Selenium-rich composite bacteria fermentation product and application thereof in photodamage repair

Abstract

The application discloses a selenium-rich compound bacteria fermentation product and application thereof in photodamage repair, inoculates animal bifidobacterium H15 and lactobacillus coryniformis ES23 after compounding into MRSc culture medium containing sodium selenite, carries out anaerobic fermentation culture, cell wall breaking, and filters and sterilizes to obtain the selenium-rich compound bacteria fermentation product. The selenium-rich compound bacteria fermentation product can efficiently remove free radicals, has good antioxidant stress resistance, the ability of repairing UVB photodamage cells and high biological safety.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a selenium-rich compound bacterial fermentation product and its application in photodamage repair. BACKGROUND

[0002] Ultraviolet rays in sunlight have a significant impact on skin sunburn, skin inflammation, skin aging and the occurrence and development of skin cancer. UVB is the main cause of skin photodamage. Keratinocytes are located in the epidermal layer of the skin and are the target cells of UVB action. UVB radiation leads to oxidative stress, resulting in excessive reactive oxygen species, damaging cell membranes, mitochondrial membranes and inactivating various cellular enzymes, and affecting related cell signal transduction and gene expression, leading to cell damage, apoptosis or carcinogenesis, thereby causing skin photoaging, inducing skin cancer and posing a great threat to skin health.

[0003] Lactic acid bacteria, as important probiotics, have a long history of use in food and clinical aspects. Current studies have found that oral administration of Bifidobacterium can improve skin aging and enhance skin barrier function. In vitro studies have found that Bifidobacterium (Saccharomyces boulardii) fermentation broth can effectively scavenge DPPH, hydroxyl radicals and superoxide anions, and can be used as an antioxidant raw material in cosmetic formulations to maintain skin homeostasis. Longum extract can promote the differentiation and regeneration of human epidermal keratinocytes and increase the content of tight junction proteins in cells, and has the effect of enhancing skin barrier function. Small molecule organic substances (such as B vitamins and amino acids) produced during Bifidobacterium fermentation can play a role in skin care. Saccharomyces boulardii fermentation products mainly play the roles of anti-aging, skin moisturizing and maintenance of skin homeostasis. In recent years, more and more studies have found that probiotics can significantly inhibit the proliferation of skin pathogenic bacteria, balance the skin epidermal flora, repair the skin barrier, effectively increase the absorption of nutrients by the skin and enhance the body's immunity.

[0004] Trace element selenium is an essential component of the important antioxidant enzyme glutathione peroxidase in the body, has strong antioxidant and anti-aging abilities, and can improve immunity and promote wound healing, and is closely related to human health. Studies have shown that selenium is closely related to the occurrence and development of some skin diseases such as psoriasis, vitiligo and skin tumors. Selenium-rich probiotics have the ability to convert inorganic selenium into high-activity nano-selenium and organic selenium, and are a hot spot in the research of selenium organic conversion. Compared with inorganic selenium and organic selenium, biological nano-selenium has the advantages of high bioavailability and low toxicity. Studies have shown that bioactive nano-selenium produced by selenium-rich probiotics has good transdermal ability, the ability to inhibit the generation of ROS in cell lines, and the ability to prevent UV-induced skin damage and slow down the aging of the body. Although these studies provide a basis for the use of nano-selenium in the preparation of biological and medical products for repairing ultraviolet damage. However, there are few reports on the direct application of selenium-rich compound bacterial fermentation products in the field of photodamage repair. SUMMARY

[0005] In order to fully tap the application value of selenium-rich microorganisms and expand the efficacy of selenium-rich microorganism fermentation products, the present application provides the following technical solutions.

[0006] In a first aspect, the present application provides a composite bacteria with antioxidant and photodamage repair functions, characterized in that the composite bacteria comprises animal bifidobacterium and lactobacillus coryniformis;

[0007] The animal bifidobacterium is animal bifidobacterium (Bifidobacterium animalis) H15, which was preserved in the China General Microbiological Culture Collection Center on June 16, 2023, and the preservation number is CGMCC NO.27654; Bifidobacterium animals The lactobacillus coryniformis is lactobacillus coryniformis (Lactobacillus coryniformis) ES23, which was preserved in the China General Microbiological Culture Collection Center on June 16, 2023, and the preservation number is CGMCC NO.27653;

[0008] Lactobacillus coryniformiss Preferably, the volume ratio of the animal bifidobacterium H15 and the lactobacillus coryniformis ES23 is 1-5:1-5, for example: 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5.

[0009] Further, the effective viable count of the animal bifidobacterium H15 is 9.46 lgCFU / mL, and the effective viable count of the lactobacillus coryniformis ES23 is 8.48 lgCFU / mL.

