Mitochondrial-function-improving agent

Abstract

The present invention addresses the problem of providing a novel mitochondrial-function-improving agent. The present invention provides a mitochondrial-function-improving agent containing lactic acid bacteria that have the ability to inhibit the production of active oxygen species in mitochondria.

Description

Mitochondrial function improver

[0001] The present invention relates to an agent for improving mitochondrial function.

[0002] Mitochondria are essential organelles for energy production through cellular respiration. During cellular respiration, oxygen acts as an electron acceptor, generating reactive oxygen species (ROS). While the amount of ROS produced in mitochondria is typically controlled by the function of the mitochondria themselves, it is known that ROS production increases with age-related cellular senescence and mitochondrial dysfunction due to inflammatory stress (Non-Patent Document 1). Increased mitochondrial ROS production has been reported to be associated with the onset and worsening of diseases such as neurodegenerative diseases, diabetes, and cancer (Non-Patent Documents 2-4). In particular, in Japan, where rising social security costs for the elderly are a growing concern, the development of preventive and therapeutic methods for these diseases is highly desirable.

[0003] International Publication No. 2021 / 241728

[0004] Biomedicines 9, 216 (2021).Cell 166, 555-566 (2016).The World Journal of Men's Health 40, 399 (2022).International Journal of Molecular Sciences 22, 6479 (2021).

[0005] The problem to be solved by the present invention is to provide a novel agent for improving mitochondrial function.

[0006] Under these circumstances, the present inventors have discovered that lactic acid bacteria capable of suppressing ROS production improve mitochondrial function. The present invention is based on these novel findings.

[0007] Therefore, the present invention provides the following items: Item 1. An agent for improving mitochondrial function, a method, a use, a lactic acid bacterium or a composition for use according to any of the following items 1-1 to 1-5: Item 1-1. An agent for improving mitochondrial function comprising lactic acid bacteria capable of suppressing the production of reactive oxygen species in mitochondria. Item 1-2. A method for improving mitochondrial function in a subject in need thereof, comprising administering to the subject an effective amount of lactic acid bacteria capable of suppressing the production of reactive oxygen species in mitochondria. Item 1-3. Use of lactic acid bacteria capable of suppressing the production of reactive oxygen species in mitochondria, for producing an agent for improving mitochondrial function. Item 1-4. Lactic acid bacteria capable of suppressing the production of reactive oxygen species in mitochondria, for use in improving mitochondrial function. Item 1-5. A composition for improving mitochondrial function, comprising lactic acid bacteria capable of suppressing the production of reactive oxygen species in mitochondria.

[0008] Item 2. The agent, method, use, lactic acid bacterium or composition for use for improving mitochondrial function according to Item 1, wherein the lactic acid bacterium suppresses the production of reactive oxygen species in mitochondria by 70% to 90%.

[0009] Item 3. The agent, method, use, lactic acid bacterium or composition for use for improving mitochondrial function according to Item 1 or 2, wherein the lactic acid bacterium comprises Ligilactobacillus salivarius.

[0010] Item 4. The agent, method, use, lactic acid bacterium or composition for use for improving mitochondrial function according to any one of Items 1 to 3, wherein the lactic acid bacterium comprises one or both of Ligilactobacillus salivarius having accession number NITE BP-03218 and Ligilactobacillus salivarius having accession number NITE ABP-03958.

[0011] Item 5. The agent, method, use, lactic acid bacterium or composition for use for improving mitochondrial function according to any one of Items 1 to 4, wherein the lactic acid bacterium has the ability to suppress the production of reactive oxygen species in mitochondria in intestinal epithelial cells or intestinal macrophages.

[0012] Item 6. The agent, method, use, lactic acid bacterium or composition for use for improving mitochondrial function according to any one of Items 1 to 5, wherein the improvement in mitochondrial function includes at least one improvement selected from the group consisting of an improvement in ATP production and an improvement in mitochondrial oxygen consumption rate.

[0013] Item 7. The agent for improving mitochondrial function, the method, the use, the lactic acid bacterium for use, or the composition for improving mitochondrial function according to any one of Items 1 to 6, wherein the agent for improving mitochondrial function is a food or drink.

[0014] Item 8. The agent for improving mitochondrial function, the method, the use, the lactic acid bacterium for use, or the composition for improving mitochondrial function according to any one of Items 1 to 7, wherein the agent for improving mitochondrial function is a pharmaceutical or quasi-drug.

[0015] According to the present invention, a novel agent for improving mitochondrial function can be provided. Specifically, mitochondrial function is improved by suppressing the production of reactive oxygen species in mitochondria. Previous research by the inventors has proposed a method for preventing and treating viral diseases using an antiviral agent containing lactic acid bacteria (Patent Document 1). However, there have been no reports to date on whether lactic acid bacteria with the ability to suppress the production of reactive oxygen species can improve mitochondrial function compared to lactic acid bacteria that do not. Many factors are known to be involved in mitochondrial function, and these factors are involved in a complex manner. Therefore, the effect of the present invention, which is to improve mitochondrial function, was unexpected.

