EA040261B1 - METHOD FOR CULTIVATION OF AKKERMANSIA MUNICIPHILA BACTERIA - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates to the cultivation of the bacteria Akkermansia muciniphila.

Prior Art

Bacteria of the genus Akkermansia, in particular the species Akkermansia muciniphila, are believed to play an important role in the prevention and/or treatment of metabolic disorders such as, for example, obesity-related disorders (WO2014/075745; WO 2014/077246). Oral administration of A. muciniphila to mice fed a control or high fat diet was found to normalize nutritional metabolic endotoxemia, obesity, and adipose tissue marker CD11c without any change in food intake (WO 2014/075745). In addition, treatment with A. muciniphila reduced body weight and improved body composition (i.e., fat to muscle ratio). In a high fat diet, treatment with A. muciniphila bacteria was found to increase the expression of adipocyte differentiation markers in mRNA and lipid oxidation without affecting lipogenesis. It was also found that as a result of colonization with A. muciniphila bacteria, alimentary hyperglycemia was completely stopped, and the insulin resistance index also decreased after treatment. Finally, A. muciniphila has been found to enhance gut barrier function (J Reunanen et al., 2015, Appl Environ Microbiol. 81:3655-62). It has been proposed to use bacteria of the genus Akkermansia, in particular the species A. muciniphila, in the food or pharmaceutical industry. At the same time, high levels of Akkermansia biomass yield are desirable.

Derrien et al. (2004, Int. J. Syst. Evol. Microbiol. 54: 1469-76) state that the MucT strain can be isolated from A. muciniphila bacteria and grown in a basal aerobic medium containing porcine gastric mucus mucin. as the sole source of carbon and nitrogen. The authors also write that A. muciniphila can be grown in fatty media such as Columbia Broth (CB) and Brain Heart Infusion (BHI) broth, but the resulting final optical density is half final optical density, which can be obtained using a mucin medium. All media known so far applicable for the cultivation of Akkermansia contain components of animal origin. BHI is isolated from animal tissue, and CB contains enzymatic breakdown products of cow's milk casein, animal tissue, and heart muscle. A. muciniphila bacteria are difficult to culture, as evidenced by their observed lack of growth in Wilkins-Chalgren Broth (WCB) (Derrien et al., 2004). WCB is designed specifically for the cultivation of anaerobic bacteria and contains, among other things, the products of the enzymatic breakdown of cow's milk casein and gelatin of animal origin.

In addition, A. muciniphila is described as being unable to grow in a basal saline medium with one of the following compounds (in each case 10 mM unless otherwise indicated): glucose, cellobiose, lactose, galalactose, xylose, fucose, rhamnose, maltose, succinate, acetate, fumarate, butyrate, lactate, casitone (0.5%), casamino acids (0.5%), tryptone (0.5%), peptone (0.5%), yeast extract (0, 5%), proline, glycine, aspartate, serine, threonine, glutamate, alanine, N-acetylglucosamine, N-acetylgalactosamine (Derrien et al., 2004).

However, N-acetylglucosamine, N-acetylgalactosamine, or glucose (10 mM each) supported growth only when 2 g/l (0.2%) of peptone, yeast extract, tryptone, and casitone were added to the basal medium, but growth was less than a quarter of that in mucin medium (Derrien et al., 2004).

In one recent study, published 10 years after it was conducted, A. muciniphila was grown in a basal medium containing 10 g/L casitone (corresponding to 1%), 5 mM glucose, 5 mM fucose, and 1 mM threonine (Lukovac et al. , 2014, MBio 12;5(4) pii: e01438-14 (doi: 10.1128/mBio. 01438-14)). Again, growth was small, about four times less than when A. muciniphila was grown in a mucus-containing medium. In addition, the medium had a high content of casitone, which is a product of the proteolytic breakdown of cow's milk protein casein and, therefore, is of animal origin. Finally, not only cheap glucose was used as a sugar in the medium, but also fucose, which is mainly found in animal products, and its cost is more than 100 times higher than that of glucose.