[0010] In a second aspect, the present application provides a selenium-rich composite bacteria fermentation product, and a preparation method of the selenium-rich composite bacteria fermentation product comprises the following steps:

[0011] (1) inoculate the animal bifidobacterium H15 and the lactobacillus coryniformis ES23 into MRSc culture medium respectively, and anaerobically culture to obtain animal bifidobacterium H15 seed liquid and lactobacillus coryniformis ES23 seed liquid;

[0012] (2) mix the H15 seed liquid and the ES23 seed liquid, inoculate into MRSc culture medium containing sodium selenite, and anaerobically culture to obtain selenium-rich composite fermentation bacteria liquid;

[0013] (3) adjust the pH of the selenium-rich composite fermentation bacteria liquid, break the cell wall, filter sterilize, and obtain the selenium-rich composite bacteria fermentation product.

[0014] Preferably, the formula of the MRSc culture medium is as follows:

[0015]

[0016] ​​Glucose 20 g, tryptone 10 g, beef extract 10 g, yeast extract 5 g, anhydrous sodium acetate 5 g, diamine citric acid 2 g, Tween 80 1 mL, potassium phosphate 2 g, magnesium sulfate heptahydrate 0.2 g, manganese sulfate heptahydrate 0.05 g, L-cysteine hydrochloride 0.5 g, distilled water 1 L, adjust pH to 6.5, sterilize at 121℃ for 15 min.

[0017] Preferably, the inoculation amount of the Bifidobacterium animalis H15 and the Lactobacillus coryniformis ES23 in step (1) is 1-5%, for example: 1%, 2%, 3%, 4%, 5%.

[0018] Preferably, the anaerobic culture temperature in step (1) is 35-40℃, for example: 35℃, 36℃, 37℃, 38℃, 39℃, 40℃.

[0019] Preferably, the anaerobic culture time in step (1) is 10-16 h, for example: 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h.

[0020] Preferably, the mixing volume ratio of the H15 seed liquid and the ES23 seed liquid in step (2) is 1-5:1-5, for example: 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5.

[0021] Further, the effective viable count of the H15 seed liquid is 9.46 lgCFU / mL, and the effective viable count of the ES23 seed liquid is 8.48 lgCFU / mL.

[0022] Preferably, the anaerobic culture temperature in step (2) is 35-40℃, for example: 35℃, 36℃, 37℃, 38℃, 39℃, 40℃.

[0023] Preferably, the anaerobic culture time in step (2) is 40-60 h, for example: 40 h, 42 h, 44 h, 46 h, 48 h, 50 h, 52 h, 54 h, 56 h, 58 h, 60 h.

[0024] Preferably, the content of sodium selenite in the MRSc medium in step (2) is 50-150 μg / mL, for example: 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL.

[0025] Preferably, the pH of the compound bacteria fermentation liquor in step (3) is 5.5-6.5, for example, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5.

[0026] Preferably, the cell wall breaking method in step (3) comprises ultrasonic disruption, high-pressure homogenizer disruption, grinding, bead beating, freeze-thawing or enzymolysis, and is further preferably ultrasonic disruption.

[0027] Further, the ultrasonic disruption is performed under ice bath condition, and the ultrasonic power is 200-500 W.

[0028] Preferably, the filter membrane pore size for filtering and sterilizing in step (3) is 0.22 μm or 0.45 μm.

[0029] Preferably, the selenium content in the selenium-rich compound fermentation product is 15-25 μg / mL, for example, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, 20 μg / mL, 21 μg / mL, 22 μg / mL, 23 μg / mL, 24 μg / mL, 25 μg / mL.

[0030] In a third aspect, the present application provides a composition comprising the compound bacteria of the first aspect or the selenium-rich compound bacteria fermentation product of the second aspect.

[0031] Preferably, the composition can further comprise bacterial bodies (such as H15 bacterial bodies, ES23 bacterial bodies), amino acids (such as arginine, lysine, leucine, isoleucine, phenylalanine), small molecule peptides.

[0032] Further, the bacterial bodies are H15 bacterial bodies and / or ES23 bacterial bodies.

[0033] Further, the amino acids are selected from one or more than two combinations of arginine, lysine, leucine, isoleucine or phenylalanine.

[0034] Further, the small molecule peptides are selected from glutathione or carnosine-histidine dipeptide.

[0035] In a fourth aspect, the present application provides use of the compound bacteria of the first aspect or the selenium-rich compound bacteria fermentation product of the second aspect in preparation of an antioxidant product.

[0036] Preferably, the product is a health food, a medicine, a cosmetic or a feed.

[0037] Further, the product comprises a permitted excipient in a health food, a medicine, a cosmetic or a feed.

[0038] In a fifth aspect, the present application provides use of the composite bacteria of the first aspect or the selenium-rich composite bacteria of the second aspect in the preparation of a product having a photodamage repair function.

[0039] Preferably, the use comprises at least one of the following:

[0040] (1) use in the preparation of a product capable of improving cell viability.

[0041] (2) use in the preparation of a product capable of improving the activity of cell antioxidant enzymes.

