Application of a Bacillus megaterium strain SI-JD-01 in preparing a whitening composition and a whitening composition prepared therefrom

Abstract

The present invention discloses an application of a Bacillus megaterium strain SI-JD-01 in the preparation of a whitening composition and a whitening composition prepared therefrom, belonging to the field of microbial application technology. The deposit number of the strain is CCTCC M2023278. Whitening includes inhibiting tyrosinase activity; a whitening composition comprising the following substances as active ingredients: Bacillus megaterium strain SI-JD-01, its lysate, its fermentation broth, or an extract of the strain, lysate or fermentation broth; Beneficial effects: The Bacillus megaterium strain adopted in the present invention, its lysate, its fermentation broth, or the strain, its lysate or its fermentation broth extract, or a composition for skin whitening comprising the same effectively inhibits melanin production, thereby having the effect of preventing, improving and / or treating symptoms or diseases caused by excessive melanin production.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, and in particular to application of a Bacillus megaterium strain SI-JD-01 in preparing a whitening composition and the whitening composition prepared using the same. Background Art

[0002] Skin color is influenced by a variety of internal and external factors, ultimately depending on the amount of melanin and its dispersion within the skin. Skin whitening effects are primarily related to melanin production inhibition and free radical scavenging capabilities. Whitening effects can also be achieved by accelerating skin metabolism and stratum corneum exfoliation. The most important mechanism is the inhibition of melanin production.

[0003] Traditional whitening drugs generally work by inhibiting melanocyte maturation, thereby reducing melanin production, but this can be prone to adverse reactions. Consequently, most whitening products developed in the cosmetics industry over the years selectively inhibit the activity of tyrosinase (TYR), reducing melanin deposition while avoiding toxicity to normal, mature melanocytes and minimizing adverse reactions. However, existing whitening products are often expensive, and some can be highly irritating, prone to discoloration, and ineffective.

[0004] The Chinese patent application document with publication number CN116004435A discloses a Bacillus megaterium and its application. The strain is named Bacillus megaterium ( Bacillus megaterium ), strain number Tu27, accession number CCTCC No. M 20221115. This strain inhibits the growth of rice pathogenic fungi when cultured in a confrontation culture; it can also synthesize silver nanoparticles with antibacterial and bactericidal activity. The nanoparticles synthesized by this strain have a small particle size of 15±6 nm and exhibit significant inhibitory effects against the growth of rice blast fungi. However, this strain does not exhibit skin-whitening activity. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to provide a Bacillus megaterium with whitening activity and a composition using the same.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] The first aspect of the present invention provides a use of a Bacillus megaterium strain SI-JD-01 in preparing a whitening composition. The deposit number of the Bacillus megaterium SI-JD-01 is CCTCC M 2023278.

[0008] Beneficial effects: The Bacillus megaterium used in the present invention ( Bacillus megaterium) strain, or a composition for skin whitening comprising the same, effectively inhibits melanin production, thereby having the effect of preventing, improving and / or treating symptoms or diseases caused by excessive melanin production.

[0009] Preferably, the whitening comprises inhibiting tyrosinase activity.

[0010] A second aspect of the present invention provides a whitening composition, the active ingredient of which comprises the lysate of the aforementioned Bacillus megaterium SI-JD-01.

[0011] Preferably, the active ingredient refers to an ingredient that can exhibit the desired activity alone or together with an inactive carrier or the like.

[0012] Preferably, the cell lysate of the strain refers to a product obtained by disrupting the strain itself by chemical or physical force.

[0013] Preferably, the protein concentration of the strain lysate is 0.125-0.5 mg / mL.

[0014] The third aspect of the present invention provides a use of the whitening composition in the preparation of food, medicine, health care product or cosmetic with whitening effect.

[0015] Preferably, the cosmetics are skin care products.

[0016] Preferably, the dosage form of the cosmetic is selected from one or more of toner, softener, toner, astringent, lotion, nourishing lotion, moisturizing lotion, nutritious lotion, massage cream, nourishing cream, moisturizing cream, hand cream, foundation, essence, nutritious essence, facial mask, soap, cleansing foam, cleansing milk, cleansing cream, body lotion, and shower gel.

[0017] The advantages of the present invention are:

[0018] 1. The Bacillus megaterium used in the present invention ( Bacillus megaterium ) strain or a composition for skin whitening containing the same effectively inhibits melanin production, thereby having the effect of preventing, improving and / or treating symptoms or diseases caused by excessive melanin production.

