Animal bifidobacterium with whitening, anti-oxidation and soothing effects and its application in skin care products

Abstract

The present invention belongs to the field of biotechnology, and in particular to a kind of animal bifidobacterium with whitening, anti-oxidation, soothing effects and its application in skin care products. The present invention is confirmed by experiments that the fermentation supernatant of animal bifidobacterium (Bifidobacterium animalis) W3 can improve the DPPH scavenging ability of human skin fibroblasts, inhibit the expression of inflammatory factors IL-1a, TNF-a and PGE2 in mouse mononuclear macrophage leukemia cells, and can also inhibit the synthesis and accumulation of melanin in mouse melanoma cells, indicating that the skin care product raw material containing the fermentation liquid of the strain can play an anti-oxidation, whitening and soothing effect, and has application ability in the raw materials of whitening, soothing and anti-oxidation effects, and therefore has the value of good practical application.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an animal bifidobacterium with whitening, anti-oxidation and soothing effects and application thereof in skin care products. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Skin inflammation is usually accompanied by the upregulation of multiple inflammatory factors. As one of the key sites of skin immune response, macrophages located in the dermis of the skin can receive different types of external stimulation, induce the immune response system inside the cell, activate the corresponding signaling pathways and release different inflammatory mediators, such as tumor necrosis factor-α (TNF-a), interleukin-1a (IL-1a), prostaglandin 2 (PGE2), etc. Melanin content is the main factor that determines the difference in human skin color. Most of the current skin care products with whitening effects use technical means to interfere with the synthesis and accumulation of melanin to achieve whitening effects. Melanin synthesis occurs in melanocytes in the basal layer of the epidermis. On average, one melanocyte is surrounded by 36 keratinocytes. The melanosomes in melanocytes are factories for producing melanin. Melanocytes transport melanin to keratinocytes through long protrusions to play a role. The degree of melanin synthesis and accumulation determines the color of human skin. Melanin synthesis starts from phenylalanine and tyrosine. Under the regulation of tyrosinase (TYR), tyrosinase related protein 1 (TRP 1), tyrosinase related protein 2 (TRP 2), α melanocyte stimulating hormone (αMSH) and adrenocorticotropic hormone (ACTH), a series of enzymatic reactions are carried out to produce melanin and pheomelanin, which are then decomposed into carbon dioxide and water by deamination reaction under the action of melanin metabolic enzyme (MME) and excreted from the body. Under the influence of ultraviolet rays, staying up late and other factors, harmless oxygen will lose an electron and become active oxygen free radicals that are harmful to cells and even the human body. Common free radicals include superoxide free radicals, hydrogen peroxide, hydroxyl free radicals, singlet oxygen and lipid peroxides. Under the action of free radicals, a series of problems such as mitochondrial damage, protein fibrosis, loss of skin elasticity, wrinkles, and dull skin tone will occur.

[0004] In the field of skin care, as people's demand for green, safe and healthy skin care products grows, fermentation technology is used to enrich the active ingredients in natural raw materials, making their molecular weight smaller and better absorbed while reducing the toxic side effects of the raw materials. Summary of the invention

[0005] In view of the prior art, the present invention aims to provide an animal bifidobacterium with whitening, anti-oxidation and soothing effects and its application in skin care products. Animal bifidobacterium (Bifidobacterium animalis) W3 is a bifidobacterium derived from the human intestinal tract. Previous studies have shown that it not only has a growth inhibitory effect on pathogenic bacteria such as pathogenic Escherichia coli, but also its fermentation supernatant also has a promoting effect on keratinocyte proliferation. Through further research, it is found that it also has whitening, anti-oxidation, anti-inflammatory and soothing effects, so as to further expand its application field and scope. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0007] In one aspect of the present invention, a bacterial agent of Bifidobacterium animalis W3 is provided for use in any one or more of the following:

[0008] (a) Use in whitening or in preparing whitening products;

[0009] (b) Application in anti-oxidation or in the preparation of anti-oxidation products;

[0010] (c) Use in anti-inflammatory and soothing effects or in the preparation of anti-inflammatory and soothing products.

[0011] Among them, the Bifidobacterium animalis W3 strain was deposited in the General Microbiology Center of China Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China) on July 8, 2022, with the deposit number CGMCC No. 25262.

[0012] In the above (a), whitening is specifically manifested in inhibiting the synthesis and accumulation of melanin and promoting the decomposition of melanin.

[0013] In the above (b), the antioxidant effect is manifested as an improvement in the ability to scavenge DDPH.

