Threonate-containing composition and method for producing the same

Abstract

A problem addressed is to provide a useful means for increasing muscle mass. A composition containing threonic acid, one or more bacteria selected from Bifidobacterium spp., Lactobacillus spp., Lacticaseibacillus spp., Lactiplantibacillus spp., Limosilactobacillus spp., Levilactobacillus spp., Ligilactobacillus spp., and Latilactobacillus spp., and one or more selected from cultures of the aforementioned bacteria is used to increase muscle mass. Such a composition is produced through, e.g., a method that includes a step for culturing one or more bacteria selected from the aforementioned bacteria in ascorbic-acid-containing medium and a step for recovering a threonic-acid-containing fraction from the culture after culturing.

Description

[Technical Field]

[0001] The present invention relates to a threonic acid-containing composition and a method for producing the same. [Background technology]

[0002] Since muscles are the basis for daily activities such as standing, walking, and maintaining posture, people of all ages and genders desire to maintain or increase muscle mass in order to maintain or improve the quality of their daily lives. In addition, people who enjoy exercise and athletes desire to increase muscle mass in order to improve their performance. Furthermore, with health consciousness growing, how to increase muscle mass has become a social issue in order to improve quality of life (QOL), alleviate frailty, and combat locomotive syndrome. "Frailty" is a term that refers to a state of frailty that occurs in old age, when vulnerability to stress increases due to a decline in physiological reserve, and lies somewhere between a healthy state and a state in which daily living functions are impaired or a state requiring nursing care (Non-Patent Document 1).

[0003] Muscles are primarily composed of proteins and free amino acids. Branched-chain amino acids are known to stimulate protein synthesis. Therefore, foods and beverages containing various proteins and amino acids have been used to increase muscle mass (Non-Patent Document 2). In addition, in recent years, it has been reported that ingesting Bifidobacterium bacteria can thicken myotubes and increase muscle mass, and they are expected to be a new active ingredient for increasing muscle mass (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2019 / 087280 [Non-patent literature]

[0005] [Non-Patent Document 1] Statement by the Japan Geriatrics Society https: / / jpn-geriat-soc.or.jp / info / topics / pdf / 20140513_01_01.pdf [Non-patent document 2] Yoshiharu Shimomura, Nutrition for Sports and Health (3rd Edition), Publisher: Nap, December 15, 2010, 3rd Edition, 1st Printing Summary of the Invention [Problem to be solved by the invention]

[0006] Conventional muscle-building compositions do not necessarily provide sufficient effects, and are sometimes difficult to take on a daily basis. Furthermore, the mechanism by which muscle mass increases involves a complex combination of various nutrients and protein synthesis systems, and some aspects remain unclear. Therefore, there is room for exploring new approaches to increasing muscle mass. In view of the above circumstances, an object of the present invention is to provide a means useful for increasing muscle mass. [Means for solving the problem]

[0007] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that threonic acid activates muscle protein synthesis-related genes and promotes muscle hypertrophy. They have also found that many Bifidobacterium species and lactic acid bacteria produce threonic acid, and that culturing these bacteria in the presence of ascorbic acid promotes threonic acid production. Based on these findings, the present inventors have concluded that threonic acid and threonic acid-producing bacteria can be used as active ingredients in compositions for muscle building, leading to the completion of the present invention.

[0008] That is, a first aspect of the present invention is a method for producing a threonic acid-containing composition or a muscle-building composition, the method comprising the steps of: culturing one or more bacteria selected from the group consisting of bacteria of the genus Bifidobacterium, bacteria of the genus Lactobacillus, bacteria of the genus Lacticaseibacillus, bacteria of the genus Lactiplantibacillus, bacteria of the genus Limosilactobacillus, bacteria of the genus Levilactobacillus, bacteria of the genus Ligilactobacillus, and bacteria of the genus Latilactobacillus in an ascorbic acid-containing medium; and recovering a threonic acid-containing fraction from the culture after the culture. In the production method of the present invention, the bacterium is preferably Bifidobacterium breve, and more preferably Bifidobacterium breve FERM BP-11175. A second aspect of the present invention is a composition for increasing muscle mass, comprising threonic acid and one or more bacteria selected from the group consisting of Bifidobacterium bacteria, Lactobacillus bacteria, Lacticaseibacillus bacteria, Lactiplantibacillus bacteria, Limosilactobacillus bacteria, Leviractobacillus bacteria, Rizilactobacillus bacteria, and Latilactobacillus bacteria, and one or more cultures of the bacteria. A third aspect of the present invention is a composition containing one or more bacteria selected from the group consisting of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Leviractobacillus, Lizilactobacillus, and Latilactobacillus, and cultures thereof, wherein the bacteria and / or cultures contain 1 μg or more threonic acid per total dry weight of the bacteria and / or cultures. The composition according to the third aspect is preferably used for increasing muscle mass. The compositions according to the second and third aspects of the present invention will hereinafter also be referred to as "compositions of the present invention." In the composition of the present invention, the bacterium is preferably Bifidobacterium breve, more preferably Bifidobacterium breve FERM BP-11175. The composition of the present invention is preferably a food or drink. The composition of the present invention is preferably a pharmaceutical product. [Effects of the Invention]

[0009] According to the present invention, a composition that can be taken continuously, is highly safe, and can efficiently increase muscle mass can be provided. Therefore, increasing muscle mass can improve daily life and exercise, and alleviate frailty and locomotive syndrome, thereby supporting healthy lifestyles in a wide range of age groups. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will now be described in detail. However, the present invention is not limited to the following embodiments and can be freely modified within the scope of the present invention.

[0011] <Production Method of the Present Invention> The present invention provides a method for producing a composition for increasing muscle mass, comprising culturing threonic acid-producing bacteria in an ascorbic acid-containing medium and recovering a threonic acid-containing fraction from the culture. Specifically, a first aspect of the present invention provides a method for producing a threonic acid-containing composition or a composition for increasing muscle mass, comprising culturing one or more bacteria selected from the group consisting of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Leviractobacillus, Lizilactobacillus, and Latilactobacillus in an ascorbic acid-containing medium and recovering a threonic acid-containing fraction from the culture. As used herein, the term "threonic acid-containing composition" is not limited as long as it contains threonic acid, but may include any of the bacterial cells themselves, a culture supernatant, a medium, purified threonic acid, or a mixture thereof. Furthermore, as used herein, the term "threonic acid-containing composition" may include a threonic acid-containing fraction, which will be described later. All bacteria other than the Bifidobacterium bacteria mentioned above were previously classified in the Lactobacillus genus, but in 2020 they were reclassified by the International Journal of Systematic and Evolutionary Microbiology (IJSEM) in accordance with the rules (ICNP) of the International Commission on Prokaryotic Nomenclature (ICSP).

