Anti-obesity agents, muscle strengthening agents, fatty liver disease treatment agents, and lipid metabolism improving agents

Abstract

To provide an anti-obesity agent and a muscle strength improving agent.SOLUTION: A lactic acid bacterium that belongs to Fructobacillus group, a culture of the lactic acid bacterium, a culture supernatant of the lactic acid bacterium, and / or an extract of the lactic acid bacterium are useful as an active ingredient of an anti-obesity agent and a muscle strength improving agent.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an anti-obesity agent, a muscle strength enhancer, a fatty liver ameliorant, and a lipid metabolism ameliorant.

Background Art

[0002] With the development of medical technology, the average life span of humans has been extended. When the average life span is extended, it is natural to hope to live a physically and mentally healthy life even in old age, that is, to extend the healthy life span. In order to extend the healthy life span, it is necessary to stop the aging phenomenon associated with aging. Such aging phenomena include obesity, decrease in muscle strength, decrease in lipid metabolism, and onset of diabetes. Therefore, research on prevention of obesity, improvement of muscle strength, promotion of lipid metabolism, prevention of diabetes, etc. is actively underway.

[0003] For example, Patent Document 1 discloses a muscle degradation inhibitor containing a lactic acid bacterial strain such as Lactobacillus curvatus or Lactobacillus amylovorus, a processed product of the lactic acid bacterial strain, or an extract thereof. Further, Patent Document 2 discloses a lactic acid bacterium belonging to Lactobacillus farciminis or Pediococcus acidilactici, a culture of the lactic acid bacterium, or a culture supernatant of the lactic acid bacterium, and is described as being used for improving mitochondrial function, improving metabolic function, improving decreased exercise ability, and suppressing decreased muscle strength.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

[0005] The present invention aims to provide an anti-obesity agent, a muscle-strengthening agent, a fatty liver disease treatment agent, and a lipid metabolism treatment agent. [Means for solving the problem]

[0006] The inventors have newly discovered that certain lactic acid bacteria and / or components derived from certain lactic acid bacteria have the effect of preventing obesity (anti-obesity effect), improving muscle strength (muscle strength improvement effect), improving fatty liver disease (especially fatty liver disease other than alcoholic fatty liver disease), and improving lipid metabolism, and have completed the present invention.

[0007] In other words, the present invention provides inventions in the following embodiments. [1] An anti-obesity agent comprising a lactic acid bacterium belonging to the genus Fructobacillus, a culture of the lactic acid bacterium, the culture supernatant of the lactic acid bacterium, and / or an extract of the lactic acid bacterium. [2] The anti-obesity agent according to [1], wherein the lactic acid bacterium is Fructobacillus fructosus. [3] The anti-obesity agent according to [1] or [2], wherein the lactic acid bacterium is Fructobacillus fructosus OS-1010 strain (accession number: NITE BP-03818). [4] An anti-obesity agent according to any one of [1] to [3], comprising nicotinamide adenine dinucleotide and nicotinamide mononucleotide, wherein the content of nicotinamide mononucleotide per 1 part by weight of nicotinamide adenine dinucleotide is 0.001 parts by weight or more and 10 parts by weight or less. Foods, cosmetics, or pharmaceuticals containing any one of the anti-obesity agents described in [5], [1], or [4]. [6] A muscle-strengthening agent comprising lactic acid bacteria belonging to the genus Fructobacillus, a culture of the lactic acid bacteria, the culture supernatant of the lactic acid bacteria, and / or an extract of the lactic acid bacteria. [7] The muscle-strengthening agent according to [6], wherein the lactic acid bacteria is Fructobacillus fructosus. [8] The muscle-strengthening agent according to [6] or [7], wherein the lactic acid bacteria is Fructobacillus fructosus OS-1010 strain (accession number: NITE BP-03818). [9] A muscle-strengthening agent according to any one of [6] to [8], comprising nicotinamide adenine dinucleotide and nicotinamide mononucleotide, wherein the content of nicotinamide mononucleotide per 1 part by weight of nicotinamide adenine dinucleotide is 0.001 parts by weight or more and 10 parts by weight or less.

[10] A muscle-strengthening agent according to any one of [6] to [9], which is for suppressing muscle mass loss, suppressing muscle weakness, increasing muscle mass, or increasing muscle strength. Food, beverages, cosmetics, or pharmaceuticals containing any one of the muscle-strengthening agents described in

[11] [6] to

[10] .

[12] A fatty liver disease treatment agent comprising lactic acid bacteria belonging to the genus Fructobacillus, a culture of the lactic acid bacteria, the culture supernatant of the lactic acid bacteria, and / or an extract of the lactic acid bacteria.

[13] The fatty liver treatment agent according to

[12] , wherein the lactic acid bacteria is Fructobacillus fructosus.

[14] The fatty liver disease treatment agent according to

[12] or

[13] , wherein the lactic acid bacterium is Fructobacillus fructosus OS-1010 strain (accession number: NITE BP-03818).

[15] A fatty liver disease improving agent according to any one of

[12] to

[14] , comprising nicotinamide adenine dinucleotide and nicotinamide mononucleotide, wherein the content of nicotinamide mononucleotide per 1 part by weight of nicotinamide adenine dinucleotide is 0.001 parts by weight or more and 10 parts by weight or less. Foods, cosmetics, or pharmaceuticals containing a fatty liver disease improving agent as described in any one of

[16] ,

[12] , or

[15] .

[17] A lipid metabolism improving agent comprising lactic acid bacteria belonging to the genus Fructobacillus, a culture of the lactic acid bacteria, the culture supernatant of the lactic acid bacteria, and / or an extract of the lactic acid bacteria.

[18] The lipid metabolism improving agent according to

[17] , wherein the lactic acid bacteria is Fructobacillus fructosus.

[19] The lipid metabolism improving agent according to

[17] or

[18] , wherein the lactic acid bacterium is Fructobacillus fructosus OS-1010 strain (accession number: NITE BP-03818).

[20] A lipid metabolism improving agent according to any one of

[17] to

[19] , comprising nicotinamide adenine dinucleotide and nicotinamide mononucleotide, wherein the content of nicotinamide mononucleotide per 1 part by weight of nicotinamide adenine dinucleotide is 0.001 parts by weight or more and 10 parts by weight or less. Foods, cosmetics, or pharmaceuticals containing a lipid metabolism improving agent as described in any one of

[21] ,

[17] , or

[19] .

