Composition for reducing d-serine level

Abstract

To optimally reduce D-serine levels.SOLUTION: A composition for reducing D-serine levels contains a bacterial cell belonging to the genus Enterocloster.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition for reducing D-serine concentration. [Background technology]

[0002] The human intestine is home to 1,000 species and 40 trillion bacteria, forming a complex symbiotic relationship known as the intestinal microbiota. The intestinal bacteria that make up the intestinal microbiota produce a variety of metabolic products, one of which is D-amino acids.

[0003] Amino acids are a general term for compounds that have an amino group and a carboxyl group in the molecule, and those in which an amino group and a carboxyl group are bonded to one carbon atom are called α-amino acids. α-amino acids have an asymmetric carbon atom and exist as enantiomers, of which those with left-handed optical rotation are called D-amino acids. Of the α-amino acids, glycine does not have an enantiomer, but the other α-amino acids exist as L-amino acids and D-amino acids. Therefore, of the 20 types of α-amino acids that make up proteins, 19 types other than glycine exist as L-amino acids and D-amino acids.

[0004] Most naturally occurring amino acids are L-amino acids, which are the building blocks of proteins, but there are also small amounts of D-amino acids. In recent years, it has become clear that D-amino acids have important physiological functions. For example, D-serine, which is found in the brain, large intestine, and kidneys, is known to contribute to higher brain functions such as memory and learning, as well as to protect against kidney damage.

[0005] There have been many reports of D-serine production, and it is known that some lactic acid bacteria and bifidobacteria in particular produce D-serine in the human intestine (Patent Documents 1 to 3). On the other hand, it is known that an excess of D-serine in the body can cause various health problems. While D-serine has a renal protective effect, it is also known that it induces damage to renal tubules when accumulated in plasma and is involved in the progression of chronic kidney disease (Non-Patent Document 1).

[0006] In addition, it has been found that D-serine and D-proline in the blood are useful indicators for evaluating changes in cognitive function, and it has been suggested that excess D-serine contributes to dysregulation of neurotransmission and the onset of dementia (Non-Patent Document 2).

[0007] Since the concentration of D-serine in the body is affected by the concentration of D-serine in the intestine, it is necessary to prevent the concentration of D-serine in the intestine from becoming too high. Therefore, when the concentration of D-serine in the intestine becomes high due to the influence of intestinal bacteria that produce D-serine, a method for reducing the concentration of D-serine in the intestine is required.

[0008] D-serine dehydratase is an enzyme that specifically degrades D-serine. It is known that this enzyme exists in some Proteobacteria bacteria, β- and γ-proteobacteria bacteria, and the eukaryote Saccharomyces cerevisiae. However, D-serine dehydratase does not exist in mammals, including humans. In addition, the known intestinal bacteria that contain D-serine dehydratase are not predominant in the human intestine, making it difficult to contribute to reducing the D-serine concentration in the human intestine.

[0009] In addition, there is D-amino acid oxidase, which metabolizes D-amino acids, as an enzyme that decomposes D-serine, but it has been reported that its catalytic activity towards D-serine is significantly inferior to that of D-serine dehydratase (Non-Patent Document 3).

[0010] Therefore, in order to effectively reduce D-serine in the intestines, it was necessary to find a bacterial species that decomposes or utilizes D-serine from the intestinal bacteria that are predominant in the human intestinal flora, and to develop a composition for reducing D-serine concentration containing the bacterial species. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Patent No. 5941445 [Patent Document 2] Patent No. 5965368 [Patent Document 3] Patent No. 6139217 [Non-patent literature]

[0012] [Non-Patent Document 1] Sci Rep. 2017 Sep 11;7(1):11168 [Non-Patent Document 2] Sci Rep. 2020 vol.10 804 [Non-Patent Document 3] D-Amino Acid Journal 2017 Vol.5 No.1 1 Summary of the Invention [Problem to be solved by the invention]

[0013] An object of the present invention is to preferably reduce the D-serine concentration. [Means for solving the problem]

[0014] The composition for reducing a D-serine concentration in embodiment 1 is characterized in that it contains a bacterium belonging to the genus Enterocloster. Aspect 2 is the composition for reducing a D-serine concentration of Aspect 1, wherein the species of the bacterial cell belonging to the genus Enterocloster is at least one of Enterocloster volteae, Enterocloster clostridioformis, Enterocloster lavalensis, Enterocloster asparagiformis, Enterocloster citronniae, and Enterocloster ardenensis. Effect of the Invention

[0015] According to the composition for reducing a D-serine concentration of the present invention, the D-serine concentration can be suitably reduced. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] An embodiment of the composition for reducing D-serine concentration according to the present invention will be described. The composition for reducing D-serine concentration contains bacteria belonging to the genus Enterocloster. In the following, the English name of the genus will be omitted.