[0019] 2. The lysate of the Bacillus megaterium strain used in the present invention, or a composition comprising the same, can prevent, improve and / or treat one or more skin pigmentations selected from dark spots, freckles, black spots, macules, melanoma, pigmentation caused by drugs, pigmentation caused by inflammation, and pigmentation caused by dermatitis that occur locally on the skin due to increased melanin production. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is a graph showing the effect of the Bacillus megaterium SI-JD-01 lysate of the present invention on free radical scavenging efficiency;

[0021] Figure 2 This figure shows the effect of the Bacillus megaterium SI-JD-01 lysate of the present invention on the safety and proliferation of skin cells;

[0022] Figure 3 This is a diagram showing the effect of the Bacillus megaterium SI-JD-01 lysate of the present invention on cell damage caused by UVA;

[0023] Figure 4 This figure shows the effect of the Bacillus megaterium SI-JD-01 lysate of the present invention on melanin production in B16 cells;

[0024] Figure 5 This is a graph showing the effect of the Bacillus megaterium SI-JD-01 lysate of the present invention on the tyrosinase inhibition rate of B16 cells. DETAILED DESCRIPTION

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0026] Example 1:

[0027] (1) Isolation and identification of strains:

[0028] The strain was discovered on May 16, 2022, in Laboratory 306, South Building, Comprehensive Laboratory Building, Hefei Institutes of Physical Science, Chinese Academy of Sciences. It was collected from indoor ambient air. Through systematic screening of strains from ambient air, a strain capable of inhibiting the growth of Staphylococcus aureus was discovered. The strain was streaked and purified on blood agar plates. The strain was preliminarily identified as Bacillus megaterium (Bacillus subtilis) using Bruker's matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI Biotyper 3.1 software. Bacillus megaterium), with a score of 2.420; 16S rRNA gene was amplified using bacterial universal primers (27F / 1492R), and the gene sequence obtained after PCR product testing (GenBank Accession: OR064032) was compared and analyzed in NCBI, and MEGA 11.0 software was used to construct the phylogenetic tree of the strain to clarify the taxonomic status of the strain.

[0029] (2) Colony morphology and bacterial structure of the strain:

[0030] On blood agar plates, the strain's colonies are light yellow, with neat edges, smooth, moist, opaque, and rounded projections, devoid of a surrounding α-hemolytic ring. Under an optical microscope, the strain's cells are round or oval, arranged in pairs or short chains, lacking capsules, spores, or flagella, and are Gram-positive.

[0031] The Bacillus megaterium strain SI-JD-01 was deposited in the China Center for Type Culture Collection (Wuhan University) on March 9, 2023, and the deposited name is Bacillus megaterium Bacillus megaterium SI-JD-01, the deposit number is CCTCC No: M 2023278.

[0032] Example 2:

[0033] Preparation of Bacillus megaterium ( Bacillus megaterium ) Fermentation broth and lysate of strain

[0034] The Bacillus megaterium SI-JD-01 strain identified in Example 1 was inoculated into a culture medium (10 g / L sodium chloride, 5 g / L yeast extract, and 10 g / L peptone) and cultured at 37°C and 200 rpm for 3 days to obtain a fermentation broth of the Bacillus megaterium strain. Furthermore, the fermentation broth was ultrasonically disrupted at 800 W for 15 minutes each time. The disrupted broth was sterilized by filtration through a 0.22 μm filter to obtain a Bacillus megaterium lysate. The effector substance content was calculated based on the relative quantification of protein concentration in the lysate (see the table below). The following batches refer to Bacillus megaterium lysate samples collected at different times under the same experimental conditions.

[0035] Table 1 Effector content in Bacillus megaterium lysates

[0036]

[0037] Example 3:

[0038] Effects of Bacillus megaterium lysate on free radical scavenging rate

[0039] 1-Diphenyl-2-trinitrobenzene (DPPH) is a stable nitrogen-centered organic free radical. The DPPH assay, first proposed in 1958, is widely used to determine the anti-aging properties of biological samples, classified substances, and foods. This assay is based on the fact that the DPPH free radical has a single electron, exhibits strong absorption at 517nm, and exhibits a purple color in ethanol solutions. In the presence of a free radical scavenger, the absorption gradually disappears due to pairing with the single electron. The degree of color fading is quantitatively related to the number of electrons accepted. Therefore, a spectrophotometer can be used for rapid quantitative analysis to monitor free radical scavenging activity and thus evaluate the anti-aging properties of a sample.

