Application of lipopolysaccharide, vitamin B5 or isoleucine in increasing the biomass of Akkermansia muciniphila

Abstract

The present invention discloses the application of lipopolysaccharide, vitamin B5 or isoleucine in increasing the biomass of Akkermansia muciniphila. Based on the whole-genome sequence information of Akkermansia muciniphila, the present invention analyzes its metabolic pathways and explores novel growth factors that can promote the growth of Akkermansia muciniphila to solve the problems of long culture period and low biomass of Akkermansia muciniphila at present. The OD of the 18-hour fermentation broth of Akkermansia muciniphila 600 can reach above 5.3, which promotes the wide application of Akkermansia muciniphila.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology and relates to the application of lipopolysaccharide, vitamin B5 and isoleucine in increasing the biomass of Akkermansia muciniphila. Background Art

[0002] Akkermansia muciniphila, hereinafter referred to as Akk, is a strictly anaerobic Gram-negative bacterium colonizing the gastrointestinal mucus layer, spherical or short rod-shaped, with good dispersion, and has the growth characteristic of using gastrointestinal mucin as the sole carbon and nitrogen source. It was first isolated from feces. Research shows that it is negatively correlated with a variety of diseases, including inflammatory bowel disease, Alzheimer's disease, obesity and diabetes, etc. Its content is one of the important markers for measuring the balance of the intestinal microecosystem. At present, the prevention and treatment of diseases by probiotics has become a research hotspot, and Akkermansia muciniphila, as a common colonizing bacterium in the human intestinal mucus layer, has gradually been regarded as one of the promising candidates among the second-generation probiotics. After A-Mansia Biotech submitted an application for this new food ingredient to the European Commission in October 2019 and through extensive scientific review, Akk was approved by the European Union and will be used for weight management. The expert group of the European Food Safety Authority concluded that under the specified usage conditions and intake levels, this ingredient is safe as a food ingredient. That is, under the condition that the number of live Akk is less than 10 CFU / g, the daily intake of 3.4×10 10 cells is safe. The European Food Safety Authority (EFSA) has approved the first new generation of probiotic Akkermansia muciniphila isolated from the human gut microbiota as a new food ingredient. Against the background of being approved by the European Union, in June 2020, Pendulum Therapeutics launched the first food containing Akk bacteria - Pendulum Glucose Control (PGC). This product has proven its safety through clinical trials, breaking the bottleneck of the "zero application" of Akk bacteria and completing a Series C financing of $54 million in 2021, indicating that Akk bacteria have huge market demand and development space. At present, many biopharmaceutical companies and research institutions have invested heavily in the research of Akk bacteria. As the next-generation probiotic that has attracted great attention, Akk bacteria have broad development prospects. Live bacteria drugs represented by the addition of Akk bacteria are the current research and application hotspots.

[0003] Akkermansia muciniphila can grow in media such as media supplemented with bicarbonate buffer, brain heart infusion broth (BHI) (containing 0.2% mucin), etc., but the growth is slow and the biomass is low when reaching the growth plateau phase, which poses a great obstacle to its industrial application. The research by Wu Zhongqin et al. showed that Akkermansia muciniphila can reach the growth stationary phase after 24 h in BHI broth (containing 0.2% mucin), and the OD 600 is 0.5 - 0.6. The Akk-G8-20211130-06 and Akk-G8-20211130-22 isolated from goat intestines by Yang Xin reached the growth stationary phase with an OD 600 of 0.5 - 0.6 in brain heart infusion broth medium. For example, CN111304133A discloses a medium for culturing Akkermansia muciniphila, its usage method and application. The medium includes brain heart infusion, porcine gastric mucin, L-cysteine hydrochloride, threonine, N-acetyl-D-glucosamine and vitamins, enabling Akkermansia muciniphila to still obtain sufficient nutrients in the medium containing a relatively low concentration of brain heart infusion and porcine gastric mucin, ensuring the normal growth and reproduction of the strain, and the OD value of the obtained bacterial liquid is 0.8211 - 0.8741.

