Composition with skin protection effect and application thereof

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a composition with a skin protection effect and application thereof. Specifically, the invention provides the composition with skin protection effects of resisting blue light, improving skin immunity, regulating skin mood pressure and the like for the first time, the composition comprises supernate of animal bifidobacterium W3 fermentation liquor and porphyra haitanensis extract, and the supernate and the porphyra haitanensis extract have a synergistic effect. The composition has the effects of resisting blue light, improving skin immunity and regulating skin mood pressure by recovering the cell viability level under blue light damage, increasing the expression quantity of VDR mRNA in skin, increasing the expression quantity of mRNA of antibacterial peptide LL-37 and increasing the content of beta-enorphin, and has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a composition and its application that has skin protective effects such as anti-blue light, enhancing skin immunity and regulating skin emotional stress. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] As the largest organ in the human body, the skin not only plays a crucial role in protecting the body from external physical, chemical, and biological damage, but also performs various physiological functions such as regulating body temperature, secretion and excretion, absorption, and sensation. Its health directly affects the overall health and external appearance of the body. In modern life, photodamage has become a common skin problem. Besides ultraviolet radiation, which is traditionally recognized, blue light in the visible light spectrum is increasingly harmful to the skin due to the widespread use of electronic devices. Studies have shown that blue light damages epidermal keratinocytes, leading to decreased cell activity and disruption of the skin barrier function. Therefore, developing safe and effective blue light protectants is an urgent need in the field of skin photoprotection.

[0004] Skin immunity is an important line of defense against pathogen invasion and the prevention of skin diseases. Vitamin D receptor (VDR) can regulate the production of antimicrobial peptides and enhance the skin's immune defense capabilities; antimicrobial peptide LL-37, as a key component of the human innate immune system, has both antibacterial and immunomodulatory functions, and the two work together to maintain skin immune homeostasis.

[0005] Furthermore, as an important sensory organ, the skin is easily affected by emotional stress. Emotional stress interferes with the function of the hypothalamus-pituitary-adrenal axis (HPA axis), altering the levels of hormones, neurotransmitters, and other active substances in the body, thereby affecting the physiological state of the skin. β-endorphin, as a neurotransmitter regulated by the HPA axis, can not only relieve emotional stress and produce a sense of pleasure, but also plays an important role in maintaining skin homeostasis. Long-term emotional stress can lead to a decrease in β-endorphin levels in the skin, affecting skin health.

[0006] Bifidobacterium animalis ( Bifidobacterium animalis Bifidobacterium animalis (Bifidobacterium) is a probiotic widely found in the intestines of humans and animals, playing an important role in maintaining gut health and enhancing immunity. In recent years, with the deepening of probiotic research, researchers have begun to explore the potential applications of probiotic fermentation products in the cosmetics field. Existing studies have shown that Bifidobacterium animalis (Bifidobacterium)... Bifidobacterium animalisThe supernatant of W3 fermentation broth inhibited the growth of pathogenic Escherichia coli and Staphylococcus epidermidis, promoted keratinocyte proliferation and filaggrin gene transcription, enhanced the antioxidant capacity of fibroblasts, inhibited the expression of inflammatory factors in macrophages, and suppressed the synthesis and accumulation of melanin in melanoma cells, indicating that it has antibacterial, repairing, antioxidant, whitening, and soothing effects (CN115786182B, CN117224462B). However, no related research has been found on its anti-blue light, skin immunity enhancement, and skin mood regulation. *Porphyra yezoensis* (also known as *Porphyra yezoensis*) Porphyra haitanensis As an economic red algae used for both food and medicine in my country, it is rich in active ingredients such as polysaccharides, phycobiliproteins, and polyphenols. Existing studies have shown that compositions containing Porphyra yezoensis have repairing, soothing, and sleep-aiding effects, suggesting its potential application in the skin care industry, but related research is still relatively scarce. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a composition and its application that possesses skin protective functions such as anti-blue light, enhancing skin immunity, and regulating skin stress. Specifically, the present invention incorporates Bifidobacterium animalis (… Bifidobacterium animalis The supernatant of W3 fermentation broth was used in combination with Porphyra yezoensis extract, and an unexpected synergistic effect was discovered. Experimental verification showed that it has anti-blue light effects, enhances skin immunity, and regulates skin stress. Based on the above research results, this invention was completed.