[22] A lipid metabolism promoter or a preventive and / or therapeutic agent for diabetes, comprising lactic acid bacteria belonging to the genus Fructobacillus, a culture of the lactic acid bacteria, the culture supernatant of the lactic acid bacteria, and / or an extract of the lactic acid bacteria. [Effects of the Invention]

[0008] The present invention provides novel materials useful as active ingredients for anti-obesity agents, muscle strengthening agents, fatty liver disease improving agents, and lipid metabolism improving agents. Specifically, the present invention provides lactic acid bacteria belonging to the genus Fructobacillus, cultures of said lactic acid bacteria, culture supernatants of said lactic acid bacteria, and / or extracts of said lactic acid bacteria as active ingredients for anti-obesity agents, muscle strengthening agents, fatty liver disease improving agents, and lipid metabolism improving agents. [Brief explanation of the drawing]

[0009] [Figure 1] This graph shows the weight gain of mice in each group in the evaluation of the anti-obesity effect. [Figure 2] This graph shows the grip strength per unit of body weight of mice in each group, used to evaluate the effect of muscle strengthening. [Figure 3] This graph shows the liver fat content in abdominal CT images of mice from each group, used to evaluate the effectiveness of treatment in improving fatty liver disease. [Modes for carrying out the invention]

[0010] The anti-obesity agent, muscle-strengthening agent, fatty liver disease-improving agent, and lipid metabolism-improving agent of the present invention are all characterized by containing lactic acid bacteria belonging to the genus Fructobacillus, a culture of said lactic acid bacteria, the culture supernatant of said lactic acid bacteria, and / or an extract of said lactic acid bacteria as active ingredients. The anti-obesity agent, muscle-strengthening agent, fatty liver disease-improving agent, and lipid metabolism-improving agent will be described in detail below.

[0011] [1. Active Ingredients] The active ingredients of the present invention's anti-obesity agent, muscle-strengthening agent, fatty liver disease-improving agent, and lipid metabolism-improving agent are lactic acid bacteria belonging to the genus Fructobacillus, cultures of the lactic acid bacteria, culture supernatants of the lactic acid bacteria, and / or extracts of the lactic acid bacteria.

[0012] [1-1. Lactic acid bacteria belonging to the genus Fructobacillus] Lactic acid bacteria belonging to the genus Fructobacillus (hereinafter also referred to as "predetermined lactic acid bacteria") are microorganisms that produce nicotinamide mononucleotide (NMN) and / or nicotinamide adenine dinucleotide (NAD). Further, the lactic acid bacteria belonging to the genus Fructobacillus are preferably microorganisms that produce at least NAD, and more preferably microorganisms that produce both NMN and NAD.

[0013] Examples of lactic acid bacteria belonging to the genus Fructobacillus include Fructobacillus durionis, Fructobacillus tropaeoil, and Fructobacillus fructosus.

[0014] More specific examples of lactic acid bacteria belonging to the genus Fructobacillus include Fructobacillus durionis RD011727 strain, Fructobacillus durionis NBRC113239 strain, Fructobacillus tropaeoil RD012353 strain, Fructobacillus tropaeoil RD012354 strain, Fructobacillus fructosus NBRC3516 strain, and Fructobacillus fructosus OS-1010 strain.

[0015] The Fructobacillus durionis RD011727 strain was internationally deposited with the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) (Room 122, 2-5-8 Kazusa Kamashima, Kisarazu City, Chiba Prefecture, Japan) on October 28, 2020, under the deposit number NITE BP-02764.

[0016] Fructobacillus tropaeoil strain RD012353 was internationally deposited with the National Institute of Technology and Evaluation (NPMD) Patent Microbial Depository Center (NPMD) (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan) on October 28, 2020, under depositary number NITE BP-02765.

[0017] Fructobacillus tropaeoil strain RD012354 was internationally deposited with the Patent Microbial Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE) (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan) on October 28, 2020, under depositary number NITE BP-02766.

[0018] Fructobacillus fructosus strain OS-1010 was internationally deposited with the National Institute of Technology and Evaluation (NPMD) Patent Microbial Depository Center (NPMD) (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan) on January 5, 2024, under accession number NITE BP-03818.

[0019] These Fructobacillus lactic acid bacteria may be used individually or in combination of multiple species.

[0020] From the viewpoint of further enhancing the anti-obesity effect, Fructobacillus tropaeoil and Fructobacillus fructosus are preferred among the Fructobacillus lactic acid bacteria, with Fructobacillus fructosus being more preferred.

[0021] Among these Fructobacillus lactic acid bacteria, from the viewpoint of further enhancing the anti-obesity effect, Fructobacillus durionis strain RD011727, Fructobacillus durionis strain NBRC113239, Fructobacillus tropaeoil strain RD012353, Fructobacillus tropaeoil strain RD012354, Fructobacillus fructosus strain NBRC3516, and Fructobacillus fructosus strain OS-1010 are preferred, and more preferably Fructobacillus tropaeoil strain RD012353, Fructobacillus tropaeoil strain RD012354, Fructobacillus fructosus strain NBRC3516, and Fructobacillus fructosus Examples include strain OS-1010, more preferably strain Fructobacillus tropaeoil RD012353, strain Fructobacillus fructosus NBRC3516, and strain Fructobacillus fructosus OS-1010, with strain Fructobacillus fructosus OS-1010 being particularly preferred.

[0022] From the perspective of further enhancing the muscle-strengthening effect, Fructobacillus tropaeoil and Fructobacillus fructosus are preferred among the Fructobacillus lactic acid bacteria, with Fructobacillus fructosus being more preferred.

[0023] Among these Fructobacillus lactic acid bacteria, from the viewpoint of further effectively improving muscle strength, Fructobacillus durionis strain RD011727, Fructobacillus durionis strain NBRC113239, Fructobacillus tropaeoil strain RD012353, Fructobacillus tropaeoil strain RD012354, Fructobacillus fructosus NBRC3516, and Fructobacillus fructosus OS-1010 are preferred, and more preferably Fructobacillus tropaeoil strain RD012353, Fructobacillus tropaeoil strain RD012354, Fructobacillus fructosus NBRC3516, and Fructobacillus fructosus Examples include strain OS-1010, more preferably strain Fructobacillus tropaeoil RD012353, strain Fructobacillus fructosus NBRC3516, and strain Fructobacillus fructosus OS-1010, with strain Fructobacillus fructosus OS-1010 being particularly preferred.

