Use of lactobacillus reuteri and / or metabolite thereof

Abstract

The use of Lactobacillus reuteri and / or a metabolite thereof in the preparation of a composition for mitigating muscle loss, enhancing muscle endurance or retarding cell aging, wherein the Lactobacillus reuteri is Lactobacillus reuteri TCI979 with the deposit number of DSM34539.

Description

Use of lactobacillus reuteri and / or metabolites thereof TECHNICAL FIELD

[0001] The present invention relates to a lactobacillus reuteri, in particular to the use of a lactobacillus reuteri and / or metabolites thereof for the preparation of a composition for slowing down muscle loss, for enhancing muscle endurance or for slowing down cell aging. BACKGROUND

[0002] Lactobacillus reuteri is an important species of lactic acid bacteria, which is usually used to improve the intestinal flora of mammals, antagonize the colonization of other harmful bacteria in the intestine, and is a potential probiotic. SUMMARY

[0003] The present invention provides a lactobacillus reuteri and / or metabolites thereof, wherein the lactobacillus reuteri is lactobacillus reuteri TCI979 with accession number DSM34539.

[0004] In some embodiments, the lactobacillus reuteri and / or metabolites thereof are used for the preparation of a composition for slowing down muscle loss.

[0005] In some embodiments, the lactobacillus reuteri and / or metabolites thereof are used for the preparation of a composition for enhancing muscle endurance.

[0006] In some embodiments, the lactobacillus reuteri and / or metabolites thereof are used for the preparation of a composition for slowing down cell aging.

[0007] In some embodiments, the lactobacillus reuteri can reduce the content of malondialdehyde in blood.

[0008] In some embodiments, the lactobacillus reuteri can enhance the total antioxidant capacity (TAC) or the content of f-Thiols in blood.

[0009] In some embodiments, the lactobacillus reuteri can inhibit the content of reactive oxygen species.

[0010] In some embodiments, the lactobacillus reuteri can reduce apoptosis.

[0011] In some embodiments, the lactobacillus reuteri can enhance the mitochondrial activity of muscle cells.

[0012] In some embodiments, the lactobacillus reuteri can slow down the rate of telomere shortening.

[0013] In some embodiments, the lactobacillus reuteri can enhance the activity of telomerase.

[0014] In summary, the Lactobacillus reuteri and / or its metabolite of any embodiment can reduce the level of malondialdehyde in blood, increase the total antioxidant capacity or the level of sulfur compounds in blood, inhibit the level of reactive oxygen species, or reduce cell apoptosis, thereby slowing down muscle loss. The Lactobacillus reuteri and / or its metabolite of any embodiment can increase muscle endurance by increasing mitochondrial activity of muscle cells, reducing the level of malondialdehyde in blood, increasing the total antioxidant capacity or the level of sulfur compounds in blood, or inhibiting the level of reactive oxygen species. The Lactobacillus reuteri and / or its metabolite of any embodiment can slow down the rate of telomere shortening, increase telomerase activity, or inhibit the level of reactive oxygen species, thereby slowing down cell aging. BRIEF DESCRIPTION OF DRAWINGS

[0015] FIG. 1 is a graph showing the results of the ROS inhibition test of Lactobacillus reuteri.

[0016] FIG. 2 is a graph showing the results of the cell apoptosis avoidance test of Lactobacillus reuteri.

[0017] FIG. 3 is a graph showing the results of the mitochondrial activity inhibition test of Lactobacillus reuteri.

[0018] FIG. 4 is a graph showing the results of the telomerase activity enhancement test of Lactobacillus reuteri.

[0019] FIG. 5 is a graph showing the results of the cell size change test of Lactobacillus reuteri.

[0020] FIG. 6 is a graph showing the results of the microscope observation of the cell size change of Lactobacillus reuteri under natural aging.

[0021] FIG. 7 is a graph showing the results of the cell size change test of Lactobacillus reuteri under external oxidative damage.

[0022] FIG. 8 is a graph showing the results of the change in blood MDA concentration in the human experiment.

[0023] FIG. 9 is a graph showing the results of the change in total antioxidant capacity in the human experiment.

[0024] FIG. 10 is a graph showing the results of the change in the level of sulfur compounds in blood in the human experiment.

[0025] FIG. 11 is a graph showing the results of the change in average upper limb muscle endurance in the human experiment.

[0026] FIG. 12 is a graph showing the results of the change in average lower limb muscle endurance in the human experiment.

[0027] Preservation of biological material

[0028] Lactobacillus reuteri TCI979 was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Germany) on February 17, 2023, and was assigned accession number DSM34539. DETAILED DESCRIPTION

[0029] Lactobacillus reuteri TCI979 is deposited at the German Collection of Microorganisms and Cell Cultures under accession number DSM 34539. In some embodiments, Lactobacillus reuteri TCI979 is a strain isolated from Wolongtan, Guizhou, China.

[0030] The Lactobacillus reuteri of the present application, also known as Lactobacillus reuteri TCI979, is commonly found in the intestines, mouth, and stomach of humans and animals, and can also be present in fermented foods (e.g., yogurt, pickles) and dairy products. Here, Lactobacillus reuteri TCI979 is a facultative anaerobic, gram-positive bacterium, but prefers low-oxygen or anaerobic environments. It has a generally elongated, rod-like appearance with curved, blunt ends, and is about 0.5 to 0.8 microns wide and 2 to 8 microns long. It lacks flagella and is non-motile, does not produce spores, and generally does not form a capsule. Its main energy source is carbohydrates such as monosaccharides (e.g., glucose, fructose) and disaccharides (e.g., lactose) as carbon sources, and it metabolizes glucose via the phosphoketolase pathway to produce metabolites such as lactic acid, ethanol, acetic acid, and carbon dioxide. Lactobacillus reuteri can metabolize glycerol to produce reuterin, a non-proteinaceous antibacterial substance that can effectively inhibit the growth of harmful bacteria and is harmless to animals.