[0012] The strains disclosed in this specification and assigned accession numbers are available from the respective depositories listed below. "NPMD" is the abbreviation for the National Institute of Technology and Evaluation (NITE) Patent Microorganism Depositary, located at Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan. In this specification, strains assigned accession numbers beginning with "NITE" are those deposited at NPMD. "IPOD" is the abbreviation for the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary. In 2012, the status of international deposit was transferred from the AIST to the National Institute of Technology and Evaluation (NITE), and patent microorganism deposit operations were centralized at NPMD. In this specification, strains assigned accession numbers beginning with "FERM" were deposited at IPOD, and their management was subsequently transferred to NPMD. "ATCC" is an abbreviation for American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110, United States of America. As used herein, strains assigned accession numbers beginning with "ATCC" have been deposited with the ATCC. "JCM" is an abbreviation for Japan Collection of Microorganisms, Microbial Materials Development Division, RIKEN BioResource Research Center, National Research and Development Agency, located at 3-1-1 Takanodai, Tsukuba, Ibaraki Prefecture, 305-0074. In this specification, strains assigned accession numbers beginning with "JCM" have been deposited at JCM. "DSMZ" is an abbreviation for Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, located at Inhoffenstr. 7B, 38124 Braunschweig, Germany. In this specification, strains assigned accession numbers beginning with "DSM" have been deposited at DSMZ.

[0013] In the production method of the present invention, when a threonic acid-containing composition containing bacterial cells is produced, the following bacteria can be used. Furthermore, in the production method of the present invention, when a threonic acid-containing composition containing a culture supernatant, a medium, or purified threonic acid is produced, the following bacteria can also be used. Examples of Bifidobacterium bacteria include Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium breve, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium pseudolongum, Bifidobacterium pseudocatenulatum, Bifidobacterium adolescentis, and Bifidobacterium bifidum.

[0014] Examples of Bifidobacterium longum subsp. longum include Bifidobacterium longum subsp. longum NITE BP-02621 (also known as BB536 or Bifidobacterium longum subsp. longum ATCC BAA-999; see, for example, JP 2012-223134 A), Bifidobacterium longum subsp. longum ATCC 15707, Bifidobacterium longum subsp. longum JCM1217, etc. can be used.

[0015] Examples of Bifidobacterium longum subsp. infantis that can be used include Bifidobacterium longum subsp. infantis M-63 (accession number NITE BP-02623) and Bifidobacterium longum subsp. infantis ATCC 15697.

[0016] Examples of Bifidobacterium breve that can be used include Bifidobacterium breve MCC1274 (accession number FERM BP-11175), Bifidobacterium breve M-16V (accession number NITE BP-02622; commercially available products such as "Bifidobacterium breve M-16V" manufactured by Morinaga Milk Industry Co., Ltd. may also be used), and Bifidobacterium breve ATCC 15700.

[0017] As Bifidobacterium animalis subsp. animalis, for example, Bifidobacterium animalis subsp. animalis ATCC25527 can be used.

[0018] As Bifidobacterium pseudolongum, for example, Bifidobacterium pseudolongum subsp. pseudolongum JCM1205T can be used.

[0019] As Bifidobacterium pseudocatenulatum, for example, Bifidobacterium pseudocatenulatum DSM20438 can be used.

[0020] As Bifidobacterium animalis subsp. lactis, for example, Bifidobacterium animalis subsp. lactis DSM10140 can be used.

[0021] As Bifidobacterium adolescentis, Bifidobacterium adolescentis JCM1275 can be used.

[0022] Examples of Bifidobacterium bifidum include Bifidobacterium bifidum MCC1092 (Accession No. NITE BP-02429), Bifidobacterium bifidum MCC1319 (Accession No. NITE BP-02431), Bifidobacterium bifidum MCC1868 (Accession No. NITE BP-02432), Bifidobacterium bifidum MCC1870 (accession number NITE BP-02433), Bifidobacterium bifidum JCM1255, etc. can be used.

[0023] Examples of the genus Lactobacillus (which was also classified in the genus Lactobacillus in the previous classification) that are a new classification in the production method of the present invention include Lactobacillus acidophillus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus helveticus, and Lactobacillus amylovorus. As the Lactobacillus acidophilus, Lactobacillus acidophilus MCC1847 (NITE BP-01695) can be used. Examples of Lactobacillus gasseri that can be used include Lactobacillus gasseri MCC1846 (NITE BP-01669) and Lactobacillus gasseri JCM1131. Examples of Lactobacillus helveticus that can be used include Lactobacillus helveticus JCM1120, Lactobacillus helveticus MCC1848 (NITE BP-01671), and Lactobacillus helveticus MCC1844 (NITE BP-02185).

[0024] Examples of the genus Lacticaseibacillus (formerly classified as the genus Lactobacillus in the old classification), which is a new classification in the production method of the present invention, include Lacticaseibacillus casei (formerly classified as Lactobacillus casei), Lacticaseibacillus paracasei (formerly classified as Lactobacillus paracasei), and Lacticaseibacillus rhamnosus (formerly classified as Lactobacillus rhamnosus). Examples of Lactobacillus casei that can be used include Lactobacillus casei JCM1134 and Lactobacillus casei ATCC393. Examples of Lacticaceae Bacillus paracasei that can be used include Lacticaceae Bacillus paracasei JCM8130, Lacticaceae Bacillus paracasei MCC1849 (NITE BP-01633), and Lacticaceae Bacillus paracasei MCC1375 (NITE BP-11313). Examples of Lacticaseibacillus rhamnosus that can be used include Lactobacillus rhamnosus JCM1136, Lactobacillus rhamnosus MCC1855 (LCS742, commercially available products such as "LCS-742" manufactured by Morinaga Milk Industry Co., Ltd. can be used), Lactobacillus rhamnosus ATCC53103, and Lactobacillus rhamnosus ATCC53103.

[0025] The genus Lactiplantibacillus (formerly classified as the genus Lactobacillus in the old classification), which is a new classification used in the production method of the present invention, includes Lactiplantibacillus plantarum (formerly classified as Lactobacillus plantarum) and Lactiplantibacillus pentosus (formerly classified as Lactobacillus pentosus). As Lactipranchibacillus plantarum, Lactipranchibacillus plantarum ATCC14917 can be used.

[0026] The genus Limosilactobacillus (formerly classified as the genus Lactobacillus in the old classification), which is a new classification used in the manufacturing method of the present invention, includes Limosilactobacillus fermentum (formerly classified as Lactobacillus fermentum) and Limosilactobacillus reuteri (formerly classified as Lactobacillus reuteri). As the Rimosilactobacillus fermentum, Rimosilactobacillus fermentum SBS-1 can be used. Examples of RimosyLactobacillus reuteri that can be used include RimosyLactobacillus reuteri JCM1112, RimosyLactobacillus reuteri DSM17938, RimosyLactobacillus reuteri ATCC PTA 6475, and RimosyLactobacillus reuteri ATCC PTA 5289.

[0027] The genus Levilactobacillus (formerly classified as the genus Lactobacillus in the old classification), which is a new classification in the production method of the present invention, includes Levilactobacillus brevis (formerly classified as Lactobacillus brevis).

[0028] The genus Ligilactobacillus (formerly classified as the genus Lactobacillus in the old classification), which is a new classification in the production method of the present invention, includes Ligilactobacillus salivarius (formerly classified as Lactobacillus salivarius).

[0029] The genus Latilactobacillus (formerly classified as Lactobacillus in the old classification), which is a new classification used in the production method of the present invention, includes Latilactobacillus curvatus (formerly classified as Lactobacillus curvatus).

