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

Abstract

The invention discloses a culture medium for colonization of intestinal flora and intestinal gas of infants. The culture medium contains a yeast extract, tryptone, mucoprotein, mycoprotein, worm protein, soybean protein, fructo-oligosaccharide, galactooligosaccharide, inulin, resistant dextrin, breast milk oligosaccharide, dietary fiber, sialic acid, algal oil DHA, potassium chloride, sodium chloride, monopotassium phosphate, sodium sulfate, 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 infants is well simulated in vitro by referring to real parameters in the infants, the intestinal flora ecology and intestinal gas distribution are reproduced, and a normal intestinal microorganism and intestinal gas database of the infants can be constructed. Accurate diagnosis and personalized treatment of the intestinal microecology of the infants are facilitated, the health state of the infants can be better evaluated, and preventive measures can be better formulated.

Description

Technical Field

[0001] The present invention relates to a culture medium for colonizing the intestinal flora and intestinal gas of infants and young children and its application, belonging to the technical field of microbiology. Background Art

[0002] The gut microbiota has become an important "microbial organ" affecting human health and diseases, and can regulate the body's metabolism, immunity and nervous system. Each person has a unique gut microbiota, and the factors causing differences between different groups of gut microbiota mainly include age, gender, geographical location, race, drugs, physiological status, lifestyle and genetic background, etc. However, its normal microbial community composition has specific characteristics and often has a similar microbiome composition. At the same time, in healthy human bodies, the microbiome forms a balanced relationship with the human body, and this balance is adapted to specific host (human) and environmental conditions. This state is called "homeostasis", which is an important factor in maintaining health. However, the internal working mechanism of the intestine is relatively complex, including not only various microorganisms but also the gases they produce, such as carbon dioxide, hydrogen, methane, hydrogen sulfide and various trace gases, which are all produced by chemical interactions and microbiota in the intestine. Analyzing these intestinal gases and their responses to dietary changes can reveal the products and functions of the gut microbiota and their impact on human health. A better understanding of the complex interactions that produce gases in the intestine will improve our ability to prevent, diagnose, treat and monitor many diseases.

[0003] The normal microbiota and intestinal gas references of a specific population can serve as an important indicator for assessing the health status of that population. By detecting the composition of an individual's microbial community and the distribution of intestinal gases, and comparing them with the normal references of the same population, it is possible to understand whether an individual's health condition is good and whether there are potential health risks. Therefore, establishing the normal microbiota and intestinal gas references of a specific population helps with accurate diagnosis and personalized treatment; better assessment of health status and formulation of prevention strategies; in-depth understanding of population differences and guidance for drug research and development; assisting in judging the effectiveness of clinical interventions and determining the disease susceptibility of a specific population. For example, for infants, establishing a healthy gut microbiota is crucial for the development of their immune system and overall health. For the elderly, maintaining the balance of the gut microbiota through diet adjustment, probiotic supplementation, etc. can prevent the occurrence of intestinal diseases and other chronic diseases. However, perfect in vitro colonization of the gut microbiome to study its mechanisms has always been an important challenge in microbiomics. In addition, most microbiome colonization strategies are designed based on the research results of Westernized populations, and there is relatively little research on microbiome colonization strategies for domestic populations, especially for infant gut colonization. The microbial communities of foreign infants have been observed. The period from birth to 2 - 3 years of age in infancy is the most important period for the formation and balance of the gut microbiota. The microbiota during this period determines the body's future immune response and metabolism. During this period, the gut microbiota is relatively fragile and has poor diversity. Multiple factors such as the pregnancy situation, birth route, feeding method, antibiotic use, and living environment can affect the colonization and succession of the gut microbiota. They are mainly composed of members of Enterobacteriaceae, Bifidobacteriaceae, Enterococcaceae, Veillonellaceae, and Staphylococcaceae. In addition, there are few domestic and foreign reports on the intestinal gases produced by infants. Summary of the Invention

[0004] Object of the Invention: The technical problem to be solved by the present invention is to provide a culture medium for colonizing the gut microbiota and intestinal gases of infants. The culture medium for the gut microbiota and intestinal gases of infants contains yeast extract, tryptone, mucin, bacterioprotein, insect protein, soy protein, fructooligosaccharide, galactooligosaccharide, inulin, resistant dextrin, human milk oligosaccharide, dietary fiber, sialic acid, algal oil DHA, potassium chloride, sodium chloride, potassium dihydrogen phosphate, sodium sulfate, bile salts, plant extract mixture, vitamin mixture, etc.