[0017] The composition for reducing a D-serine concentration preferably contains, as an active ingredient, a bacterium belonging to the genus Enterocloster or a culture of said bacterium. The above-mentioned culture of bacterial cells means a culture of bacteria in a medium such as a known preparation, food or drink, feed, or food-grade material. The culture may be a culture of the cultured bacterial cells that has been further treated with a concentration, drying, freeze-drying, or the like. The culture may be a suspension containing the cultured bacterial cells and the medium, or may be a supernatant of the culture.

[0018] The composition for reducing D-serine concentration means a composition capable of reducing D-serine concentration. Specifically, the composition for reducing D-serine concentration means a composition in which the reduction rate of D-serine concentration is less than 1. The composition for reducing D-serine concentration preferably has a reduction rate of D-serine concentration of 0.9 or less. The reduction rate of D-serine concentration is more preferably 0.5 or less, even more preferably 0.1 or less, and most preferably 0.05 or less.

[0019] The reduction rate of D-serine concentration can be defined as the value obtained by dividing the D-serine concentration after the intestinal bacteria culture by the D-serine concentration before the intestinal bacteria culture. The smaller the reduction rate of D-serine concentration, the more the D-serine concentration is reduced.

[0020] In this embodiment, the term "enterobacteria" refers to bacteria isolated from feces, small intestine, large intestine, colon, ileum, or cecum of mammals. Each enterobacteria species refers to a species having a nucleotide sequence identity of 97% or more, more preferably 98% or more, and even more preferably 99% or more of the 16S ribosomal RNA gene with each type strain.

[0021] In the present embodiment, the "composition" includes preparations, foods and drinks, feed, and other substances that can be ingested by animals (including humans). Examples of the bacterial species included in the genus Enterocloster include the following:

[0022] <Bacteria species> Examples of species of bacteria belonging to the genus Enterocloster include Enterocloster bolteae, Enterocloster clostridioformis, Enterocloster lavalensis, Enterocloster asparagiformis, Enterocloster citroniae, and Enterocloster aldenensis.

[0023] The composition for reducing D-serine concentration may contain, as an active ingredient, the bacterial cells of one of the above-mentioned bacterial species belonging to the genus Enterocloster, or may contain, as an active ingredient, the bacterial cells of two or more of the above-mentioned bacterial species in combination. In the following, the English names of the above-mentioned bacterial species will be omitted.

[0024] In the composition for reducing a D-serine concentration, the species of the bacterial cell belonging to the genus Enterocloster is preferably Enterocloster voluteae. Hereinafter, a method for culturing the bacterial cells constituting the composition for reducing a D-serine concentration will be described.

[0025] <Cultivation method> For the cultivation method of the bacterial cells constituting the composition for reducing D-serine concentration, generally, a medium used for culturing enterobacteria can be used. Examples of the medium generally used for culturing enterobacteria include YCFA medium (JCM medium No. 1130), EG medium (JCM medium No. 14), etc. Among these, GAM broth, modified GAM broth, etc. are preferable because they are easier to prepare.

[0026] The cultivation temperature is not particularly limited, but examples include a temperature around human body temperature, 36 - 38°C, and 37°C is preferable. The large intestine is in an anaerobic state, and the enterobacteria growing here are obligate anaerobes. Therefore, it is necessary to maintain an anaerobic state in the medium and the headspace of the medium with nitrogen gas. Also, since carbon dioxide gas and hydrogen gas produced by enterobacteria also exist in the intestine, it is preferable to include these. Examples of the ratio include nitrogen 80 - 90%, carbon dioxide gas 5 - 10%, and hydrogen gas 5 - 10%.

[0027] The cultivation time is not particularly limited, but it is preferably carried out until the turbidity can be visually confirmed. Various enterobacteria are added to 10 mL of the medium so as to be about 1.0E+05 cfu / mL.

