Use of lactobacillus rhamnosus tci366 to reduce skin spots, improve skin radiance and increase skin moisture content

Abstract

The present application discloses a use of lactobacillus rhamnosus TCI366 and / or its metabolite, which is a use of lactobacillus rhamnosus TCI366 and / or its metabolite in preparation of a composition for improving skin condition, wherein the lactobacillus rhamnosus TCI366 is deposited in the German Collection of Microorganisms and the deposit number is DSM33290.

Description

Technical Field

[0001] This invention relates to the use of Lactobacillus rhamnosus, particularly Lactobacillus rhamnosus TCI366 and / or its metabolites, for the preparation of compositions that improve skin condition. Background Technology

[0002] The skin consists of the epidermis, dermis, and subcutaneous tissue from the outside in. As the first layer of protection for human individuals, it provides protection against environmental factors such as ultraviolet (UV) radiation from sunlight, pathogens, and friction, thereby reducing the chance of skin damage.

[0003] Common skin damage conditions include decreased skin elasticity, insufficient skin hydration (dry, dull skin), enlarged pores, wrinkles, spots and / or an overall dull, sallow complexion.

[0004] Furthermore, besides the skin damage caused by normal aging, certain environmental factors can accelerate the process of skin damage. For example, common natural factors include ultraviolet (UV) radiation from sunlight. When exposed to UV rays, the UV radiation stimulates the skin to form melanin, further accelerating the aforementioned skin damage.

[0005] In addition, due to the significant increase in the amount of time people spend using electronic products with screens in modern life, the visible light emitted by the screens, which has a shorter wavelength and higher energy (hereinafter referred to as blue light), also accelerates the aforementioned skin damage when exposed to the skin for a long time. Summary of the Invention

[0006] In some embodiments, the use of Lactobacillus rhamnosus TCI366 and / or its metabolites in the preparation of compositions for improving skin condition, wherein Lactobacillus rhamnosus TCI366 is deposited at the German Microbiological Collection Center with accession number DSM33290.

[0007] In some embodiments, a Lactobacillus rhamnosus TCI366 is deposited at the German Microbiological Collection Center with accession number DSM33290.

[0008] In summary, *Lactobacillus rhamnosus* TCI366 and / or its metabolites from any of the embodiments can be used to prepare compositions. Furthermore, *Lactobacillus rhamnosus* TCI366 is deposited at the German Microbiological Collection Center with accession number DSM33290. In some embodiments, *Lactobacillus rhamnosus* TCI366 exhibits resistance to gastric acid and bile salts. In some embodiments, compositions prepared from *Lactobacillus rhamnosus* TCI366 and / or its metabolites can be used to improve skin condition. In some embodiments, *Lactobacillus rhamnosus* TCI366 and / or its metabolites have at least one of the following functions: reducing the expression level of the microphthalmia-associated transcription factor (MITF) gene, inhibiting tyrosinase activity, inhibiting melanin formation, reducing skin spots, reducing the formation of UV-induced pigmentation, increasing skin hydration, or a combination thereof.

[0009] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the present invention. Attached Figure Description

[0010] Figure 1 This is a graph showing the survival rate of Lactobacillus rhamnosus TCI366 in a simulated gastrointestinal environment.

[0011] Figure 2 This is a graph showing the relative ploidy rate of the MITF gene;

[0012] Figure 3 This is a graph showing the results of a tyrosinase activity experiment;

[0013] Figure 4 This is a graph showing the relative percentage of melanin content;

[0014] Figure 5 This is a graph showing the relative percentage of melanin content caused by blue light.

[0015] Figure 6 This is a graph showing the results of the spot data from week 0 and week 6;

[0016] Figure 7 This is a graph showing the results of UV spot data for weeks 0 and 6;

[0017] Figure 8 These are the skin radiance data results for weeks 0 and 6; and

[0018] Figure 9 This is a graph showing the skin hydration data for weeks 0 and 6. Detailed Implementation

[0019] *Lactobacillus rhamnosus* TCI366 is a strain of *Lactobacillus* isolated from the human intestine. It is deposited at the German Microbiological Collection (DSM-33290). This *Lactobacillus rhamnosus* TCI366 exhibits beneficial effects on improving the skin condition of both the host and recipient. These improvements include inhibiting tyrosinase activity in the host or recipient, reducing melanin formation, enhancing skin radiance, and increasing skin hydration, or combinations thereof. The host or recipient is human.

[0020] Lactobacillus rhamnosus TCI366 is a Gram-positive bacterium that can grow in anaerobic environments, classifying it as an anaerobic bacterium. Colonies of Lactobacillus rhamnosus TCI366 have smooth edges and a white appearance. The optimal growth temperature for Lactobacillus rhamnosus TCI366 is 35°C to 37°C. Furthermore, it can survive in environments with a pH value of 3 to 7.

[0021] In some embodiments, *Lactobacillus rhamnosus* TCI366 exhibits resistance to gastric acid and bile salts. For example, *Lactobacillus rhamnosus* TCI366 has a survival rate of 88% in a gastric simulated environment (pH 3-4) and a survival rate of 97% in an intestinal simulated environment (pH 7). Therefore, *Lactobacillus rhamnosus* TCI366 can colonize the human gastrointestinal tract environment.

