Culture medium for colonization of teenager intestinal flora and intestinal gas and application of culture medium

Abstract

The invention discloses a culture medium for colonization of teenager intestinal flora and intestinal gas and application thereof, and belongs to the field of microorganisms. The culture medium contains yeast extract, tryptone, mucoprotein, mycoprotein, worm protein, soybean protein, fructo-oligosaccharide, galactooligosaccharide, inulin, resistant dextrin, dietary fiber, creamer, instant tea powder, plant starch, carrageenan, cocoa butter, flavonoid, cane sugar, lactose, 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 domestic teenagers is well simulated in vitro by referring to real in-vivo parameters of the teenagers, the intestinal flora ecology and intestinal gas distribution of the teenagers are reproduced, and a normal intestinal microorganism and intestinal gas database of the teenagers can be constructed. Accurate diagnosis and personalized treatment of intestinal microecology of the teenagers are facilitated, and the health state of the teenagers can be better evaluated and preventive measures can be better formulated.

Description

Technical Field

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

[0002] The gut microbiome plays an essential role as a "microbial organ" in the human body, profoundly influencing metabolic regulation, immune function, and nervous system function. Individual gut microbiota variability is influenced by a wide range of factors, including age, sex, geographic distribution, ethnicity, drug exposure history, circadian rhythm, lifestyle, and genetic makeup. Despite this diversity, healthy gut microbial communities typically exhibit structural similarities, forming a relatively consistent microbiome framework. In a healthy state, the gut microbiome maintains a delicate balance with its host (human) and its environmental context—a state known as "homeostasis"—which is crucial for overall health. The gut is a complex biological environment, home to not only a diverse microbial population but also the metabolic production of numerous gases, such as carbon dioxide, hydrogen, methane, hydrogen sulfide, and numerous trace gases, resulting from the combined effects of biochemical interactions within the gut and microbial activity. Detailed investigations of gut gas composition and its dynamic changes in response to dietary modifications have revealed new insights into the functional properties of the gut microbiome, its metabolites, and their potential impact on host health. Deepening our understanding of the complex mechanisms of intestinal gas production is expected to provide scientific support for improving the prevention efficiency, diagnostic accuracy, treatment strategies and long-term monitoring effectiveness of various diseases.

[0003] For specific populations, their standardized microbiome structure and intestinal gas patterns constitute a critical baseline for assessing individual health status. By carefully analyzing an individual's microbiome composition and intestinal gas composition and comparing them with established normal reference ranges for that population, it is possible to accurately assess whether an individual's health status is within the normal range and whether there are any hidden health risks. Therefore, establishing a comprehensive microbiome and intestinal gas reference system for specific populations has profound implications for promoting precision medicine and developing personalized treatment plans. It not only enables a more comprehensive assessment of individual health status, providing a scientific basis for the development of preventive strategies, but also deepens understanding of interpopulation differences, opening new avenues for drug development. Furthermore, this system can effectively assist in evaluating the effectiveness of clinical interventions and help determine disease susceptibility in specific populations, providing strong support for health management. For example, for infants and young children, maintaining a healthy intestinal microbiome is crucial for the development of their immune system and overall health. For older adults, maintaining a balanced intestinal microbiome through dietary adjustments and probiotic supplementation can prevent the development of intestinal diseases and other chronic conditions. However, perfecting the in vitro colonization of the gut microbiome and studying its mechanisms and mechanisms has always been a major challenge in microbiome science. Furthermore, most microbiome colonization strategies are designed based on findings from Westernized populations, with relatively few studies targeting Chinese populations, particularly those focused on adolescent intestinal colonization. The adolescent microbiome possesses unique characteristics that reflect the growth, development, metabolic needs, and environmental influences of this particular physiological stage. The adolescent gut microbiome is highly diverse, dynamic, and metabolically active, closely related to factors such as diet, immunity, and psychology. These microbiota are primarily composed of members of the genera Lactobacillus, Clostridium, Prevotella, Bacteroides, Bifidobacterium, Escherichia, and Salmonella. Furthermore, there are few reports domestically and internationally on intestinal gas production in adolescents. Summary of the Invention

[0004] Purpose of the invention: The present invention provides a culture medium for colonizing adolescent intestinal flora and intestinal gas and its application. The culture medium for adolescent intestinal flora and intestinal gas contains yeast extract, trypsin, mucin, mycoprotein, insect protein, soy protein, oligofructose, oligogalactose, inulin, resistant dextrin, dietary fiber, creamer, instant tea powder, plant starch, carrageenan, cocoa butter, flavonoids, sucrose, lactose, sialic acid, algae oil DHA, potassium chloride, sodium chloride, potassium dihydrogen phosphate, sodium sulfate, bile salts, a plant extract mixture, a vitamin mixture, etc.

