High-density fermentation metabolism regulation process of Akkermansia muciniphila

Abstract

The invention belongs to the technical field of microorganisms, and relates to a high-density fermentation metabolism regulation and control process of Akkermansia muciniphila. The invention provides a method for fermenting Akkermansia muciniphila, which comprises the following steps: (1) carrying out activation culture on the Akkermansia muciniphila or a culture and an isolate of the Akkermansia muciniphila, so as to obtain a seed solution; and (2) inoculating the seed solution into a high-density fermentation culture medium for anaerobic fermentation culture. By adopting the high-density fermentation culture medium and the method provided by the invention, the fermentation viable count of Akkermansia muciniphila is remarkably increased, and the method and the high-density fermentation culture medium provided by the invention have good application in the production of probiotic products.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a high-density fermentation metabolism regulation process of Akkermansia muciniphila. Background Art

[0002] Probiotics are defined as living microorganisms. The right amount of probiotics will regulate the health level of the host. The effects of probiotics on the host include several modes, such as the production of antibacterial substances, competition for nutrients and adhesion sites, changes in microbial metabolism, and stimulation of allergic reactions. Probiotics can maintain the balance of intestinal flora, inhibit intestinal inflammation, protect the intestinal mucosal barrier, promote intestinal digestive function, relieve diarrhea and constipation, and play an important role in antibacterial and antiviral effects, inhibiting the growth of tumor cells, and enhancing the body's immunity. They are widely used in the food industry and healthcare in the form of probiotic preparations, and are used as fermentation agents for fermented foods. High-density fermentation is a prerequisite for probiotic preparations and high-efficiency fermentation agents.

[0003] Akkermansia muciniphila (AKK) is a strict anaerobic bacterium isolated from human feces, and uses mucin as the only source of carbon and nitrogen. Mucin is a macromolecular glycoprotein secreted by intestinal epithelial goblet cells and is the main component of the intestinal mucosal mucus layer. The energy sources and environmental conditions required for the growth and reproduction of AKK bacteria are complex. It is necessary to clarify its absorption efficiency of various energy sources, integrate different nutritional configurations throughout the separation and screening processes, and match appropriate environmental conditions.

[0004] Chinese patent CN112342151A discloses an anaerobic culture method for Akkermansia muciniphila, wherein the mixed gas ratio used in the anaerobic culture is 85-94% N2, 5% H2, and 1-10% CO2, and the culture medium used is a modified BHI culture medium, wherein Mucin and Yeast Exctact are added to the modified BHI culture medium. The combination of the culture medium and culture conditions of the invention makes the growth and reproduction of Akk bacteria not limited by the height of the liquid level, and can grow well in a shake bottle or on a flat plate surface, thereby reducing the difficulty of culturing Akk bacteria, and at the same time reducing the growth rate of Akk bacteria from 3-4d in the prior art to 24-36h, greatly shortening the culture time, improving the activity of Akk bacteria and promoting the growth of Akk bacteria, which is of great significance to the expanded culture and in-depth research of Akk bacteria.

[0005] Chinese patent CN115975885A discloses a preparation of a culture medium for Akkermansia muciniphila using fish processing byproducts as a nitrogen source, wherein the raw materials are: brain heart infusion broth, L-cysteine ​​hydrochloride, fish processing byproduct hydrolyzate and water; the culture medium for Akkermansia muciniphila contains 1.5-2.5g of brain heart infusion broth, 0.01-0.05g of L-cysteine ​​hydrochloride and 0.05-2g of fish processing byproduct hydrolyzate per 60mL of water; the preparation method of the fish processing byproduct hydrolyzate comprises: crushing, sterilizing and removing fat from the fish processing byproducts, and then enzymolyzing them with alkaline protease and papain, filtering the enzymolysis products to obtain the fish processing byproduct hydrolyzate; the fish processing byproducts are silver carp processing byproducts. While reducing the cost of the culture medium, solving the waste of silver carp processing byproduct resources and the environmental pollution after the disposal of the silver carp processing byproducts, the culture efficiency of Akk bacteria can also be improved.

