Fermentation product of bifidobacterium animalis subsp. Lactis BAPerfectus006 and application of fermentation product in inflammation resistance, light aging resistance and oil control

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a fermentation product of bifidobacterium animalis subsp. Lactis (Bifidobacterium animalis subsp. Lactis) BAPerfectus006 and application of the fermentation product in anti-inflammation, anti-light aging and oil control of the fermentation product of the bifidobacterium animalis subsp. Lactis (Bifidobacterium animalis subsp. Lactis) BAPerfectus006. According to the present invention, the RBGR value of the animal bifidobacterium subsp. Lactis BAPerfectus006 is higher than the RBGR value of other types of animal bifidobacteria, such that the oxygen resistance of the animal bifidobacterium subsp. Lactis BAPerfectus006 is good; the fermentation product lysate or filtrate containing the animal bifidobacterium subsp. Lactis BAPerfectus006 has the effects of resisting inflammation, resisting blue light, resisting light aging, controlling oil and the like.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to an application of a fermentation product of animal Bifidobacterium lactis subspecies BAPerfectus006 in anti-inflammatory, anti-blue light, anti-photoaging and oil control. Background Art

[0002] Bifidobacterium animalis subsp. lactis is a probiotic that is widely found in the intestines of humans and animals. It plays an important role in maintaining intestinal health and enhancing immunity. In recent years, with the deepening of probiotic research, people have begun to explore the potential application of its fermentation products in the field of skin photoaging and oil control. Some studies have shown that probiotic fermentation products may help reduce skin damage caused by photoaging and reduce the secretion of sebaceous glands.

[0003] Anti-photoaging refers to a series of measures to prevent and reduce skin aging caused by exposure to light sources such as ultraviolet (UV) and blue light. Long-term exposure to sunlight or other light sources can lead to premature aging phenomena such as dry skin, spots, wrinkles and sagging. Ultraviolet rays are mainly divided into two types: UVA and UVB. UVA can penetrate deep into the dermis of the skin, causing degradation of collagen and elastic fibers, causing skin sagging and wrinkles; UVB mainly affects the epidermis, causing immediate sunburn and long-term skin damage. Blue light is the shorter wavelength part of the visible spectrum, which can penetrate deep into the skin, aggravate the skin's oxidative stress response, lead to an increase in free radicals, and then promote skin aging. Radiation from ultraviolet and blue light can also cause inflammatory reactions and aggravate skin damage.

[0004] At present, publication number CN 115634189A discloses a bifidobacterium fermentation product lysate and its preparation method and application. The application of the bifidobacterium fermentation product lysate in anti-photoaging cosmetics has the effect of down-regulating the expression of inflammatory factors IL-1a, IL-6, and TNF-α of keratinocytes after ultraviolet irradiation. Publication number CN111235058B discloses a short bifidobacterium with the effects of repairing ultraviolet damage, relieving inflammation, and preventing skin photoaging, and its preparation method and application. The short bifidobacterium has the effect of repairing skin damage and inflammation caused by ultraviolet rays, can improve the skin photoaging phenomenon caused by long-term and repeated ultraviolet irradiation, reduce the content of ROS, IL-1β, IL-6 and TNFα, and increase the content of type I and type III collagen, hydroxyproline, and hyaluronic acid.

[0005] However, the research on the fermentation products of Bifidobacterium animalis subsp. lactis in the field of photoaging is still in its infancy. Most studies mainly use probiotic liquid or its fermentation product lysate, focusing on anti-photoaging, oxidative damage and inflammatory response caused by ultraviolet radiation. With the development of science and technology and the popularization of electronic products, blue light is increasingly damaging the skin. Existing studies do not provide a comprehensive photoaging protection strategy and lack multifunctional applications.

[0006] Oil control refers to taking measures to slow down the secretion and deposition of sebum and reduce the oiliness of the skin. Sebum is mainly secreted by keratinocytes and sebaceous glands to form a mixture of various lipids including squalene, monoester wax, free fatty acids, triglycerides, etc., which reach the skin surface through hair follicles. In existing research, publication number CN116747185B discloses a fermented complex for dandruff removal and oil control, and its preparation method and application. The fermented complex can exert excellent dandruff removal and oil control effects when applied to the scalp, and is suitable as a daily scalp care product.

[0007] At present, most existing studies are about compounding or co-fermenting probiotic fermentation products with plant extracts, and there are few studies on using fermentation products alone to achieve the effect of skin oil control. The application of probiotics in the field of skin care is mainly its fermentation product lysate or fermentation product filtrate. However, in the process of industrialization, only one of them is often developed and used alone, because few probiotic fermentation products can be used simultaneously in different skin care directions.

[0008] Therefore, there is an urgent need for an animal Bifidobacterium lactis subspecies that can comprehensively deal with multiple photoaging factors such as ultraviolet rays and blue light, and provide new solutions and technical support for the field of photoaging protection and oil control. Summary of the invention

[0009] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and to provide an animal Bifidobacterium lactis subspecies BAPerfectus006 or an animal Bifidobacterium lactis subspecies BAPerfectus006 fermentation product and its application in anti-inflammatory, anti-blue light, anti-photoaging and oil control.

[0010] To achieve the above purpose, the technical solution adopted by this application is:

[0011] The present application provides an application of animal Bifidobacterium lactis subspecies BAPerfectus006 or a fermentation product lysate or filtrate containing animal Bifidobacterium lactis subspecies BAPerfectus006 for non-disease treatment purposes in at least one of the following aspects: 1) anti-inflammatory; 2) anti-blue light; 3) anti-photoaging; 4) oil control;

[0012] The preservation number of the animal Bifidobacterium lactis subspecies BAPerfectus006 is GDMCC No: 62539, and the preservation classification name is Bifidobacterium animalis subsp.lactis. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2022, and the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0013] The animal Bifidobacterium lactis subspecies BAPerfectus006 of the present application is cultured in a reinforced clostridial culture medium and anaerobically cultured at 37° C. for 48 hours. The colonies are white, round, moist, opaque, and have neat edges.

[0014] Animal Bifidobacterium lactis subspecies BAPerfectus006 is irregular rod-shaped under a microscope, 0.6-0.9μm×1.1-4.8μm, arranged singly or in pairs, and Gram-positive.

[0015] The 16S rDNA gene sequence of the above-mentioned Bifidobacterium animalis subspecies lactis BAPerfectus006 is shown in SEQ ID NO: 1, and the groEL gene sequence is shown in SEQ ID NO: 2.

[0016] The 16S rDNA sequence and groEL gene sequence of Bifidobacterium animalis subsp. lactis BAPerfectus006 have a similarity of 100.0% with those of Bifidobacterium animalis subsp. lactis DSM 10140T (CP001606), and the similarity of groEL gene sequence with Bifidobacterium animalis subsp. animalis ATCC 25527T (CP002567) is 96.9%, so it can be determined to be Bifidobacterium animalis subsp. lactis.

[0017] Experiments have shown that the RBGR value of animal Bifidobacterium lactis subspecies BAPerfectus006 is higher than that of other types of animal Bifidobacterium, indicating that animal Bifidobacterium lactis subspecies BAPerfectus006 has better oxygen resistance, which ensures the stability of the bacteria during fermentation.

[0018] As a preferred embodiment of the application described in the present application, the animal Bifidobacterium lactis subspecies BAPerfectus006, the fermentation product lysate or filtrate containing animal Bifidobacterium lactis subspecies BAPerfectus006 is made into a microbial preparation.

[0019] Preferably, in the microbial preparation, the concentration of the fermentation product lysate of Bifidobacterium animalis subspecies BAPerfectus006 is 0.1% to 2%, and the concentration of the fermentation product filtrate of Bifidobacterium animalis subspecies BAPerfectus006 is 1 to 20%.

[0020] In some specific embodiments, the concentration of the fermentation product lysate of Bifidobacterium animalis subsp. lactis is 1% to 2%, or 0.3 to 0.6%, or 0.5%, or 0.1%.

