Agent for inducing SIRT1 gene expression

Abstract

To provide a preparation for inducing SIRT1 gene expression that allows consumers to feel a sense of Japanese style and can be blended in food, cosmetics or pharmaceuticals to induce the expression of human sirtuin 1 gene expression induction activity.SOLUTION: A preparation for inducing SIRT1 gene expression includes lactic acid bacteria derived from Japanese cherries, known for their SIRT1 gene expression induction activity, or the bacteria's crushed cells or a culture solution of these cells. The lactic acid bacteria are one or more selected from Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paraplantarum, and Lactobacillus brevis.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to a preparation for inducing SIRT1 gene expression.

Background Art

[0002] Since ancient times, foods using lactic acid bacteria such as yogurt and pickles have been widely consumed.

[0003] In addition to its intestinal regulating effect, lactic acid bacteria have recently been found to have an immune enhancing effect and a function of inducing the expression of the sirtuin gene, and foods using lactic acid bacteria are attracting increasing attention.

[0004] Among them, the function of inducing the expression of the sirtuin gene is an action that promotes the expression of the sirtuin gene, which is considered to support healthy longevity, and it is expected that the intake of foods using lactic acid bacteria may contribute to maintaining healthy longevity.

[0005] Therefore, conventionally, a life-prolonging substance containing lactic acid bacteria having a function of enhancing the promoter activity of the sirtuin gene has been proposed (see, for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] However, a functional preparation that enhances the promoter activity of the sirtuin gene, which is specialized for lactic acid bacteria derived from plants, has not been proposed so far.

[0008] Microorganisms derived from healthy vegetables, fruits, beautiful flowers, and trees can give a favorable impression corresponding to the plants from which they are derived to those who consume foods, cosmetics, pharmaceuticals, etc., expecting life-prolonging effects by lactic acid bacteria.

[0009] Among them, cherry blossoms are a symbol of Japan and have a strong image of harmony. If lactic acid bacteria separated from cherry blossoms can achieve these functions, an appealing effect can be expected that they are safe, reassuring, and healthy preparations for consumers.

[0010] The present invention has been made in view of such circumstances, and provides a preparation for inducing the expression of the human sirtuin 1 gene (hereinafter also referred to as the SIRT1 gene) that can make consumers feel an image of harmony and can promote the expression of the expression-inducing activity of the SIRT1 gene when contained in foods, cosmetics, pharmaceuticals, etc. Further, the present invention also provides foods, cosmetics, and pharmaceuticals that can be expected to induce the expression of the SIRT1 gene by containing the preparation.

Means for Solving the Problems

[0011] In order to solve the above conventional problems, the preparation for inducing the expression of the SIRT1 gene according to the present invention is (1) At least one selected from Lactobacillus pentosus strain A51 (NITE AP-03922), Lactobacillus pentosus strain A55 (NITE AP-03923), Lactobacillus fermentum strain A89 (NITE AP-03924), Lactobacillus plantarum strain B15 (NITE AP-03928), Lactobacillus casei strain C24 (NITE AP-03929) It contains lactic acid bacteria derived from cherry blossoms having SIRT1 gene expression-inducing activity, a disrupted product of the bacterial cells, or a culture solution of the bacterial cells.

[0012] Further, the preparation for inducing the expression of the SIRT1 gene according to the present invention also has the following features. (2) The lactic acid bacteria are Lactobacillus pentosus Strain A55 (NITE AP-03923) and is. (3 ) It is lactic acid bacteria derived from cherry blossoms having SIRT1 gene expression-inducing activity LIt contains a cell mixture of Lactobacillus plantarum strain B15 (NITE AP-03928) and Lactobacillus casei strain C24 (NITE AP-03929), a crushed product of the cell mixture, or a mixture of culture solutions of each cell.

[0013] Also, the For inducing SIRT1 gene expression food product is (4) The lactic acid bacterium is Lactobacillus fermentum strain A89 (NITE AP-03924) or Lactobacillus casei strain C24 (NITE AP-03929) described above (1 ) to and contains the preparation for inducing SIRT1 gene expression described above. In addition, in the cosmetic for inducing SIRT1 gene expression according to the present invention, (5) the lactic acid bacterium is at least one selected from Lactobacillus pentosus strain A51 (NITE AP-03922), Lactobacillus pentosus strain A55 (NITE AP-03923), Lactobacillus fermentum strain A89 (NITE AP-03924), Lactobacillus plantarum strain B15 (NITE AP-03928), and the preparation for inducing SIRT1 gene expression described in (1) above or the preparation for inducing SIRT1 gene expression described in (2) above is contained. In addition, in the pharmaceutical for inducing SIRT1 gene expression according to the present invention, (6) the preparation for inducing SIRT1 gene expression described in (1) above, wherein the lactic acid bacterium is Lactobacillus fermentum strain A89 (NITE AP-03924) or Lactobacillus casei strain C24 (NITE AP-03929) is contained. In the present invention, in the production of a food for inducing SIRT1 gene expression, a cosmetic for inducing SIRT1 gene expression, or a pharmaceutical for inducing SIRT1 gene expression, Lactobacillus pentosus A55 strain (NITE AP-03923) is used in combination with Lactococcous lactis having bifidobacteria-activating activity, antibacterial activity, skin resident flora balance improving effect, or fibroblast-activating effect as a main component for inducing at least any one selected from SIRT1 gene expression-inducing activity, GABA productivity, bifidobacteria-activating activity, antibacterial activity, skin resident flora balance improving effect, or fibroblast-activating effect.

Advantages of the Invention

[0014] According to the lactic acid bacteria of the present invention, since it is a preparation for inducing SIRT1 gene expression containing lactic acid bacteria derived from cherry blossoms having SIRT1 gene expression induction activity, a crushed product of the cells, or a culture solution of the cells, it can make consumers feel an image of harmony, and can provide a preparation for inducing SIRT1 gene expression that can be contained in foods, cosmetics, pharmaceuticals, etc. to promote the expression of SIRT1 gene expression induction activity.