[0005] Technical Solution: To solve the above technical problem, the present invention provides the following technical solution:

[0006] The present invention provides a culture medium for colonizing the intestinal flora and intestinal gas of infants. The content of each component in every 1 L of the culture medium is as follows: the addition amount of yeast extract is 1 - 3 g, the addition amount of tryptone is 1 - 2 g, the addition amount of mucin is 1 - 2 g, the addition amount of mycoprotein is 0.5 - 1.5 g, the addition amount of insect protein is 0.2 - 0.4 g, the addition amount of soy protein is 0.5 - 1 g, the addition amount of fructooligosaccharide is 3 - 6 g, the addition amount of galactooligosaccharide is 1 - 2 g, the addition amount of inulin is 0.5 - 1.5 g, the addition amount of resistant dextrin is 1 - 2 g, the addition amount of human milk oligosaccharide is 0.5 - 1.5 g, the addition amount of dietary fiber is 1 - 2 g, the addition amount of sialic acid is 0.5 - 1.5 g, the addition amount of algal oil DHA is 0.5 - 1.5 g, the addition amount of potassium chloride is 0.5 - 1.5 g, the addition amount of sodium chloride is 0.5 - 1.5 g, the addition amount of potassium dihydrogen phosphate is 1 - 2 g, the addition amount of sodium sulfate is 0.5 - 1.5 g, the addition amount of bile salts is 0.2 - 0.6 g, the addition amount of plant extract mixture is 0.4 - 0.8 g, the addition amount of vitamin mixture is 1 - 2 mL. All the above reagents are of analytical pure concentration.

[0007] Preferably, the content of each component in every 1 L of the culture medium is as follows: the addition amount of yeast extract is 2 g, the addition amount of tryptone is 1.5 g, the addition amount of mucin is 1.5 g, the addition amount of mycoprotein is 1 g, the addition amount of insect protein is 0.3 g, the addition amount of soy protein is 0.75 g, the addition amount of fructooligosaccharide is 4 g, the addition amount of galactooligosaccharide is 1.5 g, the addition amount of inulin is 1 g, the addition amount of resistant dextrin is 1.5 g, the addition amount of human milk oligosaccharide is 1 g, the addition amount of dietary fiber is 1.5 g, the addition amount of sialic acid is 1 g, the addition amount of algal oil DHA is 1 g, the addition amount of potassium chloride is 1 g, the addition amount of sodium chloride is 1 g, the addition amount of potassium dihydrogen phosphate is 1.5 g, the addition amount of sodium sulfate is 1 g, the addition amount of bile salts is 0.4 g, the addition amount of plant extract mixture is 0.6 g, the addition amount of vitamin mixture is 1.5 mL.

[0008] Further, the mucin includes porcine mucin, bovine mucin, and mussel mucin.

[0009] Further, the mycoprotein includes lentinus edodes powder, ganoderma lucidum powder, auricularia auricula powder, boletus edulis powder, dictyophora indusiata powder, agaricus blazei powder, hericium erinaceus powder, pleurotus nebrodensis powder, cordyceps sinensis powder.

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

[0011] Further, the plant extract mixture includes puerarin, carotene, bioflavonoids, phytoandrogens, soy isoflavones, limonin, lycopene, catechins, anthocyanins, resveratrol, etc.

[0012] Further, in every 1 L of the vitamin mixture, the addition amounts of vitamin B1, vitamin B2, niacin, folic acid, and pantothenic acid are 0.5 g, 0.5 g, 5 g, 10 g, and 15 g, respectively.