[0016] In Example 1 (2), (a) the results of verifying the effect of the addition of MitoSOX on cultured cells (cell detachment), (b) the results of verifying the effect of the addition of MitoROS 580 on cultured cells (cell detachment), and (c) the results of detecting and quantifying ROS induced by Antimycin A using MitoROS 580 are shown. In Example 1 (3), (a) the results of examining ROS-inducing pro-inflammatory substances used in a model of mitochondrial dysfunction, (b), (c), and (d) the results of measuring ROS production 24 hours, 3 hours, and 30 minutes after adding candidate substances to cultured cells are shown. In Example 1 (5), the results of verifying the effect of adding killed lactic acid bacteria cells on the ability to suppress the production of reactive oxygen species in mitochondria are shown. In Example 2 (3), the results of verifying the effect of adding FFIG35 on oxygen consumption rate are shown. In Example 2 (4), the results of verifying the effect of adding FFIG35 on ATP production are shown. 1 shows the results of verifying the effect of addition of FFIG35 on ROS production in mitochondria of porcine intestinal macrophages in Example 3.

[0017] Mitochondrial function improver The present invention provides a mitochondrial function improver comprising lactic acid bacteria having the ability to suppress the production of reactive oxygen species in mitochondria.

[0018] Ability to Inhibit the Production of Reactive Oxygen Species in Mitochondria Reactive oxygen species refer to oxygen species that are more reactive and active than the commonly occurring ground-state triplet oxygen. In the present invention, the term "reactive oxygen species" includes superoxide, hydrogen peroxide, hydroxyl radical, and singlet oxygen. In a typical embodiment of the present invention, lactic acid bacteria capable of inhibiting the production of reactive oxygen species refer to lactic acid bacteria capable of inhibiting the production of at least one of the above-mentioned reactive oxygen species. Therefore, the amount of reactive oxygen species produced can be measured by detecting at least one species selected from the group consisting of superoxide, hydrogen peroxide, hydroxyl radical, and singlet oxygen, and preferably by detecting superoxide.

[0019] Most reactive oxygen species in living cells are localized in mitochondria or present in the cytosol. In the present invention, reactive oxygen species in mitochondria refer to reactive oxygen species localized in mitochondria.

[0020] Inhibition of reactive oxygen species production refers to a tendency to reduce the amount of reactive oxygen species produced. For example, when comparing 1) cultured cells to which an inflammation-inducing substance has been added to increase the amount of reactive oxygen species produced in mitochondria with 2) cultured cells to which lactic acid bacteria, which are the active ingredient of the present invention, have been added, followed by an inflammation-inducing substance, to increase the amount of reactive oxygen species produced in mitochondria, if the amount of reactive oxygen species produced in the latter 2) is lower, then the lactic acid bacteria can be said to have the ability to inhibit the production of reactive oxygen species.

[0021] In the present invention, the mitochondria targeted for the inhibition of reactive oxygen species production are not particularly limited, and can be, for example, mitochondria in cultured cells (including cells in tissue culture) and in biological tissues. In an embodiment of cultured cells, preferably, cells derived from the intestine can be used, and more preferably, intestinal epithelial cells or intestinal macrophages can be used. In an embodiment of biological tissue, the target tissue is preferably the intestine, and more preferably, intestinal epithelium or intestinal macrophages. The organism from which the cells or tissues are derived is not particularly limited, but is preferably mammalian. Examples of the mammals include humans, pigs, monkeys, mice, rats, rabbits, cats, dogs, cows, horses, sheep, etc., and preferably humans or pigs.

[0022] Lactic Acid Bacteria In this specification, lactic acid bacteria refer to bacteria that produce lactic acid through metabolism, that are Gram-positive bacteria, and that have a rod-shaped or spherical shape.

[0023] In a typical embodiment, the present invention uses lactic acid bacteria capable of inhibiting the production of reactive oxygen species in mitochondria. The percentage of inhibition of reactive oxygen species production is not particularly limited, but in one embodiment of the present invention, the percentage of inhibition of reactive oxygen species production in mitochondria is preferably 70% to 90%, more preferably 80% to 90%, and even more preferably 80% to 85%. The percentage of inhibition of reactive oxygen species production can be calculated as the arithmetic mean of the ratios calculated from two or more measured values, and it is preferable that there is a significant difference in the Dunnett test. That is, the p-value in the Dunnett test is preferably p<0.05, more preferably p<0.01, and even more preferably p<0.001.

[0024] The method for measuring the amount of reactive oxygen species produced in mitochondria is not particularly limited, and can be performed using a reagent and measurement device known to be capable of measuring the amount of reactive oxygen species produced in mitochondria. Such known measurement methods can also be modified as appropriate. From the perspective of throughput, measurement of the amount of reactive oxygen species produced in mitochondria is preferably performed using a fluorescent detection reagent. In measurement methods using a fluorescent detection reagent, for example, the amount of reactive oxygen species produced can be measured by detecting the fluorescence intensity exhibited by the reaction of a reagent added to a sample with the reactive oxygen species in mitochondria using a fluorescent detection device such as a plate reader or flow cytometer. Examples of the fluorescent detection reagent include MitoROS 580 (AAT Bioquest) and MitoSOX (Invitrogen), with MitoROS 580 being preferred. MitoROS 580 contains a superoxide-sensitive fluorescent dye that immediately translocates to mitochondria upon entry into living cells, and therefore can be used as a reagent for measuring the amount of reactive oxygen species produced in mitochondria.