[0015] Also, according to the foods, cosmetics, and pharmaceuticals of the present invention, since they contain the preparation for inducing SIRT1 gene expression of the present invention, it is possible to provide foods, cosmetics, and pharmaceuticals in which the induction of SIRT1 gene expression is expected.

Brief Description of the Drawings

[0016]

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Modes for Carrying Out the Invention

[0017] The present invention provides a preparation for inducing SIRT1 gene expression, which comprises a lactic acid bacterium derived from cherry blossoms having an activity of inducing SIRT1 gene expression, a disrupted product of the bacterial cells, or a culture solution of the bacterial cells.

[0018] The SIRT1 gene is a gene encoding a histone deacetylase, and its relationship with anti-aging effects and cellular stress resistance is known. It is also known that an increase in its expression level extends the lifespan of cells and animals that are the test subjects.

[0019] Moreover, as the lactic acid bacterium derived from cherry blossoms used in the preparation for inducing SIRT1 gene expression according to the present embodiment, for example, a lactic acid bacterium isolated from cherry blossoms (Prunus × yedoensis) can be used.

[0020] In addition, the preparation for inducing SIRT1 gene expression according to the present embodiment may contain a lactic acid bacterium derived from cherry blossoms, but may also contain a disrupted product of the lactic acid bacterium derived from cherry blossoms or a culture solution of the lactic acid bacterium derived from cherry blossoms (hereinafter, also collectively referred to as cherry blossom lactic acid bacterium components).

[0021] That is, the preparation for inducing SIRT1 gene expression contains the cherry blossom lactic acid bacterium components as the main components for inducing functions. For example, it can be the bacterial cells obtained by culturing, growing, and collecting in a predetermined medium, or can contain disrupted products obtained by physically or chemically treating these, such as homogenizing or enzyme-treating them.

[0022] Moreover, for example, it may contain a culture solution obtained by liquid-culturing a lactic acid bacterium derived from cherry blossoms. The culture solution obtained by this liquid culture may be in a state containing the bacterial cells, or may be one from which the bacterial cells have been removed.

[0023] The preparation for inducing SIRT1 gene expression according to the present embodiment can be composed only of the above-described cherry blossom lactic acid bacterium components, but in addition to these cherry blossom lactic acid bacterium components, it can also have a configuration containing other auxiliary components.

[0024] The auxiliary components are not particularly limited as long as they do not prevent the induction of SIRT1 gene expression to the extent of losing practicality, and may include excipients and additives according to the dosage form, food form, etc., and components for adding other functions.

[0025] As the excipient, for example, when the dosage form is a solid preparation, lactose, crystalline cellulose, starch, etc. can be used.

[0026] Examples of the additives include stabilizers, surfactants, solubilizers, plasticizers, sweeteners, antioxidants, flavoring agents, coloring agents, preservatives, inorganic fillers, etc.

[0027] Examples of the components for adding other functions include, for example, other lactic acid bacteria derived from cherry blossoms having the activity of inducing SIRT1 gene expression and lactic acid bacteria derived from cherry blossoms having useful physiological activities but not having the activity of inducing SIRT1 gene expression, as well as lactic acid bacteria other than lactic acid bacteria derived from cherry blossoms having useful physiological activities.

[0028] Note that "contained as the main component for inducing a function" means, when expressed by replacing it with a pharmaceutical product, something like "contained as an active ingredient". That is, assuming a health functional food, in a functional indication food, it is a functional related component, in a nutritional functional food, it is a nutritional component, and in a specific health food, it is a related component, etc. Although the expressions are different, it means containing the cells of lactic acid bacteria or the crushed product of the same cells, or the culture solution of lactic acid bacteria as a component for exerting functions and effects. Also, in general foods that do not require special permits, etc. for sale, it also means containing the above-mentioned cells, crushed products, and culture solutions as the main component for inducing the function of inducing SIRT1 gene expression.

[0029] Also, in the present application, a pharmaceutical product containing the above-mentioned preparation for inducing SIRT1 gene expression is also provided. The pharmaceutical product according to this embodiment is characterized in that it contains the above-mentioned preparation for inducing SIRT1 gene expression as the main component for inducing a function. The pharmaceutical product in the present application may be orally ingested or may be used as a topical skin preparation.

[0030] The present application also provides a cosmetic containing the above-described agent for inducing SIRT1 gene expression.

[0031] In the present application, the cosmetic includes, in addition to so-called makeup cosmetics, toiletry products such as soaps, detergents, and bath agents used for basic cosmetics, hair tonics, perfumes, toothpastes, shampoos, rinses, and body washing, etc., and also includes pharmaceutical cosmetics that claim preventive effects, etc.

[0032] The present application also provides a food, a general food, or a functional food containing the above-described agent for inducing SIRT1 gene expression, and in particular, provides a food that is expected to induce the expression of the SIRT1 gene by oral ingestion.

[0033] Functional food is a concept that encompasses all foods that are said to contribute to maintaining and promoting health without containing pharmaceutical ingredients. For example, in addition to dietary supplements, health supplements, and nutritional adjustment foods, it also includes general foods such as so-called supplements, as well as health functional foods such as foods for specified health uses, nutritional functional foods, and foods with function claims.

[0034] Also, when in the form of a supplement, its dosage form is not particularly limited, and any dosage form such as tablets, capsules, fine granules, pills, troches, liquids, jelly-like substances, etc. can be selected.