[0040] LB broth was used to prepare lysate solutions of Bacillus megaterium and a control strain (Escherichia coli standard strain OP50) with protein concentrations of 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, respectively. 0.1 mL of the lysate was added to a test tube, followed by 0.1 mL of DPPH ethanol solution. The mixture was mixed and allowed to react in the dark at room temperature for 30 min. The OD value was measured at 525 nm, and the clearance rate was calculated according to the following formula: Clearance rate (%) = [1-(T-T0) / (C-C0)] × 100%, where: T0: absorbance of 0.1 mL of sample solution plus 0.1 mL of 95% ethanol; T: absorbance of 0.1 mL of sample solution plus 0.1 mL of DPPH solution; C0: absorbance of 0.1 mL of water plus 0.1 mL of 95% ethanol; C: absorbance of 0.1 mL of water plus 0.1 mL of DPPH solution.

[0041] from Figure 1 The results showed that the free radical scavenging ability of Bacillus megaterium lysate was excellent, and the free radical scavenging rate was better than that of Escherichia coli standard strain ( P <0.05), the difference was statistically significant.

[0042] Example 4:

[0043] Effects of Bacillus megaterium lysate on the safety and cell proliferation rate of skin cells

[0044] Keratinocytes are the most important cells in the epidermis, participating in the formation of the skin's physical barrier, protecting the skin from physical, chemical, and microbial invasions. A key characteristic of skin aging is a thinning of the epidermis and slower wound healing, primarily due to a decrease in the regenerative capacity of keratinocytes in the epidermis and a reduction in the proliferation capacity of the keratinocyte system. Human skin fibroblasts are the most important cells in the reticular layer of the dermis and are one of the primary repair cells following skin aging and cell damage. They not only promote the migration, proliferation, and differentiation of epidermal cells, but also secrete large amounts of collagen, elastin, and a variety of cell repair factors, possessing a powerful self-renewal capacity to repair aging skin.

[0045] The effects of Bacillus megaterium lysate on skin cell safety and proliferation were tested using the following methods. The aforementioned human immortalized epidermal cells (HaCAT) are an immortalized human keratinocyte cell line, whose morphology and function are similar to those of normal human keratinocytes. Compared to cell lines, primary human fibroblasts (FB cells) better mimic the functions of human skin.

[0046] Specifically, the fermentation liquid of the Bacillus megaterium strain isolated from the air obtained in Example 1 was ultrasonically disrupted and then prepared into solutions of different concentrations using LB broth medium, with LB broth medium without sample as blank control. 5 Skin cells were seeded at 100 μL per well of a 96-well cell culture plate at 50 cells / mL. After 24 hours of incubation, 100 μL of Bacillus megaterium lysate at protein concentrations of 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL was added and incubated for another 24 hours. The experiment was repeated three times for each concentration. The bioactivity of the cells was compared using the MTT assay. After staining, the absorbance at 550 nm was measured using a microplate reader. The effect on the proliferation of HaCAT cells or FB cells was evaluated against a blank control. The bioactivity of the blank control was considered 100%.

[0047] The results showed that Bacillus megaterium lysate had a certain proliferation-promoting effect on skin epidermal HaCAT cells and fibroblasts.

[0048] Example 5:

[0049] Protective Effect of Bacillus megaterium Lysate against UV Damage

[0050] Skin aging can be divided into intrinsic and extrinsic aging. Sunlight, especially ultraviolet (UV) radiation, is the primary factor in exogenous aging, hence exogenous aging is also known as photoaging. UVA (320-400 nm) and a smaller amount of UVB (280-320 nm) are primarily responsible for skin photoaging. Numerous data indicate that 95% of UV radiation reaching the skin is absorbed by keratinocytes. Therefore, we established a skin photodamage model by irradiating immortalized human epidermal HaCAT cells with UVA. This model was used to evaluate the protective effects of Bacillus megaterium lysate against UV-induced skin damage. A concentration of 5.0×10 5 Immortalized human epidermal HaCaT cells were seeded into 96-well cell culture plates at a concentration of 100 μL per well and incubated at 37°C in a 5% CO2 incubator for 24 hours. Before experimental treatment, cells were starved of serum for 6 hours. After the medium was replaced with PBS, the cells were irradiated with UVA (365 nm) at a dose of 10 J / cm2. 2 The cells were irradiated for 15 minutes, the PBS was discarded, and 100 μL of the test sample (protein concentrations of 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, respectively) and 100 μL of cell culture medium were added. Culture was continued for 24 hours, and cell viability was assessed using the MTT assay. A group without UVA irradiation served as the negative control (UVA-), and a group irradiated with UVA alone served as the model group (UVA+). Vitamin C (Vc) and vitamin E (Ve) were used as positive controls, with the final concentrations of both Vc and Ve being 10 μM.