[0004] In summary, there are still problems such as long culture period and low biomass in the current culture of Akkermansia muciniphila. Therefore, it is of great significance to develop a method for efficiently culturing Akkermansia muciniphila. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art and actual needs, the present invention provides the application of lipopolysaccharide, vitamin B5 or isoleucine in improving the biomass of Akkermansia muciniphila, and explores new growth factors that can promote the growth of Akkermansia muciniphila, so as to increase the biomass of culturing Akkermansia muciniphila and shorten the culture period.

[0006] To achieve this purpose, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides the application of lipopolysaccharide, vitamin B5 or isoleucine in the preparation of a medium for improving the biomass of Akkermansia muciniphila.

[0008] In the present invention, based on the whole genome sequence information of Akkermansia muciniphila, its metabolic pathway is analyzed. Based on the analysis results, new growth factors that can promote the growth of Akkermansia muciniphila are explored to solve the problems of long culture period and low biomass of Akkermansia muciniphila at present, and to promote the wide application of Akkermansia muciniphila.

[0009] It can be understood that the lipopolysaccharide refers to a complex of lipid and polysaccharide.

[0010] It can be understood that the present invention analyzes the metabolic pathway of Akkermansia muciniphila based on the whole-genome sequence information to mine novel growth factors, with wide applicability and theoretically applicable to any Akkermansia muciniphila strain, such as Akkermansia muciniphila Akk007, etc.

[0011] In a second aspect, the present invention provides a medium for increasing the biomass of Akkermansia muciniphila, and the medium for increasing the biomass of Akkermansia muciniphila contains growth factors, a carbon source, a nitrogen source, and inorganic salts; the growth factors include any one or a combination of at least two of lipopolysaccharide, vitamin B5, or isoleucine.

[0012] Based on the mined novel growth factors, the present invention further designs a medium for increasing the biomass of Akkermansia muciniphila, with the expectation of being applied to the cultivation of Akkermansia muciniphila to increase the biomass and shorten the cultivation cycle.

[0013] In the present invention, it is also found that the synergistic cooperation among lipopolysaccharide, vitamin B5, and isoleucine can further increase the biomass of Akkermansia muciniphila.

[0014] Preferably, the carbon source and nitrogen source include brain heart infusion powder, mucin, peptone, and glucose.

[0015] Preferably, the inorganic salts include sodium chloride and disodium hydrogen phosphate.

[0016] It can be understood that the brain heart infusion powder, mucin, peptone, etc. are all well-known culture medium components in the art, and those skilled in the art can know and obtain them (prepared by existing methods or directly purchased below). For example, the brain heart infusion powder refers to a reagent obtained by screening, refining, digesting, and drying the brains and hearts of animals (such as cows, etc.), which can be in a mixed package or independently packaged as brain infusion powder and heart infusion powder, and commercially available such reagents are all applicable to the present invention. Mucin refers to a complex composed of oligosaccharides and amino acids and contains one or more of N-acetylglucosamine, N-acetylgalactosamine, galactose, and fucose.

[0017] In a specific embodiment of the present invention, the growth factors can also be added to the traditional BHI medium to obtain the medium for increasing the biomass of Akkermansia muciniphila.

[0018] Preferably, the mass percentage of lipopolysaccharide in the medium for increasing the biomass of Akkermansia muciniphila is 0.5% - 3.5%, including but not limited to 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.5%, 2.8%, 3%, 3.1%, 3.2%, 3.3%, or 3.4%, preferably 1.5% - 3.5%, and further preferably 2.0% - 3.0%.

[0019] Preferably, the mass percentage of vitamin B5 in the medium for increasing the biomass of Akkermansia muciniphila is 0.01% to 0.3%, including but not limited to 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.1%, 0.12%, 0.15%, 0.18%, 0.2%, 0.22%, 0.25%, 0.26%, 0.28% or 0.29%, and preferably 0.04% to 0.2%.

[0020] Preferably, the mass percentage of isoleucine in the medium for increasing the biomass of Akkermansia muciniphila is 0.01% to 0.8%, including but not limited to 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.72%, 0.75%, 0.78% or 0.79%, and preferably 0.05% to 0.6%, and more preferably 0.1% to 0.4%.

[0021] Preferably, the concentration of brain heart infusion powder in the medium for increasing the biomass of Akkermansia muciniphila is 30 to 40 g / L, including but not limited to 31, 32, 33, 34, 35, 36, 37, 38 or 39 g / L, etc.