[0008] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a composition having a skin-protective effect, comprising, by weight, Bifidobacterium animalis (… Bifidobacterium animalis The supernatant of W3 fermentation broth contains 0.05-10 parts, and Porphyra yezoensis extract contains 0.002-0.6 parts. The skin protective effect is specifically manifested in any one or more of the following: anti-blue light, enhanced skin immunity, and regulation of skin emotional stress.

[0009] A second aspect of the present invention provides a method for preparing the above-mentioned composition having skin protective effect, the method comprising the step of mixing the supernatant of the fermentation broth of Bifidobacterium animalis W3 with Porphyra yezoensis extract.

[0010] A third aspect of the invention provides the use of the above-described composition with skin-protective properties in the preparation of a product. In this invention, the product can be a pharmaceutical or a cosmetic; further, the cosmetic is a skin care product.

[0011] In a fourth aspect, the present invention provides a cosmetic product comprising at least the above-described composition having a skin-protective effect.

[0012] The beneficial technical effects of one or more of the above technical solutions are as follows: The aforementioned technical solution provides, for the first time, a composition with skin-protective effects such as anti-blue light, enhanced skin immunity, and regulation of skin-related emotional stress. This composition comprises the supernatant of Bifidobacterium animalis W3 fermentation broth and Porphyra yezoensis extract, which have a synergistic effect. This composition exerts its anti-blue light, skin-immunity-enhancing, and skin-related emotional stress-regulating effects by restoring cell viability levels damaged by blue light, upregulating VDR mRNA expression in the skin, increasing the expression of the antimicrobial peptide LL-37 mRNA, and increasing β-endorphin content, thus demonstrating significant practical application value. Attached Figure Description

[0013] Figure 1 The graph shows the effects of Examples 1-4 and Comparative Examples 1-3 on cell viability levels. Figure 2 The graph shows the effects of Examples 1-4 and Comparative Examples 1-3 on VDR mRNA expression levels. Figure 3 The graph shows the effect of Examples 1-4 and Comparative Examples 1-3 on the expression level of antimicrobial peptide LL-37 mRNA; Figure 4 The graph shows the effect of Examples 1-4 and Comparative Examples 1-3 on β-endorphin content; in, Figures 1 - 4 In the study, significance was indicated by # for comparison with the blank control group, 0.01 < P ≤ 0.05 was indicated by #, and P ≤ 0.01 was indicated by ##; significance was indicated by * for comparison with the negative control group, 0.01 < P ≤ 0.05 was indicated by *, and P ≤ 0.01 was indicated by **. Detailed Implementation

[0014] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0015] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0016] In a typical embodiment of the present invention, a composition is provided that has skin protective effects such as anti-blue light, enhancing skin immunity, and regulating skin emotional stress, wherein the composition comprises, by weight, Bifidobacterium animalis (… Bifidobacterium animalis 0.05-10 parts of the supernatant of W3 fermentation broth and 0.002-0.6 parts of Porphyra yezoensis extract.

[0017] Among them, the animal bifidobacteria ( Bifidobacterium animalis The supernatant of the W3 fermentation broth is the liquid remaining after centrifugation, filtration, or sedimentation of the fermentation broth containing *Bifidobacterium animalis* W3, separating the bacterial cells from the liquid and removing the bacterial cells. The fermentation medium used during fermentation is BBL medium. The formula of the BBL medium is: 15.0 g peptone, 2.0 g yeast extract, 20.0 g glucose, 0.5 g soluble starch, 5.0 g sodium chloride, 0.5 g cysteine, 400.0 mL tomato extract, 1.0 mL Tween 80, 80.0 mL liver extract, 20.0 g agar, 5.0 g tomato extract powder, 2.0 g liver extract powder, 1000.0 mL distilled water, pH 7.0. Sterilize at 115℃ for 20 minutes and cool before use.

