Culture medium and method for culturing akkermansia muciniphila

Abstract

Disclosed are a method for optimizing a bacterial culture medium based on a metabolic network model, an obtained Akkermansia muciniphila culture medium, and a related use. Compared with culture mediums in the prior art, the culture medium optimized by means of the method achieves reduced redundant components, simple components, and low costs, and is capable of directly and effectively promoting high-density culture of bacteria. Therefore, the obtained Akkermansia muciniphila culture medium can increase the number of viable bacteria to 1010 cfu / mL or above, thereby making industrial production of Akkermansia muciniphila possible.

Description

Culture medium and method for culturing Akkermansia muciniphila Technical Field

[0001] The present invention relates to the field of microbial culture, and in particular to a high-density culture medium for Akkermansia muciniphila (A. muciniphila), a method for obtaining the culture medium, and related applications thereof. Background Art

[0002] Akkermansia muciniphila is a Gram-negative bacterium isolated from the human intestine. It is anaerobic but can tolerate nanomolar oxygen levels. It is abundant in the intestine, comprising approximately 3-5% of the total intestinal microbial population. It resides in the intestinal mucus layer and can utilize mucin as its sole carbon and nitrogen source. Because Akkermansia muciniphila is the only member of the Verrucomicrobia phylum in the mammalian intestine and is easily detected using its 16S rRNA gene sequence, numerous reports have shown that changes in the abundance of Akkermansia in the intestine are associated with host health and disease.

[0003] The intestinal microbiota is closely linked to various host physiological processes, such as energy intake, immune system maturation, and hormone secretion. As a key member of the intestinal microbiota, Akkermansia muciniphila is considered one of the "next-generation probiotics," and changes in its abundance are significantly associated with various physiological disorders, including obesity, diabetes, and inflammatory bowel disease. Numerous studies have shown that administration of live or pasteurized Akkermansia muciniphila to mice reduces weight and fat mass gain, decreases serum triglyceride and fasting blood glucose levels, and improves insulin sensitivity. The mechanism by which Akkermansia muciniphila regulates lipid metabolism can be explained by its role in barrier function and immune responses, with its metabolites serving as a substrate. Furthermore, potential cross-feeding and nutritional interactions between Akkermansia muciniphila and butyrate-producing bacteria may also promote butyrate production. These short-chain fatty acids may activate GPR43 or GPR41, further regulating lipid and glucose metabolism.

[0004] Many studies have shown that colitis and inflammatory bowel disease (IBD) are associated with altered abundance of Akkermansia muciniphila. The abundance of Akkermansia muciniphila is significantly decreased in patients with IBD. Higher levels of Akkermansia muciniphila have been observed in the gut of patients with constipation-predominant irritable bowel syndrome (C-IBS) compared with healthy individuals. Animals harboring the C-IBS microbiota exhibit recovery from DSS colitis, and pretreatment of conventional C57BL / 6 mice or human microbiota-associated rats with Akkermansia muciniphila also reduces the severity of colitis. These findings suggest an anti-inflammatory role for the gut microbiota, particularly Akkermansia muciniphila, in patients with C-IBS.

[0005] Meanwhile, studies suggest that Akkermansia muciniphila may play a role in the development and regulation of autoimmune diseases. NOD mice fed a gluten-free diet showed a lower incidence of spontaneous type 1 diabetes and an increased abundance of Akkermansia muciniphila. Akkermansia muciniphila was particularly abundant in cancer patients who responded to PD-1 therapy compared to those who did not.

[0006] Although Akkermansia muciniphila has many health benefits, the bottleneck problem of large-scale cultivation of Akkermansia muciniphila needs to be solved. Derrien et al. disclosed a culture method, and Chinese Patent No. CN107849093B and Chinese Patent No. CN107384828B were optimized based on the method published by Derrien et al., but these three culture media are all based on the basic culture medium provided by Derrien et al. The composition of this basic culture medium is extremely complex, with about 30 kinds of compounds. After the fermentation is completed, it is difficult to centrifuge in the downstream processing process, the centrifugal yield is extremely low, and the loss is too large, which is not conducive to industrial production. In addition, the culture medium is rich in Ca 2+, calcium salt precipitation is produced after high-temperature and high-pressure sterilization, which is extremely detrimental to production equipment. In addition, the culture method disclosed by Derrien et al. and Chinese Patent No. CN107384828B also involves animal-derived ingredients, containing potential allergens and other ingredients that are not desired in food and drug production. In addition, when using it for conventional culture, the inventor found that the growth of Akkermansia muciniphila was affected by the initial liquid level of the culture medium. Chinese Patent No. CN112342151B discloses a culture method. Although it is not restricted by the liquid level, its improvement is based on BHI culture medium, which has complex ingredients, is also rich in animal-derived ingredients, and the cost is still high. In addition, no in-depth research has been conducted on the high-density fermentation of Akkermansia muciniphila. Another Chinese Patent No. CN201910265701.0 discloses a culture medium. Although the culture medium composition is slightly simpler, the culture time is as long as 3 days, the bacterial count is low, and high-density fermentation has not been explored. Plovier H et al. (A purified membrane protein from Akkermansia muciniphila or the pasteurized bacterium improves metabolism in obese and diabetic mice [J]. Nature medicine, 2017, 23(1): 107-113) disclosed a synthetic culture medium that achieves the same culture efficiency as mucin without affecting the physiological effects of Akkermansia muciniphila. However, the culture medium contains 33 compounds and still remains unresolved, including complex composition, large amounts of precipitation after sterilization, low centrifugation yield, and difficulties in downstream processing. Furthermore, the culture medium contains Na2S·9H2O, which is strictly prohibited for use in food and drug production.

[0007] Due to the unique nature of Akkermansia muciniphila isolation (using a culture medium containing mucin), researchers' exploration of culture media for this species has been stuck in a rut. To date, published patents and patent applications have never explored the use of mucin derivatives: glucose, galactose, fucose, N-acetyl-D-glucosamine, and N-acetyl-D-galactosamine. Glucose and N-acetyl-D-glucosamine are particularly prevalent. However, during high-temperature and high-pressure sterilization of culture media, these substances undergo intense Maillard reactions with proteins and amino acids in the nitrogen source, resulting in significant nutrient loss and severe browning of the culture medium. Furthermore, the Maillard reaction products also have a certain antibacterial effect, potentially adversely affecting the growth of Akkermansia muciniphila.

[0008] Traditional methods for optimizing culture media for Akkermansia muciniphila are relatively unsophisticated and fail to accurately identify the nutrients required for Akkermansia muciniphila growth, significantly consuming the time and energy of researchers. Currently, mucin media and modified BHI media are commonly used for culturing Akkermansia muciniphila both domestically and internationally. These are expensive, and most cultures take 3-5 days, severely hindering the large-scale production and application of Akkermansia muciniphila. Furthermore, both mucin and the main component of BHI media (brain heart extract) are animal-derived products, posing other potential risks and making them unsuitable for use in human food or pharmaceuticals.

[0009] Therefore, there is a need in the art for a large-scale, low-cost, low-loss, and highly efficient non-animal-derived culture medium for Akkermansia muciniphila. Summary of the Invention

[0010] To address these challenges, the inventors of the present invention applied a genome-scale metabolic network model to optimize Akkermansia muciniphila culture medium for the first time. Combining theory with experiment, they ultimately developed a low-cost, simple, non-animal-derived culture medium for Akkermansia muciniphila that enables high-density fermentation production of Akkermansia muciniphila. This led to the completion of the present invention.