[0025] More specifically, the lactic acid bacteria include, for example, the genus Ligilactobacillus, the genus Lactiplantibacillus, the genus Lactobacillus, the genus Amylolactobacillus, the genus Holzapfelia, the genus Bombilactobacillus, the genus Companilactobacillus, and the genus Lapis. genus dilactobacillus, genus Agrilactobacillus, genus Schleiferilactobacillus, genus Lacticaseibacillus, genus Paralactobacillus, genus Latilactobacillus, genus Loigolactobacillus, genus Dellaglioa, Liq Examples of the bacteria include bacteria of the genera Ligilactobacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Secundilactobacillus, Levilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus and Lentilactobacillus, and preferably bacteria of the genus Ligilactobacillus and Lactiplantibacillus, and more preferably bacteria of the genus Ligilactobacillus.

[0026] The bacteria of the genus Ligilactobacillus can preferably include Ligilactobacillus salivarius.

[0027] In one preferred embodiment, the lactic acid bacteria may include one or both of Ligilactobacillus salivarius (FFIG35) having the accession number NITE BP-03218 and Ligilactobacillus salivarius (FFIG56) having the accession number NITE ABP-03958, and more preferably Ligilactobacillus salivarius (FFIG35) having the accession number NITE BP-03218. These strains were deposited under the Budapest Treaty at the Patent Microorganisms Depositary Center, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818) on May 19, 2020 and August 4, 2023, respectively.

[0028] Lactic acid bacteria of the genus Lactiplantibacillus may preferably include Lactiplantibacillus plantarum, and more preferably include one or both of the CS91 strain and the CS96 strain. The CS91 strain and the CS96 strain are stored in the laboratory of the present inventors, and the present inventors are prepared to provide the CS91 strain and the CS96 strain to those skilled in the art upon request.

[0029] The lactic acid bacteria of the present invention can be cultured using known methods.

[0030] When culturing lactic acid bacteria using a medium, known media for culturing lactic acid bacteria can be used, or such known media can be modified as appropriate. Examples of media include, but are not limited to, MRS medium (de Man, Rogosa, Sharpe, Becton Dickinson and Company). The medium may also be a culture solution. Amino acids or salts thereof, or combinations thereof, may be added to the medium as appropriate. Food ingredients or foods may be used instead of the medium.

[0031] The culture temperature is, for example, 15°C to 60°C, preferably 25°C to 50°C, and more preferably 35°C to 40°C, for good bacterial growth. The pH of the medium or culture solution is, for example, 4.8 to 6.8, and preferably 6.0 to 6.5. The culture time is, for example, 10 hours to 3 days, and preferably 16 hours to 24 hours. Lactic acid bacteria can be cultured under either anaerobic or aerobic conditions. The above culture may be carried out on a laboratory scale or on an industrial scale.

[0032] Improving mitochondrial function Mitochondria perform cellular respiration using oxygen as an electron acceptor and produce adenosine triphosphate (ATP) through coupling with the electron transport chain. Therefore, mitochondrial function can be evaluated by measuring the activity of the electron transport chain. In other words, when mitochondrial function decreases, the oxygen consumption rate (OCR) decreases. Conversely, when mitochondrial function is improved, the oxygen consumption rate recovers.

[0033] In the present invention, the method for measuring the oxygen consumption rate is not particularly limited, but examples include methods using an extracellular flux analyzer, a plate reader capable of detecting fluorescence, etc., and methods using an extracellular flux analyzer are preferred. An example of an extracellular flux analyzer is the Agilent Seahorse XF24 Analyzer (Seahorse Bioscience).

[0034] The Agilent Seahorse XF24 analyzer can be used to evaluate basal respiration and maximal respiration in terms of oxygen consumption rate. More specifically, oxygen consumption rate can be measured and evaluated, for example, by the method described in the Examples of the present application. The basal respiration oxygen consumption rate is preferably 105% or higher, more preferably 110% or higher, and even more preferably 120% or higher, compared to a control not containing the mitochondrial function improver of the present invention. While there is no upper limit to the oxygen consumption rate, examples include mitochondrial oxygen consumption rates of 140% or lower, 130% or lower, or 125% or lower, compared to a control not containing the mitochondrial function improver of the present invention. The maximal respiration oxygen consumption rate is preferably 105% or higher, more preferably 110% or higher, compared to a control not containing the mitochondrial function improver of the present invention. There is no upper limit to the oxygen consumption rate, but examples include cases where the mitochondrial oxygen consumption rate is 130% or less, 120% or less, 115% or less, etc., compared to a control to which the mitochondrial function improver of the present invention has not been added.

[0035] Furthermore, 80-90% of ATP production in eukaryotes comes from the mitochondrial electron transport chain, and a decrease in intracellular ATP reflects the activity of the electron transport chain, which is an indicator of mitochondrial activity. Therefore, mitochondrial function can also be evaluated by directly measuring intracellular ATP production. In other words, when mitochondrial function decreases, ATP production decreases. Conversely, when mitochondrial function improves, ATP production recovers.