[0042] (3) use in the preparation of a product capable of improving the content of cell hydroxyproline.

[0043] (4) use in the preparation of a product capable of reducing the level of cell inflammation.

[0044] (5) use in the preparation of a product capable of inhibiting UVB-induced skin damage.

[0045] (6) use in the preparation of a product having a collagen protection function.

[0046] Preferably, the product is a pharmaceutical product, a cosmetic product or a feed.

[0047] Advantages of the present application:

[0048] The present application combines animal bifidobacterium H15 and lactobacillus coryniformis ES23, and through optimization of selenium-rich concentration, strain composite ratio, culture time and low-temperature physical wall breaking, obtains a composite bacteria fermentation product rich in Bio-SeNPs (biological nano selenium) with high biological activity, which can efficiently scavenge free radicals, has good antioxidant stress and the ability to repair UVB photodamaged cells, and has high biological safety. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 The appearance of the composite bacteria fermentation broth and the selenium-rich composite bacteria fermentation broth is shown, A is the selenium-rich composite bacteria fermentation broth, and B is the control composite bacteria fermentation broth;

[0050] Figure 2 The scanning electron microscope image of biological nano selenium in the selenium-rich composite bacteria fermentation product is shown;

[0051] Figure 3 The DPPH free radical scavenging rate of different concentrations of selenium-rich composite bacteria fermentation products is shown;

[0052] Figure 4 The ABTS free radical scavenging rate of different concentrations of composite bacteria fermentation products is shown. +

[0053] Figure 5 ​Effect of different concentrations of selenium-rich compound bacteria fermentation products on HaCaT cell viability

[0054] Figure 6 Effect of different UVB irradiation intensities on HaCaT cell viability

[0055] Figure 7 Effect of selenium-rich compound bacteria fermentation products on HaCaT cell viability after UVB irradiation damage

[0056] Figure 8 Effect of selenium-rich compound bacteria fermentation products on HaCaT cell reactive oxygen species level after UVB irradiation damage

[0057] Figure 9 Effect of selenium-rich compound bacteria fermentation products on HaCaT GSH content after UVB irradiation damage

[0058] Figure 10 Effect of selenium-rich compound bacteria fermentation products on HaCaT MMP-1 content after UVB irradiation damage

[0059] Figure 11 Effect of selenium-rich compound bacteria fermentation products on HaCaT HYP content after UVB irradiation damage

[0060] Figure 12 Effect of selenium-rich compound bacteria fermentation products on HaCaT IL-1β content after UVB irradiation damage

[0061] Figure 13 Effect of selenium-rich compound bacteria fermentation products on HaCaT TNF-α content after UVB irradiation damage

[0062] Figure 14 Effect of selenium-rich compound bacteria fermentation products on HaCaT IL-6 content after UVB irradiation damage

[0063] Figure 15 Flowchart of repair of light-damaged mouse skin experiment by selenium-rich compound bacteria fermentation products

[0064] Figure 16 Repair of light-damaged mouse skin tissue by selenium-rich compound bacteria fermentation products and detection results of skin antioxidant indexes, A is the mouse skin damage; B is the mouse skin damage degree; C is the mouse skin HE staining, scale 100 μm; D is Masson staining, scale 100 μm; E is the skin epidermis thickness; F is the collagen fiber ratio; G is the MDA level; H is the SOD enzyme activity. DETAILED DESCRIPTION

[0065] The technical solutions of the present application will be further described below in combination with examples and drawings, and the advantages and characteristics of the present application will be more apparent with the description. However, it should be understood that the examples are only exemplary and do not limit the scope of the present application.

[0066] In addition, unless otherwise specified, the experimental materials and reagents used in the present application are all conventional materials and reagents in the art.

[0067] Example 1 Preparation of fermentation products of each group

[0068] 1.1 Preparation of MRSc medium:

[0069] Glucose 20 g, tryptone 10 g and beef extract 10 g, yeast extract 5 g, anhydrous sodium acetate 5 g, citric acid diamine 2 g, Tween 80 1 ml, potassium phosphate dibasic 2 g, magnesium sulfate heptahydrate 0.2 g, manganese sulfate heptahydrate 0.05 g, L-cysteine hydrochloride 0.5 g, distilled water 1 L, mix, adjust pH to 6.5, sterilize at 121℃ for 15 min.

[0070] 1.2 Selenium-rich culture:

[0071] The activated animal Bifidobacterium H15 and Lactobacillus coryniformis ES23 were inoculated into sterile MRSc medium at an inoculation amount of 2%, and anaerobically cultured to the logarithmic phase (about 12 h). The Ⅱ generation fermentation broth of the cultured H15 (effective viable count 9.46 lgCFU / mL) and ES23 (effective viable count 8.48 lgCFU / mL) was mixed at a volume ratio of 1:1, and then inoculated into MRSc medium containing 100 μg / mL sodium selenite at an inoculation amount of 2%. The culture was incubated at 37℃ under constant temperature anaerobic conditions for 48 h to obtain selenium-rich composite fermentation broth. At the same time, MRSc medium was used as a control to obtain composite bacteria fermentation broth.