Therefore, it is clear that the bacteria A. muciniphila is difficult to culture, and all media and growth conditions described so far involve the use of compounds of animal origin. Animal products may contain viral, prion or bacterial contaminants or allergens, antigenic peptides or other objectionable substances, or may otherwise be considered unacceptable for cultivation of Akkermansia for human food or pharmaceutical use, for example because they are not kosher or halal . Until now it was believed that media containing mucus provide the highest biomass yield. Since only animals are the source of mucus, this imposes restrictions on the cultivation of A. muciniphila and prevents the use of these bacteria to improve the health of humans and animals, as proposed in the existing scientific and patent literature. In addition, no isolates of A. muciniphila other than the MucT strain have been described. It is recognized that

- 1 040261 culturing and isolating A. muciniphila is a difficult task, as recently demonstrated in one study in which, despite considerable effort, only an enrichment rather than a pure culture of A. muciniphila was obtained (Caputo et al., 2015, Biol Direct 10 : 5 (doi:10.1186/s13062015-0041-1)), which indicates that its cultivation is associated with serious difficulties.

The object of the present invention is to provide a composition that can be used to culture the bacteria Akkermansia muciniphila to achieve a high final optical density (high biomass yield) and/or that is less complex than known compositions for the cultivation of bacteria of the genus Akkermansia and preferably does not contains products of animal origin. Such a culture medium would make it possible to obtain large quantities of Akkermansia bacteria applicable to humans, for example for use in the food, feed or pharmaceutical industry.

Brief summary of the invention

The present invention provides a method for cultivating Akkermansia muciniphila bacteria, comprising the steps of using a composition containing a monosaccharide, a nitrogen-containing monosaccharide derivative, and an amino acid source; inoculation of said composition with Akkermansia muciniphila bacteria; and reproduction of Akkermansia bacteria.

The nitrogen-containing monosaccharide derivative is preferably N-acetyl-glucosamine (Glc-Nac). Glc-NAc may preferably be present in an amount of 25-40 mm.

The monosaccharide is preferably glucose. Glucose may be present, for example, in an amount of 10-25 mM.

The composition may additionally contain threonine, for example, in an amount of 2-4 mm.

The source of the amino acid in the composition can be selected from a vegetable source of amino acid, a microbial source of amino acid, or a combination of alanine, glutamate, proline and serine. In one embodiment, the source of the amino acid is a vegetable protein hydrolyzate, such as soy protein hydrolyzate, pea protein hydrolyzate, wheat protein hydrolyzate, rice protein hydrolyzate, cotton protein hydrolyzate, and the like.

The vegetable protein hydrolyzate may be present in an amount of from about 0.01 g/L to about 1 kg/L, for example, from about 0.05 to about 500 g/L, from about 0.1 to about 250 g/L, from about 0.5 to about 150 g/L, about 1 to about 100 g/L, or about 2 to about 80 g/L.

Detailed description of the invention

The present inventors have found that Akkermansia muciniphila can be cultured to achieve very high optical density in a composition containing glucose, N-acetylglucosamine and an amino acid source. The source of the amino acid can ideally be an entirely plant or microbial source.

Thus, the present invention also provides a composition for cultivating bacteria, namely the genus Akkermansia, in particular Akkermansia muciniphila, containing a monosaccharide, a nitrogen-containing monosaccharide derivative and an amino acid source.

The monosaccharide may be any monosaccharide, in particular a monosaccharide which is widely used for economically efficient cultivation of bacteria, preferably glucose.

The nitrogen-containing monosaccharide derivative is preferably N-acetylglucosamine.

The composition may contain a monosaccharide such as glucose in an amount of 10-25 mM.

The composition may contain a nitrogen-containing monosaccharide derivative such as Glc-NAc in an amount of 25-40 mM.

The composition according to the present invention may not contain products of animal origin.

The amino acid source can be any amino acid source known to those skilled in the art, including, without limitation, an animal source, a plant source, or a source isolated from a microorganism. The source of the amino acid may be, for example, a protein hydrolysate, such as vegetable protein hydrolysates. Hydrolysates are obtained from protein sources by (partial) hydrolysis, and they usually consist of a mixture of peptides, amino acids, carbohydrates and lipids, as well as many unidentified components with unclear biological activity. They are often obtained by enzymatic, alkaline or acidic digestion of a given feedstock from a variety of sources, such as, but not limited to, plant sources such as soybeans, wheat, peas, chickpeas, or cotton.