[0018] Specifically, the animal bifidobacteria ( Bifidobacterium animalis The W3 strain was inoculated into BBL medium and fermented at 30-40℃ (preferably 37℃) for 30-40 (preferably 36) hours. The fermentation broth was centrifuged to remove the bacterial cells, and then further filtered through a 0.22 µm filter membrane. The filtrate was collected as the supernatant of the fermentation broth for later use.

[0019] In this invention, the animal bifidobacteria ( Bifidobacterium animalis The supernatant of the W3 fermentation broth can be prepared according to the method described in Chinese Patent CN 115786182B.

[0020] The animal bifidobacteria ( Bifidobacterium animalis strain W3 was deposited on July 8, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25262. It is clearly already disclosed in Chinese patent CN115786182B.

[0021] The preparation method of the *Porphyra yezoensis* extract includes: After crushing the laver, water was added and mixed evenly. The homogenate was then heated to obtain a seaweed swelling solution. After adjusting the temperature of the seaweed swelling solution, β-galactosidase and pectinase were added successively to carry out enzymatic hydrolysis to obtain seaweed hydrolysate. The seaweed hydrolysate was then separated and dried to obtain the final product.

[0022] The ratio of laver to water is 1:15-30, preferably 1:20.

[0023] Heating to 90-110℃ (preferably 105℃) and maintaining the temperature for 10-60 min (preferably 30 min) yields a seaweed swelling solution; After cooling to 25-45℃ (preferably 30℃), the pH is adjusted to weakly acidic or neutral (e.g., pH 6.0), and β-galactosidase is added for the first enzymatic hydrolysis reaction. Then, the temperature is raised to 40-60℃ (preferably 50℃), the pH is adjusted to acidic (e.g., pH 4.0), and pectinase is added for the second enzymatic hydrolysis reaction. Finally, the temperature is raised to a high temperature (e.g., 90℃) to inactivate the enzyme solution, obtaining the seaweed enzymatic hydrolysate. The mass ratio of β-galactosidase to pectinase is 10:0.5-5, preferably 10:1; the enzyme activity ratio of β-galactosidase to pectinase is 1:30-50, preferably 1:40; and the mass ratio of β-galactosidase to *Porphyra yezoensis* raw material is 1:10-30, preferably 1:20.

[0024] The specific steps for separating and drying the seaweed enzymatic hydrolysate include: centrifuging to remove solid residue to obtain seaweed supernatant, adding dextrin and homogenizing to dissolve, and then spray drying to obtain the final product. The amount of dextrin added is 10-20% of the seaweed supernatant. w / w (), preferably 15%.

[0025] In another specific embodiment of the present invention, a method for preparing the above composition is provided, the method comprising the step of mixing the supernatant of the fermentation broth of Bifidobacterium animalis W3 with Porphyra yezoensis extract.

[0026] In another specific embodiment of the present invention, the application of the above composition in the preparation of a product is provided. In this invention, the product can be a pharmaceutical or a cosmetic; further, the cosmetic is a skincare product. Through experimental verification, the present invention demonstrates that the above composition can exert its effects of anti-blue light, enhancing skin immunity, and regulating skin emotional stress by restoring cell viability levels under blue light damage, upregulating the expression level of VDR mRNA in the skin, increasing the expression level of the antimicrobial peptide LL-37 mRNA, and increasing the content of β-endorphin.

[0027] In another specific embodiment of the present invention, a cosmetic is provided, the cosmetic comprising at least the above-mentioned composition having skin protective effects such as anti-blue light, enhancing skin immunity and regulating skin emotional stress.

[0028] The cosmetics may also contain other ingredients permitted in any skincare product field, including but not limited to emulsifiers, emollients, moisturizers, cooling agents, skin conditioning agents, thickeners, and preservatives.

[0029] Furthermore, by rationally adding the above-mentioned raw material components, this invention can also be used to prepare different cosmetic formulations, such as essence water, essence lotion, essence cream, etc., without making specific limitations here.