[0024] From the viewpoint of further enhancing the effectiveness of improving fatty liver disease, Fructobacillus tropaeoil and Fructobacillus fructosus are preferred among the Fructobacillus lactic acid bacteria, with Fructobacillus fructosus being more preferred.

[0025] Among these Fructobacillus lactic acid bacteria, from the viewpoint of further improving the effect on improving fatty liver disease, Fructobacillus durionis strain RD011727, Fructobacillus durionis strain NBRC113239, Fructobacillus tropaeoil strain RD012353, Fructobacillus tropaeoil strain RD012354, Fructobacillus fructosus strain NBRC3516, and Fructobacillus fructosus strain OS-1010 are preferred, and more preferably Fructobacillus tropaeoil strain RD012353, Fructobacillus tropaeoil strain RD012354, Fructobacillus fructosus strain NBRC3516, and Fructobacillus fructosus Examples include strain OS-1010, more preferably strain Fructobacillus tropaeoil RD012353, strain Fructobacillus fructosus NBRC3516, and strain Fructobacillus fructosus OS-1010, with strain Fructobacillus fructosus OS-1010 being particularly preferred.

[0026] From the viewpoint of further enhancing the lipid metabolism-improving effect, Fructobacillus tropaeoil and Fructobacillus fructosus are preferred among the Fructobacillus lactic acid bacteria, with Fructobacillus fructosus being more preferred.

[0027] Among these Fructobacillus lactic acid bacteria, from the viewpoint of more effectively improving lipid metabolism, Fructobacillus durionis strain RD011727, Fructobacillus durionis strain NBRC113239, Fructobacillus tropaeoil strain RD012353, Fructobacillus tropaeoil strain RD012354, Fructobacillus fructosus strain NBRC3516, and Fructobacillus fructosus strain OS-1010 are preferred, and more preferably Fructobacillus tropaeoil strain RD012353, Fructobacillus tropaeoil strain RD012354, Fructobacillus fructosus strain NBRC3516, and Fructobacillus fructosus Examples include strain OS-1010, more preferably strain Fructobacillus tropaeoil RD012353, strain Fructobacillus fructosus NBRC3516, and strain Fructobacillus fructosus OS-1010, with strain Fructobacillus fructosus OS-1010 being particularly preferred.

[0028] [1-2. Form of the active ingredient] The active ingredient may take the form of at least one of the following: the specified lactic acid bacteria, the culture of the lactic acid bacteria, the culture supernatant of the lactic acid bacteria, or an extract of the lactic acid bacteria. The active ingredient may be used alone in any one of these forms, or in combination of multiple forms.

[0029] [1-2-1.Lactic acid bacteria] If the active ingredient is in the form of lactic acid bacteria, the active ingredient includes the cellular components of the lactic acid bacteria and the products that are or were originally present in the lactic acid bacteria. The lactic acid bacteria may be either live or dead. In the case of dead bacteria, the cellular components may be crushed. In the case of crushed bacteria, some of the cellular components may be removed. Furthermore, the lactic acid bacteria may be in the form of a cellular powder dried by means of freeze-drying, shelf-drying, spray-drying, etc. Among these, freeze-dried cellular powder is preferred from the viewpoint of further enhancing the anti-obesity effect, muscle-strengthening effect, fatty liver disease improvement effect, and lipid metabolism improvement effect.

[0030] Lactic acid bacteria can be obtained by a manufacturing method that includes the steps of culturing one or more of the above-mentioned specified lactic acid bacteria and separating and recovering the bacterial cells. The method used in the separation and recovery step includes solid-liquid separation and bacterial cell washing. The manufacturing method may include other steps in addition to the above-mentioned steps. Other steps include a dormant bacterial cell reaction step (which can be performed after the culturing step, preferably after the separation and recovery step), a drying step (which can be performed after the separation and recovery step or the dormant bacterial cell reaction step), and a bacterial cell crushing step (which can be performed after the separation and recovery step, the dormant bacterial cell reaction step or the drying step).

[0031] Culture media that can be used in the culture process include those used for expansion culture (pre-culture media) and those used for production culture (main culture media). The main culture media can be prepared by adding additives to the media used as the pre-culture media. The media is preferably a liquid medium, but it may also be an agar medium. In addition to a carbon source, the media generally contains a nitrogen source, minerals, etc.

[0032] Carbon sources include carbohydrates and carbohydrate materials. Carbohydrates include sugars (monosaccharides, disaccharides, oligosaccharides), polysaccharides, and sugar alcohols. Examples of carbohydrates include lactose, sucrose, glucose, starch, xylitol, and dextrose. Carbohydrate materials can be any organic composition containing carbohydrates, such as milk and its processed products (skim milk powder, whey, milk powder, condensed milk, etc.), soy milk and its processed products (soy milk hydrolysate, etc.), grains, fruits, and vegetables. Milk can be derived from any mammal such as cows, goats, sheep, buffalo, camels, llamas, donkeys, yaks, horses, and reindeer. Carbohydrates may be isolated or contained in carbohydrate materials. For example, fructose (carbohydrate) may be used in the form contained in fruit (carbohydrate material). These carbon sources may be used individually or in combination of multiple types. Among these carbon sources, glucose is preferred.

[0033] The concentration of the carbon source in the culture medium is not particularly limited and can be set appropriately depending on the type of medium and culture method, but for example, 0.5 to 4 w / w%, preferably 1 to 3 w / w%, and more preferably 1.5 to 2.5 w / w% are given.

[0034] Any inorganic or organic nitrogen source can be used as the nitrogen source. Examples include yeast extract (such as brewer's yeast), meat extract, proteins such as casein; protein hydrolysates such as peptone (such as protease peptone), peptides, and nitrogen-containing salts such as ammonium salts (such as ammonium citrate) and nitrates. These nitrogen sources may be used individually or in combination.