[0014] In (c), the anti-inflammatory and soothing effects are specifically manifested as inhibiting the expression of relevant inflammatory factors, including but not limited to IL-1a, TNF-a and PGE2.

[0015] The second aspect of the present invention provides a skin care product with whitening, anti-oxidation, anti-inflammatory and soothing effects, wherein the skin care product contains a bacterial agent.

[0016] Wherein, the bacterial agent comprises the supernatant of the above-mentioned Bifidobacterium animalis W3 fermentation broth.

[0017] Beneficial technical effects of one or more of the above technical solutions:

[0018] The above technical scheme discloses a strain of Bifidobacterium animalis W3 and its fermentation supernatant. Experiments have confirmed that the fermentation supernatant can improve the DPPH scavenging ability of human skin fibroblasts (HSF), inhibit the expression of inflammatory factors IL-1a, TNF-a and PGE2 in mouse mononuclear macrophage leukemia cells (RAW264.7), and can also inhibit the synthesis and accumulation of melanin in mouse melanoma cells (B16), indicating that the skin care product raw materials containing the fermentation liquid of this strain can have antioxidant, whitening and soothing effects, and have application capabilities in raw materials with whitening, soothing and antioxidant effects, and therefore have good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for use in the description of the embodiment will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 This is the proliferation inhibitory effect of Bifidobacterium animalis W3 fermentation broth on RAW264.7 cells in Example 2 of the present invention.

[0021] Figure 2 This is the inhibitory effect of Bifidobacterium animalis W3 fermentation broth on the expression of inflammatory factors in Example 3 of the present invention.

[0022] Figure 3 This is the proliferation inhibitory effect of Bifidobacterium animalis W3 fermentation broth on B16 cells in Example 4 of the present invention.

[0023] Figure 4 This is the effect of Bifidobacterium animalis W3 fermentation broth on the relative content of melanin in B16 cells in Example 5 of the present invention.

[0024] Figure 5 This is the regulatory effect of Bifidobacterium animalis W3 fermentation broth on melanin metabolism in B16 cells in Example 6 of the present invention.

[0025] Figure 6This is the proliferation inhibitory effect of Bifidobacterium animalis W3 fermentation broth on HSF cells in Example 7 of the present invention.

[0026] Figure 7 This is the promoting effect of Bifidobacterium animalis W3 fermentation broth on the removal of DPPH in HSF cells in Example 8 of the present invention. DETAILED DESCRIPTION

[0027] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0028] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0029] In a typical embodiment of the present invention, a bacterial agent of Bifidobacterium animalis W3 is provided for use in any one or more of the following:

[0030] (a) Use in whitening or in preparing whitening products;

[0031] (b) Application in anti-oxidation or in the preparation of anti-oxidation products;

[0032] (c) Use in anti-inflammatory and soothing effects or in the preparation of anti-inflammatory and soothing products.

[0033] in,

[0034] The Bifidobacterium animalis W3 is derived from the feces of healthy adults and has been verified by morphology, physiology and biochemistry, and gene sequencing (16S rDNA). The strain has been deposited in the General Microbiology Center of the China Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China) on July 8, 2022, with the deposit number CGMCC No. 25262.

[0035] The bacterial agent can be the supernatant of the fermentation broth of the above-mentioned Bifidobacterium animalis W3.

[0036] In the present invention, the supernatant is specifically the fermentation liquid of the animal Bifidobacterium W3, which is centrifuged, filtered, settled or other means known in the art to separate the bacterial cells grown in the fermentation liquid from the liquid, and remove the remaining liquid when the bacterial cells are removed. During the fermentation process, the fermentation medium used is not specifically limited, as long as it can be used as a medium for culturing bacteria, and of course preferably a medium for culturing bifidobacteria. In a specific embodiment of the present invention, the medium is a BBL medium.

[0037] Of course, the above-mentioned bacterial agent can also exist in solid form, such as lyophilized powder.By freeze-drying animal bifidobacterium (Bifidobacterium animalis) W3 or its fermentation product, the corresponding lyophilized agent is obtained.The above-mentioned freeze-drying process can be carried out by existing conventional methods, and freeze-drying protective agents can also be used to protect during the freeze-drying process. The freeze-drying protective agent includes but is not limited to polyols (such as glycerol, mannitol, polyethylene glycol, etc.), sugars (such as mannose, maltose, glucose, heparin, etc.), surfactants (such as Tween-80, Tween-20, sodium dodecyl sulfate, etc.), which are not specifically limited here.