[0005] Technical solution: The present invention provides a culture medium for colonizing intestinal flora and intestinal gas in adolescents. The content of each component in each 1L of the culture medium is as follows: 1-4g of yeast extract, 1-3g of tryptone, 1-3g of mucin, 1-1.5g of mycoprotein, 0.1-0.3g of insect protein, 0.5-1.5g of soy protein, 4-8g of oligofructose, 1.5-2g of oligogalactose, 1-1.5g of inulin, 1-2.5g of resistant dextrin, 2-3g of dietary fiber, 1-2g of creamer, 2-3g of instant tea powder, and 1-2g of plant starch. The added amount of powder is 1-2g, the added amount of carrageenan is 1-1.5g, the added amount of cocoa butter is 2-3g, the added amount of flavonoids is 0.3-0.5g, the added amount of sucrose is 1-1.5g, the added amount of lactose is 1-1.5g, the added amount of sialic acid is 0.5-2g, the added amount of algal oil DHA is 0.5-1g, the added amount of potassium chloride is 0.5-1g, the added amount of sodium chloride is 0.5-2g, the added amount of potassium dihydrogen phosphate is 1.5-2g, the added amount of sodium sulfate is 0.5-1g, the added amount of bile salt is 0.3-0.7g, the added amount of plant extract mixture is 0.5-0.9g, and the added amount of vitamin mixture is 1.5-2mL. All of the above reagents are analytical grade concentrations.

[0006] Preferably, the content of each component in each 1L of culture medium is as follows: 2.5g of yeast extract, 2g of tryptone, 2g of mucin, 1.25g of mycoprotein, 0.2g of insect protein, 1g of soy protein, 6g of oligofructose, 1.75g of oligogalactose, 1.25g of inulin, 1.75g of resistant dextrin, 2.5g of dietary fiber, 1.5g of creamer, 2.5g of instant tea powder, 1.5g of plant starch, g, 1.25g of carrageenan, 2.5g of cocoa butter, 0.4g of flavonoids, 1.25g of sucrose, 1.25g of lactose, 1.25g of sialic acid, 0.75g of algal oil DHA, 0.75g of potassium chloride, 1.25g of sodium chloride, 1.75g of potassium dihydrogen phosphate, 0.75g of sodium sulfate, 0.5g of bile salts, 0.7g of plant extract mixture, and 1.75mL of vitamin mixture.

[0007] In one embodiment of the present invention, the mucin includes porcine mucin, bovine mucin, and mussel mucin, preferably bovine mucin.

[0008] In one embodiment of the present invention, 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 boletus powder.

[0009] In one embodiment of the present invention, the insect protein includes proteins extracted from eggs, larvae, adults, pupae, moths, etc., such as bean worm powder, silkworm pupae, etc., preferably silkworm pupae.

[0010] In one embodiment of the present invention, the instant tea powder includes black tea, green tea, white tea, dark tea, etc.

[0011] In one embodiment of the present invention, the plant starch includes cassava starch, sweet potato starch, potato starch, glutinous rice flour, preferably cassava starch.

[0012] In one embodiment of the present invention, the plant extract mixture comprises puerarin, carotene, bioflavonoids, phytoandrogens, soy isoflavones, limonene, lycopene, catechins, anthocyanidins, and resveratrol, preferably carotene and phytoandrogens.

[0013] In one embodiment of the present invention, per 1 L of the vitamin mixture, the added amount of vitamin B1 is 0.6 g, the added amount of vitamin B2 is 0.6 g, the added amount of niacin is 6 g, the added amount of folic acid is 12 g, and the added amount of pantothenic acid is 18 g.