[0022] Preferably, the concentration of mucin in the medium for increasing the biomass of Akkermansia muciniphila is 0.1 to 1.0 g / L, including but not limited to 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 g / L, etc.

[0023] Preferably, the concentration of peptone in the medium for increasing the biomass of Akkermansia muciniphila is 5 to 20 g / L, including but not limited to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 g / L, etc.

[0024] Preferably, the concentration of glucose in the medium for increasing the biomass of Akkermansia muciniphila is 10 to 20 g / L, including but not limited to 11, 12, 13, 14, 15, 16, 17, 18 or 19 g / L, etc.

[0025] Preferably, the concentration of sodium chloride in the medium for increasing the biomass of Akkermansia muciniphila is 5 to 15 g / L, including but not limited to 6, 7, 8, 9, 10, 11, 12, 13 or 14 g / L, etc.

[0026] Preferably, the concentration of disodium hydrogen phosphate in the medium for increasing the biomass of Akkermansia muciniphila is 1 to 5 g / L, including but not limited to 2, 3 or 4 g / L, etc.

[0027] Preferably, the medium for increasing the biomass of Akkermansia muciniphila contains 30 - 40 g / L of brain heart infusion powder, 0.1 - 1.0 g / L of mucin, 10 - 20 g / L of peptone, 10 - 20 g / L of glucose, 5 - 15 g / L of sodium chloride, 1 - 5 g / L of disodium hydrogen phosphate, and growth factors; the growth factors include any one or a combination of at least two of lipopolysaccharide, vitamin B5, or isoleucine, the mass percentage of lipopolysaccharide is 0.5% - 3.5%, the mass percentage of vitamin B5 is 0.01% - 0.3%, and the mass percentage of isoleucine is 0.01% - 0.8%.

[0028] Thirdly, the present invention provides the application of lipopolysaccharide, vitamin B5, isoleucine, or the medium for increasing the biomass of Akkermansia muciniphila described in the second aspect in increasing the biomass of Akkermansia muciniphila.

[0029] Fourthly, the present invention provides a method for increasing the biomass of Akkermansia muciniphila, the method includes inoculating Akkermansia muciniphila in a medium containing any one or a combination of at least two of lipopolysaccharide, vitamin B5, and isoleucine and culturing.

[0030] Preferably, the medium for culturing includes the medium for increasing the biomass of Akkermansia muciniphila described in the second aspect.

[0031] Preferably, the culturing includes anaerobic culturing, and the gas ratio of the anaerobic culturing is 90 - 99% N 2 、0 - 5% H 2 、0 - 5% CO 2 .

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] Based on the whole genome sequence information of Akkermansia muciniphila, the present invention analyzes its metabolic pathway, and excavates new growth factors that can promote the growth of Akkermansia muciniphila to solve the problems of long culture period and low biomass of Akkermansia muciniphila at present. The OD of the 18 - hour fermentation broth of Akkermansia muciniphila 600 can reach above 5.3, promoting the wide application of Akkermansia muciniphila. Description of the Drawings

[0034] Figure 1 is the annotation result diagram of the metabolic pathway information of Akkermansia muciniphila;

[0035] Figure 2A is the UDP - glucose metabolic pathway diagram of Akkermansia muciniphila;

[0036] Figure 2BIt is the cysteine metabolic pathway diagram of Akkermansia muciniphila;

[0037] Figure 2C It is the lipopolysaccharide metabolic pathway diagram of Akkermansia muciniphila;

[0038] Figure 2D It is the pantothenate metabolic pathway diagram of Akkermansia muciniphila;

[0039] Figure 2E It is the isoleucine metabolic pathway diagram of Akkermansia muciniphila;

[0040] Figure 3 It is the biomass result diagram of Akkermansia muciniphila under different growth factor conditions;

[0041] Figure 4 It is the result diagram of the effect of lipopolysaccharide concentration on the growth of Akkermansia muciniphila;

[0042] Figure 5 It is the result diagram of the effect of vitamin B5 concentration on the growth of Akkermansia muciniphila;

[0043] Figure 6 It is the result diagram of the effect of isoleucine concentration on the growth of Akkermansia muciniphila;

[0044] Figure 7 Result diagram of the effect of the combination of lipopolysaccharide, vitamin B5 and isoleucine on the growth of Akkermansia muciniphila;

[0045] Figure 8 It is the result diagram of the pilot-scale culture of Akkermansia muciniphila. Detailed implementation manners

[0046] To further elaborate on the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with embodiments and drawings. It can be understood that the specific implementation manners described herein are only used to explain the present invention, rather than limiting the present invention.