[0006] Currently, there is still a need in the art for more high-density fermentation methods of Akkermansia muciniphila. Summary of the invention

[0007] In order to achieve the above technical objectives, the present invention proposes the following technical solutions:

[0008] In one aspect, the present invention provides a method for fermenting Akkermansia muciniphila, the method comprising the following steps:

[0009] (1) activating Akkermansia muciniphila or a culture or isolate thereof to obtain a seed solution;

[0010] (2) inoculating the seed liquid into a high-density fermentation medium for anaerobic fermentation;

[0011] The high-density fermentation medium comprises the following components:

[0012] Galacto-oligosaccharides, maltose, peptone, beef extract, yeast extract, diammonium citrate, Tween 80, sodium acetate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, threonine, serine, aspartic acid and phenylalanine.

[0013] In some preferred embodiments, the high-density fermentation medium comprises the following components:

[0014] Galacto-oligosaccharides 2-6g / L, maltose 1-4g / L, peptone 8-15g / L, beef extract 8-15g / L, yeast extract 3-8g / L, diammonium hydrogen citrate 1-3g / L, Tween 80 0.5-1.5mL / L, sodium acetate 3-8g / L, dipotassium hydrogen phosphate 1-3g / L, magnesium sulfate 0.4-0.6g / L, manganese sulfate 0.2-0.3g / L, threonine 0.2-1g / L, serine 1-2g / L, aspartic acid 0.1-0.5g / L and phenylalanine 0.01-0.1g / L.

[0015] In some embodiments, the step (2) further comprises:

[0016] When Akkermansia muciniphila in the fermentation broth reached the logarithmic growth phase, feed medium was added to carry out fed-batch culture.

[0017] In some embodiments, the logarithmic growth phase is the period of Akkermansia muciniphila during fermentation culture for 6-8 hours.

[0018] In some embodiments, the feed medium comprises glucose, maltose and peptone.

[0019] In some embodiments, the feed medium comprises 180-220 g / L glucose, 80-120 g / L maltose and 180-220 g / L peptone.

[0020] In some embodiments, the anaerobic fermentation in step (2) is strictly anaerobic fermentation.

[0021] In some preferred embodiments, the conditions for anaerobic fermentation in step (2) include:

[0022] 1) The dissolved oxygen in the culture medium is less than 1 mg / L;

[0023] 2) Culture temperature 37-38°C;

[0024] 3) Constantly control pH at 6.5-7.0.

[0025] In some embodiments, when the fermentation culture is carried out in a fermenter, the stirring speed is 120-150 r / min.

[0026] In some embodiments, the activation culture in step (1) may be:

[0027] Akkermansia muciniphila or a culture or isolate thereof is inoculated into a BHI medium and cultured for one or two generations.

[0028] In another aspect, the present invention provides a high-density fermentation medium, the high-density fermentation medium comprising the following components:

[0029] Galacto-oligosaccharides, maltose, peptone, beef extract, yeast extract, diammonium citrate, Tween 80, sodium acetate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, threonine, serine, aspartic acid and phenylalanine.

[0030] In some preferred embodiments, the high-density fermentation medium comprises the following components:

[0031] Galacto-oligosaccharides 2-6g / L, maltose 1-4g / L, peptone 8-15g / L, beef extract 8-15g / L, yeast extract 3-8g / L, diammonium hydrogen citrate 1-3g / L, Tween 80 0.5-1.5mL / L, sodium acetate 3-8g / L, dipotassium hydrogen phosphate 1-3g / L, magnesium sulfate 0.4-0.6g / L, manganese sulfate 0.2-0.3g / L, threonine 0.2-1g / L, serine 1-2g / L, aspartic acid 0.1-0.5g / L and phenylalanine 0.01-0.1g / L.

[0032] In some embodiments, the high-density fermentation medium may further include L-cysteine ​​or a salt thereof.

[0033] In some embodiments, the high density fermentation medium may further include mupirocin lithium salt.

[0034] In yet another aspect, the present invention provides use of any of the aforementioned high-density fermentation media in high-density fermentation culture of Akkermansia muciniphila.