[0021] In some specific embodiments, the concentration of the fermentation product filtrate of Bifidobacterium animalis subsp. lactis is 0.1-0.15%, or 1-2%, or 5%-20%, or 1.25%-5%.

[0022] The fermentation product lysate or filtrate of Bifidobacterium animalis subspecies lactis of the present application has better anti-inflammatory, anti-blue light, anti-photoaging and oil control effects.

[0023] The present application provides the use of the above-mentioned animal Bifidobacterium lactis subspecies BAPerfectus006 or a fermentation product lysate or filtrate containing animal Bifidobacterium lactis subspecies BAPerfectus006 in the preparation of a product, wherein the product is used in at least one of the following aspects: 1) anti-inflammatory; 2) anti-blue light; 3) anti-photoaging; 4)

[0024] Oil control;

[0025] The preservation number of the animal Bifidobacterium lactis subspecies BAPerfectus006 is GDMCC No: 62539, and the preservation classification name is Bifidobacterium animalis subsp.lactis. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2022, and the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0026] As a preferred embodiment of the application described in this application, the product includes cosmetics, health foods or medicines.

[0027] The present application provides a cosmetic, which includes Bifidobacterium animalis subspecies BAPerfectus006 or a fermentation product lysate or filtrate containing Bifidobacterium animalis subspecies BAPerfectus006.

[0028] The present application provides a healthy food, which includes Bifidobacterium animalis subspecies BAPerfectus006 or a fermentation product lysate or filtrate containing Bifidobacterium animalis subspecies BAPerfectus006.

[0029] The present application provides a medicine, which includes animal Bifidobacterium lactis subspecies BAPerfectus006 or a fermentation product lysate or filtrate containing animal Bifidobacterium lactis subspecies BAPerfectus006.

[0030] In the technical scheme of the present application, the animal bifidobacterium lactis subspecies BAPerfectus006 fermentation product lysate of the present application can significantly reduce the neutrophil fluorescence intensity of zebrafish embryos, and has anti-inflammatory soothing effect; animal bifidobacterium lactis subspecies BAPerfectus006 fermentation product lysate enhances the ability of skin to resist blue light radiation damage by reducing the effect of ROS; adding animal bifidobacterium lactis subspecies BAPerfectus006 fermentation product lysate, 8-OHdG content also showed a significant decrease (P < 0.01), indicating that animal bifidobacterium lactis subspecies BAPerfectus006 fermentation product lysate has a certain degree of anti-DNA damage function when cells are damaged by ultraviolet light. Animal bifidobacterium lactis subspecies BAPerfectus006 fermentation product lysate can significantly reduce MMP-1 content, significantly increase Collagen Ι and Elastin content, indicating that the fermentation product lysate of animal bifidobacterium lactis subspecies can reduce the degradation of skin collagen and elastin by reducing MMP-1 content, thereby enhancing the skin's ability to resist photoaging.

[0031] The relative expression of the AQP3 gene of the animal Bifidobacterium lactis subspecies BAPerfectus006 fermentation product lysate was significantly increased (P<0.01), and the expression of the AQP3 gene reached a significant difference compared with the competing product (P<0.01), indicating that the animal Bifidobacterium lactis subspecies BAPerfectus006 fermentation product lysate prepared by the present application has the effect of moisturizing. At the same time, the relative expression of the LOR and TGM1 genes was significantly increased (P<0.01) by adding the animal Bifidobacterium lactis subspecies BAPerfectus006 fermentation product lysate, and the expression of the LOR and TGM1 genes reached a significant difference (P<0.01) compared with the competing product, indicating that the animal Bifidobacterium lactis subspecies BAPerfectus006 fermentation product lysate prepared by the present application has the effect of barrier repair.

[0032] In the technical scheme of the present application, the fermentation product filtrate of animal Bifidobacterium lactis subspecies BAPerfectus006 of the present application can significantly inhibit the production of lipid droplets by sebaceous gland cells and has the effect of oil control. In addition, the fermentation product filtrate of the present application also has the effects of skin barrier repair, moisturizing, anti-inflammatory and the like.

[0033] Compared with the prior art, this application has the following beneficial effects:

[0034] The present application provides an application of animal Bifidobacterium lactis subspecies and its fermentation products in anti-inflammatory, anti-blue light, anti-photoaging and oil control. The RBGR value of animal Bifidobacterium lactis subspecies BAPerfectus006 of the present application is higher than that of other types of animal Bifidobacterium, indicating that animal Bifidobacterium lactis subspecies BAPerfectus006 has better oxygen resistance; the fermentation product lysate or filtrate containing animal Bifidobacterium lactis subspecies BAPerfectus006 has anti-inflammatory, anti-blue light, anti-photoaging and oil control effects, so that animal Bifidobacterium lactis subspecies BAPerfectus006 has a wider application prospect in the field of skin health care. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the colony morphology of Bifidobacterium animalis subspecies lactis BAPerfectus006;

[0036] Figure 2 This is a microscopic bacterial morphology of animal Bifidobacterium lactis subspecies BAPerfectus006;

[0037] Figure 3 It is a 16s rDNA sequence alignment diagram of Bifidobacterium animalis lactis subspecies BAPerfectus006;

[0038] Figure 4 This is a comparison diagram of the groEL sequence of Bifidobacterium animalis subspecies lactis BAPerfectus006;

[0039] Figure 5 The graph is a result of the change in the fluorescence intensity of neutrophils in different samples in Example 4;

[0040] Figure 6 The results of the changes in ROS content of different samples in Example 5;

[0041] Figure 7 8-OHdG content variation results of different samples in Example 6;

[0042] Figure 8 This is a graph showing the changes in the MMP-1 content of different samples in Example 7;

[0043] Fig. 9 The figure is the result of the change of Collagen 1 content of different samples in Example 7;

[0044] Fig.10 This is a graph showing the changes in Elastin content of different samples in Example 7;

[0045] Fig.11 is a graph showing the relative expression of the AQP3 gene in different samples in Example 8;

[0046] Fig.12 The relative expression level of LOR genes in different samples in Example 9;

[0047] Fig.13 is a graph showing the relative expression of the TGM1 gene in different samples in Example 9;

[0048] Fig.14 The scratch repair results of different groups in Example 10 are shown;

[0049] Fig.15 This is a diagram showing the distribution of lipid droplets in different groups before and after the action in Example 11 (magnification: 10×);

[0050] Fig.16 This is a statistical analysis result of the fluorescence intensity of Nile red staining of cells in different groups in Example 11. DETAILED DESCRIPTION

[0051] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0052] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0053] In the following embodiments, the preservation number of Bifidobacterium animalis subsp. lactis F829 is GDMCC No: 64783, the preservation classification name is Bifidobacterium animalis subsp. lactis, and it has been deposited in Guangdong Provincial Microbiological Culture Collection Center on June 21, 2024, and the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0054] The preservation number of Bifidobacterium animalis subsp. lactis F830 is GDMCC No: 64784, and the preservation classification name is Bifidobacterium animalis subsp. lactis. It was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 21, 2024. The preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0055] Example 1: Isolation and identification of animal Bifidobacterium lactis subspecies BAPerfectus006

[0056] This embodiment provides a method for isolating and identifying animal Bifidobacterium lactis subspecies BAPerfectus006, comprising the following steps:

[0057] 1. Bifidobacterium animalis subsp. lactis

[0058] Separation of BAPerfectus006:

[0059] Feces of healthy male infants aged 3.5 months were collected as samples. 1 g of sample was added to 10 mL of sterile saline and mixed. The mixture was centrifuged at 800 rpm for 5 min to remove large impurities. 1 mL of the supernatant bacterial suspension was diluted 10 times with sterile saline and the appropriate dilution gradient (10 -5 , 10 -6 , 10 -7 ) was spread on a plate of Bifidobacterium culture medium BBL (Haibo Biotechnology Co., Ltd., Qingdao High-tech Industrial Park, HB0397) and placed in an anaerobic incubator (37°C) for 24 to 48 hours. White opaque circular colonies were selected with an inoculation loop and streaked 3-5 times continuously until a single colony was purified. Staining and microscopic examination were performed according to the instructions of the Gram staining kit. After confirming that it was a Gram-positive bacterium, it was sent for 16s rDNA sequencing and identification. After enrichment culture, the pure colonies were mixed with sterile glycerol (final concentration was 20% glycerol) and stored at -80°C.