[0035] Note that the descriptions of the above-mentioned pharmaceuticals, cosmetics, and functional foods are examples of articles corresponding to these for the purpose of understanding the present invention, and the interpretation of each phrase is not limited to these listed articles, etc. However, when the applicant of the present application seeks to obtain patent rights for the present application, it does not prevent the present invention from being limited to these articles, etc.

[0036] Hereinafter, regarding the agent for inducing SIRT1 gene expression according to the present embodiment, it will be further described with reference to the experimental process and results.

[0037] [1. Isolation of plant-derived lactic acid bacteria] The inflorescence part collected from Yoshino cherry was placed in a sterilized bag and immersed in sake mash medium, then left standing at room temperature for 1 to 2 weeks to perform pre-culture of the microbial community adhering to the inflorescence.

[0038] Next, the pre-cultured sake mash medium was streaked on white MRS agar medium and cultured at 30 °C for 3 days.

[0039] Next, the strains showing the formation of clear zones in the above culture were isolated and re-pure cultured on MRS agar medium at 30 °C for 3 days. The bacteria obtained by the culture were collected and stored at -80 °C as glycerol stocks. In this way, 166 strains of lactic acid bacteria were isolated from cherry blossoms.

[0040] The identification of lactic acid bacteria was performed by MALDI-TOF MS. As a result, among the 166 strains of lactic acid bacteria, there were 2 strains of the genus Enterococcus, 41 strains of the genus Lactobacillus, 10 strains of the genus Lactococcus, 58 strains of the genus Leuconostoc, and 55 strains of the genus Pediococcus.

[0041] More specifically, among the Enterococcus genus, there were 2 strains of Enterococcus mundtii. Among the Lactobacillus genus, there were 4 strains of Lactobacillus brevis (Levilactobacillus brevis), 3 strains of Lactobacillus casei (Lacticaseibacillus casei), 2 strains of Lactobacillus fermentum (Limosilactobacillus fermentum), 1 strain of Lactobacillus paraplantarum (Lactiplantibacillus paraplantarum), 12 strains of Lactobacillus pentosus (Lactiplantibacillus pentosus), and 19 strains of Lactobacillus plantarum (Lactiplantibacillus plantarum), for a total of 41 strains. Among the Lactococcus genus, there were 10 strains of Lactococcus lactis. Among the Leuconostoc genus, there were 50 strains of Leuconostoc mesenteroides, 4 strains of Leuconostoc pseudomesenteroides, 3 strains of Leuconostoc holzapheii, and 1 strain of Leuconostoc citreum-holzapheii, for a total of 58 strains. Among the Pediococcus genus, there were 55 strains of Pediococcus pentosaceus. Each isolated strain was assigned a number for identification, and the strain names were based on the same numbers. Figure 1 shows a list of the isolated and identified lactic acid bacteria.

[0042] [2. Confirmation test for induction of SIRT1 gene expression] Next, among the 166 strains isolated and identified from cherry blossoms as described above, a confirmation test for the induction activity of SIRT1 gene expression was conducted on 18 strains. The strains used in the test are shown in Figure 2.

[0043] In this section, tests were conducted on two types of tests: the "test by the direct method", which simulates the case of topically applying the preparation for inducing SIRT1 gene expression according to this embodiment, such as cosmetics or topical medications, and the "test by the indirect method", which simulates the case of orally ingesting the preparation for inducing SIRT1 gene expression according to this embodiment, such as foods or oral medications.

[0044] (2-1) Test by the direct method In the test by the direct method, a method developed by the present inventor for confirming the induction of SIRT1 gene expression was used. This method uses a phSIRT1p-GFP stable expression animal cell line (HaCaT cells: human epidermal keratinocyte cell line) established using a vector (phSIRT1p-GFP) constructed by inserting a human SIRT1 promoter instead of the CMV promoter based on the pEGFP-C3 vector. For the culture wells of this cell line, the cells of any one of the above 18 strains of lactic acid bacteria were added, and the transcriptional activity of human SIRT1 was traced by the GFP fluorescence intensity. Figure 3 shows the results of the test by the direct method.

[0045] As shown in Figure 3, among the 18 strains tested by the direct method, significant differences were confirmed for 8 strains, namely Lactobacillus pentosus A43 strain, Lactobacillus pentosus A51 strain, Lactobacillus plantarum A52 strain, Lactobacillus plantarum A53 strain, Lactobacillus paraplantarum A54 strain, Lactobacillus pentosus A55 strain, Lactobacillus pentosus A56 strain, and Lactobacillus brevis A95 strain (hereinafter also referred to as SIRT1 direct method induction strains). The control was a similar test conducted by adding PBS (Phosphate-buffered saline) instead of the cells.

[0046] Among them, Lactobacillus pentosus strain A51 has an induction activity relative to the control of 1.19 times at the first time, 1.08 times at the second time, and 1.01 times at the third time. Compared with other strains, the variation is small, and the average is about 1.09 times, which is a relatively high value. Therefore, it was shown to be a particularly useful strain.

[0047] Similarly, for Lactobacillus pentosus strain A55, the induction activity relative to the control is 1.11 times at the first time, 1.07 times at the second time, and 1.09 times at the third time. Compared with other strains, the variation is small, and the average is about 1.09 times, which is a relatively high value. Therefore, it was shown to be a particularly useful strain.

[0048] Similarly, for Lactobacillus brevis strain A95, the induction activity relative to the control is 1.13 times at the first time, 1.18 times at the second time, and 1.08 times at the third time. Compared with other strains, the variation is small, and the average is about 1.13 times, which is a relatively high value. Therefore, it was shown to be a particularly useful strain.

[0049] From these results, it was suggested that the SIRT1 direct method-induced strains can promote the expression of the induction activity of the human SIRT1 gene, and it is possible to provide a preparation for inducing SIRT1 gene expression that can promote the expression of the same activity when contained in foods, cosmetics, pharmaceuticals, etc., and particularly can promote the expression of the same activity by external application such as cosmetics and topical medications.