[0013] The present invention also provides a method for colonizing the intestinal flora and intestinal gas of infants and young children in vitro, and the method includes the following steps:

[0014] (1) Collect 6 g of fresh feces of infants and young children, put them into 30 mL of PBS and stir evenly, and then filter the solids with a gauze to obtain a fecal suspension;

[0015] (2) Inject the fecal suspension obtained in step (1) into the ascending colon reactor, transverse colon reactor, and descending colon reactor of the above medium, and carry out colonization culture for 24 h under anaerobic conditions to respectively obtain the intestinal flora and intestinal gas of infants and young children at the ascending colon, transverse colon, and descending colon stages;

[0016] In step (1), the fresh feces are collected by a fecal collector, and then every 1 g of feces is suspended in 5 mL of PBS, and the time from feces collection to injection into the reactor is controlled within 10 min.

[0017] In step (2), under the anaerobic conditions, dynamic culture is carried out at 37 °C for 24 h, the simulated peristaltic frequency of each reactor is 3 times per minute, the stable pH of the ascending colon reactor is 6.0, the stable pH of the transverse colon reactor is 6.5, the stable pH of the descending colon reactor is 7.0, and the addition amount of the fecal suspension in 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 intestinal flora of infants and young children for a long time, fresh 10% medium can be injected every 8 h for stable growth.

[0020] The headspace gas of each reactor consists of 100% nitrogen to ensure an anaerobic environment. As the culture time extends, colonization samples and intestinal gas are regularly taken out from the reactor for measurement.

[0021] Beneficial effects:

[0022] The bionic colon reactor of the present invention is based on gastrointestinal physiology, biotechnology and fermentation engineering. By referring to the real parameters in infants, it well simulates the real intestinal environment of domestic infants in vitro and reproduces their intestinal flora ecology and intestinal gas distribution. The method of the present invention uses an infant intestinal flora culture medium combined with a bionic colon reactor to establish a colonization strategy for infant intestinal microorganisms and intestinal gas, and can construct a database of normal infant intestinal microorganisms and intestinal gas, which helps to accurately diagnose and personalized treatment of the infant intestinal microecology, better evaluate the health status of infants and formulate preventive measures. Description of the Drawings

[0023] Figure 1 : Comparison of species richness and gas components after culturing with ordinary culture medium and infant intestinal flora and intestinal gas culture medium. Detailed Embodiments

[0024] The descriptions of the components of the culture medium involved in the following embodiments are as follows:

[0025] The yeast extract, tryptone, mucin, mycoprotein, fructooligosaccharide, galactooligosaccharide, inulin, resistant dextrin, human milk oligosaccharide, dietary fiber, sialic acid, algal oil DHA, potassium chloride, sodium chloride, potassium dihydrogen phosphate, sodium sulfate, bile salt, vitamin mixture, and plant extract are all of analytical pure concentration.

[0026] The mucin includes porcine mucin, bovine mucin, mussel mucin, etc.

[0027] The mycoprotein includes lentinus edodes powder, ganoderma lucidum powder, auricularia auricula powder, boletus edulis powder, dictyophora indusiata powder, agaricus blazei powder, hericium erinaceus powder, pleurotus nebrodensis powder, cordyceps sinensis powder.

[0028] The insect protein includes proteins extracted from eggs, larvae, adults, pupae, moths, etc., such as bean caterpillar powder, silkworm pupa.

[0029] The plant extract mixture includes puerarin, carotene, bioflavonoids, phytoandrogens, soy isoflavones, limonin, lycopene, catechins, anthocyanins, resveratrol.

[0030] The addition amount of vitamin B1 in the vitamin mixture per 1 L is 0.5 g, the addition amount of vitamin B2 is 0.5 g, the addition amount of nicotinic acid is 5 g, the addition amount of folic acid is 10 g, and the addition amount of pantothenic acid is 15 g. When the culture medium is a solid culture medium, the addition amount of agar powder is 1-1.4%.

[0031] Traditional culture medium: 4 g / L of bovine brain extract powder, 4 g / L of bovine heart extract powder, 5 g / L of peptone, 16 g / L of casein peptone, 5 g / L of sodium chloride, 2 g / L of glucose, 2.5 g / L of disodium hydrogen phosphate, 13.5 g / L of agar.