[0014] The present invention also provides a method for in vitro colonization of adolescent intestinal flora and intestinal gas, the method comprising the following steps:

[0015] (1) 6 g of fresh adolescent feces was collected and placed in 30 mL of PBS, stirred evenly, and then the solid was filtered with gauze to obtain a fecal suspension;

[0016] (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 to obtain the intestinal flora and intestinal gas of adolescents in the ascending colon, transverse colon, and descending colon stages, respectively;

[0017] In one embodiment of the present invention, the application is that fresh feces are collected by a feces collector, and then each gram of feces is suspended in 5 mL of PBS, and the time from feces collection to injection into the reactor is controlled within 10 minutes.

[0018] In one embodiment of the present invention, 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 4-7 times per minute, the simulated peristalsis frequency of the transverse colon reactor is 2-4 times per minute, and the simulated peristalsis frequency of the descending colon reactor is 6-9 times per minute; the rate of transfer of the contents of the ascending colon into the transverse colon is 2-5 ml / min, and the rate of transfer of the contents of the transverse colon into the descending colon is 4-7 ml / min; the stable pH of the ascending colon reactor is 5.0-6.5, the stable pH of the transverse colon reactor is 6.0-7.5, and the stable pH of the descending colon reactor is 6.5-8.0, and the amount of fecal suspension added to each reactor is 5%.

[0019] In one embodiment of the present invention, 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.

[0020] In one embodiment of the present invention, in step (2), if it is necessary to maintain a steady state of adolescent intestinal flora for a long time, fresh 10% culture medium can be injected every 8 hours for stable growth.

[0021] 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.

[0022] Beneficial effects:

[0023] This biomimetic colon bioreactor, based on gastrointestinal physiology, biotechnology, and fermentation engineering, effectively simulates the actual intestinal environment of adolescents in China in vitro by referencing actual in vivo parameters, reproducing their intestinal flora ecology and intestinal gas distribution. The method of this invention utilizes adolescent intestinal flora culture media combined with the biomimetic colon bioreactor to establish a strategy for colonization of adolescent intestinal microorganisms and intestinal gas. This can construct a database of normal adolescent intestinal microorganisms and intestinal gas, facilitating accurate diagnosis and personalized treatment of adolescent intestinal microbiomes, better assessing adolescent health status, and developing preventive measures. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Comparison of species richness of intestinal flora and intestinal gas cultured on common culture medium and in adolescents.

[0025] Figure 2 : Comparison of gas composition after culture of intestinal flora and intestinal gas culture medium in adolescents. DETAILED DESCRIPTION

[0026] The culture medium components involved in the following examples are described as follows:

[0027] The yeast extract, tryptone, mucin, mycoprotein, insect protein, soy protein, fructooligosaccharide, galacto-oligosaccharide, inulin, resistant dextrin, dietary fiber, creamer, instant tea powder, plant starch, carrageenan, cocoa butter, flavonoids, sucrose, lactose, 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.

[0028] The mucin includes porcine mucin, bovine mucin and mussel mucin.

[0029] 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.

[0030] The insect protein includes proteins extracted from eggs, larvae, adults, pupae, moths, etc., such as bean worm powder and silkworm pupae.

[0031] The instant tea powder includes black tea, green tea, white tea and dark tea.

[0032] The plant starch includes cassava starch, sweet potato starch, potato starch and glutinous rice flour.

[0033] The plant extract mixture includes puerarin, carotene, bioflavonoids, phytoandrogen, soy isoflavones, limonene, lycopene, catechin, anthocyanidin, and resveratrol.

[0034] The vitamin mixture contains 0.56g of vitamin B1, 0.6g of vitamin B2, 6g of niacin, 12g of folic acid, and 18g of pantothenic acid per 1 L. When the culture medium is a solid culture medium, the amount of agar powder added is 1.2-1.6%.