[0036] In the present invention, the method for measuring ATP production is not particularly limited, but examples include a method for quantifying the amount of ATP in cultured cells using a luciferase luminescence assay. Examples of luminescent enzymes that can be used in the luciferase luminescence assay include firefly luciferase, Renilla luciferase, and Cypridina luciferase, with firefly luciferase being preferred. Therefore, measurement is preferably performed using a plate reader capable of detecting chemiluminescence, such as ARVO X3 (PerkinElmer). Detection reagents are not particularly limited, but the ATP Assay Kit-Luminescence (DOJINDO) is preferred. More specifically, ATP production can be measured and evaluated, for example, by the method described in the Examples below. The ATP production is preferably 110% or more, and more preferably 115%, of the mitochondrial ATP production in a control without the mitochondrial function improver of the present invention. There is no upper limit to the amount of ATP production, but examples include cases where the amount of mitochondrial ATP production is 140% or less, 130% or less, 120% or less, etc., compared to a control to which the mitochondrial function improving agent of the present invention has not been added.

[0037] In the present invention, the lactic acid bacteria having the ability to inhibit the production of reactive oxygen species in mitochondria, which is the active ingredient of the present invention, may be used as an agent for improving mitochondrial function, or may be used as a composition in combination with various carriers (e.g., isotonicity agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, thickeners, etc.), food materials, etc., within a range that does not impair the effects of the present invention.

[0038] Examples of the isotonicity agent include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol, polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol, and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride. These isotonicity agents can be used alone or in combination of two or more.

[0039] Examples of the chelating agent include edetate salts such as disodium edetate, calcium disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or a salt thereof, sodium hexametaphosphate, citric acid, etc. These chelating agents may be used alone or in combination of two or more.

[0040] The stabilizer may, for example, be sodium hydrogen sulfite.

[0041] Examples of pH adjusters include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or hydrogen carbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, and bases such as trometamol. These pH adjusters can be used alone or in combination of two or more.

[0042] Examples of preservatives include sorbic acid, potassium sorbate, parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate, quaternary ammonium salts such as chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride, alkylpolyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine. These preservatives can be used alone or in combination of two or more.

[0043] Examples of antioxidants include sodium hydrogen sulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherols, etc. These antioxidants may be used alone or in combination of two or more.

[0044] Examples of solubilizing agents include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol, etc. These solubilizing agents can be used alone or in combination of two or more.

[0045] Examples of thickeners include polyethylene glycol, methyl cellulose, ethyl cellulose, carmellose sodium, xanthan gum, sodium chondroitin sulfate, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, etc. These thickeners may be used alone or in combination of two or more.

[0046] The mitochondrial function improver of the present invention may further contain a component known to have an effect of improving mitochondrial function. Examples of such components include taurine, vitamin C, and catechins. Such components may be used alone or in combination of two or more.

[0047] In the present invention, the mitochondria targeted by the mitochondrial function improver are not particularly limited, and can be, for example, mitochondria in cultured cells (including cells in tissue culture) and in biological tissues. In an embodiment of cultured cells, preferably, cells derived from the intestinal tract can be used, and more preferably, intestinal epithelial cells or intestinal macrophages can be used. In an embodiment of biological tissue, the target tissue is preferably the intestinal tract, and more preferably, intestinal epithelium or intestinal macrophages. The organism from which the cells or tissue are derived is not particularly limited, but is preferably a mammal. Examples of mammals include humans, pigs, monkeys, mice, rats, rabbits, cats, dogs, cows, horses, sheep, etc., and preferably humans or pigs.

[0048] The mitochondrial function improver of the present invention can also be used as an experimental reagent in cell experiments (including tissue culture experiments) or in vivo cell experiments. Furthermore, the method for providing the mitochondrial function improver of the present invention is not particularly limited, and it can be used, for example, as a food or drink, or a pharmaceutical or quasi-drug.

[0049] In the embodiment of a food or drink, the mitochondrial function improver of the present invention can be prepared into a desired form either as is or in combination with other food materials or additives. Such foods and drinks include general foods, as well as foods for specified health uses, nutritional supplements, functional foods, and foods for patients.

[0050] Examples of these foods and beverages include, but are not limited to, supplements such as capsules (soft capsules, hard capsules), tablets, granules, powders, jellies, and liposome preparations; beverages such as energy drinks, fruit juice drinks, carbonated drinks, and lactic acid drinks; luxury items such as dumplings, ice cream, sherbet, gummies, and candies; etc. Among these foods and beverages, beverages or supplements are preferred.

[0051] The content of the lactic acid bacteria capable of inhibiting the production of reactive oxygen species in mitochondria, which is the active ingredient of the present invention, varies depending on the intake route, the patient's age, weight, symptoms, etc., and cannot be generally defined, but can be an amount such that the daily intake of compound (I) for an adult is usually about 10 to 5,000 mg, more preferably about 100 to 1,000 mg. In an embodiment of the composition, the content of the lactic acid bacteria capable of inhibiting the production of reactive oxygen species in mitochondria in the composition is not particularly limited and can be appropriately set based on conditions such as, for example, 90% by mass or more, 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, or 1% by mass or more.