[0060] 2. Bifidobacterium animalis subsp. lactis

[0061] Identification of BAPerfectus006:

[0062] According to the "FMIC-QO01-001-2015 Microbiological Detection of Bacterial Multiphase Identification Detection Method", animal Bifidobacterium lactis subspecies BAPerfectus006 was identified. The results are as follows:

[0063] (1) Morphological observation:

[0064] a. Macroscopic morphology: Animal Bifidobacterium lactis subspecies BAPerfectus006 was cultured in a reinforced Clostridium medium and cultured anaerobically at 37°C for 48 hours. The colonies were white, round, moist, opaque, and had neat edges. Figure 1 .

[0065] b. Microscopic morphology: The bacteria are irregular rod-shaped under the microscope, 0.6-0.9μm×1.1-4.8μm, arranged singly or in pairs, and Gram-positive, such as Figure 2 .

[0066] (2) 16S rDNA identification and groEL sequence identification:

[0067] The 16S rDNA gene sequence of the strain claimed in the present application is shown in SEQ ID NO: 1.

[0068] The National Center for Biotechnology Information (NCBI) database was used for sequence alignment and phylogenetic analysis. Figure 3 From the results of 16S rDNA, it showed 100% similarity with Bifidobacterium animalis subsp. lactis DSM10140T (CP001606), and it was preliminarily determined to be Bifidobacterium animalis subsp. lactis.

[0069] In order to further confirm the accuracy of its type, its groEL sequence was tested, and the sequence result is shown in SEQ ID NO:2.

[0070] The groEL gene sequences were compared and the results were as follows Figure 4 As shown, the similarity between animal Bifidobacterium lactis subsp. BAPerfectus006 and animal Bifidobacterium lactis subsp. (Bifidobacterium animalis subsp. lactis) DSM10140T (CP001606) reached 100.0%, and the similarity between animal Bifidobacterium animalis subsp. (Bifidobacterium animalis subsp. animalis) ATCC 25527T (CP002567) was 96.9%, which can be determined to be animal Bifidobacterium lactis subsp.

[0071] The preservation number of Bifidobacterium animalis subsp. lactis BAPerfectus006 is GDMCC No: 62539, and the preservation classification name is Bifidobacterium animalis subsp. lactis. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2022. The preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0072] Example 2: Evaluation of oxygen tolerance of Bifidobacterium animalis subsp. lactis BAPerfectus006

[0073] In this example, two strains of animal Bifidobacterium lactis subspecies F830 and F829 [derived from the strain library of Perfect (Guangdong) Daily Necessities Co., Ltd. and sent to the Guangdong Provincial Microbial Culture Collection Center for preservation] were selected for comparison. Animal Bifidobacterium lactis subspecies F830 and F829 were both independently isolated, purified and identified by Perfect, and the test method was as follows:

[0074] Animal Bifidobacterium lactis subspecies BAPerfectus006 and animal Bifidobacterium lactis subspecies F830 and F829 were subcultured to the second generation, 2.5 mL (5% addition) of the second generation bacterial solution was taken into a centrifuge tube filled with 50 mL of oxygen-resistant culture medium, shaken evenly, placed in an anaerobic culture box, and cultured in a biochemical incubator at 37°C for about 24 h (stable period, 3 parallel samples for each strain), and when the strains grew to the stable period, the OD of this period was measured. 600 value.

[0075] Take another 5 mL of the second-generation bacterial solution (5% addition) and put it into a 250 mL conical flask filled with 100 mL BBL medium, seal it with kraft paper, put it in a shaker, adjust the speed to 100 rpm, and culture it at 37 ° C for 24 h (stable period, 3 parallel samples for each strain), and measure the OD 600 The RBGR value (specific growth rate, RBGR = aerobic conditions OD 600 Value / strict anaerobic conditions OD 600 The larger the RBGR value, the better the oxygen tolerance of the bacteria (RBGR>0.7). The RBGR value is referenced in the following literature:

[0076] [1] Wang Yuanchi, Yao Guoqiang, Zhang Wenyi, et al. Study on oxygen tolerance acclimation of Bifidobacterium lactis Probio-M8[J]. Journal of Chinese Institute of Food Science and Technology, 2020, 20(12): 7. DOI: 10.16429 / j.1009-7848.2020.12.001.

[0077] [2]Talwalkar A, Kailasapathy K, Peiris P, et al.Application of RBGR--asimple way for screening of oxygen tolerance in probiotic bacteria[J].International Journal of Food Microbiology,2001,71(2-3):245-248.DOI:10.1016 / S0168-1605(01)00563-3.

[0078] The test results are shown in Table 1.

[0079] Table 1 Results of oxygen tolerance test of animal Bifidobacterium lactis subspecies BAPerfectus006

[0080]

[0081] As can be seen from Table 1 above, the RBGR values ​​of Bifidobacterium animalis subspecies BAPerfectus006 are higher than those of Bifidobacterium animalis subspecies F830 and F829, which means that Bifidobacterium animalis subspecies BAPerfectus006 has better oxygen resistance, thus ensuring the stability of the bacteria during fermentation.

[0082] Example 3, Preparation method of fermentation product of Bifidobacterium animalis subsp. lactis BAPerfectus006

[0083] This embodiment provides a method for preparing a fermentation product of Bifidobacterium animalis subspecies BAPerfectus006. The fermentation product includes two types, one is a lysate of the fermentation product of Bifidobacterium animalis subspecies BAPerfectus006 (i.e., bifid yeast), and the other is a filtrate of the fermentation product of Bifidobacterium animalis subspecies BAPerfectus006 (i.e., bifid yeast filtrate).

[0084] The specific preparation method is referred to below, comprising the following steps:

[0085] Seed expansion: Under sterile conditions, take the animal Bifidobacterium lactis subsp. BAPerfectus006 ampule tube freeze-dried powder stored at -80°C and inoculate it into the first-level seed culture medium at a rate of 1%-10%. After inoculation, place it in an anaerobic incubator and culture it at 36-38°C for 16-48h to obtain the first-level seed solution. Continue seed expansion with the same operation steps to obtain the second-level seed culture solution and the third-level seed culture medium.

[0086] Fermentation: Take the third-level seed liquid and inoculate it into the fermentation medium at an inoculation rate of 1%-10%, and culture it anaerobically at 36-38° C. and a rotation speed of 50-100 rpm for 16-48 hours to obtain the animal Bifidobacterium lactis subsp. BAPerfectus006 fermentation liquid.

[0087] Fermentation product lysate (bifid yeast): Use a disc centrifuge to centrifuge the animal Bifidobacterium lactis subspecies BAPerfectus006 fermentation liquid at a speed of 8000-12000rpm, and collect the centrifuged supernatant and bacterial mud respectively. The centrifuged supernatant is used for the preparation of the fermentation product filtrate (bifid yeast filtrate). The bacterial mud is diluted with the composite solution at a ratio of 1%-10%, stirred at a speed of 100-500rpm for 10-60min, and then the bacterial mud composite liquid is homogenized by a high-pressure homogenizer, the homogenization pressure is 200-1200bar, and the number of homogenizations is 1-10 times. Heat treat the homogenized broken liquid at 60-120℃ for 10-100min, centrifuge it at a speed of 8000-12000rpm, collect the centrifuged supernatant, and perform pasteurization and preservative addition to obtain bifid yeast. The pasteurization parameters are 60-120℃ and the time is 15-100min.

[0088] Fermentation product filtrate (binofibrate yeast filtrate): Take the first centrifugation supernatant in the production process of the fermentation product lysate (binofibrate yeast), and use membrane separation equipment such as microfiltration and nanofiltration to decolorize and filter until the OD value of the decolorized liquid is less than 2; after decolorization, perform 0.22μm sterilization filtration and add a preservative to obtain the binofibrate yeast filtrate.