[0035] Example 1: In vitro spatiotemporal dynamic colonization of adolescent intestinal flora and intestinal gas

[0036] The specific steps are as follows:

[0037] (1) Preparation of fecal suspension

[0038] Fresh stool samples were collected from five adolescents aged 16 years. The adolescents 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. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0039] (2) Preparation of culture medium

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

[0041] Table 1 In vitro colonization culture medium of adolescent intestinal flora and intestinal gas

[0042]

[0043] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0044] 200 mL of culture medium was added to the ascending colon reactor, transverse colon reactor, and 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 of the ascending colon reactor was 4-7 times per minute, the simulated peristalsis frequency of the transverse colon reactor was 2-4 times per minute, and the simulated peristalsis frequency of the descending colon reactor was 6-9 times per minute. The rate of transfer of ascending colon contents into the transverse colon was 2-5 ml / min, and the rate of transfer of transverse colon contents into the descending colon was 4-7 ml / min. The stable pH of the ascending colon reactor was 5.0-6.5, the stable pH of the transverse colon reactor was 6.0-7.5, and the stable pH of the descending colon reactor was 6.5-8.0. The stable pH of each reactor was adjusted up and down using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For adolescent 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.

[0045] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0046] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0047] The results showed that: in terms of intestinal flora structure, the culture medium of the present invention can significantly increase the colonization abundance of Lactobacillus, Clostridium, Prevotella, Bacteroides, Bifidobacterium, Escherichia, and Salmonella 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 teenagers. 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 teenagers. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0048] Example 2: In vitro spatiotemporal dynamic colonization of adolescent intestinal flora and intestinal gas

[0049] The specific steps are as follows:

[0050] (1) Preparation of fecal suspension

[0051] Fresh stool samples were collected from five adolescents aged 16 years. The adolescents 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. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0052] (2) Preparation of culture medium

[0053] Prepare the culture medium according to the formula in Table 2:

[0054] Table 2 In vitro colonization culture medium of adolescent intestinal flora and intestinal gas

[0055]

[0056]

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

[0058] 200 mL of culture medium was added to the ascending colon reactor, transverse colon reactor, and 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 of the ascending colon reactor was 4-7 times per minute, the simulated peristalsis frequency of the transverse colon reactor was 2-4 times per minute, and the simulated peristalsis frequency of the descending colon reactor was 6-9 times per minute. The rate of transfer of ascending colon contents into the transverse colon was 2-5 ml / min, and the rate of transfer of transverse colon contents into the descending colon was 4-7 ml / min. The stable pH of the ascending colon reactor was 5.0-6.5, the stable pH of the transverse colon reactor was 6.0-7.5, and the stable pH of the descending colon reactor was 6.5-8.0. The stable pH of each reactor was adjusted up and down using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For adolescent 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.

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

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

[0061] The results showed that: in terms of intestinal flora structure, the culture medium of the present invention can significantly increase the colonization abundance of Lactobacillus, Clostridium, Prevotella, Bacteroides, Bifidobacterium, Escherichia, and Salmonella 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 teenagers. 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 teenagers. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0062] Example 3: In vitro spatiotemporal dynamic colonization of adolescent intestinal flora and intestinal gas

[0063] The specific steps are as follows:

[0064] (1) Preparation of fecal suspension

[0065] Fresh stool samples were collected from five adolescents aged 16 years. The adolescents 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. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0066] (2) Preparation of culture medium

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

[0068] Table 3 In vitro colonization culture medium of adolescent intestinal flora and intestinal gas

[0069]

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

[0071] 200 mL of culture medium was added to the ascending colon reactor, transverse colon reactor, and 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 of the ascending colon reactor was 4-7 times per minute, the simulated peristalsis frequency of the transverse colon reactor was 2-4 times per minute, and the simulated peristalsis frequency of the descending colon reactor was 6-9 times per minute. The rate of transfer of ascending colon contents into the transverse colon was 2-5 ml / min, and the rate of transfer of transverse colon contents into the descending colon was 4-7 ml / min. The stable pH of the ascending colon reactor was 5.0-6.5, the stable pH of the transverse colon reactor was 6.0-7.5, and the stable pH of the descending colon reactor was 6.5-8.0. The stable pH of each reactor was adjusted up and down using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For adolescent 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.