[0030] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Test methods in the following examples, unless otherwise specified, are generally performed under conventional conditions.

[0031] Bifidobacterium animalis used in the examples and comparative examples ( Bifidobacterium animalis The supernatant of the W3 fermentation broth was prepared in accordance with the description in Example 2 of Chinese Patent CN 115786182 B.

[0032] The preparation method of the *Porphyra yezoensis* extract used in the examples and comparative examples is as follows: (1) Heating and swelling: 20 kg of dried laver is crushed and mixed with 400 kg of water at a material-to-liquid ratio of 1:20. The homogenate is then pumped into a reaction vessel and heated to 105 °C. After maintaining the temperature for 30 min, laver swelling solution is obtained.

[0033] (2) Enzyme-assisted extraction: The seaweed swelling solution was transferred to an enzymatic hydrolysis tank and cooled to 30 °C. The pH was adjusted to 6.0. 1,000 g of β-galactosidase (enzyme activity of 250 U / g) was added and reacted for 4 h. The temperature was then raised to 50 °C and the pH was adjusted to 4.0. 100 g of pectinase (enzyme activity of 10,000 U / g) was added and reacted for 1 h. The temperature was then raised to 90 °C and held for 20 min to completely inactivate the enzyme solution and obtain the seaweed enzymatic hydrolysate.

[0034] (3) Solid-liquid separation: a butterfly centrifuge was used to remove most of the solid residue, followed by a tubular centrifuge (speed > 13,000 rpm) to remove the solid residue, and the supernatant was collected to obtain seaweed supernatant.

[0035] (4) Mixing: Add 15% ( ) to the seaweed supernatant. w / w Dextrin was added and homogenized to obtain a seaweed mixture.

[0036] (5) Spray drying: The seaweed mixture was dried using a centrifugal atomizer. The inlet air temperature was set to 140-155 ℃ and the outlet air temperature to 80-85 ℃ to obtain seaweed extract powder.

[0037] The preparation method of Porphyra yezoensis extract-2 in Comparative Example 3 differs from that of Porphyra yezoensis extract only in the enzyme-assisted extraction step. The pH of the β-galactosidase hydrolysis reaction in the enzyme extraction step is 8.0, while the other steps are the same.

[0038] Examples 1-4 provide a multi-effect composition with anti-blue light, skin immunity enhancement and skin emotional stress regulation effects. The raw material composition is shown in Table 1 below. For comparison, Comparative Examples 1-3 are also provided in Table 1. The difference between them and the examples is that they contain only one substance in the composition or contain compositions with different preparation processes of extracts.

[0039] Table 1. Raw material composition and proportions of the compositions of Examples 1-4 and Comparative Examples 1-3 (unit: wt%)

[0040] Experimental example: The performance of the products in the above embodiments and comparative examples was tested, as follows: Preparation of experimental materials: The primary human keratinocytes used in the following tests were purchased from Guangdong Boxi Biotechnology Co., Ltd.; the immortalized human keratinocytes (HaCaT cells) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; and the β-endorphin ELISA kit was purchased from Wuhan Elite Biotechnology Co., Ltd. Unless otherwise specified, all other reagents and materials are commercially available products.

[0041] I. Effects of Examples 1-4 and Comparative Examples 1-3 on Cell Viability Levels 1. Experimental Methods: HaCaT cells in good growth condition were collected and prepared into a cell suspension using DMEM high-glucose complete cell culture medium (hereinafter referred to as complete culture medium), and the cell concentration was adjusted to 1.5 × 10⁻⁶. 5 Cells / mL were added to 100 μL / well of cell suspension in a 96-well cell culture plate. A blank control group and a negative control group (45 J / cm²) were included in the experiment. 2 Blue light), sample group (45 J / cm) 2 Blue light was used in Examples 1-4 and Comparative Examples 1-3, with three replicates per group. The 96-well cell culture plates were cultured in a cell culture incubator (5% CO2, 37°C) for 24 h. When the cell confluence reached 50%-60%, the culture medium was discarded. 100 μL of PBS buffer was added to each 96-well cell culture plate, and all plates except the blank control group were irradiated with blue light at a dose of 45 J / cm². 2 After irradiation, discard the PBS buffer. Add 100 μL of complete culture medium to each well of the blank control group and negative control group, and add 100 μL of complete culture medium containing Examples 1-4 / Comparative Examples 1-3 to the other groups. Incubate in a cell culture incubator for 24 h, discard the culture medium, wash twice with PBS buffer, add 100 μL of CCK-8 working solution to each well, incubate in a cell culture incubator in the dark for 1 h, and then detect the OD value at 450 nm using a microplate reader.