[0052] When the mitochondrial function improving agent of the present invention is provided as a pharmaceutical or quasi-drug, the formulation is not particularly limited, and examples thereof include various formulation forms such as orally administered agents such as tablets, pills, capsules, powders, granules, syrups, and sublingual preparations; and parenterally administered agents such as injections (intravenous injection, intramuscular injection, local injection, and the like), mouthwashes, infusions, topical preparations (ointments, creams, patches, inhalants), and suppositories. Among the above-mentioned formulation forms, preferred examples include injections, orally administered agents, and topical preparations, and in particular inhalants (drugs administered via the respiratory tract).

[0053] In an embodiment of a pharmaceutical or quasi-drug, the content of the lactic acid bacteria having the ability to inhibit the production of reactive oxygen species in mitochondria, which is the active ingredient of the present invention, varies depending on the route of administration, the age, weight, symptoms, etc. of the patient and cannot be generally defined, but can be an amount such that the daily dose of compound (I) for an adult is usually about 10 to 5000 mg, more preferably about 100 to 1000 mg.

[0054] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0055] In this example, unless otherwise specified, special grade or first-grade reagents manufactured by Wako Pure Chemical Industries, Ltd. (Osaka, Japan) were used, and MilliQ water (Merck Millipore, Germany) was used for all water.

[0056] Example 1 Verification of the Ability of Lactic Acid Bacteria to Inhibit Mitochondrial Production of Reactive Oxygen Species (1) Porcine Intestinal Epithelial Cells (INV1-3) ​​In this example, the ability of Lactic Acid Bacteria to Inhibit Mitochondrial Production of Reactive Oxygen Species was verified using porcine intestinal epithelial cells (INV1-3). INV1-3 cells were cloned from the small intestine of Duroc piglets and reflect a NOD2 gene polymorphism (hyperfunction type) in the genome.

[0057] INV1-3 cells were cultured in collagen (Type I)-coated 250 mL flasks (Sumiron) in DMEM / F-12 medium (10% FCS, 1% penicillin-streptomycin, 1% ITS (100x), 0.05% rec Human EGF (10 μg / mL), high glucose, L-glutamine, and 0.11 ng / mL sodium pyruvate; Thermo Fisher Scientific, USA) (hereafter referred to as DMEM / F-12 medium (+FCS)).

[0058] The cells were maintained in a 5% CO2 incubator at 37°C. When the cells reached 80-90% confluence, they were washed once with PBS and treated with epithelial buffer (0.1 M Na2HPO4 / 12H2O, 0.45 M sucrose, 0.36% EDTA / 4Na, BSA) at 37°C for 5 minutes. The cells were then detached from the flask using trypsin buffer (0.25% trypsin, 0.02% EDTA in PBS) at 37°C for 5 minutes and centrifuged at 12,000 rpm for 5 minutes at 4°C. A cell count of 3.5 × 10 5 The cells were seeded at 100 cells / 250 mL flask. The day of seeding was designated as day 0 of culture. On day 2 of culture, the culture supernatant was aspirated and removed using an aspirator, and 10 mL of fresh DMEM / F-12 medium was added and cultured. In this example, cells from passages 8 to 18 were used.

[0059] (2) Reagents for measuring the amount of reactive oxygen species produced in mitochondria We investigated two types of reagents, MitoSOX and MitoROS 580, as candidate reagents that can be used in plate assays that can examine multiple samples simultaneously and that can confirm the effects of long-term stimulation with food components.

[0060] To confirm the effect of the ROS production measurement reagents (MitoSOX and MitoROS 580) on INV1-3 cells, each reagent was added to the cells, incubated at 37°C for 10 minutes, and then the cells were observed under a microscope. MitoSOX and MitoROS 580 were both dissolved in 50 μL of DMSO and diluted 1 / 500 with assay buffer before addition. As a result, MitoSOX induced cell detachment, as shown in Figure 1(a), whereas MitoROS 580 did not, as shown in Figure 1(b).

[0061] Next, to verify whether MitoROS 580 can detect mitochondrial ROS in INV1-3, we added antimycin A, a known inducer of ROS production, to INV1-3 to promote ROS production. Antimycin A was added at 50 μM as specified in the MitoROS 580 protocol. MitoROS 580 was then added to INV1-3 and incubated at 37°C for 1 hour. Fluorescence intensity (Excitation: 540 nm / Emission: 590 nm / Cutoff: 570 nm) was measured using an ARVO X3 (PerkinElmer) in bottom read mode. MitoROS 580 was dissolved in 50 μL of DMSO and diluted 1 / 500 with assay buffer before being added to the cells. A non-treated sample was prepared using the same procedure, except for the addition of antimycin A. The fluorescence intensity of the antimycin A-treated sample and the non-treated sample were measured. The same experiment, from the cell culture to the measurement of fluorescence intensity, was carried out three times (three wells).