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

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

[0074] The results showed that: in terms of intestinal flora structure, the culture medium of the present invention can significantly increase the colonization abundance of Lactobacillus, Clostridium, Prevotella, Bacteroides, Bifidobacterium, Escherichia, and Salmonella 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 teenagers. 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 teenagers. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

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

[0076] The specific steps are as follows:

[0077] (1) Preparation of fecal suspension

[0078] Fresh stool samples were collected from five adolescents aged 16 years. The adolescents 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. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0079] (2) Preparation of culture medium

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

[0081] Table 4 In vitro colonization culture medium of adolescent intestinal flora and intestinal gas

[0082]

[0083]

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

[0085] 200 mL of culture medium was added to the ascending colon reactor, transverse colon reactor, and 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 of the ascending colon reactor was 4-7 times per minute, the simulated peristalsis frequency of the transverse colon reactor was 2-4 times per minute, and the simulated peristalsis frequency of the descending colon reactor was 6-9 times per minute. The rate of transfer of ascending colon contents into the transverse colon was 2-5 ml / min, and the rate of transfer of transverse colon contents into the descending colon was 4-7 ml / min. The stable pH of the ascending colon reactor was 5.0-6.5, the stable pH of the transverse colon reactor was 6.0-7.5, and the stable pH of the descending colon reactor was 6.5-8.0. The stable pH of each reactor was adjusted up and down using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For adolescent 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.

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

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

[0088] The results showed that in terms of intestinal flora structure, compared with the real intestinal environment of adolescents, the colonization abundance of Lactobacillus, Clostridium, Prevotella, Bacteroides, Bifidobacterium, Escherichia, and Salmonella in the fermentation liquid was significantly reduced. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide, and volatile gases (VOCs) were the main gases, but the gas production was different from the real intestinal gas of adolescents. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

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

[0090] The specific steps are as follows:

[0091] (1) Preparation of fecal suspension

[0092] Fresh stool samples were collected from five adolescents aged 16 years. The adolescents 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. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0093] (2) Preparation of culture medium

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

[0095] Table 5 In vitro colonization culture medium of adolescent intestinal flora and intestinal gas

[0096]

[0097]

[0098] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0099] 200 mL of culture medium was added to the ascending colon reactor, transverse colon reactor, and 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 of the ascending colon reactor was 4-7 times per minute, the simulated peristalsis frequency of the transverse colon reactor was 2-4 times per minute, and the simulated peristalsis frequency of the descending colon reactor was 6-9 times per minute. The rate of transfer of ascending colon contents into the transverse colon was 2-5 ml / min, and the rate of transfer of transverse colon contents into the descending colon was 4-7 ml / min. The stable pH of the ascending colon reactor was 5.0-6.5, the stable pH of the transverse colon reactor was 6.0-7.5, and the stable pH of the descending colon reactor was 6.5-8.0. The stable pH of each reactor was adjusted up and down using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For adolescent 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.

[0100] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0101] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0102] The results showed that in terms of intestinal flora structure, compared with the real intestinal environment of adolescents, there were certain differences in the abundance of colonization of Lactobacillus, Clostridium, Prevotella, Bacteroides, Bifidobacterium, Escherichia, and Salmonella in the fermentation liquid. In terms of intestinal gas, carbon dioxide decreased and volatile gases (VOC) increased. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0103] Comparative Example 3: In vitro spatiotemporal dynamic colonization culture of adolescent intestinal flora and intestinal gas (without addition of bovine mucin)

[0104] The specific steps are as follows:

[0105] (1) Preparation of fecal suspension

[0106] Fresh stool samples were collected from five adolescents aged 16 years. The adolescents 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. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0107] (2) Preparation of culture medium

[0108] The culture medium was prepared according to the formula in Table 6:

[0109] Table 6 In vitro colonization culture medium of adolescent intestinal flora and intestinal gas

[0110]

[0111]

[0112] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0113] 200 mL of culture medium was added to the ascending colon reactor, transverse colon reactor, and 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 of the ascending colon reactor was 4-7 times per minute, the simulated peristalsis frequency of the transverse colon reactor was 2-4 times per minute, and the simulated peristalsis frequency of the descending colon reactor was 6-9 times per minute. The rate of transfer of ascending colon contents into the transverse colon was 2-5 ml / min, and the rate of transfer of transverse colon contents into the descending colon was 4-7 ml / min. The stable pH of the ascending colon reactor was 5.0-6.5, the stable pH of the transverse colon reactor was 6.0-7.5, and the stable pH of the descending colon reactor was 6.5-8.0. The stable pH of each reactor was adjusted up and down using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For adolescent 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.

