descending colon reactor was 5-7 times per minute. 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 the ascending colon reactor was 5.5-6.5, the stable pH of the transverse colon reactor was 6.0-7.0, and the stable pH of the descending colon reactor was 6.5-7.5. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For middle-aged and elderly intestinal flora that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0043] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0044] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0045] The results showed that: in terms of intestinal flora structure, the culture medium of the present invention can significantly increase the colonization abundance of Bacteroides, Prevotella, Clostridium, Ruminococcus, Faecalibacterium, Bifidobacterium, Escherichia, Klebsiella, and Akkermansia compared to the traditional culture medium (main ingredients: calf brain extract powder, beef heart extract powder, peptone, glucose, sodium chloride, disodium hydrogen phosphate, agar), which is similar to the real intestinal environment of middle-aged and elderly 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 middle-aged and elderly people. Specific parameters such as Figure 1 , as shown in Tables 9 and 10.

[0046] Example 2: In vitro spatiotemporal dynamic colonization and cultivation of intestinal flora and intestinal gas in middle-aged and elderly people

[0047] The specific steps are as follows:

[0048] (1) Preparation of fecal suspension

[0049] Fresh stool samples were collected from five middle-aged and elderly individuals aged 50 years. The subjects 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.

[0050] (2) Preparation of culture medium

[0051] Prepare the culture medium according to the formula in Table 2:

[0052] Table 2 In vitro colonization culture medium of intestinal flora and intestinal gas in middle-aged and elderly people

[0053]

[0054]

[0055] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0056] 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 incubated at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in the ascending colon reactor was 4-6 times per minute, the simulated peristalsis frequency in the transverse colon reactor was 2-3 times per minute, and the simulated peristalsis frequency in the descending colon reactor was 5-7 times per minute. 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 the ascending colon reactor was 5.5-6.5, the stable pH of the transverse colon reactor was 6.0-7.0, and the stable pH of the descending colon reactor was 6.5-7.5. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For middle-aged and elderly intestinal flora that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0057] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0058] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0059] The results showed that: in terms of intestinal flora structure, the culture medium of the present invention can significantly increase the colonization abundance of Bacteroides, Prevotella, Clostridium, Ruminococcus, Faecalibacterium, Bifidobacterium, Escherichia, Klebsiella, and Akkermansia compared to the traditional culture medium (main ingredients: calf brain extract powder, beef heart extract powder, peptone, glucose, sodium chloride, disodium hydrogen phosphate, agar), which is similar to the real intestinal environment of middle-aged and elderly 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 middle-aged and elderly people. Specific parameters such as Figure 1 , as shown in Tables 9 and 10.

[0060] Example 3: In vitro spatiotemporal dynamic colonization and cultivation of intestinal flora and intestinal gas in middle-aged and elderly people

[0061] The specific steps are as follows:

[0062] (1) Preparation of fecal suspension

[0063] Fresh stool samples were collected from five middle-aged and elderly individuals aged 50 years. The subjects 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.

[0064] (2) Preparation of culture medium

[0065] Prepare the culture medium according to the formula in Table 3:

[0066] Table 3 In vitro colonization culture medium of intestinal flora and intestinal gas in middle-aged and elderly people

[0067]

[0068] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0069] 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 incubated at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in the ascending colon reactor was 4-6 times per minute, the simulated peristalsis frequency in the transverse colon reactor was 2-3 times per minute, and the simulated peristalsis frequency in the descending colon reactor was 5-7 times per minute. 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 the ascending colon reactor was 5.5-6.5, the stable pH of the transverse colon reactor was 6.0-7.0, and the stable pH of the descending colon reactor was 6.5-7.5. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For middle-aged and elderly intestinal flora that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0070] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0071] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0072] The results showed that: in terms of intestinal flora structure, the culture medium of the present invention can significantly increase the colonization abundance of Bacteroides, Prevotella, Clostridium, Ruminococcus, Faecalibacterium, Bifidobacterium, Escherichia, Klebsiella, and Akkermansia compared to the traditional culture medium (main ingredients: calf brain extract powder, beef heart extract powder, peptone, glucose, sodium chloride, disodium hydrogen phosphate, agar), which is similar to the real intestinal environment of middle-aged and elderly 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 middle-aged and elderly people. Specific parameters such as Figure 1 , as shown in Tables 9 and 10.