[0032] Example 1: In vitro spatio-temporal dynamic colonization culture of infant intestinal flora and intestinal gas

[0033] The specific steps are as follows:

[0034] (1) Preparation of fecal suspension

[0035] Collect fresh fecal samples from 5 healthy infants under 3 years old. The infants have not taken antibiotics and other drugs that interfere with the intestinal microecology and have no gastrointestinal digestive system diseases in the past 3 months; the fresh feces are collected by a fecal collector, and then every 1 gram of feces is suspended in 5 mL of PBS buffer, and then the solids are filtered with gauze to obtain a fecal 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 for infant intestinal flora and intestinal gas

[0039]

[0040] (3) In vitro spatio-temporal dynamic colonization of intestinal flora and intestinal gas; specifically as follows:

[0041] Add 200 mL of culture medium to the ascending colon reactor, transverse colon reactor and descending colon reactor, inject 10 mL of fecal suspension respectively, and culture dynamically at 37 °C for 24 h under anaerobic conditions. The simulated peristaltic frequency of each reactor is 3 times per minute. The stable pH of the ascending colon reactor is 6.0, the stable pH of the transverse colon reactor is 6.5, and the stable pH of the descending colon reactor is 7.0. 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. For the infant intestinal flora that maintains a steady state for a long time, fresh 10% culture medium can be injected every 8 h for stable growth.

[0042] Anaerobic condition: The headspace gas of the reactor consists of 100% nitrogen

[0043] Measure the 16s RNA and gas components of the fermentation broth and intestinal gas after culturing in each reactor respectively:

[0044] Results show that: in terms of the gut microbiota structure, the culture medium of the present invention can significantly increase the colonization abundance of Enterobacteriaceae, Bifidobacteriaceae, Enterococcaceae, Veillonellaceae, and Staphylococcaceae compared to traditional culture media, which is similar to the real gut environment of infants. In terms of gut gases, it mainly consists of carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide, and volatile organic compounds (VOCs), which is similar to the real gut gases of infants. Specific parameters are as Figure 1 shown in Table 8 and Table 9.

[0045] Example 2: In vitro spatio-temporal dynamic colonization culture of infant gut microbiota and gut gases

[0046] The specific steps are as follows:

[0047] (1) Prepare fecal suspension

[0048] Collect fresh fecal samples from 5 healthy infants under 3 years old. The infants have not taken antibiotics or other drugs that interfere with the gut microecology and have no gastrointestinal digestive system diseases in the past 3 months; the fresh feces are collected by a fecal collector, and then 1 gram of feces is suspended in 5 mL of PBS buffer, and then the solid is filtered with gauze to obtain a fecal suspension;

[0049] (2) Prepare the culture medium

[0050] Prepare the culture medium according to the formula in Table 2:

[0051] Table 2 In vitro colonization culture medium for infant gut microbiota and gut gases

[0052]

[0053]

[0054] (3) In vitro spatio-temporal dynamic colonization of gut microbiota and gut gases; specifically as follows:

[0055] Add 200 mL of culture medium to the ascending colon reactor, transverse colon reactor, and descending colon reactor, respectively inject 10 mL of fecal suspension, and perform dynamic culture at 37 °C for 24 h under anaerobic conditions. The simulated peristaltic frequency of each reactor is 3 times per minute. The stable pH of the ascending colon reactor is 6.0, the stable pH of the transverse colon reactor is 6.5, and the stable pH of the descending colon reactor is 7.0. The stable pH of each reactor is adjusted up and down with 0.5 mol of hydrochloric acid and 0.5 mol of sodium hydroxide. For the infant intestinal flora that maintains a steady state for a long time, fresh 10% culture medium can be injected every 8 h for stable growth.

[0056] Anaerobic condition: The headspace gas of the reactor consists of 100% nitrogen

[0057] Measure the 16s RNA and gas components of the fermented liquid and intestinal gas after culturing in each of the above reactors respectively:

[0058] The results show that: in terms of the intestinal flora structure, the culture medium of the present invention can significantly increase the colonization abundance of Enterobacteriaceae, Bifidobacteriaceae, Enterococcaceae, Veillonellaceae, and Staphylococcaceae compared with the traditional culture medium, which is similar to the real intestinal environment of infants. In terms of intestinal gas, it is mainly composed of carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide, and volatile organic compounds (VOCs), which is similar to the real intestinal gas of infants. The specific parameters are as Figure 1 shown in Table 8 and Table 9.