[0031] In some embodiments, Lactobacillus reuteri TCI979 and / or its metabolites have the effect of slowing muscle loss. In other words, Lactobacillus reuteri TCI979 and / or its metabolites are suitable for use in the preparation of a muscle loss slowing composition.

[0032] In some embodiments, Lactobacillus reuteri TCI979 and / or its metabolites have the effect of enhancing muscle endurance. In other words, the use of Lactobacillus reuteri and / or its metabolites for the preparation of a muscle endurance enhancing composition.

[0033] In some embodiments, Lactobacillus reuteri TCI979 and / or its metabolites have the effect of slowing cellular aging. In other words, the use of Lactobacillus reuteri or its metabolites for the preparation of a cellular aging slowing composition.

[0034] In some embodiments, Lactobacillus reuteri can reduce the amount of malondialdehyde (MDA) in the blood. Malondialdehyde, also referred to as MDA, is generally formed as a result of lipid peroxidation. Malondialdehyde is highly reactive in the body and readily binds to DNA or proteins to damage normal cell development. Generally, the higher the amount of malondialdehyde in the body, the worse the antioxidant capacity. Studies have also shown that the amount of MDA in muscle significantly increases with age, and gradually increases as muscle mass decreases.

[0035] In some embodiments, the Lactobacillus reuteri can increase the Total Antioxidant Capacity (TAC). The TAC is the body's ability to reduce oxidants, and the higher the TAC, the better the body's ability to mitigate the damage caused by reactive oxygen species. Increasing the TAC can further increase muscle strength and muscle mass.

[0036] In some embodiments, the Lactobacillus reuteri can increase the level of f-Thiols in the blood. f-Thiols are sulfur-containing compounds in the blood that act as free radical scavengers, either by directly neutralizing free radicals or by reducing oxidative stress through redox signaling pathways, such as increasing glutathione reductase activity. Reducing oxidative stress helps maintain normal cellular functions, including energy metabolism, DNA repair, and protein synthesis, which can increase muscle strength and mass.

[0037] In some embodiments, the Lactobacillus reuteri can inhibit the level of reactive oxygen species.

[0038] In some embodiments, the Lactobacillus reuteri can reduce apoptosis. Apoptosis is a natural form of cell death that occurs under genetic control in response to environmental stimuli, also known as programmed cell death. It is different from necrosis, which is an abnormal form of cell death that occurs when a cell is damaged beyond repair. Under normal circumstances, any abnormalities that occur during cell formation are eliminated through apoptosis. For example, the growth of cancer cells into tumors is inhibited by apoptosis.

[0039] In some embodiments, the Lactobacillus reuteri can increase mitochondrial activity in muscle cells.

[0040] In some embodiments, the Lactobacillus reuteri can slow down the rate of telomere shortening. In some embodiments, the Lactobacillus reuteri can increase telomerase activity. Telomeres are highly repetitive DNA sequences or protein complexes located at the ends of chromosomes that are essential for maintaining chromosome integrity and stability. That is, when telomeres shorten with cell division, this shortening phenomenon can easily cause cell aging or disease, and telomerase can effectively lengthen and maintain telomere length, slowing down the rate of telomere shortening.

[0041] In some embodiments, the specific amount of Lactobacillus reuteri is 100 mg per day.

[0042] In some embodiments, the aforementioned composition comprises a specific amount of Lactobacillus reuteri or its metabolite. That is, the amount of Lactobacillus reuteri TCI979 used is 100 mg per day. Specifically, assuming that one serving of the composition is administered per day and the Lactobacillus reuteri is in dry powder form, the composition comprises at least 100 mg of Lactobacillus reuteri.

[0043] In some embodiments, the aforementioned composition can be a health product, a food product, or a food additive for non-medical purposes. In other words, the health product, the food product, or the food additive comprises a specific amount of Lactobacillus reuteri.

[0044] In some embodiments, the aforementioned health product, the food product, or the food additive can further comprise a food industry acceptable carrier that is widely used in food manufacturing technology. For example, the food industry acceptable carrier can comprise one or more of the following agents: a solvent, a buffer, an emulsifier, a suspending agent, a decomposer, a disintegrating agent, a dispersing agent, a binding agent, an excipient, a stabilizing agent, a chelating agent, a diluent, a gelling agent, a preservative, a wetting agent, a lubricant, an absorption delaying agent, a liposome, and the like. The selection and amount of these agents are within the ordinary skill and routine techniques of one of ordinary skill in the art.

[0045] In some embodiments, the aforementioned food industry acceptable carrier of the health product, the food product, or the food additive comprises a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), and an aqueous solution containing alcohol.

[0046] In some embodiments, the food product can be, but is not limited to, beverages, fermented foods, bakery products, health foods for non-medical purposes, and dietary supplements.

[0047] Example 1: Isolation and identification of the bacterial strain

[0048] First, an appropriate amount of water from Wolong Lake in Guizhou, China was added to a liquid Lactobacilli MRS medium (BD Difco™ Lactobacilli MRS Broth) and incubated at 37°C in an anaerobic environment (i.e., the oxygen concentration in the culture environment was 1% by volume) for 24 hours to form a bacterial solution. Next, the bacterial solution was subjected to serial dilution and then plated onto a solid Lactobacilli MRS medium (BD Difco™ Lactobacilli MRS Broth with 1.5% agar) and incubated at 37°C in an anaerobic environment (i.e., the oxygen concentration in the culture environment was 1% by volume) until colonies were formed on the culture plate. One of the colonies was selected for subsequent testing.

[0049] Next, the strain of the colony was subjected to bacterial strain identification. The 16S ribosomal gene (16S rDNA) sequence (i.e., SEQ ID NO: 1) of the isolated strain was obtained through polymerase chain reaction (PCR), and the total length of the gene sequence was 1,050. Next, using the website of the National Center for Biotechnology Information (NCBI), the indicated gene sequence was subjected to sequence alignment with the 16S rDNA sequences of other Lactobacilli strains, and the similarity of the 16S rDNA sequences is shown in Table 1 below. Based on this, it was found that the 16S rDNA sequence of the isolated strain had the highest similarity to that of Lactobacillus reuteri (accession number NBRC 15892).