[0038] In (a), whitening is specifically manifested as inhibiting the synthesis and accumulation of melanin and promoting the decomposition of melanin. Specifically, the present invention has proved through experiments that the supernatant of the above-mentioned animal Bifidobacterium W3 fermentation broth has a certain inhibitory effect on genes related to melanin synthesis, and an up-regulating effect on genes related to melanin decomposition metabolism.

[0039] In (b), the antioxidant effect is manifested as improving the DDPH scavenging ability. Specifically, the present invention proves through experiments that the supernatant of the above-mentioned animal Bifidobacterium W3 fermentation broth can improve the DPPH scavenging ability of human skin fibroblasts (HSF).

[0040] In (c), the anti-inflammatory and soothing effect is specifically manifested as inhibiting the expression of related inflammatory factors, including but not limited to IL-1a, TNF-a and PGE2. Specifically, the present invention has proved through experiments that the supernatant of the above-mentioned animal Bifidobacterium W3 fermentation broth has the effect of inhibiting the expression of inflammatory factors IL-1a, TNF-a and PGE2 in mouse mononuclear macrophage leukemia cells (RAW264.7), thereby helping to relieve skin redness, swelling and stinging, and having a soothing effect.

[0041] The product may be a cosmetic, more specifically a skin care product.

[0042] In another specific embodiment of the present invention, a skin care product with whitening, anti-oxidation, anti-inflammatory and soothing effects is provided, and the skin care product contains a bacterial agent.

[0043] Wherein, the bacterial agent comprises the supernatant of the above-mentioned Bifidobacterium animalis W3 fermentation broth.

[0044] Obviously, the skin care products may also contain any auxiliary materials allowed to be added in the cosmetics field, including but not limited to solvents, solubilizers, penetration enhancers, moisturizers, stabilizers and preservatives, etc., which are not specifically limited here.

[0045] The present invention is further elaborated in detail below in conjunction with specific embodiments. The following embodiments are not intended to limit the present invention, but are only intended to illustrate the present invention. The animal bifidobacterium (Bifidobacteriumanimalis) W3 involved in the following embodiments has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, and the deposit number is CGMCCNo.25262. The animal bifidobacterium (Bifidobacterium animalis) W3 fermentation liquid in Examples 2-8 is taken from the supernatant of the fermentation liquid obtained in Example 1. The HSF cells involved in the following embodiments are purchased from Guangdong Boxi Biotechnology Co., Ltd., the RAW264.7 and B16 cells involved in the following examples are purchased from Wuhan Punosai Biological Co., Ltd., the DPPH kit involved in the following examples is purchased from Nanjing Jiancheng Biotechnology Co., Ltd., and the IL-1a, TNF-a, and PGE2 ELISA kits involved in the following examples are purchased from Jiangsu Jingmei Biotechnology Co., Ltd.

[0046] Example 1 Preparation of Bifidobacterium animalis W3 fermentation broth

[0047] The activated Bifidobacterium animalis W3 strain was inoculated into BBL medium at a volume ratio of 3% and cultured anaerobically at 37°C for 36 hours to obtain a viable count of 6.6 x 10 8 CFU / mL. Then centrifuge at 8000-10000g for 10-15min, filter through a 0.22μm filter to remove the bacteria, and collect the supernatant of the fermentation broth.

[0048] The BBL medium formula includes: peptone 15.0g, yeast powder 2.0g, glucose 20.0g, soluble starch 0.5g, sodium chloride 5.0g, cysteine ​​0.5g, tomato extract 400.0mL, Tween 80 1.0mL, liver extract 80.0mL, agar 20.0g, tomato extract powder 5.0g, liver powder 2.0g, distilled water 1000.0mL, pH 7.0, sterilized at 115℃ for 20 minutes, cooled for use.

[0049] Example 2 Inhibitory effect of Bifidobacterium animalis W3 fermentation broth on the proliferation of RAW264.7 cells.

[0050] RAW264.7 cells were cultured at 1×10 4 Cells were inoculated into 96-well plates at a seeding density of 100 / well, and incubated in an incubator (37°C, 5% CO2) for 24 hours. When the cell plating rate in the 96-well plate reached 40%-60%, a zero adjustment group, a blank control group, a positive control group and a sample group were set. A gradient dilution was set in the sample group according to the final concentration of 1:1000-1:100, and the cells were cultured in the incubator for 24 hours. CCK-8 working solution was added, and the cells were incubated at 37°C in the dark for 1.5 hours. After the incubation, the OD value was read at 450nm, and the relative cell viability (%) was calculated according to the formula: (sample well OD-decay well OD) / (solvent control well OD-decay well OD)*100%. The cytotoxicity was determined, and the results were as follows: Figure 1 As shown, there was no cytotoxicity within the intervention concentration range of 1:250 dilution, so subsequent experiments were performed at dilution concentrations of 1:250 and 1:500.

