A special culture medium for AKK and its application and culture method

Abstract

The present invention discloses a special culture medium for AKK and its application and culture method, and relates to the field of biotechnology. The present invention provides an AKK fermentation medium, wherein the fermentation medium is made of the following components: 15-18 g / L soy peptone, 0.5-5 g / L threonine, 4-6 g / L glucose, 4.5-5.5 g / L sodium chloride, 0.1-1 g / L cysteine, 1-3 g / L potassium dihydrogen phosphate and 0.5-5 g / L N-acetylglucosamine. The fermentation medium has high safety and low cost, and the culture medium of the present invention is used to culture AKK bacteria so that the AKK bacteria has a good fermentation effect. The present invention also provides a freeze-drying protectant for AKK bacteria, and the freeze-drying protectant can better protect the activity of AKK bacteria.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a special culture medium for AKK and an application and a culture method thereof. Background Art

[0002] Akkermansia muciniphila (AKK) belongs to the Akkermansia genus of the family Akkermansiaceae of the phylum Verrucomicrobia. It is an oval, strictly anaerobic, non-motile, Gram-negative, non-endospore-forming intestinal commensal bacterium that colonizes the gastrointestinal mucosal layer. It was first isolated from healthy human feces in a culture medium with mucin as the only carbon and nitrogen source. Studies have shown that AKK is closely related to human health and has good intestinal probiotic properties. AKK is very important for maintaining normal intestinal function. Studies have found that AKK has an ameliorative effect on a variety of intestinal diseases. The abundance of AKK is reduced in diseases such as IBS, inflammatory bowel disease, appendicitis, and allergic diarrhea. The gut-liver axis participates in regulating host health by tightly connecting the intestine, microorganisms, and liver. AKK can reduce liver damage by restoring the diversity of intestinal flora. AKK can act on the human nervous system and has an ameliorative effect on a variety of nervous system diseases. Due to a series of important discoveries on the physiological functions of Akkermansia muciniphila, the cultivation of Akkermansia muciniphila as a candidate probiotic for new biological living therapeutic drugs is particularly important.

[0003] Chinese patent CN117821554A discloses a selective culture medium for AKK bacteria and its application. The culture medium is made of the following raw materials: weigh 38.5g brain heart infusion broth, 2-4g mucin, 5-10g N-acetyl-glucosamine, 2.5-5g L-threonine, 0.5-1g L-cysteine ​​hydrochloride, 0.01-0.05g hemin chloride, 0.1-0.5g vitamin K1, 0.002-0.008g vancomycin, 0.001-0.005g gentamicin and 0.002-0.006g kanamycin and add water to 1L. The invention provides a selective culture medium for AKK bacteria, which can selectively screen out AKK bacteria and improve the screening efficiency of AKK bacteria. Compared with conventional AKK bacteria screening culture medium, the culture medium of the invention has simple components and can greatly simplify the culture medium preparation process.

[0004] Chinese patent CN114540246A discloses a selective culture medium and a separation and identification method for AKK bacteria. The selective culture medium for AKK bacteria of the invention is prepared by adding clarified sterile colonic fluid, porcine gastrointestinal mucin and vancomycin hydrochloride to a bicarbonate buffer medium. The volume percentage concentration of clarified sterile colonic fluid in the bicarbonate buffer medium is 0.4-1%, the mass percentage concentration of porcine gastrointestinal mucin in the bicarbonate buffer medium is 0.15-0.3%, and the mass percentage concentration of vancomycin hydrochloride in the bicarbonate buffer medium is 0.002-0.008‰; each 1L of bicarbonate buffer medium contains 0.38-0.55g KH2PO4, 0.0025-0.005g FeSO4, 0.45-0.55g Na2HPO4, 0.26-0.35g C6H 14 N2O7, 0.25-0.35gNaCl, 0.05-0.15g MgCl 2· 6H2O, 0.04-0.13g CaCl2, 1.4-3.0ml trace element solution, 0.7-1.5ml vitamin solution, 0.0003-0.001g resazurin sodium salt, 3.5-4.2g NaHCO3 and 0.15-0.35g Na2S·7-9H2O.