[0089] Example 4. Anti-inflammatory ability test of lysate (Bifidobacterium animalis subsp. lactis) BAPerfectus006 fermentation product

[0090] 1. Test principle:

[0091] Sodium dodecyl sulfate can induce epidermal inflammatory cell infiltration after stimulating the epidermis, which can lead to symptoms such as edema, erythema, itching, and pain. Sodium dodecyl sulfate can also induce an inflammatory response when exposed to the skin surface of zebrafish embryos, causing neutrophils to undergo an immune response and migrate and aggregate to the site of inflammation. Therefore, an inflammation model can be established using transgenic neutrophil green fluorescent strain zebrafish embryos, and the fluorescence changes of zebrafish embryo neutrophils before and after treatment with the test substance can be used to detect whether the sample has anti-inflammatory effects.

[0092] 2. Test methods:

[0093] Normally developed 72hpf transgenic neutrophil green fluorescent strain zebrafish embryos were pre-screened under a stereomicroscope and randomly assigned to 96-well plates, one per well, and 8 replicates per group. The embryo culture medium in the 96-well plate was removed without harming the young fish. The embryo culture medium was prepared as follows: NaCl 7.000g, NaHCO3 0.400g, KCl0.100g, CaCl2 0.235g, and each reagent was added to a measuring cup in sequence according to the formula dosage. The volume was adjusted to 2000mL with water that met the requirements of GB / T 6682, and filtered using a vacuum filter (pore size 0.2μM), sterilized using an autoclave, and kept at room temperature for use. Then, 200 μL of working solution of different concentrations was quickly added to each well of the sample group (see Table 2 for details, sample group bifidum yeast (animal Bifidobacterium lactis subsp. BAPerfectus006 fermentation product lysate), competitor group Repair Complex CLR (a long bifidobacterium fermentation product lysate, purchased from CLR, Germany); the working solution was prepared by adding the corresponding solution to the embryo culture solution and diluting it to the corresponding concentration), the blank group and the model group were added with 200 μL of embryo culture solution, and the positive group was added with 200 μL of dexamethasone embryo culture solution (concentration 10 μmol / L). After incubation in a biochemical incubator at 28.5±0.5℃ for 120 minutes, the solution in the 96-well plate was removed without harming the zebrafish embryos, and 200 μL of embryo culture solution was quickly added to the blank group, and 200 μL of modeling agent sodium dodecyl sulfate was added to the model group, positive group, competitor group and sample group, and incubated in a biochemical incubator at 28.5±0.5℃ for 120 minutes. After the incubation, 4 zebrafish embryos were randomly selected from the zebrafish embryos with normal phenotype and behavior, fixed with 4% methylcellulose, observed and photographed under a fluorescence microscope, and the fluorescence intensity of the obtained zebrafish embryo pictures was statistically analyzed using the image analysis software Image J to represent the severity of inflammation. The data are expressed as Mean ± SD. The comparison between the groups was analyzed by t-test. The statistical analysis was two-tailed. P < 0.05 was considered to be significantly different, and the smaller the P value, the greater the significant difference. # represents P < 0.05, ## represents P < 0.01, and ### represents P < 0.001 (# represents comparison with the blank group); * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001 (* represents comparison with the model group).

[0094] Table 2 Zebrafish anti-inflammatory test design and grouping

[0095]

[0096] 3. Test results

[0097] Fluorescence intensity of inflammatory neutrophils in zebrafish embryos Figure 5As shown. The results show that compared with the blank group, the fluorescence intensity of neutrophils in the model group increased significantly (P < 0.01), indicating that the stimulation conditions of this test were effective and the model was successfully established. Compared with the model group, the fluorescence intensity of neutrophils in the positive group was significantly reduced (P < 0.01), indicating that the positive control test was effective. Compared with the model group, 1% and 2% concentrations of the competitor and sample group bifid yeast can significantly reduce the fluorescence intensity of neutrophils in zebrafish embryos (P < 0.01), among which the sample group bifid yeast has a better effect.

[0098] This experiment was based on the zebrafish embryo model. Compared with the blank group, the sample group's bifid yeast (lysate of the fermentation product of Bifidobacterium animalis subspecies BAPerfectus006) at concentrations of 1% and 2% could significantly reduce the fluorescence intensity of neutrophils in the skin (P < 0.01), and had anti-inflammatory and soothing effects. The anti-inflammatory effect was better than that of competing products.

[0099] Example 5. Anti-blue light experiment of lysate of fermentation product of Bifidobacterium animalis subsp. lactis BAPerfectus006 (Bifidobacterium animalis subsp. lactis)

[0100] 1. Test principle:

[0101] The free radicals produced by mitochondria during the electron transfer process will attack cell membranes, proteins and even DNA, leading to cell aging and even death, and are closely related to a variety of skin diseases. In daily life, the skin is often exposed to blue light radiation, which is recognized and absorbed by four "photoreceptors", causing living cells to produce a large number of free radicals. Free radicals further damage living cells, leading to excessive ROS in skin cells, which are toxic and cause oxidative stress damage to cells. Therefore, an oxidative stress model was established by irradiating humanized fibroblasts with blue light, and the changes in cell reactive oxygen species (ROS) before and after treatment with the test substance were used to detect whether the sample has anti-blue light efficacy.

[0102] 2. Test methods:

[0103] Humanized fibroblasts in the logarithmic growth phase were selected and routinely digested with 1 ml of 0.25% trypsin. The trypsin was removed after about 3 minutes, and 3 ml of cell culture medium (DMEM, purchased from Gibco) was immediately added to resuspend the cells. After pipetting and mixing evenly, the cells were counted and the cell concentration was adjusted to 3.0×10 5 / well were inoculated in a 6-well plate. After culturing for 24 h, the culture medium was aspirated, and the positive group and the sample group were added with the working solution of corresponding concentration, that is, the bifid yeast (lysate of the fermentation product of Bifidobacterium animalis subsp. lactis BAPerfectus006) or vitamin E in the sample group was added to DMEM culture medium and diluted to the corresponding concentration, as shown in Table 3, and the blank group and the model group were added with the same amount of culture medium.

[0104] Table 3 Fibroblasts anti-blue light test design grouping

[0105]

[0106] Reactive oxygen species (ROS) were detected using the Bio-Tech Reactive Oxygen Detection Kit (S0033S), and the specific method is as follows. After the administration, the 6-well plate was placed in a CO2 incubator (37°C, 5% CO2) and cultured for 24 hours, washed once with phosphate buffered saline (PBS), and 1 mL / well of DCFH-DA fluorescent probe (probe DCFH-DA is derived from Bio-Tech's Reactive Oxygen Detection Kit (S0033S)) working solution was added (the probe was diluted 1:1000 with DMEM culture medium), incubated at 37°C for 20 minutes, the probe working solution was aspirated, the cells were washed 3 times with PBS, and 1 mL of PBS was added to each well. All groups except the blank group were irradiated with blue light, and the irradiation dose was 15 J / cm 2 After the radiation, the DMEM culture medium was directly replaced, 2 mL / well, and incubated for 30 minutes. After the incubation, the cells were digested with trypsin (0.25%), washed with PBS, and tested on the machine. The data are expressed as Mean ± SD. The comparison between the groups was analyzed by t-test. All statistical analyses were two-tailed. P < 0.05 was considered to be significantly different, and the smaller the P value, the greater the significant difference. # represents P < 0.05, ## represents P < 0.01, and ### represents P < 0.001 (# represents comparison with the blank group); * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001 (* represents comparison with the model group).

[0107] 3. Test results:

[0108] The bar graph of ROS content is as follows Figure 6 As shown. The results show that compared with the blank group, the ROS content in the model group increased significantly (P < 0.01), indicating that blue light radiation can significantly cause an increase in ROS, indicating that the blue light radiation modeling conditions tested in this test are effective. Compared with the model group, the ROS content in the positive group decreased significantly (P < 0.01), indicating that this positive control test is effective. Compared with the model group, 0.5% concentration of bifid yeast can significantly reduce the ROS content (P < 0.01), which is better than vitamin E.