[0059] Example 3: In vitro spatio-temporal dynamic colonization culture of infant intestinal flora and intestinal gas

[0060] The specific steps are as follows:

[0061] (1) Prepare fecal suspension

[0062] Collect fresh fecal samples from 5 healthy infants under 3 years old. The infants have not taken antibiotics or other drugs that interfere with the intestinal microecology in the past 3 months and have no gastrointestinal digestive system diseases, etc.; the fresh feces are collected by a fecal collector, and then each 1 g of feces is suspended in 5 mL of PBS buffer, and then the solid is filtered with gauze to obtain fecal suspension;

[0063] (2) Prepare the culture medium

[0064] Prepare the culture medium according to the formula in Table 3:

[0065] Table 3 In vitro colonization culture medium for infant intestinal flora and intestinal gas

[0066]

[0067] (3) Spatiotemporal dynamic colonization of intestinal flora and intestinal gas in vitro; specifically as follows:

[0068] Add 200 mL of culture medium to the ascending colon reactor, transverse colon reactor and descending colon reactor, inject 10 mL of fecal suspension respectively, and culture dynamically at 37 °C for 24 h under anaerobic conditions. The simulated peristaltic frequency of each reactor is 3 times per minute. The stable pH of the ascending colon reactor is 6.0, the stable pH of the transverse colon reactor is 6.5, and the stable pH of the descending colon reactor is 7.0. The stable pH of each reactor is adjusted up and down with 0.5 mol of hydrochloric acid and 0.5 mol of sodium hydroxide. For the intestinal flora of infants and young children that maintains a steady state for a long time, fresh 10% culture medium can be injected every 8 h for stable growth.

[0069] Anaerobic condition: The headspace gas of the reactor consists of 100% nitrogen

[0070] Measure the 16S RNA and gas components of the fermentation broth and intestinal gas after culturing in each reactor respectively:

[0071] The results show that: in terms of the intestinal flora structure, the culture medium of the present invention can significantly increase the colonization abundance of Enterobacteriaceae, Bifidobacteriaceae, Enterococcaceae, Veillonellaceae and Staphylococcaceae compared with the traditional culture medium, which is similar to the real intestinal environment of infants and young children. In terms of intestinal gas, it is mainly composed of carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide and volatile organic compounds (VOC), which is similar to the real intestinal gas of infants and young children. The specific parameters are as Figure 1 shown in Table 8 and Table 9.

[0072] Comparative Example 1: Spatiotemporal dynamic colonization culture of intestinal flora and intestinal gas in infants and young children (the addition amount of culture medium components is lower than the minimum value)

[0073] The specific steps are as follows:

[0074] (1) Preparation of fecal suspension

[0075] Collect fresh fecal samples from 5 healthy infants and young children under 3 years old. The infants and young children have not taken antibiotics and other drugs that interfere with the intestinal microecology and have no gastrointestinal digestive system diseases in the past 3 months; the fresh feces are collected by a fecal collector, and then each 1 g of feces is suspended in 5 mL of PBS buffer, and then the solid is filtered with gauze to obtain a fecal suspension;

[0076] (2) Prepare the culture medium

[0077] Prepare the culture medium according to the formula in Table 4:

[0078] Table 4 In vitro colonization culture medium for infant intestinal flora and intestinal gas

[0079]

[0080] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically as follows:

[0081] Add 200 mL of the culture medium to the ascending colon reactor, transverse colon reactor and descending colon reactor, inject 10 mL of the fecal suspension respectively, and perform dynamic culture at 37 °C for 24 h under anaerobic conditions. The simulated peristaltic frequency of each reactor is 3 times per minute. The stable pH of the ascending colon reactor is 6.0, the stable pH of the transverse colon reactor is 6.5, and the stable pH of the descending colon reactor is 7.0. 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. For the infant intestinal flora that maintains a steady state for a long time, fresh 10% culture medium can be injected every 8 h for stable growth.