[0022] In some embodiments, by culturing *Lactobacillus rhamnosus* TCI366 and separating the *Lactobacillus rhamnosus* TCI366 cells and supernatant by centrifugation, the supernatant can be filtered to obtain the metabolites of *Lactobacillus rhamnosus* TCI366. In other words, the metabolites of *Lactobacillus rhamnosus* TCI366 include substances secreted into the culture medium after metabolism by *Lactobacillus rhamnosus* TCI366. In some embodiments, the metabolites of *Lactobacillus rhamnosus* TCI366 further include the culture medium used to culture *Lactobacillus rhamnosus* TCI366, and this culture medium has been used to culture *Lactobacillus rhamnosus* TCI366, but this culture medium does not contain *Lactobacillus rhamnosus* TCI366 cells. In one example, *Lactobacillus rhamnosus* TCI366 was cultured on Lactobacillus MRS medium (BD Difco™ Lactobacilli MRS Broth) at 37°C for 16 hours to obtain a bacterial culture containing *Lactobacillus rhamnosus* TCI366 cells and its metabolites. Next, the bacterial culture was centrifuged at 5000 rpm for 15 minutes to separate Lactobacillus rhamnosus TCI366 cells and the supernatant containing Lactobacillus rhamnosus TCI366 metabolites. The supernatant was then filtered through a 0.22-micrometer (μm) filter to obtain the Lactobacillus rhamnosus TCI366 metabolites.

[0023] Here, the term "metabolite" means the substance secreted into the culture medium after being metabolized by Lactobacillus rhamnosus, or the substance secreted into the culture medium after being metabolized by Lactobacillus rhamnosus and the Lactobacillus culture medium used to culture Lactobacillus rhamnosus, but does not include Lactobacillus rhamnosus itself.

[0024] In some embodiments, *Lactobacillus rhamnosus* TCI366 and / or its metabolites can reduce the expression of melanin-forming genes in the host or receptor. These melanin-forming genes can be, but are not limited to, microphthalmia-associated transcription factor (MITF) genes. Therefore, when a host takes *Lactobacillus rhamnosus* TCI366 and / or its metabolites, the expression of MITF genes in the host can be suppressed, thereby reducing melanin formation.

[0025] In some embodiments, Lactobacillus rhamnosus TCI366 and / or its metabolites have the function of inhibiting the tyrosinase activity of the host or receptor. For example, when a host takes Lactobacillus rhamnosus TCI366 and / or its metabolites, tyrosinase activity in the host's body can be inhibited, thereby reducing melanin formation and thus reducing the number of spots on the host's skin and / or reducing the formation of ultraviolet-induced pigmentation on the host's skin.

[0026] Therefore, in some embodiments, *Lactobacillus rhamnosus* TCI366 and / or its metabolites have the function of inhibiting melanin formation. Melanin formation can be caused by natural processes (e.g., natural aging, natural light exposure) and blue light exposure. Accordingly, when a host ingests *Lactobacillus rhamnosus* TCI366 and / or its metabolites, melanin formation can be inhibited, thereby reducing the number of spots on the host's skin and / or reducing the formation of ultraviolet-induced pigmentation. This melanin formation includes melanin formation caused by natural aging, melanin formation caused by natural light exposure, melanin formation caused by blue light exposure, or a combination thereof.

[0027] In some embodiments, Lactobacillus rhamnosus TCI366 has the function of improving the radiance of the host's skin. For example, when a host takes Lactobacillus rhamnosus TCI366, the radiance of the host's skin can be improved.

[0028] In some embodiments, Lactobacillus rhamnosus TCI366 has the function of increasing the moisture content of the host's skin. For example, when a host takes Lactobacillus rhamnosus TCI366, the moisture content of the host's skin can be increased.

[0029] Therefore, in some embodiments, *Lactobacillus rhamnosus* TCI366 and / or its metabolites can be used to prepare compositions that improve skin condition. These improvements may include enhancing skin radiance, increasing skin hydration, reducing melanin formation, reducing the formation of freckles, reducing the number of freckles, reducing the formation of UV-induced pigmentation, or combinations thereof.

[0030] In some embodiments, any of the aforementioned compositions may be a food product. In other words, the food product contains a specific amount of Lactobacillus rhamnosus TCI366 and / or its metabolites. In some embodiments, the aforementioned food product may be an edible product or a food additive. In some embodiments, edible products may be, but are not limited to: beverages, dairy products, fermented foods, bakery products, health foods, and dietary supplements.

[0031] In some embodiments, any of the foregoing compositions may be a cosmetic or skincare product. In other words, the cosmetic or skincare product contains a specific amount of Lactobacillus rhamnosus TCI366 and / or its metabolites.

[0032] In some embodiments, the aforementioned cosmetics or skincare products may be any of the following forms: toner, gel, gel mask, mud mask, lotion, cream, lipstick, foundation, pressed powder, loose powder, cleansing oil, cleansing milk, facial cleanser, body wash, shampoo, conditioner, sunscreen, hand cream, nail polish, perfume, serum, and face mask. In some embodiments, the aforementioned cosmetics or skincare products may, as needed, include topical acceptable ingredients. In some embodiments, topical acceptable ingredients may be, for example, emulsifiers, penetration enhancers, softeners, solvents, excipients, antioxidants, or combinations thereof.