[0050] (2-2) Test by indirect method The test by the indirect method also shares something in common with the test by the direct method in that it uses the method developed by the present inventor for confirming the induction of SIRT1 gene expression. However, in the direct method, for the phSIRT1p-GFP stable expression animal cell line (HaCaT) cultured in a microplate, lactic acid bacteria cells were directly added to the medium to observe the expression level of GFP via the human SIRT1 promoter. In contrast, in the indirect method, as shown in Fig. 4, lactic acid bacteria cells were added to and cultured with Caco-2 cells, an intestinal-derived cell line seeded in another microplate, and the culture supernatant containing this metabolite was added to the phSIRT1p-GFP stable expression animal cell line (HaCaT) to observe the expression level of GFP, which is different. To add, it can be said that the indirect method mimics the case of oral ingestion of the SIRT1 gene expression-inducing preparation by mediating the metabolism by Caco-2 cells in advance. The experiment was conducted 3 times for each lactic acid bacterial strain, and the addition amount of lactic acid bacteria cells to Caco-2 cells was 30 μg / ml for the first time and 90 μg / ml for the second and third times.

[0051] Also, considering the results through the first to third times for each strain, a strain in which stronger induction than the control was observed in 2 or more of the 3 tests and which was relatively good among them was judged as a strain for which the effect was confirmed. Fig. 5 shows the results of the test by the indirect method.

[0052] As shown in Fig. 5, among the 18 strains subjected to the indirect method test, effects were confirmed in 11 strains, namely Lactobacillus brevis A46 strain, Lactobacillus plantarum A53 strain, Lactobacillus paraplantarum A54 strain, Lactobacillus pentosus A55 strain, Lactobacillus fermentum A89 strain, Lactobacillus fermentum A90 strain, Lactobacillus brevis A94 strain, Lactobacillus brevis A95 strain, Lactobacillus brevis A96 strain, Lactobacillus plantarum B15 strain, and Lactobacillus casei C24 strain (hereinafter also referred to as SIRT1 indirect method induction strains).

[0053] Among them, Lactobacillus brevis A46 strain, Lactobacillus pentosus A55 strain, Lactobacillus fermentum A89 strain, Lactobacillus brevis A94 strain, Lactobacillus brevis A95 strain, Lactobacillus brevis A96 strain, Lactobacillus plantarum B15 strain, and Lactobacillus casei C24 strain were considered to be particularly useful strains.

[0054] Specifically, the induction activity of Lactobacillus brevis A46 strain relative to the control was 1.01 times at the first time, 1.05 times at the second time, and 1.06 times at the third time. Compared with other strains, the variation was small, and the average value was 1.04 times, which is a slightly higher value. Therefore, it was shown to be a particularly useful strain.

[0055] In addition, Lactobacillus pentosus strain A55 had induction activities of 1.01-fold at the first time, 1.08-fold at the second time, and 1.00-fold at the third time compared to the control. Since the value at the second time was slightly higher than that of other strains and the average value was 1.03-fold, it was shown to be a particularly useful strain.

[0056] In addition, Lactobacillus fermentum strain A89 had induction activities of 1.05-fold at the first time, 1.06-fold at the second time, and 1.12-fold at the third time compared to the control. Although it was relatively stable in all three times, the value at the third time was slightly higher than that of other strains and the average value was 1.076-fold, so it was shown to be a particularly useful strain.

[0057] In addition, Lactobacillus brevis strain A94 had induction activities of 1.045-fold at the first time, 1.12-fold at the second time, and 1.045-fold at the third time compared to the control. Since the value at the second time was slightly higher than that of other strains and the average value was 1.07-fold, it was shown to be a particularly useful strain.

[0058] In addition, Lactobacillus brevis strain A95 had induction activities of 1.03-fold at the first time, 1.04-fold at the second time, and 1.04-fold at the third time compared to the control. It had less variation compared to other strains and the average value was 1.036-fold, so it was shown to be a particularly useful strain.

[0059] In addition, Lactobacillus brevis strain A96 had induction activities of 1.03-fold at the first time, 1.16-fold at the second time, and 1.07-fold at the third time compared to the control. Since the value at the second time was higher than that of other strains and the average value was about 1.086-fold, it was shown to be a particularly useful strain.

[0060] In addition, Lactobacillus plantarum strain B15 had an induction activity relative to the control of 1.03-fold at the first time, 1.10-fold at the second time, and 1.03-fold at the third time. Since the value at the second time was higher than that of other strains and the average value was 1.05-fold, it was shown to be a particularly useful strain.

[0061] In addition, Lactobacillus casei strain C24 had an induction activity relative to the control of 1.045-fold at the first time, 1.06-fold at the second time, and 1.05-fold at the third time. Since it was overall stable and the average value was 1.05-fold, it was shown to be a particularly useful strain.

[0062] From these results, it was suggested that the SIRT1 indirect induction strains could promote the expression of the induction activity of the human SIRT1 gene, and it was possible to provide a preparation for inducing SIRT1 gene expression that could promote the expression of the same activity when contained in foods, cosmetics, pharmaceuticals, etc., and particularly could promote the expression of the same activity by oral ingestion such as foods and oral medications.

[0063] [3. Evaluation of GABA productivity] Next, among the 166 identified lactic acid bacteria strains derived from cherry blossoms, in order to find lactic acid bacteria having the ability to produce GABA (γ-aminobutyric acid), the GABA productivity was evaluated.

[0064] Specifically, glutamic acid with a final concentration of 100 mM was added to the culture solution obtained by inoculating each strain into MRS liquid medium and culturing it, and 2 μL of the centrifuged supernatant was subjected to thin layer chromatography to observe the color development by the ninhydrin reaction to evaluate the GABA productivity.