[0050] Table 1

[0051] In this case, the 16S rDNA sequence of the strain isolated from the water of Wolong Lake in Guizhou, China had a high similarity of 99.42% to the sequence of Lactobacillus reuteri. Therefore, the isolated strain was named Lactobacillus reuteri TCI979 (Limosilactobacillus reuteri TCI979).

[0052] Example 2: Preservation and cultivation experiment of Lactobacillus reuteri

[0053] The isolated Lactobacillus reuteri was inoculated at a concentration of 1% (about 1x10 4CFU / mL) were inoculated in MRS medium (BD Difco TM TCI979 bacterial liquid was formed after Lactobacilli MRS Broth, 1% (v / v) was inoculated in MRS medium (BD Difco

[0054] The supernatant and Lactobacilli reuteri cells were separated by centrifuging the TCI979 bacterial liquid at 5000 rpm for 20 minutes. The supernatant was filtered by a 0.2 μm filter membrane. The obtained filtrate was the TCI979 sample (i.e., the TCI979 sample contained Lactobacilli reuteri metabolites).

[0055] The above TCI979 bacterial liquid was added to 5% (W / W) soy milk powder, 5% (W / W) trehalose, 10% (W / W) sorbitol, and 20% (W / W) indigestible malt dextrin, mixed uniformly, freeze-dried, and ground into powder to form TCI979 bacterial powder.

[0056] Example 3: Active oxygen content test

[0057] This test simulates the situation in which cells are damaged by external oxidation by using hydrogen peroxide (H2O2). The change in the active oxygen substance (ROS) content of human peripheral blood mononuclear cells treated with the TCI979 sample was determined by using a probe DCFH-DA in combination with flow cytometry.

[0058] 3-1. Materials and instruments:

[0059] Cells: Human peripheral blood mononuclear cells (purchased from lifeline, model HC-002, hereinafter referred to as PBMC cells).

[0060] Culture medium: RPMI 1640 (Roswell Park Memorial Institute, purchased from Gibco), added with 10% FBS (Fetal Bovine Serum, purchased from Gibco) and 1% antibiotic-antimycotic (purchased from Gibco).

[0061] PBS buffer and DPBS buffer (purchased from Gibco).

[0062] Hydrogen peroxide H2O2 (purchased from Sigma).

[0063] 2,7-dichloro-dihydro-fluorescein diacetate (DCFH-DA) (purchased from Sigma / SI-D6883-50MG). The DCFH-DA was dissolved in DMSO before use to dilute to a concentration of 5 pg / mL DCFH-DA solution.

[0064] Flow cytometer (purchased from BD company, model Accuri TM C6 Plus).

[0065] 3-2. Test procedure:

[0066] First, 1 x 10 5 PBMC cells were taken to a six-well cell culture dish containing 2 mL of medium per well, and incubated at 37°C, 5% CO2 constant temperature for 24 hours.

[0067] After the PBMC cells in each well were suspended in the cell culture dish, the PBMC cells were separated into a blank group, a control group and an experimental group.

[0068] Blank group: only add medium, treat at 37°C for 2 hours.

[0069] Control group: add medium, treat at 37°C for 1 hour, then add 1 mM hydrogen peroxide, continue to treat at 37°C for 1 hour.

[0070] Experimental group: add medium containing 0.125% TCI979 sample prepared in Example 2, treat at 37°C for 1 hour, then add 1 mM hydrogen peroxide, continue to treat at 37°C for 1 hour.

[0071] Next, add 5 pg / mL DCFH-DA solution to each group of cells, treat at 37°C for 15 minutes.

[0072] After moving the cells in each group to a 1.5 mL centrifuge tube, centrifuge (400 x g) for 5 minutes, remove the supernatant, wash with 1X PBS buffer, and then centrifuge (400 x g) for 5 minutes again. After centrifugation, remove the supernatant and add 200 pL of 1X DPBS buffer to each tube to resuspend the PBMC cells, forming the test cell solution.

[0073] The fluorescence signal of DCFH-DA in each group of test cell solution was detected using a flow cytometer (set excitation light: 450-490 nm; scattering light: 510-550 nm). The fluorescence intensity of PBMC cells treated with DCFH-DA can reflect the content of intracellular reactive oxygen species (ROS), and can be converted to a relative value.

[0074] 3-3. Test results:

[0075] It is particularly noted that the results in Figure 1 are presented in relative fold, i.e. the quantification results of the experimental groups are converted into the performance relative to the blank group by taking the quantification results of the blank group as 100. Among them, Student T-Test is used to analyze whether there is a statistically significant difference. In the figure, "#" represents that the p value is less than 0.05 relative to the blank group, "##" represents that the p value is less than 0.01 relative to the blank group, and "###" represents that the p value is less than 0.001 relative to the blank group, "*" represents that the p value is less than 0.05 relative to the control group, "**" represents that the p value is less than 0.01 relative to the control group, and "***" represents that the p value is less than 0.001 relative to the control group.

[0076] Referring to Figure 1, as shown in Figure 1, the control group shows that the oxidative damage simulation indeed induces the intracellular generation of a large amount of ROS, and the ROS content of the control group increases by 1262% relative to the blank group. In contrast, the ROS content of the experimental group is 1169%, which is decreased by 93% relative to the control group, so it can be known that Lactobacillus reuteri TCI979 can help cells effectively resist the oxidative damage of hydrogen peroxide. It is known that high concentration of ROS can trigger oxidative stress, which can cause mitochondrial dysfunction, thereby damaging skeletal muscle cell components, and the increase of ROS can interfere with skeletal muscle differentiation and reduce the efficiency of myoblast generation.

[0077] Example 4: Apoptosis test

[0078] This test observes whether the sample can help cells avoid apoptosis under the condition that the cells are subjected to external oxidative damage simulated by hydrogen peroxide (H2O2). Here, the cells are stained with FITC-fluorescent Annexin V, and PI is used as a nuclear dye, and fluorescence microscopy or flow cytometry is used to detect apoptosis and the stage of apoptosis.