[0114] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0115] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0116] The results showed that in terms of intestinal flora structure, the colonization abundance of Prevotella, Bacteroides, and Bifidobacterium in the fermentation broth was significantly reduced compared with the real intestinal environment of adolescents. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide, and volatile gases (VOCs) were the main gases, but there were certain differences from the real intestinal gas of adolescents. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0117] Comparative Example 4: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in adolescents (without sodium sulfate addition)

[0118] The specific steps are as follows:

[0119] (1) Preparation of fecal suspension

[0120] Fresh stool samples were collected from five adolescents aged 16 years. The adolescents 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. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0121] (2) Preparation of culture medium

[0122] The culture medium was prepared according to the formula in Table 7:

[0123] Table 7 In vitro colonization culture medium of adolescent intestinal flora and intestinal gas

[0124]

[0125]

[0126] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0127] 200 mL of culture medium was added to the ascending colon reactor, transverse colon reactor, and 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 of the ascending colon reactor was 4-7 times per minute, the simulated peristalsis frequency of the transverse colon reactor was 2-4 times per minute, and the simulated peristalsis frequency of the descending colon reactor was 6-9 times per minute. The rate of transfer of ascending colon contents into the transverse colon was 2-5 ml / min, and the rate of transfer of transverse colon contents into the descending colon was 4-7 ml / min. The stable pH of the ascending colon reactor was 5.0-6.5, the stable pH of the transverse colon reactor was 6.0-7.5, and the stable pH of the descending colon reactor was 6.5-8.0. The stable pH of each reactor was adjusted up and down using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For adolescent 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.

[0128] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0129] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0130] The results showed that in terms of intestinal flora structure, compared with the real intestinal environment of adolescents, there were certain differences in the abundance of colonization of Lactobacillus, Clostridium, Prevotella, Bacteroides, Bifidobacterium, Escherichia, and Salmonella 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 Table 12 and Table 13.

[0131] Comparative Example 5: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in adolescents (without adding milk tea ingredients: creamer, instant tea powder, plant starch, carrageenan)

[0132] The specific steps are as follows:

[0133] (1) Preparation of fecal suspension

[0134] Fresh stool samples were collected from five adolescents aged 16 years. The adolescents 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. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0135] (2) Preparation of culture medium

[0136] The culture medium was prepared according to the formula in Table 7:

[0137] Table 9 In vitro colonization culture medium of adolescent intestinal flora and intestinal gas

[0138]

[0139]

[0140] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0141] 200 mL of culture medium was added to the ascending colon reactor, transverse colon reactor, and 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 of the ascending colon reactor was 4-7 times per minute, the simulated peristalsis frequency of the transverse colon reactor was 2-4 times per minute, and the simulated peristalsis frequency of the descending colon reactor was 6-9 times per minute. The rate of transfer of ascending colon contents into the transverse colon was 2-5 ml / min, and the rate of transfer of transverse colon contents into the descending colon was 4-7 ml / min. The stable pH of the ascending colon reactor was 5.0-6.5, the stable pH of the transverse colon reactor was 6.0-7.5, and the stable pH of the descending colon reactor was 6.5-8.0. The stable pH of each reactor was adjusted up and down using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For adolescent 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.

[0142] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0143] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0144] The results showed that in terms of intestinal flora structure, the abundance of Escherichia and Salmonella in the fermentation broth was reduced compared with the real intestinal environment of adolescents. In terms of intestinal gas, carbon dioxide and hydrogen sulfide decreased, and volatile organic compounds (VOCs) increased. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0145] Comparative Example 6: In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas in adolescents (without added chocolate ingredients: cocoa butter, flavonoids, sucrose, lactose, minerals)

[0146] The specific steps are as follows:

[0147] (1) Preparation of fecal suspension

[0148] Fresh stool samples were collected from five adolescents aged 16 years. The adolescents 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. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0149] (2) Preparation of culture medium

[0150] The culture medium was prepared according to the formula in Table 7:

[0151] Table 11 In vitro colonization culture medium of adolescent intestinal flora and intestinal gas

[0152]

[0153]

[0154] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0155] 200 mL of culture medium was added to the ascending colon reactor, transverse colon reactor, and 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 of the ascending colon reactor was 4-7 times per minute, the simulated peristalsis frequency of the transverse colon reactor was 2-4 times per minute, and the simulated peristalsis frequency of the descending colon reactor was 6-9 times per minute. The rate of transfer of ascending colon contents into the transverse colon was 2-5 ml / min, and the rate of transfer of transverse colon contents into the descending colon was 4-7 ml / min. The stable pH of the ascending colon reactor was 5.0-6.5, the stable pH of the transverse colon reactor was 6.0-7.5, and the stable pH of the descending colon reactor was 6.5-8.0. The stable pH of each reactor was adjusted up and down using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For adolescent 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.