[0033] Example 1: Strain Identification

[0034] First, the isolate from the human gut was identified using the 16S ribosomal gene (16S rDNA) sequence of lactic acid bacteria. The 16S rDNA sequence of this isolate (SEQ ID NO:1) was obtained by polymerase chain reaction (PCR). Next, using the National Center for Biotechnology Information (NCBI) website, the gene sequence shown in SEQ ID NO:1 was compared with the 16S rDNA sequences of other *Lactobacillus rhamnosus* subspecies. The similarity of the 16S rDNA sequence of this isolate to other *Lactobacillus rhamnosus* subspecies is shown in Table 1. Therefore, this isolate was named *Lactobacillus rhamnosus* TCI366. Furthermore, *Lactobacillus rhamnosus* TCI366 was deposited at the German Microbiological Collection Center (DSM33290).

[0035] Table 1

[0036]

[0037] Example 2: Gastric acid and bile salt tolerance test

[0038] To confirm the acid-base tolerance of *Lactobacillus rhamnosus* TCI366 in the gastrointestinal tract of organisms, buffer solution, simulated gastric fluid (pH 3), and simulated intestinal fluid (pH 7) were tested. The buffer solution consisted of 0.2 M potassium chloride (KCl, Sigma-Aldrich) at pH 7. The simulated gastric fluid consisted of 0.2 M potassium chloride at pH 3. The simulated intestinal fluid consisted of 0.2 M potassium chloride and 0.3% by weight ox bile salts (Difco™ Oxgall) at pH 7.

[0039] An activation procedure was performed to activate Lactobacillus rhamnosus TCI366. First, frozen Lactobacillus rhamnosus TCI366 was cultured at a volume of 10% in liquid Lactobacillus MRS medium (BD Difco™ Lactobacilli MRS Broth) and incubated at 37°C for 16 hours to activate Lactobacillus rhamnosus TCI366.

[0040] Next, a 1% (v / v) concentration of the activated *Lactobacillus rhamnosus* TCI366 culture was inoculated into 20 mL of the test solution, and then cultured at 37°C with shaking at 50 rpm for 3 hours. The test solutions for the three groups were artificial gastric fluid (experimental group), artificial intestinal fluid (experimental group), and potassium chloride buffer (control group). 100 μL of the cultured culture was spread onto a plate and incubated at 28°C for 3 days. After 3 days, the viable bacterial count for each group was visually counted (the number of colonies on the solid *Lactobacillus* MRS medium was calculated).

[0041] Please see Figure 1 The survival rate of *Lactobacillus rhamnosus* TCI366 in the diagram is the number of viable bacteria after counting, expressed as log CFU / mL. Here, log CFU / mL represents the colony-forming units (CFU) per milliliter of bacterial culture, expressed logarithmically. 10 (This is a direct translation of the Chinese text.) Figure 1 The survival rate of *Lactobacillus rhamnosus* TCI366 in the control group was 7.79 log CFU / mL, in the artificial gastric fluid experimental group it was 6.85 log CFU / mL, and in the artificial intestinal fluid experimental group it was 7.59 log CFU / mL. Therefore, the viable bacterial count in the artificial gastric fluid experimental group was 88% of that in the control group, and the viable bacterial count in the artificial intestinal fluid experimental group was 97% of that in the control group.

[0042] Therefore, Lactobacillus rhamnosus TCI366 has the function of being resistant to gastric acid and bile salts, which means that Lactobacillus rhamnosus TCI366 can colonize the host's intestines through the acid-base pressure of the host's gastrointestinal tract.

[0043] Example 3: Preparation of metabolites from Lactobacillus rhamnosus TCI366

[0044] After activating Lactobacillus rhamnosus TCI366 using the aforementioned activation procedure, 10% (v / v) of the activated Lactobacillus rhamnosus TCI366 was inoculated into liquid Lactobacillus MRS medium and cultured at 37°C for 16 hours. Then, the cells of Lactobacillus rhamnosus TCI366 and the supernatant containing its metabolites were separated by centrifugation at 5000 rpm for 15 minutes. The supernatant was collected and filtered through a 0.22 μm filter to obtain the metabolites of Lactobacillus rhamnosus TCI366.

[0045] Therefore, it can be concluded that the metabolites of Lactobacillus rhamnosus TCI366 are substances secreted into the culture medium after Lactobacillus rhamnosus TCI366 undergoes metabolism, as well as the liquid Lactobacillus MRS medium (but without bacterial cells) used to culture this Lactobacillus rhamnosus TCI366.

[0046] Example 4: Expression analysis of melanin formation genes

[0047] The culture medium used was MEM medium containing 90% Eagle's Minimum Basic Medium - Non-Essential Amino Acids (Gibco), 10% fetal bovine serum albumin (FBS, Gibco), and 1 mM sodium pyruvate (Gibco). The cell line used was human melanoma cells (ATCC®, CRL-1872), hereinafter referred to as A375.S2 cells. The melanin-forming gene analyzed was the microphthalmia-associated transcription factor (MITF) gene.

[0048] A375.S2 cells were added at a rate of 1 × 10⁶ cells per well. 5 Each sample was inoculated into a 6-well culture dish containing 2 ml of MEM medium per well and incubated at 37°C for 24 hours, divided into experimental and control groups. Next, the MEM medium was replaced with the experimental medium, and the mixture was incubated for another 24 hours. The experimental group used MEM medium containing 0.25% (v / v) of the *Lactobacillus rhamnosus* TCI366 metabolite prepared in Example 3. The control group used MEM medium only (i.e., without the *Lactobacillus rhamnosus* TCI366 metabolite).