[0030] In the production method of the present invention, any one or more of the above-mentioned bacteria can be used in combination, preferably one or more selected from the group consisting of Bifidobacterium breve, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium animalis subsp. animalis, Bifidobacterium pseudolongum, Bifidobacterium pseudocatenulatum, Bifidobacterium adolescentis, Bifidobacterium animalis subsp. lactis, Lactobacillus gasseri, Lactobacillus helveticus, Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, and Lactobacillus reuteri. More preferably, the active ingredient is one or more selected from Bifidobacterium breve, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Lactobacillus gasseri, Lactobacillus helveticus, Lacticaseibacillus paracasei, and Lacticaseibacillus rhamnosus. Even more preferably, the active ingredient is Bifidobacterium breve or Bifidobacterium longum subsp. longum. Most preferably, the active ingredient is Bifidobacterium breve FERM BP-11175 or Bifidobacterium longum NITE BP-02621.

[0031] The bacteria identified by the above-mentioned exemplified bacterial names are not limited to the strains deposited or registered with a designated institution under those bacterial names (hereinafter, for convenience of explanation, also referred to as "deposited strains"), but also include strains substantially equivalent thereto (also referred to as "derived strains" or "derived strains"). That is, they are not limited to the strains deposited with the depository institution under the above-mentioned accession numbers, but also include strains substantially equivalent thereto. With respect to each bacterium, "a strain substantially equivalent to the deposited strain" refers to a strain that belongs to the same species as the deposited strain, whose 16S rRNA gene nucleotide sequence is preferably 97% or more, more preferably 98% or more, even more preferably 99% or more, and even more preferably 100% identical to the 16S rRNA gene nucleotide sequence of the deposited strain, and preferably has the same bacteriological properties as the deposited strain. With respect to each bacterium, a strain substantially equivalent to the deposited strain may be, for example, a derivative strain derived from the deposited strain. Derivative strains include strains bred from the deposited strain and strains that have arisen naturally from the deposited strain. Breeding methods include modification by genetic engineering techniques and modification by mutation treatment. Mutation treatments include irradiation with X-rays, irradiation with ultraviolet light, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and methyl methanesulfonate. Strains that have arisen naturally from the deposited strain include strains that have arisen naturally during use of the deposited strain. Such strains include mutants that have arisen naturally through cultivation (e.g., subculturing) of the deposited strain. Derivative strains may be constructed by a single modification, or by two or more modifications.

[0032] In the production method of the present invention, ascorbic acids are added to the medium when culturing the bacteria. As used herein, "ascorbic acids" refers to one or more selected from ascorbic acid, ascorbic acid derivatives, and salts thereof. L-ascorbic acids can usually be used. Ascorbic acid, L-ascorbic acid can be used, and erythorbic acid, which is a stereoisomer of L-ascorbic acid, is also included. Preferred examples of the ascorbic acid derivative include salts of inorganic acid esters of L-ascorbic acid, such as L-ascorbic acid phosphate and L-ascorbic acid sulfate, and glycosides of L-ascorbic acid, such as L-ascorbic acid-2-glucoside. The salt of ascorbic acid or a derivative thereof is not particularly limited, but preferred examples thereof include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium and magnesium; organic amine salts such as ammonium salts, triethanolamine salts and triethylamine salts; and basic amino acid salts such as lysine salts and arginine salts. Specific preferred examples of ascorbic acids in the present invention include sodium L-ascorbate, potassium L-ascorbate, calcium L-ascorbate, magnesium L-ascorbate, sodium erythorbate, potassium erythorbate, calcium erythorbate, magnesium erythorbate, and L-ascorbic acid-2-glucoside. The amount of ascorbic acids to be added is not particularly limited, but is preferably 1 mg / L to 100 g / L, more preferably 10 mg / L to 10 g / L, and even more preferably 100 mg / L to 1 g / L relative to the medium. The timing of adding ascorbic acids is not particularly limited, but they are usually added at the start of culture.

[0033] The culturing step in the production method of the present invention is not particularly limited as long as it allows the bacteria to grow. For example, a method commonly used for culturing the bacteria can be used as is, or with appropriate modifications. The culture temperature may be, for example, 25 to 50°C, preferably 35 to 42°C. The culture is preferably carried out under anaerobic conditions, for example, while aerating with an anaerobic gas such as carbon dioxide. The culture can also be carried out under microaerobic conditions, such as liquid static culture. The culture can be carried out, for example, until threonic acid is accumulated intracellularly and / or secreted extracellularly to a desired extent, or until the bacteria grow to a desired extent. The culture may be for example, 12 to 72 hours.

[0034] The medium used for the culture is not particularly limited as long as it contains ascorbic acids and allows the bacteria to grow. For example, a medium typically used for culturing the bacteria can be used as is or with appropriate modifications. Specifically, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysates, and blackstrap molasses can be used as carbon sources depending on the assimilation potential. Nitrogen sources include ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, as well as nitrates. Inorganic salts include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Organic components such as peptone, soybean flour, defatted soybean meal, meat extract, and yeast extract can also be used. Specific examples of media commonly used for culturing Bifidobacterium include reinforced clostridial medium, de Man, Rogosa, and Sharpe (MRS) medium, modified MRS (mMRS) medium, TOS propionate (TOSP) medium, and TOS propionate mupirocin (TOSP Mup) medium.

[0035] The production method of the present invention includes a step of recovering a threonic acid-containing fraction from the culture after the culturing step. As used herein, the "threonic acid-containing fraction" may be any of the bacterial cells themselves, the culture supernatant, the medium, or a mixture thereof, as long as it contains threonic acid. As will be described in the Examples below, threonic acid is an active ingredient that exerts a muscle-building effect. Furthermore, when ascorbic acids or similar compounds were added to a medium and threonic acid-producing bacteria were cultured, the presence of threonic acid was confirmed in the culture supernatant and the supernatant of the disrupted bacterial cells. This indicates that ascorbic acid is metabolized by the bacterial cells to threonic acid, which is accumulated within the bacterial cells and is also secreted outside the bacterial cells. In the production method of the present invention, when a threonic acid-containing composition containing bacterial cells is produced, the method may include a step of treating the recovered threonic acid-containing fraction by centrifugation, crushing, washing, freeze-drying, spray-drying, or the like. In the production method of the present invention, when a threonic acid-containing composition containing bacterial cells is produced, the method may include a step of obtaining a disrupted composition, a washed composition, a freeze-dried composition, or a spray-dried composition.

[0036] In the production method of the present invention, when a threonic acid-containing composition containing a culture supernatant is produced, the method may include a step of treating the recovered threonic acid-containing fraction by centrifugation, supernatant recovery, concentration, freeze-drying, spray-drying, or the like. In the production method of the present invention, when a threonic acid-containing composition containing a culture supernatant is produced, the method may include a step of obtaining a concentrated composition, a freeze-dried composition, or a spray-dried composition.