[0028] Hereinafter, the method for measuring the D-serine concentration will be described. <Method for measuring D-serine concentration> The method for measuring the D-serine concentration is not particularly limited, and a known measurement method can be adopted. Examples of the known measurement method include measurement by high performance liquid chromatography (hereinafter also referred to as HPLC), ultra-high performance liquid chromatography (hereinafter also referred to as UHPLC), etc.

[0029] <Other components> The composition for reducing D-serine concentration may contain other ingredients, such as excipients, stabilizers, binders, disintegrants, lubricants, flavorings, suspending agents, coating agents, colorants, solvents, and drugs, within the scope of not impairing the effect of the composition for reducing D-serine concentration.

[0030] Hereinafter, application forms of the composition for reducing D-serine concentration will be described. <Application form> The application form of the composition for reducing D-serine concentration is not particularly limited, and it is applied, for example, as a feed composition for reducing D-serine concentration (hereinafter also referred to as a feed composition), a pharmaceutical composition for reducing D-serine concentration (hereinafter also referred to as a pharmaceutical composition), or a food composition for reducing D-serine concentration (hereinafter also referred to as a food composition). Pharmaceutical compositions include quasi-drug compositions.

[0031] Hereinafter, applications of the composition for reducing D-serine concentration will be described. <Application> The use of the composition for reducing D-serine concentration is not particularly limited, and the composition may be incorporated into feed or food and drink, or may be added to raw materials during the production process of feed or food and drink.

[0032] Specific examples of foods and beverages include, but are not limited to, dairy products such as cheese, fermented milk, dairy lactic acid bacteria drinks, lactic acid bacteria drinks, butter, margarine, etc. Other examples include beverages such as milk drinks, fruit juice drinks, and soft drinks, egg products such as jellies, candies, puddings, and mayonnaise, and sweets and breads such as butter cakes. In addition, various types of milk powder, foods for infants, nutritional compositions, etc. can also be mentioned.

[0033] The classification of the above-mentioned food and drink is not particularly limited, and the food and drink can be general food, health functional food, special purpose food, etc. In addition, the health functional food can be a specific health food, a nutrient functional food, a functional food, etc.

[0034] When indicating the use of the composition for reducing D-serine concentration, examples of the indication include indications stipulated by various laws, enforcement regulations, guidelines, etc. Indications of use include indications on packages such as wrapping and containers, as well as indications on advertising media such as pamphlets.

[0035] The subject of administration of the composition for reducing D-serine concentration is not particularly limited, and it can be administered to humans. The subject of administration may be an animal other than a human (e.g., a dog, a cat, a horse, or a rabbit, etc.). When the subject of administration is a human, it can be administered to, for example, a minor under 20 years old, an adult, or an elderly person over 65 years old.

[0036] <Actions and Effects of the Embodiments> The operation and effects of this embodiment will be described. (1) The composition for reducing D-serine concentration contains bacteria belonging to the genus Enterocloster. By containing bacteria belonging to the genus Enterocloster, the D-serine concentration can be suitably reduced. Therefore, the D-serine concentration can be suitably reduced by using intestinal bacteria that are predominant in the human intestinal flora. In addition, the suitable reduction of the D-serine concentration can contribute to the maintenance of good health.

[0037] (2) The species of the bacteria belonging to the genus Enterocloster is at least one of Enterocloster volteae, Enterocloster clostrigioformis, Enterocloster lavalensis, Enterocloster asparagiformis, Enterocloster citronniae, and Enterocloster ardenensis. Therefore, the D-serine concentration can be more suitably reduced.

[0038] <Example of change> This embodiment can be modified as follows. This embodiment and the following modifications can be combined to the extent that they are not technically inconsistent. The composition for reducing D-serine concentration may contain bacteria with a D-serine concentration reduction rate of 1 or more, as long as the D-serine concentration reduction rate of the entire composition is less than 1. EXAMPLES

[0039] In the following, examples will be given in order to more specifically describe the configuration and effects of the present invention, but the present invention is not limited to these examples. Unless otherwise specified, % indicates % by volume.

[0040] <Test Example 1> 1. Selection of Enterobacteria and Preparation of Freeze-Concentrated Bacterial Cells (1) Obtaining intestinal bacteria Enterocloster volteae, Enterocloster clostrigioformis, and Enterocloster lavalensis, which are types of intestinal bacteria that are predominant in the human intestinal flora, were obtained from the Microorganism Materials Development Section of the RIKEN BioResource Research Center (hereinafter referred to as JCM).