[0035] The concentration of the nitrogen source in the culture medium is not particularly limited and can be set appropriately according to the type of medium and culture method, but for proteins, for example, 0.3 to 4 w / w%, preferably 0.5 to 3 w / w%, and more preferably 1 to 2 w / w%; for peptides, for example, 0.1 to 2 w / w%, preferably 0.3 to 1.8 w / w%, and more preferably 0.5 to 1.5 w / w%; and for nitrogen-containing salts, for example, 0.03 to 1.5 w / w%, preferably 0.05 to 1 w / w%, and more preferably 0.1 to 0.5 w / w%.

[0036] Examples of minerals include manganese (e.g., manganese salts such as manganese sulfate), zinc, iron, sodium (e.g., sodium salts such as sodium acetate), potassium (e.g., potassium salts such as dipotassium bisulfate and dipotassium hydrogen phosphate), magnesium (e.g., magnesium salts such as magnesium sulfate), calcium, phosphorus (e.g., phosphates such as dipotassium hydrogen phosphate), sulfur (e.g., sulfates such as manganese sulfate, potassium bisulfate, and magnesium sulfate), and trace elements. These minerals may be used individually or in combination. Among these minerals, manganese, sodium, magnesium, and potassium are preferred.

[0037] The concentration of minerals in the culture medium is not particularly limited and can be set appropriately according to the type of culture medium and culture method, but examples include: for manganese salts, 0.001 to 0.01 w / w%, preferably 0.003 to 0.008 w / w%; for sodium salts, 0.05 to 1.5 w / w%, preferably 0.1 to 1 w / w%; for magnesium salts, 0.001 to 0.02 w / w%, preferably 0.005 to 0.015 w / w%; for potassium salts, 0.05 to 1 w / w%, preferably 0.1 to 0.5 w / w%; for phosphates, 0.05 to 1 w / w%, preferably 0.1 to 0.5 w / w%; and for sulfates, 0.001 to 0.04 w / w%, preferably 0.005 to 0.02 w / w%.

[0038] In addition to the components mentioned above, the culture medium may also contain other components such as vitamins (e.g., B vitamins), surfactants (e.g., nonionic surfactants (e.g., Tween), anionic surfactants (e.g., SDS), antibacterial agents (e.g., triclosan), and antibiotics (e.g., monesin). These other components may be used individually or in combination. Among these other components, surfactants are preferred, and nonionic surfactants are more preferred.

[0039] The concentrations of other components in the culture medium are not particularly limited and can be set appropriately depending on the type of other components, the type of culture medium, the culture method, etc. However, if a surfactant is included, the concentration of the surfactant can be, for example, 0.01 to 0.5 w / w%, preferably 0.05 to 0.3 w / w%.

[0040] The culture conditions are not particularly limited as long as they are conditions under which Fructobacillus lactic acid bacteria can grow.

[0041] The culture temperature can be any temperature that is optimal for the Fructobacillus lactic acid bacteria to be cultured, for example, 26-40°C, preferably 27-38°C, more preferably 28-36°C, and even more preferably 29-34°C. The culture time can be set appropriately according to the type of Fructobacillus lactic acid bacteria to be cultured, for example, 4-48 hours, preferably 8-36 hours, and more preferably 12-24 hours.

[0042] Regarding the operations during cultivation, it is not necessary to stir the culture medium during cultivation. Furthermore, as an example of a specific cultivation procedure, the above cultivation can be performed as a production culture (main culture) for a certain period of time, and before that, an expansion culture (pre-culture) can be performed in a small amount of medium (for example, 1 / 6 to 1 / 4 of the main culture medium by volume). The culture conditions for the pre-culture can be set appropriately according to the type of Fructobacillus lactic acid bacteria, and the above conditions can be adopted. In addition, in the main culture, the culture obtained in the pre-culture can be inoculated into the main culture medium so that the OD660 is, for example, 0.01 to 0.04, preferably 0.01 to 0.03.

[0043] In the separation and recovery process, the culture medium can be separated into solid and liquid components by methods such as filtration using filter paper, centrifugation, decantation, screw press, roller press, rotary drum screen, belt screen, vibrating screen, multi-plate vibrating filter, vacuum dehydration, pressure dehydration, belt press, centrifugal concentration dehydration, and multi-disc dehydration, and the obtained bacterial cells can be washed.

[0044] The resting cell process can increase the nicotinamide mononucleotide content ratio in lactic acid bacteria.

[0045] The liquid used in the resting cell reaction step can be any liquid that can enable lactic acid bacteria belonging to the genus Fructobacillus to undergo the resting cell reaction, without any particular restrictions. For example, water, buffer solutions, organic solvents, etc., can be used.

[0046] Examples of the pH of the liquid used in the resting bacterial cell reaction include 4.0 to 10.0, preferably 5.0 to 9.0, and more preferably 5.5 to 7.5.

[0047] When the liquid used in the resting bacterial cell reaction is a buffer solution, examples of buffer solutions include acetate buffer, phosphate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, and HEPES buffer.

[0048] More specific examples of buffers include KHC8H4O4-NaOH (pH 4.0), CH3COOH-CH3COONa (pH 4.0), MES-NaOH (pH 5.0), CH3COOH-CH3COONa (pH 5.0), KH2PO4-K2HPO4 (pH 6.0), MES-NaOH (pH 6.0), KH2PO4-K2HPO4 (pH 7.0), and PIPES-NaOH. Examples include (pH 7.0), HEPES-NaOH (pH 8.0), H3BO4-NaOH (pH 8.0), CHES-NaOH (pH 9.0), H3BO4-NaOH (pH 9.0), H2CO3-NaHCO3 (pH 10.0), CHES-NaOH (pH 10.0), and preferably CH3COOH-CH3COONa, KH2PO4-K2HPO4, H3BO4-NaOH, and more preferably KH2PO4-K2HPO4.