[0011] Therefore, in a first aspect of the present disclosure, there is provided a culture medium for culturing Akkermansia muciniphila, comprising:

[0012] -2-200 g / L of sugars, wherein the sugars are selected from disaccharides, oligosaccharides, polysaccharides, and any combination thereof;

[0013] -2-200 g / L glucosamine and / or glucosamine polymers, wherein the glucosamine and / or glucosamine polymers are selected from D-glucosamine, D-glucosamine hydrochloride, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetyl-D-mannosamine, chitosan oligosaccharides, chitosan, chitin, and any combination thereof;

[0014] -5-200 g / L of a non-animal nitrogen source, wherein the non-animal nitrogen source is selected from soy peptone, wheat peptone, yeast extract powder, yeast peptone, and any combination thereof;

[0015] -0.1-20g / L L-threonine, L-tryptophan, L-phenylalanine;

[0016] -0-50 mM reducing agent, the reducing agent is selected from L-cysteine, L-cysteine ​​hydrochloride, ascorbic acid, glutathione, SO3 2- 、S2O3 2- 、S2O42- , thiourea dioxide (CH4N2O2S), and any combination thereof;

[0017] -0-1g / L cobalamin;

[0018] -0-10 mM Mg 2+ ;as well as

[0019] - 0-10 g / L of buffer salt, wherein the buffer salt is selected from dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, sodium bicarbonate, sodium carbonate, and any combination thereof.

[0020] In a second aspect, the present disclosure provides a method for culturing Akkermansia muciniphila (A. muciniphila), comprising culturing Akkermansia muciniphila in the culture medium of the first aspect.

[0021] In summary, the present invention obtains a set of metabolic uptake substances suitable for the growth of microorganisms such as Akkermansia muciniphila based on a genome-scale metabolic network model. This set provides reference components for optimizing the culture medium. Then, based on experiments, components that promote microbial growth in the metabolic uptake substances are obtained, thereby ultimately providing a high-density culture medium for Akkermansia muciniphila. This culture medium reduces redundant components in the culture medium of the prior art, is simple in composition, low in cost, and does not contain animal-derived components, and can also increase the viable count of Akkermansia muciniphila to 10 10 cfu / mL or more, which makes it possible to realize the industrial production of Akkermansia muciniphila. The Akkermansia muciniphila culture medium of the present invention avoids the inclusion of various inorganic salts, especially Ca 2+ , thereby solving the problem of precipitation during high-temperature and high-pressure sterilization of Akkermansia muciniphila culture media in the prior art, avoiding damage to fermentation equipment. Furthermore, the culture medium does not contain ingredients such as Na2S·9H2O that are strictly prohibited for use in food and drug production. It has fewer Maillard reactions and less nutrient loss, resulting in good bacterial growth, rapid proliferation, short culture times, superior downstream processing performance, higher centrifugation yields, and virtually no processing losses. Furthermore, the non-animal-derived culture medium of the present invention avoids the presence of pollutants or allergens, has higher safety, and can meet the production requirements of the food and pharmaceutical industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific implementation or the description of the prior art.

[0023] FIG1 shows some metabolic uptake substances obtained by simulating the normal growth of Akkermansia muciniphila using a genome-scale metabolic network model.

[0024] FIG2 shows the effect of different pH on high-density fermentation of Akkermansia muciniphila.

[0025] FIG3 shows the exemplary improved culture medium of the present invention at 3×10 7 cfu / mL, 6×10 7 cfu / mL and 1.2×10 8 cfu / mL and the inoculum size of 1.2×10 8 cfu / mL inoculum size, and the effect of fed-batch fermentation on high-density fermentation of Akkermansia muciniphila.

[0026] FIG4 shows the difference in downstream centrifugation processing using a control medium and an exemplary modified medium of the present invention.

[0027] FIG5 shows the effects of Akkermansia muciniphila cultured in a control medium and an exemplary improved medium of the present invention on the body weight of hyperlipidemic mice.

[0028] FIG6 shows a comparison of the collinearity of the Akkermansia muciniphila genome sequences cultured using a control medium and an exemplary improved medium of the present invention (the upper figure shows the results obtained using the control medium, and the lower figure shows the results obtained using the exemplary improved medium of the present invention).

[0029] FIG7 shows a single colony of Akkermansia muciniphila cultured on a plate prepared according to one embodiment of the present invention. DETAILED DESCRIPTION

[0030] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the following description is merely illustrative of the present invention and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the appended claims. Furthermore, those skilled in the art will appreciate that the technical solutions of the present invention may be modified without departing from the spirit and purpose of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the present invention belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

[0032] Where a numerical range is provided, such as a concentration range, a percentage range, or a ratio range, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of the range and any other stated or intervening values ​​in the stated range are encompassed within the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the subject matter.

[0033] In the context of the present invention, many embodiments use the expressions "comprising", "including" or "consisting essentially / mainly of..." The expressions "comprising", "including" or "consisting essentially / mainly of..." can generally be understood as open-ended expressions, indicating that in addition to the elements, components, assemblies, method steps, etc. specifically listed after the expression, other elements, components, assemblies, method steps, etc. are also included. In addition, in this document, the expressions "comprising", "including" or "consisting essentially / mainly of..." can also be understood as closed-ended expressions in some cases, indicating that only the elements, components, assemblies, method steps specifically listed after the expression are included, and no other elements, components, assemblies, method steps are included. In this case, the expression is equivalent to the expression "consisting of..."

[0034] As described above, the present invention obtains a set of metabolic uptake substances suitable for the growth of microorganisms such as Akkermansia muciniphila based on a genome-scale metabolic network model. This set provides reference components for optimizing the culture medium. Then, based on experiments, components in the set of metabolic uptake substances that promote microbial growth are obtained, thereby ultimately providing a high-density culture medium for Akkermansia muciniphila.

[0035] Therefore, in a first aspect of the present disclosure, the present disclosure provides a culture medium for culturing Akkermansia muciniphila, comprising:

[0036] -2-200 g / L of sugars, wherein the sugars are selected from disaccharides, oligosaccharides, polysaccharides, and any combination thereof;

[0037] -2-200 g / L of glucosamine and / or a polymer, wherein the glucosamine and / or glucosamine polymer is selected from D-glucosamine, D-glucosamine hydrochloride, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetyl-D-mannosamine, chitosan oligosaccharide, chitosan, chitin, and any combination thereof;

[0038] -5-200 g / L of a non-animal nitrogen source, wherein the non-animal nitrogen source is selected from soy peptone, wheat peptone, yeast extract powder, yeast peptone, and any combination thereof;

[0039] -0.1-10g / L L-threonine, L-phenylalanine, L-tryptophan;

[0040] -0-50 mM reducing agent, the reducing agent is selected from L-cysteine, L-cysteine ​​hydrochloride, ascorbic acid, reduced glutathione, SO3 2- 、S2O3 2- 、S2O4 2- , thiourea dioxide (CH4N2O2S), and any combination thereof;

[0041] -0-1g / L cobalamin;

[0042] -0-10 mM Mg 2+ ;as well as

[0043] - 0-10 g / L of buffer salt, wherein the buffer salt is selected from dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, sodium bicarbonate, sodium carbonate, and any combination thereof.

[0044] In the context of the present disclosure, those skilled in the art will appreciate that both sugars and glucosamine and / or glucosamine polymers can serve as carbon sources for culturing Akkermansia muciniphila.