[0005] At present, there are few studies on the culture medium of AKK bacteria. The traditional culture medium for culturing Akkermansia muciniphila contains animal-derived components, including culture medium prepared with bovine brain heart infusion agar powder and porcine gastric mucin, as well as Columbia liquid medium and brain heart infusion liquid medium. Although the culture effect is acceptable, the cost is high, and brain heart infusion agar powder and porcine gastric mucin are both derived from animal tissues or digests. The components derived from animals may contain contaminants from viruses, prions or bacteria, or contain antiallergens, antigenic peptides or other undesirable components, and may also carry unknown viruses and other dangerous components. Therefore, the safety of culture medium with animal-derived components cannot be fully guaranteed. Summary of the invention

[0006] The invention aims to provide a special culture medium for AKK and an application and a culture method thereof.

[0007] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] On the one hand, the present invention provides an AKK fermentation medium, which is made of the following components: 15-32 g / L soy peptone, 0.5-5 g / L threonine, 4-6 g / L glucose, 4.5-5.5 g / L sodium chloride, 0.1-2 g / L cysteine, 1-3 g / L potassium dihydrogen phosphate and 0.5-5 g / L N-acetylglucosamine.

[0009] Specifically, the fermentation medium is made of the following components: 16 g / L soy peptone, 0.5-5 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 0.5-5 g / L N-acetylglucosamine.

[0010] Furthermore, the fermentation medium is made of the following components: 16 g / L soy peptone, 0.5 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 1 g / L N-acetylglucosamine.

[0011] According to some embodiments of the present invention, the fermentation medium consists of the following components: 16 g / L soy peptone, 0.5 g / L threonine, 0.5 g / L cysteine, 4.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine.

[0012] According to some embodiments of the present invention, the fermentation medium consists of the following components: 16 g / L soy peptone, 1 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine.

[0013] According to some embodiments of the present invention, the fermentation medium consists of the following components: 16 g / L soy peptone, 2.5 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine.

[0014] According to some embodiments of the present invention, the fermentation medium consists of the following components: 16 g / L soy peptone, 5 g / L threonine, 4.5 g / L glucose, 0.5 g / L cysteine, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine.

[0015] According to some embodiments of the present invention, the fermentation medium consists of the following components: 16 g / L soy peptone, 0.5 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 1 g / L N-acetylglucosamine.

[0016] According to some embodiments of the present invention, the fermentation medium consists of the following components: 16 g / L soy peptone, 0.5 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 2 g / L N-acetylglucosamine.

[0017] According to some embodiments of the present invention, the fermentation medium consists of the following components: 16 g / L soy peptone, 0.5 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 5 g / L N-acetylglucosamine.

[0018] Specifically, the pH of the fermentation medium is 7.2±0.2.

[0019] Furthermore, the pH of the fermentation medium is 7.2.

[0020] In another aspect, the present invention provides a fermentation culture method of AKK bacteria, which comprises inoculating AKK bacteria into the above-mentioned fermentation medium for fermentation culture.

[0021] Specifically, the inoculation amount of the AKK bacteria is 5-15%;

[0022] Furthermore, the inoculation amount of the AKK bacteria is 10%.

[0023] Furthermore, the fermentation time is 16-46h;

[0024] Furthermore, the fermentation time is 16 hours.

[0025] Specifically, the fermentation temperature is 35-38°C; further, the fermentation temperature is 37°C.

[0026] Specifically, the fermentation and cultivation method of AKK bacteria comprises the following steps:

[0027] (1) Inoculate AKK into culture medium and culture anaerobically at 35-38°C for 48-72 hours;

[0028] (2) inoculating the activated bacterial solution into a shake flask fermentation medium for anaerobic fermentation;

[0029] (3) The seed liquid cultured in step (2) is inoculated into a fermentation medium for anaerobic fermentation.

[0030] Furthermore, the culture medium in step (1) is BHI+mucin liquid culture medium;

[0031] Further, the inoculation amount described in step (1) is 10%;

[0032] Furthermore, in step (1), the culture is carried out anaerobically at 37° C. for 72 hours.

[0033] Furthermore, step (2) includes subjecting the activated bacterial solution to Gram staining microscopy to ensure that there are no foreign bacteria before inoculation.

[0034] Furthermore, the inoculation amount in step (2) is 10%.

[0035] Furthermore, the temperature of the anaerobic fermentation in step (2) is 37°C.

[0036] Furthermore, step (3) includes subjecting the cultured seed solution to Gram staining microscopic examination to ensure that there are no foreign bacteria before inoculation.

[0037] Furthermore, the inoculation amount in step (3) is 10%, and nitrogen is filled to maintain pressure for anaerobic fermentation at 37°C.

[0038] In another aspect, the present invention provides use of the above culture medium in high-density fermentation of AKK bacteria.