[0065] As a result, as shown in Fig. 6, remarkable GABA productivity was confirmed in five strains, namely Lactobacillus brevis A46 strain, Lactobacillus pentosus A55 strain, Leuconostoc mesenteroides A93 strain, Lactobacillus brevis A95 strain, and Lactobacillus brevis A96 strain.

[0066] [4. Bifidobacterium activation activity confirmation test] Next, among the 166 identified strains of cherry-derived lactic acid bacteria, a confirmation test was conducted to find lactic acid bacteria having the activation activity of Bifidobacterium.

[0067] In this test, as shown in Fig. 7, first, a culture solution (final concentration 5%) of Bifidobacterium cultured in thioglycolic acid liquid medium and a heat-killed cell lysate of each cherry-derived lactic acid bacterium (final concentration 10%) were added to 3 mL of skim milk medium, and the mixture was cultured at 37 °C for 5 days under anaerobic conditions.

[0068] Next, the pH of the obtained culture solution was measured, and the viable cell count was measured by serially diluting the culture solution and spreading it on TOS propionic acid agar medium for culture.

[0069] The heat-killed cell lysate of each cherry-derived lactic acid bacterium is a solution obtained by culturing a predetermined cherry-derived lactic acid bacterium strain in MRS liquid medium at 30 °C for 3 days, collecting the cells, washing the collected cells with sterilized water, heating them at 90 °C for 30 minutes to obtain dead cells, freeze-drying them, adding sterilized water to the obtained freeze-dried cells to adjust the concentration to 10 mg / mL, and performing ultrasonic disruption and reheating, as shown in Fig. 8.

[0070] As a result of this test, as shown in Fig. 9, activation activity of Bifidobacterium was confirmed in six strains, namely Lactococcus lactis strain A19, Lactococcus lactis strain A26, Lactococcus lactis strain A27, Lactobacillus brevis strain A46, Lactobacillus brevis strain A95, and Lactobacillus brevis strain A96.

[0071] In addition, based on the results of this test and the inventors' experience, there was not much tendency for the degree of Bifidobacterium activation activity to depend on the genus or species. Even within the same genus or species, there were strains with high activation activity, and in some cases, there were also strains that suppressed the activation.

[0072] Among them, Lactobacillus brevis strain A46, Lactobacillus brevis strain A95, and Lactobacillus brevis strain A96, which had high activation activity against the control, were found to be particularly useful strains.

[0073] Specifically, since the activation activity against the control was slightly higher at 1.17-fold for Lactobacillus brevis strain A46, 2.01-fold higher for Lactobacillus brevis strain A95, and very high at 2.52-fold for Lactobacillus brevis strain A96, they were judged to be useful strains.

[0074] 〔5. Antibacterial Activity Confirmation Test〕 Next, a confirmation test was conducted to find lactic acid bacteria among the 166 identified cherry-derived lactic acid bacteria strains that had antibacterial activity against Staphylococcus aureus subsp. aureus NBRC12732.

[0075] As shown in Fig. 10, 1 mL of the culture solution obtained by culturing Staphylococcus aureus, which is the test bacterium, in broth medium was added to 4 mL of 0.8% agar broth medium, and after thorough stirring, it was mixed with 10 mL of 1.5% agar medium.

[0076] Also, the culture supernatant of lactic acid bacteria derived from cherry blossoms, which is the activity confirmation bacterium, was added to the dilution medium prepared in this way, and the formation of an inhibition zone was confirmed.

[0077] As a result, antibacterial activity against Staphylococcus aureus was confirmed in four strains, namely Lactococcus lactis A19 strain, Lactococcus lactis A26 strain, Lactococcus lactis A27 strain, and Lactococcus lactis A33 strain.

[0078] Next, regarding these four strains in which antibacterial activity against Staphylococcus aureus was confirmed, the antibacterial activity against other test bacteria was confirmed. Specifically, 10 strains, namely Lactobacillus brevis NBRC3345, Latilactobacillus sakei subsp. sakei NBRC15893, Lactococcus lactis subsp. lactis NBRC100933, Enterococcus faecalis NBRC12966, Bacillus subtilis subsp. subtilis NBRC3108, Bacillus coaglans NBRC12583, Cutibacterium acnes NBRC107605, Staphylococcus epidermidis NBRC100911, Escherichia coli NBRC3972, and Pseudomonas aeruginosa NBRC3080, were tested as test bacteria. The test method was the same as that for Staphylococcus aureus.

[0079] Figure 11 is an explanatory diagram showing the antibacterial activity of four strains of lactic acid bacteria derived from cherry blossoms against a total of 11 strains, including Staphylococcus aureus and 10 strains of test bacteria for additional tests. As can be seen from Figure 11, Lactococcus lactis A19 strain and Lactococcus lactis A26 strain have antibacterial activity against Lactobacillus brevis NBRC3345, Latilactobacillus sakei subsp. sakei NBRC15893, Enterococcus faecalis NBRC12966, Bacillus subtilis subsp. subtilis NBRC3108, Bacillus coaglans NBRC12583, Cutibacterium acnes NBRC107605 in addition to Staphylococcus aureus (Staphylococcus aureus subsp. aureus NBRC12732).

[0080] In addition, Lactococcus lactis A27 strain has been shown to have antibacterial activity against Lactobacillus brevis NBRC3345, Latilactobacillus sakei subsp. sakei NBRC15893, Bacillus subtilis subsp. subtilis NBRC3108, Bacillus coaglans NBRC12583, Cutibacterium acnes NBRC107605 in addition to Staphylococcus aureus.

[0081] In addition, Lactococcus lactis A33 strain has been shown to have antibacterial activity against Latilactobacillus sakei subsp. sakei NBRC15893, Bacillus coaglans NBRC12583, Cutibacterium acnes NBRC107605 in addition to Staphylococcus aureus.