[0045] In a preferred embodiment, the concentration of the carbohydrate can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 150 or 200 g / L or any value therebetween. In a preferred embodiment, the concentration of the carbohydrate can be 2-50 g / L. In a more preferred embodiment, the concentration of the carbohydrate can be 4-20 g / L.

[0046] In a specific embodiment, the disaccharide may be selected from lactose, maltose, isomaltulose, cellobiose, turanose, melibiose, and any combination thereof, but is not limited thereto.

[0047] In another specific embodiment, the oligosaccharide can be selected from galacto-oligosaccharides, fructo-oligosaccharides, mannose oligosaccharides, isomalto-oligosaccharides, maltooligosaccharides, soybean oligosaccharides, human milk oligosaccharides, and any combination thereof, but is not limited thereto.

[0048] In a more specific embodiment, the human milk oligosaccharide may be selected from 2'-salulosyl lactose, 3'-sialyllactose, lacto-N-neotetraose or lacto-N-triose, and any combination thereof, but is not limited thereto.

[0049] In another specific embodiment, the polysaccharide can be selected from dextrin, starch, modified starch, dextran, and any combination thereof, but is not limited thereto. Among them, dextrin can be maltodextrin, resistant dextrin, cyclodextrin, etc. Modified starch can be, for example, pregelatinized starch, soluble starch, etc.

[0050] In a preferred embodiment, the concentration of the glucosamine and / or glucosamine polymer can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 150 or 200 g / L or any numerical value therebetween. In a preferred embodiment, the concentration of the glucosamine and / or glucosamine polymer can be 2-50 g / L. In a more preferred embodiment, the concentration of the glucosamine and / or glucosamine polymer can be 5-20 g / L.

[0051] In another preferred embodiment, the glucosamine and / or glucosamine polymer may be N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, chitosan, or any combination thereof, but is not limited thereto.

[0052] In a preferred embodiment, the non-animal nitrogen source may be yeast extract.

[0053] The concentration of the non-animal derived nitrogen source can be 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 150 or 200 g / L or any value therebetween. In a preferred embodiment, the concentration of the non-animal derived nitrogen source can be 5-50 g / L.

[0054] In a more preferred embodiment, the concentration of the non-animal derived nitrogen source may be 10-20 g / L.

[0055] In the context of the present disclosure, those skilled in the art will appreciate that, in addition to the nitrogen sources listed above, glucosamine and / or glucosamine polymers can also be used as nitrogen sources for culturing Akkermansia muciniphila.

[0056] In a preferred embodiment, the concentration of the L-threonine can be 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 g / L or any value therebetween. In a preferred embodiment, the concentration of the L-threonine can be 2-10 g / L. In a more preferred embodiment, the concentration of the L-threonine can be 4-8 g / L.

[0057] In a further preferred embodiment, the culture medium may further include other amino acids in addition to L-threonine, and the other amino acids may be selected from L-phenylalanine, L-tryptophan, or any combination thereof. The present inventors have discovered that, compared with the addition of other amino acids to the culture medium, the addition of L-threonine to the culture medium can significantly promote the growth of Akkermansia muciniphila; and that the addition of L-phenylalanine and / or L-tryptophan to L-threonine can further significantly promote the growth of Akkermansia muciniphila.

[0058] Therefore, in a more preferred embodiment, the amino acid may be a combination of L-threonine and L-phenylalanine, or a combination of L-threonine and L-tryptophan, or a combination of L-threonine, L-phenylalanine, and L-tryptophan.

[0059] In the context of the present disclosure, the concentration of the other amino acids can be 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.0 g / L or any value therebetween. In a preferred embodiment, the concentration of the other amino acids can be 0.5-2.0 g / L.

[0060] In a preferred embodiment, the concentration of cobalamin can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 150, 200, 350, 500, 800 or 1000 mg / L or any value therebetween. In a preferred embodiment, the concentration of cobalamin can be 0.1-10 mg / L. The present inventors have found that, compared to adding other vitamins to the culture medium, the addition of only cobalamin to the culture medium can significantly promote the growth of Akkermansia muciniphila. Therefore, in the culture medium of the present invention, only cobalamin can be included without other vitamins.

[0061] In another preferred embodiment, the concentration of the reducing agent can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 mM or any value therebetween. In a preferred embodiment, the concentration of the reducing agent can be 0.1-10 mM. In a more preferred embodiment, the concentration of the reducing agent can be 1-10 mM.

[0062] The inventors found that Akkermansia muciniphila hardly grows in a culture medium without adding a reducing agent. Commonly used Na2S and its hydrates such as Na2S·9H2O are prohibited from being used in food and drug production. Therefore, it is necessary to find a reducing agent that can replace Na2S and its hydrates. Reducing agents such as ascorbic acid, reduced glutathione, L-cysteine ​​hydrochloride, which are widely used in probiotic culture, and S2O3 2- (such as Na2S2O3·5H2O), S2O3 2- 、S2O4 2- , thiourea dioxide (CH4N2O2S), etc., can achieve a reduction effect equivalent to that of Na2S·9H2O in the selected concentration range, and can achieve indiscriminate replacement of Na2S·9H2O.

[0063] In a preferred embodiment, the reducing agent may be S2O3 2- .

[0064] In addition, it can be understood that in the present invention, S2O3 2- and S2O4 2- It can be derived from its sodium salt, potassium salt or hydrate thereof, such as Na2S2O3·5H2O, Na2S2O4, but is not limited thereto.

[0065] Inorganic salt Mg 2+ The concentration of Mg can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mM or any value therebetween. In a preferred embodiment, the Mg 2+ The concentration of Mg can be 0-5mM. It is understood that in the present invention, Mg 2+ It may be derived from magnesium chloride, magnesium sulfate, or a hydrate thereof, but is not limited thereto.

[0066] The inventors of the present invention have found that the inorganic salt ion Ca commonly used in the culture medium of Akkermansia muciniphila 2+ The growth of Akkermansia muciniphila is less affected by Ca, so it is not necessary to add Ca to the culture medium. 2+ , thereby avoiding precipitation under high temperature and high pressure sterilization conditions, and thus avoiding damage to the culture equipment.

[0067] In addition, the inventors of the present invention also found that acidic trace element solutions (such as FeCl2, H3BO3, ZnCl2, CuCl2·2H2O, MnCl2, CoCl2, NiCl2, etc.) and alkaline trace element solutions (such as Na2SeO3, Na2WO4·2H2O, Na2MoO4·2H2O, etc.) commonly used in the cultivation of Akkermansia muciniphila have little effect on the growth of Akkermansia muciniphila, and therefore do not need to be added to the culture medium.

[0068] In yet another specific embodiment, the fermentation medium may further include 2-7 g / L of buffer salt.

[0069] In yet another specific embodiment, the concentration range of potassium dihydrogen phosphate, disodium hydrogen phosphate and dipotassium hydrogen phosphate is 0-4.5 g / L respectively. In a preferred embodiment, the concentration range of potassium dihydrogen phosphate, disodium hydrogen phosphate and dipotassium hydrogen phosphate is 0-2 g / L respectively.

[0070] In yet another specific embodiment, the concentration range of sodium bicarbonate is 1-10 g / L. In a preferred embodiment, the concentration range of sodium bicarbonate is 1-5 g / L.