[0039] In another aspect, the present invention provides a freeze-drying protectant for AKK bacteria, wherein the freeze-drying protectant comprises 0.5-0.8 parts of lactose, 0.01-0.05 parts of maltodextrin, 0.01-0.05 parts of glycine, and 0.01-0.05 parts of mannitol.

[0040] Specifically, the lyophilization protective agent includes 0.5-0.6 parts of lactose, 0.01-0.02 parts of maltodextrin, 0.01-0.02 parts of glycine, and 0.01-0.02 parts of mannitol.

[0041] Furthermore, the lyophilization protective agent includes 0.517 parts of lactose, 0.0166 parts of maltodextrin, 0.0166 parts of glycine, and 0.0166 parts of mannitol.

[0042] In another aspect, the present invention provides a freeze-drying method for AKK bacteria, wherein the freeze-drying method comprises adding a freeze-drying protectant to an AKK bacteria suspension for freeze-drying.

[0043] The beneficial effects of the present invention are:

[0044] The present invention provides a fermentation medium for AKK bacteria, wherein the fermentation medium does not contain animal-derived components, the medium has high safety, and low cost, and the use of the medium of the present invention to culture AKK bacteria enables the AKK bacteria to have a good fermentation effect. The present invention also provides a freeze-drying protective agent for AKK bacteria, and the freeze-drying protective agent can better protect the activity of AKK bacteria. DETAILED DESCRIPTION

[0045] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0046] AKK bacteria originate from the feces of healthy humans. A strain of Akkermansia muciniphila CCTCC.M.2020950 was isolated, cultured and identified using selective culture medium and named Akkermansia muciniphila SZ, or AKK bacteria for short.

[0047] Example 1

[0048] Preparation of culture medium:

[0049] Culture medium formula No. 1: 38.5 g / L BHI broth + 2 g / L mucin (BHI formula: 16 g / L peptone, 12.5 g / L dehydrated calf brain extract powder, 5 g / L dehydrated buffalo heart and brain extract powder, 5 g / L sodium chloride, 2 g / L glucose, 2.5 g / L disodium hydrogen phosphate, PH7.4±0.2).

[0050] Medium No. 2 formula: 16 g / L soy peptone, 0.5 g / L threonine, 4.5 g / L glucose and 0.5 g / L N-acetylglucosamine, pH 7.2.

[0051] Medium No. 3 formula: 16 g / L soy peptone, 0.5 g / L threonine, 4.5 g / L glucose, 0.5 g / L cysteine ​​and 0.5 g / L N-acetylglucosamine, pH 7.2.

[0052] Medium No. 4 formula: 16 g / L soy peptone, 4 g / L threonine, 4.5 g / L glucose, 0.5 g / L cysteine, 5 g / L sodium chloride and 0.5 g / L N-acetylglucosamine, pH 7.2.

[0053] Medium No. 5 formula: 16g / L soy peptone, 0.5g / L threonine, 0.5g / L cysteine, 4.5g / L glucose, 5g / L sodium chloride, 2.5g / L disodium hydrogen phosphate and 0.5g / L N-acetylglucosamine, pH 7.2.

[0054] Table 1 Culture medium material addition information

[0055]

[0056]

[0057] Note: “+” means the material is added, “-” means the material is not added.

[0058] Table 2 Development of AKK non-animal derived fermentation medium

[0059]

[0060] From the above results, it can be seen that no growth was observed in culture media No. 2 and No. 3 after 72 hours of strict anaerobic culture with an AKK inoculation rate of 10%, but growth was observed in culture media No. 1, No. 4 and No. 5. The growth of No. 5 was better than that of No. 4, and culture media No. 1 contained an animal-derived formula, so culture media No. 5 was determined, and the next step of optimization culture was carried out based on this culture media.

[0061] Example 2

[0062] Medium No. 6 formula: 16 g / L soy peptone, 0.5 g / L threonine, 0.5 g / L cysteine, 4.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine.

[0063] Table 3 AKK non-animal origin fermentation medium culture development

[0064]

[0065] Culture medium No. 6 replaced the disodium hydrogen phosphate in culture medium No. 5 with potassium dihydrogen phosphate, and its AKK growth OD value was the best, reaching 1.571 at 24 hours and a maximum of 2.259 at 40 hours, which was higher than BHI+mucin culture (1.676), indicating that potassium dihydrogen phosphate was more conducive to the growth of AKK than disodium hydrogen phosphate.

[0066] Example 3 Test of different addition amounts of threonine

[0067] Medium No. 7: 16 g / L soy peptone, 0.25 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine, pH 7.2.