[0042] The test concentrations of the composition samples in Examples 1-4 and Comparative Examples 1-3 are shown in Table 1, and the solvent is DMEM high-glucose complete cell culture medium.

[0043] 2. Experimental Results: The harmful effects of blue light on the skin are mainly manifested in the inhibition of cell growth and development, damage to cell structure, reduction of cell vitality, and ultimately cell death. For example... Figure 1 As shown in Table 2, after blue light stimulation, cell viability in the negative control group was significantly lower than that in the blank control group. P <0.01), proving the successful modeling of blue light damage; after blue light stimulation, the cell viability of the damaged cells was significantly increased compared to the negative control group after adding the compositions of Examples 1-4 and Comparative Examples 1-3. P The improvement rates were 54.21%, 81.17%, 57.37%, 64.42%, 43.90%, 20.54%, and 25.69%, respectively (<0.01), demonstrating that the compositions of Examples 1-4 and Comparative Examples 1-3 have the effect of resisting blue light damage and restoring cell viability. The results of Comparative Examples 1-2 show that using either *Porphyra yezoensis* extract or the supernatant of *Bifidobacterium animalis* W3 fermentation broth alone can improve cell viability. However, compared with the results of Example 2, it can be seen that the improvement effect is more significant when the two are used in combination, indicating that the composition formed by *Porphyra yezoensis* extract and the supernatant of *Bifidobacterium animalis* W3 fermentation broth has a synergistic effect in resisting blue light damage and improving cell viability. At the same time, compared with the results of Example 2, Comparative Example 3 shows that the combined use of *Porphyra yezoensis* extract and the supernatant of *Bifidobacterium animalis* W3 fermentation broth in the embodiments of the present invention has a better effect.

[0044] Table 2. Effects of Examples 1-4 and Comparative Examples 1-3 on cell viability levels

[0045] II. Effects of Examples 1-4 and Comparative Examples 1-3 on VDR mRNA Expression Level 1. Experimental Methods: Human primary keratinocytes in good growth condition were collected and a cell suspension was prepared using KcGrowth complete cell culture medium (hereinafter referred to as complete culture medium). The cell concentration was adjusted to 1.25 × 10⁻⁶ cells / mL. 5 Cells / mL were added to 2 mL / well of a 6-well cell culture plate. A blank control group and a negative control group (300 mJ / cm²) were included in the experiment. 2 UVB), sample group (300 mJ / cm) 2UVB+ Examples 1-4 / Comparative Examples 1-3), with 3 replicates per group. The 6-well plates were incubated in a cell culture incubator (5% CO2, 37°C) for 24 h. When the cell confluence reached 50%-60%, the culture medium was discarded. 2 mL of PBS buffer was added to each 6-well cell culture plate, and all plates except the blank control group were irradiated with UVB at a dose of 300 mJ / cm². 2 After irradiation, discard the PBS buffer. Add 2 mL of complete culture medium to each well of the blank control group and negative control group. Add 2 mL of complete culture medium containing Examples 1-4 / Comparative Examples 1-3 to the remaining groups. Incubate in a cell culture incubator for 24 h. Discard the culture medium. Extract total RNA from the cells of each group according to the RNA extraction kit instructions. Reverse transcribe the total RNA from the cells of each group into cDNA according to the RNA reverse transcription kit instructions. Detect using an RT-qPCR kit with primer sequences in Table 3 below. Pre-denaturation: 95℃, 30 s; Cycling reaction: 40 cycles, 60℃~95℃; Melting curve: obtained using the instrument's default program. Calculate the CT value of each sample using a 2... -△△Ct The results were analyzed using the method.