[0047] For those not specifying specific techniques or conditions in the embodiments, they shall be in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product specifications. For those reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained through regular commercial channels.

[0048] In the specific embodiment of the present invention, Akkermansia muciniphila Akk007 is taken as an example to verify the method of the present invention. Akkermansia muciniphila Akk007 is preserved in the Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CCTCC NO.M20231117.

[0049] In the specific embodiments of the present invention, UDP-glucose was purchased from Guangzhou Huahui Biotechnology Co., Ltd.; cysteine was purchased from Hebei Huaheng Biotechnology Co., Ltd.; lipopolysaccharide was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; vitamin B5 was purchased from Anhui Huaheng Biotechnology Co., Ltd.; isoleucine was purchased from Hebei Huaheng Biotechnology Co., Ltd.

[0050] Example 1

[0051] In this example, a culture protocol for Akkermansia muciniphila was designed.

[0052] The complete genome sequence information of Akkermansia muciniphila was obtained through a sequencing platform and its metabolic pathway information was annotated. As Figure 1 shown, where different colors represent the proportion of data information classified under the KEGG database.

[0053] Metabolic pathways with complete metabolic pathway genes were selected for verification: verified including a: uridine diphosphate glucose (UDP-glucose) metabolic pathway; b: cysteine metabolic pathway; c: lipopolysaccharide metabolic pathway; d: pantothenate metabolic pathway; e: isoleucine metabolic pathway. The relevant information diagrams of the metabolic pathways are as Figures 2A - 2E shown.

[0054] Fermentation culture protocol: Six groups of BHI synthetic media were prepared. The ck group was not treated. Group a was added with 0.1% UDP-glucose, group b was added with 0.1% cysteine, group c was added with 0.1% lipopolysaccharide, group d was added with 0.1% vitamin B5, and group e was added with 0.1% isoleucine. After sterilization at 121°C for 20 min, it was ready for use.

[0055] Akkermansia muciniphila (Akk007) stored in an ultra-low temperature freezer at -80°C was first placed at 4°C for 2 h and then restored to 25°C, and inoculated into 200 mL of BHI liquid medium at an inoculation amount of 5%, and continuously activated 3 times.

[0056] Inoculate into 1.2 L of medium in sequence at an inoculation amount of 5%. In each group of medium, anaerobic fermentation culture was carried out at 37°C (using nitrogen for pressure maintenance during the process). After collecting samples using a BODS microbial fermentation on-line detection system, the biomass OD 600 value was measured using a spectrophotometer and the data was recorded. The qualitative results of growth factors are as Figure 3 shown.

[0057] From Figure 3It can be seen that compared with the basal BHI medium, the addition of lipopolysaccharide, vitamin B5 or isoleucine can all increase the biomass of Akkermansia muciniphila.

[0058] Example 2

[0059] In this example, Akkermansia muciniphila was cultured.

[0060] (1) The Akkermansia muciniphila (Akk007) stored in an ultra-low temperature freezer at -80°C was first placed at 4°C for 2 h and then restored to 25°C, and inoculated into 200 mL of BHI liquid medium at an inoculation amount of 3% and continuously activated 3 times.

[0061] (2) Fermentation culture: Quantitative analysis of lipopolysaccharide was carried out. Eight groups of BHI media were prepared and lipopolysaccharide with contents of 0, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0% and 3.5% was added in sequence. After sterilization at 121°C for 20 min, it was ready for use. The strains were inoculated into each 1.2 L of the grouped media at an inoculation amount of 5% in sequence. After anaerobic fermentation culture at 37°C for 18 h, the biomass OD 600 value was measured using a spectrophotometer and the data were recorded. The results are as Figure 4 shown. Adding lipopolysaccharide to the BHI medium can increase the biomass of Akkermansia muciniphila. In addition, controlling the addition amount of lipopolysaccharide can further increase the biomass, and the optimal concentration is 2.5%.