[0082] Anaerobic condition: The headspace gas of the reactor consists of 100% nitrogen

[0083] Measure the 16S RNA and gas components of the fermentation broth and intestinal gas after culturing in each of the above reactors respectively:

[0084] The results show that: in terms of the intestinal flora structure, compared with the real infant intestinal environment, the colonization abundances of Enterobacteriaceae, Bifidobacteriaceae, Enterococcaceae, Veillonellaceae and Staphylococcaceae in the fermentation broth are significantly reduced. In terms of intestinal gas, it is mainly composed of carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide and volatile organic compounds (VOC), but the gas production is different from that of real infant intestinal gas. The specific parameters are as Figure 1 shown in Table 8 and Table 9.

[0085] Comparative Example 2: In vitro spatiotemporal dynamic colonization culture of infant intestinal flora and intestinal gas (the addition amount of culture medium components is higher than the maximum value)

[0086] The specific steps are as follows:

[0087] (1) Prepare the fecal suspension

[0088] Fresh fecal samples were collected from 5 infants under 3 years old. The infants had not taken antibiotics or other drugs that interfere with the intestinal microecology and had no gastrointestinal digestive system diseases in the past 3 months; the fresh feces were collected by a fecal collector, and then 1 gram of feces was suspended in 5 mL of PBS buffer, and then the solids were filtered with gauze to obtain a fecal suspension;

[0089] (2) Prepare the culture medium

[0090] Prepare the culture medium according to the formula in Table 5:

[0091] Table 5 In vitro colonization culture medium for infant intestinal flora and intestinal gas

[0092]

[0093]

[0094] (3) In vitro spatiotemporal dynamic colonization of intestinal flora and intestinal gas; specifically as follows:

[0095] Add 200 mL of the culture medium to the ascending colon reactor, transverse colon reactor and descending colon reactor, inject 10 mL of the fecal suspension respectively, and culture dynamically at 37 °C for 24 h under anaerobic conditions. The simulated peristaltic frequency of each reactor is 3 times per minute. The stable pH of the ascending colon reactor is 6.0, the stable pH of the transverse colon reactor is 6.5, and the stable pH of the descending colon reactor is 7.0. The stable pH of each reactor is adjusted up and down with 0.5 mol of hydrochloric acid and 0.5 mol of sodium hydroxide. For the infant intestinal flora that maintains a steady state for a long time, fresh 10% culture medium can be injected every 8 h for stable growth.

[0096] Anaerobic condition: The headspace gas of the reactor consists of 100% nitrogen

[0097] Measure the 16s RNA and gas components of the fermentation broth and intestinal gas after culturing in each reactor respectively:

[0098] The results show that: in terms of the intestinal flora structure, compared with the real infant intestinal environment, there is no significant difference in the colonization abundance of Enterobacteriaceae, Bifidobacteriaceae, Enterococcaceae, Veillonellaceae and Staphylococcaceae in the fermentation broth. In terms of intestinal gas, carbon dioxide decreases and volatile organic compounds (VOC) increase. The specific parameters are as Figure 1 , shown in Table 8 and Table 9.

[0099] Comparative Example 3: In vitro spatio-temporal dynamic colonization culture of infant intestinal flora and intestinal gas (without adding mucin)

[0100] The specific steps are as follows:

[0101] (1) Preparation of fecal suspension

[0102] Collect fresh fecal samples from 5 healthy infants under 3 years old. The infants have not taken antibiotics or other drugs that interfere with the intestinal microecology and have no gastrointestinal digestive system diseases in the past 3 months; the fresh feces are collected by a fecal collector, and then 1 gram of feces is suspended in 5 mL of PBS buffer, and then the solid is filtered with gauze to obtain a fecal suspension;.