[0049] When the liquid used in the resting cell reaction is an organic solvent, examples of organic solvents include aromatic compounds such as benzene and benzonitrile, ketones such as acetone, acetylacetone, and methyl ethyl ketone, fatty acid esters such as ethyl acetate, butyl acetate, ethyl butyrate, and ethyl formate, ethers such as diethyl ether, dipropyl ether, dibutyl ether, tetrahydrofuran, and 1,4-dioxane, halogenated hydrocarbons such as dichloromethane, chloroform, and dichloroethane, and 1,2-propanediol, 1,2- Examples include diols such as butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 1,2-hexanediol, 1,6-hexanediol, 1,2-pentanediol, 1,5-pentanediol, 2-methyl-2,4-pentanediol, and 3-methyl-1,5-pentanediol; alcohols having linear or branched alkyl groups with 1 to 7 carbon atoms; and alcohols such as cyclohexanol, 3-methoxy-3-methyl-1-butanol, and 3-methoxy-1-butanol.

[0050] Among the liquids mentioned above, water and buffer solutions are preferred as the liquids used in the resting bacterial cell reaction.

[0051] Examples of reaction temperatures for the resting bacterial cell reaction include 21 to 37°C, preferably 24 to 28°C. Examples of reaction times include 0.1 to 24 hours, preferably 6 to 12 hours.

[0052] The resting cell reaction can be carried out by suspending lactic acid bacteria belonging to the genus Fructobacillus in the above liquid and allowing it to stand, stir, or shake.

[0053] Furthermore, the resting cell reaction may be carried out without using the above-mentioned liquid by adjusting the pH of a culture medium containing lactic acid bacteria belonging to the genus Fructobacillus to a range of 4.0 to 10.0, preferably 5.0 to 9.0, and more preferably 5.5 to 7.5.

[0054] In the drying process, the microbial cells can be dried using drying methods such as freeze-drying, shelf drying, and spray drying.

[0055] In the cell disruption step, the dried cells can be disrupted by any method. In the cell disruption step, some of the cell components may be removed from the disrupted cells.

[0056] [1-2-2. Lactic acid bacteria culture] If the active ingredient is in the form of a lactic acid bacteria culture, the active ingredient includes the lactic acid bacteria along with the culture medium components containing the products of the lactic acid bacteria. The lactic acid bacteria culture may be a culture prepared using one of the specified lactic acid bacteria alone, a culture prepared using a combination of multiple species, or a mixture of a culture prepared using one species alone and a culture prepared using another species alone.

[0057] The culture medium components in the culture may have some of the components used to cultivate the lactic acid bacteria removed. Furthermore, the lactic acid bacteria in the culture may be either live or dead. In the case of dead bacteria, the cells may be crushed. If the cells are crushed, some of the cell components may be removed.

[0058] A culture of lactic acid bacteria can be obtained by a manufacturing method that includes a step of culturing one or more of the specified lactic acid bacteria described above. This manufacturing method may include other steps in addition to the above steps. Other steps may include a step of removing a portion of the culture medium components, a step of subjecting the lactic acid bacteria to a resting cell reaction, and / or a drying step. Details of each step are as described in "1-2-1. Lactic Acid Bacteria" above.

[0059] [1-2-3. Culture supernatant of lactic acid bacteria] If the active ingredient is in the form of a culture supernatant of lactic acid bacteria, the active ingredient includes a culture medium component containing the products of the lactic acid bacteria. The culture supernatant of lactic acid bacteria may be a culture supernatant prepared using one of the specified lactic acid bacteria alone, a culture supernatant prepared using a combination of multiple species, or a mixture of a culture supernatant prepared using one species alone and a culture supernatant prepared using another species alone.

[0060] The culture medium components contained in the culture supernatant may have some of the culture medium components used to cultivate the lactic acid bacteria removed.

[0061] The culture supernatant of lactic acid bacteria can be obtained by a manufacturing method that includes the steps of culturing one or more of the specified lactic acid bacteria described above, and separating and recovering the culture supernatant. This manufacturing method may include other steps in addition to the steps described above. Other steps may include the steps of removing some of the culture medium components, subjecting the lactic acid bacteria to a resting cell reaction, and / or a drying step. Details of each step are as described in "1-2-1. Lactic Acid Bacteria" above.

[0062] [1-2-4. Lactic acid bacteria extract] When the active ingredient is in the form of a lactic acid bacteria extract, the active ingredient is a multi-component composition obtained by subjecting the lactic acid bacteria or lactic acid bacteria culture to an extraction process. Examples of extraction solvents used in preparing the extract include water; monohydric lower alcohols having 1 to 4 carbon atoms such as ethanol and isopropanol; polyhydric alcohols such as 1,3-butylene glycol, propylene glycol, and glycerin; and polar solvents such as mixtures thereof. The temperature conditions during extraction are preferably room temperature (for example, 5 to 35°C, preferably 20 to 30°C). Specific methods for preparing the extract include, for example, preparing a suspension containing the lactic acid bacteria or lactic acid bacteria culture or pulverized thereof in an extraction solvent, and obtaining an extract by solid-liquid separation of the suspension, or, if necessary, drying the extract to obtain a powder.

[0063] [1-3. Ingredients contained in the active ingredient] The active ingredients used in this invention—lactic acid bacteria belonging to the genus Fructobacillus, cultures of the lactic acid bacteria, culture supernatants of the lactic acid bacteria, and extracts of the lactic acid bacteria—all contain not only NMN and / or NAD produced by the lactic acid bacteria, but also other metabolites (other than NMN and NAD) and / or cellular components produced by the lactic acid bacteria. The excellent anti-obesity effect, muscle-strengthening effect, fatty liver disease-improving effect, and lipid metabolism-improving effect of the present invention are thought to be achieved by the combined effect of these components.

[0064] When the active ingredients used in the present invention include NAD and NMN, the NMN content is not particularly limited, but the NMN content (content ratio) per 1 part by weight of NAD is, for example, 0.001 parts by weight or more and 10 parts by weight or less. From the viewpoint of more effectively improving at least one selected from the group consisting of anti-obesity effect, muscle strength improvement effect, fatty liver disease improvement effect, and lipid metabolism improvement effect, the NMN content per 1 part by weight of NAD is preferably 0.005 parts by weight or more and 5.0 parts by weight or less, more preferably 0.005 parts by weight or more and 3.0 parts by weight or less, even more preferably 0.008 parts by weight or more and 2.0 parts by weight or less, particularly preferably 0.01 parts by weight or more and 1.5 parts by weight or less, and even more preferably 0.03 parts by weight or more and 1.0 part by weight or less.