[0079] 4-1. Materials and instruments:

[0080] Cells: Human mononuclear cells (obtained from ATCC, TIB202, hereinafter referred to as THP-1 cells).

[0081] Culture medium: RPMI 1640 (Roswell Park Memorial Institute, purchased from Gibco), added with 10% FBS (Fetal Bovine Serum, purchased from Gibco) and 1% antibiotic-antimycotic (purchased from Gibco).

[0082] PBS buffer and DPBS buffer (purchased from Gibco).

[0083] Hydrogen peroxide H2O2 (purchased from Sigma).

[0084] Nucleus dye (Propidium iodide, PI, purchased from BD, Bioscience).

[0085] Annexin V, FITC Apoptosis Detection Kit (purchased from Enzo).

[0086] Flow cytometer (purchased from BD, model Accuri TM C6 Plus).

[0087] 4-2. Test procedure:

[0088] First, 1 x 10 5 THP-1 cells were implanted in a 24-well cell culture plate and cultured at 37°C, 5% CO2 for 24 hours. The THP-1 cells were separated into a blank group, a control group, and an experimental group.

[0089] Blank group: only medium was added and treated at 37°C for 2 hours.

[0090] Control group: only medium was added and treated at 37°C for 1 hour, then 1 mM hydrogen peroxide was added and treated at 37°C for another 1 hour.

[0091] Experimental group: medium containing 0.125% TCI979 sample prepared in Example 2 was added and treated at 37°C for 1 hour, then 1 mM hydrogen peroxide was added and treated at 37°C for another 1 hour.

[0092] The supernatant in the culture plate was removed and washed twice with 1 mL of 1X PBS solution. Then, PI dye and reagents in the test kit (Annexin V) were added to the cells in each group and treated at 37°C for 5 minutes.

[0093] After the cells in each group were transferred to a 1.5 mL centrifuge tube, centrifugation (400 x g) was performed for 5 minutes, the supernatant was removed, and the cells were washed with 1X PBS buffer and centrifuged again (400 x g) for 5 minutes. After centrifugation, the supernatant was removed, 200 μL of 1X DPBS buffer was added to each tube to resuspend the PBMC cells, and the resulting cell suspension was obtained.

[0094] The fluorescence signals of FITC and PE in the cell suspension in each group were detected using a flow cytometer (excitation light: 450-490 nm; scattered light: 510-550 nm). The fluorescence intensity of the cells can reflect the apoptosis of the cells, and the relative values were calculated.

[0095] 4-3. Test results:

[0096] It is particularly noted that the results shown in Figure 2 are presented in relative ratios, i.e., the quantitative results of the blank group are taken as 100 to convert the quantitative results of the experimental group and the control group into the performance relative to the blank group. Among them, Student T-Test is used to analyze whether there is a statistically significant difference. In the figure, "#" represents that the p value is less than 0.05 relative to the blank group, "##" represents that the p value is less than 0.01 relative to the blank group, and "###" represents that the p value is less than 0.001 relative to the blank group, "*" represents that the p value is less than 0.05 relative to the control group, "**" represents that the p value is less than 0.01 relative to the control group, and "***" represents that the p value is less than 0.001 relative to the control group.

[0097] Referring to Figure 2, as shown in Figure 2, the control group shows that the oxidative damage simulation does indeed cause a large amount of apoptosis, and the number of cell apoptosis in the control group increases by 1201% relative to the blank group. In contrast, the number of cell apoptosis in the experimental group is 1122%, which is 79% lower than that of the control group. It can be seen that Lactobacillus reuteri TCI979 can help cells effectively avoid apoptosis under external damage. That is, Lactobacillus reuteri TCI979 can alleviate cell apoptosis caused by oxidative stress, with an inhibition rate of 79%.

[0098] Example 5: Mitochondrial activity experiment in skeletal muscle cells

[0099] Mitochondria are important organelles for oxidative metabolism and energy production in cells. Poor mitochondrial function can lead to insufficient ATP production. Inactive and inefficient mitochondria are a kind of cell waste, and excessive accumulation of which can cause cell swelling and aging, thereby leading to reduced muscle exercise tolerance. In this test, hydrogen peroxide (H2O2) is added to simulate the condition of cells being subjected to external oxidative damage, and the resulting low mitochondrial activity. In this test, the lower the calculated mitochondrial activity ratio, the better, indicating that Lactobacillus reuteri TCI979 can improve the metabolic rate of cells and reduce the aging phenomenon caused by waste accumulation.

[0100] 5-1. Materials and instruments:

[0101] Cell strain: Mouse myoblast C2C12 (hereinafter referred to as C2C12 cells) was used. C2C12 cells were purchased from the C2C12 cell strain (Cat. 60083) of the Bioresource Collection and Research Center (BCRC).

[0102] Culture medium: Dulbecco's modified Eagle's medium, purchased from Gibco, USA, Cat. 11965-092. Added 10% fetal bovine serum, purchased from Gibco, USA, Cat. 10437-028. Added 1% antibiotic-antimycotic, purchased from Gibco, USA, Cat. 15240-062.

[0103] Reagents: PBS buffer (purchased from Gibco, Cat. 14200-075), Trypan blue dead cell stain (purchased from Lonza, Cat. 17-942E), trypsin: 10X Trypsin-EDTA (purchased from Gibco), MitoScreen flow cytometry mitochondrial membrane potential assay kit (BD; Cat. BDB551302) including: JC-1 dye, 10X Assay buffer; hydrogen peroxide H2O2 (purchased from Sigma).

[0104] 5-2. Test procedure:

[0105] First, 1 x 10 5 C2C12 cells were taken to a six-well cell culture dish containing 2 mL of culture medium per well, and incubated at 37°C, 5% CO2, for 24 hours. The C2C12 cells were divided into three groups: experimental group, blank group, and control group.

[0106] Blank group: only added culture medium, incubated at 37°C, 5% CO2, for 25 hours.

[0107] Control group: added 1 mM of hydrogen peroxide, incubated at 37°C, 5% CO2, for 25 hours.