[0068] Medium No. 8: 16 g / L soy peptone, 0.5 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine, pH 7.2.

[0069] Medium No. 9: 16 g / L soy peptone, 1 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine, pH 7.2.

[0070] Medium No. 10: 16 g / L soy peptone, 2.5 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine, pH 7.2.

[0071] Medium No. 11: 16 g / L soy peptone, 5 g / L threonine, 0.5 g / L cysteine, 4.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine, pH 7.2.

[0072] Table 4

[0073]

[0074] The above results show that the addition of threonine in the range of 0.5g / L-5g / L has no significant effect on the growth of AKK.

[0075] Example 4 Experiments with different amounts of N-acetylglucosamine added

[0076] Medium No. 12: 16 g / L soy peptone, 0.5 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 0.25 g / L N-acetylglucosamine, pH 7.2.

[0077] Medium No. 13: 16 g / L soy peptone, 0.5 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine, pH 7.2.

[0078] Medium No. 14: 16 g / L soy peptone, 0.5 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 1 g / L N-acetylglucosamine, pH 7.2.

[0079] Medium No. 15: 16 g / L soy peptone, 0.5 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 2 g / L N-acetylglucosamine, pH 7.2.

[0080] Medium No. 16: 16 g / L soy peptone, 0.5 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 5 g / L N-acetylglucosamine, pH 7.2.

[0081] Table 5

[0082]

[0083] The above results show that the addition of N-acetylglucosamine in the range of 0.5g / L-5g / L has no significant effect on the growth of AKK.

[0084] Example 5 Experiments with different pH values ​​of culture medium

[0085] Medium No. 17: 16 g / L soy peptone, 0.5 g / L threonine, 0.5 g / L cysteine, 4.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine; (pH 7.40).

[0086] Medium No. 18: 16 g / L soy peptone, 0.5 g / L threonine, 0.5 g / L cysteine, 4.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine; (pH 7.20).

[0087] Medium No. 19: 16 g / L soy peptone, 0.5 g / L threonine, 0.5 g / L cysteine, 4.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine; (pH 7.0).

[0088] Medium No. 20: 16 g / L soy peptone, 0.5 g / L threonine, 0.5 g / L cysteine, 4.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine; (pH 6.50).

[0089] Medium No. 21: 16 g / L soy peptone, 0.5 g / L threonine, 0.5 g / L cysteine, 4.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine; (pH 6.00).

[0090] Table 6

[0091]

[0092]

[0093] The above results show that the optimal pH for culturing AKK in this culture medium is 7.2±0.2.

[0094] Comparative Example 1

[0095] 1) Experimental group culture medium configuration: 16 g / L soy peptone, 0.5 g / L threonine, 0.5 g / L cysteine, 4.5 g / L glucose, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate and 0.5 g / L N-acetylglucosamine. Add purified water and stir well. Stir well and sterilize at 121°C for 15 min. Set aside. pH 7.2.

[0096] 2) Comparative group culture medium preparation: Take a 250ml conical flask, add 200mL of pure water, then add 1.44g of glucose, 6.4g of soy peptone, 1.6g of threonine, 1.056g of N-acetylglucosamine, 1g of sodium chloride and 0.5g of disodium hydrogen phosphate, stir well, and sterilize at 121℃ for 15min for later use.

[0097] 3) Comparison of AKK culture OD value determination between the experimental group and the control group: The activated AKK fresh culture fluid was inoculated into the above-mentioned two sterilized culture media (experimental group and control group) for cultivation, with an inoculation amount of 10%. Three parallels were set for each group. After 3 days of strict anaerobic culture, the culture fluid was taken for Gram staining microscopy. After confirming the pure bacteria, a certain amount of bacterial fluid was taken and diluted a certain multiple with sterile water, and the OD value was determined. The OD value reading of the bacterial dilution was within the range of 0.3-0.5. The final OD value was the OD value of the bacterial dilution multiplied by the dilution multiple, and the result was recorded. The OD value of the experimental group was 2.288, and the OD value of the control group was 1.479. The experimental group was more suitable for AKK bacterial fermentation culture than the control group. The specific data are shown in the table below.

[0098] Table 7

[0099]

[0100] Example 6 AKK lyophilization protectant

[0101] 1. Different freeze-drying protective agent formulas

[0102] Table 8 AKK different freeze-drying protective agent formula

[0103]

[0104]

[0105] Add 5 ml of AKK centrifuged bacterial suspension to the above freeze-dried protective agent solid, mix well, pre-freeze at -80 degrees for 3 hours, and freeze-dry in a Leaf Freeze Dryer.