[0156] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0157] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0158] The results showed that in terms of intestinal flora structure, the abundance of Escherichia and Salmonella in the fermentation broth was reduced compared with the real intestinal environment of adolescents. In terms of intestinal gas, hydrogen and carbon dioxide decreased, while hydrogen sulfide and volatile organic compounds (VOCs) increased. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0159] Comparative Example 7: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in adolescents (without adding porcini powder)

[0160] The specific steps are as follows:

[0161] (1) Preparation of fecal suspension

[0162] Fresh stool samples were collected from five adolescents aged 16 years. The adolescents 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. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0163] (2) Preparation of culture medium

[0164] The culture medium was prepared according to the formula in Table 6:

[0165] Table 6 In vitro colonization culture medium of adolescent intestinal flora and intestinal gas

[0166]

[0167]

[0168] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0169] 200 mL of culture medium was added to the ascending colon reactor, transverse colon reactor, and 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 of the ascending colon reactor was 4-7 times per minute, the simulated peristalsis frequency of the transverse colon reactor was 2-4 times per minute, and the simulated peristalsis frequency of the descending colon reactor was 6-9 times per minute. The rate of transfer of ascending colon contents into the transverse colon was 2-5 ml / min, and the rate of transfer of transverse colon contents into the descending colon was 4-7 ml / min. The stable pH of the ascending colon reactor was 5.0-6.5, the stable pH of the transverse colon reactor was 6.0-7.5, and the stable pH of the descending colon reactor was 6.5-8.0. The stable pH of each reactor was adjusted up and down using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For adolescent 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.

[0170] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0171] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0172] The results showed that in terms of intestinal flora structure, the colonization abundance of Bacteroides and Bifidobacterium in the fermentation broth was significantly reduced compared with the real intestinal environment of adolescents. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide and volatile gases (VOCs) were the main gases, but there were certain differences from the real intestinal gas of adolescents. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0173] Comparative Example 8: In vitro spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in adolescents (without addition of carotene and phytoandrogens)

[0174] The specific steps are as follows:

[0175] (1) Preparation of fecal suspension

[0176] Fresh stool samples were collected from five adolescents aged 16 years. The adolescents 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. Each gram of stool was suspended in 5 mL of PBS buffer, and the solids were filtered through gauze to obtain a stool suspension.

[0177] (2) Preparation of culture medium

[0178] The culture medium was prepared according to the formula in Table 6:

[0179] Table 6 In vitro colonization culture medium of adolescent intestinal flora and intestinal gas

[0180]

[0181] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically:

[0182] 200 mL of culture medium was added to the ascending colon reactor, transverse colon reactor, and 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 of the ascending colon reactor was 4-7 times per minute, the simulated peristalsis frequency of the transverse colon reactor was 2-4 times per minute, and the simulated peristalsis frequency of the descending colon reactor was 6-9 times per minute. The rate of transfer of ascending colon contents into the transverse colon was 2-5 ml / min, and the rate of transfer of transverse colon contents into the descending colon was 4-7 ml / min. The stable pH of the ascending colon reactor was 5.0-6.5, the stable pH of the transverse colon reactor was 6.0-7.5, and the stable pH of the descending colon reactor was 6.5-8.0. The stable pH of each reactor was adjusted up and down using 0.5 mol hydrochloric acid and 0.5 mol sodium hydroxide. For adolescent 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.