[0065] Furthermore, the NMN content (content ratio) per mole of NAD is not particularly limited, but for example, it can be 0.001 moles or more and 10 moles or less, preferably 0.005 moles or more and 5 moles or less. In a more preferred form, the NMN content per mole of NAD can be preferably 0.01 moles or more and 3 moles or less, more preferably 0.02 moles or more and 2 moles or less, and even more preferably 0.03 moles or more and 1.5 moles or less.

[0066] [1-4. Content of active ingredients] The amount of active ingredients contained in each of the anti-obesity agent, muscle-strengthening agent, fatty liver disease-improving agent, and lipid metabolism-improving agent of the present invention is not particularly limited and can be appropriately determined according to the anti-obesity effect, muscle-strengthening effect, fatty liver disease-improving effect, and lipid metabolism-improving effect to be imparted.

[0067] The content of the active ingredients in each of the anti-obesity agent, muscle-strengthening agent, fatty liver disease-improving agent, and lipid metabolism-improving agent of the present invention is, for example, 0.00001 to 10% by weight, preferably 0.0001 to 1% by weight, based on the dry weight of Fructobacillus lactic acid bacteria. The above dry weight equivalent refers to the dry weight of the active ingredient if the active ingredient is lactic acid bacteria or a culture of lactic acid bacteria; if the active ingredient is a culture supernatant of lactic acid bacteria, it refers to the dry weight of the lactic acid bacteria used to obtain the culture supernatant; and if the active ingredient is an extract, it refers to the dry weight of the lactic acid bacteria used to obtain the extract.

[0068] [2. Other ingredients] The anti-obesity agent, muscle-strengthening agent, fatty liver disease-improving agent, and lipid metabolism-improving agent of the present invention may contain, or may not contain, other pharmacological components in addition to the above-mentioned active ingredients, as needed. Examples of such pharmacological components include anti-inflammatory agents, antioxidants, bactericides, cooling agents, vitamins, mucopolysaccharides, and the like.

[0069] Furthermore, the anti-obesity agent, muscle-strengthening agent, fatty liver disease-improving agent, and lipid metabolism-improving agent of the present invention may or may not contain bases and / or additives as necessary to achieve the desired formulation. Such bases and / or additives are not particularly limited to the extent that they are pharmaceutically acceptable, but examples include aqueous bases such as water and monohydric lower alcohols having 1 to 4 carbon atoms (ethanol, isopropanol, etc.); oily bases such as naturally derived oils (vegetable oils, animal oils, processed oils thereof), mineral oils, ester oils, fatty acid alkyl esters, fatty acids, fatty acid esters, and higher alcohols; surfactants; polyhydric alcohols (glycerin, propylene glycol, dipropylene glycol, 1,3-butylene glycol, etc.); and additives such as cooling agents, preservatives, flavoring agents, coloring agents, viscosity adjusters, pH adjusters, wetting agents, stabilizers, antioxidants, UV absorbers, chelating agents, adhesives, buffering agents, solubilizers, and preservatives.

[0070] [3. Formulation and Product Classification] The formulation form of the anti-obesity agent, muscle-strengthening agent, fatty liver disease-improving agent, and lipid metabolism-improving agent of the present invention is not particularly limited and may be liquid, semi-solid (cream, gel, ointment, paste), solid (granules, fine granules, powder, tablet, capsule), etc. Furthermore, the anti-obesity agent, muscle-strengthening agent, fatty liver disease-improving agent, and lipid metabolism-improving agent of the present invention may be a non-emulsified formulation such as an aqueous formulation or an oily formulation, or an emulsified formulation such as an oil-in-water emulsion or a water-in-oil emulsion.

[0071] Furthermore, product classifications for the anti-obesity agent, muscle-strengthening agent, fatty liver disease-improving agent, and lipid metabolism-improving agent of the present invention include foods and beverages, cosmetics, and pharmaceuticals.

[0072] While not particularly limited to food and beverages, examples include fermented milk (such as drinkable yogurt), lactic acid bacteria beverages, milk beverages (such as coffee milk and fruit milk), tea-based beverages (such as green tea, black tea, and oolong tea), fruit and vegetable-based beverages (beverages containing fruit juices such as orange, apple, and grape, and vegetable juices such as tomato and carrot), alcoholic beverages (such as beer, sparkling wine, and wine), carbonated beverages, soft drinks, and water-based beverages; and processed foods such as fermented milk (such as set-type yogurt and soft yogurt), confectionery, instant foods, and seasonings.

[0073] Furthermore, functional foods can also be cited as food and beverages. Functional foods refer to foods that have a certain function on the living body, and examples include health functional foods such as Foods for Specified Health Uses (including conditionally designated FOSHU [Foods for Specified Health Uses]) and nutrient function foods, foods with functional claims, foods for special dietary uses, nutritional supplements, health supplements, supplements (for example, in various dosage forms such as tablets, coated tablets, sugar-coated tablets, capsules and liquids), and beauty foods (for example, diet foods). Moreover, functional foods may also be foods for special dietary uses such as foods for the sick, powdered milk for pregnant and lactating women, infant formula, foods for the elderly, and foods for nursing care.

[0074] Cosmetics include topical preparations, including those for the skin and mucous membranes. More specifically, these include basic cosmetics such as lotions, emulsions, creams, essences, gels, packs, sheet masks, and lip balms; skin cleansing products such as facial cleansers, makeup removers (including cleansing agents), exfoliants, and body shampoos; body care cosmetics such as sunscreens, body gels, body massagers, antiperspirants, deodorants, hair removal agents, and bath additives; makeup cosmetics such as foundations, face powders, lipsticks, blushes, eyeshadows, eyeliners, mascaras, and eyebrow pencils; nail cosmetics such as manicures and nail removers; hair cosmetics such as hair styling products, shampoos, conditioners, rinses, and hair growth products; and oral cosmetics such as liquid toothpaste, toothpaste, mouthwash, and mouth sprays.

[0075] Pharmaceuticals include oral preparations, as well as topical preparations (including quasi-drugs) that include skin preparations and mucosal preparations. Dosage forms of oral preparations include tablets, coated tablets, sugar-coated tablets, capsules, and liquids, while dosage forms of topical preparations include liquids (including lotions, sprays, aerosols, and emulsions), foams, ointments, creams, gels, and patches.