[0051] Example 3 Inhibitory effect of Bifidobacterium animalis W3 fermentation broth on inflammatory factors

[0052] RAW264.7 cells were cultured at 1×10 4 Cells were inoculated into 96-well plates at a seeding density of 100 / well. After incubation in an incubator (37°C, 5% CO2) for 24 hours, when the cell plating rate in the 96-well plate reached 40%-60%, a zero adjustment group, a blank control group, a positive control group and a sample group were set. A gradient dilution of 1:5000-1:100 was set in the sample group. The cells were cultured in the incubator for 24 hours, and CCK-8 working solution was added. The cells were incubated at 37°C in the dark for 1.5 hours. After the incubation, the OD value was read at 450nm. The relative cell viability (%) was calculated according to the formula: (sample well OD-decay well OD) / (solvent control well OD-decay well OD)*100%. The cytotoxicity was determined and the concentration of inflammatory factors was selected. After the drug administration concentration was determined, 1×10 5 The cells were inoculated into 6-well plates at a seeding density of 1:1 / well and cultured in a 5% CO2, 37°C incubator for 24 h. Then, a blank group, a positive control group (LPS+DEX), a negative control group (LPS) and a sample group (LPS+sample) were set up. The expression levels of inflammatory factors were detected by IL-1a, TNF-a and PGE2 ELISA kits. The results are shown in Figure 2 As shown in the results, lactic acid bacteria fermentation broth can significantly reduce the expression of inflammatory factors PGE2, IL-1a and TNF-a, and play a soothing role.

[0053] Example 4 Inhibitory effect of Bifidobacterium animalis W3 fermentation broth on the proliferation of B16 cells

[0054] B16 cells were cultured at 1×10 4 Cells were inoculated into 96-well plates at a seeding density of 100 cells / well, and incubated in an incubator (37°C, 5% CO2) for 24 hours. When the cell plating rate in the 96-well plate reached 40%-60%, a zero adjustment group, a blank control group, a positive control group and a sample group were set. A gradient dilution was set in the sample group according to a final concentration of 0.025%-10%. The cells were cultured in the incubator for 24 hours, and CCK-8 working solution was added. The cells were incubated at 37°C in the dark for 1.5 hours. After the incubation, the OD value was read at 450nm. The relative cell viability (%) was calculated according to the formula: (sample well OD-decay well OD) / (solvent control well OD-decay well OD)*100%. The cytotoxicity was determined. The results are shown in Figure 3 As shown, there was no cytotoxicity within the intervention concentration range of 10%, so the subsequent experiments were performed at concentrations of 0.5% and 5%.

[0055] Example 5 Effect of Bifidobacterium animalis W3 fermentation broth on the relative content of melanin in B16 cells

[0056] B16 cells were cultured at 1×10 5 Cells were inoculated into 6-well plates at a density of 100 / well. After incubation in an incubator (37°C, 5% CO2) for 24 hours, the cell plating rate reached 40% to 60%. A blank control group and sample groups with different final concentrations were set up and continued to be cultured in the incubator for 24 hours. After the incubation, the cells were digested and collected, and 20 μl of the cell suspension was taken for counting, centrifuged at 500 rpm / min for 10 minutes, the supernatant was discarded, 2 ml of PBS was added to resuspend the cells, 500 μl of a 1:1 volume ratio of ethanol and ether was added, and the mixture was placed at room temperature for 30 minutes, centrifuged at 3000 rpm / min for 5 minutes, the supernatant was discarded, 1 ml of 1M NaOH solution containing 10% DMSO was added, mixed evenly, and placed in a water bath at 80 degrees for 45 minutes, and A490 was measured. The results are as follows Figure 4 As shown, compared with the control group, the fermentation broth at concentrations of 0.5% and 5% significantly reduced the melanin content in the corresponding B16 cells, indicating that it has an inhibitory effect on melanin production.

[0057] Example 6 Regulation of melanin metabolism in B16 cells by Bifidobacterium animalis W3 fermentation broth

[0058] B16 cells were cultured at 1×10 5Cells were inoculated into 6-well plates at a seeding density of 100 cells / well. After incubation in an incubator (37°C, 5% CO2) for 24 hours, the cell plating rate reached 40% to 60%. A blank control group and sample groups with different final concentrations were set up and cultured in the incubator for another 24 hours. After the incubation, the cells were digested and collected, and the total genomic RNA was extracted and then reversed into cDNA as a template for real-time fluorescence quantitative PCR to measure the expression changes of the target genes (TYR, TRP-1, TRP-2, ACTH, a-MSH, MME). The results are shown in the figure. Figure 5 As shown, genes related to melanin synthesis, such as TYR, TRP-1, TRP-2, ACTH, and a-MSH, all showed a certain inhibitory effect, and genes related to melanin catabolism were upregulated.