[0037] In the production method of the present invention, when a threonic acid-containing composition containing a medium is produced, the method may include a step of treating the recovered threonic acid-containing fraction by centrifugation, medium recovery, concentration, freeze-drying, spray-drying, or the like. In the production method of the present invention, when a threonic acid-containing composition containing a culture medium is produced, the method may include a step of obtaining a concentrated composition, a freeze-dried composition, or a spray-dried composition.

[0038] In the production method of the present invention, when a threonic acid-containing composition containing purified threonic acid is produced, the method may include a step of purifying threonic acid contained in the threonic acid-containing fraction. The step of purifying threonic acid is not particularly limited as long as it can purify threonic acid, and may include a step of treating the threonic acid by disrupting the bacterial cells, centrifugation, membrane separation, solvent extraction, or the like. When the production method of the present invention includes a step of purifying threonic acid from a threonic acid-containing fraction, the production method of the present invention can be used as a method for producing threonic acid. When the production method of the present invention is used as a method for producing threonic acid, the recovered threonic acid-containing fraction may be included. The threonic acid-containing fraction thus recovered typically contains threonic acid in an amount of preferably 0.01 μg / g or more, more preferably 0.1 μg / g or more, even more preferably 1 μg / g or more, still more preferably 5 μg / g or more, and even more preferably 10 μg / g or more, based on the total dry weight of the fraction. In this specification, "threonic acid" generally refers to the L-form. In the composition of the present invention, threonic acid may be contained in the form of a threonate salt, and examples of such salts include, but are not limited to, sodium salts, potassium salts, and calcium salts. Threonate can be identified and quantified using known analytical techniques, such as liquid chromatography mass spectrometry (LC / MS), as described in the Examples below, using, for example, Prominence (Shimadzu Corporation) for HPLC and TSQ Quantum Discovery MAX (Thermo Corporation) for mass spectrometry.

[0039] As described above, the production method of the present invention may include a step of treating the recovered threonic acid-containing fraction after the step of recovering the threonic acid-containing fraction. The method for treating the threonic acid-containing fraction is not particularly limited as long as it does not impair the muscle-building effect, and examples of the method include dilution, concentration, heating, freeze-drying, spray-drying, crushing, fractionation, etc. That is, in the production method of the present invention, the steps of treating the recovered threonic acid-containing fraction may include a step of separating and recovering bacterial cells, a step of separating and recovering a culture supernatant, a step of separating and recovering a medium, and a step of purifying threonic acid from the threonic acid-containing fraction. Furthermore, when the production method of the present invention includes a step of purifying threonic acid from the threonic acid-containing fraction, the production method of the present invention can be used as a method for producing threonic acid.

[0040] The production method of the present invention may include a step of mixing the recovered threonic acid-containing fraction with a food raw material. The production method of the present invention may include a step of converting the recovered threonic acid-containing fraction into a form such as powder, granules, paste, emulsion, wetting agent, capsule, or tablet.

[0041] The production method of the present invention may include a step of sterilizing the recovered threonic acid-containing fraction after the step of recovering the threonic acid-containing fraction. The step of sterilizing the threonic acid-containing fraction is not particularly limited as long as it does not impair the muscle-building effect, and examples thereof include heat treatment, crushing treatment, pressure treatment, and treatment with a chemical. In particular, when bacterial cells are recovered as the threonic acid-containing fraction, a step of sterilizing the bacterial cells may be included, and the sterilization may be heat treatment, crushing treatment, or pressure treatment. That is, when bacterial cells are contained as the threonic acid-containing fraction in the resulting muscle-building composition, the bacterial cells may be viable, killed, or a mixture of viable and killed cells. The steps of treating the recovered threonic acid-containing fraction, mixing with a food material, forming into a liquid or other form, and sterilizing are optional, and the order of these steps can be changed as appropriate.

[0042] <Composition of the Present Invention> A composition according to a second aspect of the present invention contains threonic acid and one or more bacteria selected from the group consisting of Bifidobacterium bacteria, Lactobacillus bacteria, Lacticaseibacillus bacteria, Lactiplantibacillus bacteria, Limosilactobacillus bacteria, Leviractobacillus bacteria, Rizilactobacillus bacteria, and Latilactobacillus bacteria, and one or more cultures of the above bacteria, and is used for increasing muscle mass. A composition according to a second alternative embodiment of the present invention contains purified threonic acid or a threonic acid-containing fraction and is used for increasing muscle mass.

[0043] The composition of the present invention can be preferably produced by the production method of the present invention described above. In this case, threonic acid is usually contained in the bacterium and / or its culture. Therefore, from another viewpoint, the composition of the present invention, as a third aspect, is a composition containing one or more bacteria selected from the group consisting of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Leviractobacillus, Rizilactobacillus, and Latilactobacillus, and cultures of the bacteria, wherein the bacteria and / or the cultures contain 1 μg / g or more of threonic acid per total dry weight. Such a composition is suitable for muscle building applications. The type of the bacterium in the composition of the present invention is the same as that described above in the production method of the present invention. The bacterium and / or its culture to be contained in the composition of the present invention may refer to the threonic acid-containing fraction recovered in the production method.

[0044] The content of the bacteria and / or its culture in the composition of the present invention is not particularly limited, but for example, it may be 1.0 × 10 per 1 g of the composition in terms of the amount of bacterial cells. 7 cfu or more, more preferably 1.0 × 10 8 cfu or more, more preferably 1.0 × 10 9The composition may contain at least cfu of bacterial cells. Here, cfu refers to colony forming unit. In this specification, the value may be, for example, the value when cultured at 38°C in a solid medium containing 10% by mass of reconstituted skim milk powder. When the bacterial cells are killed, cfu can be substituted for individual cells. The content of bacterial cells, converted into the solid matter amount of the culture, is preferably 0.1 mg or more per 1 g of the composition, more preferably 1 mg or more, and even more preferably 10 mg or more.

[0045] The content of threonic acid in the composition of the present invention is not particularly limited, but is preferably 0.01 μg or more per gram of composition, more preferably 0.1 μg or more, even more preferably 1 μg or more, even more preferably 5 μg or more, and even more preferably 10 μg or more. These may be in the range of contents typically used when distributed as oral compositions.

[0046] The form of the composition of the present invention is not particularly limited, and for example, the bacteria and / or culture thereof may be a diluted composition, concentrated composition, heated composition, freeze-dried composition, spray-dried composition, crushed composition, fractionated composition, or sterilized composition. The form of the composition of the present invention may also be a liquid, powder, granule, paste, emulsion, moisturizer, capsule, tablet, etc. When the composition of the present invention is a sterilized composition, the sterilization method is not limited, and the composition may be a heat-sterilized composition, a chemically sterilized composition, or a crushing treatment sterilized composition.

[0047] The composition of the present invention has the effect of increasing muscle mass in animals, including humans. "Muscle mass" includes an increase in muscle volume and / or weight and suppression of muscle volume and / or weight loss. The increase in muscle volume and / or weight and suppression of muscle volume and / or weight loss may be due to muscle hypertrophy (thickening of myotubes or muscle fibers) or an increase in the number of myotubes or muscle fibers. The muscle-massing effect of the composition of the present invention is thought to be due to the activation of protein synthesis-related genes such as p70S6K by threonic acid, which promotes muscle protein synthesis.