[0108] Experimental group: added 4% TCI979 sample prepared in Example 2 to the cell culture medium, incubated at 37°C, 5% CO2, for 24 hours. Then added 1 mM of hydrogen peroxide, and continued to incubate at 37°C, 5% CO2, for 1 hour.

[0109] The supernatant in the culture dish was removed and rinsed with 1 mL of 1X PBS solution twice. Then 200 μL of trypsin was added to each well and reacted for 5 minutes in the dark. After the reaction was completed, the cells in each well were collected into a separate corresponding 1.5 mL centrifuge tube, and the centrifuge tube containing the cells was centrifuged at 400 x g for 5 minutes. After centrifugation, the supernatant was removed, and the cells were resuspended with 1 mL of PBS solution, and the centrifuge tube containing the cells was again centrifuged at 400 x g for 5 minutes. After the second centrifugation, the supernatant in each centrifuge tube was removed, and 100 μL of JC-1 working reagent was added to each centrifuge tube and left to stand for 15 minutes in the dark. After 15 minutes, each centrifuge tube was centrifuged at 400 x g for 5 minutes. After centrifugation, the supernatant in each centrifuge tube was removed, and the cells were washed with 1 mL of buffer solution and centrifuged at 400 x g for 5 minutes, and this step was repeated twice. After the second centrifugation, the supernatant in each centrifuge tube was removed, and 500 μL of 1X PBS solution (with 2% FBS added) was added to each centrifuge tube to resuspend the cells to obtain the test cell solution. Finally, the fluorescence signal of each well of the test cell solution was measured by flow cytometry (excitation light: 488 nm; scattered light: 527 nm & 590 nm) to calculate the mitochondrial membrane potential of the cells for mitochondrial activity analysis.

[0110] 5-3. Test Results:

[0111] It should be noted that the results shown in Figure 3 are presented in relative ratios, i.e., the quantitative results of the blank group are taken as 100 to convert the quantitative results of the experimental and control groups into performance relative to the blank group. The Student T-Test was used to analyze whether there was a statistically significant difference. In the figure, “*” represents a p-value less than 0.05 relative to the control group, “**” represents a p-value less than 0.01 relative to the control group, and “***” represents a p-value less than 0.001 relative to the control group.

[0112] Referring to Figure 3, when the mitochondrial activity reduction measured for the blank group is taken as 100%, the mitochondrial activity reduction of the control group relative to the blank group is 173.5%. That is, the blank group shows normal (no external oxidative damage) conditions, and when the control group is added with hydrogen peroxide (H2O2) to simulate the case where cells are subjected to external oxidative damage, the resulting mitochondrial activity reduction is significantly higher.

[0113] On the other hand, the mitochondrial activity reduction of the experimental group relative to the blank group is 73.1%, and is a significant decrease of 100.4% compared to the control group. That is, Lactobacillus reuteri TCI979 can help cells effectively resist mitochondrial activity reduction caused by hydrogen peroxide, and restore the mitochondrial activity reduction performance caused by external oxidative damage.

[0114] Example 6: Telomerase Activity Test

[0115] Telomerase is a reverse transcriptase with an RNA segment, which is generally believed to repair the DNA of telomeres by reverse transcription, extend the length of telomeres, and maintain the integrity and stability of chromosomes. The test uses the ability of cells to maintain the length of telomeres to infer the influence on cell proliferation, aging, or potential stemness characteristics. The higher the telomerase activity, the stronger the ability to repair the DNA at the end of the chromosome.

[0116] 6-1. Materials and instruments:

[0117] Cells: Human chorionic mesenchymal cells (cbMSC-hTERT-RFP, BCRC, No. 60605, hereinafter referred to as mesenchymal cells).

[0118] Culture medium: 80% minimum essential medium (purchased from Eagle), added with 20% FBS (Fetal Bovine Serum, purchased from Gibco), 1% penicillin-streptomycin (purchased from Gibco), and 1 mM sodium pyruvate (purchased from Thermo Fisher) and 4 ng / mL human basic fibroblast growth factor (Basic Fibroblast Growth Factor, bFGF, purchased from Gibco, 13256-029).

[0119] PBS buffer and DPBS buffer (purchased from Gibco).

[0120] Flow cytometer (purchased from BD Company, model Accuri TM C6Plus).

[0121] 6-2. Test procedure:

[0122] First, 1×10 5 mesenchymal cells were planted in a 24-well cell culture plate and cultured at 37°C and 5% CO2 for 24 hours. After the mesenchymal cells in each well were grown, the mesenchymal cells were separated into a blank group and an experimental group.

[0123] Blank group: only added with culture medium and cultured at 37°C for three days.

[0124] Experimental group: added with cell culture medium with a concentration of 0.125% TCI979 sample prepared in Example 2 and cultured at 37°C for three days.

[0125] Next, the supernatant of each group of cells was removed, and the cells were washed twice with 1X PBS buffer. The interstitial cells were removed from the culture plate by adding trypsin and transferring them to a 1.5 mL centrifuge tube. A 2% fetal bovine serum neutralization reaction was added, and the cells were centrifuged (400 x g) for 5 minutes. The supernatant was removed after centrifugation, and the cells were washed again with 1X PBS buffer. The cells were again centrifuged (400 x g) for 5 minutes, and the medium was added to the centrifuge tube to resuspend the interstitial cells, which formed the test cell solution.

[0126] The red fluorescence signal in the test cell solution of each group was detected using a flow cytometer (wavelength set to 632-647 nm). The fluorescence intensity can reflect the telomerase activity state, and the relative value was calculated.

[0127] 6-3. Test results:

[0128] It should be particularly noted that the results in Figure 4 are presented in relative multiples, i.e., the quantitative results of the blank group are taken as 100 to convert the quantitative results of the experimental and control groups into performance values relative to the blank group. Student's T-Test was used to analyze whether there was a statistically significant difference. In the figure, “*” represents a p-value less than 0.05 relative to the blank group, “**” represents a p-value less than 0.01 relative to the blank group, and “***” represents a p-value less than 0.001 relative to the blank group.