[0073] Comparative Example 1: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in middle-aged and elderly people (the amount of culture medium components added is lower than the minimum value)

[0074] The specific steps are as follows:

[0075] (1) Preparation of fecal suspension

[0076] Fresh stool samples were collected from five middle-aged and elderly individuals aged 50 years. The subjects 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.

[0077] (2) Preparation of culture medium

[0078] Prepare the culture medium according to the formula in Table 4:

[0079] Table 4 In vitro colonization culture medium of intestinal flora and intestinal gas in middle-aged and elderly people

[0080]

[0081] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0082] 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 incubated at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in the ascending colon reactor was 4-6 times per minute, the simulated peristalsis frequency in the transverse colon reactor was 2-3 times per minute, and the simulated peristalsis frequency in the descending colon reactor was 5-7 times per minute. 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 the ascending colon reactor was 5.5-6.5, the stable pH of the transverse colon reactor was 6.0-7.0, and the stable pH of the descending colon reactor was 6.5-7.5. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For middle-aged and elderly intestinal flora that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0083] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0084] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0085] The results showed that in terms of intestinal flora structure, compared with the real intestinal environment of middle-aged and elderly people, the colonization abundance of Bacteroides, Prevotella, Clostridium, Ruminococcus, Faecalibacterium, Bifidobacterium, Escherichia, Klebsiella, and Akkermansia in the fermentation broth was significantly reduced. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide and volatile gases (VOC) are the main gases, but the gas production is different from the real intestinal gas of middle-aged and elderly people. Specific parameters such as Figure 1 , as shown in Tables 9 and 10.

[0086] Comparative Example 2: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in middle-aged and elderly people (the amount of culture medium components added is higher than the maximum value)

[0087] The specific steps are as follows:

[0088] (1) Preparation of fecal suspension

[0089] Fresh stool samples were collected from five middle-aged and elderly individuals aged 50 years. The subjects 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.

[0090] (2) Preparation of culture medium

[0091] Prepare the culture medium according to the formula in Table 5:

[0092] Table 5 In vitro colonization culture medium of intestinal flora and intestinal gas in middle-aged and elderly people

[0093]

[0094]

[0095] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0096] 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 incubated at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in the ascending colon reactor was 4-6 times per minute, the simulated peristalsis frequency in the transverse colon reactor was 2-3 times per minute, and the simulated peristalsis frequency in the descending colon reactor was 5-7 times per minute. 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 the ascending colon reactor was 5.5-6.5, the stable pH of the transverse colon reactor was 6.0-7.0, and the stable pH of the descending colon reactor was 6.5-7.5. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For middle-aged and elderly intestinal flora that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0097] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0098] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0099] The results showed that in terms of intestinal flora structure, compared with the real intestinal environment of middle-aged and elderly people, there were certain differences in the colonization abundance of Bacteroides, Prevotella, Clostridium, Ruminococcus, Faecalibacterium, Bifidobacterium, Escherichia, Klebsiella, and Akkermansia in the fermentation broth. In terms of intestinal gas, carbon dioxide decreased and volatile gases (VOCs) increased. Specific parameters such as Figure 1 , as shown in Tables 9 and 10.

[0100] Comparative Example 3: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in middle-aged and elderly people (without adding porcine mucin)

[0101] The specific steps are as follows:

[0102] (1) Preparation of fecal suspension

[0103] Fresh stool samples were collected from five middle-aged and elderly individuals aged 50 years. The subjects 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.

[0104] (2) Preparation of culture medium

[0105] The culture medium was prepared according to the formula in Table 6:

[0106] Table 6 In vitro colonization culture medium of intestinal flora and intestinal gas in middle-aged and elderly people

[0107]

[0108]

[0109] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0110] 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 incubated at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in the ascending colon reactor was 4-6 times per minute, the simulated peristalsis frequency in the transverse colon reactor was 2-3 times per minute, and the simulated peristalsis frequency in the descending colon reactor was 5-7 times per minute. 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 the ascending colon reactor was 5.5-6.5, the stable pH of the transverse colon reactor was 6.0-7.0, and the stable pH of the descending colon reactor was 6.5-7.5. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For middle-aged and elderly intestinal flora that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0111] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0112] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0113] The results showed that in terms of intestinal flora structure, compared with the real intestinal environment of middle-aged and elderly people, the colonization abundance of Clostridium, Ruminococcus, Faecalibacterium, Bifidobacterium, and Akkermansia in the fermentation liquid was reduced. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide, and volatile gases (VOCs) were the main components, which were different from the real intestinal gas of middle-aged and elderly people. Specific parameters such as Figure 1 , as shown in Tables 9 and 10.