[0046] Table 3. Primer sequences

[0047] The test concentrations of the composition samples of Examples 1-4 and Comparative Examples 1-3 are shown in Table 1, and the solvent is KcGrowth complete cell culture medium.

[0048] 2. Experimental Results: VDR exerts its regulatory effect on skin immunity by inducing the expression of antimicrobial peptides. For example... Figure 2 As shown in Table 4, after UVB stimulation, the expression level of VDR mRNA in the negative control group was significantly lower than that in the blank control group. P <0.01), proving the successful modeling of the damage; after UVB stimulation, the expression level of VDR mRNA was significantly increased compared with the negative control group after adding the compositions of Examples 1-4 and Comparative Examples 1-3 to the damaged cells. P<0.05), with enhancement rates of 76.74%, 93.25%, 65.11%, 58.37%, 32.29%, 39.82%, and 39.20%, respectively. The results of Comparative Examples 1 and 2 indicate that using either *Porphyra yezoensis* extract or the supernatant of *Bifidobacterium animalis* W3 fermentation broth alone can enhance VDR mRNA expression. However, compared to the results of Example 2, the enhancement effect is more significant when the two are used in combination, indicating that the combination of *Porphyra yezoensis* extract and the supernatant of *Bifidobacterium animalis* W3 fermentation broth has a synergistic effect in promoting VDR mRNA expression. Furthermore, compared to the results of Example 2, Comparative Example 3 shows that the combined use of *Porphyra yezoensis* extract and the supernatant of *Bifidobacterium animalis* W3 fermentation broth in the embodiments of this invention is more effective.

[0049] Table 4. Effects of Examples 1-4 and Comparative Examples 1-3 on VDR mRNA expression levels

[0050] III. Effects of Examples 1-4 and Comparative Examples 1-3 on the expression level of antimicrobial peptide LL-37 mRNA 1. Experimental Methods: HaCaT cells in good growth condition were collected and prepared into a cell suspension using DMEM high-glucose complete cell culture medium (hereinafter referred to as complete culture medium), and the cell concentration was adjusted to 1.5 × 10⁻⁶. 5 Cells / mL were added to 2 mL / well of cell suspension in a 6-well cell culture plate. A blank control group and a negative control group (300 mJ / cm²) were included in the experiment. 2 UVB), sample group (300 mJ / cm) 2 UVB+ Examples 1-4 / Comparative Examples 1-3), with 3 replicates per group. The 6-well plates were incubated in a cell culture incubator (5% CO2, 37°C) for 24 h. When the cell confluence reached 50%-60%, the culture medium was discarded. 2 mL of PBS buffer was added to each 6-well cell culture plate, and all plates except the blank control group were irradiated with UVB at a dose of 300 mJ / cm². 2 After irradiation, discard the PBS buffer. Add 2 mL of complete culture medium to each well of the blank control group and negative control group. Add 2 mL of complete culture medium containing Examples 1-4 / Comparative Examples 1-3 to the remaining groups. Incubate in a cell culture incubator for 24 h. Discard the culture medium. Extract total RNA from the cells of each group according to the RNA extraction kit instructions. Reverse transcribe the total RNA from the cells of each group into cDNA according to the RNA reverse transcription kit instructions. Detect using an RT-qPCR kit with primer sequences in Table 5 below. Pre-denaturation: 95℃, 30 s; Cycling reaction: 40 cycles, 60℃~95℃; Melting curve: obtained using the instrument's default program. Calculate the CT value of each sample using a 2...-△△Ct The results were analyzed using the method.

[0051] Table 5. Primer sequences

[0052] The test concentrations of the composition samples in Examples 1-4 and Comparative Examples 1-3 are shown in Table 1, and the solvent is DMEM high-glucose complete cell culture medium.