[0129] Referring to Figure 4, as shown in Figure 4, the relative telomerase activity of the experimental group was increased to 103.05% relative to the 100% of the blank group. It can be seen that Lactobacillus reuteri TCI979 can effectively increase the telomerase activity by 3.9%, slowing down the rate of telomere shortening.

[0130] Example 7: Cell size and morphology test under natural aging

[0131] This test observes the effect of Lactobacillus reuteri on cell natural aging. Changes in cell size and morphology are one of the indicators for observing cell aging in vitro cell experiments. When the cell swells and the cell fibers increase, it indicates that the cell is aging seriously. Generally speaking, the main reason for cell natural aging is that cell division causes telomeres to become shorter and more reactive oxygen species accumulate in cells.

[0132] 7-1. Test materials and equipment description:

[0133] Cell line: Human vascular endothelial cells (EA.hy926, hereinafter referred to as endothelial cells) were purchased from ATCC, CRL-2922.

[0134] Medium: 90% DMEM medium (Dulbecco's Modified Eagle Medium, brand: Gibco), 10% Fetal Bovine Serum (FBS, brand: Gibco), 1% Penicillin-streptomycin (brand: Gibco).

[0135] Reagent: Dulbecco's phosphate buffered saline (Gibco, hereinafter referred to as PBS), Trypsin.

[0136] Staining agent: ActinRed TM 555 ReadyProbes TM Reagent (Thermo; Cat. R37112), Hoechst 33342 (Thermo; Cat. 62249).

[0137] Equipment: Fluorescence microscope (brand ZEISS, Vert. Al), Flow cytometer (purchased from BD company, model Accuri TM C6 Plus).

[0138] 7-2. Test procedure:

[0139] First, endothelial cells were seeded in each well of a 6-well culture plate containing 2 mL of medium at a cell number of 2 x 10 5 cells per well, and incubated at 37°C for 24 hours, and then the cells were divided into a blank group and an experimental group.

[0140] Experimental group: incubated with medium containing 0.125% TCI979 sample prepared in Example 2, and incubated at 37°C for 7 days. The medium was changed every 2-3 days during the 7-day cell treatment.

[0141] Blank group: only medium was added, and incubated at 37°C for 7 days. The medium was changed every 2-3 days during the 7-day cell treatment.

[0142] First, trypsin was added to remove endothelial cells from the culture plate, and 4 / 5 of the cells were transferred to a 1.5 mL centrifuge tube. Each centrifuge tube was washed with DPBS to wash the endothelial cells, and then centrifuged (400 x g) for 5 minutes. After centrifugation, the supernatant was removed, 200 μL of DPBS was added to each centrifuge tube to resuspend the cells, and then the test cell solution was formed. Each test cell solution was measured using a flow cytometer. First, the cell size distribution was quantified, then the software was used to frame the large cell group in each group, and the percentage in the framed range was recorded. The results are shown in Figure 5.

[0143] The remaining 1 / 5 cells were seeded in a 24-well culture plate, and 1 drop of ActinRed was added to each well TM 555 ReadyProbes TM The reagent and 300 μL of Hoechst 33342 (1:20,000 dilution) were stained for 15 minutes in the dark. The results were observed and recorded under a fluorescence microscope in the dark, as shown in FIG. 6.

[0144] 7-3. Test results:

[0145] Here, FIG. 5 shows the results in relative multiples, i.e., the quantitative results of the experimental group are converted into the performance relative to the blank group by taking the quantitative results of the blank group as 100. The Student T-Test is used to analyze whether there is a statistically significant difference. In the figure, "*" represents that the p value is less than 0.05 relative to the blank group, "**" represents that the p value is less than 0.01 relative to the blank group, and "***" represents that the p value is less than 0.001 relative to the blank group.

[0146] Referring to FIG. 5, the relative cell size of the experimental group is significantly reduced to 43.1% under the condition that the cell size of the blank group is 100%. Therefore, it can be known that the Lactobacillus reuteri TCI979 can effectively inhibit cell swelling and has a cell aging prevention function.

[0147] Referring to FIG. 6, it can be seen from the same frame range of the blank group and the experimental group that the number of cell nuclei of the blank group is less, the overall cell swelling is obvious, and the number of cell fibers is large. On the contrary, in the frame range of the experimental group, the number of cell nuclei is large, the cell presents an elongated state without swelling, and the number of cell fibers is small and dense. It can be known that the Lactobacillus reuteri can effectively inhibit cell swelling and inhibit excessive proliferation of cell fibers, and has a cell aging prevention function.

[0148] Example 8: Cell size and morphology test under external oxidative damage

[0149] In this test, hydrogen peroxide (H2O2) was added to simulate the changes in cell size and morphology under external oxidative damage to cells.

[0150] 8-1. Test materials and equipment description:

[0151] Cell strain: Human vascular endothelial cells (EA.hy926, hereinafter referred to as endothelial cells) were purchased from ATCC, CRL-2922.

[0152] Medium: 90% DMEM medium (Dulbecco's Modified Eagle Medium, brand: Gibco), 10% fetal bovine serum (Fetal Bovine Serum, brand: Gibco), 1% antibiotic (Penicillin-streptomycin, brand: Gibco).

[0153] Reagent: Phosphate buffer (Dulbecco's phosphate buffered saline brand Gibco, hereinafter referred to as PBS), trypsin, hydrogen peroxide H2O2 (purchased from Sigma).

[0154] Staining agent: ActinRed TM 555 ReadyProbes TM Reagent (Thermo; Cat. R37112), Hoechst 33342 (Thermo; Cat. 62249).

[0155] Equipment: Fluorescence microscope (brand ZEISS, Vert. Al), flow cytometer (purchased from BD company, model Accuri TM C6 Plus).

[0156] 8-2. Test procedure:

[0157] First, the endothelial cells were seeded in each well of a 6-well culture plate containing 2 mL of medium per well at a cell number of 2 x 10 5 cells per well, and incubated at 37°C for 24 hours, and then the cells were divided into a blank group, a control group and an experimental group.