[0114] Comparative Example 4: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in middle-aged and elderly people (without sodium sulfate addition)

[0115] The specific steps are as follows:

[0116] (1) Preparation of fecal suspension

[0117] Fresh stool samples were collected from five healthy individuals aged 50 years and older. The individuals had not taken antibiotics or other medications that could disrupt the intestinal microbiome in the past three months, nor had any gastrointestinal diseases. Fresh stool was collected using a stool collector, and each gram of stool was suspended in 5 mL of PBS buffer. The solids were then filtered through gauze to obtain a stool suspension.

[0118] (2) Preparation of culture medium

[0119] The culture medium was prepared according to the formula in Table 7:

[0120] Table 7 In vitro colonization culture medium of intestinal flora and intestinal gas in middle-aged and elderly people

[0121]

[0122] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0123] 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 incubated at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in the ascending colon reactor was 4-6 times per minute, the simulated peristalsis frequency in the transverse colon reactor was 2-3 times per minute, and the simulated peristalsis frequency in the descending colon reactor was 5-7 times per minute. 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 the ascending colon reactor was 5.5-6.5, the stable pH of the transverse colon reactor was 6.0-7.0, and the stable pH of the descending colon reactor was 6.5-7.5. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For middle-aged and elderly intestinal flora that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0124] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0125] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0126] The results showed that in terms of intestinal flora structure, compared with the real intestinal environment of middle-aged and elderly people, there were certain differences in the colonization abundance of Bacteroides, Prevotella, Clostridium, Ruminococcus, Faecalibacterium, Bifidobacterium, Escherichia, Klebsiella, and Akkermansia in the fermentation broth. 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 Tables 9 and 10.

[0127] Comparative Example 5: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in middle-aged and elderly people (without adding grains: sorghum, buckwheat, oats, and coix seed)

[0128] The specific steps are as follows:

[0129] (1) Preparation of fecal suspension

[0130] Fresh stool samples were collected from five healthy individuals aged 50 years and older. The individuals had not taken antibiotics or other medications that could disrupt the intestinal microbiome in the past three months, nor had any gastrointestinal diseases. Fresh stool was collected using a stool collector, and each gram of stool was suspended in 5 mL of PBS buffer. The solids were then filtered through gauze to obtain a stool suspension.

[0131] (2) Preparation of culture medium

[0132] Prepare the culture medium according to the formula in Table 8:

[0133] Table 8 In vitro colonization culture medium of intestinal flora and intestinal gas in middle-aged and elderly people

[0134]

[0135]

[0136] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0137] 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 incubated at 37°C under anaerobic conditions for 24 hours. The simulated peristalsis frequency in the ascending colon reactor was 4-6 times per minute, the simulated peristalsis frequency in the transverse colon reactor was 2-3 times per minute, and the simulated peristalsis frequency in the descending colon reactor was 5-7 times per minute. 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 the ascending colon reactor was 5.5-6.5, the stable pH of the transverse colon reactor was 6.0-7.0, and the stable pH of the descending colon reactor was 6.5-7.5. The stable pH of each reactor was adjusted upward and downward using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For middle-aged and elderly intestinal flora that maintain a steady state for a long time, fresh 10% culture medium can be injected every 8 hours to maintain stable growth.

[0138] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0139] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0140] The results showed that compared with the real intestinal environment of middle-aged and elderly people, the colonization abundance of Bifidobacterium in the fermentation broth decreased, while the colonization abundance of Escherichia and Klebsiella increased. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide and volatile gases (VOCs) were the main components, which were somewhat different from the real intestinal gas of middle-aged and elderly people. Specific parameters such as Figure 1 , as shown in Tables 9 and 10.

[0141] Table 9 Comparison of species richness of intestinal flora and intestinal gas culture medium in middle-aged and elderly people.

[0142]

[0143] Table 10 Comparison of gas composition after culture of intestinal flora and intestinal gas culture medium in the middle-aged and elderly.