[0041] (2) Preparation of freeze-concentrated bacterial cells The above bacterial species were cultured in modified GAM bouillon medium (product code 05433, Nissui). The composition of the modified GAM bouillon medium used is shown in Table 1.

[0042] [Table 1]

[0043] The modified GAM bouillon medium was sterilized by heating at 121°C for 15 minutes. The above-mentioned species of enterobacteria were inoculated into the sterilized medium and anaerobically cultured at 37°C using an anaerobic workstation (Concept 400 manufactured by Central Scientific Trading Co., Ltd.). The anaerobic workstation operation was performed by aerating with a mixed gas. The proportions of anaerobic gas were 80% (v / v) nitrogen, 10% (v / v) carbon dioxide, and 10% (v / v) hydrogen. The obtained culture was concentrated by centrifugation, and glycerol was added to a concentration of 10% (v / v) to obtain concentrated bacterial cells. The obtained concentrated bacterial cells were frozen at -80°C.

[0044] The frozen concentrated cells were then thawed, serially diluted, and smeared on modified GAM bouillon agar medium to measure the viable cell count. It was confirmed that the cells contained a viable cell count of 1.0E+07 cfu / mL or more.

[0045] (3) Cultivation of various intestinal bacteria The above-mentioned frozen concentrated bacteria were thawed in an anaerobic workstation, inoculated into 10 mL of modified GAM bouillon medium at 2% and cultured for 24 or 72 hours at 37°C in an incubator attached to the workstation. The culture time was determined to be close to either 24 or 72 hours when the formation of a suspended solid was confirmed by visual inspection.

[0046] The proportions of anaerobic gases aerated were 80% (v / v) nitrogen, 10% (v / v) carbon dioxide, and 10% (v / v) hydrogen. After cultivation, the bacteria were collected by centrifugation. Then, chromosomal DNA was extracted using the DNeasy Blood & Tissue kit (QIAGEN). The base sequence of the 16S rRNA gene was determined to confirm that the target bacterial species had been cultivated.

[0047] (4) Preparation of culture supernatant The enterobacteria cultured in the anaerobic workstation were removed from the anaerobic workstation. Centrifugation was performed at 14,000 rpm, 10 minutes, and 4°C under atmospheric conditions, and about 9 mL of crude culture supernatant was collected. The crude culture supernatant was further applied to a column (3 kDa Molcut; Millipore Merck) equipped with a 3 kDa ultrafiltration membrane. Centrifugation was performed at 14,000 rpm, 30 minutes, and 4°C to remove the high molecular weight fraction. The fraction that passed through the ultrafiltration membrane was used as the culture supernatant for D-serine measurement. The culture supernatant for D-serine measurement was used as the composition for reducing D-serine concentration of Examples 1 to 3 shown in Table 2.

[0048] In addition, as Comparative Example 1, a culture supernatant for D-serine measurement that does not contain enterobacteria was prepared. The culture supernatant for D-serine measurement was prepared in the same manner as the culture supernatant. The D-serine concentration measured in Comparative Example 1 means the D-serine concentration contained before the enterobacteria are cultured. In addition, as Comparative Example 2, a composition for reducing D-serine concentration was prepared by the same method as in Example 1, except that Ruminococcus gnavus was used instead of bacteria belonging to the genus Enterocoloster.

[0049] 2. Measurement of D-serine concentration o-Phthalaldehyde and N-isobutyryl-L-cysteine ​​were added to the compositions for reducing D-serine concentration of Examples 1 to 3 and Comparative Examples 1 and 2 to convert D- and L-serine contained in each sample into fluorescently labeled diastereomers. Then, the samples were subjected to UHPLC manufactured by Shimadzu Corporation for separation.

[0050] To confirm that the peak obtained was D-serine, the above sample was treated with D-amino acid oxidase in advance to convert it to a keto acid that does not react with the derivatization reagent. After derivatization under the same conditions, it was subjected to UHPLC again. The value obtained by subtracting the value obtained after enzyme treatment from the value obtained without enzyme treatment was calculated.

[0051] Because the measurement sample was diluted 5-fold during the enzymatic treatment, the calculated value was multiplied by 5 to obtain the D-serine concentration. A calibration curve was created using 0.1, 0.5, 2.5, 12.5, and 25 μM D-serine as the standard substance, and quantification was performed after confirming that the measured values ​​were within this range.