[0158] Blank group: only medium was added, and treated at 37°C for 3 hours.

[0159] Control group: only medium was added, and treated at 37°C for 1 hour, then 30 μM of hydrogen peroxide cell culture medium was added, and treated at 37°C for 2 hours.

[0160] Experimental group: 0.125% TCI979 sample prepared in Example 2 was added to the cell culture medium, and treated at 37°C for 1 hour, then 30 μM of hydrogen peroxide was added, and treated at 37°C for 2 hours.

[0161] Next, trypsin was added to detach the endothelial cells from the culture dish, and the cells were transferred to 1.5 mL centrifuge tubes. Each centrifuge tube was washed with DPBS and centrifuged (400 x g) for 5 minutes. The supernatant was removed, and 200 μL of DPBS was added to each centrifuge tube to resuspend the cells, and the resulting cell suspension was used for the measurement. The flow cytometer was used to measure each cell suspension. First, the cell size distribution was quantified, and then the software was used to select the large cell population in each group, and the percentage of the selected range was recorded. The results are shown in FIG. 7.

[0162] 8-3. Test results:

[0163] It should be noted that the results shown in FIG. 7 are presented in relative ratios, i.e., the quantification results of the blank group are taken as 100 to convert the quantification results of the experimental and control groups into performance relative to the blank group. The Student T-Test was used to analyze whether there was a statistically significant difference. In the figure, "#" represents that the p-value of the control group relative to the blank group is less than 0.05, "##" represents that the p-value of the control group relative to the blank group is less than 0.01, and "###" represents that the p-value of the control group relative to the blank group is less than 0.001, "*" represents that the p-value of the experimental group relative to the control group is less than 0.05, "**" represents that the p-value of the experimental group relative to the control group is less than 0.01, and "***" represents that the p-value of the experimental group relative to the control group is less than 0.001.

[0164] Referring to FIG. 7, the relative cell size of the control group increased to 119% under oxidative damage, while the relative cell size of the experimental group decreased significantly to 93%, with the cell size of the blank group taken as 100%. That is, Lactobacillus reuteri TCI979 can effectively resist cell aging caused by external oxidative damage. Therefore, Lactobacillus reuteri TCI979 can effectively inhibit cell swelling and has a cell aging prevention function.

[0165] Example 9: Human test

[0166] 9-1. Sample: Capsules prepared using the Lactobacillus reuteri powder prepared in Example 2, containing 100 mg of TCI979 powder.

[0167] 9-2. Subjects: 5 subjects. Each subject was an elderly person over 60 years old.

[0168] 9-3. Test items: Blood malondialdehyde (MDA), total antioxidant capacity (TAC), sulfur-containing compounds (f-Thiols), upper limb muscle endurance, and lower limb muscle endurance.

[0169] Among them, blood malondialdehyde (MDA), total antioxidant capacity (TAC), sulfur compounds (f-Thiols) are measured after blood by the commission of the medical laboratory.

[0170] Among them, the test method of upper limb muscle endurance: each subject uses the number of times of lifting dumbbells in 30 seconds with the dominant hand, wherein lifting dumbbells refers to the state of bending and straightening the elbow with the palm holding the dumbbell, and the dumbbell used by females is 5 pounds and the dumbbell used by males is 8 pounds.

[0171] Among them, the test method of lower limb muscle endurance: each subject stands up and sits down from a chair in 30 seconds.

[0172] 9-4. Test procedure:

[0173] Five subjects were instructed to take 100 mg of the capsule prepared from the Lactobacillus reuteri powder in Example 2 every day, and the intake was continued for eight weeks. Measurements were taken before the intake began (i.e., at week 0, also referred to as the blank group), after four weeks of drinking (i.e., at week 4, also referred to as experimental group A), and after eight weeks of drinking (i.e., at week 8, also referred to as experimental group B).

[0174] Blood malondialdehyde, total antioxidant capacity, and sulfur compounds were calculated using the values provided in the report from the medical laboratory, and the average values of the five subjects were presented.

[0175] The upper limb muscle endurance and lower limb muscle endurance test results were based on week 0, and the fourth week and the eighth week were presented in relative multiples, that is, the quantitative results of the fourth week and the eighth week were converted into relative percentages based on the quantitative results of week 0 being considered as 100%, in order to more clearly present the improvement status of each test item from the beginning to the end of the test.

[0176] 9-5. Test results:

[0177] Please refer to FIG. 8. Under the condition of taking 100 mg of Lactobacillus reuteri every day and maintaining daily diet and routine, the average blood malondialdehyde (MDA) concentration of the five subjects decreased significantly from 1.52 mmol / L (blank group) to 1.17 mmol / L (experimental group A) after four weeks of intake, and although it adjusted slightly to 1.28 mmol / L (experimental group B) after eight weeks of intake, a overall downward trend was still observed after taking Lactobacillus reuteri TCI979. Among them, the blood malondialdehyde (MDA) concentrations of the five subjects all decreased, that is, the proportion of improved people reached 100%. It can be observed that the difference in average blood malondialdehyde before and after using the Lactobacillus reuteri of the present application for only eight weeks reached 15.8%. That is, taking 100 mg of Lactobacillus reuteri every day can effectively reduce the blood malondialdehyde concentration in the blood and improve the antioxidant capacity.

[0178] Referring to FIG. 9, the average total antioxidant capacity (TAC) of 5 subjects was increased from 0.56 (blank group) to 0.60 (experimental group A) after 4 weeks of ingestion of 100 mg of Lactobacillus reuteri per day and maintained the daily diet and routine, and was maintained at 0.60 (experimental group B) after 8 weeks of ingestion, and it was observed that the overall trend was increased after ingestion of Lactobacillus reuteri TCI979. Among the 5 subjects, 3 subjects showed an increase in total antioxidant capacity, that is, the proportion of improved persons was 60%. It was observed that the difference in average blood total antioxidant capacity before and after the use of Lactobacillus reuteri of the present application for only 8 weeks was 7.1%. That is, the ingestion of 100 mg of Lactobacillus reuteri per day can effectively improve the antioxidant capacity.