[0072] As shown in Figure 1 , compared with the yellow control composite bacteria fermentation broth, the selenium-rich composite fermentation broth was red, indicating that inorganic selenium was converted to nano selenium in the culture system containing selenium-rich sodium selenite, and red bioactive nano selenium particles were generated.

[0073] 1.3 Preparation of selenium-rich composite bacteria (Se-F) fermentation product:

[0074] The selenium-rich composite bacteria fermentation broth was collected, the pH value of the fermentation broth was adjusted to 6.0-6.5, and then the cells were fully broken by low-temperature high-pressure homogenization. Under ice bath conditions, the nano selenium on the cell wall was separated from the bacteria by using an ultrasonic disrupter, the ultrasonic power was 300 W, 10 s stop 10 s, ultrasonic for 20 min, and then 0.45 μm sterile filter membrane was used for filtration and sterilization, to obtain the selenium-rich composite bacteria fermentation product.

[0075] 1.4 Preparation of fermentation product of composite bacteria (F)

[0076] The fermentation broth of the composite bacteria is collected, the pH value of the fermentation broth is adjusted to 6.0-6.5, and then the cells are fully broken by low-temperature high-pressure homogenization. The cells are broken by using an ultrasonic disrupter under ice bath conditions, the ultrasonic power is 300 W, 10 s stop 10 s, ultrasonic for 20 min, and then filtered by using a sterile filter membrane of 0.45 μm to remove bacteria, thereby obtaining the fermentation product of the composite bacteria (F).

[0077] 1.5 Preparation of fermentation product of H15 (H)

[0078] The activated animal Bifidobacterium H15 is inoculated into sterile MRSc culture medium at an inoculation amount of 2%, and then incubated at 37°C under anaerobic conditions for 24 h to obtain the second-generation H15 fermentation broth (the effective viable bacterial number is 9.46 lgCFU / mL). The second-generation animal Bifidobacterium H15 fermentation broth is collected, the pH value of the fermentation broth is adjusted to 6.0-6.5, and then the cells are fully broken by low-temperature high-pressure homogenization. The cells are broken by using an ultrasonic disrupter under ice bath conditions, the ultrasonic power is 300 W, 10 s stop 10 s, ultrasonic for 20 min, and then filtered by using a sterile filter membrane of 0.45 μm to remove bacteria, thereby obtaining the fermentation product of H15 (H).

[0079] 1.6 Preparation of selenium-rich H15 (Se-H) fermentation product

[0080] The second-generation H15 fermentation broth is inoculated into MRSc culture medium containing 100 μg / mL sodium selenite at an inoculation amount of 2%. The broth is incubated at 37°C under anaerobic conditions for 48 h to obtain the selenium-rich H15 fermentation broth. The selenium-rich H15 fermentation broth is collected, the pH value of the fermentation broth is adjusted to 6.0-6.5, and then the cells are fully broken by low-temperature high-pressure homogenization. The nanometer selenium on the cell wall is separated from the bacteria by using an ultrasonic disrupter under ice bath conditions, the ultrasonic power is 300 W, 10 s stop 10 s, ultrasonic for 20 min, and then filtered by using a sterile filter membrane of 0.45 μm to remove bacteria, thereby obtaining the selenium-rich H15 fermentation product (Se-H).

[0081] 1.7 Preparation of fermentation product of ES23 (E)

[0082] The activated Lactobacillus coryniformis ES23 was inoculated into sterile MRS medium at an inoculation amount of 2%, and incubated at 37°C for 24 h to obtain the second-generation ES23 fermentation broth (effective viable cell number was 8.48 lgCFU / mL). The second-generation Lactobacillus coryniformis ES23 fermentation broth was collected, and the pH value of the fermentation broth was adjusted to 6.0-6.5, and then the cells were fully broken by low-temperature high-pressure homogenization. Under ice bath conditions, the cells were broken by using an ultrasonic disrupter, the ultrasonic power was 300 W, 10 s stop 10 s, ultrasonic for 20 min, and then filtered by using a sterile filter membrane with a pore size of 0.45 μm to remove bacteria, to obtain the ES23(E) fermentation product filtrate.

[0083] 1.8 Preparation of selenium-rich ES23(Se-E) fermentation product

[0084] The activated ES23 second-generation fermentation broth was inoculated into MRSc medium containing 100 μg / mL sodium selenite at an inoculation amount of 2%. It was incubated at 37°C for 48 h to obtain the selenium-rich ES23 fermentation broth. The selenium-rich ES23 fermentation broth was collected, and the pH value of the fermentation broth was adjusted to 6.0-6.5, and then the cells were fully broken by low-temperature high-pressure homogenization. Under ice bath conditions, the nanometer selenium on the cell wall was separated from the bacteria by using an ultrasonic disrupter, the ultrasonic power was 300 W, 10 s stop 10 s, ultrasonic for 20 min, and then filtered by using a sterile filter membrane with a pore size of 0.45 μm to remove bacteria, to obtain the selenium-rich ES23 fermentation product (Se-E).