[0062] Example 3

[0063] In this example, Akkermansia muciniphila was cultured.

[0064] (1) Strain activation: The Akkermansia muciniphila (Akk007) stored in an ultra-low temperature freezer at -80°C was first placed at 4°C for 2 h and then restored to 25°C, and inoculated into 200 mL of BHI liquid medium at an inoculation amount of 6% and continuously activated 3 times.

[0065] (2) Fermentation culture: Quantitative analysis of vitamin B5 was carried out. Eight groups of BHI media were prepared and vitamin B5 with contents of 0, 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.10% and 0.20% was added in sequence. After sterilization at 121°C for 20 min, it was ready for use. The strains were inoculated into each 1.2 L of the grouped media at an inoculation amount of 5% in sequence. After anaerobic fermentation culture at 37°C for 18 h, the biomass OD 600 value was measured using a spectrophotometer and the data were recorded. The results are shown in Figure 5, adding vitamin B5 to the BHI medium can increase the biomass of Akkermansia muciniphila. In addition, controlling the addition amount of vitamin B5 can further increase the biomass, and the optimal concentration is 0.04%.

[0066] Example 4

[0067] In this example, Akkermansia muciniphila was cultured.

[0068] (1) Strain activation: The Akkermansia muciniphila (Akk007) stored in a -80°C ultra-low temperature refrigerator was first placed at 4°C for 2 h and then restored to 20°C, and inoculated into 200 mL of BHI liquid medium at an inoculation amount of 4%, and continuously activated 3 times.

[0069] (2) Fermentation culture: Quantitative analysis of isoleucine was carried out, and eight groups of BHI media were prepared and isoleucine with contents of 0, 0.01%, 0.05%, 0.1%, 0.2%, 0.4%, 0.6% and 0.8% were added in turn. After sterilization at 121°C for 20 min, they were inoculated into each 1.2 L of the grouped media at an inoculation amount of 5% in turn. After anaerobic fermentation culture at 37°C for 18 h, the biomass OD 600 value was measured using a spectrophotometer and the data was recorded. The results are as Figure 6 shown. Adding isoleucine to the BHI medium can increase the biomass of Akkermansia muciniphila. In addition, controlling the addition amount of isoleucine can further increase the biomass, and the optimal concentration is 0.2%.

[0070] Example 5

[0071] In this example, Akkermansia muciniphila was cultured.

[0072] (1) Strain activation: The strain stored in a -80°C ultra-low temperature refrigerator was first placed at 4°C for 2 h and then restored to 30°C, and inoculated into 200 mL of BHI liquid medium at an inoculation amount of 5%, and continuously activated 3 times.

[0073] (2) Fermentation culture: According to the quantitative analysis results of Examples 2 - 4, 25 g / L lipopolysaccharide, 0.4 g / L vitamin B5 and 2 g / L isoleucine were mixed in a weight ratio of 1:1:1 to obtain mixture H. Eight groups of BHI media were prepared and mixture H with contents of 0, 0.2%, 0.4%, 0.8%, 1.0%, 1.2%, 1.4% and 1.6% were added in turn. After sterilization at 121°C for 20 min, they were inoculated into 1.2 L of the medium at an inoculation amount of 5% in turn. After anaerobic fermentation culture at 37°C for 18 h for each grouped medium, the biomass OD 600 value was measured using a spectrophotometer and the data was recorded. The results are as Figure 7 shown.

[0074] By Figure 7 It can be seen that the quantitative analysis of the mixed substance of lipopolysaccharide, vitamin B5 and isoleucine, which have a promoting effect on growth, showed that the mixed substance showed similar results to the single factor results, and the synergistic combination of lipopolysaccharide, vitamin B5 and isoleucine can further increase the biomass.

[0075] In addition, the biomass growth of Akk in the mixture below the optimal addition amount is directly proportional to its concentration, and the correlation coefficient R 2 is 0.9389. When the addition amount is greater than the optimal growth concentration, the effect of promoting Akk growth is reduced. High concentration will inhibit the biomass growth of Akk. The optimal addition amount is 2.5% lipopolysaccharide, 0.04% vitamin B5 and 0.2% isoleucine.