[0049] Next, RNA was extracted from A375.S2 cells cultured in experimental medium using an RNA extraction kit (brand: GeneAid). After translating the RNA into cDNA, it was analyzed using MITF-F (SEQ ID NO:2) and MITF-R (SEQ ID NO:3) primers (as shown in Table 2) in conjunction with the nCounter Analysis Systems. The analysis results are as follows: Figure 2 As shown. It should be noted that... Figure 2 Gene expression was presented as relative ploidy, with standard deviation calculated using the STDEV formula in Excel software, and statistically significant differences between groups were statistically analyzed using student t-tests.

[0050] Table 2

[0051]

[0052] In the sequence name, R (REVERSE) represents reverse and F (FORWARD) represents forward.

[0053] Please see Figure 2The expression level of the MITF gene in the control group was considered as 1, representing that the expression level of the MITF gene in A375.S2 cells not treated with Lactobacillus rhamnosus TCI366 metabolites was considered as 100%. Therefore, the expression level of the MITF gene in the experimental group was 0.31. In other words, the expression level of the MITF gene in A375.S2 cells treated with Lactobacillus rhamnosus TCI366 metabolites was suppressed by 69%.

[0054] Therefore, the metabolites of Lactobacillus rhamnosus TCI366 secreted by Lactobacillus rhamnosus TCI366 have the effect of reducing the expression level of the MITF gene. In other words, Lactobacillus rhamnosus TCI366 and / or its metabolites can be used to reduce the expression level of the MITF gene, thereby reducing melanin synthesis.

[0055] Example 5: Tyrosinase activity detection

[0056] The culture medium used was Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum albumin (FBS; brand: Gibco) and 1% penicillin / streptomycin (Gibco), hereinafter referred to as the basal medium. The cell line used was the mouse melanoma cell line B16F10 (purchased from the American Type Culture Collection Center (ATCC), catalog number CRL-6475), hereinafter referred to as B16F10 cells. The solutions used included lysis buffer, which was prepared with 0.1M phosphate buffer, 0.1mM phenylmethylsulfonyl fluoride (PMSF; brand: Sigma), and 1% Triton X-100 (pH 6.8; brand: JTBaker).

[0057] B16F10 cells were planted at a density of 1.5 × 10⁶ cells per well. 5 Cells were seeded into each well of a 6-well culture dish containing 2 mL of basal culture medium and incubated at 37°C for 24 hours.

[0058] After 24 hours of culture, B16F10 cells were divided into three groups: an experimental group, a control group, and a control group. The basal medium for each group was removed and replaced with 2 mL of experimental medium per well, then cultured at 37°C for another 48 hours. The experimental group received basal medium containing 0.25% (v / v) of the *Lactobacillus rhamnosus* TCI366 metabolite prepared in Example 3. The control group received basal medium containing 0.25% (v / v) kojic acid. The control group received only basal medium (i.e., without *Lactobacillus rhamnosus* TCI366 metabolites or kojic acid).

[0059] After 48 hours of culture, the experimental medium in each well was removed, and the cells were rinsed twice with 1x phosphate-buffered saline (PBS, Gibco). Following rinsing, trypsin was added to each well to treat the cells for 3 minutes. After 3 minutes of treatment, the suspended cells from each well were individually collected into 15 mL centrifuge tubes and centrifuged at 400xg for 5 minutes to separate the first cell pellet from the supernatant. The cell pellet was resuspended in 1xPBS and centrifuged twice. The cell pellet was then resuspended in 50 μL of lysis buffer to obtain a cell suspension. The centrifuge tubes containing the cell suspension were shaken and centrifuged at 12,000xg for 20 minutes to separate the second cell pellet from the cell solution. After protein quantification of each cell solution, 400 μg / well of cell solution was transferred to a 96-well plate and diluted to 90 μL per well with lysis buffer. Next, 10 μL of 10 mM L-DOPA was added to each well to form a reaction solution. The solution was observed every 10 minutes at 37°C in the dark until the color of the reaction solution in each well changed from transparent to dark. Then, the absorbance (OD) of each well in the 96-well culture dish was read at a wavelength of 405 nm using an ELISA (enzyme-linked immunosorbent assay) reader (brand: BioTek). 405 ).

[0060] After measurement, the measured absorbance values ​​were substituted into the following formula (1) to calculate the percentage of tyrosinase activity (%). In other words, the tyrosinase activity of the control group was considered as 100% to calculate the relative percentage of tyrosinase activity (%) for each group. Furthermore, statistically significant differences between groups were statistically analyzed using a student t-test, such as... Figure 3 As shown. In Figure 3In the figure, "***" indicates that the p-value is less than 0.001 when compared with the control group, and "▲▲▲" indicates that the p-value is less than 0.001 when compared with the control group.

[0061] Formula (1)

[0062] Relative tyrosinase activity percentage (%) = (OD 405 sample / OD 405 control (1) × 100%

[0063] Among them, OD 405 sample The absorbance value represents the group to be converted, while the OD value represents the absorbance of the group to be converted. 405 control The absorbance value represents the control group.

[0064] Please refer to Figure 3 Compared to the control group, the experimental group showed a tyrosinase activity percentage of 33.21%, representing a 66.79% inhibition of tyrosinase activity, while the control group showed a tyrosinase activity percentage of 78.04%, representing a 21.96% inhibition of tyrosinase activity. Furthermore, the experimental group exhibited a significantly lower tyrosinase activity percentage compared to the control group. In other words, compared to kojic acid, the metabolite of *Lactobacillus rhamnosus* TCI366 effectively inhibits tyrosinase activity, thereby effectively suppressing melanin production.