[0144]

[0145] 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 middle-aged and elderly people, characterized in that: The content of each component in 1L of culture medium is as follows: Yeast extract added in an amount of 2-3g, tryptone added in an amount of 2-3g, mucin added in an amount of 1.5-2g, mycoprotein added in an amount of 1-2g, insect protein added in an amount of 0.2-0.3g, soy protein added in an amount of 1-1.5g, oligofructose added in an amount of 3-7g, oligogalactose added in an amount of 1-2.5g, inulin added in an amount of 0.5-2g, resistant dextrin added in an amount of 1.5-2g, dietary fiber added in an amount of 1.5-2g, sorghum added in an amount of 1-2g, buckwheat added in an amount of 1-2g, oats added in an amount of 1-2g, coix seed added in an amount of 1-2g, corn germ oil added in an amount of 1-2g The amount of added sugars is 0.5-1.5g, the amount of olive oil added is 0.5-1.5g, the amount of linseed oil added is 0.5-1.5g, the amount of walnut oil added is 0.5-1.5g, the amount of sialic acid added is 0.5-1g, the amount of algal oil DHA added is 1-1.5g, the amount of potassium chloride added is 1-1.5g, the amount of sodium chloride added is 1-1.5g, the amount of potassium dihydrogen phosphate added is 1.5-2.5g, the amount of sodium sulfate added is 0.5-2g, the amount of bile salt added is 0.4-0.6g, the amount of plant extract mixture added is 0.5-1g, and the amount of vitamin mixture added is 0.5-1.5mL.

2. The culture medium for colonizing intestinal flora and intestinal gas in middle-aged and elderly people according to claim 1, characterized in that The mucin includes one or a mixture of porcine mucin, bovine mucin and mussel mucin.

3. The culture medium for colonizing intestinal flora and intestinal gas in middle-aged and elderly people according to claim 1, characterized in that The mycoprotein comprises one or a mixture of several 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.

4. The culture medium for colonizing intestinal flora and intestinal gas in middle-aged and elderly 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.

5. The culture medium for colonizing intestinal flora and intestinal gas in middle-aged and elderly people according to claim 1, characterized in that The plant extract mixture comprises one or a mixture of several of puerarin, carotene, bioflavonoids, phytoandrogen, soy isoflavones, limonene, lycopene, catechin, anthocyanidin and resveratrol.

6. The culture medium for colonizing intestinal flora and intestinal gas in middle-aged and elderly people according to claim 1, characterized in that The vitamin mixture contains 0.4 g of vitamin B1, 0.4 g of vitamin B2, 4 g of niacin, 8 g of folic acid, and 12 g of pantothenic acid per 1 L.

7. A method for in vitro colonization of intestinal flora and intestinal gas in middle-aged and elderly people, characterized by: The method comprises the following steps: (1) Fresh feces of middle-aged and elderly people 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 the ascending colon reactor, the transverse colon reactor and the descending colon reactor of the above-mentioned culture medium, and colonized and cultured under anaerobic conditions to obtain the intestinal flora and intestinal gas of the middle-aged and elderly people in the ascending colon, transverse colon and descending colon stages, respectively.

8. The method for in vitro colonization of intestinal flora and intestinal gas in middle-aged and elderly people according to claim 7, characterized in that: 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.

9. The method for in vitro colonization of intestinal flora and intestinal gas in middle-aged and elderly people according to claim 7, characterized in that: In step (2), the colonization culture is a dynamic culture at 37°C for 24 hours under anaerobic conditions; The simulated peristalsis frequency of the ascending colon reactor is 4-6 times per minute, the simulated peristalsis frequency of the transverse colon reactor is 2-3 times per minute, and the simulated peristalsis frequency of the descending colon reactor is 5-7 times per minute; The rate of transfer of the contents of the ascending colon into the transverse colon is 2-4 ml / min, and the rate of transfer of the contents of the transverse colon into the descending colon is 4-6 ml / min; The stable pH of the ascending colon reactor is 5.5-6.5, the stable pH of the transverse colon reactor is 6.0-7.0, and the stable pH of the descending colon reactor is 6.5-7.

5. The amount of feces suspension added to each reactor is 5%.

10. The method for in vitro colonization of intestinal flora and intestinal gas in middle-aged and elderly people according to claim 9, characterized in that: In step (2), the pH of each reactor is adjusted by 0.5 mol of hydrochloric acid and 0.5 mol of sodium hydroxide.

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