[0048] It is known that ascorbic acid contributes to maintaining muscle mass in skeletal muscles (S. Takisawa et al., Scientific Reports vol.9, Art. no.4702, (2019)). However, because threonic acid has a significantly different structure from ascorbic acid, it is thought that threonic acid exerts its muscle-building effect through a different mechanism than ascorbic acid. Bifidobacterium bacteria and cultures thereof are also known to have muscle-building effects (Patent Document 1), and the muscle-building effect can be further enhanced by combining these with threonic acid or by using bacteria that produce threonic acid or cultures thereof as active ingredients.

[0049] When the composition of the present invention is intended to be orally ingested (administered), it is preferably in the form of a food or drink. That is, another aspect of the present invention is use of a composition containing threonic acid and one or more bacteria selected from Bifidobacterium bacteria, Lactobacillus bacteria, Lacticaseibacillus bacteria, Lactiprantibacillus bacteria, Limocilactobacillus bacteria, Leviractobacillus bacteria, Rizilactobacillus bacteria, and Latilactobacillus bacteria, as well as one or more cultures of the above bacteria, in increasing muscle mass. Another aspect of the present invention is the use of one or more bacteria selected from the group consisting of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Leviractobacillus, Rizilactobacillus, and Latilactobacillus, and cultures of these bacteria, in muscle mass building, wherein the bacteria and / or cultures contain 1 μg or more of threonic acid per total dry weight thereof. Another aspect of the present invention is a method for increasing muscle mass, comprising administering to a subject one or more bacteria selected from Bifidobacterium bacteria, Lactobacillus bacteria, Lacticaseibacillus bacteria, Lactiplantibacillus bacteria, Limosilactobacillus bacteria, Leviractobacillus bacteria, Rizilactobacillus bacteria, and Latilactobacillus bacteria, and cultures of said bacteria, wherein the bacteria and / or the cultures contain 1 μg / g or more of threonic acid per total dry weight thereof.

[0050] The present specification also discloses, as a fourth aspect, a composition for increasing muscle mass, which contains threonic acid as an active ingredient. This invention is based on the fact that threonic acid activates protein synthesis-related genes in skeletal muscle cells, promotes muscle hypertrophy, and exerts a muscle-building effect. This invention can also be rephrased as the use of threonic acid in increasing muscle mass. The invention can also be rephrased as a method for increasing muscle mass, which comprises administering threonic acid to a subject.

[0051] The food and beverage composition of the present invention is capable of increasing muscle mass, and is therefore expected to prevent and / or improve symptoms such as muscle atrophy and diseases caused by muscle mass loss, as well as prevent and / or improve muscle mass loss associated with lack of exercise and aging. Examples of diseases accompanied by symptoms such as muscle atrophy include hypotonia, muscle atrophy, muscle dystrophy, muscle degeneration, inflammatory muscle diseases, myasthenia, and sarcopenia. Examples of diseases caused by muscle mass loss include osteoporosis, fractures, diabetes, chronic obstructive pulmonary disease, chronic kidney disease, and dementia. In addition, since a decrease in muscle mass due to lack of exercise and aging can lead to sarcopenia, frailty, locomotive syndrome, etc., it is expected that this technology will also help prevent and / or improve these conditions. The food and drink composition of the present invention is also useful for healthy people and people who play sports, as it can maintain and improve daily life and improve exercise efficiency. Here, "amelioration" of a symptom or disease as used herein includes curing the disease, alleviating the symptom, reducing the severity of the disease or symptom, and delaying the progression of the disease or symptom. Furthermore, "prevention" of a symptom or disease includes preventing the onset of the symptom or disease, delaying the onset, and reducing the risk of the onset.

[0052] The subjects to which the food and beverage compositions of the second to fourth aspects of the present invention are administered (recipients) and those to which they are ingested (consumers) are not particularly limited as long as they are animals, but are usually humans. They may be adults, children, infants, newborns (including low birth weight infants), etc. Furthermore, their gender is not particularly limited.

[0053] In this specification, "administering to a subject" may be synonymous with "intake by a subject." Intake is usually voluntary (ad libitum intake), but may also be forced (forced intake).

[0054] The timing of ingestion (administration) of the food and drink composition of the present invention is not particularly limited, and can be appropriately selected depending on the condition of the subject to be ingested (administered).

[0055] The intake (administration) amount of the food and drink composition of the present invention is appropriately selected depending on the age, sex, condition, and other conditions of the subject to be ingested (administered). The intake (administration) amount of the food and beverage composition of the present invention, converted into the solid content of the bacterium and / or its culture, is, for example, preferably in the range of 100 μg / day to 10 g / day, more preferably 1 mg / day to 1 g / day, and even more preferably 10 mg / day to 500 mg / day for adults, and converted into the amount of threonic acid, is, for example, preferably in the range of 1 ng / day to 100 μg / day, more preferably 1 ng / day to 1 μg / day, and even more preferably 10 ng / day to 500 ng / day for adults. Regardless of the amount or period of ingestion (administration), the food and drink composition of the present invention can be ingested (administered) once a day or in divided doses multiple times a day.

[0056] The intake (administration) period of the food and drink composition of the present invention is not particularly limited. Furthermore, there is no particular upper limit to the intake (administration) period, and continuous, long-term intake (administration) is possible.

[0057] The form and properties of the food and drink products are not particularly limited as long as they do not impair the effects of the present invention and can be orally ingested (administered). Except for containing threonic acid and the bacterium and / or a culture thereof, the food and drink products can be produced by a conventional method using raw materials typically used for food and drink products.

[0058] Food and drink products include, regardless of their form, liquid, paste, gel-like solid, powder, etc., such as nutritional supplements, tablet confectionery; liquid food (nutritional food for tube feeding); wheat flour products such as bread, macaroni, spaghetti, noodles, cake mix, fried chicken flour, breadcrumbs, etc.; instant noodles, cup noodles, retort / prepared foods, canned foods, microwave foods, instant soup / stew, instant miso soup / cleaning liquid, canned soup, freeze-dried foods, other instant foods, etc.; agricultural products Agricultural processed products such as canned goods, canned fruit, jams and marmalades, pickles, boiled beans, dried agricultural goods, and cereals (processed grain products); processed seafood products such as canned seafood, fish ham and sausage, fish paste products, seafood delicacies, and tsukudani (fish stews); processed livestock products such as canned livestock paste, livestock ham and sausage; dairy products such as processed milk, milk drinks, yogurt, lactic acid bacteria drinks, cheese, ice cream, infant formula, cream, and other dairy products; fats and oils such as butter, margarines, and vegetable oils Basic seasonings such as soy sauce, miso, sauces, processed tomato seasonings, mirin, vinegars, etc.; Complex seasonings such as cooking mixes, curry bases, sauces, dressings, noodle soups, spices, and other complex seasonings; Frozen foods such as frozen ingredients, semi-cooked frozen foods, and cooked frozen foods; Caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice snacks, bean snacks, desserts, jellies and other sweets; carbonated drinks, natural fruit juices, fruit juice drinks, soft drinks with fruit juice, fruit pulp drinks, fruit drinks with fruit pieces, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, and other beverages; other commercially available foods such as baby food, furikake, and ochazuke nori seaweed; infant formula; enteral nutritional foods; and health functional foods (foods for specified health uses, foods with nutrient functions, foods with functional claims).