[0065] Therefore, Lactobacillus rhamnosus TCI366 and / or its metabolites can be used to inhibit tyrosinase activity, thereby improving skin condition.

[0066] Example 6: Melanin content detection

[0067] The culture medium used was Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum (FBS; brand: Gibco) and 1% penicillin / streptomycin (Gibco), hereinafter referred to as the basal medium. The cell line used was the mouse melanoma cell line B16F10 (purchased from the American Type Culture Collection (ATCC), catalog number CRL-6475), hereinafter referred to as B16F10 cells.

[0068] B16F10 cells were planted at a density of 1.5 × 10⁶ cells per well. 5 Cells were seeded into each well of a 6-well culture dish containing 3 mL of basal culture medium and incubated at 37°C for 24 hours.

[0069] After 24 hours of culture, B16F10 cells were divided into three groups: an experimental group, a control group, and a control group. The basal medium for each group was removed and replaced with 3 mL of experimental medium per well, then cultured at 37°C for another 48 hours. The experimental group received basal medium containing 0.25% (v / v) of the *Lactobacillus rhamnosus* TCI366 metabolites prepared in Example 3. The control group received basal medium containing 0.25% (v / v) kojic acid. The control group received only basal medium (i.e., without *Lactobacillus rhamnosus* TCI366 metabolites or kojic acid).

[0070] After 48 hours of culture, the culture medium in each well was removed, and the cells were rinsed twice with 1x phosphate-buffered saline (PBS, Gibco). Following rinsing, trypsin was added to each well to treat the cells for 3 minutes. After 3 minutes, the suspended cells from each well were individually collected into 15 mL centrifuge tubes and centrifuged at 400xg for 5 minutes to separate the cell pellet from the supernatant. The cell pellet was resuspended in 1xPBS and centrifuged twice. The cell pellet was then resuspended in 200 μL of 1xPBS to obtain a cell solution. The cell solution was then placed in liquid nitrogen for 10 minutes, followed by thawing at room temperature for 30 minutes. After complete thawing, each centrifuge tube was centrifuged at 12,000xg for 30 minutes. After centrifugation for 30 minutes, the supernatant in each centrifuge tube was removed, and 120 μL of 1N NaOH (prepared with ddH2O) was added to mix with the precipitate in each tube. After thorough mixing, each centrifuge tube containing the mixed solution was incubated in a 60°C dry bath for 1 hour. Then, 100 μL of the mixed solution was transferred from each centrifuge tube to a 96-well culture dish, and the absorbance (OD) of each well in the 96-well culture dish was read at a wavelength of 405 nm using an ELISA (enzyme-linked immunosorbent assay) reader (brand: BioTek). 405 ).

[0071] After measurement, the measured absorbance values ​​were substituted into the following formula (2) to calculate the relative percentage (%) of melanin content. In other words, the melanin content of the control group was considered as 1 (i.e., the relative percentage of melanin content in the control group was 100%) to calculate the relative percentage (%) of melanin content in each group. Furthermore, statistically significant differences between groups were statistically analyzed using a student t-test, such as... Figure 4 As shown. In Figure 4In the diagram, "*" indicates that the p-value is less than 0.05 when compared with the control group, and "**" indicates that the p-value is less than 0.01 when compared with the control group.

[0072] Formula (2)

[0073] Relative percentage of melanin content (%) = (OD) 405 sample / OD 405 control )×100%(2)

[0074] Please refer to Figure 4 Compared to the control group, the experimental group had a melanin content of 79.51% relative percentage, while the control group had a melanin content of 98.62% relative percentage. In other words, the experimental group reduced melanin production by 21.49%, while the control group reduced melanin production by 1.38%. Therefore, the experimental group showed a significant decrease in the relative percentage of melanin content compared to both the control and control groups. This indicates that, compared to kojic acid, the metabolites of *Lactobacillus rhamnosus* TCI366 can effectively inhibit melanin production.

[0075] Therefore, Lactobacillus rhamnosus TCI366 and / or its metabolites can be used to inhibit melanin production, thereby improving skin condition.

[0076] Example 7: Detection of melanin content caused by blue light

[0077] The culture medium used was Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum (FBS; brand: Gibco) and 1% penicillin / streptomycin (Gibco), hereinafter referred to as the basal medium. The cell line used was the mouse melanoma cell line B16F10 (purchased from the American Type Culture Collection (ATCC), catalog number CRL-6475), hereinafter referred to as B16F10 cells.

[0078] B16F10 cells were planted at a density of 1.5 × 10⁶ cells per well. 5 Cells were seeded into each well of a 6-well culture dish containing 3 mL of basal culture medium and incubated at 37°C for 24 hours.

[0079] After 24 hours of culture, B16F10 cells were divided into three groups: an experimental group, a control group, and a control group. The basal medium for each group was removed and replaced with 3 mL of experimental medium per well. The control and experimental groups were then irradiated with blue LED light for 3 hours (the control group was not irradiated with blue LED light). Each group was then cultured at 37°C for another 24 hours. The experimental medium for the experimental group contained 0.25% (v / v) of the *Lactobacillus rhamnosus* TCI366 metabolite prepared in Example 3. The experimental medium for the control and control groups was simple basal medium (i.e., without the *Lactobacillus rhamnosus* TCI366 metabolite).