[0103] (2) Preparation of culture medium

[0104] Prepare the culture medium according to the formula in Table 6:

[0105] Table 6 In vitro colonization culture medium for infant intestinal flora and intestinal gas

[0106]

[0107] (3) In vitro spatio-temporal dynamic colonization of intestinal flora and intestinal gas; specifically as follows:

[0108] Add 200 mL of culture medium to the ascending colon reactor, transverse colon reactor and descending colon reactor, inject 10 mL of fecal suspension respectively, and culture dynamically at 37 °C for 24 h under anaerobic conditions. The simulated peristaltic frequency of each reactor is 3 times per minute. The stable pH of the ascending colon reactor is 6.0, the stable pH of the transverse colon reactor is 6.5, and the stable pH of the descending colon reactor is 7.0. 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. For the infant intestinal flora that maintains a steady state for a long time, 10% fresh culture medium can be injected every 8 h for stable growth.

[0109] Anaerobic condition: The headspace gas of the reactor consists of 100% nitrogen

[0110] Measure the 16s RNA and gas components of the fermentation broth and intestinal gas after culturing in each reactor respectively:

[0111] The results show that: in terms of the intestinal flora structure, compared with the real infant intestinal environment, the colonization abundances of Bifidobacteriaceae and Veillonellaceae in the fermentation broth are significantly reduced. In terms of intestinal gas, it is mainly composed of carbon dioxide, hydrogen, methane, nitric oxide, hydrogen sulfide and volatile organic compounds (VOC), and there is no difference from the real infant intestinal gas. The specific parameters are as Figure 1as shown in Table 8 and Table 9.

[0112] Comparative Example 4: In vitro spatio-temporal dynamic colonization culture of infant intestinal flora and intestinal gas (without adding sodium sulfate)

[0113] The specific steps are as follows:

[0114] (1) Preparation of fecal suspension

[0115] Collect fresh fecal samples from 5 healthy infants under 3 years old. The infants have not taken antibiotics or other drugs that interfere with the intestinal microecology and have no gastrointestinal digestive system diseases in the past 3 months; the fresh feces are collected by a fecal collector, and then 1 gram of feces is suspended in 5 mL of PBS buffer, and then the solid is filtered with gauze to obtain a fecal suspension.

[0116] (2) Preparation of culture medium

[0117] Prepare the culture medium according to the formula in Table 7:

[0118] Table 7 In vitro colonization culture medium for infant intestinal flora and intestinal gas

[0119]

[0120]

[0121] (3) In vitro spatio-temporal dynamic colonization of intestinal flora and intestinal gas; specifically as follows:

[0122] Add 200 mL of culture medium to the ascending colon reactor, transverse colon reactor and descending colon reactor, inject 10 mL of fecal suspension respectively, and culture dynamically at 37 °C for 24 h under anaerobic conditions. The simulated peristaltic frequency of each reactor is 3 times per minute. The stable pH of the ascending colon reactor is 6.0, the stable pH of the transverse colon reactor is 6.5, and the stable pH of the descending colon reactor is 7.0. 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. For the infant intestinal flora that maintains a steady state for a long time, 10% fresh culture medium can be injected every 8 h for stable growth.

[0123] Anaerobic condition: The headspace gas of the reactor consists of 100% nitrogen

[0124] Measure 16s RNA and gas components of the fermentation broth and intestinal gas after culturing in each reactor respectively:

[0125] The results showed that: in terms of the gut microbiota structure, compared with the real infant gut environment, there were no significant differences in the colonization abundances of Enterobacteriaceae, Bifidobacteriaceae, Enterococcaceae, Veillonellaceae, and Staphylococcaceae in the fermentation broth. In terms of gut gases, no hydrogen sulfide was produced because the medium lacked sulfur elements and the gut microbiota could not utilize sulfur for metabolism to produce hydrogen sulfide. The specific parameters are as Figure 1 shown in Table 8 and Table 9.