[0035] In another aspect, the present invention provides use of the Akkermansia muciniphila fermentate obtained by any of the aforementioned methods, or the Akkermansia muciniphila fermentate cultured in any of the aforementioned high-density fermentation medium, in the preparation of a probiotic product.

[0036] In some embodiments, the probiotic preparation further comprises a food-acceptable excipient, which comprises erythritol, D-mannitol, fumaric acid, glycerin, pectin, potassium alginate, sodium alginate, talc, sodium pyrophosphate, polydextrose, carrageenan, sodium ascorbate, ascorbyl palmitate, L-malic acid, L(+)-tartaric acid, maltitol, gelatin, xylitol, citric acid, potassium citrate, sodium citrate, citric acid fatty acid glyceride, agar, lactic acid, sodium lactate, sorbic acid and its potassium salt, sorbitol, acid red, calcium carbonate, sodium carbonate, sodium bicarbonate, beet red, vitamin C, vitamin E, oxidized starch, ethanol, sodium acetate, stearic acid, calcium stearate, magnesium stearate or dextrin.

[0037] In some embodiments, the dosage form of the probiotic preparation includes a liquid preparation, a solid preparation, a semi-solid preparation or a gaseous preparation.

[0038] In some embodiments, the probiotic product includes recombined milk, sterilized milk, fermented milk, condensed milk, cream, light cream, anhydrous butter, concentrated milk, compound protein beverages, coffee beverages, plant beverages, flavored beverages, sports drinks, nutrient beverages, energy drinks, electrolyte beverages, whole milk powder, skimmed milk powder, partially skimmed milk powder, recombined milk powder, whey powder, cheese, processed cheese, popsicles, slush, popsicles, edible ice, sweetened ice or ice cream.

[0039] Aiming at the special metabolic pathway (phosphoketolase pathway) of Akkermansia muciniphila, the present invention also provides a feed medium, which can not only ensure the stable supply of carbon source but also reduce the acetic acid produced by metabolism by adding maltose to the feed medium to replace a part of glucose. The feed medium has a significant effect on promoting the number of viable fermentation bacteria of Akkermansia muciniphila, and compared with only using glucose feed, the number of viable fermentation bacteria is increased by about 50%. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The changes of OD600 in different experimental groups during the fermentation process are shown. DETAILED DESCRIPTION

[0041] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0042] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0043] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0044] As used herein, the term "culture" refers to a living cell group or living growth of a microorganism in a certain time and space, such as a slant culture of a microorganism, a shake flask culture, etc. In some examples, it refers to a living microbial population cultivated and propagated under artificially specified conditions.

[0045] As used herein, the term "isolate" refers to a living microbial substance that is separated from a population or a mixed population of microorganisms and exists in a certain medium, including fermentation broth supernatant, fermentation broth precipitate, bacterial suspension, freeze-dried powder, etc.

[0046] As used herein, the term "probiotic preparation" refers to a preparation prepared according to the principles of microecology using normal beneficial microorganisms or their promoting substances that are beneficial to the host, which has the function of maintaining or adjusting the balance of microecology, preventing and treating diseases and promoting the health of the host.

[0047] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment. If the specific conditions are not specified in the embodiment, it is carried out according to the conditions recommended by normal conditions or manufacturers. If the manufacturer is not specified in all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. Such structures and technologies are also described in many publications.

[0048] The experimental materials mainly involved in the present invention are as follows:

[0049] AKK ONE comes from Shan En Kang Biotechnology Co., Ltd., with the deposit number CGMCC No.20954, which has been disclosed in patent CN18286268A.

[0050] AKK PROBIO comes from Shan En Kang Biotechnology Co., Ltd., with the deposit number CGMCC No.20955, which has been disclosed in patent CN116898883A.

[0051] BHI liquid medium:

[0052] Peptone 10.0g, dehydrated calf brain extract powder 12.5g, dehydrated beef heart extract powder 5.0g, sodium chloride 5.0g, glucose 2.0g,

[0053] 2.5 g of disodium hydrogen phosphate, pH 7.4 ± 0.2, 25 °C, dilute to 1000 mL with distilled water.