[0080] After 24 hours of culture, the culture medium in each well was removed, and the cells were rinsed twice with 1x phosphate-buffered saline (PBS, Gibco). Following rinsing, trypsin was added to each well to treat the cells for 3 minutes. After 3 minutes of treatment, the suspended cells from each well were individually collected into 15 mL centrifuge tubes and centrifuged at 400xg for 5 minutes to separate the cell pellet from the supernatant. The cell pellet was resuspended in 1xPBS and centrifuged twice. The cell pellet was then resuspended in 200 μL of 1xPBS to obtain a cell solution. The cell solution was then rapidly frozen in liquid nitrogen for 30 seconds, followed by thawing at room temperature for 30 minutes. After complete thawing, each centrifuge tube was centrifuged at 14,000xg for 30 minutes. After centrifugation for 30 minutes, the supernatant in each centrifuge tube was removed, and 120 μL of 1N NaOH (prepared with ddH2O) was added to mix with the precipitate in each tube. After thorough mixing, each centrifuge tube containing the mixed solution was incubated in a 60°C dry bath for 1 hour. Then, 100 μL of the mixed solution was transferred from each centrifuge tube to a 96-well culture dish, and the absorbance (OD) of each well in the 96-well culture dish was read at a wavelength of 405 nm using an ELISA (enzyme-linked immunosorbent assay) reader (brand: BioTek). 405 ).

[0081] After measurement, the measured absorbance value is substituted into the following formula (3) to calculate the relative percentage (%) of melanin content caused by blue light, such as Figure 5 As shown ( Figure 5And formula (3) is denoted as "relative percentage of melanin content"). In other words, here, the melanin content of the control group is considered as 1 (i.e., the relative percentage of melanin content in the control group is 100%) to calculate the relative percentage of melanin content (%) of each group. Furthermore, statistically significant differences between groups are statistically analyzed using a student t-test. Figure 5 In the figure, "▲▲" indicates that the p-value is less than 0.01 when compared with the control group, and "***" indicates that the p-value is less than 0.001 when compared with the control group.

[0082] Formula (3)

[0083] Relative percentage of melanin content (%) = (OD) 405 sample / OD 405 control )×100%(3)

[0084] Please refer to Figure 5 Compared to the control group, the experimental group had a melanin content of 108.08% relative percentage, while the control group had a melanin content of 130.28% relative percentage. In other words, after blue light treatment, the control group's melanin content increased by 30.28% relative percentage compared to the control group, while the experimental group, treated with both blue light and metabolites of *Lactobacillus rhamnosus* TCI366, only saw an 8.08% increase in melanin content relative percentage compared to the control group. Therefore, compared to the control group, the blue light-induced melanin production in the experimental group was significantly inhibited by 22.2%, indicating that the metabolites of *Lactobacillus rhamnosus* TCI366 can effectively inhibit blue light-induced melanin production.

[0085] Therefore, Lactobacillus rhamnosus TCI366 and / or its metabolites can be used to inhibit melanin production caused by blue light, thereby improving skin condition.

[0086] Example 8: Human Experimentation

[0087] To further confirm the effects of Lactobacillus rhamnosus TCI366 on humans, each capsule contains 5 × 10⁻⁶ cells / mL. 9 CFU (i.e., 5×10) 9 Live Lactobacillus rhamnosus TCI366 capsules (CFU / cap) were administered to subjects daily (50 mg of bacterial powder / day, meaning one capsule containing 50 mg of Lactobacillus rhamnosus TCI366 bacterial powder per day) for 6 weeks. Skin analyses were performed before (week 0) and after 6 weeks (week 6) using the DermaLab® Combo SkinAnalyzer. The analyses included skin pigmentation, UV-induced dark spots, skin radiance, and skin hydration.

[0088] The participants consisted of eight individuals, both women and men, aged 20-55. Furthermore, these participants had dull skin and freckles on their faces.

[0089] The test results were compared together with the values ​​from week 0 and week 6. Week 0 represents the pre-trial measurements, indicating the skin condition of the subjects before taking the live bacteria capsules containing Lactobacillus rhamnosus TCI366. Week 6 represents the measurements after six consecutive weeks of use, indicating the skin condition of the subjects after taking the live bacteria capsules containing Lactobacillus rhamnosus TCI366. It is important to note that statistically significant differences between groups were determined using a student t-test. In the graph, "*" indicates a p-value less than 0.05 compared to week 0, and "**" indicates a p-value less than 0.01 compared to week 0.

[0090] Please see Figure 6 The average number of skin spots measured in week 0 of the 8 subjects was taken as 100%, and the average number of skin spots measured in week 6 of the 8 subjects was 92.7% lower than the number in week 0. In other words, after taking live bacteria capsules containing Lactobacillus rhamnosus TCI366 for 6 consecutive weeks, the number of skin spots in the 8 subjects decreased by 7.3%. Therefore, Lactobacillus rhamnosus TCI366 can reduce the number of skin spots in the subjects and has the effect of improving skin condition.