[0062] The ratio of the mean fluorescence intensity of the non-treated samples (n = 3) to the mean fluorescence intensity of the samples treated with Antimycin A (n = 3) was calculated. As a result, as shown in Figure 1(c), it was confirmed that MitoROS 580 can detect ROS induced by Antimycin A.

[0063] Based on the above, we decided to use the fluorescent reagent MitoROS 580 as the reagent for measuring the amount of reactive oxygen species produced in mitochondria. We also decided to dissolve MitoROS 580 in 50 μL of DMSO, dilute it to 1 / 500 with assay buffer, and then add it to the cells.

[0064] (3) Examination of ROS-inducing inflammatory substances to be used in a model of mitochondrial dysfunction To determine the mitochondrial ROS-inducing inflammatory substances to be used in a model representing a state of mitochondrial dysfunction in intestinal epithelial cells INV1-3, we tested the cell viability and ROS production induction ability when candidate substances were added, and examined their concentration and duration of action.

[0065] Three candidate substances were selected: dextran sulfate sodium (DSS), which is used to produce an inflammatory bowel disease model; killed cells of Fusobacterium nucleatum, which are confirmed to grow during the development of inflammatory bowel disease; and lipopolysaccharide (LPS), which is used in immune-related inflammation induction models. The effective concentration of each candidate substance was determined by assessing cell viability after 24 hours of stimulation using Cell counting kit-8 (Dojindo, Kumamoto).

[0066] Specifically, INV1-3 cells were plated on a collagen Type I collagen-coated 96-well plate (SUMILON) at 3.0 × 10 4 After seeding at 1000 cells / mL, the cells were cultured for 4 days. After medium replacement, DSS, LPS, and killed Fusobacterium nucleatum cells were added and cultured for 24 hours at 37°C. After thorough washing with PBS, 105 μL of DMEM / F-12 medium (+FCS) supplemented with 8% Cell Counting Kit-8 (Dojindo) was added and incubated at 37°C for 2.5 hours. Absorbance (450 nm) was measured using an ARVO X3 (PerkinElmer). Non-treated samples were prepared using the same procedure, except for the addition of ROS-inducing pro-inflammatory substances. The same experiment, from cell culture to absorbance measurement, was performed in triplicate (triplicate wells).

[0067] The ratio of the mean absorbance of each sample treated with ROS-inducing inflammatory agents (n = 3) to the mean absorbance of the untreated samples (n = 3) was calculated as the cell viability. The changes in cell viability after 24 hours of exposure are shown in Figure 2. DSS at 1% or higher, killed F. nucleatum cells at 100 MOI, and LPS at 0.1 μg / mL or higher reduced cell viability to 80-90% ( / Control) (data for LPS at 0.1 μg / mL not shown) [Figure 2(a)].

[0068] Using these concentrations as a reference, ROS production was measured 24 hours, 3 hours, and 30 minutes after the addition of the mitochondrial ROS-inducing inflammation-promoting candidate substances, as shown in Figure 2(b)-(d). ROS production was measured using ROS580 and ARVO X3, as in Example 1(2). The same experiment, from the cell culture to the fluorescence intensity measurement, was performed three times (three wells each).

[0069] As a result, only DSS increased mitochondrial ROS production among the candidate substances. Specifically, as shown in Figure 2(b)-(d), an increase in ROS production was confirmed after 24 hours of stimulation with 1-5% DSS, and after 30 minutes and 3 hours of stimulation with 5% DSS.

[0070] Based on the above, the stimulus used in the model of mitochondrial dysfunction in INV1-3 cells was DSS, with a concentration of 5% and an exposure time of 3 hours.

[0071] (4) Preparation of strains used and preparation of killed bacteria As candidates for substances capable of inhibiting the production of reactive oxygen species in mitochondria, lactic acid bacteria [Ligilactobacillus salivarius (FFIG35) (Accession No. NITE BP-03218), Ligilactobacillus salivarius (FFIG56) (Accession No. NITE ABP-03958), Ligilactobacillus salivarius (FFIG110), Ligilactobacillus salivarius (FFIG130), Lactiplantibacillus plantarum (CS91), and Lactiplantibacillus plantarum (CS96)] were cultured.

[0072] The strains used were those isolated from pigs in our laboratory. The isolation source and genus of the strains used are shown in Table 1. Samples stocked in 30% glycerol in our laboratory were spread onto MRS agar medium using a platinum loop.

[0073]

[0074] A single colony of lactic acid bacteria was picked with a sterilized toothpick, inoculated into 1 mL of MRS liquid medium, and cultured at 37°C for 24 hours. The culture was then stirred, and 20 μL was added to 1 mL of MRS liquid medium and cultured at 37°C for 1 day. Similarly, the culture was stirred, and 200 μL was added to 10 mL of MRS liquid medium and cultured at 37°C for 16 hours. Killed lactic acid bacteria cells were prepared using the following procedure.