[0076] Among these product categories, food and beverages are preferred from the viewpoint of further enhancing anti-obesity effects, muscle-strengthening effects, fatty liver disease improvement effects, and lipid metabolism improvement effects.

[0077] [4.Application] The anti-obesity agent, muscle-strengthening agent, fatty liver disease-improving agent, and lipid metabolism-improving agent of the present invention are used for applications aimed at anti-obesity effects, muscle-strengthening effects, fatty liver disease-improving effects, and lipid metabolism-improving effects, respectively. Applications of the muscle-strengthening agent include suppressing muscle mass loss, suppressing muscle weakness, increasing muscle mass, or increasing muscle strength.

[0078] The fatty liver disease improving agent and lipid metabolism improving agent of the present invention can also be used for purposes aimed at promoting lipid metabolism, or for purposes aimed at preventing and / or treating diabetes. That is, it can be used as a lipid metabolism promoting agent or a preventive and / or therapeutic agent for diabetes (referred to as "diabetes preventive agent, etc.").

[0079] The above-mentioned lipid metabolism promoter is characterized by containing lactic acid bacteria belonging to the genus Fructobacillus, a culture of the lactic acid bacteria, the culture supernatant of the lactic acid bacteria, and / or an extract of the lactic acid bacteria. The lactic acid bacteria are preferably Fructobacillus fructosus, and more preferably Fructobacillus fructosus strain OS-1010 (accession number: NITE BP-03818). The above-mentioned lipid metabolism promoter can be described using the descriptions of [1. Active ingredient], [2. Other ingredients], [3. Formulation and product classification], and [5. Dosage], similar to the anti-obesity agent, muscle strengthening agent, fatty liver disease improving agent, and lipid metabolism improving agent of the present invention.

[0080] The above-mentioned diabetes preventive agent, etc., is characterized by containing lactic acid bacteria belonging to the genus Fructobacillus, a culture of the lactic acid bacteria, the culture supernatant of the lactic acid bacteria, and / or an extract of the lactic acid bacteria. The lactic acid bacteria are preferably Fructobacillus fructosus, and more preferably Fructobacillus fructosus strain OS-1010 (accession number: NITE BP-03818). The above-mentioned diabetes preventive agent, etc., can be described using the same descriptions as the anti-obesity agent and muscle-strengthening agent of the present invention, including [1. Active ingredient], [2. Other ingredients], [3. Formulation and product classification], and [5. Dosage].

[0081] [5.Dose] When the anti-obesity agent, muscle-strengthening agent, fatty liver disease-improving agent, and lipid metabolism-improving agent of the present invention are administered orally, the amount of the active ingredient is, for example, 0.01 to 200 mg / kg / day, preferably 0.1 to 20 mg / kg / day, and can be administered orally 1 to 5 times a day. [Examples]

[0082] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0083] [Preparation of sample (lactic acid bacteria powder)] Fructobacillus fructosus OS-1010 strain was inoculated into 30 ml of Difco MRS medium (pre-culture medium) and cultured statically at 30°C for 24 hours. The resulting culture was inoculated into 1 L of MRS medium (main culture medium) to achieve an OD660 of 0.02 and cultured with stirring at 30°C for 12 hours at pH 6.0-7.0. The resulting culture was centrifuged to collect the cells. The collected cells were washed with 1 L of 0.85 w / w% KCl aqueous solution. The washed cells were centrifuged again to collect the cells. The collected cells were suspended in 50 ml of distilled water, sterilized at 65°C for 30 minutes, and then freeze-dried to obtain a sample (lactic acid bacteria powder).

[0084] Using the HPLC analysis conditions described later, the production amounts of nicotinamide mononucleotide (NMN) and nicotinamide adenine dinucleotide (NAD) in the recovered lactic acid bacteria powder were measured. The ratio of NMN to 1 part by weight of NAD was 0.03 parts by weight (0.06 moles of NMN per mole of NAD).

[0085] (Composition of MRS medium) 2w / w% glucose 1 w / w% protease peptone 1 w / w% beef extract 0.5 w / w% yeast extract 0.2 w / w% ammonium citrate 0.1w / w% Tween80 0.5 w / w% sodium acetate 0.01 w / w% magnesium sulfate 0.005 w / w% manganese sulfate 0.2 w / w% dipotassium hydrogen phosphate

[0086] (HPLC analysis conditions) • NMN measurement method Columns: DaisoPak SP-100-5-ODS-P (4.6 x 150 mm) x 2 Column temperature: 25℃ Eluent: 75 mM ammonium phosphate aqueous solution (pH 6.0) Flow rate: 0.6ml / min Detector: UV detector (260nm) Detection time: 14.5 minutes • Method for measuring NAD Columns: DAISOPAK SP-100-5-ODS-P (4.6 x 250 mm) x 2 Column temperature: 25℃ Eluent: 75 mM ammonium phosphate aqueous solution (pH 6.0): methanol = 95:5 (w:w) Flow rate: 0.7mL / min Detector: UV detector (260nm) Detection time: 33 minutes

[0087] (Method for quantifying NMN and NAD) A calibration curve was created using purchased NMN and NAD as standard samples, and the production amounts of NMN and NAD in the lactic acid bacteria powder were quantified from the peak area ratio of their respective HLPCs.

[0088] [Preparing the evaluation mice] Thirty-six five-week-old male C57BL / 6J mice (Nippon Crea Co., Ltd.) were purchased and pre-fed for three weeks on commercially available solid feed CRF-1 (Oriental Yeast Co., Ltd.). After that, they were divided into four groups of nine mice each to ensure a uniform average weight. The mice in the four groups described above were given free access to (1) D12450J (Research Diet Co., Ltd.) as a "standard diet," (2) D12492 (Research Diet Co., Ltd.) as a "high-fat diet," (3) D12492 formulated with the above sample (lactic acid bacteria powder) at 0.05% by weight as a "high-fat diet + 0.05% lactic acid bacteria," and (4) D12492 formulated with the above sample (lactic acid bacteria powder) at 2.0% by weight as a "high-fat diet + 2.0% lactic acid bacteria." The experimental animals were raised in an environment with a rearing temperature of 22±2℃ and a 12-hour light-dark cycle (light period from 7 to 19:00).