[0054] BHI solid medium: Add 1.5% agar to BHI medium and sterilize at 121°C for 15 min.

[0055] The main experimental methods of the present invention are:

[0056] Determination of viable bacteria count: The plate count method was used to calculate the bacterial concentration in the fermentation broth (Zhang Yan, Liu Jun'e, Zhang Jing, et al. Detection of intestinal flora in feces by plate count method [J]. Journal of Capital Medical University, 2008, 29(1): 8586). The results are expressed in CFU / mL.

[0057] Determination of bacterial density: The bacterial density of Akkermansia muciniphila was measured at a wavelength of 600nm using a multifunctional microplate detector for each fermentation broth sample. The bacterial density value was valid when it was 0.2-0.8. If the bacterial density was greater than 0.8, the bacterial solution must be diluted. The detection parameters were set to oscillate for 30s before detection, with a consistent oscillation speed. 200μL of fermentation broth was added to each well in turn, and it was blown evenly. Each sample was measured three times, and three parallels (N=3) were established.

[0058] Example 1 Fermentation culture of AKK PROBIO

[0059] 1.1 Preparation of high-density culture medium

[0060] High density culture medium:

[0061] Galactooligosaccharide 4g, maltose 2g, peptone 10g, beef extract 10g, yeast extract 5g, diammonium hydrogen citrate 2g, Tween 80 1mL, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.5g, manganese sulfate 0.25g, threonine 0.5g, serine 1.5g, aspartic acid 0.4g and phenylalanine 0.05g, make up to 1000mL with distilled water.

[0062] 1.2 Bacteria Activation

[0063] Take out the pre-frozen AKK PROBIO from the -80℃ refrigerator, streak it on BHI solid culture medium, place the plate in a 37℃ constant temperature incubator and culture it for 72h, pick a single colony, inoculate it into 5mL BHI liquid culture medium, activate it twice in succession, and take the bacterial solution cultured to the logarithmic growth phase as the seed culture medium.

[0064] 1.3 Inoculation and culture

[0065] Nitrogen was introduced into the vent of the fermenter to reduce the dissolved oxygen to below 1 mg / L. The fermentation parameters were set as follows: the culture temperature was 37.0°C, the stirring speed was 150 r / min, the pH was constantly controlled at 6.8, and an ammonia solution was fed.

[0066] The seed culture solution was inoculated into a smart fermenter containing 5L high-density culture medium in a sterile environment, with an inoculation volume of 4% (v / v) for strict anaerobic fermentation. The absorbance value of the culture solution at 600nm was monitored every hour. When the strain reached the logarithmic growth phase (12-18h), feed medium was added for feed culture. The components of the feed medium include: 200g / L glucose, 100g / L maltose and 200g / L peptone.

[0067] After the fermentation culture is completed, the number of viable bacteria in the fermentation broth is detected.

[0068] Example 2 Fermentation culture of AKK PROBIO

[0069] 2.1 Preparation of high-density fermentation medium

[0070] High-density fermentation medium: 2 g galacto-oligosaccharide, 1 g maltose, 8 g peptone, 8 g beef extract, 3 g yeast extract, 1 g diammonium hydrogen citrate, 0.5 mL Tween 80, 3 g sodium acetate, 1 g dipotassium hydrogen phosphate, 0.4 g magnesium sulfate, 0.2 g manganese sulfate, 0.2 g threonine, 1.0 g serine, 0.1 g aspartic acid and 0.01 g phenylalanine, make up to 1000 mL with distilled water.

[0071] 2.2 Bacteria Activation

[0072] Take out the pre-frozen AKK PROBIO from the -80℃ refrigerator, streak it on BHI solid culture medium, place the plate in a 37℃ constant temperature incubator and culture it for 72h, pick a single colony, inoculate it into 5mL BHI liquid culture medium, activate it twice in succession, and take the bacterial solution cultured to the logarithmic growth phase as the seed culture medium.