[0071] In a more preferred embodiment, the culture medium may include 4-15 g / L sugars, 10-20 g / L glucosamine and / or glucosamine polymers, 10-20 g / L yeast extract, 1-1.2 g / L phenylalanine, 0.25 g / L sodium thiosulfate pentahydrate or its corresponding amount of sodium thiosulfate or 0.11 g / L CH4N2O2S, 4-8 g / L L-threonine, 0.1 mg / L cobalamin, and optionally 0.5 g / L tryptophan, 0.1-0.12 g / L magnesium chloride, and 6-7 g / L buffer salts.

[0072] Without wishing to be bound by theory, a person skilled in the art would appreciate that the culture medium of the present invention can be used not only for high-density culture of Akkermansia muciniphila but also for static culture.

[0073] As commonly used in the art, the term "static culture" refers to a technique of culturing cells in a container containing liquid culture medium without aeration or shaking.

[0074] As commonly used in the art, the term "high-density culture" is also known as high-density fermentation, which refers to a culture technique in which microorganisms grow in a liquid culture at a cell population density that is more than 10 times that of conventional culture.

[0075] As commonly used in the art, high-density culture can be carried out using a fed-batch fermentation method. For example, a continuous fed-batch method can be used for feeding in high-density culture. As commonly used in the art, the term "fed-batch fermentation" refers to a fermentation technique in which, during the batch fermentation process of microorganisms, certain materials are added to the fermentation system in a certain manner, but the fermentation liquid is not continuously discharged. This fermentation technique is a fermentation technique between batch fermentation and continuous fermentation. Depending on whether the feeding time is continuous, it can be divided into intermittent feeding and continuous feeding. Those skilled in the art will know that high concentrations of substrate in the culture medium will inhibit the growth of microorganisms, while fed-batch fermentation is to first ferment under conditions of relatively low nutrient concentrations, and then continuously add fresh nutrients to allow the bacteria to grow normally and continuously accumulate metabolites, thereby avoiding substrate inhibition caused by excessive one-time feeding in batch fermentation.

[0076] As commonly used in the art, the feed can be a complete feed medium or a semi-fed batch medium. The so-called "complete feed medium" refers to a medium supplemented with complete nutrients, while the so-called "semi-fed batch medium" refers to an incomplete medium supplemented with only one or more nutrients, for example, an incomplete medium supplemented with only a carbon source (such as maltose, maltodextrin, N-acetylglucosamine, lactose, N-acetylmannosamine, and any combination thereof), or an incomplete medium supplemented with a carbon source (such as maltose, maltodextrin, N-acetylglucosamine, lactose, N-acetylmannosamine, and any combination thereof) and a nitrogen source (such as soy peptone, wheat peptone, yeast extract powder, yeast peptone, and any combination thereof), or a medium with several times (such as 1-10 times) the concentration but without a buffer.

[0077] In a second aspect of the present disclosure, a method for culturing Akkermansia muciniphila is provided, the method comprising culturing Akkermansia muciniphila in the culture medium according to the first aspect.

[0078] As mentioned above, without wishing to be bound by theory, it is understood that the culture medium of the present invention can be used not only for high-density culture but also for static culture.

[0079] In a specific embodiment, the culture is static culture, high-density culture, or static culture followed by high-density culture.

[0080] In another specific embodiment, the culture is carried out under anaerobic conditions. Herein, the "anaerobic conditions" are well known to those skilled in the art, such as 5-10% H2, 5-10% CO2 and 80-85% N2.

[0081] In another specific embodiment, the static culture comprises culturing at 36° C.-38° C. for 12-36 hours and performing 2-3 subcultures. In a preferred embodiment, the 2nd-3rd subculture is performed using the culture medium described in the second aspect.

[0082] In another specific embodiment, in the high density culture, the Akkermansia muciniphila is cultured at a density of 3×10 7 cfu / mL-3×10 8 The inoculum size of cfu / mL was inoculated into the culture medium.

[0083] In a preferred embodiment, the Akkermansia muciniphila is expressed in 3×10 7 cfu / mL-1.5×10 8 The inoculum size of cfu / mL was inoculated into the culture medium.

[0084] In a more preferred embodiment, the Akkermansia muciniphila is present at a concentration of 3×10 7 cfu / mL-1.2×10 8 The inoculum size of cfu / mL was inoculated into the culture medium.

[0085] In yet another specific embodiment, the high-density culture is carried out at a temperature of 20° C. to 40° C. In a preferred embodiment, the high-density culture is carried out at a temperature of 36° C. to 38° C. In a further preferred embodiment, the high-density culture is carried out at a temperature of 37° C.

[0086] In another specific embodiment, the high-density culture time is 24h-36h.

[0087] In another specific embodiment, the high-density culture further comprises controlling the pH of the culture medium during the culture process to be between 6 and 8. In a preferred embodiment, the pH of the culture medium is controlled to be between 6.0 and 7.5.

[0088] In another specific embodiment, the high-density culture is carried out at a rotation speed of 50-300 rpm. In a preferred embodiment, the high-density culture is carried out at a rotation speed of 50-150 rpm.

[0089] Without wishing to be bound by theory, the culture medium for static culture before high-density culture may also be different from the culture medium for high-density culture. In an exemplary embodiment, the culture medium for static culture before high-density culture may include, but is not limited to, BHI culture medium, mucin culture medium, and Columbia culture medium.

[0090] In another specific embodiment, the high density culture is carried out in a fed-batch fermentation mode. For example, a continuous feeding mode can be used for feeding in the high density culture.

[0091] In a further specific embodiment, the feed can be a complete feed medium or a semi-fed batch medium. As mentioned above, the so-called "complete feed medium" refers to a medium supplemented with complete nutrients, while the so-called "semi-fed batch medium" refers to an incomplete medium supplemented with only one or more nutrients, for example, an incomplete medium supplemented with only a carbon source (such as maltose, maltodextrin, D-glucosamine, N-acetylglucosamine, lactose, N-acetylmannosamine, and any combination thereof), or an incomplete medium supplemented with a carbon source and a nitrogen source (such as soy peptone, wheat peptone, yeast extract powder, yeast peptone, and any combination thereof), or a medium with a 1-10 times (e.g., 3-5 times) concentration but no buffer.

[0092] In a specific embodiment, the feed may be a semi-fed-batch medium. As an example, the semi-fed-batch medium may include, but is not limited to, a carbon source at a concentration of 1-10 times, such as 240 g / L. The carbon source may be maltose, maltodextrin, D-glucosamine, N-acetylglucosamine, lactose, N-acetylmannosamine, or any combination thereof.

[0093] In a preferred embodiment, during high-density culture, 1 mL·L -1 ·h -1 to 6mL·L -1 ·h -1 Continuous feeding is performed at a feed flow rate of 1.2 mL·L, where mL is the volume of the feed medium (ml), L is the volume of the original fermentation system (liter), and h is the feeding time (hour). In a further preferred embodiment, during high-density culture, a feed flow rate of 1.2 mL·L can be used. -1 ·h -1 Continuous feeding was performed at a feeding rate of .

[0094] Those skilled in the art will appreciate that, in the present invention, by supplementing the culture medium of Akkermansia muciniphila with one or more nutrients required for Akkermansia muciniphila fermentation, such as a carbon source, the bacteria can be grown and multiplied continuously and in large quantities, thereby achieving high-density culture of Akkermansia muciniphila and avoiding the problem of decreased bacterial volume caused by prolonged centrifugation after fermentation. Furthermore, the use of a fed-batch method can further reduce fermentation time, shorten the fermentation cycle, improve fermentation efficiency, simplify fermentation steps, reduce equipment investment, and lower production costs, which is very beneficial for achieving large-scale production of Akkermansia muciniphila.