[0053] 2. Experimental Results: LL-37 is part of the human innate immune system, possessing two main functions: killing bacteria and regulating immunity. Therefore, all three play an important role in regulating skin immunity. Figure 3 As shown in Table 6, after UVB stimulation, the expression level of LL-37 mRNA in the negative control group was significantly lower than that in the blank control group. P <0.01), proving the successful modeling of the damage; after UVB stimulation, the expression level of LL-37 mRNA was significantly increased compared with the negative control group after adding the compositions of Examples 1-4 and Comparative Examples 1-3 to the damaged cells. P <0.05), with enhancement rates of 86.61%, 102.02%, 80.41%, 70.16%, 40.83%, 57.93%, and 60.34%, respectively. The results of Comparative Examples 1 and 2 indicate that using either *Porphyra yezoensis* extract or the supernatant of *Bifidobacterium animalis* W3 fermentation broth alone can enhance LL-37 mRNA expression. However, compared to the results of Example 2, the enhancement effect is more significant when the two are used in combination, indicating that the combination of *Porphyra yezoensis* extract and the supernatant of *Bifidobacterium animalis* W3 fermentation broth has a synergistic effect in promoting LL-37 mRNA expression. Furthermore, compared to the results of Example 2, Comparative Example 3 shows that the combination of *Porphyra yezoensis* extract and the supernatant of *Bifidobacterium animalis* W3 fermentation broth used in the embodiments of this invention has a better effect.

[0054] Table 6. Effects of Examples 1-4 and Comparative Examples 1-3 on LL-37 mRNA expression levels

[0055] IV. Effects of the compositions of Examples 1-4 and Comparative Examples 1-3 on β-endorphin content 1. Experimental Methods: HaCaT cells in good growth condition were collected and prepared into a cell suspension using DMEM high-glucose complete cell culture medium (hereinafter referred to as complete culture medium). The cell density of the suspension was adjusted to 3 × 10⁻⁶ cells / mL. 5 Cells / mL were added to 1 mL of cell suspension in a 12-well cell culture plate. A blank control group and a negative control group (300 mJ / cm²) were included in the experiment. 2 UVB), sample group (300 mJ / cm)2 UVB+ Examples 1-4 / Comparative Examples 1-3), with 3 replicates per group. The 6-well plates were incubated in a cell culture incubator (5% CO2, 37℃) for 24 h until cell confluence reached 40-60%, at which point the culture medium was discarded. 1 mL of PBS buffer was added to each 12-well cell culture plate, and all plates except the blank control were irradiated with UVB at a dose of 300 mJ / cm². 2 After irradiation, discard the PBS buffer, add 1 mL of complete culture medium to each well of the blank control group and the negative control group, and add 1 mL of complete culture medium containing Examples 1-4 / Comparative Examples 1-3 to the other groups. Incubate in a cell culture incubator for 24 h, discard the culture medium, collect the cells, break the cells by repeated freeze-thaw cycles, centrifuge at 1500g for 10 min, and take the supernatant to detect the β-endorphin content.

[0056] The test concentrations of the compositions in Examples 1-4 and Comparative Examples 1-3 are shown in Table 1, and the solvent is DMEM high-glucose complete cell culture medium.

[0057] 2. Experimental Results: β-Endorphin is a key molecule with dual functions of "mood regulation" and "skin protection." Promoting the production of β-Endorphin in skin tissue helps alleviate the negative effects of emotional stress on the skin and helps the skin restore a healthy state. Figure 4 As shown in Table 7, after UVB stimulation, the β-endorphin content in the negative control group was significantly lower than that in the blank control group. P <0.01), proving the successful modeling of the damage; after UVB stimulation, the β-endorphin content was significantly increased compared to the negative control group after adding the compositions of Examples 1-4 and Comparative Examples 1-3 to the damaged cells. P <0.01), with enhancement rates of 45.62%, 80.27%, 63.61%, 68.28%, 28.96%, 20.96%, and 23.43%, respectively. The results of Comparative Examples 1 and 2 indicate that using either the supernatant of *Bifidobacterium animalis* W3 fermentation broth or *Porphyra yezoensis* extract alone has a certain effect on increasing β-endorphin content. Compared with the results of Example 2, it can be seen that the enhancement effect is more significant when the two are used in combination, indicating that the composition of *Porphyra yezoensis* extract and the supernatant of *Bifidobacterium animalis* W3 fermentation broth has a synergistic effect in increasing β-endorphin content. Meanwhile, comparing the results of Comparative Example 3 with those of Example 2, it can be seen that the combined use of *Porphyra yezoensis* extract and the supernatant of *Bifidobacterium animalis* W3 fermentation broth in the embodiments of this invention has a better effect.