[0085] Example 2 Separation, purification and characterization of biological nanometer selenium

[0086] 2.1 The selenium-rich composite bacteria fermentation product prepared in 1.3 was centrifuged at 12000 rpm for 10 min, washed with sterile PBS for 3 times, resuspended in PBS and adjusted to the concentration. 5 μL was added on a 200-mesh carbon-supported copper mesh, and dried at room temperature for 2 h, then the sample was measured by TEM at an acceleration voltage of 15 KV. At the same time, the total selenium content in the composite bacteria fermentation product was detected according to the first method of GB5009.93-2017.

[0087] As shown in Figure 2 , the biological nanometer selenium (Bio-SeNPs) in the selenium-rich composite bacteria fermentation product was monodispersed and uniform in spherical structure, with a particle size of 40-400 nm and an average particle size of about 220.81 nm±28.25. In addition, the Bio-SeNPs had a negative potential (-28.50 mV±0.69) and a larger dispersion (0.03±0.01) consistent with most Bio-SeNPs. The high negative charge indicated that the Bio-SeNPs had higher stability.

[0088] 2.2 The total selenium content of the selenium-rich composite bacteria fermentation product was detected according to the first method of GB5009.93-2017, and the results showed that the total selenium content of the selenium-rich composite bacteria fermentation product was 22 μg / mL.

[0089] Example 3 Determination of the in vitro antioxidant capacity of the fermentation product

[0090] 3.1 Preparation of the test sample; according to the culture method of Example 1, H15 and ES23 were respectively subjected to single-strain fermentation, single-strain selenium-rich fermentation, composite bacteria fermentation, and composite bacteria selenium-rich fermentation to obtain the corresponding fermentation products. Then the test sample was prepared according to Table 1.

[0091] Table 1 Sample test concentration setting table

[0092]

[0093] 3.2 Determination of the DPPH free radical scavenging capacity of the sample

[0094] According to Table 2, the sample was added, and after thorough mixing, the absorbance of each well at 517 nm was measured under room temperature and light shielding for 30 min, wherein the positive control was 0.01% V C . The DPPH free radical scavenging rate was calculated according to the following formula, and the results are shown in Figure 3 .

[0095] DPPH free radical scavenging rate (%) = [1-(A1-A2) / A0] x 100%

[0096] Table 2 DPPH free radical scavenging experiment sample table

[0097]

[0098] As shown in Figure 3 , the fermentation product has strong scavenging activity on DPPH free radicals, and the scavenging rate gradually increases with the increase of concentration. When the concentration of the fermentation product is 10%, 20% and 100% respectively, the DPPH free radical scavenging rate of the composite bacteria fermentation product is 69.82%, 70.40% and 72.99% respectively, and the DPPH free radical scavenging rate of the selenium-rich composite bacteria fermentation product is 75.90%, 79.32% and 80.26% respectively, indicating that when the concentration of the fermentation product is more than 10%, the DPPH free radical scavenging activity of the composite bacteria fermentation product and the selenium-rich composite bacteria fermentation product tends to be stable, and is significantly better than that of the single bacteria fermentation product and the selenium-rich single bacteria fermentation product. Therefore, the animal bifidobacterium H15 and coryneform lactobacillus ES23 can synergistically enhance each other in terms of antioxidant activity.

[0099] 3.3 Determination of the ABTS + free radical scavenging capacity of the sample

[0100] According to Edison ABTS + The ABTS working solution was prepared using the free radical scavenging reagent kit (ADS-W-AB001). Samples were added according to Table 3, mixed thoroughly, and incubated at room temperature in the dark for 6 minutes. The absorbance of each well was then read at 734 nm. ABTS was calculated using the following formula. + Clearance rate, results are shown in Figure 4 .

[0101] ABTS + Clearance rate (%) = [1 - (A1 - A0) / A2] × 100%

[0102] Table 3 ABTS + Free radical scavenging experiment sample loading table

[0103]

[0104] like Figure 4 As shown, the fermentation products affect ABTS + Free radicals exhibit strong scavenging activity, and the scavenging rate gradually increases with increasing concentration. When the fermentation product concentration is above 1%, the ABTS of the selenium-enriched complex bacteria fermentation product... + The free radical scavenging activity of the fermentation products was higher than that of other groups. At fermentation product concentrations of 5%, 10%, and 20%, the ABTS of the selenium-enriched complex fermentation products were... + The free radical scavenging rates were 61.97%, 83.33%, and 85.17%, respectively, indicating that when the concentration of fermentation products reached above 10%, the ABTS of the selenium-enriched complex bacteria fermentation products was significantly reduced. + The changes in free radical scavenging activity tend to stabilize.