[0073] 2.3 Inoculation and culture

[0074] Nitrogen was introduced into the vent of the fermenter to reduce the dissolved oxygen to below 1 mg / L. The fermentation parameters were set as follows: the culture temperature was 37.0°C, the stirring speed was 120 r / min, the pH was constantly controlled at 6.5, and sodium hydroxide solution was added continuously.

[0075] The seed culture solution was inoculated into a smart fermentation tank containing 5L high-density fermentation medium in a sterile environment, with an inoculation volume of 4% (v / v) for strict anaerobic fermentation. The absorbance value of the culture solution at 600nm was monitored every hour. When the strain reached the logarithmic growth phase (12-18h), feed medium was added for feed culture. The feed medium components included: 180g / L glucose, 80g / L maltose and 180g / L peptone.

[0076] After the fermentation culture is completed, the number of viable bacteria in the fermentation broth is detected.

[0077] Example 3 Fermentation culture of AKK PROBIO

[0078] 3.1 Preparation of high-density fermentation medium

[0079] High-density fermentation medium: 6 g galacto-oligosaccharide, 4 g maltose, 15 g peptone, 15 g beef extract, 8 g yeast extract, 3 g diammonium hydrogen citrate, 1.5 mL Tween 80, 8 g sodium acetate, 3 g dipotassium hydrogen phosphate, 0.6 g magnesium sulfate, 0.3 g manganese sulfate, 1.0 g threonine, 2.0 g serine, 0.5 g aspartic acid and 0.1 g phenylalanine, make up to 1000 mL with distilled water.

[0080] 3.2 Bacteria Activation

[0081] Take out the pre-frozen AKK PROBIO from the -80℃ refrigerator, streak it on BHI solid culture medium, place the plate in a 37℃ constant temperature incubator and culture it for 72h, pick a single colony, inoculate it into 5mL BHI liquid culture medium, activate it twice in succession, and take the bacterial solution cultured to the logarithmic growth phase as the seed culture medium.

[0082] 3.3 Inoculation and culture

[0083] Nitrogen was introduced into the vent of the fermenter to reduce the dissolved oxygen to below 1 mg / L; the fermentation parameters were set as follows: the culture temperature was 37°C, the stirring speed was 130 r / min, the pH was constantly controlled at 7.0, and sodium carbonate solution was fed.

[0084] The seed culture solution was inoculated into a GS-MFC intelligent fermenter containing 5L high-density fermentation medium in a sterile environment, with an inoculation amount of 4% (v / v) for strict anaerobic fermentation. The absorbance value of the culture solution at 600nm was monitored every 1 hour. When the strain reached the logarithmic growth phase (12-18h), feed medium was added for fed culture. The components of the feed medium include: 220g / L glucose, 120g / L maltose and 220g / L peptone.

[0085] After the fermentation culture is completed, the number of viable bacteria in the fermentation broth is detected.

[0086] Example 4 Fermentation culture of AKK ONE

[0087] The only difference from Example 1 is that the strain used for fermentation culture is different, and the rest are the same. The strain used for fermentation culture in Example 4 is AKK ONE.

[0088] Comparative Example 1

[0089] Compared with Example 1, the only difference is that the high-density fermentation medium does not contain threonine, serine, aspartic acid and phenylalanine.

[0090] Comparative Example 2

[0091] Compared with Example 1, the only difference is that serine, aspartic acid and phenylalanine in the high-density fermentation medium are replaced by threonine.

[0092] Comparative Example 3

[0093] Compared with Example 1, the only difference is that the galacto-oligosaccharide in the high-density fermentation medium is replaced by glucose.

[0094] Comparative Example 4

[0095] Compared with Example 1, the only difference is that the concentrations of the components in the high-density fermentation medium are different. The high-density medium in Comparative Example 4 includes the following components:

[0096] Galacto-oligosaccharides 8g / L, maltose 5g / L, peptone 16g / L, beef extract 16g / L, yeast extract 10g / L, diammonium hydrogen citrate 5g / L, Tween 80 2.0mL / L, sodium acetate 10g / L, dipotassium hydrogen phosphate 5g / L, magnesium sulfate 1.0g / L, manganese sulfate 0.5g / L, threonine 2g / L, serine 3g / L, aspartic acid 1.0g / L and phenylalanine 0.2g / L.