[0095] In the third aspect of the present disclosure, a strain Akk-101 of Akkermansia muciniphila cultured according to the second aspect of the present disclosure is provided, which was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) on September 13, 2023, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC NO.40786.

[0096] In other aspects of the present disclosure, a method for optimizing microbial culture medium based on a genome-scale metabolic network model is provided, comprising the following steps:

[0097] 1) constructing a genome-scale metabolic network model based on all protein sequences in the microbial genome, wherein the genome-scale metabolic network model involves biochemical reactions at three locations: the extracellular space, the periplasm, and the cytoplasm of the microbial cell, and the biochemical reactions include bacterial growth simulation reactions;

[0098] 2) Based on the genome-scale metabolic network model, multiple flow balance analyses are performed under different intake conditions consisting of multiple intake substances involved in biochemical reactions to simulate normal growth of the microorganism, thereby obtaining a set of metabolic intake substances involved in the normal growth of the microorganism;

[0099] 3) For each metabolic uptake substance in the set of metabolic uptake substances, the effect of the metabolic uptake substance on microbial growth is determined by comparing the microbial growth in a reference culture medium containing and excluding the metabolic uptake substance, thereby obtaining a metabolic uptake substance that promotes microbial growth, which is a candidate culture medium component for the microbial culture medium.

[0100] In the context of the present disclosure, the "metabolic network model" refers to the genome scale "metabolic network model (GSMM)", which includes the vast majority of biochemical reactions occurring in a given microbial cell (such as bacteria). The metabolic regulation of microbial cells occurs at the levels of gene-gene, gene-protein, protein-protein, protein-metabolite and physical interactions. Through integration with other omics data, such as transcriptomes and proteomes, GSMM is used to fully understand the regulatory mechanisms within microbial cells. Therefore, GSMM can provide an efficient platform to understand the physiological metabolic functions of microorganisms from a global level, and is widely used in qualitative and quantitative predictions of cell growth phenotypes under different metabolic or environmental perturbation conditions. "Flux balance analysis (FBA)" is a mathematical method proposed in recent years that can be used to construct and simulate the analysis of GSMM.

[0101] In a specific embodiment, the method further comprises: classifying the candidate culture medium components, and simplifying the components in the microbial culture medium based on the classification.

[0102] Those skilled in the art will appreciate that the method for optimizing microbial culture media of the present invention is a universal method that can be applied to a variety of bacterial species, including Akkermansia, Lactobacillus, Blautia, and Bifidobacterium, but is not limited thereto. In the context of the present disclosure, the inventors used the above method to optimize the culture medium of Akkermansia muciniphila within the genus Akkermansia, thereby providing a high-density culture medium and culture process for Akkermansia muciniphila.

[0103] Hereinafter, the present invention will be described in more detail in conjunction with exemplary embodiments. However, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be considered as intended to interpret the scope of the present invention.

[0104] Example

[0105] The following examples describe methods for optimizing Akkermansia muciniphila culture media using the metabolic network model analysis method of the present invention, as well as the optimized Akkermansia muciniphila culture media, high-density culture methods, and related characterization. Unless otherwise specified, all experimental methods employed are conventional methods, and all experimental materials used in the following examples were purchased from conventional reagent stores. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains.

[0106] It should be noted that the terms used in the description of the present invention are intended only to describe specific embodiments and are not intended to limit the present invention. The above summary of the invention and the detailed description below are intended only to illustrate the present invention and are not intended to limit the present invention in any way. Without departing from the spirit and purpose of the present invention, the scope of the present invention is determined by the appended claims.

[0107] Example 1: Obtaining candidate culture medium components from a metabolic network model of the Akkermansia muciniphila genome

[0108] Step 1: Retrieve all protein sequences of the Akkermansia muciniphila genome from NCBI (https: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCF / 000 / 020 / 225 / GCF_000020225.1_ASM2022v1 / file "GCF_000020225.1_ASM2022v1_protein.faa.gz" is the protein sequence file of Akkermansia muciniphila).

[0109] Step 2: Based on all protein sequences of the Akkermansia muciniphila genome obtained above, a genome-scale metabolic network model was constructed using the [carveme] software (available from https: / / github.com / cdanielmachado / carveme). The method for constructing this genome-scale metabolic network model can be found at https: / / carveme.readthedocs.io / en / latest / (the metabolic network model file is in SBML format, http: / / sbml.org / ).

[0110] The results showed that the genome-scale metabolic network model of Akkermansia muciniphila simulates the flow of substances in the extracellular space, periplasm, and cytosol of the bacteria, involving 737 compounds and 1,490 biochemical reactions. The specific reaction types include:

[0111] Biochemical reactions involving 904 enzymes

[0112] Transport reactions of 431 simulated compounds transported in different bacterial compartments

[0113] 151 exchange reactions simulating bacterial ingestion and secretion of compounds

[0114] 1 bacterial growth simulation reaction

[0115] 1 ATP synthesis reaction that simulates the cell's energy maintenance

[0116] Two pseudo-reactions simulating the absorption and production of compounds within bacteria

[0117] Step 3: Using the genome-scale metabolic network model of Akkermansia muciniphila, we simulated the normal growth of Akkermansia muciniphila under different environmental conditions, obtaining a variety of possible ingested and secreted substances for subsequent experimental analysis. The specific ingested and secreted substances are shown in Figure 1.

[0118] Example 2: Effects of different sugars on the growth of Akkermansia muciniphila

[0119] The control medium was prepared according to the medium formula provided by Plovier H et al., as follows: KH2PO4 0.4 g / L, Na2HPO4 0.53 g / L, NH4Cl 0.3 g / L, NaCl 0.3 g / L, MgCl2 0.1 g / L, CaCl2 0.11 g / L, NaHCO3 4 g / L, Na2S·9H2O 0.25 g / L, resazurin 0.5 mg / L, acidic trace element solution 1 mL / L, alkaline trace element solution 1 mL / L, vitamin solution 1 mL / L, soy peptone 16 g / L, threonine 4 g / L, glucose 4.5 g / L, acetylglucosamine 5.5 g / L, wherein: acidic trace element solution components: FeCl2 0.95 g / L, H3BO3 0.62 g / L, ZnCl2 0.068g / L, CuCl2·2H2O 0.017g / L, MnCl2 0.063g / L, CoCl2 0.065g / L, NiCl2 0.013g / L, HCl 50mmol / L; alkaline trace element solution composition: Na2SeO3 0.017g / L, Na2WO4·2H2O 0.033g / L, Na2MoO4·2H2O 0.024g / L, NaOH 0.4g / L; vitamin solution composition: thiamine 0.2g / L, riboflavin 0.1g / L, niacin 0.2g / L, calcium pantothenate 0.1g / L, pyridoxine 0.5g / L, biotin 0.2g / L, cobalamin 0.1g / L, p-aminobenzoic acid 0.1g / L.

[0120] Based on the above control medium, the glucose in the control medium was replaced by the sugars or sugar alcohols listed in Table 1 below, with the concentration of 4.5 g / L. The OD values ​​of the control medium were compared after culture. 600 Comparisons were made to determine the effects of each candidate culture medium component on the growth of Akkermansia muciniphila.