[0105] Example 4: Keratinocyte toxicity test of selenium-enriched compound bacteria fermentation products

[0106] 4.1 Cell Culture: Immortalized human keratinocytes (HaCaT cells) were cultured in a cell culture incubator at 37°C and 5% CO2 using DMEM medium containing 10% fetal bovine serum. Cells in a sub-fusion state were passaged by digestion with 0.25% trypsin and 0.03% EDTA, counted using a cell counting chamber, and cultured at a rate of 1×10⁻⁶ cells / cells. 4 Cells were seeded at a density of 100 μL / well in 96-well plates, with 100 μL of cell suspension added to each well using a pipette. After seeding, the 96-well plates were incubated overnight at 37°C in a 5% CO2 incubator until the cells adhered.

[0107] 4.2 Experimental grouping: Sample solutions of different concentrations were prepared according to Table 4, diluted with serum-free DMEM cell culture medium as solvent, and prepared into test sample solutions with final concentrations of 1%, 2%, 5%, 10%, and 20%, respectively.

[0108] Table 4. Concentration Setting Table for Cytotoxicity Test

[0109]

[0110] 4.3 Drug administration method: After the cells were incubated overnight in the incubator, they were treated according to the groups in Table 6. 100 µL of the corresponding sample solution was added to each group, and then the 96-well plate was placed in the incubator and cultured for 24 h.

[0111] 4.4 Detection: After 24 h of cell culture, discard the supernatant from each well, wash each well three times with 100 μL of PBS, and then add 100 μL of serum-free DMEM containing 10% CCK-8 reagent to each well. Gently shake the culture plate to avoid generating air bubbles. Incubate the 96-well plate at 37℃ for 1 h, and measure the absorbance at 450 nm using a microplate reader. Calculate cell viability according to the following formula.

[0112] Cell viability (%) = (As-Ab) / (Ac-Ab) × 100%

[0113] Ab blank well: culture medium

[0114] Ac control wells: Culture medium + cells

[0115] As experimental wells: culture medium + cells + drug treatment

[0116] like Figure 5 As shown, compared with the fermentation products of the compound bacteria, the fermentation products of the selenium-enriched compound bacteria have no obvious toxicity to HaCaT cells in the 20% concentration range, and have the effect of promoting cell proliferation at 10% and 20%. Therefore, 10% was selected as the experimental concentration for photodamage repair of the fermentation products of the selenium-enriched compound bacteria.

[0117] Example 5: Protective effect of selenium-enriched compound bacteria fermentation products on UVB-damaged HaCaT cells

[0118] 5.1 Construction of a UVB-induced photodamage model in HaCaT cells

[0119] To determine the UVB intensity used to construct the HaCaT cell oxidative damage model, 20-200 mJ / cm² was used. 2 HaCaT cells were treated with irradiation.

[0120] like Figure 6As shown, the survival rate of HaCaT cells gradually decreased with increasing UVB irradiation intensity. When the UVB irradiation intensity reached 200 mJ / cm², the survival rate of HaCaT cells decreased further. 2 At that time, under the microscope, the cells were observed to be severely shrunken and dispersed, with a survival rate of only 15.50%. This indicates that 200 mJ / cm² is the optimal concentration for cell survival. 2 UVB has a strong inhibitory effect on the survival of HaCaT cells. When the UVB irradiation intensity is less than 20 mJ / cm², the effect is more pronounced. 2 Under the microscope, the cells were observed to be densely packed, with smooth edges, uniform cytoplasm, and a cell viability rate close to 100%, indicating that the irradiation intensity was less than 20 mJ / cm². 2 It has no or only a slight inhibitory effect on cell survival. When the UVB irradiation intensity is 100 mJ / cm²... 2 Under the microscope, some cells were observed to be irregularly densely packed, some cells were shrunken and atypical, and some cells had uniformly round cytoplasm. The cell viability rate was 74.44%. This indicates that the irradiation intensity was 100 mJ / cm². 2 At that time, most cells were still viable, which provided an experimental opportunity to study the repair effect of selenium-enriched bacterial fermentation products on UVB-damaged cells. Therefore, a concentration of 100 mJ / cm² was used. 2 A photodamage model was established based on the UVB irradiation intensity.

[0121] 5.2 Effects of Selenium-Enriched Compound Bacterial Fermentation Products on Photodamaged Cell Viability

[0122] 5.2.1 Analysis of the protective effect of selenium-enriched complex bacterial fermentation products on photodamage of HaCaT cells based on CCK8 assay. HaCaT cells were fermented at 1×10⁻⁶... 4 Cells were seeded per well in 96-well cell culture plates and cultured for 24 h. After culturing, the culture medium was aspirated, and the cells were washed once with PBS and set aside. DMEM medium containing 10% (v / v) E, H, F, Se-E, Se-H, and Se-F fermentation products were added to verify the protective effect of the selenium-enriched complex bacterial fermentation products on HaCaT. Untreated cells served as blank control (NC), and cells cultured without fermentation products but treated with UVB served as model control (UVB). Samples were collected after treatment for subsequent analysis, and cell morphology was observed using electron microscopy.