[0051] Depend on Figure 3 The results showed that Bacillus megaterium lysate (0.25 mg / mL and 0.5 mg / mL) had a significant protective effect on cell damage caused by UVA, and the cell viability after treatment was higher than that of the model group ( P <0.05), the difference was statistically significant.

[0052] Example 6:

[0053] Skin whitening effect of Bacillus megaterium lysate:

[0054] The lysate of the Bacillus megaterium strain isolated from the air obtained in Example 1 was treated on melanin-producing cells (B16 melanoma cells) to evaluate the whitening effect. 5B16 cells were seeded into 24-well cell culture plates and cultured for 24 hours for use in the experiment. To induce melanin production, they were treated with 1M α-MSH (α-Melanocyte-stimulating hormone) and treated with lysates of Bacillus megaterium at concentrations ranging from 0.125 mg / mL to 0.5 mg / mL. The experiment was repeated three times for each concentration. After another 72 hours of culture, the absorbance at 405 nm was measured to compare the melanin content in the culture medium. Based on the absorbance of the model group inducing melanin production being considered 100%, the degree of melanin induction in the lysate-treated group was expressed as %.

[0055] The inhibitory effect of the samples on tyrosinase was determined by L-Dopa oxidation method. Mouse melanoma B16 cells were cultured at 1.0×10 5 Cells were cultured at high density in 96-well plates. After 24 hours, Bacillus megaterium lysate at concentrations of 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL was added to the cells and cultured for 48 hours. The culture medium was then discarded, and 100 μL of PBS buffer containing 1% Triton X-100 was added to each well, followed by 50 μL of 0.2 mg / mL L-Dopa. The cells were treated at 37°C for 3 hours, and the absorbance at 490 nm was measured. Enzyme activity was calculated using the following formula: Tyrosinase inhibition rate = [1 - (OD value of experimental group / OD value of control group)] × 100%

[0056] The results showed that the use of Bacillus megaterium lysate could significantly inhibit the melanin production and tyrosinase activity of B16 cells ( P <0.05), the difference was statistically significant and concentration-dependent, indicating that the lysate of Bacillus megaterium strain has a skin whitening effect.

[0057] As described above, the specific parts of this aspect have been described in detail. It should be understood by those skilled in the art that these specific technologies are only preferred embodiments and the scope of the present invention is not limited thereto. Therefore, the scope of protection of the present invention will be limited by the appended claims and their equivalents.

[0058] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of a lysate of a Bacillus megaterium strain SI-JD-01 in the preparation of a cosmetic composition having whitening, anti-aging and anti-ultraviolet damage effects, characterized in that: The deposit number of the Bacillus megaterium SI-JD-01 is CCTCCM 2023278.

2. The use according to claim 1, characterized in that The whitening includes inhibiting tyrosinase activity; the anti-aging includes scavenging free radicals.

3. A whitening, anti-aging and anti-ultraviolet damage cosmetic composition, characterized in that: The active ingredient comprises a lysate of Bacillus megaterium strain SI-JD-01, and the deposit number of the Bacillus megaterium SI-JD-01 is CCTCC M2023278.

4. The whitening, anti-aging and anti-ultraviolet damage cosmetic composition according to claim 3, characterized in that: The active ingredient refers to an ingredient that exhibits the desired activity alone or together with a carrier that is inactive by itself.

5. The whitening, anti-aging and anti-ultraviolet damage cosmetic composition according to claim 3, characterized in that: The lysate refers to a product obtained by disrupting the strain itself by chemical or physical force.

6. The whitening, anti-aging and anti-ultraviolet damage cosmetic composition according to claim 4, characterized in that: The protein concentration of the lysate was 0.125-0.5 mg / mL.

7. The whitening, anti-aging and anti-ultraviolet damage cosmetic composition according to claim 6, characterized in that: The protein concentration of the lysate was 0.125 mg / mL.

8. The whitening, anti-aging and anti-ultraviolet damage cosmetic composition according to claim 6, characterized in that: The protein concentration of the lysate was 0.5 mg / mL.

9. The whitening, anti-aging and anti-ultraviolet damage cosmetic composition according to claim 3, characterized in that: Cosmetics are skin care products.

10. The whitening, anti-aging and anti-ultraviolet damage cosmetic composition according to claim 3, characterized in that: The dosage form of the cosmetic is selected from one or more of toner, softener, toner, astringent, moisturizing lotion, moisturizing lotion, nutritious lotion, massage cream, moisturizing cream, foundation, essence, facial mask, soap, cleansing foam, cleansing milk, cleansing cream, body lotion, and shower gel.

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