[0109] Blue light is a shorter wavelength part of the visible spectrum, which can penetrate deep into the skin, aggravate the oxidative stress response of the skin, lead to an increase in free radicals, and then promote skin aging. This experiment is based on human fibroblasts. Compared with the model group, the sample group of bifid yeast (the fermentation product lysate of animal Bifidobacterium lactis subspecies BAPerfectus006) significantly reduced the ROS content at a concentration of 0.5% (P < 0.01), indicating that the bifid yeast prepared in this application may enhance the skin's ability to resist blue light radiation damage by reducing the effect of ROS.

[0110] Example 6. DNA damage repair ability experiment of lysate (Bifidobacterium animalis subsp. lactis) BAPerfectus006 fermentation product

[0111] 1. Test principle:

[0112] Ultraviolet rays can be divided into three categories according to their wavelength: long-wave ultraviolet rays (UVA, 400-315nm), medium-wave ultraviolet rays (UVB, 315-280nm) and short-wave ultraviolet rays (UVC, 280-100nm). Since UVC is almost completely absorbed by the atmospheric ozone layer, about 95% of UVA and only 5% of UVB can reach the earth's surface. Therefore, UVA and UVB are the main light sources that cause skin damage. UVB has a shorter wavelength, but its energy is much higher than UVA. It mainly causes lesions in the epidermis and superficial dermis of the skin, can directly induce DNA damage, and is the main spectrum that induces oxidative stress and skin cancer, and also has a certain effect on skin aging. Therefore, a DNA damage model can be established based on UVB irradiation of human immortalized keratinocytes (HaCaT) cells, and the anti-DNA damage efficacy of the test substance can be further explored.

[0113] 8-Hydroxydeoxyguanosine (8-OHdG) is an oxidative adduct produced by reactive oxygen free radicals such as light free radicals and singlet oxygen attacking the 8th carbon atom of the guanine base in the DNA molecule. 8-OHdG can be detected by highly sensitive and selective detection methods and has become the most commonly used biomarker for DNA oxidative damage.

[0114] 2. Test methods:

[0115] Human immortalized keratinocytes (HaCat) in the logarithmic growth phase were selected and rinsed twice with PBS. 3 mL of DMEM culture medium was immediately added. The cells were gently blown off with a pipette tip and collected into a centrifuge tube. After blowing and beating evenly, the cells were counted and 2 × 10 5The cells were inoculated into 12-well plates at a density of 100 / mL, with 1 mL of cell suspension per well, and incubated in an incubator (37°C, 5% CO2) for 24 h. The sample group of bifid yeast (lysate of fermentation product of animal Bifidobacterium lactis subspecies BAPerfectus006) or the competitor group was added to the embryo culture medium and diluted to the corresponding concentration (Table 4). The data are expressed as Mean±SD. The comparison between the groups was analyzed by t-test. The statistical analysis was two-tailed. P<0.05 was considered to be significantly different, and the smaller the P value, the greater the significant difference. # represents P<0.05, ## represents P<0.01, ### represents P<0.001 (# represents comparison with the blank group); * represents P<0.05, ** represents P<0.01, *** represents P<0.001 (* represents comparison with the model group).

[0116] Table 4 DNA damage repair test design grouping

[0117]

[0118]

[0119] According to the specific experimental design in Table 4, when the cell plating rate in the 12-well plate reached 50% to 60%, the drugs were divided into groups, and the dosage per well was 1 mL. Each group had 3 replicate wells. After culturing in an incubator (37°C, 5% CO2) for 12 h, the culture medium was carefully aspirated, and 1 mL of pre-cooled PBS buffer was added to each well. The blank control group was covered with aluminum foil. After the instrument was preheated, 1 J / cm 2 UVB was used to irradiate the model group, competitor group and sample group at the same time. After irradiation, PBS was discarded, and the competitor group and sample group were added with solutions of corresponding concentrations (Table 4). The model group and blank group were added with equal amounts of culture medium and cultured in the dark for 24 hours. The cell supernatant of each group was collected in an EP tube and the specific operation was performed according to the corresponding 8-hydroxydeoxyguanosine (8-OHdG) ELISA detection kit (purchased from BBI Company); the standard curve was drawn using the "four-parameter logistics model" with the standard concentration as the horizontal axis and the absorbance value as the vertical axis. The OD value measured at a wavelength of 450nm on the microplate reader was substituted into the standard curve to calculate the final 8-OHdG content.

[0120] 3. Test results:

[0121] The results are as follows Figure 7 As shown, compared with the blank group, HaCaT cells were 2After UVB irradiation, the 8-OHdG content of the model group cells increased significantly (P < 0.01), and the production amount was 1646.7pg / mL, indicating that the modeling was effective. Compared with the model group, the 8-OHdG content of the positive group decreased significantly (P < 0.01). Compared with the model group, the 8-OHdG content of the bifid yeast at 0.3% and 0.6% concentrations also decreased significantly (P < 0.01). This shows that the bifid yeast (lysate of the fermentation product of Bifidobacterium animalis subspecies lactis BAPerfectus006) prepared in this application has a certain degree of anti-DNA damage function when the cells are damaged by ultraviolet light.

[0122] Example 7: Experiment on the anti-photoaging ability of the lysate (Bifidobacterium animalis subsp. lactis) BAPerfectus006 fermentation product

[0123] 1. Test principle:

[0124] Blue light is the main cause of skin photoaging. Compared with ultraviolet rays, blue light has a longer wavelength and is easier to penetrate the skin epidermis and dermis, causing the skin to produce excessive reactive oxygen species (ROS), causing skin photoaging. Collagen I is type I collagen, and collagen accounts for about 80% of the weight of the skin dermis, making the skin full and full, promoting the increase of Collagen I content, and can achieve the effect of resisting wrinkles. MMP is a matrix metalloproteinase, and its main function is to degrade collagen. After the MMP-1 content is reduced, the degree of collagen degradation is reduced, thereby achieving a certain anti-wrinkle effect. Elastin is the main protein constituting elastic fibers, and the reduced content causes elastic fibers to become sparse, and the skin elasticity is reduced. Therefore, this test uses blue light irradiation fibroblasts, and by detecting the changes in ROS, type I collagen (Collagen Ι), matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 3 (MMP-3) and elastin (Elastin) content, the anti-photoaging effect of the sample to be tested is evaluated.

[0125] 2. Test methods:

[0126] Human fibroblasts in the logarithmic growth phase were selected and inoculated in 6-well plates. After 24 hours of cell culture, the culture medium was carefully aspirated, and the bifid yeast (lysate of the fermentation product of Bifidobacterium animalis subsp. lactis BAPerfectus006) in the positive group and the sample group was added with DMEM culture medium and diluted to the corresponding concentration of working solution (Table 5), and the model group and the blank group were added with an equal amount of DMEM culture medium.

[0127] Table 5 Anti-light aging test design grouping

[0128]

[0129]

[0130] After the administration, the 6-well plate was placed in a CO2 incubator (37°C, 5% CO2) and cultured for 24 h. All groups except the blank control group were irradiated with blue light at a dose of 15 J / cm 2 After irradiation, continue to culture in a CO2 incubator (37°C, 5% CO2) for 24 hours. Collect the culture supernatant in a centrifuge tube and store it in a -80°C refrigerator. Perform the test and analysis according to the operating instructions of the ELISA test kit. The data are expressed as Mean±SD. The t-test statistical analysis was used for comparison between the groups. All statistical analyses were two-tailed. P<0.05 is considered to be significantly different, and the smaller the P value, the greater the significant difference. # represents P<0.05, ## represents P<0.01, and ### represents P<0.001 (# represents comparison with the blank group); * represents P<0.05, ** represents P<0.01, and *** represents P<0.001 (* represents comparison with the model group).