[0121] The specific steps were as follows: the preserved bacteria were taken from the -80°C freezer, activated in the above control medium for 24 h, then inoculated at 1% inoculum, cultured anaerobically (10% H2, 10% CO2, 80% N2) for 36 h, and then the OD was measured using a microplate reader (INFINITE M NANO, Tecan, Switzerland). 600 (200 μL / well), the results are shown in Table 1.

[0122] Table 1: Effects of candidate sugars and sugar alcohols on the growth of Akkermansia muciniphila Note: * indicates P < 0.05 compared with the control group; ** indicates P < 0.01 compared with the control group.

[0123] As shown in Table 1, the monosaccharides showed no significant increase compared with the control. On the contrary, fucose and galactose had a more serious Maillard reaction, resulting in an OD 600 Among the disaccharides, except for isomerized lactose, other sugars can significantly increase OD 600 , indicating that these disaccharides can better promote the growth of Akkermansia muciniphila than glucose. In this example, only six types of oligosaccharides, including oligofructose, oligogalactose, oligoisomaltodextrin, soluble starch and glucan, are listed. They can significantly increase the OD 600 , indicating that they can greatly promote the growth of Akkermansia muciniphila compared with glucose.

[0124] Example 3: Effects of different reducing agents on the growth of Akkermansia muciniphila

[0125] A control medium was prepared according to the recipe provided by Plovier H et al., as given in Example 2, in which the reducing agent was 0.25 g / L (1 mM) Na2S·9H2O. Culture media containing different reducing agents were prepared by replacing Na2S·9H2O with Na2S2O3·5H2O, Na2S2O4, CH4N2O2S, Na2SO3, L-cysteine ​​hydrochloride, ascorbic acid, and reduced glutathione, respectively, and compared with the control medium.

[0126] The specific steps were as follows: the preserved bacteria were taken from the -80°C freezer, activated in the above control medium for 24 h, inoculated at 1% inoculum, cultured anaerobically (10% H2, 10% CO2, 80% N2) for 36 h, and then the OD was measured using a microplate reader (INFINITE M NANO, Tecan, Switzerland). 600 (200 μL / well), the results are shown in Table 2.

[0127] Table 2: Effects of different reducing agents on the growth of Akkermansia muciniphila Note: * indicates P < 0.05 compared with the control group; ** indicates P < 0.01 compared with the control group.

[0128] The results in Table 2 show that Akkermansia muciniphila hardly grows in the culture medium without reducing agent (blank group). The experimental groups with Na2S2O3·5H2O, Na2S2O4, and CH4N2O2S have a significantly higher OD value than the control group. 600There is no significant difference. Na2S·9H2O is prohibited from being used in food and drug production, while Na2S2O3·5H2O, Na2S2O4, and CH4N2O2S are widely used in food and drug production, so they can replace the reducing agent in the original culture medium. Although Na2SO3 can be used in food and drug production, its effect is not as good as Na2S·9H2O. Although ascorbic acid, reduced glutathione, and L-cysteine ​​hydrochloride are widely used in probiotic culture, their reducing effects are also not as good as Na2S·9H2O. In the present invention, S2O3 obtained by flow equilibrium analysis 2- (Na2S2O3·5H2O) can replace Na2S·9H2O without any difference.

[0129] Example 4: Effects of different amino acids on the growth of Akkermansia muciniphila

[0130] A control medium was prepared according to the recipe provided by Plovier H et al., as given in Example 2, with 4 g / L threonine. In addition to threonine, various amino acids listed in Table 3 were additionally added to the control medium at 0.5 g / L to prepare media containing various amino acids, which were then compared with the control medium.

[0131] The specific steps were as follows: the preserved bacteria were taken from a -80°C freezer, activated in a control medium for 24 h, inoculated at a 1% inoculum volume, cultured anaerobically (10% H2, 10% CO2, 80% N2) for 36 h, and the OD was measured using a microplate reader (INFINITE M NANO, Tecan, Switzerland). 600 (200 μL / well), the results are shown in Table 3.

[0132] Table 3: Effects of different amino acids on the growth of Akkermansia muciniphila Note: * indicates P < 0.05 compared with the control group; ** indicates P < 0.01 compared with the control group.

[0133] The results in Table 3 show that, among the various amino acids, the addition of tryptophan or phenylalanine both promoted the growth of Akkermansia muciniphila. Cysteine ​​and cysteine ​​hydrochloride, commonly used reducing agents in probiotic cultures, promote the growth of anaerobic bacteria. However, they did not promote the growth of Akkermansia muciniphila in this culture medium. This is presumably because the Na₂S·9H₂O in the original culture medium provides sufficient reducing power, eliminating the need for the addition of cysteine. The addition of phenylalanine and tryptophan, as determined by flow equilibrium analysis in the present invention, can promote the growth of Akkermansia muciniphila.

[0134] Example 5: Effects of different vitamins on the growth of Akkermansia muciniphila

[0135] A control culture medium was prepared according to the recipe provided by Plovier H et al. in Example 2, wherein the vitamin solution was added in an amount of 1 mL. The final concentrations of the various vitamins contained in the vitamin solution were: 0.2 mg / L thiamine, 0.1 mg / L riboflavin, 0.2 mg / L niacin, 0.1 mg / L calcium pantothenate, 0.5 mg / L pyridoxine, 0.2 mg / L biotin, 0.1 mg / L cobalamin, and 0.1 mg / L para-aminobenzoic acid. A blank culture medium without vitamins and an experimental culture medium containing only one of the vitamin solutions were prepared. Separately, a culture medium containing 1 mg / L hemin and a culture medium containing 5 μg / L folic acid were prepared as experimental culture media.

[0136] Table 4: Effects of different vitamins on the growth of Akkermansia muciniphila Note: * indicates P < 0.05 compared with the blank group; ** indicates P < 0.01 compared with the blank group.

[0137] From the results in Table 4, we can see that compared with the blank group, the control group had a higher OD 600 The OD value of the control group was significantly increased, indicating that the vitamin solution in the control group had a promoting effect on the growth of Akkermansia muciniphila. After adding many vitamins separately, the OD value of the blank group was significantly increased when only cobalamin was added. 600 The significant difference indicates that among the vitamins in the vitamin solution, only cobalamin promotes the growth of Akkermansia muciniphila. The other vitamins showed no significant difference compared to the blank control, indicating that these vitamins have no effect on the growth of Akkermansia muciniphila. Furthermore, hemin and folic acid also had no effect on the growth of Akkermansia muciniphila.

[0138] Example 6. Comparison of optimized culture medium and control culture medium

[0139] The control culture medium was prepared according to the formula provided by Plovier H et al. in Example 2, and the OD values ​​of the optimized culture medium and the control culture medium were compared. 600 difference.

[0140] The specific steps were as follows: the preserved bacteria were taken from a -80°C freezer, activated in a control medium for 24 h, inoculated at a 1% inoculum size, cultured anaerobically (10% H2, 10% CO2, 80% N2) in the control medium and the optimized medium for 36 h, and the OD values ​​were measured using an enzyme-labeled instrument (INFINITE M NANO, Tecan, Switzerland). 600 (200 μL / well), the results are shown in Table 5.