The amino acid source may include commercial amino acid sources such as yeast extracts, eg ultrafiltered HyPep™ YE or UltraPep™ YE or HyYest™; dairy hydrolysates e.g. derived from casein, lactalbumin and milk solids hydrolysates such as Amicase™, Hy-Case™ Amino, Hy-Case™ SF, N-Z-Amine™ A, NZ-Amine™ AS, N-Z-Amine™ EKC, N-Z-Case™ Plus, N-Z-Case™ TT, Edamin F, or trypton (a set of peptides derived from the digestion of casein with proteolytic trypsin); vegetable hydrolysates

- 2 040261 proteins such as HyPer 1510™ (enzymatic hydrolysate of soy), HyPer 1511™ (Ultrafiltered enzymatic digest of soy), HyPer 1512™ (Enzymatic digest of soy), HyPer 4601N™ (Ultrafiltered enzymatic digest of wheat gluten) , HyPep 5603™ (Ultrafiltered enzymatic digest of rice protein and wheat gluten), HyPer 7504™ (Ultrafiltered enzymatic digest of cotton protein), UltraPep Cotton™, Amisoy (acid digest of soy isolate as a mixture of amino acids and small molecular weight peptides, but without tryptophan), Phytone or Soytone Peptones (Difco™ and BBL brand peptones) or UltraPep Soy™. The amino acid source can also be an amino acid composition containing a single amino acid or a combination of single amino acids. In one embodiment, the amino acid source is a plant derived amino acid source.

The amino acid source may be present in the composition of the invention in an amount of from about 0.01 g/L to about 1 kg/L, for example, from about 0.05 to about 500 g/L, from about 0.1 to about 250 g/L , from about 0.5 to about 150 g/l, from about 1 to about 100 g/l, or from about 2 to about 80 g/l. For example, HySoy or Amisoy may be present in the composition according to the invention, preferably in an amount of 16 g/l.

The composition according to the invention preferably contains threonine, for example in an amount of 24 mm. The threonine may be present in the form of L-threonine or D,L-threonine.

In addition, the composition according to the invention may contain alanine, glutamate, proline and serine, preferably each in an amount of from about 0.01 to about 100 mM, preferably from about 0.05 to about 50 mM, more preferably from about 0.1 to about 25 mm, even more preferably from about 0.5 to about 15 mm, most preferably from about 1 to about 10 mm, for example, from about 1 mm to about 8 mm.

In one embodiment, the composition according to the invention further comprises a buffer system to maintain the pH in the range of 5.5-8.0, preferably 6.0-7.0, more preferably about 6.6. The person skilled in the art will be able to select a buffer system for this purpose.

In one embodiment, the composition of the invention further comprises about 0.1 to about 2%, such as about 0.3 to about 1.5%, or about 0.5 to about 1.3%, or about 1.0 % cysteine.

Although not required, vitamins can be added to the composition of the invention. Useful vitamins that may be added include, without limitation, biotin, cobalamin, PABA, folic acid, pyridoxamine, and the like. For example, cobalamin can be used when the desired end product of metabolism by cobalamin-dependent methylmalonyl-CoA mutase is propionate rather than succinate.

The present invention also provides a method for cultivating Akkermansia municiphila bacteria, comprising the steps of: using a composition according to the invention, inoculating said composition with Akkermansia municiphila bacteria; and reproduction of bacteria of the genus Akkermansia.

Applicable cultivation conditions for Akkermansia muciniphila include, but are not limited to, temperature in the range of 20-40°C and pH in the range of 5.5-8.0, with temperatures of about 36-38°C and pH 6.0-7.0 being optimal for growth. , preferably about 6.5. Because Akkermansia muciniphila are strictly anaerobic bacteria (Derrien et al. 2004. Int J System Evol Microbiol 54:1469-1476), anaerobic conditions should ideally be maintained as much as possible and contact with air should be avoided as much as possible. Anaerobic culture methods are known to those skilled in the art.

The present invention is further illustrated, but not limited, by the following examples.

From the foregoing description and these examples, those skilled in the art will be able to recognize the essential features of the present invention and will be able to suggest various changes and modifications, without departing from the spirit and scope thereof, to adapt it to various applications and conditions. Thus, from the above description, those skilled in the art will recognize various modifications of the invention beyond those discussed and described therein. Such modifications are also intended to be within the scope of the appended claims.

Examples

Example 1 Glucose and N-acetylglucosamine stimulate the growth of A. muciniphila.

A. muciniphila Muc ^T (ATTC BAA-835) was grown in the previously described basal anaerobic medium (see above Derrien et al., 2004). The medium was supplemented with purified porcine mucus alone (type III; Sigma; 0.5%), mucus alone (0.25%), or mucus supplemented with sugars or tryptone (Difco; 1%) supplemented with sugars. As Sugars used only D-glucose (glucose), D-fucose (fucose) or Nacetylglucosamine (GlcNac) at a concentration of 20 mm or a combination of both at a final concentration of 10 mm, respectively. When incubated with sugars, 1 mM D,L threonine (threonine) was also used. Incubation in all cases was carried out in whey broth bottles sealed with butyl rubber stoppers at a temperature of 37°C under anaerobic conditions provided with a gaseous phase.