[0131] 2. Test results

[0132] The content values ​​of matrix metalloproteinase 1 (MMP-1), type I collagen (Collagen Ι) and elastin (Elastin) are as follows Figure 8-10 As shown. The results show that compared with the blank group, the content of MMP-1 in the model group increased significantly (P < 0.01), and the content of Collagen Ι and Elastin decreased significantly (P < 0.01), indicating that the blue light radiation modeling conditions in this test are effective. Compared with the model group, the content of MMP-1 in the positive group decreased significantly (P < 0.01), and the content of Collagen Ι and Elastin increased significantly (P < 0.01), indicating that the positive control test is effective. Compared with the model group, 0.5% concentration of bifid yeast can significantly reduce the content of MMP-1 (P < 0.01) and significantly increase the content of Collagen Ι and Elastin (P < 0.01).

[0133] This experiment was based on human fibroblasts. Compared with the model group, the bifid yeast at a concentration of 0.5% significantly reduced the MMP-1 content and significantly increased the Collagen Ι and Elastin contents (P < 0.01), indicating that the bifid yeast in the sample group prepared in the present application may reduce the degradation of skin collagen and elastin by reducing the MMP-1 content, thereby enhancing the skin's ability to resist photoaging caused by blue light.

[0134] Example 8: Experiment on the Moisturizing Capacity of Lysate (Bifidobacterium animalis subsp. lactis) BAPerfectus006 Fermentation Product

[0135] 1. Test principle:

[0136] Aquaporins (AQPs) are channels that facilitate the passage of water through cell membranes, of which AQP3 is primarily responsible for the transport of water and glycerol. Glycerol transported by AQP3 plays an important role in the hydration of the stratum corneum, improving barrier repair and moisturizing functions by promoting the biosynthesis of various lipids and promoting peeling. Ultraviolet light UVB (315-280nm) can cause damage to skin integrity and premature aging of the skin, while inducing epidermal cell inflammation, oxidative stress, DNA damage and apoptosis, and leading to skin cancer. Therefore, the AQP3 gene expression level can be evaluated based on UVB irradiation of HaCaT cells to explore the skin moisturizing effect of the test substance.

[0137] 2. Test methods:

[0138] Human immortalized keratinocytes (HaCat) in the logarithmic growth phase were selected and cultured at 3.0×10 5 Cells were inoculated into 6-well plates at an inoculation density of 100 cells / mL, 2 mL of cell suspension was added to each well, and incubated in an incubator (37°C, 5% CO2) for 24 h. The working solution was prepared according to the experimental design (Table 6), and the bifid yeast (lysate of the fermentation product of Bifidobacterium animalis subsp. lactis BAPerfectus006) in the positive group and the sample group was diluted to the corresponding concentration of the working solution by adding DMEM culture solution, and the model group and the blank group were added with an equal amount of DMEM culture solution.

[0139] Table 6 Moisturizing ability test design grouping

[0140]

[0141] When the cell plating rate in the 6-well plate reaches 50% to 60%, the grouping and administration are carried out, and the dosage per well is 2 mL. Each group has 3 replicate wells, and the culture is continued for 48 hours in the incubator (37°C, 5% CO2). Add 1 ml of phosphate buffered saline (PBS) to each well for washing once. For cells that are tightly attached to the wall, add 100 μl of trypsin to each well to digest the cells and fully collect the cells in a 1.5 ml centrifuge tube, and then add RNA Easy lsolation Reagent 0.5 ml-1 ml for treatment. For specific RNA extraction operations, please refer to the instructions (reagent model: R701-01). TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit was used for cDNA reverse transcription. For specific operations and reaction conditions, please refer to the instructions (reagent model: AT311-03, Quanshijin). The RNA loading concentration refers to the kit. If the RNA concentration is low, the reversal concentration can be appropriately reduced. The TransStart TopGreen qPCR SuperMix (reagent model: AQ141-03, full gold) kit was used for the experiment. The cDNA template of each sample was diluted 5 times. The specific reaction system is shown in Table 7 below.

[0142] Table 7qPRC reaction system

[0143]

[0144] Reaction conditions: 50℃2min, 95℃10min; 95℃30sec, 60℃30sec, 40cycles. Melting curve was performed, and the expression was calculated according to the CT value of the fluorescence curve generated by the experiment. The final data was analyzed by relative quantification using 2-△△Ct. The data were expressed as Mean±SD. The t-test statistical analysis was used for comparison between the groups. All statistical analyses were two-tailed. P<0.05 was considered to be significantly different, and the smaller the P value, the greater the significant difference. # represents P<0.05, ## represents P<0.01, and ### represents P<0.001 (# represents comparison with the blank group); * represents P<0.05, ** represents P<0.01, and *** represents P<0.001 (* represents comparison with the model group).

[0145] 3. Test results:

[0146] The results show Fig.11As shown, compared with the blank group, the relative expression of the AQP3 gene in the model group was significantly downregulated (P<0.01), and the experimental modeling was considered to be effective; compared with the model group, the relative expression of the AQP3 gene in the positive group was significantly upregulated (P<0.01), and the experimental system was considered to be effective. On the basis that the experiment meets the validity verification, compared with the blank group, the relative expression of the AQP3 gene in the sample group bifid yeast (animal bifidobacterium lactis subspecies BAPerfectus006 fermentation product lysate) was significantly upregulated (P<0.01). It shows that the sample group bifid yeast (animal bifidobacterium lactis subspecies BAPerfectus006 fermentation product lysate) prepared in this application has the effect of moisturizing at the tested concentration, and the effect is better than that of competing products.

[0147] Example 9: Experiment on the cell barrier repair ability of the lysate (Bifidobacterium animalis subsp. lactis) BAPerfectus006 fermentation product

[0148] 1. Test principle:

[0149] The skin barrier can be divided into broad and narrow barriers in terms of structure. Among them, the broad skin barrier mainly includes barriers related to the structure of each layer of the skin, such as physical barriers, pigment barriers, neural barriers, and immune barriers. The narrow skin barrier mainly refers to the physical barrier, which mainly involves barriers related to the structure of the skin's stratum corneum. The keratin envelope (CE) is an insoluble and tough outer membrane formed by cross-linking between cell membranes during the upward migration and differentiation of keratinocytes from the basal layer to the stratum corneum. The formation of CE is a key way for the skin barrier to exert its function. Proteins such as loricrin (LOR) and transglutaminase 1 (TGM1) are the main precursor components of several keratin envelopes that have been studied relatively clearly.

[0150] 2. Test methods:

[0151] The specific operation of this embodiment 9 is the same as that of embodiment 8, and the detection indicators are LOR and TGM1. According to the experimental design (Table 8), the working solution of the test substance is configured, and the bifid yeast (animal Bifidobacterium lactis subspecies BAPerfectus006 fermentation product lysate) of the positive group, the competitive product group and the sample group is added with DMEM culture fluid to be diluted to the corresponding concentration of the working solution, and the model group and the blank group are added with DMEM culture fluid of equal amount. The data are expressed as Mean±SD. The comparison between the groups was analyzed by t-test statistics. The statistical analysis was two-tailed. P<0.05 is considered to have a significant difference, and the smaller the P value, the greater the significant difference. # represents P<0.05, ## represents P<0.01, and ### represents P<0.001 (# represents comparison with the blank group); * represents P<0.05, ** represents P<0.01, and *** represents P<0.001 (* represents comparison with the model group).

[0152] Table 8 Barrier repair test design grouping

[0153]

[0154] 3. Test results:

[0155] The results show that Figure 12-13 As shown, compared with the blank group, the relative expression of LOR and TGM1 genes in the model group was significantly downregulated (P<0.01), and the experimental modeling was considered effective; compared with the model group, the relative expression of LOR and TGM1 genes in the positive group was significantly upregulated (P<0.01), and the experimental system was considered effective. On the basis that the experiment meets the validity verification, compared with the blank group, the relative expression of LOR and TGM1 genes in the sample group bifid yeast (animal Bifidobacterium lactis subspecies BAPerfectus006 fermentation product lysate) was significantly upregulated (P<0.01), and compared with the competing products, the expression of LOR and TGM1 genes reached a significant difference (P<0.01). It shows that the bifid yeast prepared in this application has the effect of barrier repair at the tested concentration.