[0123] 5.2.2 Cell viability was assessed according to the CCK-8 assay kit instructions. HaCaT cells were incubated in DMEM medium containing each fermentation product, with DMEM medium supplemented with no fermentation product serving as the control group (NC). Incubation lasted 2 h. The medium was then aspirated, and 1 mL of PBS buffer was added to each well. UVB irradiation was then applied at a dose of 100 mJ / cm². 2After UVB treatment, cells were cultured in DMEM medium containing fermentation extract for 24 h, and cell viability was detected according to the CCK-8 kit instructions.

[0124] Experimental groupings: 10% fermentation product of compound bacteria (F), 10% fermentation product of selenium-enriched compound bacteria (Se-F), 10% fermentation product of H15 (H), 10% fermentation product of ES23 (E), 10% selenium-enriched fermentation product of H15 (Se-H), and 10% selenium-enriched fermentation product of ES23 (Se-E). Each group had 3 replicate wells. The methods for solution preparation, drug administration, detection, and calculation were the same as in Example 4.

[0125] Under an electron microscope, HaCaT cells in the normal control group showed typical epithelial-like structures with tightly packed cells and clear outlines. Cells in the UVB model group suffered significant damage, manifested as rounded and shrunken cells, with an increase in suspended cells and cell debris. The degree of cell damage in each group treated with the bacterial fermentation product recovered to varying degrees compared to the model group, with fewer suspended cells and more cells retaining their original morphology.

[0126] like Figure 7 As shown, cell viability testing revealed that the cell viability in the UVB group was significantly lower than that in the normal control group, with a statistically significant difference (P<0.05), indicating that the cell photodamage model was successfully established at 100 mJ / cm². 2 After 24 h of UVB irradiation, the proliferation activity of HaCaT cells decreased. Adding H, F, Se-H, Se-E, and Se-E to photodamaged cells restored cell activity to levels similar to the normal control group, significantly increasing cell activity compared to the UVB group (P<0.05). The Se-F group showed the most significant effect on cell activity, indicating that the fermentation products of the combined bacteria and selenium-enriched combined bacteria have good repair effects on photodamaged cells. Furthermore, the effect of the selenium-enriched combined bacteria fermentation product is significantly better than that of the single selenium-enriched bacteria fermentation product. Therefore, Bifidobacterium animalis H15 and Lactobacillus corynebacterium ES23 can synergistically enhance each other in the repair of photodamaged cells.

[0127] Example 6 Evaluation of cellular antioxidant effects

[0128] 6.1 Oxidative stress refers to the imbalance in the scavenging of oxygen free radicals in the body, resulting in the production of a large amount of highly reactive reactive oxygen species (ROS). ROS are a class of single-electron reduction products of oxygen in the body, mainly originating from cellular oxidative metabolism and ultraviolet radiation. According to the free radical aging theory, ROS plays a major role in skin aging, and excessive ROS can directly damage cells. Flow cytometry was used to quantitatively detect the intracellular ROS content.

[0129] 6.1.1 Cell Culture: After digestion of HaCaT cells, the cell suspension was diluted 10-fold and counted, with 3 × 10⁶ cells per well. 5 Cells were placed in six-well plates. The cells were incubated at 37°C for 24 h. After observing cell adhesion under a microscope, the supernatant was discarded, and 1 mL of bacterial fermentation product (final concentration 10%) was added to each well. The cells were then subjected to a 100 mJ / cm² reaction. 2 After irradiation treatment, continue culturing for 24 hours;

[0130] 6.1.2 Probe Incubation: Prepare a final concentration of 10 μmol / L for the fluorescent probe DCFH-DA by diluting it 1:1000 with serum-free medium. Then, aspirate the supernatant from the six-well plates and add 1 mL of 10 μmol / L DCFH-DA to each well (except the blank control wells). Incubate the six-well plates at 37°C in the dark for 20 min. After incubation, wash three times with serum-free culture medium to remove excess DCFH-DA from the cell surface. Observe the six-well plates under a fluorescence microscope to check if the fluorescent probe has been successfully implanted. Collect cells by trypsin digestion and centrifuge at 24°C and 1000 r / min for 5 min. Adjust the cell concentration with PBS after centrifugation.