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N2/CO2 at 182 kPa (1.8 atm). Growth was determined by spectrophotometric methods as optical density at a wavelength of 600 nm (OD600), while the results are presented in table. A.

In addition, the previously described HPLC analysis was used (see above Derrien et al., 2004;

see above Luzovac et al., 2014) to determine the concentration of these Sugars as well as products including acetate, propionate, 1,2-propanediol.

Table A. Growth of A. muciniphila on various substrates

Glucose fucose GlcNac Trip Slime Height (mm) (mm) (mm) tone (g/l) (g/l) OD600 - - - - 5 2.5 - - - - 2.5 0.8 - - - - 1.25 0.4 20 - 2.5 2.3 - 20 - - 2.5 1.6 - - 20 - 2.5 2.3 20 - 10 - 0.6 - 20 - 10 - 0.15 - - 20 10 - 1.0 10 10 - 10 0.4 10 - 10 10 - 1.0

The compounds added to the basal medium are listed with their concentrations (units in parentheses), as in the case of OD600, which was determined after 24-60 hours of incubation. The data were obtained as a result of repeated experiments, while the value of their deviation in different experiments reached about 25%. Explanation of abbreviations is given in the text. Various series of experiments are presented with a breakdown for clarity.

The results confirm earlier observations (see supra Derrien et al., 2004) that mucus at 5 g/L supports good growth of A. muciniphila at a relatively high rate (generation lifetime of approximately 2 hours) and a high final OD600 of 2.5, which corresponds to about 5.10 ⁹ cells/ml. However, a decrease in mucus concentration has a dramatic effect on the growth of A. muciniphila. Combinations of glucose and other sugars were tested to investigate the growth of A. muciniphila. It turned out that glucose or N-acetylglucosamine, but to a lesser extent fucose, are able to compensate for a twofold decrease in the concentration of mucus and support the growth of A. muciniphila to an OD600 of more than 2.0. In addition, individual sugars, especially Nacetylglucosamine and, to a lesser extent, glucose, but not fucose, could support the growth of A. muciniphila in the presence of an additional source of nitrogen, in this case tryptone, a product of tryptic digestion of casein. Similar results were obtained using casein, which is a product of the breakdown of casein by pancreatic enzymes. However, growth was several times slower compared to growth in basal medium containing 0.5% mucus, as previously described (see above Derrien et al., 2004).

Based on these results, it can be concluded that N-acetylglucosamine is the best sugar to support the growth of A. muciniphila; however, due to the scarcity and high cost of this sugar, it would be more beneficial to use glucose. It was unexpectedly found that when using the combination of glucose with N-acetylglucosamine, the growth of A. muciniphila was significantly better than when using only glucose (Table A).

Example 2 Threonine stimulates the growth of A. muciniphila.

In addition to the fact that N-acetylglucosamine could be used to increase the final optical density when growing A. muciniphila on glucose, it was found that an external source of protein is important to maintain good growth. It turned out that with increasing tryptone concentrations, the growth of A. muciniphila was significantly stimulated to a level that exceeded the level of growth that could be obtained in media containing mucus (Table B). The amount of tryptone used was 32 g/l, which is similar to the protein concentration in normal cow's milk.

The experiment was carried out essentially as described in Example 1.

In addition, the effect of increasing the concentration of threonine in a basal medium containing glucose and N-acetylglucosamine was tested. At threonine concentrations above 2 mM, preferably above 4 mM, an unexpected effect was observed, providing a very high OD600 of A. muciniphila over 7.0, close to industrial scale.

Interestingly, both L-threonine and D,L-threonine can be used. Since D,L-threonine is more readily available and less expensive, D,L-threonine is preferably added to the medium according to the invention.

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Table B. Growth of A. muciniphila on various substrates, optionally containing threonine

Glucose GlcNac Threonine trypton Height (mm) (mm) (mm) (g/l) OD600 12.5 12.5 0 8 0.2 12.5 12.5 0 16 0.5 12.5 12.5 0 32 3.5 25 25 0 32 4.0 25 25 1 32 5.6 25 25 2 32 7.1 25 25 4 32 7.2

Example 3 Growth of A. muciniphila in non-animal and synthetic media.