[0091] Please see Figure 7 The average value of UV-induced pigmentation on the skin of the 8 subjects measured at week 0 was taken as 100%, and the average value of UV-induced pigmentation on the skin of the 8 subjects measured at week 6 was 94.4% compared to the value at week 0. In other words, after taking live bacteria capsules containing Lactobacillus rhamnosus TCI366 for 6 consecutive weeks, the UV-induced pigmentation on the skin of the 8 subjects decreased by 5.6%. Therefore, it can be concluded that Lactobacillus rhamnosus TCI366 can reduce the distribution of UV-induced pigmentation on the skin of the subjects and has the effect of improving skin condition.

[0092] Please see Figure 8 The average skin radiance measured by the 8 subjects at week 0 was taken as 100%, and the average skin radiance measured by the 8 subjects at week 6 was 109.8% higher than the value at week 0. In other words, after taking live bacteria capsules containing Lactobacillus rhamnosus TCI366 for 6 consecutive weeks, the skin radiance of the 8 subjects improved by 9.8%. Therefore, it can be concluded that Lactobacillus rhamnosus TCI366 can improve the skin radiance of the subjects and has the effect of improving skin condition.

[0093] Please see Figure 9The average skin hydration level measured by the 8 subjects at week 0 was taken as 100%, and the average skin hydration level measured by the 8 subjects at week 6 was 105.8% higher than that at week 0. In other words, after taking live bacteria capsules containing Lactobacillus rhamnosus TCI366 for 6 consecutive weeks, the skin hydration level of the 8 subjects increased by 5.8%. Therefore, Lactobacillus rhamnosus TCI366 can increase the skin hydration level of the subjects and has the effect of improving skin condition.

[0094] Thus, as shown in Example 2, *Lactobacillus rhamnosus* TCI366 can adapt to the human gut environment. As shown in Example 4, the metabolites of *Lactobacillus rhamnosus* TCI366 can reduce the expression levels of melanin-forming genes (e.g., the MITF gene). As shown in Example 5, the metabolites of *Lactobacillus rhamnosus* TCI366 can reduce tyrosine activity. As shown in Examples 6 and 7, the metabolites of *Lactobacillus rhamnosus* TCI366 can inhibit melanin formation (naturally formed, caused by natural light, and caused by blue light). As shown in Example 8, *Lactobacillus rhamnosus* TCI366 can improve skin condition, including reducing the number of spots, reducing UV-induced pigmentation, improving skin radiance, and increasing skin hydration, or a combination thereof.

[0095] In summary, *Lactobacillus rhamnosus* TCI366 according to any embodiment of the present invention can be used to prepare compositions that improve skin condition. Furthermore, *Lactobacillus rhamnosus* TCI366 is resistant to gastric acid and bile salts, allowing it to survive in the gastrointestinal environment. Because *Lactobacillus rhamnosus* TCI366 can reduce the expression of the MITF gene in receptors and / or inhibit tyrosinase activity, it can effectively inhibit melanin formation, thereby improving the skin condition of receptors (e.g., reducing skin spots, reducing UV-induced pigmentation, etc.). In some embodiments, *Lactobacillus rhamnosus* TCI366 can be used to increase the radiance and / or moisture content of receptor skin, thereby improving the skin condition of receptors.

[0096] Although the technical content of the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any modifications and refinements made by those skilled in the art without departing from the spirit of the present invention should be included within the scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

[0097] Of course, the present invention may have other various embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and modifications according to the present invention, but these corresponding changes and modifications should all fall within the protection scope of the claims of the present invention.

[0098] [Biomaterial Storage]

[0099] German Microbial Collection (Germany); September 19, 2019; Deposit No.: DSM33290.

[0100] [Sequence List]

[0101] <110> TCI Biotech Co., Ltd.

[0102] <120> Uses of Lactobacillus rhamnosus TCI366 to reduce skin blemishes, improve skin radiance, and increase skin hydration