[0058] Table 7. Effect of the compositions of Examples 1-4 and Comparative Examples 1-3 on β-endorphin content

[0059] The above results indicate that, including Bifidobacterium animalis ( Bifidobacterium animalis The combination of the supernatant of W3 fermentation broth and Porphyra yezoensis extract has a synergistic effect in resisting blue light, enhancing skin immunity, and regulating skin emotional stress.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A composition having a skin-protective effect, characterized in that, By weight: Bifidobacterium animalis ( Bifidobacterium animalis 0.05-10 parts of the supernatant of W3 fermentation broth and 0.002-0.6 parts of Porphyra yezoensis extract.

2. The composition according to claim 1, characterized in that, The animal bifidobacteria ( Bifidobacterium animalis The supernatant of the W3 fermentation broth is the liquid remaining after centrifugation, filtration or sedimentation of the fermentation broth of Bifidobacterium animalis W3, in which the bacterial cells growing in the fermentation broth are separated from the liquid and the bacterial cells are removed.

3. The composition according to claim 2, characterized in that, The fermentation medium used during the fermentation process is BBL medium.

4. The composition according to claim 1, characterized in that, The preparation method of the *Porphyra yezoensis* extract includes: After crushing the laver, water was added and mixed evenly. The homogenate was then heated to obtain a seaweed swelling solution. After adjusting the temperature of the seaweed swelling solution, β-galactosidase and pectinase were added to carry out enzymatic hydrolysis to obtain seaweed hydrolysate. The seaweed hydrolysate was then separated and dried to obtain the final product.

5. The composition according to claim 4, characterized in that, The ratio of laver to water is 1:15-30; Heat the solution to 90-110℃ and maintain the temperature for 10-60 minutes to obtain the seaweed swelling solution. After adjusting the temperature and pH, β-galactosidase and pectinase were added sequentially to carry out the enzymatic hydrolysis reaction; Further, after cooling to 25-45℃, the pH is adjusted to weakly acidic or neutral, and β-galactosidase is added for the first enzymatic hydrolysis reaction. Then, the temperature is raised to 40-60℃, the pH is adjusted to acidic, and pectinase is added for the second enzymatic hydrolysis reaction. Finally, the enzyme solution is inactivated by heating to obtain seaweed hydrolysate. The mass ratio of β-galactosidase to pectinase is 10:0.5-5; the enzyme activity ratio of β-galactosidase to pectinase is 1:30-50; and the mass ratio of β-galactosidase to laver raw material is 1:10-30.

6. The composition according to claim 4, characterized in that, The specific steps for separating and drying the seaweed enzymatic hydrolysate include: after centrifuging to remove solid residue, adding dextrin for homogenization and dissolution, and then spray drying to obtain the final product.

7. A method for preparing the composition with skin-protective effect according to any one of claims 1-6, characterized in that, The preparation method includes the step of mixing the supernatant of the fermentation broth of Bifidobacterium animalis W3 with Porphyra yezoensis extract.

8. The use of the composition having a skin-protective effect according to any one of claims 1-6 in the preparation of a product.

9. The application as described in claim 8, characterized in that, The product is a pharmaceutical or a cosmetic; further, the cosmetic is a skincare product. The skin protection effect is specifically manifested in one or more of the following: anti-blue light, enhancing skin immunity, and regulating skin emotional stress.

10. A cosmetic product, characterized in that, The cosmetic product comprises at least the skin-protecting composition according to any one of claims 1-6.

Images