[0126] Table 8 Comparison of species richness after culturing in normal medium and infant gut microbiota and gut gas medium

[0127]

[0128]

[0129] Table 9 Comparison of gas components after culturing in normal medium and infant gut microbiota and gut gas medium

[0130]

[0131] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A culture medium for colonizing the intestinal flora and intestinal gas of infants and young children, characterized in that, It includes the following components: 1 - 3 g / L of yeast extract, 1 - 2 g / L of tryptone, 1 - 2 g / L of mucin, 0.5 - 1.5 g / L of bacterial protein, 0.2 - 0.4 g / L of insect protein, 0.5 - 1 g / L of soy protein, 3 - 6 g / L of fructooligosaccharide, 1 - 2 g / L of galactooligosaccharide, 0.5 - 1.5 g / L of inulin, 1 - 2 g / L of resistant dextrin, 0.5 - 1.5 g / L of human milk oligosaccharide, 1 - 2 g / L of dietary fiber, 0.5 - 1.5 g / L of sialic acid, 0.5 - 1.5 g / L of algal oil DHA, 0.5 - 1.5 g / L of potassium chloride, 0.5 - 1.5 g / L of sodium chloride, 1 - 2 g / L of potassium dihydrogen phosphate, 0.5 - 1.5 g / L of sodium sulfate, 0.2 - 0.6 g / L of bile salt, 0.4 - 0.8 g / L of plant extract mixture, 1 - 2 mL / L of vitamin mixture, and the balance is water.

2. The culture medium for colonizing the intestinal flora and intestinal gas of infants and young children according to claim 1, wherein The mucin includes one or a mixture of several of porcine mucin, bovine mucin, and mussel mucin; The bacterial protein includes one or a mixture of several of lentinus edodes powder, ganoderma lucidum powder, auricularia auricula powder, boletus edulis powder, dictyophora indusiata powder, agaricus blazei powder, hericium erinaceus powder, pleurotus nebrodensis powder, cordyceps sinensis powder; 3. The culture medium for colonizing the intestinal flora and intestinal gas of infants and young children according to claim 1, wherein, The insect protein includes one or a mixture of several of proteins extracted from eggs, larvae, adults, pupae, moths, etc.

4. The culture medium for colonizing the intestinal flora and intestinal gas of infants and young children according to claim 1, wherein The plant extract mixture includes one or a mixture of several of puerarin, carotene, bioflavonoids, phytoandrogens, soy isoflavones, limonin, lycopene, catechins, anthocyanins, resveratrol.

5. The culture medium for colonizing the intestinal flora and intestinal gas of infants and young children according to claim 1, wherein In the vitamin mixture, the addition amount of vitamin B1 per 1 L is 0.5 g, the addition amount of vitamin B2 is 0.5 g, the addition amount of niacin is 5 g, the addition amount of folic acid is 10 g, and the addition amount of pantothenic acid is 15 g.

6. A method for colonizing the intestinal flora and intestinal gas of infants in vitro, characterized in that, It includes the following steps: (1) Collect infant feces, put them into PBS and stir evenly, then filter the solids with a gauze to obtain a fecal suspension; (2) Inject the fecal suspension obtained in step (1) into the ascending colon reactor, transverse colon reactor, and descending colon reactor of the above medium, and carry out colonization culture under anaerobic conditions for 24 h to obtain the intestinal flora and intestinal gas of infants at the ascending colon, transverse colon, and descending colon stages respectively.

7. The method for in vitro colonization of intestinal flora and intestinal gas in infants according to claim 6, wherein, In step (1), collect the feces of obese people with a fecal collector, suspend every 1 g of feces in 5 mL of PBS, and control the time from feces collection to injection into the reactor within 10 min.

8. The method for colonizing the intestinal flora and intestinal gas of infants in vitro according to claim 6, wherein, In step (2), for the anaerobic condition colonization culture, the culture temperature is 37 °C; The simulated peristaltic frequencies of the ascending colon reactor, transverse colon reactor, and descending colon reactor are 3 times per minute. The stable pH of the ascending colon reactor is 6.0, the stable pH of the transverse colon reactor is 6.5, and the stable pH of the descending colon reactor is 7.

0. The ratio of the fecal suspension to the medium in each reactor is 5 mL:100 mL.

9. The method for in vitro colonization of intestinal flora and intestinal gas in infants according to claim 6, characterized in that, In step (2), the stable pH of each reactor is adjusted up and down with 0.5 mol of hydrochloric acid and 0.5 mol of sodium hydroxide.

10. The method for colonizing the intestinal flora and intestinal gas of infants in vitro according to claim 8, characterized in that, In step (2), inject fresh 10% medium every 8 h.

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