[0156] Example 10: Experiment on the cell barrier repair ability of the fermentation product filtrate (Bifidobacterium animalis subsp. lactis) BAPerfectus006 (Bifidobacterium animalis subsp. lactis)

[0157] 1. Test principle:

[0158] Keratinocytes (HaCat) are the main cell type that constitutes the epidermis of the skin, and have functions such as epidermal formation, wound repair, cell keratinization, cytokine secretion and immune surveillance. They are often used in the biomedical field as a cell line for in vitro skin research, such as skin repair and healing. In the early stages of wound healing, keratinocytes in the epidermis are activated and migrate to the wound site to induce wound contraction, followed by cell proliferation to repair and rebuild the skin. Therefore, keratinocytes play an important role in wound healing. A scratch test model was established using human immortalized keratinocytes HaCaT to detect whether the test article has the effect of promoting cell proliferation and repairing scratches, as a basis for whether it has the ability to repair skin barrier damage.

[0159] 2. Test methods:

[0160] Line the bottom of the plate: Use a marker pen to draw three parallel lines on the outside bottom of the 6-well plate at intervals of about 1 cm. Cell inoculation: Select human keratinocytes HaCat in the logarithmic growth phase, use 1 mL of 0.25% trypsin for routine digestion, remove the trypsin after about 3 minutes, immediately add 3 mL of DMEM culture medium to resuspend the cells, pipette and evenly count the cells, and adjust the cell concentration to 1×10 6 Cells were inoculated into 6-well plates at a seeding density of 100 / mL, with 2mL of cell suspension per well, and incubated in an incubator (37°C, 5% CO2) for 24h. Scratching and dosing: When the cell plating rate reaches 90% to 100%, the culture medium in the well plate is retained during the scratching. A 200μL sterile gun tip is used to scratch, and 3 scratches are made vertically at intervals of about 1cm on the bottom of the plate. Rinse with PBS 3 times to remove residual cells in the scratch area. After scratching, the cells were randomly divided into a positive group, a blank group, and a sample group. Each group was set up with 3 parallel wells for group dosing, and the dosing volume per well was 2mL. The cells were cultured in an incubator (37°C, 5% CO2) for 24h. Photography: Observe and take pictures under an inverted microscope, with a microscope eyepiece magnification of 25× and an objective lens magnification of 4×. The first photo was taken 0h after the scratch. While finding a suitable field of view, the field of view was recorded in the notebook by the cross-point method. The second photo was taken 48h after the scratch to ensure that the field of view was the same as the first time. Calculation of scratch area: The healing rate of scratch injury area was calculated using the software Image J. Migration rate = ((original area - area after scratching) / original area) × 100%.

[0161] Statistical methods: In the experiment, the software Image J was used to analyze the pictures, and the software GraphPad Prism was used to perform statistical analysis and plot the data. The statistical methods were T-test, two-sided test, or one-way ANOVA. The mean (Mean) and standard error (Standard error of the mean, SEM) of each group of experiments were calculated, and the statistical analysis results were expressed as Mean ± SEM. Each group of experiments was repeated at least 3 times, and P < 0.05 was considered statistically significant, indicated by *.

[0162] 3. Test results:

[0163] Table 9 Average cell migration

[0164]

[0165]

[0166] The average cell migration rate is shown in Table 9 and Fig.14 As shown. The results show that compared with the blank group, the average cell migration rate of the positive group was significantly increased (P < 0.001), indicating that the positive control test was effective. Compared with the blank group, 0.1% and 0.15% concentrations of bifid yeast filtrate can significantly increase the average cell migration rate (P < 0.01), indicating that the sample group bifid yeast filtrate (animal Bifidobacterium lactis subspecies BAPerfectus006 fermentation product filtrate) has the effect of promoting the migration and fusion of HaCaT cells at the tested concentration, thereby repairing the mechanical damage of the skin barrier.

[0167] Example 11. Experiment on the oil control ability of the fermentation product filtrate (Bifidobacterium animalis subsp. lactis) BAPerfectus006 (Bifidobacterium animalis subsp. lactis)

[0168] 1. Test principle:

[0169] Sebum is mainly derived from keratinocytes and sebaceous glands. The right amount of sebum secretion has a positive effect. Excessive sebum secretion first forms simple oily skin, and usually increased sebum is also accompanied by the occurrence of some diseases, such as easy formation of acne, causing acne, and may also cause seborrheic dermatitis, seborrheic alopecia, etc. Human sebaceous gland cells are the main site of androgen synthesis and metabolism in the skin, and are also the cells with the highest androgen receptor content in the skin. Androgens have been shown to stimulate the differentiation of sebaceous gland cells, and increased androgen content can cause increased sebum secretion. Testosterone and 5α-dihydrotestosterone (5α-DHT) in facial sebaceous glands are active androgens that can stimulate lipid synthesis and promote the proliferation and differentiation of sebaceous cells. The in vitro method can be used to detect the oil control effect, which can be evaluated based on the 5α-DHT stimulation SZ95 sebaceous gland cell model, and the Nile red staining method can be used to detect the intracellular lipid droplet content to evaluate the oil control effect of the test object.

[0170] 2. Test methods:

[0171] Cell seeding: 5×10 5 Inoculate cells into 12-well plates at a seeding density of 1 / mL, add 1 mL of cell suspension to each well, and incubate in an incubator (37°C, 5% CO2) for 24 hours. Solution preparation: Prepare the test substance working solution according to the experimental design (Table 10). Drug administration: When the cell plating rate in the 12-well plate reaches 50% to 60%, group drug administration is carried out, with a dosage of 1 mL per well, and 2 replicate wells are set for each group. 5α-DHT stimulation: According to the group design, add 5α-DHT working solution prepared from the corresponding test substance working solution to the well plate that has been administered, shake the well plate left and right to mix the drugs in the well plate, and the final concentration of 5α-DHT is 1600nM. Continue to culture in an incubator (37°C, 5% CO2) for 24 hours. Nile red staining: After incubation for 24 hours, discard the culture medium, wash twice with 1×PBS, and fix with 4% paraformaldehyde at room temperature for 10 minutes. After fixation, stain with Nile red dye (100 μg / mL) in dark, incubate in a 37°C incubator in dark for 10 min, remove the dye, wash with PBS 3 to 5 times, and evenly add anti-fluorescence quenching sealing agent to each well. Observe the staining under a fluorescence microscope and take pictures. Cells with strong red fluorescence are lipid-rich positive cells. When the t-test method was used for statistical analysis, compared with the model group: *P < 0.05, **P < 0.01, ***P < 0.001.

[0172] Table 10 Experimental design

[0173]

[0174] 3. Test results:

[0175] like Fig.15 and 16As shown in the figure, compared with the blank group, the amount of lipid droplets in the model group increased significantly (P < 0.001), indicating that the stimulation conditions of this experiment are effective. Compared with the model group, the amount of lipid droplets in the positive group decreased significantly (P < 0.01), indicating that the positive control test is effective. Fig.16 It can be seen that the red fluorescence intensity representing the lipid droplet content in the model group cells is 1.4×10 7 ±844575, the fluorescence intensity of the sample group bifid yeast filtrate (1%) was 1.8×10 7 ±1105784, the fluorescence intensity of the sample group bifid yeast filtrate (2%) was 8.3×10 6 ±169793. Compared with the model group, the bifid yeast filtrate (1%) in the sample group had no effect on reducing lipid droplets, while the red fluorescence intensity of the bifid yeast filtrate (2%) in the sample group was significantly weakened (P < 0.05), indicating that the bifid yeast filtrate (filtrate of the fermentation product of Bifidobacterium animalis subsp. lactis BAPerfectus006) in the sample group had the effect of controlling oil.

[0176] Example 12: Experiment on the Moisturizing Capacity of the Fermentation Product Filtrate (Bifidobacterium animalis subsp. lactis) BAPerfectus006 (Bifidobacterium animalis subsp. lactis)

[0177] 1. Test principle:

[0178] When zebrafish are treated with sodium chloride, the skin surface will lose water and shrink due to osmotic pressure, and the tail area will become smaller due to shrinkage. The moisturizing effect of the test substance is evaluated by evaluating the effect on the tail area of ​​the zebrafish.