[0076] Example 6

[0077] In this example, Akkermansia muciniphila was cultured.

[0078] (1) Activation of bacterial strains: Place the bacterial strains stored in a -80℃ ultra-low temperature freezer at 4℃ for 2 hours and then restore to 25℃. Inoculate them into 200mL BHI liquid culture medium at a 5% inoculation rate and activate them three times in a row.

[0079] (2) Fermentation scale-up test: Two groups of BHI culture medium were prepared, and 0% (CK group) and 0.3% (EG group) of the mixture H described in Example 5 were added respectively. The mixture was sterilized at 121°C for 20 min and then inoculated into each group in turn at a 5% inoculation rate. The scale-up fermentation volume was 30 L. After anaerobic fermentation at 37°C for 18 h, the process biomass OD was measured using a spectrophotometer. 600 values ​​and record data, repeat the verification test for four groups, the pilot scale-up results are as follows Figure 8 As shown.

[0080] The results of the small test were verified by enlarged test. The results of the four groups of experiments all showed that the experimental group EG had a higher biological growth rate than the control group ck and showed good consistency.

[0081] Example 7

[0082] In this example, Akkermansia muciniphila was cultured. Compared with the culture scheme in Example 5 in which 1.0% mixture H was added, the only difference was that vitamin B5 was replaced with an equal amount of vitamin K3.

[0083] Example 8

[0084] ​​​​In this example, Akkermansia muciniphila was cultured. Compared with the culture protocol in Example 5 with the addition of 1.0% of mixture H, the only difference is that vitamin B5 was replaced with an equal amount of vitamin K1.

[0085] Example 9

[0086] In this example, Akkermansia muciniphila was cultured. Compared with the culture protocol in Example 5 with the addition of 1.0% of mixture H, the only difference is that isoleucine was replaced with an equal amount of threonine.

[0087] Example 10

[0088] In this example, Akkermansia muciniphila was cultured. Compared with the culture protocol in Example 5 with the addition of 1.0% of mixture H, the only difference is that isoleucine was replaced with an equal amount of leucine.

[0089] Example 11

[0090] In this example, Akkermansia muciniphila was cultured. Compared with the culture protocol in Example 5 with the addition of 1.0% of mixture H, the only difference is that lipopolysaccharide was replaced with an equal amount of trehalose.

[0091] Analyzing the results of Examples 7 - 11, the DO of the culture fermentation broth 600 was 3.95, 4.05, 4.23, 4.15, and 3.28 respectively. It can be seen that based on the metabolic pathway information of Akkermansia muciniphila, the present invention has discovered biological factors that promote its growth, and found that the synergistic combination of lipopolysaccharide, vitamin B5, and isoleucine can further increase the biomass of Akkermansia muciniphila, and the OD of the fermentation broth 600 can reach above 5.3.

[0092] In summary, based on the whole - genome sequence information of Akkermansia muciniphila, the present invention analyzed its metabolic pathway, and discovered novel growth factors that can promote the growth of Akkermansia muciniphila, so as to solve the problems of the long culture period and low biomass of Akkermansia muciniphila at present. The OD of the 18 - hour fermentation broth of Akkermansia muciniphila 600 can reach above 5.3, which can effectively improve the efficiency of producing Akkermansia muciniphila, reduce costs, and promote the wide application of Akkermansia muciniphila.

[0093] The applicant declares that the present invention uses the above - mentioned examples to illustrate the detailed method of the present invention, but the present invention is not limited to the above - mentioned detailed method, that is, it does not mean that the present invention must rely on the above - mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A culture medium for increasing the biomass of Akkermansia muciniphila, characterized in that: The culture medium for increasing the biomass of Akkermansia muciniphila contains growth factors, carbon and nitrogen sources, and inorganic salts; The growth factor is a combination of lipopolysaccharide, vitamin B5 and isoleucine; The culture medium for increasing the biomass of Akkermansia muciniphila has a lipopolysaccharide mass percentage of 0.5% to 3.5%, a vitamin B5 mass percentage of 0.01% to 0.3%, and a isoleucine mass percentage of 0.01% to 0.8%.