[0097] Comparative Example 5

[0098] Compared with Example 1, the only difference is that no maltose is added to the feed medium, and the content of glucose is increased to 300 g / L.

[0099] Comparative Example 6

[0100] Compared with Example 1, the only difference is that maltose in the feed medium is replaced by an equal amount of fructose.

[0101] The number of viable bacteria in the fermentation broth of Examples 1-4 and Comparative Examples 1-6 was detected.

[0102] The results are shown in Table 1. The number of viable bacteria cultured using the fermentation medium of Example 1 was the highest, reaching 7.62×10 10CFU / mL. Threonine, serine, aspartic acid and phenylalanine were not added to the high-density fermentation medium (Comparative Example 1), and the number of viable fermentation bacteria decreased by 1 order of magnitude, which shows that the addition of threonine, serine, aspartic acid and phenylalanine to the culture medium has a significant growth-promoting effect on Akkermansia muciniphila. When only threonine was added to the high-density culture medium, or when oligosaccharides were replaced with glucose (Comparative Examples 2 and 3), the number of viable fermentation bacteria of Akkermansia muciniphila recovered to a certain extent, but the number of viable fermentation bacteria was still less than that in Example 1. When the concentrations of each component in the fermentation medium were adjusted outside the protection scope of the present invention (Comparative Example 4), the number of viable fermentation bacteria decreased by 1 order of magnitude, and the fermentation effect was far less than that in Example 1. When maltose was not added to the feed medium and the glucose content was increased to 300 g / L (Comparative Example 5), the number of viable fermentation bacteria was only about half of that in Example 1 due to the influence of the accumulation of metabolites; after maltose was replaced with an equal amount of fructose (Comparative Example 6), the number of viable fermentation bacteria was also poor due to the influence of the accumulation of metabolites.

[0103] Table 1

[0104] Experimental Group Viable bacteria count (CFU / mL) Example 1 <![CDATA[(7.62±0.32)×10 10 ]]> Example 2 <![CDATA[(5.85±0.12)×10 10 ]]> Example 3 <![CDATA[(6.12±0.16)×10 10 ]]> Example 4 <![CDATA[(7.03±0.22)×10 10 ]]> Comparative Example 1 <![CDATA[(5.30±0.27)×10 9 ]]> Comparative Example 2 <![CDATA[(2.58±0.08)×10 10 ]]> Comparative Example 3 <![CDATA[(3.16±0.15)×10 10 ]]> Comparative Example 4 <![CDATA[(8.10±0.18)×10 9 ]]> Comparative Example 5 <![CDATA[(3.14±0.26)×10 10 ]]> Comparative Example 6 <![CDATA[(3.64±0.30)×10 10 ]]>

[0105] After testing the OD600 during the fermentation process, the test results were consistent with the above conclusions: the addition of threonine, serine, aspartic acid and phenylalanine to the culture medium significantly promoted the growth of Akkermansia muciniphila, which was better than adding only one amino acid; when the oligosaccharide in the high-density fermentation medium was replaced with glucose or the concentration of each component was adjusted to outside the protection scope of the present invention, the fermentation effect was poor ( Figure 1 ).

[0106] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for fermenting Akkermansia muciniphila, characterized in that: The method comprises the following steps: (1) activating Akkermansia muciniphila or a culture or isolate thereof to obtain a seed solution; (2) inoculating the seed liquid into a high-density fermentation medium for anaerobic fermentation; The high-density fermentation medium comprises the following components: Galacto-oligosaccharides, maltose, peptone, beef extract, yeast extract, diammonium citrate, Tween 80, sodium acetate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, threonine, serine, aspartic acid and phenylalanine.