[0052] <Test Example 2> 1. Selection of Enterobacteria and Preparation of Freeze-Concentrated Bacterial Cells (1) Obtaining intestinal bacteria Six strains of five species, namely, Enterocloster clostrigioformis, Enterocloster volteae, Enterocloster citronniae, Enterocloster ardenensis, and Enterocloster asparagiformis, shown in Table 3, were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (hereinafter also referred to as DSMZ), a strain preservation and distribution organization in Germany.

[0053] (2) Preparation of freeze-concentrated bacterial cells Using the above six types of strains, freeze-concentrated bacterial cells were prepared in the same manner as in Test Example 1. (3) Cultivation of various intestinal bacteria Each intestinal bacterium was cultured in the same manner as in Test Example 1.

[0054] (4) Preparation of culture supernatant Culture supernatants for D-serine measurement were prepared in the same manner as in Test Example 1. These culture supernatants for D-serine measurement were used as compositions for reducing D-serine concentration in Examples 4 to 9 shown in Table 3. In addition, as Comparative Example 3, culture supernatants for D-serine measurement that did not contain enterobacteria were prepared.

[0055] 2. Measurement of D-serine concentration In the same manner as in Test Example 1, the D-serine concentrations in the compositions for reducing a D-serine concentration of Examples 4 to 9 and Comparative Example 3 were measured.

[0056] <Test Results> Table 2 shows the enterobacteria species, D-serine concentrations, and reduction rates contained in the D-serine concentration-reducing compositions of Examples 1 to 3 and Comparative Examples 1 and 2. Table 3 shows the enterobacteria species, D-serine concentrations, and reduction rates contained in the D-serine concentration-reducing compositions of Examples 4 to 9 and Comparative Example 3.

[0057] In Table 2, each D-serine concentration-reducing composition, its strain number, D-serine concentration, and D-serine concentration reduction rate are listed in the "Containing bacterial species" column, "JCM strain number" column, "D-serine concentration (μM)" column, and "Reduction rate" column, respectively.

[0058] In Table 3, each D-serine concentration-reducing composition, its strain number, D-serine concentration, and D-serine concentration reduction rate are listed in the "Containing bacterial species" column, "DSM strain number" column, "D-serine concentration (μM)" column, and "Reduction rate" column, respectively.

[0059] [Table 2]

[0060] [Table 3]

[0061] In Table 2, the "reduction rate" was calculated based on the D-serine concentration in Comparative Example 1 and was calculated as the reduction rate of the D-serine concentration in Examples 1 to 3 and Comparative Example 2. In Table 3, the "reduction rate" was calculated based on the D-serine concentration in Comparative Example 3 and was calculated as the reduction rate of the D-serine concentration in Examples 4 to 9.

[0062] As shown in Table 2, it was confirmed that the composition for reducing a D-serine concentration of Comparative Example 2 did not have the effect of reducing the D-serine concentration because the reduction rate exceeded 1. In contrast, the compositions for reducing a D-serine concentration of Examples 1 to 3 had a D-serine concentration reduction rate of less than 1, and it was confirmed that the D-serine concentration could be reduced more suitably. Moreover, the reduction rate of Enterocloster volteae of Example 1 was 0.04, and it was confirmed that the D-serine concentration could be reduced more suitably.

[0063] Furthermore, as shown in Table 3, it was confirmed that the compositions for reducing the D-serine concentration of Examples 4 to 9 had a D-serine concentration reduction rate of less than 1, and thus could more suitably reduce the D-serine concentration. Furthermore, the reduction rate of Enterocloster volteae of Example 6 was 0.050, and thus it was confirmed that the D-serine concentration could be more suitably reduced. [Industrial Applicability]

[0064] According to the present invention, the D-serine concentration can be suitably reduced by using intestinal bacteria that are predominant in the human intestinal flora.

Claims

1. A composition for reducing D-serine concentration, comprising a bacterium belonging to the genus Enterocloster.

2. The composition for reducing D-serine concentration according to claim 1, wherein the species of the bacteria belonging to the genus Enterocloster is at least one of Enterocloster volteae, Enterocloster clostrigioformis, Enterocloster lavalensis, Enterocloster asparagiformis, Enterocloster citronniae, and Enterocloster ardenensis.

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