[0082] 〔6. Evaluation Test of the Effect of Improving the Balance of Skin Resident Bacteria〕 Next, for the four strains whose antibacterial activity against Staphylococcus aureus was confirmed in [5. Antibacterial Activity Confirmation Test], a test was conducted to evaluate the effect of improving the balance of skin commensal bacteria.

[0083] Specifically, as shown in Figure 12, 0.25 mL of 1M phosphate buffer, 0.65 mL of the culture supernatant of any one of the four strains, 0.05 mL of a suspension of Staphylococcus aureus adjusted to a concentration of 1×10 6 CFU / mL, and 0.05 mL of a suspension of Staphylococcus epidermidis adjusted to a concentration of 1×10 6 CFU / mL were mixed, and cultured with shaking at 30°C for 24 hours. Then, it was serially diluted and spread on standard agar medium, and cultured at 37°C for 2 days to count the colonies of Staphylococcus aureus and Staphylococcus epidermidis. Note that a control was prepared by adding 0.65 mL of sterilized water instead of the culture supernatant. The results are shown in Figure 13.

[0084] As shown in Figure 13, the Lactococcus lactis A19 strain and Lactococcus lactis A26 strain, which showed good antibacterial activity against Staphylococcus aureus in the previous [5. Antibacterial Activity Confirmation Test], did not show antibacterial activity in this test, but rather showed a tendency to increase. Also, for Staphylococcus epidermidis, no particular effect was observed.

[0085] On the other hand, the Lactococcus lactis A27 strain and Lactococcus lactis A33 strain, which had relatively weak antibacterial activity against Staphylococcus aureus in the previous [5. Antibacterial Activity Confirmation Test], showed a significant inhibitory effect against Staphylococcus aureus in this test. In particular, for the Lactococcus lactis A33 strain, further growth of Staphylococcus epidermidis was observed, and an interesting result was obtained that Staphylococcus aureus was inhibited while Staphylococcus epidermidis was promoted to grow.

[0086] [7. Fibroblast Activation Confirmation Test] Next, using normal human dermal fibroblasts (NHDF), the effects of the heat-killed cell lysates of the 166 strains of cherry-derived lactic acid bacteria identified above on cell proliferation in the presence of the heat-killed cell lysates described in [4. Confirmation test for bifidogenic activity] and Figure 8 were examined to confirm whether the cells of the lactic acid bacteria could activate fibroblasts.

[0087] Specifically, as shown in Figure 14, a predetermined number of normal human dermal fibroblasts were seeded per well in a 96-well plate, cultured at 37°C for 1 day in the presence of 5% CO2, the medium was removed, and a 1% FBS mixed DMEM medium containing the heat-killed cell lysate at a final concentration of 1% was added. The cells were cultured again at 37°C for 3 days in the presence of 5% CO2, and the activation activity of the fibroblasts was measured by measuring the absorbance using a Cell Counting Kit-8 (manufactured by Dojindo Laboratories).

[0088] As a result, as shown in Figure 15, among the 166 strains of cherry-derived lactic acid bacteria identified, activation activity against NHDF of approximately 1.2-fold, exceeding the equivalent, was observed in 7 strains: Lactococcus lactis A27 strain, Lactococcus lactis A33 strain, Leuconostoc mesenteroides A34 strain, Lactobacillus plantarum A53 strain, Lactobacillus pentosus A56 strain, Leuconostoc mesenteroides A80 strain, and Leuconostoc mesenteroides A83 strain.

[0089] 〔8. Collagen and hyaluronic acid gene expression analysis〕 Next, to confirm the effects of the 7 strains of cherry-derived lactic acid bacteria with activation activity against NHDF on collagen production and hyaluronic acid production in cells, the transcription levels of type I collagen gene (COL1A1) and hyaluronic acid synthase gene (HAS3) were confirmed.

[0090] Specifically, as shown in Fig. 16, normal human dermal fibroblasts (NHDF) were cultured at 37°C for 2 days in a 10% FBS mixed DMEM medium containing a heat-killed bacterial lysate with a final concentration of 1%, and mRNA was extracted using the RNAeasy (registered trademark) Plus Mini kit (manufactured by QIAGEN).

[0091] Next, the transcription levels of each gene were quantified by real-time RT-PCR. For the reverse transcription reaction, ReverTra AceTM qPCR RT Master Mix (manufactured by Toyobo Co., Ltd.) was used, and the reaction was carried out with the following treatments: 37°C for 20 minutes → 50°C for 5 minutes → 98°C for 5 minutes. For the real-time PCR reaction, FastStart SYBR Green Master (ROX) (manufactured by Roche Diagnostics Deutschland GmbH) was used, and the reaction was carried out with the following treatments: 95°C for 10 minutes → (95°C for 30 seconds → 58°C for 30 seconds → 72°C for 1 minute) × 40 cycles. The results are shown in Fig. 17.

[0092] As shown in Fig. 17, for the type I collagen gene (COL1A1), the Lactococcus lactis A33 strain had a transcription level almost equivalent to that of the control, and the transcription levels of most other strains were lower. However, for the Leuconostoc mesenteroides A80 strain, significant induction activity of the type I collagen gene was observed.

[0093] Regarding the hyaluronic acid synthase gene (HAS3), although slight induction activity was observed in the Leuconostoc mesenteroides A80 strain, transcription was suppressed in all other strains.

[0094] [9. Evaluation of Collagen and Hyaluronic Acid Production] Next, to further confirm the effects of the 7 strains of cherry-derived lactic acid bacteria, which were examined for transcriptional induction activity, on cell collagen production and hyaluronic acid production, the amounts of collagen and hyaluronic acid contained in the culture cell supernatant were confirmed.