[0075] The culture medium was centrifuged (6000 rpm, 5 minutes). The supernatant was discarded, and 5 mL of PBS was added. The mixture was stirred until the pellet disappeared, and then centrifuged (6000 rpm, 5 minutes). The supernatant was discarded, and 5 mL of PBS was added and stirred. The mixture was then heat-treated in a water bath heated to 70°C for 90 minutes. The mixture was then centrifuged (6000 rpm, 5 minutes), the supernatant was discarded, and 6 mL of PBS was added and stirred. 1 mL of this was dispensed and diluted 2, 10, and 50 times with PBS. 10 μL was placed on a bacterial counting chamber (AS ONE), crushed with a cover glass until it no longer moved, and examined under a microscope. The number of bacteria was counted, and the total number of bacteria was calculated using the following formula: Formula: Total number of bacteria C / mL = Counted number c × 250,000 × [Dilution factor] After measuring the number of bacteria, 2.5 × 10 9 The bacterial solution was prepared with PBS to a concentration of 100 cells / mL, dispensed into microtubes, and stored frozen at -20°C.

[0076] (5) Verification of the ability to suppress the production of reactive oxygen species in mitochondria. INV1-3 was added at 3.0 × 10 4 100 μL of the culture medium was seeded onto an XF24 cell culture plate (Agilent Technologies) at a concentration of 100 cells / mL and cultured at 37°C for 24 hours to allow adhesion. 24 hours after the start of culture, 150 μL of DMEM / F-12 medium (plus FCS) was added to bring the volume to 250 μL. 72 hours after the start of culture, the medium was replaced. 96 hours after the start of culture, killed cells of the candidate lactic acid bacteria prepared in Example 1(4) were added to INV1-3 cells and cultured at 37°C for 24 hours. Because previous research in this laboratory has shown that a density of 100 MOI (multiplicity of infection) does not significantly affect cell viability, the killed cells were added at a concentration of 100 MOI.

[0077] The INV1-3 cells were then washed with PBS, and then exposed to 5% DSS for 3 hours. A control sample was prepared in the same manner as in Example 1(4), except that killed cells of the candidate lactic acid bacteria were not added.

[0078] Thereafter, the fluorescence intensity of the sample containing killed lactic acid bacteria cells and the fluorescence intensity of the control sample were measured using ROS 580 and ARVO X3, as in Example 1(2). The same experiment, from the cell culture to the fluorescence intensity measurement, was performed six times (six wells each).

[0079] The ratio of the average fluorescence intensity measurements of the control sample (n = 6) to the average fluorescence intensity measurements of each sample (n = 6) was calculated and used as the amount of ROS produced in the mitochondria of each sample (Table 2 and Figure 3).

[0080]

[0081] Example 2 Verification of the mitochondrial function-improving effect of lactic acid bacteria capable of suppressing the production of reactive oxygen species in mitochondria (1) Cells used and culture conditions INV1-3 cells, which are immortalized porcine intestinal epithelial cells, were used as in Example 1. The culture method was also similar to that in Example 1(1).

[0082] (2) Test strain and preparation method Ligilactobacillus salivarius (FFIG35), which showed significant suppression of ROS production in Example 1, was used. Killed cell samples were prepared in the same manner as in Example 1 (4) and (5).

[0083] (3) Measurement of mitochondrial oxygen consumption rate using an extracellular flux analyzer. Preparation of measurement sample: INV1-3 at 3.0 × 10 4 100 μL of the cells / mL were seeded onto an XF24 cell culture plate (Agilent Technologies) and cultured at 37°C for 24 hours to allow adhesion.

[0084] Twenty-four hours after the start of culture, 150 μL of DMEM / F-12 medium (plus FCS) was added to bring the total volume to 250 μL. The medium was replaced 72 hours after the start of culture, and killed FFIG35 cells were added 96 hours after the start of culture and cultured at 37°C for 24 hours. Killed cells were added at an MOI of 100. After washing the INV1-3 cells with PBS, DSS was added to a concentration of 5% and the cells were exposed for 3 hours.

[0085] A DSS-only sample was prepared in the same manner except that killed FFIG35 cells were not added.

[0086] Measurement of Oxygen Consumption Rate (OCR). The 5% DSS-supplemented medium was replaced with 500 μL of XF assay medium (25 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate, serum-free, Agilent Technologies) per well. The reagents from the Cell Mito Stress Test Kit (Agilent Technologies), 10 μM Oligomycin, 20 μM FCCP, and 5 μM Rot / AA, were added to ports A, B, and C, respectively, and steady-state mitochondrial oxygen consumption rate (basal respiration) and maximal respiratory capacity (maximal respiration) were measured using an Agilent Seahorse XF24 analyzer (Seahorse Bioscience). Protein content was then measured using a BCA Protein Assay Kit (Thermo Fisher Scientific). The DSS-only sample was subjected to five replicate experiments (5 wells), and the FFIG35-killed cell-supplemented sample was subjected to four replicate experiments (4 wells).

[0087] The measured values ​​of oxygen consumption rate in basal respiration and oxygen consumption rate in maximal respiration were corrected by the protein amount ratio, with the protein amount in one well of the DSS-only sample being set to 1. The average values ​​of the corrected measured values ​​of oxygen consumption rate in basal respiration and maximal respiration (DSS only: n = 5, FFIG35: n = 4) were used as the oxygen consumption rates in basal respiration and maximal respiration, respectively.