[0059] Furthermore, one aspect of the food and drink product may be feed, such as pet food, livestock feed, and fish feed. The form of the feed is not particularly limited, and may contain, in addition to threonic acid and the bacterium and / or a culture thereof, for example, grains such as corn, wheat, barley, rye, milo, etc.; vegetable oil cakes such as soybean oil cake, rapeseed oil cake, palm oil cake, and linseed oil cake; bran such as wheat bran, wheat bran, rice bran, and defatted rice bran; manufacturing residues such as corn gluten meal and corn jam meal; animal feeds such as fish meal, skim milk powder, casein, yellow grease, and tallow; yeasts such as torula yeast and brewer's yeast; mineral feeds such as calcium phosphate and calcium carbonate; oils and fats; simple amino acids; and sugars.

[0060] When the composition of the present invention is in the form of a food or drink (including feed), it can be provided and sold as a food or drink labeled for use in increasing muscle mass.

[0061] Such "indication" acts include all acts for informing consumers of the aforementioned uses, and any expression that can recall or infer the aforementioned uses falls under the category of "indication" acts in this invention, regardless of the purpose of the indication, the content of the indication, the object or medium on which it is displayed, etc. Furthermore, it is preferable that the "labeling" be done in an expression that allows consumers to directly recognize the intended use. Specifically, this includes acts of transferring, delivering, displaying for the purpose of transferring or delivering, or importing food and beverage products or product packaging on which the intended use is stated, displaying or distributing advertisements, price lists, or transaction documents related to the products and including the intended use, or providing information containing the above content by electromagnetic means (such as the Internet).

[0062] On the other hand, it is preferable that the content of the labeling be one approved by the government, etc. (for example, a labeling approved based on various systems established by the government and made in a manner based on such approval.) It is also preferable that such content of the labeling be affixed to promotional materials at the point of sale, such as packaging, containers, catalogs, pamphlets, POP displays, and other documents.

[0063] "Labeling" also includes labeling as health food, functional food, enteral nutritional food, special dietary food, health functional food, food for specified health uses, food with nutrient functions, food with functional claims, quasi-drug, etc. Among these, labeling approved by the Consumer Affairs Agency, such as labeling approved under systems related to foods for specified health uses, foods with nutrient functions, or foods with functional claims, or similar systems, can be cited. Specific examples include labeling as a food for specified health uses, labeling as a conditional food for specified health uses, labeling that indicates an effect on the structure or function of the body, labeling that reduces disease risk, and labeling of functionality based on scientific evidence. More specifically, typical examples include labeling as a food for specified health uses (especially labeling of health uses) and similar labeling as defined in the Cabinet Office Ordinance on Permission for Labeling for Special Uses Provided in the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009).

[0064] Examples of such claims include "builds muscle," "for those who want to build muscle strength," "for preventing frailty," "for improving quality of life," and "supports muscle building for those who exercise."

[0065] The composition of the present invention can also be in the form of a pharmaceutical product. That is, another aspect of the present invention is a composition used for increasing muscle mass, which contains threonic acid and one or more bacteria selected from the group consisting of Bifidobacterium bacteria, Lactobacillus bacteria, Lacticaseibacillus bacteria, Lactiprantibacillus bacteria, Limosilactobacillus bacteria, Leviractobacillus bacteria, Rizilactobacillus bacteria, and Latilactobacillus bacteria, and one or more cultures of the above bacteria. Another aspect of the present invention is the use of threonic acid and one or more bacteria selected from the group consisting of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Leviractobacillus, Rizilactobacillus, and Latilactobacillus, and cultures of the above bacteria, in the production of a composition for increasing muscle mass. Another aspect of the present invention is a composition used for increasing muscle mass, which contains threonic acid and one or more bacteria selected from the group consisting of Bifidobacterium bacteria, Lactobacillus bacteria, Lacticaseibacillus bacteria, Lactiprantibacillus bacteria, Limosilactobacillus bacteria, Leviractobacillus bacteria, Rizilactobacillus bacteria, and Latilactobacillus bacteria, and one or more cultures of the above bacteria. Another aspect of the present invention is a method for increasing muscle mass, comprising administering to a subject threonic acid and one or more bacteria selected from Bifidobacterium bacteria, Lactobacillus bacteria, Lacticaseibacillus bacteria, Lactiplantibacillus bacteria, Limosilactobacillus bacteria, Leviractobacillus bacteria, Lizilactobacillus bacteria, and Latilactobacillus bacteria, as well as cultures of the above bacteria. Another aspect of the present invention is a composition used for increasing muscle mass, comprising one or more bacteria selected from the group consisting of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Leviractobacillus, Rizilactobacillus, and Latilactobacillus, and cultures of the bacteria, wherein the bacteria and / or the cultures contain 1 μg or more of threonic acid per total dry weight of the composition. Another aspect of the present invention is the use of one or more bacteria selected from the group consisting of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Leviractobacillus, Rizilactobacillus, and Latilactobacillus, and cultures of such bacteria, in the production of a composition for increasing muscle mass, wherein the bacteria and / or cultures contain 1 μg or more of threonic acid per total dry weight thereof. Another aspect of the present invention is a method for increasing muscle mass, comprising administering to a subject one or more bacteria selected from Bifidobacterium bacteria, Lactobacillus bacteria, Lacticaseibacillus bacteria, Lactiplantibacillus bacteria, Limosilactobacillus bacteria, Leviractobacillus bacteria, Rizilactobacillus bacteria, and Latilactobacillus bacteria, and cultures of said bacteria, wherein the bacteria and / or the cultures contain 1 μg / g or more of threonic acid per total dry weight thereof.

[0066] The present specification also discloses, as a fourth aspect, a composition for increasing muscle mass, which contains threonic acid as an active ingredient. This invention is based on the fact that threonic acid activates protein synthesis-related genes in skeletal muscle cells, promotes muscle hypertrophy, and exerts a muscle-building effect. This invention can also be rephrased as threonic acid used for muscle mass building. This invention can also be rephrased as the use of threonic acid in the production of a composition for increasing muscle mass. The invention can also be rephrased as a method for increasing muscle mass, which comprises administering threonic acid to a subject.

[0067] The pharmaceutical composition of the present invention is capable of increasing muscle mass and is therefore expected to prevent and / or improve symptoms such as muscle atrophy and diseases caused by muscle mass loss, as well as prevent and / or improve muscle mass loss associated with lack of exercise and aging. Examples of diseases accompanied by symptoms such as muscle atrophy include hypotonia, muscle atrophy, muscle dystrophy, muscle degeneration, inflammatory muscle diseases, myasthenia, and sarcopenia. Examples of diseases caused by muscle mass loss include osteoporosis, fractures, diabetes, chronic obstructive pulmonary disease, chronic kidney disease, and dementia. In addition, since a decrease in muscle mass due to lack of exercise and aging can lead to sarcopenia, frailty, locomotive syndrome, etc., it is expected that this technology will also help prevent and / or improve these conditions.