[0095] As shown in Fig. 18, in this test, a 1% FBS mixed DMEM medium containing a heat-killed bacteria lysate at a final concentration of 1% was added to a 96-well plate, a predetermined number of normal human dermal fibroblasts were seeded per well, and the cells were cultured at 37°C for 3 days in the presence of 5% CO2. The amounts of collagen and hyaluronic acid contained in the obtained culture supernatant were measured by the ELISA method.

[0096] As a result, as shown in Fig. 19, regarding the production amount of collagen, about 1.1-fold increase was observed in Leuconostoc mesenteroides A83 strain compared to the control. For other strains, the results were equivalent or decreased.

[0097] Regarding the production amount of hyaluronic acid, all the results were lower than the control.

[0098] [10. Summary] Fig. 20 shows a table summarizing the above-described test results. In the table, the solid circles indicate the strains used in the test, the black circles indicate that the effects in each test were confirmed, and the white circles indicate that the effects could not be confirmed as much as the results of the black circles.

[0099] As shown in Fig. 20, regarding the confirmation test of SIRT1 gene expression induction activity conducted on 18 strains out of 166 strains isolated and identified from cherry blossoms, by the direct method, significant expression was observed in 8 strains: Lactobacillus pentosus A43 strain, Lactobacillus pentosus A51 strain, Lactobacillus plantarum A52 strain, Lactobacillus plantarum A53 strain, Lactobacillus paraplantarum A54 strain, Lactobacillus pentosus A55 strain, Lactobacillus pentosus A56 strain, and Lactobacillus brevis A95 strain.

[0100] In the indirect method, the effects were confirmed in 11 strains, namely Lactobacillus brevis A46 strain, Lactobacillus plantarum A53 strain, Lactobacillus paraplantarum A54 strain, Lactobacillus pentosus A55 strain, Lactobacillus fermentum A89 strain, Lactobacillus fermentum A90 strain, Lactobacillus brevis A94 strain, Lactobacillus brevis A95 strain, Lactobacillus brevis A96 strain, Lactobacillus plantarum B15 strain, and Lactobacillus casei C24 strain.

[0101] Moreover, based on the results of these direct and indirect methods, four strains, Lactobacillus plantarum A53 strain, Lactobacillus paraplantarum A54 strain, Lactobacillus pentosus A55 strain, and Lactobacillus brevis A95 strain, in which the effects were confirmed by both the direct and indirect methods, were shown to have SIRT1 gene expression-inducing activity in both cases of external application or oral ingestion.

[0102] Among the lactic acid bacteria derived from cherry blossoms having such SIRT1 gene expression-inducing activity, Lactobacillus brevis A46 strain, Lactobacillus brevis A95 strain, Lactobacillus brevis A96 strain, Lactobacillus plantarum B15 strain, and Lactobacillus casei C24 strain generally tend to be suitable for actual production based on the experimental results of the present inventors and experiences such as the culture conditions in the operation process. Therefore, they were considered to be particularly useful in realizing the preparation for inducing SIRT1 gene expression, foods, cosmetics, and pharmaceuticals containing the same according to this embodiment.

[0103] Among the lactic acid bacteria derived from cherry blossoms that have the activity of inducing SIRT1 gene expression, Lactobacillus brevis A46 strain has the effects of GABA productivity and bifidobacterium activation, Lactobacillus plantarum A53 strain has fibroblast activation, Lactobacillus pentosus A55 strain has GABA productivity, Lactobacillus pentosus A56 strain has fibroblast activation, Lactobacillus brevis A95 strain has the effects of GABA productivity and bifidobacterium activation, and Lactobacillus brevis A96 strain has both the effects of GABA productivity and bifidobacterium activation. It was considered to contribute to the improvement of the added value of the preparation for inducing SIRT1 gene expression according to this embodiment.

[0104] In addition, although the activity of inducing SIRT1 gene expression was not observed, as strains expected to contribute to multifunctionalization in constituting the foods, cosmetics, and pharmaceuticals according to this embodiment, Lactococcus lactis A19 strain (bifidobacterium activation and antibacterial activity), Lactococcus lactis A26 strain (bifidobacterium activation and antibacterial activity), Lactococcus lactis A27 strain (bifidobacterium activation, antibacterial activity, effect of improving the balance of skin resident bacteria, and fibroblast activation), Lactococcus lactis A33 strain (antibacterial activity, effect of improving the balance of skin resident bacteria, and fibroblast activation), Leuconostoc mesenteroides A34 strain (fibroblast activation), Leuconostoc mesenteroides A80 strain (fibroblast activation and promotion of collagen gene transcription), Leuconostoc mesenteroides A83 strain (fibroblast activation and collagen production), and Leuconostoc mesenteroides A93 strain (GABA productivity) were found to exist. In particular, six strains, namely A19 strain, A26 strain, A27 strain, A33 strain, A80 strain, and A83 strain, were found to have two or more added value effects at present, and were considered useful in constituting the foods, cosmetics, and pharmaceuticals according to this embodiment.

[0105] Furthermore, based on these results, when manufacturing a preparation for inducing SIRT1 gene expression, a food, a cosmetic, or a pharmaceutical according to this embodiment, it is also possible to realize a product with higher added value by using a plurality of lactic acid bacteria derived from cherry blossoms in combination.

[0106] For example, if a mixture of Lactobacillus brevis A95 strain, Lactobacillus plantarum B15 strain, and Lactobacillus casei C24 strain is added to a preparation for inducing SIRT1 gene expression, a food, a cosmetic, or a pharmaceutical and used in combination, it is possible to manufacture a preparation with high SIRT1 gene expression induction activity while combining strains of different species.