[0088] The data were analyzed for oxygen consumption rate (OCR) using Seahorse Wave Desktop Software (Agilent Technologies) (Table 3 and Figure 4).

[0089]

[0090] (4) Measurement of mitochondrial ATP production using a plate reader. Preparation of measurement samples: 3.0 x 10 4 Cells were seeded at 100 μL / well at 100 cells / mL and cultured at 37°C for 24 hours to allow adhesion. DMEM / F-12 medium (plus FCS) was added 24 hours after the start of culture. The medium was replaced 72 hours after the start of culture, and killed FFIG35 cells were added 96 hours after the start of culture and cultured at 37°C for 24 hours. Killed cells were added at an MOI of 100. After washing the INV1-3 cells with PBS, DSS was added to a concentration of 5% and exposed for 3 hours. A DSS-only sample was prepared using the same procedure, except that killed FFIG35 cells were not added. A non-treated sample was prepared using the same procedure, except that neither DSS nor killed FFIG35 cells were added.

[0091] Measurement of ATP Production: The working solution for the ATP Assay Kit-Luminescence (DOJINDO) was prepared according to the recommended protocol. After removing the 5% DSS-supplemented medium and washing twice with PBS, 50 μL of DMEM / F-12 medium (without FCS) was added to each well. For the standard, 50 μL of ATP standard solution (5, 2.5, 0.625, 0.313, 0.078, or 0 μmol / L) was added to unseeded wells. 50 μL of the working solution was added to each well, and the wells were shaken at 25°C for 2 minutes in an ARVO X3 shaker. The wells were then incubated at 25°C for 10 minutes without opening. Luminescence (RLU) was measured once every 0.1 seconds. Protein content in each well was then measured using a BCA Protein Assay Kit (Thermo Fisher Scientific). The same experiment, from cell culture to protein content measurement, was performed six times (six wells).

[0092] The measured luminescence intensity of each sample was corrected by the protein amount of each sample, assuming that the protein amount of the DSS-only sample was 1. Furthermore, the ratio of the average (n = 6) of the corrected luminescence intensity measurements of the non-treatment sample and the sample with FFIG35 added to the average (n = 6) of the luminescence intensity measurements of the DSS-only sample was calculated and used as the amount of ATP produced by the cells (Table 4 and Figure 5).

[0093]

[0094] Example 3: Inhibitory effect of FFIG-35 on ROS production in intestinal macrophages. Porcine intestinal macrophages were stimulated with post-immunobiotics (FFIG35) (48 hr). Specifically, porcine intestinal macrophages (Takenouchi et al., Front Vet Sci. 9:919077. 2022) were used. 6.0 × 10 4 100 μL of the culture medium was seeded onto XF24 cell culture plates (Agilent Technologies) at a concentration of 100 μM cells / mL and cultured at 37°C for 72 hours. After changing the medium, killed FFIG35 cells prepared as described in Example 1 (4) were added to porcine intestinal macrophages and cultured at 37°C for 24 hours. Killed cells were added at an MOI of 100. Subsequently, stimulation was performed with antimycin A (AMA), an inhibitor of mitochondrial electron transport chain complex III. Specifically, porcine intestinal macrophages were washed with PBS, and AMA was added to a concentration of 50 μM and exposed for 3 hours. A control sample was prepared using the same procedure, except that killed FFIG35 cells were not added. Mitochondrial ROS production levels were measured using mitROS. The results are shown in Figure 6.

[0095] AMA stimulation was shown to induce ROS production. On the other hand, pre-stimulation with FFIS35 significantly suppressed AMA-induced ROS production (significant differences between different signs). This indicates that FFIS35 can suppress ROS production not only in intestinal epithelial cells but also in intestinal macrophages.

[0096] [Rule 26 Amendment 11.10.2024]

Claims

1. A mitochondrial function improver comprising lactic acid bacteria having the ability to inhibit the production of reactive oxygen species in mitochondria.

2. The mitochondrial function improving agent according to claim 1, wherein the lactic acid bacteria suppresses the production of reactive oxygen species in mitochondria by 70% to 90%.

3. The mitochondrial function improving agent according to claim 1, wherein the lactic acid bacteria includes Ligilactobacillus salivarius.

4. The mitochondrial function improver according to Item 1, wherein the lactic acid bacteria include one or both of Ligilactobacillus salivarius having the accession number NITE BP-03218 and Ligilactobacillus salivarius having the accession number NITE ABP-03958.

5. The mitochondrial function improving agent according to claim 1, wherein the lactic acid bacteria has the ability to inhibit the production of reactive oxygen species in mitochondria in intestinal epithelial cells.

6. The mitochondrial function improving agent according to claim 1, wherein the improvement in mitochondrial function includes at least one improvement selected from the group consisting of an improvement in ATP production amount and an improvement in mitochondrial oxygen consumption rate.

7. A mitochondrial function improving agent according to any one of claims 1 to 6, which is in the form of a food or drink.

8. The mitochondrial function improving agent according to any one of claims 1 to 6, which is a drug or quasi-drug.

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