[0183] Anaerobic conditions: The reactor headspace gas consists of 100% nitrogen

[0184] The fermentation liquid and intestinal gas after cultivation in each of the above reactors were respectively subjected to 16s RNA and gas composition determination:

[0185] The results showed that in terms of intestinal flora structure, compared with the real intestinal environment of adolescents, there were certain differences in the colonization abundance of Lactobacillus, Clostridium, Prevotella, Bacteroides, Bifidobacterium, Escherichia, and Salmonella in the fermentation liquid. In terms of intestinal gas, carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide, and volatile gases (VOCs) were the main gases, but there were certain differences from the real intestinal gas of adolescents. Specific parameters such as Figure 1 , as shown in Table 12 and Table 13.

[0186] Table 12 Comparison of species richness of intestinal flora and intestinal gas cultured in ordinary culture medium and adolescent culture medium

[0187]

[0188] Table 13 Comparison of gas composition after culture in common culture medium and adolescent intestinal flora and intestinal gas culture medium

[0189]

[0190] 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 adolescent intestinal flora and intestinal gas, characterized in that: The content of each component in 1L of culture medium is as follows: Yeast extract 1-4g, tryptone 1-3g, mucin 1-3g, mycoprotein 1-1.5g, insect protein 0.1-0.3g, soy protein 0.5-1.5g, oligofructose 4-8g, oligogalactose 1.5-2g, inulin 1-1.5g, resistant dextrin 1-2.5g, dietary fiber 2-3g, creamer 1-2g, instant tea powder 2-3g, plant starch 1-2g, carrageenan 1-1.5g , cocoa butter 2-3g, flavonoids 0.3-0.5g, sucrose 1-1.5g, lactose 1-1.5g, sialic acid 0.5-2g, algal oil DHA 0.5-1g, potassium chloride 0.5-1g, sodium chloride 0.5-2g, potassium dihydrogen phosphate 1.5-2g, sodium sulfate 0.5-1g, bile salts 0.3-0.7g, plant extract mixture 0.5-0.9g, vitamin mixture 1.5-2mL.

2. The culture medium for colonizing adolescent intestinal flora and intestinal gas according to claim 1, characterized in that The mucin includes one or more of porcine mucin, bovine mucin, and mussel mucin; The mycoprotein comprises one or more 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; The insect protein includes proteins extracted from eggs, larvae, adults, pupae, and moths; The instant tea powder includes black tea powder, green tea powder, white tea powder and dark tea powder.

3. The culture medium for colonizing adolescent intestinal flora and intestinal gas according to claim 1, characterized in that The plant starch includes one or more of cassava starch, sweet potato starch, potato starch and glutinous rice flour.

4. The culture medium for colonizing intestinal flora and intestinal gas in adolescents 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.

5. The method for colonizing intestinal flora and intestinal gas in adolescents according to claim 1, characterized in that: The vitamin mixture contains 0.6 g of vitamin B1, 0.6 g of vitamin B2, 6 g of niacin, 12 g of folic acid, and 18 g of pantothenic acid per 1 L.

6. A method for in vitro colonization of adolescent intestinal flora and intestinal gas, characterized in that: The method comprises the following steps: (1) 6 g of fresh adolescent feces was collected and placed in 30 mL of PBS, stirred evenly, and then the solid was filtered with gauze to obtain a fecal suspension; (2) The fecal suspension obtained in step (1) is injected into the ascending colon reactor, transverse colon reactor and descending colon reactor of the above culture medium, and cultured under anaerobic conditions for 24 hours to obtain the intestinal flora and intestinal gas of adolescents in the ascending colon, transverse colon and descending colon stages, respectively.

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

8. The method for in vitro colonization of adolescent intestinal flora and intestinal gas according to claim 6, characterized in that: 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 4-7 times per minute, the simulated peristalsis frequency of the transverse colon reactor is 2-4 times per minute, and the simulated peristalsis frequency of the descending colon reactor is 6-9 times per minute; the rate of transfer of the contents of the ascending colon into the transverse colon is 2-5 ml / min, and the rate of transfer of the contents of the transverse colon into the descending colon is 4-7 ml / min; the stable pH of the ascending colon reactor is 5.0-6.5, the stable pH of the transverse colon reactor is 6.0-7.5, and the stable pH of the descending colon reactor is 6.5-8.0, and the amount of fecal suspension added to each reactor is 5%.

9. The method for in vitro colonization of adolescent intestinal flora and intestinal gas according to claim 6, characterized in that: 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.

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

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