[0068] The subjects to which the pharmaceutical compositions of the second to fourth aspects of the present invention are administered (recipients) and those to which the pharmaceutical compositions are ingested (consumers) are not particularly limited as long as they are animals, but are usually humans. They may be adults, children, infants, newborns (including low birth weight infants), etc. Furthermore, their gender is not particularly limited.

[0069] The timing of intake (administration) of the pharmaceutical composition of the present invention is not particularly limited, and can be appropriately selected depending on the condition of the subject to be taken (administered).

[0070] The amount of intake (administration) of the pharmaceutical composition of the present invention is appropriately selected depending on the age, sex, condition, and other conditions of the subject to be ingested (administered). The intake (administration) amount of the pharmaceutical composition of the present invention, converted into the solid content of the bacterium and / or its culture, is, for example, preferably in the range of 100 μg / day to 10 g / day for adults, more preferably in the range of 1 mg / day to 1 g / day, and even more preferably in the range of 10 mg / day to 500 mg / day, and converted into the amount of threonic acid, for example, in adults, is preferably in the range of 1 ng / day to 100 μg / day, more preferably in the range of 1 ng / day to 1 μg / day, and even more preferably in the range of 10 ng / day to 500 ng / day. Regardless of the amount or duration of ingestion (administration), the pharmaceutical composition of the present invention can be ingested (administered) once or multiple times a day. The timing of ingestion (administration) of the pharmaceutical of the present invention is not particularly limited, and may be, for example, before meals, after meals, between meals, or before bedtime.

[0071] The period for taking (administering) the pharmaceutical composition of the present invention is not particularly limited. In addition, there is no particular upper limit to the period for taking (administering), and continuous, long-term taking (administration) is possible.

[0072] The route of administration of pharmaceuticals may be oral or parenteral, with oral administration being preferred. Parenteral administration includes transdermal, intravenous, rectal, and inhalation routes. The pharmaceutical form can be formulated into a desired dosage form depending on the method of ingestion (administration). For example, in the case of oral ingestion (administration), it can be formulated into solid preparations such as powders, granules, tablets, and capsules; or liquid preparations such as solutions, syrups, suspensions, and emulsions. In addition, in the case of parenteral ingestion (administration), it can be formulated into suppositories, ointments, injections, etc. When preparing the formulation, ingredients commonly used in formulations, such as excipients, pH adjusters, colorants, and flavoring agents, can be used. It is also possible to use other pharmaceutical ingredients, or other drugs, such as ingredients with known or future muscle-building effects, in combination. In addition, formulation can be carried out by a known method as appropriate depending on the dosage form. When formulating, carriers commonly used in formulations may be appropriately blended to form the formulation. Such carriers include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, etc.

[0073] Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, and carboxymethyl cellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; and sulfate derivatives such as calcium sulfate.

[0074] Examples of binders include gelatin, polyvinylpyrrolidone, macrogol, and the like, in addition to the above-mentioned excipients.

[0075] Examples of disintegrants include the above-mentioned excipients, as well as chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and cross-linked polyvinylpyrrolidone.

[0076] Examples of lubricants include talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as veegum and gaelt; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; and starch derivatives.

[0077] Examples of stabilizers include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; sorbic acid; and the like.

[0078] Examples of flavoring agents include sweeteners, acidulants, and fragrances. In the case of a liquid preparation for oral ingestion (administration), examples of the carrier to be used include solvents such as water. [Example]

[0079] The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples.

[0080] <Test Example 1> Examination of the muscle-building effect of threonic acid (1) Sample preparation 155 g of calcium L-threonate (Tokyo Chemical Industry Co., Ltd.) was suspended in 90 mL of water, and an appropriate amount of 6 N hydrochloric acid (Nacalai Tesque) was added until completely dissolved. The suspension was neutralized with 4 N sodium hydroxide (Nacalai Tesque), and the volume was adjusted to 100 mL with water to obtain a 1 M threonic acid sample.

[0081] (2) Evaluation of protein synthesis-related gene activity Rat myoblast cell line L6 cells (hereafter referred to as "L6") obtained from the American Type Culture Collection (ATCC) were cultured at 1.5 × 104 cells / cm 2 The cells were seeded onto 6-well plates so that the total volume was 100 μg / well and cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C under 5% CO2 for 24 hours. To induce differentiation, the DMEM medium (containing 2% horse serum and 1% penicillin-streptomycin; hereafter referred to as "differentiation medium") was replaced with fresh medium every two days and the cells were cultured for 7 days. Then, the 1 M threonic acid sample prepared in (1) above was added to a final concentration of 1 or 10 μM. After culturing for 1 hour, lysates were prepared using RIPA buffer. Activation (phosphorylation) of the protein synthesis-related gene p70S6K was evaluated by Western blotting. Specifically, the fluorescence intensity of the bands of phosphorylated p70S6K and total p70S6K was measured using a ChemiDoc (registered trademark) MP Imaging System (Bio-Rad Laboratories), and the ratio of phosphorylated p70S6K to total p70S6K was calculated as the activity value of p70S6K. The same procedure as above was carried out except that the threonic acid sample was not added, and this was used as a control.

[0082] (3) Evaluation of myotube cell thickness L6 to 1.5 x 10 4 cells / cm 2 The cells were seeded onto a 12-well plate so that the total volume was 100 μL and cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin streptomycin) at 37°C under 5% CO2 for 24 hours. The 1 M threonic acid sample prepared in (1) above was then added to the DMEM medium (differentiation medium) to a final concentration of 1 or 10 μM, and cultured at 37°C under 5% CO2 for 7 days. The medium was replaced with fresh medium every two days. The cells obtained by this procedure are hereinafter referred to as "myotube cells." The same procedure was carried out except that the threonic acid sample was not added, and this was used as a control. Thereafter, hematoxylin-eosin staining (HE staining) was performed, and the thickness of 50 randomly selected myotubes was measured using an optical microscope BX53 (Olympus).

[0083] (4) Results (4-1) Evaluation of protein synthesis-related gene activity The activity of a protein synthesis-related gene (p70S6K) in L6 cells treated with threonic acid is shown in Tables 1 and 2. The activity (degree of phosphorylation) of p70S6K increased 3.37-fold in the 1 μM threonic acid-treated group and 3.03-fold in the 10 μM threonic acid-treated group compared to the control.

[0084] [Table 1]

[0085] [Table 2]

[0086] (4-2) Evaluation of myotube cell thickness The diameters (average values) of myotubes obtained by the procedure (3) above are shown in Table 3. The diameter of myotubes was larger than the control by 2.58 μm or more in the 1 μM threonic acid treatment group and by 1.85 μm or more in the 10 μM threonic acid treatment group, confirming an increase in muscle mass.

[0087] [Table 3]

[0088] (4-3) These results demonstrate that threonic acid activates muscle protein synthesis-related genes and promotes muscle hypertrophy.