2. The method according to claim 1, characterized in that: The high-density fermentation medium comprises the following components: 2-6 g / L galacto-oligosaccharide, 1-4 g / L maltose, 8-15 g / L peptone, 8-15 g / L beef extract, 3-8 g / L yeast extract, 1-3 g / L diammonium hydrogen citrate, 0.5-1.5 mL / L Tween 80, 3-8 g / L sodium acetate, 1-3 g / L dipotassium hydrogen phosphate, 0.4-0.6 g / L magnesium sulfate, 0.2-0.3 g / L manganese sulfate, 0.2-1 g / L threonine, 1-2 g / L serine, 0.1-0.5 g / L aspartic acid and 0.01-0.1 g / L phenylalanine.

3. The method according to claim 1 or 2, characterized in that: The step (2) further comprises: When Akkermansia muciniphila in the fermentation broth reached the logarithmic growth phase, feed medium was added to carry out fed-batch culture.

4. The method according to claim 3, characterized in that The feed medium includes 180-220 g / L glucose, 80-120 g / L maltose and 180-220 g / L peptone.

5. The method according to claim 1 or 4, characterized in that: The conditions for anaerobic fermentation in step (2) include: 1) The dissolved oxygen in the culture medium is less than 1 mg / L; 2) Culture temperature 37-38°C; 3) Constantly control pH at 6.5-7.

0.

6. A high-density fermentation medium, characterized in that: The high-density fermentation medium comprises the following components: Galacto-oligosaccharides, maltose, peptone, beef extract, yeast extract, diammonium citrate, Tween 80, sodium acetate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, threonine, serine, aspartic acid and phenylalanine.

7. The high-density fermentation medium according to claim 6, characterized in that The high-density fermentation medium comprises the following components: 2-6 g / L galacto-oligosaccharide, 1-4 g / L maltose, 8-15 g / L peptone, 8-15 g / L beef extract, 3-8 g / L yeast extract, 1-3 g / L diammonium hydrogen citrate, 0.5-1.5 mL / L Tween 80, 3-8 g / L sodium acetate, 1-3 g / L dipotassium hydrogen phosphate, 0.4-0.6 g / L magnesium sulfate, 0.2-0.3 g / L manganese sulfate, 0.2-1 g / L threonine, 1-2 g / L serine, 0.1-0.5 g / L aspartic acid and 0.01-0.1 g / L phenylalanine.

8. Use of the high-density fermentation medium according to claim 6 or 7 in high-density fermentation culture of Akkermansia muciniphila.

9. Use of the Akkermansia muciniphila fermentation product obtained by the method according to any one of claims 1 to 5, or the Akkermansia muciniphila fermentation product cultured in the high-density fermentation medium according to any one of claims 6 to 7 in the preparation of a probiotic product.

10. The use according to claim 9, characterized in that: The probiotic preparation further comprises a food-acceptable auxiliary material, wherein the auxiliary material comprises erythritol, D-mannitol, fumaric acid, glycerin, pectin, potassium alginate, sodium alginate, talc, sodium pyrophosphate, polydextrose, carrageenan, sodium ascorbate, ascorbyl palmitate, L-malic acid, L(+)-tartaric acid, maltitol, gelatin, xylitol, citric acid, potassium citrate, sodium citrate, citric acid fatty acid glyceride, agar, lactic acid, sodium lactate, sorbic acid and its potassium salt, sorbitol, acid red, calcium carbonate, sodium carbonate, sodium bicarbonate, beet red, vitamin C, vitamin E, oxidized starch, ethanol, sodium acetate, stearic acid, calcium stearate, magnesium stearate or dextrin; and / or, the dosage form of the probiotic preparation includes a liquid preparation, a solid preparation, a semisolid preparation or a gaseous preparation; And / or, the probiotic product includes recombined milk, sterilized milk, fermented milk, condensed milk, cream, light cream, anhydrous butter, concentrated milk, compound protein beverage, coffee beverage, plant beverage, flavored beverage, sports drink, nutrient beverage, energy drink, electrolyte beverage, whole milk powder, skimmed milk powder, partially skimmed milk powder, recombined milk powder, whey powder, cheese, processed cheese, popsicle, slush, popsicle, edible ice, sweet ice or ice cream.

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