[0106] 2. Freeze-drying of AKK with different freeze-drying protectants

[0107] The method for detecting live bacteria of freeze-dried powder samples is as follows: pour plate culture and count: take 1g of freeze-dried powder and dissolve it in sterile saline, perform 10-fold gradient dilution, select 3-5 dilution multiples, take 1ml respectively and add it to the plate, then pour it into BHI+mucin agar medium at about 45°C (that is, add 15g / L agar to medium No. 1), shake it evenly and wait for it to cool and solidify, cover it with a lid and place it in a 37°C anaerobic incubator for 72h-96h for colony counting, and the total live bacteria count unit is CFU / g.

[0108] The test results are shown in the following table:

[0109] Table 9 Results of live bacteria detection of freeze-dried powder samples

[0110]

[0111]

[0112] As can be seen from the above table, the 1D formula: 0.517 g lactose, 0.0166 g maltodextrin, 0.0166 g glycine, and 0.0166 g mannitol has the best freeze-drying protective agent effect.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An AKK fermentation medium, characterized in that The fermentation medium is made of the following components: 15-18 g / L soy peptone, 0.5-5 g / L threonine, 4-6 g / L glucose, 4.5-5.5 g / L sodium chloride, 0.1-1 g / L cysteine, 1-3 g / L potassium dihydrogen phosphate and 0.5-5 g / L N-acetylglucosamine.

2. The fermentation medium according to claim 1, characterized in that The fermentation medium is made of the following components: 16 g / L soy peptone, 0.5-5 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 0.5-5 g / L N-acetylglucosamine.

3. The fermentation medium according to claim 2, characterized in that The fermentation medium is made of the following components: 16 g / L soy peptone, 0.5 g / L threonine, 4.5 g / L glucose, 5 g / L sodium chloride, 0.5 g / L cysteine, 2.5 g / L potassium dihydrogen phosphate and 1 g / L N-acetylglucosamine.

4. The fermentation medium according to any one of claims 1 to 3, characterized in that The pH of the fermentation medium is 7.2±0.

2.

5. The fermentation medium according to claim 4, characterized in that The pH of the fermentation medium is 7.

2.

6. A fermentation and culture method of AKK bacteria, characterized in that: The method comprises inoculating AKK bacteria into the fermentation medium according to any one of claims 1 to 5 for fermentation culture.

7. The fermentation culture method according to claim 6, characterized in that: The inoculation amount of the AKK bacteria is 5-15%.

8. The fermentation culture method according to claim 7, characterized in that: The inoculation amount of the AKK bacteria is 10%.

9. The fermentation culture method according to claim 6, characterized in that: The fermentation culture time is 16-46h.

10. The fermentation culture method according to claim 9, characterized in that: The fermentation time is 16 hours.

11. The fermentation culture method according to claim 6, characterized in that: The fermentation temperature is 35-38°C.

12. The fermentation culture method according to any one of claims 7 to 11, characterized in that: The fermentation and culture method of AKK bacteria comprises the following steps: (1) Inoculate AKK into culture medium and culture anaerobically at 35-38°C for 48-72 hours; (2) inoculating the activated bacterial solution into a shake flask fermentation medium for anaerobic fermentation; (3) The seed liquid cultured in step (2) is inoculated into a fermentation medium for anaerobic fermentation.

13. Use of the culture medium according to any one of claims 1 to 5 in high-density fermentation of AKK bacteria.

14. A freeze-drying protective agent for AKK bacteria, characterized in that: The freeze-drying protective agent comprises 0.5-0.8 parts of lactose, 0.01-0.05 parts of maltodextrin, 0.01-0.05 parts of glycine and 0.01-0.05 parts of mannitol.

15. The lyoprotectant according to claim 14, characterized in that The freeze-drying protective agent comprises 0.5-0.6 parts of lactose, 0.01-0.02 parts of maltodextrin, 0.01-0.02 parts of glycine and 0.01-0.02 parts of mannitol.

16. The lyoprotectant according to claim 15, characterized in that The freeze-drying protective agent includes 0.517 parts of lactose, 0.0166 parts of maltodextrin, 0.0166 parts of glycine and 0.0166 parts of mannitol.

17. A freeze-drying method for AKK bacteria, characterized in that: The freeze-drying method is to add the freeze-drying protectant described in any one of claims 14 to 16 to the AKK bacterial suspension for freeze-drying.

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