Since casein is an animal protein source, it was tested whether plant or microbial protein sources could be used to support the growth of A. muciniphila. Since soy is one of the most abundant and complete sources of protein, a number of commercial preparations of hydrolysed soy have been tested. Good growth of A. muciniphila has been observed in basal media containing glucose and N-acetylglucosamine supplemented with HySoy and AmiSoy, both obtained from Quest International, which have previously been found to support the growth of pathogenic bacteria (see US Pat. No. 6,558,926 and WO 1998054296). ). However, in all cases, the addition of threonine supported growth, as shown in Table 1. B. Highly efficient growth of A. muciniphila was observed at 2 mM threonine and even better growth at 4 mM in the presence of 16 g/l HySoy.

Changing the ratio of glucose to N-acetylglucosamine in the presence of HySoy and threonine affected the growth of A. muciniphila and demonstrated that both sugars are also required when using these soy-based media, but their ratio may differ (Table B). This has also been observed with casein-derived trypton, suggesting that optimization of the glucose to N-acetylglucosamine ratio can be achieved regardless of the nitrogen source.

To test whether a microbial source of protein could also support the growth of A. muciniphila, yeast extract (Difco) was added. Because yeast extracts are generally more expensive than soy-based media (Kwon et al., Enzyme Microb Technol. 2000 Feb 1; OD600 4.4, a relatively small amount of yeast extract, and found that it further enhanced the growth of A. muciniphila to an OD600 of 6.5 (Table C). This suggests that yeast extract can be used to support growth, and also that increasing the amount of protein source can further increase the growth of A. muciniphila in non-animal derived media.

Since it is clear that the most cost effective is to grow the bacteria in a synthetic medium, it was tested which amino acids in the soy hydrolyzate were able to support the growth. It was found that a mixture of 4 amino acids including alanine, glutamate, proline and serine (4 mM each) could support the growth of A. muciniphila without the need for amino acids other than threonine in the presence of glucose and N-acetylglucosamine (Table B). Since the observed growth exceeded that on mucus alone (see above), this opens up possibilities for further optimizing the cost-effective rearing of A. muciniphila. As such, seri can also be replaced by threonine, and the combination of threonine and proline alone has been found to serve as a highly efficient nitrogen source for A. muciniphila.

Table B. Growth of A. muciniphila on various substrates, HySoy, yeast extract (YE) at 10 g/l and 4 amino acids (AA) including alanine, glutamate, proline and serie (4 mM each). Experiments were carried out essentially as described in Example 5.

Glucose GlcNac Threonine HySoy Additives Height (mm) (mm) (mm) (g/l) OD600 25 25 0 16 - 0.2 25 25 2 16 - 1.8 25 25 4 16 - 4.4 50 0 2 16 - 0.1 0 50 2 16 - 3.9 40 10 2 16 - 0.3 10 40 2 16 - 2.8 25 25 4 16 YE (10 g/l) 6.5 25 25 4 - AA (4 mM) 2.9

Example 4 Fermentation of A. muciniphila on an industrial scale.

To illustrate the possibility of obtaining A. muciniphila on an industrial scale, A. muciniphila was grown in the basal medium described above containing 32 g HySoy, 25 g glucose, 4.4 g

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N-acetylglucosamine, 4 g threonine, 0.5 g cysteine per 1 kg of medium, as well as vitamin solution (see above Dement et al., 2004), in a 600 liter fermenter at pH 7.0. After fermentation, the OD600 of the culture was 7.2, indicating that the medium containing glucose, N-acetyl-glucosamine and a non-animal protein source is capable of supporting excellent growth of A. muciniphila on an industrial scale.

Claims (7)

CLAIM 1. A method for cultivating Akkermansia municiphila bacteria, characterized in that a basal medium is used for cultivation with the addition of a composition containing glucose, N-acetide glucosamine, and threonine and/or a source of amino acids of animal origin, or threonine and sources of amino acids of plant and microbial origin, or threonine and a plant source of amino acids or a mixture of amino acids derived from a plant source, 2. The method according to claim 1, characterized in that the composition contains glucose in an amount of 10-25 mm. 3. The method according to claim 1, characterized in that the composition contains N-acetyl-glucosamine in an amount of 25-40 mm. 4. The method according to claim 1, characterized in that the composition contains threonine in an amount of 2-4 mm. 5. The method according to claim 1, characterized in that the plant source of amino acids is a soy protein hydrolyzate. 6. The method according to claim 5, characterized in that the composition contains soy protein hydrolyzate in an amount of 16 g/l. 7. The method according to claim 5, characterized in that the mixture of amino acids obtained from said soy protein hydrolyzate is a mixture of alanine, glutamate, proline and serine. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2