[0141] The results in Table 5 show that compared with formula 1 (control), culture medium formula 2 does not contain CaCl2, NaCl, NH4Cl, resazurin, acidic trace element solution (including: FeCl2, H3BO3, ZnCl2, CuCl2·2H2O, MnCl2, CoCl2, NiCl2) and alkaline trace element solution (including: Na2SeO3, Na2WO4·2H2O, Na2MoO4·2H2O), and replaces the vitamin solution (including: thiamine, riboflavin, niacin, calcium pantothenate, pyridoxine, biotin, para-aminobenzoic acid) with cobalamin. The results show that OD 600 The number of viable bacteria did not decrease significantly. The OD 600 Table 5: Effects of optimized culture medium and original culture medium on the growth of Akkermansia muciniphila Note: * indicates P < 0.05 compared with the control group; ** indicates P < 0.01 compared with the control group.

[0142] Example 7: Effect of pH on high-density fermentation of Akkermansia muciniphila

[0143] High-density fermentation was carried out using Formulation 3 in Table 5 of Example 6 (hereinafter also referred to as Medium No. 3) as a high-density culture medium.

[0144] The specific steps are as follows: a strain stored at -80°C was inoculated at a 1% inoculum into a test tube containing 5 mL of a control culture medium. After activation for 24 hours, the inoculum was transferred to 100 mL of Medium No. 3 at a 1% inoculum. After activation for an additional 24 hours, the inoculum was transferred to a fermenter containing Medium No. 3 at a 1% inoculum. Culture conditions were: 37°C, 150 rpm, with a slow aeration of 10% H2, 10% CO2, and 80% N2. The pH was controlled at 7.5, 7.0, 6.5, 6.0, or no pH control was performed.

[0145] The initial pH of the fermentation medium was about 8.5. 3M hydrochloric acid was used to adjust the pH to the set value and the culture was inoculated and fermented overnight. After 12 hours of fermentation, samples were taken every 2 hours and diluted 4 times before measuring the OD value using a microplate reader. 600 The results are shown in Figure 2. As shown in Figure 2, except for pH 6.0, the amount of Akkermansia muciniphila increased with decreasing pH, and the best growth was at pH 6.5. Under this pH condition, the cell reached a stable stage after 28 hours of growth, and the OD 600 The fermentation was extended to 40h, and the OD 600The pH value of the group with pH 7.0 was 2.6992 (not shown). The growth of the group with pH 7.0 slowed down after 26 hours of growth and reached a stable stage after 30 hours of growth. The growth curves of pH 6.0 and pH 7.5 had a high degree of overlap, but the growth rate was not as good as that of pH 7.0. Without controlling the pH, the pH of Akkermansia muciniphila decreased to around 5.35 after 40 hours of growth, and the OD 600 is 1.5183 (growth curve not shown).

[0146] Example 8. Effects of inoculum size and fed-batch fermentation on high-density fermentation of Akkermansia muciniphila

[0147] In this example, medium No. 3 was still used as the high-density medium for high-density fermentation.

[0148] The specific steps are as follows: take the bacteria stored at -80℃, and 7 cfu / mL inoculation volume was inoculated into a 5 mL test tube of the control culture medium system. After activation for 36 h, it was transferred to 100 mL of No. 10 culture medium at a 1% inoculation volume. After activation for another 36 h, it was transferred to 3×10 7 cfu / mL, 6×10 7 cfu / mL, 1.2×10 8 cfu / mL was transferred to a fermentor containing Medium No. 3. After reaching stationary phase, feed was initiated with 300 mL of a carbon source (maltose:N-acetylglucosamine = 4:6, final concentration 240 g / L). Culture conditions were: 37°C, 150 rpm, slowly aerated with 10% H2, 10% CO2, and 80% N2, and pH maintained at 6.5.

[0149] The results are shown in Figure 3. It can be seen from the figure that the larger the inoculum size, the faster the growth and the earlier the stationary phase is reached. 7 cfu / mL, the fermentation did not reach the stable phase until 28h; at 6×10 7 cfu / mL, the fermentation reached the stable period after 20 h; at 1.2×10 8 cfu / mL inoculation amount, fermentation reached the stable period after 16h, and feeding was started at 18h. 600 Rapidly increased, fermentation to 22h, OD 600 The highest value was 4.52, and then it stopped growing. At this time, the maximum number of viable bacteria reached 2x10 10 cfu / mL.

[0150] Example 9: Comparison of precipitation after sterilization of culture medium

[0151] The pH of the control medium in Example 6 after sterilization and cooling to room temperature was about 8.2.2+ A large amount of precipitation was produced. As shown in Example 7, the optimal pH for the growth of Akkermansia muciniphila is 6.5. After the pH of the control medium was adjusted to 6.5, some precipitation still remained in the medium. When the pH was further adjusted to 5.5, the medium became clear and transparent. In contrast, the OD value of medium No. 3 after sterilization was 0. 600 The blank OD values ​​obtained in the control medium and medium No. 3 at different pH values ​​were only 0.0068, and no precipitation was produced. 600 The results are shown in Table 6. As shown in Table 6, medium No. 3 can effectively solve the problem of precipitation during high temperature and high pressure sterilization.

[0152] Table 6: OD after sterilization of control medium and medium No. 3 600 Changes with pH

[0153] Example 10: Effects of different culture media on downstream processing techniques

[0154] A control medium and a modified medium (medium 3) were prepared according to the medium formulations in Table 5 of Example 6. A 1% inoculum was inoculated into a 5 mL test tube containing the control medium. After 36 h of incubation, the cells were transferred to 100 mL of the control medium and medium 10, respectively, at 1% inoculum. The incubation conditions were: 37°C, 10% H₂, 10% CO₂, and 80% N₂. After 36 h of incubation, the cells were transferred to 50 mL centrifuge tubes and centrifuged at 10,000 rpm for 15 min using a centrifuge (Xiangyi Instruments H1750R, China).

[0155] As shown in Example 7, when the pH was not controlled until the end of the fermentation, the pH of the fermentation broth was 5.35, which was lower than 5.5. At this time, the precipitate in the fermentation broth had been completely dissolved, and there was no calcium salt precipitation in the bacterial sludge obtained by centrifugation. 600 The OD value of the supernatant cultured in medium No. 3 was 0.2433, indicating that a large number of bacteria were not harvested by centrifugation in the supernatant. 600 The value is 0.0095, which is less than 0.01, indicating that there is almost no loss of bacteria. The centrifugation effect is shown in Figure 4. The left centrifuge tube shows the results obtained in the control medium. As can be seen from the figure, the edges of the precipitate are rough and scattered, indicating that the centrifugation sedimentation effect is poor; the right centrifuge tube shows the results obtained in medium No. 3. As can be seen from the figure, the edges of the precipitate are round and smooth, indicating that the centrifugation sedimentation effect is good.

[0156] Example 11: Effects of bacteria cultured in different media on mouse body weight

[0157] A control medium and a modified medium (medium 3) were prepared according to the medium formula in Table 5 of Example 6. Bacteria stored at -80°C were inoculated into a 5 mL test tube of the control medium system at a 1% inoculum size. After activation for 36 hours, the inoculum size was transferred to 100 mL of the control medium and medium 3, respectively, at a 1% inoculum size. The activation culture was carried out for 36 hours in an atmosphere of 10% H₂, 10% CO₂, and 80% N₂.

[0158] The fermentation broth obtained in the control medium and medium No. 3 was centrifuged separately, the precipitate washed three times with PBS, and then frozen in 25% glycerol for later use. Three groups of 10 mice were used to establish a hyperlipidemia model in mice, each fed a high-fat diet. Two of the groups were fed a high-fat diet and given an oral gavage of 2×10 mice with Akkermansia muciniphila cultured in the control medium or medium No. 3, respectively. 8 cfu / day / mouse. Another group, serving as a high-fat control, received an equivalent dose of PBS by gavage. Body weights were measured weekly. The effects of bacterial cultures grown in the two media on mouse body weight were compared, and the results are shown in Figure 5 (Akk in the figure is an abbreviation for Akkermansia muciniphila).