[0179] 2. Test methods:

[0180] According to the well plate setting, the blank group was added with standard dilution water (Table 11), the sample group was added with a mixture of bifid yeast filtrate and sodium chloride, and the positive group was added with a mixture of glycerol and sodium chloride, and placed in a biochemical incubator at 28.5±0.5℃ for 20 minutes. Observation and photography: 10 zebrafish were randomly selected from each group for observation, photography and preservation under a stereomicroscope to count the tail area of ​​zebrafish in each group. Image analysis: The photographed images were analyzed using graphic analysis software, the tail area of ​​zebrafish from the cloaca to the end of the caudal fin was counted, and the inhibition rate of tail area reduction was calculated; the data were expressed as Mean±SD. The comparison between the groups was analyzed by t-test statistics. Statistical analysis was two-tailed. P<0.05 was considered to be significantly different, and the smaller the P value, the greater the significant difference. # represents P < 0.05, ## represents P < 0.01, ### represents P < 0.001 (# represents comparison with the blank group); * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001 (* represents comparison with the model group).

[0181]

[0182] Result judgment: The inhibition rate of tail area reduction was positive and had statistically significant difference compared with the model group (P<0.05), which indicated that the test substance had moisturizing effect.

[0183] 3. Test results:

[0184] Table 11 Zebrafish tail area

[0185]

[0186] The tail area of ​​zebrafish is shown in Table 11. The results show that compared with the blank group, the tail area of ​​zebrafish in the model group was significantly reduced (P < 0.001), indicating that sodium chloride can significantly cause a reduction in the tail area of ​​zebrafish, indicating that the sodium chloride modeling conditions tested in this test are effective. Compared with the model group, the inhibition rate of tail area reduction in the positive group was significantly increased (P < 0.05), indicating that the positive control test was effective. Compared with the model group, the 5% concentration of the bifid yeast filtrate in the sample group can significantly increase the inhibition rate of zebrafish tail area reduction (P < 0.05), indicating that the bifid yeast filtrate in the sample group (animal Bifidobacterium lactis subspecies BAPerfectus006 fermentation product filtrate) has a moisturizing effect at this tested concentration.

[0187] Example 13. Anti-inflammatory efficacy experiment of the fermentation product filtrate (Bifidobacterium animalis subsp. lactis) BAPerfectus006 (Bifidobacterium animalis subsp. lactis)

[0188] 1. Test principle:

[0189] Sodium dodecyl sulfate can induce epidermal inflammatory cell infiltration after stimulating the epidermis, which can lead to symptoms such as edema, erythema, itching, and pain. Sodium dodecyl sulfate can also induce an inflammatory response when exposed to the skin surface of zebrafish embryos, causing neutrophils to undergo an immune response and migrate and aggregate to the site of inflammation. Therefore, an inflammation model can be established using transgenic neutrophil green fluorescent strain zebrafish embryos, and the fluorescence changes of zebrafish embryo neutrophils before and after treatment with the test substance can be used to detect whether the sample has anti-inflammatory effects.

[0190] 2. Test methods:

[0191] According to the experimental requirements, transgenic neutrophil green fluorescent strain zebrafish embryos were pre-screened and randomly assigned to 6-well plates. Different concentrations of bifid yeast filtrate (Table 12) were added to each well of the sample group, dexamethasone solution was added to the positive group, and embryo culture medium was added to each well of the blank group and the model group. Incubate in a 28.5±0.5℃ biochemical incubator for 120min. After incubation for 120min, the solution in the 6-well plate was removed without harming the zebrafish, and embryo culture medium was added to the blank group, and the modeling agent sodium dodecyl sulfate was added to the model group, positive group and sample group. Cover the culture plate panel and incubate in a 28.5±0.5℃ biochemical incubator for 90min. Observation and photography: After the modeling was completed, 10 zebrafish were selected from each group to observe, photograph and save pictures under a fluorescence microscope. Image analysis: The obtained zebrafish pictures were analyzed using image analysis software, and the fluorescence intensity of neutrophils in the tail of the zebrafish was counted.

[0192] Statistical analysis: The fluorescence intensity of zebrafish neutrophils was counted, and the mean (Mean) and standard deviation (Standard Deviation) of each group of experiments were calculated. The statistical analysis results were expressed as Mean±SD. Statistical analysis software was used to perform statistical analysis on the data, compare the fluorescence intensity of neutrophils in each experimental group, and calculate the anti-inflammatory effect. # represents P < 0.05, ## represents P < 0.01, ### represents P < 0.001 (# represents comparison with the blank group); * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001 (* represents comparison with the model group).

[0193] 3. Test results:

[0194] Table 12 Zebrafish fluorescence area

[0195]

[0196] The fluorescence area of ​​zebrafish is shown in Table 12. The results show that compared with the blank group, the fluorescence area of ​​zebrafish in the model group increased significantly (P < 0.05), indicating that the sodium dodecyl sulfate modeling conditions tested in this test are effective. Compared with the model group, the fluorescence area of ​​the positive group was significantly reduced (P < 0.05), indicating that the positive control test was effective. Compared with the model group, the sample group bifid yeast filtrate at a concentration of 1.25%, 2.5%, and 5% can significantly reduce the fluorescence area of ​​zebrafish (P < 0.05), indicating that the sample group bifid yeast filtrate (animal Bifidobacterium lactis subspecies BAPerfectus006 fermentation product filtrate) has anti-inflammatory effects at the tested concentration.

[0197] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. Application of animal Bifidobacterium lactis subspecies BAPerfectus006 or fermentation product lysate or filtrate containing animal Bifidobacterium lactis subspecies BAPerfectus006 for non-disease treatment purposes in at least one of the following aspects: 1) anti-inflammatory; 2) anti-blue light; 3) anti-photoaging; 4) oil control; The preservation number of the animal Bifidobacterium lactis subspecies BAPerfectus006 is GDMCC No: 62539. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2022. The preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The use according to claim 1, characterized in that The animal Bifidobacterium lactis subspecies BAPerfectus006, the fermentation product lysate or the filtrate containing animal Bifidobacterium lactis subspecies BAPerfectus006 is made into a microbial preparation.

3. The use according to claim 2, characterized in that In the microbial preparation, the concentration of the fermentation product lysate of Bifidobacterium animalis subspecies BAPerfectus006 is 0.1% to 2%, and the concentration of the fermentation product filtrate of Bifidobacterium animalis subspecies BAPerfectus006 is 1 to 20%.

4. Use of animal Bifidobacterium lactis subspecies BAPerfectus006 or a fermentation product lysate or filtrate containing animal Bifidobacterium lactis subspecies BAPerfectus006 in preparing a product, wherein the product is used in at least one of the following aspects: 1) anti-inflammatory; 2) anti-blue light; 3) anti-photoaging; 4) oil control; The preservation number of the animal Bifidobacterium lactis subspecies BAPerfectus006 is GDMCC No: 62539. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2022. The preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

5. The use according to claim 4, characterized in that The products include cosmetics, health foods or medicines.

6. A cosmetic, characterized in that: The cosmetics include animal Bifidobacterium lactis subspecies BAPerfectus006 or a fermentation product lysate or filtrate containing animal Bifidobacterium lactis subspecies BAPerfectus006; The preservation number of the animal Bifidobacterium lactis subspecies BAPerfectus006 is GDMCC No: 62539. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2022. The preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

7. A health food, characterized in that: The health food comprises animal Bifidobacterium lactis subspecies BAPerfectus006 or a fermentation product lysate or filtrate containing animal Bifidobacterium lactis subspecies BAPerfectus006; The preservation number of the animal Bifidobacterium lactis subspecies BAPerfectus006 is GDMCC No: 62539. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2022. The preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

8. A drug, characterized in that The drug comprises animal Bifidobacterium lactis subspecies BAPerfectus006 or a fermentation product lysate or filtrate containing animal Bifidobacterium lactis subspecies BAPerfectus006; The preservation number of the animal Bifidobacterium lactis subspecies BAPerfectus006 is GDMCC No: 62539. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2022. The preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

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