[0107] Also, for example, regarding the five particularly useful strains mentioned above, namely Lactobacillus brevis A46 strain, Lactobacillus brevis A95 strain, Lactobacillus brevis A96 strain, Lactobacillus plantarum B15 strain, and Lactobacillus casei C24 strain, by freely combining at least two or more strains selected from these, it is possible to manufacture a preparation with excellent productivity while having SIRT1 gene expression induction activity. That is, the applicant of the present application may seek to obtain rights for some or all of all combinations of two or more strains selected from these five strains in the future.

[0108] Also, for example, as an example of a combination with a lactic acid bacterium whose SIRT1 gene expression induction activity is unknown, a mixture of Lactococcus lactis A27 strain and Lactobacillus pentosus A55 strain may be added to a preparation for inducing SIRT1 gene expression, a food, a cosmetic, or a pharmaceutical. With such a configuration, it is possible to manufacture a preparation for which various effects such as SIRT1 gene expression induction activity, GABA productivity, bifidobacterium activation, antibacterial activity, skin resident flora balance improvement effect, and fibroblast activation effect can be expected.

[0109] Similarly, as an example of a combination with lactic acid bacteria with unknown SIRT1 gene expression-inducing activity, a mixture of Lactococcus lactis strain A33 and Lactobacillus brevis strain A95 may be added to a preparation, food, cosmetic, or pharmaceutical for inducing SIRT1 gene expression. Even with such a configuration, it is possible to produce preparations and the like that can be expected to have various effects such as SIRT1 gene expression-inducing activity, GABA productivity, bifidobacteria activation, antibacterial activity, skin resident flora balance improvement effect, and fibroblast activation effect.

[0110] In addition, although it is possible to perform co-culture for the combined use of these strains, from the perspective of production stability and the like, it is also an option to mix and use the dead cells of each strain, preferably dried powders.

[0111] Regarding the following strains, they have been deposited at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation, and the accession numbers are as follows. Lactobacillus brevis strain A46, NITE AP-03921 Lactobacillus pentosus strain A51, NITE AP-03922 Lactobacillus pentosus strain A55, NITE AP-03923 Lactobacillus fermentum strain A89, NITE AP-03924 Lactobacillus brevis strain A94, NITE AP-03925 Lactobacillus brevis strain A95, NITE AP-03926 Lactobacillus brevis strain A96, NITE AP-03927 Lactobacillus plantarum strain B15, NITE AP-03928 Lactobacillus casei strain C24, NITE AP-03929

[0112] As described above, according to the preparation for inducing SIRT1 gene expression according to the present embodiment, since it contains lactic acid bacteria derived from cherry blossoms, disrupted cells of the same, or a culture solution of the same, it is possible to provide foods, cosmetics, and pharmaceuticals in which induction of SIRT1 gene expression is expected.

[0113] Finally, the description of each of the above-described embodiments is an example of the present invention, and the present invention is not limited to the above-described embodiments. Therefore, it goes without saying that various modifications can be made according to the design and the like as long as they are within the scope not departing from the technical idea of the present invention even if they are other than the above-described embodiments.

Claims

1. A preparation for inducing SIRT1 gene expression, comprising at least one lactic acid bacterium derived from cherry blossoms having SIRT1 gene expression-inducing activity selected from Lactobacillus pentosus strain A51 (NITE AP-03922), Lactobacillus pentosus strain A55 (NITE AP-03923), Lactobacillus fermentum strain A89 (NITE AP-03924), Lactobacillus plantarum strain B15 (NITE AP-03928), Lactobacillus casei strain C24 (NITE AP-03929), a disrupted product of the bacterial cells, or a culture solution of the bacterial cells.

2. The preparation for inducing SIRT1 gene expression according to Claim 1, wherein the lactic acid bacterium is Lactobacillus pentosus strain A55 (NITE AP-03923).

3. A preparation for inducing SIRT1 gene expression, comprising a mixture of bacterial cells of Lactobacillus plantarum strain B15 (NITE AP-03928) and Lactobacillus casei strain C24 (NITE AP-03929), which are lactic acid bacteria derived from cherry blossoms having SIRT1 gene expression-inducing activity, a disrupted product of the bacterial cell mixture, or a mixture of culture solutions of the respective bacterial cells.

4. A food for inducing SIRT1 gene expression, comprising the preparation for inducing SIRT1 gene expression according to Claim 1, wherein the lactic acid bacterium is Lactobacillus fermentum strain A89 (NITE AP-03924) or Lactobacillus casei strain C24 (NITE AP-03929).

5. A cosmetic for inducing SIRT1 gene expression, comprising the preparation for inducing SIRT1 gene expression according to Claim 1 or the preparation for inducing SIRT1 gene expression according to Claim 2, wherein the lactic acid bacterium is at least one selected from Lactobacillus pentosus strain A51 (NITE AP-03922), Lactobacillus pentosus strain A55 (NITE AP-03923), Lactobacillus fermentum strain A89 (NITE AP-03924), and Lactobacillus plantarum strain B15 (NITE AP-03928). The pharmaceutical for inducing SIRT1 gene expression according to claim 1, wherein the lactic acid bacterium is Lactobacillus fermentum A89 strain (NITE AP-03924) or Lactobacillus casei C24 strain (NITE AP-03929), containing the preparation for inducing SIRT1 gene expression.

7. Use of Lactobacillus pentosus A55 strain (NITE AP-03923) in combination with Lactococcous lactis having bifidobacteria activating activity, antibacterial activity, skin resident flora balance improving effect and fibroblast activating effect as a main component for inducing at least any one selected from SIRT1 gene expression inducing activity, GABA productivity, bifidobacteria activating activity, antibacterial activity, skin resident flora balance improving effect or fibroblast activating effect in the production of a food for inducing SIRT1 gene expression, a cosmetic for inducing SIRT1 gene expression or a pharmaceutical for inducing SIRT1 gene expression.

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