[0179] Referring to FIG. 10, the average f-thiols in the blood of 5 subjects was increased from 235.2 μg / mL (blank group) to 263.2 μg / mL (experimental group A) after 4 weeks of ingestion of 100 mg of Lactobacillus reuteri per day and maintained the daily diet and routine, and was increased to 275.6 μg / mL (experimental group B) after 8 weeks of ingestion, and it was observed that the overall trend was increased after ingestion of Lactobacillus reuteri TCI979. Among the 5 subjects, 4 subjects showed a significant increase in f-thiols in the blood, that is, the proportion of improved persons was 80%. It was observed that the difference in average f-thiols in the blood before and after the use of Lactobacillus reuteri of the present application for only 8 weeks was 17.2%. That is, the ingestion of 100 mg of Lactobacillus reuteri per day can effectively improve the f-thiols in the blood to effectively alleviate the damage caused by active oxygen free radicals.

[0180] Referring to FIG. 11, the average upper limb muscle endurance of 5 subjects was increased from 100% (blank group) to 142.7% (experimental group A) after 4 weeks of ingestion of 100 mg of Lactobacillus reuteri per day and maintained the daily diet and routine, and was continuously increased to 169.8% (experimental group B) after 8 weeks of ingestion, and it was observed that the overall trend was significantly increased after ingestion of Lactobacillus reuteri TCI979. Among the 5 subjects, all subjects showed a significant increase in upper limb muscle endurance, that is, the proportion of improved persons was 100%. It was observed that the difference in average upper limb muscle endurance before and after the use of Lactobacillus reuteri of the present application for only 8 weeks was 69.8%. That is, the ingestion of 100 mg of Lactobacillus reuteri per day can effectively improve the upper limb muscle endurance.

[0181] Referring to FIG. 12, the average lower limb muscle endurance of the 5 subjects increased from 100% (blank group) to 129.0% (experimental group A) after 4 weeks of ingestion of 100 mg of Lactobacillus reuteri per day and maintaining the daily diet and routine, and continuously increased to 151.5% (experimental group B) after 8 weeks of ingestion. It can be seen that the overall trend of Lactobacillus reuteri TCI979 is significantly increased after ingestion. Among them, the lower limb muscle endurance of 4 of the 5 subjects showed a significant increase, that is, the proportion of the number of people improved reached 80%. It can be observed that the average lower limb muscle endurance difference before and after using the Lactobacillus reuteri of the present application for only 8 weeks reached 51.5%. That is, the daily ingestion of 100 mg of Lactobacillus reuteri can effectively improve the lower limb muscle endurance.

[0182] Of course, the present application can have other various embodiments, and those skilled in the art can make various corresponding changes and modifications according to the present application without departing from the spirit and essence of the present application. These corresponding changes and modifications should all belong to the protection scope of the claims of the present application.

[0183] Industrial applicability

[0184] In summary, the Lactobacillus reuteri and / or metabolites thereof of any embodiment can reduce the content of malondialdehyde in blood, improve the total antioxidant capacity or the content of sulfur compounds in blood, inhibit the content of reactive oxygen species or reduce cell apoptosis, thereby slowing down muscle loss. The Lactobacillus reuteri and / or metabolites thereof of any embodiment can improve muscle cell mitochondrial activity, reduce the content of malondialdehyde in blood, improve the total antioxidant capacity or the content of sulfur compounds in blood, or inhibit the content of reactive oxygen species, thereby improving muscle endurance. The Lactobacillus reuteri and / or metabolites thereof of any embodiment can slow down the rate of telomere shortening, improve telomerase activity, or inhibit the content of reactive oxygen species, thereby slowing down cell aging.

Claims

1. Use of Lactobacillus reuteri and / or metabolites thereof for the manufacture of a composition for slowing down muscle loss, wherein the Lactobacillus reuteri is Lactobacillus reuteri having the deposit accession number DSM 34539.

2. Use according to claim 1, wherein the Lactobacillus reuteri and / or metabolites thereof are for reducing the level of malondialdehyde in blood.

3. Use according to claim 1, wherein the Lactobacillus reuteri and / or metabolites thereof are for increasing the total antioxidant capacity (TAC) or the level of f-Thiols in blood.

4. Use according to claim 1, wherein the Lactobacillus reuteri and / or metabolites thereof are for inhibiting the level of reactive oxygen species.

5. Use according to claim 1, wherein the Lactobacillus reuteri and / or metabolites thereof are for reducing apoptosis.

6. Use of Lactobacillus reuteri and / or metabolites thereof for the manufacture of a composition for increasing muscle endurance, wherein the Lactobacillus reuteri is Lactobacillus reuteri having the deposit accession number DSM 34539.

7. Use according to claim 6, wherein the Lactobacillus reuteri and / or metabolites thereof are for increasing mitochondrial activity in muscle cells.

8. Use according to claim 6, wherein the Lactobacillus reuteri and / or metabolites thereof are for reducing the level of malondialdehyde in blood.

9. Use according to claim 6, wherein the Lactobacillus reuteri and / or metabolites thereof are for increasing the total antioxidant capacity (TAC) or the level of f-Thiols in blood.

10. Use according to claim 6, wherein the Lactobacillus reuteri and / or metabolites thereof are for inhibiting the level of reactive oxygen species.

11. Use of Lactobacillus reuteri and / or metabolites thereof for the manufacture of a composition for slowing down cellular aging, wherein the Lactobacillus reuteri is Lactobacillus reuteri having the deposit accession number DSM 34539.

12. Use according to claim 11, wherein the Lactobacillus reuteri and / or metabolites thereof are for slowing down the rate of telomere shortening.

13. Use according to claim 12, wherein the Lactobacillus reuteri and / or metabolites thereof are for increasing the activity of telomerase.

14. Use according to claim 11, wherein the Lactobacillus reuteri and / or metabolites thereof are for inhibiting the level of reactive oxygen species.

Images