[0159] As shown in Figure 5, compared to the weight of mice in the high-fat control group, Akkermansia muciniphila cultured in both the control medium and Medium No. 3 reduced the weight of mice, and the difference in weight compared to the high-fat control group was significant (p < 0.05). There was no significant difference in weight between the two groups of mice fed with bacteria cultured in the control medium and Medium No. 3 (p > 0.05). This indicates that the physiological functions of Akkermansia muciniphila cultured in Medium No. 3 were unchanged compared to those cultured in the control medium.

[0160] Example 12: Comparison of Akkermansia muciniphila genomes

[0161] According to the method described in Example 6, Akkermansia muciniphila was cultured in a control medium and in Medium No. 3. The resulting fermentation broth was then centrifuged to obtain bacterial cells. These cells were used for whole-genome sequencing and collinearity comparison (collinearity refers to the distribution or arrangement of homologous genes within or between species). The results are shown in Figure 6. In Figure 6, the upper panel shows the results obtained for Akkermansia muciniphila cultured in the control medium, while the lower panel shows the results obtained for Akkermansia muciniphila cultured in Medium No. 3. As can be seen in Figure 6, the species annotation results of the genomes from the two bacterial sources are identical. The Fastani value of the genome comparison of the two bacterial sources was 99.9999%, indicating that the average nucleotide identity of the genomes from the two bacterial sources is almost identical, thus confirming that the optimized culture medium does not cause changes in the Akkermansia muciniphila genome.

Claims

1. A culture medium for culturing Akkermansia muciniphila, comprising: - 2-200 g / L, preferably 2-50 g / L, more preferably 4-20 g / L of sugars, wherein the sugars are selected from disaccharides, oligosaccharides, polysaccharides, and any combination thereof; -2-200 g / L, preferably 2-50 g / L, more preferably 5-20 g / L of glucosamine and / or glucosamine polymers, wherein the glucosamine and / or glucosamine polymers are selected from D-glucosamine, D-glucosamine hydrochloride, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetyl-D-mannosamine, chitosan oligosaccharides, chitosan, chitin, and any combination thereof; -5-200 g / L, preferably 5-50 g / L, more preferably 10-20 g / L of a non-animal nitrogen source, wherein the non-animal nitrogen source is selected from soy peptone, wheat peptone, yeast extract powder, yeast peptone, and any combination thereof; - 0.1-20 g / L, preferably 2-10 g / L, more preferably 4-8 g / L of L-threonine; -0-50 mM, preferably 0.1-10 mM, more preferably 1-10 mM reducing agent, the reducing agent is selected from L-cysteine, L-cysteine ​​hydrochloride, SO3 2- 、S2O3 2- 、S2O4 2- , thiourea dioxide (CH4N2O2S), and any combination thereof; - 0-1 g / L, preferably 0.1-10 mg / L of cobalamin; - 0-10 mM, preferably 0-5 mM Mg 2+ ;as well as - 0-10 g / L, preferably 2-7 g / L, of a buffer salt selected from the group consisting of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, sodium bicarbonate, sodium carbonate, and any combination thereof.

2. The culture medium according to claim 1, wherein The disaccharide is selected from lactose, maltose, isomaltulose, cellobiose, turanose, melibiose, and any combination thereof; The oligosaccharide is selected from galacto-oligosaccharide, fructo-oligosaccharide, manno-oligosaccharide, isomalto-oligosaccharide, maltooligosaccharide, soybean oligosaccharide, breast milk oligosaccharide (such as 2'-salinalgyl lactose, 3'-sialyllactose, lacto-N-neotetraose, lacto-N-triose), and any combination thereof; The polysaccharide is selected from dextrin (maltodextrin, resistant dextrin, cyclodextrin), starch and modified starch (such as pregelatinized starch, soluble starch, etc.), dextran, and any combination thereof; The aminosugar or aminosugar polymer is N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, chitosan, chitosan oligosaccharide, chitin, and any combination thereof; The non-animal nitrogen source is yeast extract powder; The reducing agent is S2O3 2- , or its sodium salt, potassium salt or hydrate, S2O4 2- , or its sodium salt, potassium salt or hydrate, thiourea dioxide (CH4N2O2S), and any combination thereof; preferably, the reducing agent is S2O3 2- , or its sodium salt, potassium salt or hydrate, such as Na2S2O3·5H2O.

3. The culture medium according to claim 1 or 2, wherein The culture medium further comprises 0-2 g / L, preferably 0.5-2 g / L, of other amino acids selected from L-phenylalanine, L-tryptophan, or any combination thereof.

4. The culture medium according to claim 1 or 2, wherein The concentration range of potassium dihydrogen phosphate, dipotassium hydrogen phosphate or sodium dihydrogen phosphate is 0-4.5 g / L, preferably 0-2 g / L; for example, the concentration range of sodium bicarbonate is 1-10 g / L, preferably 1-5 g / L.

5. The culture medium according to claim 4, wherein The culture medium comprises 4-15 g / L sugar, 10-20 g / L aminosugar and / or aminosugar polymer, 10-20 g / L yeast extract, 0.5-1.2 g / L phenylalanine, 0.25 g / L sodium thiosulfate pentahydrate or its corresponding amount of sodium thiosulfate or 0.11 g / L CH4N2O2S, 4-8 g / L L-threonine, 0.1 mg / L cobalamin, and optionally 0.5 g / L tryptophan, 0.1-0.12 g / L magnesium chloride, and 6-7 g / L buffer salt.

6. A method for culturing Akkermansia muciniphila, the method comprising culturing Akkermansia muciniphila in the culture medium according to any one of claims 1 to 5; preferably, the culturing is static culture, high-density culture, or static culture followed by high-density culture.

7. The method according to claim 6, wherein: The static culture comprises culturing at 36° C.-38° C. for 12-36 hours and performing 2-3 subcultures; preferably, the 2nd-3rd subcultures are performed using the culture medium described in any one of claims 1-6.

8. The method according to claim 6 or 7, wherein: The high-density culture includes culturing Akkermansia muciniphila at 3×10 7 cfu / mL-3×10 8 cfu / mL, preferably 3×10 7 cfu / mL-1.5×10 8 cfu / mL is inoculated into the culture medium and cultured at a temperature of, for example, 20°C-40°C, preferably 36°C-38°C for a period of time, for example, 24h-36h; more preferably, the high-density culture further comprises controlling the pH of the culture medium during the culture process to 6-8, preferably to 6.0-7.5, and most preferably to 6.

5.

9. The method according to claim 6 or 7, wherein: The high-density culture adopts a fed-batch fermentation method; the feed is, for example, a complete feed medium or a semi-batch feed medium, such as an incomplete medium comprising a carbon source (such as sugars, amino sugars and / or amino sugar polymers) and a nitrogen source; for example, the feed flow rate is 1-6 mL·L -1 ·h -1 , preferably 1.2mL·L -1 ·h -1 Continuous feeding is carried out at a feeding rate of.

10. A strain of Akkermansia muciniphila cultured by the method described in any one of claims 6 to 9, which was deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration of Microorganisms on September 13, 2023, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.40786.

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