[0103] <150> 62 / 886,397

[0104] <151> 2019-08-14

[0105] <160> 3

[0106] <170> PatentIn version 3.5

[0107] <210> 1

[0108] <211> 1203

[0109] <212> DNA

[0110] <213> Lactobacillus rhamnosus TCI366

[0111] <400> 1

[0112] ctaagcggta tgcctaatac atgcaagtcg aacgagttct gattattgaa aggtgcttgc 60

[0113] atcttgattt aattttgaac gagtggcgga cgggtgagta acacgtgggt aacctgccct 120

[0114] taagtgggga taacatttgg aaacagatgc taataccgca taaatccaag aaccgcatgg 180

[0115] ttcttggctg aaagatggcg taagctatcg cttttggatg gacccgcggc gtattagcta 240

[0116] gttggtgagg taacgctcac caaggcaatg atacgtagcc gaactgagag gttgatcggc 300

[0117] cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtagg gaatcttcca 360

[0118] caatggacgc aagtctgatg ggcaacgccg cgtgagtgaa gaaggctttc gggtcgtaaa 420

[0119] actctgttgt tggagaagaa tggtcggcag agtaactgtt gtcggcgtga cggtatccaa 480

[0120] ccagaaagcc acggctaact acgtgccaca gccgcggtaa tacgtaggtg gcaagcgtta 540

[0121] tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat gtgaaagccc 600

[0122] tcggcttaac cgaggaagtg catcggaaac tgggaacttg agtgcagaag aggacagtgg 660

[0123] aactccatgt gtagcggtga aatgcgtaga tatatggaag aacaccagtg gcgaaggcgg 720

[0124] ctgtctggtc tgtaactgac gctgaggctc gaaagcatgg gtgcgaacag gattagatac 780

[0125] cctggtagtc catgccgtaa acgatgaatg ctaggtgttg gagggtttcc gcccttcagt 840

[0126] gccgcagcta acgcattaag cattccgcct ggggagtacg accgcaaggt gaaactcaaa 900

[0127] ggaattgacg ggggcccgca caagcggtgg agcatggtgg tttaattcga agcaacgcga 960

[0128] agaaccttac cagtcttgac atcttttgat cactgaagat cagtttcccc ttcgggcaaa1020

[0129] tgacaggtgg tgcatgttgt cgtcagcctc gtgtctgaga atgttggtaa gtcccgccaa1080

[0130] cgagcgccac cctatgacta ggtggcagca atttagtggc acctctagta gactggcgtg1140

[0131] acacgtagaa ggtgggatga cgtcaatcat catgccttag aacttggact tacaccggtg1200

[0132] ctc 1203

[0133] <210> 2

[0134] <211> 20

[0135] <212> DNA

[0136] <213> Artificial sequence

[0137] <220>

[0138] <223> MITF-F

[0139] <400> 2

[0140] gcctccaagc ctccgataag 20

[0141] <210> 3

[0142] <211> 21

[0143] <212> DNA

[0144] <213> Artificial sequence

[0145] <220>

[0146] <223> MITF-R

[0147] <400> 3

[0148] gcactctctg ttgcatgaac t 21 [Sequence List] <110> TCI Biotech Co., Ltd. <120> Uses of Lactobacillus rhamnosus TCI366 and / or its metabolites for improving skin condition <150> US62886397 <151> 2019-08-14 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 1203 <212> DNA <213> Lactobacillus rhamnosus TCI366 <400> 1 ctaagcggta tgcctaatac atgcaagtcg aacgagttct gattattgaa aggtgcttgc 60 atcttgattt aattttgaac gagtggcgga cgggtgagta acacgtgggt aacctgccct 120 taagtgggga taacatttgg aaacagatgc taataccgca taaatccaag aaccgcatgg 180 ttcttggctg aaagatggcg taagctatcg cttttggatg gacccgcggc gtattagcta 240 gttggtgagg taacgctcac caaggcaatg atacgtagcc gaactgagag gttgatcggc 300 cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtagg gaatcttcca 360 caatggacgc aagtctgatg ggcaacgccg cgtgagtgaa gaaggctttc gggtcgtaaa 420 actctgttgt tggagaagaa tggtcggcag agtaactgtt gtcggcgtga cggtatccaa 480 ccagaaagcc acggctaact acgtgccaca gccgcggtaa tacgtaggtg gcaagcgtta 540 tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat gtgaaagccc 600 tcggcttaac cgaggaagtg catcggaaac tgggaacttg agtgcagaag aggacagtgg 660 aactccatgt gtagcggtga aatgcgtaga tatatggaag aacaccagtg gcgaaggcgg 720 ctgtctggtc tgtaactgac gctgaggctc gaaagcatgg gtgcgaacag gattagatac 780 cctggtagtc catgccgtaa acgatgaatg ctaggtgttg gagggtttcc gcccttcagt 840 gccgcagcta acgcattaag cattccgcct ggggagtacg accgcaaggt gaaactcaaa 900 ggaattgacg ggggcccgca caagcggtgg agcatggtgg tttaattcga agcaacgcga 960 agaaccttac cagtcttgac atcttttgat cactgaagat cagtttcccc ttcgggcaaa 1020 tgacaggtgg tgcatgttgt cgtcagcctc gtgtctgaga atgttggtaa gtcccgccaa 1080 cgagcgccac cctatgacta ggtggcagca atttagtggc acctctagta gactggcgtg 1140 acacgtagaa ggtgggatga cgtcaatcat catgccttag aacttggact tacaccggtg 1200 ctc 1203 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> MITF‑F <400> 2 gcctccaagc ctccgataag 20 <210> 3 <211> 21 <212> DNA <213> Artificial sequence <220> <223> MITF‑R <400> 3 gcactctctg ttgcatgaac t 21

Claims

1. A use of Lactobacillus rhamnosus TCI366, characterized in that, This describes the use of Lactobacillus rhamnosus TCI366 in the preparation of compositions for reducing skin blemishes. Lactobacillus rhamnosus TCI366 is deposited at the German Microbiological Collection Center with accession number DSM33290.

2. The use according to claim 1, characterized in that, This Lactobacillus rhamnosus TCI366 exhibits resistance to gastric acid and bile salts.

3. The use according to claim 1, characterized in that, This Lactobacillus rhamnosus TCI366 has the function of reducing the formation of UV-induced pigmentation on the skin.

4. A use of Lactobacillus rhamnosus TCI366, characterized in that, This describes the use of Lactobacillus rhamnosus TCI366 in the preparation of compositions that improve skin radiance. Lactobacillus rhamnosus TCI366 is deposited at the German Microbiological Collection Center with the accession number DSM33290.

5. A use of Lactobacillus rhamnosus TCI366, characterized in that, This refers to the use of Lactobacillus rhamnosus TCI366 in the preparation of compositions that improve skin hydration. Lactobacillus rhamnosus TCI366 is deposited at the German Microbiological Collection Center with the accession number DSM33290.

6. The use according to any one of claims 1, 4, and 5, characterized in that, The composition is a food or health product.

Images