[0059] Example 7 Effect of Bifidobacterium animalis W3 fermentation broth on the proliferation of HSF cells

[0060] HSF cells were cultured at 1×10 4 Cells were inoculated into 96-well plates at a seeding density of 100 cells / well, and incubated in an incubator (37°C, 5% CO2) for 24 hours. When the cell plating rate in the 96-well plate reached 40%-60%, a zero adjustment group, a blank control group, a positive control group and a sample group were set. A gradient dilution was set in the sample group according to a final concentration of 0.025%-10%. The cells were cultured in the incubator for 24 hours, and CCK-8 working solution was added. The cells were incubated at 37°C in the dark for 1.5 hours. After the incubation, the OD value was read at 450nm. The relative cell viability (%) was calculated according to the formula: (sample well OD-decay well OD) / (solvent control well OD-decay well OD)*100%. The cytotoxicity was determined. The results are shown in Figure 6 As shown, there was no cytotoxicity within the intervention concentration range of 10%, so the subsequent experiments were performed at concentrations of 0.5% and 5%.

[0061] Example 8 Effect of Bifidobacterium animalis W3 fermentation broth on the DPPH scavenging ability of HSF cells

[0062] HSF cells were cultured at 1×10 5 Cells were inoculated into 6-well plates at a seeding density of 100 cells / well. After incubation in an incubator (37°C, 5% CO2) for 24 hours, the cell plating rate reached 40% to 60%. A blank control group and sample groups with different final concentrations were set up and cultured in the incubator for another 24 hours. After the incubation, the cells were digested and collected, centrifuged at 2000 rpm for 10 minutes, and the supernatant was taken. The cells were grouped according to the figure below, and the OD value was detected at 517 nm to detect the changes in the DPPH scavenging ability of HSF cells. The results are shown in FIG. Figure 7As shown, when the fermentation broth was intervened with 0.5% concentration, its DPPH scavenging ability increased by 27%, and when the concentration was intervened with 5%, its scavenging ability increased by 38.5%.

[0063] Blank Group Control group Sample set Cell culture supernatant (μL) 400 400 / 80% methanol (μL) 600 / 400 DPPH working solution (μL) / 600 600

[0064] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. Bifidobacterium animalis ( Bifidobacterium animalis )Application of W3 bacterial agent in any one or more of the following: (a) preparing whitening cosmetics; (b) preparing antioxidant cosmetics; (c) preparing anti-inflammatory soothing cosmetics; in, The animal Bifidobacterium ( Bifidobacterium animalis )W3 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 8, 2022, with the deposit number CGMCC No.25262; The bacterial agent is the supernatant of the fermentation broth of Bifidobacterium animalis W3; The supernatant is the liquid remaining after the bacterial cells grown in the fermentation broth of the animal Bifidobacterium W3 are separated from the liquid by centrifugation, filtration or sedimentation, and the bacterial cells are removed; During the fermentation process, the fermentation medium used was BBL medium; The BBL medium formula includes: peptone 15.0 g, yeast powder 2.0 g, glucose 20.0 g, soluble starch 0.5 g, sodium chloride 5.0 g, cysteine ​​0.5 g, tomato extract 400.0 mL, Tween 80 1.0 mL, liver extract 80.0 mL, agar 20.0 g, tomato extract powder 5.0 g, liver extract powder 2.0 g, distilled water 1000.0 mL, pH 7.0, sterilized at 115℃ for 20 minutes, and cooled for use.

2. The use as claimed in claim 1, It is characterized in that The bacterial agent is a solid preparation.

3. The use as claimed in claim 1, It is characterized in that The bacterial agent is a freeze-dried powder.

4. The use as claimed in claim 1, It is characterized in that In the above (a), whitening is specifically manifested in inhibiting the synthesis and accumulation of melanin and promoting the decomposition of melanin.

5. The use as claimed in claim 1, It is characterized in that In the above (b), the antioxidant effect is manifested as improving the ability to scavenge DDPH.

6. The use as claimed in claim 1, It is characterized in that In (c), the anti-inflammatory and soothing effect is specifically manifested by inhibiting the expression of relevant inflammatory factors, which include IL-1a, TNF-a and PGE2.

7. The use as claimed in claim 1, It is characterized in that The cosmetics are skin care products.

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