[0089] <Test Example 2> Examination of extracellular threonic acid production by probiotics (1) Cultivation of probiotics using ascorbic acid-containing medium 5.5 g of Difco Lactobacilli MRS Broth (BD) and 50 mg of L-cysteine ​​monohydrochloride monohydrate (Wako Pure Chemical Industries, Ltd.) were dissolved in purified water to make 100 mL, adjusted to pH 6.5 with hydrochloric acid, and sterilized at 121°C for 15 minutes to prepare MRS liquid medium. Bifidobacterium breve FERM BP-11175, Bifidobacterium pseudolongum subsp. pseudolongum JCM1205T, and Limocilactobacillus reuteri JCM1112T, which had been frozen and stored in an aqueous solution containing 10% skim milk powder, were thawed and inoculated into the MRS liquid medium at 1% (v / v) each and cultured anaerobically at 37°C (preculture). After 16 hours of culturing, the preculture solution was inoculated at 1% (v / v) into an ascorbic acid-containing medium prepared by adding an L-ascorbic acid (Wako Pure Chemical Industries, Ltd.) aqueous solution to the MRS liquid medium to a final concentration of 1 g / L, and the medium was anaerobically cultured at 37°C (main culture). After 16 hours of culturing, the culture supernatant was collected by centrifugation. As a control, bacteria were cultured in the same manner except that ascorbic acid was not added.

[0090] (2) Identification and quantification of threonic acid The culture supernatant collected in (1) was mixed with an equal volume of 0.2% formic acid water, filtered (φ=0.22 μm, Millipore), and analyzed for threonic acid content. Threonate analysis was performed by LC / MS. A Prominence (Shimadzu) HPLC system was used, and tandem mass spectrometry was performed using a TSQ Quantum Discovery MAX (Thermo). An XBridge C18 column (Waters) was used. The mobile phase consisted of 0.1% formic acid water and 0.1% formic acid-containing acetonitrile. The acetonitrile concentration was increased from 2% to 95% over 25 min after sample injection to elute threonic acid. Threonate eluted approximately 7.8 min after sample injection. The precursor ion (m / z) was 137.050, the product ion (m / z) was 119.042, and the collision energy was 5.25 eV. Threonate was identified and quantified. Identification and quantification of threonic acid can be performed by LC / MS using a standard sample.

[0091] (3) Results The threonic acid content in the medium for each strain is shown in Table 4. It was confirmed that the amount of threonic acid in the culture supernatant increased with the addition of ascorbic acid for all strains.

[0092] [Table 4]

[0093] <Test Example 3> Examination of the muscle-building effect of probiotic culture supernatant Using the culture supernatant of probiotics cultured in the presence of ascorbic acid, we evaluated the promotion of protein synthesis-related gene (p70S6K) activity in L6 and examined its muscle-building effect. (1) Sample preparation Sodium hydroxide was added to the culture supernatants of the three bacterial strains (Bifidobacterium breve FERM BP-11175, Bifidobacterium pseudolongum subsp. pseudolongum JCM1205T, and Limocilactobacillus reuteri JCM1112T) prepared in Test Example 2 (1), to adjust the pH to 7.0±0.05, and the mixture was filter-sterilized.

[0094] (2) Evaluation of protein synthesis-related gene activity L6 was cultured in the same manner as in Test Example 1 (2), except that the culture supernatants of the three strains were added instead of adding 1 M threonic acid sample. The same procedure was repeated except that no culture supernatant was added, and this was used as a control. The control group with ascorbic acid added was cultured in a differentiation medium containing 1 g / L of ascorbic acid at 1% (v / v). As in Test Example 1, the activity value of p70S6K was calculated.

[0095] (3) Results The activity of the protein synthesis-related gene (p70S6K) in L6 of each group is shown in Table 5. In all strains, p70S6K activity was greater when cultured in the presence of ascorbic acid than when cultured in the absence of ascorbic acid. The culture supernatants of Bifidobacterium breve FERM BP-11175, Bifidobacterium pseudolongum subsp. pseudolongum JCM1205T, and Limocylic Lactobacillus reuteri JCM1112T cultured in the presence of ascorbic acid were found to promote muscle protein synthesis.

[0096] [Table 5]

[0097] <Test Example 4> Examination of threonic acid production in probiotic bacteria (1) Probiotic culture and quantification of threonic acid The probiotics shown in Table 6 were cultured using the same procedure as in Test Example 2. The culture was centrifuged to collect the bacterial cells, which were then washed with ultrapure water and suspended to a concentration of 10 mg / mL (wet weight of bacterial cells / ultrapure water). The cells were then disrupted using a FastPrep-24 5G Homogenizer (MP Biomedicals). The resulting crude disrupted solution was centrifuged to recover the supernatant (hereinafter referred to as "disrupted bacterial cell supernatant"). The amount of threonic acid in the disrupted bacterial cell supernatant was then measured using the same procedure as in Test Example 2.

[0098] (2) Results The threonic acid content in the supernatant of each bacterial cell lysate is shown in Table 6 (ascorbic acid added) and Table 7 (ascorbic acid not added). All strains were confirmed to produce threonic acid (>5 ng / mL) upon addition of ascorbic acid. All strains except Bifidobacterium bifidum JCM1255 were confirmed to produce threonic acid at levels of 100 ng / mL or higher. Among all strains, Lactobacillus reuteri JCM1112T produced the highest amount of threonic acid upon addition of ascorbic acid. Although Bifidobacterium bifidum JCM1255 produced threonic acid, the production level was only 51.2 ng / mL. When ascorbic acid was not added, threonic acid production was observed in Bifidobacterium pseudolongum subsp. pseudolongum JCM1205T, but it was below the detection limit (<5 ng / mL) in all other strains. From the above results, it was revealed that the addition of ascorbic acid during the cultivation of the bacteria shown in Table 6 significantly increased the amount of threonic acid produced within the bacteria.

[0099] [Table 6]

[0100] [Table 7]

Claims

1. The present invention relates to a method for producing a lactobacillus-derived bacterium, ... Cultivating one or more types of bacteria in a medium containing ascorbic acids; A method for producing a threonic acid-containing composition, comprising the step of recovering a threonic acid-containing fraction from the culture after the culture.

2. The bacterium is Bifidobacterium breve. The manufacturing method according to claim 1.

3. The method according to claim 2, wherein the bacterium is Bifidobacterium breve FERM BP-11175.

4. threonic acid, A composition for muscle building containing one or more bacteria selected from Lactobacillus bacteria, Lacticaseibacillus bacteria, Lactiprantibacillus bacteria, Limocilactobacillus bacteria, Leviractobacillus bacteria, Rizilactobacillus bacteria, and Latilactobacillus bacteria, as well as one or more cultures of the above bacteria.

5. A composition containing one or more bacteria selected from the group consisting of Lactobacillus bacteria, Lacticaseibacillus bacteria, Lactiprantibacillus bacteria, Limocilactobacillus bacteria, Leviractobacillus bacteria, Rizilactobacillus bacteria, and Latilactobacillus bacteria, and one or more bacteria selected from cultures of said bacteria, A threonic acid-containing composition for muscle building, wherein the bacterium and / or the culture contains 1 μg / g or more of threonic acid per total dry weight thereof.

6. The composition according to claim 4 or 5, which is a food or drink.

7. The composition according to claim 4 or 5, which is a pharmaceutical product.

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