[0089] [Evaluation of anti-obesity effects] The body weight of each mouse was measured 91 days after the start of ad libitum feeding, and the weight gain was calculated using the following formula (X). The weight gain for each group is shown in Figure 1. The results shown in Figure 1 are expressed as the mean ± standard error for each group. Weight gain = Weight on day 91 of free intake - Weight before starting free intake ... Formula (X)

[0090] Statistical analysis revealed that the group fed a high-fat diet showed significantly greater weight gain compared to the group fed a normal diet. Furthermore, compared to the high-fat diet group, the high-fat diet + 0.05% lactic acid bacteria group and the high-fat diet + 2.0% lactic acid bacteria group showed significantly suppressed weight gain, confirming the anti-obesity effect of lactic acid bacteria intake (Figure 1).

[0091] [Evaluation of the effect on muscle strength improvement] On the 84th day after the start of free-feeding, the grip strength of each mouse was measured using the following method. (Measurement of grip strength) Using a rat / mouse grip strength meter GPM-101B / V (Melquest Co., Ltd.), the grip strength was measured five times per mouse by pulling its tail horizontally. The maximum value of the five measurements was divided by the mouse's body weight to calculate the grip strength per unit of body weight. The grip strength per unit of body weight for each group is shown in Figure 2. The results shown in Figure 2 are expressed as the mean ± standard error for each group.

[0092] Statistical analysis revealed that grip strength was significantly reduced in the group fed a high-fat diet compared to the group fed a normal diet. Furthermore, compared to the high-fat diet group, the high-fat diet + 0.05% lactic acid bacteria group and the high-fat diet + 2.0% lactic acid bacteria group showed significantly suppressed grip strength reduction, confirming the muscle-strengthening effect, or in other words, the muscle-strength-suppressing effect, of lactic acid bacteria intake (Figure 2).

[0093] [Evaluation of anti-steatohepatitis effect] On day 85 after the start of ad libitum feeding, each mouse was anesthetized, and abdominal CT images were taken using an X-ray CT scanner for experimental animals (Hitachi Aloka Medical Latheta LCT-200SE). Liver fat content was measured using the accompanying analysis software (Figure 3). The results shown in Figure 3 are the mean ± standard error for each group. Compared to the group fed normal diet, the group fed high-fat diet showed a significantly increased liver fat content. Furthermore, compared to the high-fat diet group, the high-fat diet + 0.05% lactic acid bacteria group and the high-fat diet + 2.0% lactic acid bacteria group showed a decrease in liver fat content, confirming the inhibitory effect of lactic acid bacteria intake on fat accumulation in the liver (Figure 3).

[0094] Furthermore, after measuring the body weight of each mouse 91 days after the start of ad libitum intake, serum was collected from each mouse and liver enzyme levels (ALT) were measured (Table 1). The results shown in Table 1 are expressed as the mean ± standard error for each group. Compared to the group fed a normal diet, the group fed a high-fat diet showed a significant increase in ALT levels. Furthermore, compared to the high-fat diet group, the groups fed a high-fat diet with 0.05% lactic acid bacteria and the high-fat diet with 2.0% lactic acid bacteria showed a decrease in ALT levels, confirming the anti-steatohepatitis effect of lactic acid bacteria intake (Table 1).

[0095] [Table 1]

[0096] [Confirmation of the effect on improving lipid metabolism] On day 91 after the start of ad libitum intake, the body weight of each mouse was measured, and serum was collected from each mouse to measure total cholesterol, HDL cholesterol, and LDL cholesterol (Table 2). The arteriosclerosis index was calculated using the following formula. Arteriosclerosis index = (Total cholesterol - HDL cholesterol) / HDL cholesterol The results shown in Table 2 are expressed as the mean ± standard error for each group.

[0097] [Table 2]

[0098] As shown in Table 2, LDL cholesterol and arteriosclerosis index increased in the group that consumed a high-fat diet compared to the group that consumed a normal diet. Furthermore, compared to the high-fat diet group, LDL cholesterol and arteriosclerosis index decreased in the high-fat diet + 0.05% lactic acid bacteria group and the high-fat diet + 2.0% lactic acid bacteria group, confirming the effect of lactic acid bacteria intake on improving lipid metabolism.

Claims

1. An anti-obesity agent containing the lactic acid bacterium Fructobacillus fructosus OS-1010 strain (accession number: NITE BP-03818).

2. The anti-obesity agent according to claim 1, comprising nicotinamide adenine dinucleotide and nicotinamide mononucleotide, wherein the content of nicotinamide mononucleotide per 1 part by weight of nicotinamide adenine dinucleotide is 0.001 parts by weight or more and 10 parts by weight or less.

3. A muscle-strengthening agent containing the lactic acid bacterium Fructobacillus fructosus OS-1010 strain (accession number: NITE BP-03818).

4. The muscle-strengthening agent according to claim 3, comprising nicotinamide adenine dinucleotide and nicotinamide mononucleotide, wherein the content of nicotinamide mononucleotide per 1 part by weight of nicotinamide adenine dinucleotide is 0.001 parts by weight or more and 10 parts by weight or less.

5. A muscle-strengthening agent according to claim 3, which is for suppressing muscle mass loss, suppressing muscle weakness, increasing muscle mass, or increasing muscle strength.

6. A liver enzyme (ALT) lowering agent containing the lactic acid bacterium Fructobacillus fructosus strain OS-1010 (accession number: NITE BP-03818).

7. A liver enzyme (ALT) lowering agent according to claim 6, comprising nicotinamide adenine dinucleotide and nicotinamide mononucleotide, wherein the content of nicotinamide mononucleotide per 1 part by weight of nicotinamide adenine dinucleotide is 0.001 parts by weight or more and 10 parts by weight or less.

8. A lipid metabolism improving agent containing the lactic acid bacterium Fructobacillus fructosus OS-1010 strain (accession number: NITE BP-03818).

9. A lipid metabolism improving agent according to claim 8, comprising nicotinamide adenine dinucleotide and nicotinamide mononucleotide, wherein the content of nicotinamide mononucleotide per 1 part by weight of nicotinamide adenine dinucleotide is 0.001 parts by weight or more and 10 parts by weight or less.

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