2. The culture medium for increasing the biomass of Akkermansia muciniphila according to claim 1, characterized in that: The carbon source and nitrogen source include brain heart extract powder, mucin, peptone and glucose.

3. The culture medium for increasing the biomass of Akkermansia muciniphila according to claim 1, characterized in that: The inorganic salts include sodium chloride and disodium hydrogen phosphate.

4. The culture medium for increasing the biomass of Akkermansia muciniphila according to claim 1, characterized in that: The mass percentage of lipopolysaccharide in the culture medium for increasing the biomass of Akkermansia muciniphila is 1.5% to 3.5%.

5. The culture medium for increasing the biomass of Akkermansia muciniphila according to claim 4, characterized in that: The mass percentage of lipopolysaccharide in the culture medium for increasing the biomass of Akkermansia muciniphila is 2.0% to 3.0%.

6. The culture medium for increasing the biomass of Akkermansia muciniphila according to claim 1, characterized in that: The mass percentage of vitamin B5 in the culture medium for increasing the biomass of Akkermansia muciniphila is 0.04% to 0.2%.

7. The culture medium for increasing the biomass of Akkermansia muciniphila according to claim 1, characterized in that: The mass percentage of isoleucine in the culture medium for increasing the biomass of Akkermansia muciniphila is 0.05% to 0.6%.

8. The culture medium for increasing the biomass of Akkermansia muciniphila according to claim 7, characterized in that: The mass percentage of isoleucine in the culture medium for increasing the biomass of Akkermansia muciniphila is 0.1% to 0.4%.

9. The culture medium for increasing the biomass of Akkermansia muciniphila according to claim 2, characterized in that: The concentration of brain heart extract powder in the culture medium for increasing the biomass of Akkermansia muciniphila is 30-40 g / L.

10. The culture medium for increasing the biomass of Akkermansia muciniphila according to claim 2, characterized in that: The concentration of mucin in the culture medium for increasing the biomass of Akkermansia muciniphila is 0.1-1.0 g / L.

11. The culture medium for increasing the biomass of Akkermansia muciniphila according to claim 2, characterized in that: The concentration of peptone in the culture medium for increasing the biomass of Akkermansia muciniphila is 5-20 g / L.

12. The culture medium for increasing the biomass of Akkermansia muciniphila according to claim 2, characterized in that: The concentration of glucose in the culture medium for increasing the biomass of Akkermansia muciniphila is 10-20 g / L.

13. The culture medium for increasing the biomass of Akkermansia muciniphila according to claim 3, characterized in that: The concentration of sodium chloride in the culture medium for increasing the biomass of Akkermansia muciniphila is 5-15 g / L.

14. The culture medium for increasing the biomass of Akkermansia muciniphila according to claim 3, characterized in that: The concentration of disodium hydrogen phosphate in the culture medium for increasing the biomass of Akkermansia muciniphila is 1-5 g / L.

15. The culture medium for increasing the biomass of Akkermansia muciniphila according to claim 1, characterized in that: The culture medium for increasing the biomass of Akkermansia muciniphila contains 30-40 g / L brain heart extract powder, 0.1-1.0 g / L mucin, 5-20 g / L peptone, 10-20 g / L glucose, 5-15 g / L sodium chloride, 1-5 g / L disodium hydrogen phosphate and growth factors; The growth factor is a combination of lipopolysaccharide, vitamin B5 and isoleucine, wherein the mass percentage of lipopolysaccharide is 0.5%-3.5%, the mass percentage of vitamin B5 is 0.01%-0.3%, and the mass percentage of isoleucine is 0.01%-0.8%.

16. Use of the culture medium for increasing the biomass of Akkermansia muciniphila according to any one of claims 1 to 15 in increasing the biomass of Akkermansia muciniphila.

17. A method for increasing the biomass of Akkermansia muciniphila, characterized in that: The method comprises inoculating Akkermansia muciniphila into the culture medium for increasing the biomass of Akkermansia muciniphila according to any one of claims 1 to 15 and culturing the culture medium.

18. The method for increasing the biomass of Akkermansia muciniphila according to claim 17, characterized in that: The culture includes anaerobic culture, and the gas ratio of the anaerobic culture is 90% N2, 5% H2, and 5% CO2 based on 100%.

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