[0131] 6.1.3 Quantitative detection of intracellular ROS content by flow cytometry: such as Figure 8 As shown, at 100 mJ / cm 2 After 24 h of treatment alone on HaCaT cells, the fluorescence intensity in the model group increased by 7.59 times compared with the control group, and the difference was statistically significant (P < 0.001), indicating that the photo-induced oxidative damage model was successfully established. Compared with the model group, the fluorescence intensity in the cells decreased to varying degrees after treatment with the bacterial fermentation products. There were no significant differences in the E, H, and UVB groups, while the other groups showed significant differences compared with the UVB group. The selenium-enriched compound bacterial fermentation products showed the best reduction in reactive oxygen species (ROS) levels and had a good ability to scavenge intracellular ROS.

[0132] 6.2 Glutathione (GSH) is an important intracellular metabolic regulator containing active sulfhydryl groups in its structure. It participates in various important biochemical reactions in the body, protecting the sulfhydryl groups of important enzyme proteins from oxidation and inactivation, thus ensuring cellular energy metabolism. Simultaneously, glutathione can bind to free radicals in the body through its sulfhydryl groups, reducing them to acidic substances, thereby accelerating the excretion of free radicals and reducing their damage to the body. GSH content was measured in cells under photodamage.

[0133] 6.2.1 Photodamaged HaCaT cells treated with bacterial fermentation products were collected and sonicated to obtain cell extract. The cell extract was centrifuged at 4℃ and 1500×g for 10 min, and the supernatant was used to detect GSH content according to the instructions of Jiangsu Enzyme Immunosorbent Assay Kit (MM-0458H2).

[0134] like Figure 9 As shown, after UVB treatment, the GSH content in cells was significantly lower than that in the normal treatment group (p<0.01), indicating that UVB treatment reduced the antioxidant capacity of cells. The GSH content in cells increased after treatment with the fermentation products of each group of bacteria. Compared with the UVB model group, the treatment with selenium-enriched compound bacteria fermentation products (Se-F), selenium-enriched bacteria H15 fermentation products (Se-H), and ES23 selenium-enriched fermentation products (Se-E) significantly increased the GSH content in cells (p<0.05), indicating that the three groups of selenium-enriched bacteria fermentation products could improve the antioxidant capacity of cells, and the effect of Se-F was better than that of Se-H and Se-E. Therefore, the selenium-enriched compound bacteria fermentation products can significantly increase the antioxidant enzyme activity of HaCaT cells and reduce oxygen free radicals, providing photoprotection against UVB-damaged human keratinocytes. Furthermore, its performance is significantly better than that of selenium-enriched single-strain fermentation products, indicating that Bifidobacterium animalis H15 and Lactobacillus corynebacterium ES23 can synergistically enhance photodamage repair.

[0135] Example 7 Evaluation of the in vitro anti-photoaging effect of selenium-enriched compound bacteria fermentation products

[0136] MMPs are a group of zinc-dependent endopeptidases that specifically degrade almost all of the extracellular matrix and play an important role in skin photoaging. UVB radiation mainly promotes the expression of MMP-1, accelerates collagen fiber degradation, leads to the disappearance of the extracellular matrix, and causes skin cell aging. Hydroxyproline (HYP) is a unique amino acid in collagen and one of the main components of collagen tissue. HYP content is an important indicator of cellular anti-photoaging activity.

[0137] HaCaT cells damaged by light and treated with bacterial fermentation products were cultured and collected according to method 6.1.1. Cells were then disrupted by sonication to obtain cell extracts. The cell extracts were centrifuged at 4℃ and 1500×g for 10 min. The supernatant was collected and the contents of matrix metalloproteinase-1 (MMP-1) and hydroxyproline (HYP) in the cells were detected according to the instructions of Jiangsu Enzyme Immunosorbent Assay Kits (catalog numbers: MM-0072H2 and MM-1500H2).

[0138] like Figure 10As shown, UVB treatment significantly increased MMP-1 expression in cells (P < 0.0001), indicating that UVB treatment increased MMP-1 levels, thereby exacerbating collagen tissue damage. Treatment with bacterial fermentation products reversed the increase in MMP-1, and all fermentation products significantly reduced MMP-1 expression. The fermentation products of selenium-enriched composite bacteria showed the strongest trend in reversing MMP-1 expression, outperforming the fermentation products of selenium-enriched single bacteria. This suggests that Bifidobacterium animalis H15 and Lactobacillus corynebacterium ES23 have a synergistic effect in delaying collagen fiber degradation and slowing skin cell aging.

[0139] like Figure 11 As shown, the content of HYP in cells decreased significantly after UVB treatment (p<0.001), indicating that UVB treatment affects the synthesis and metabolism of collagen tissue. However, the content of HYP increased significantly after treatment with bacterial fermentation products. The HYP concentration of selenium-enriched compound bacterial fermentation products (Se-F) increased the most significantly, which was better than the HYP concentration after treatment with selenium-enriched single bacterial fermentation products. This indicates that Bifidobacterium animalis H15 and Lactobacillus corynebacterium ES23 can synergistically enhance the anti-photoaging ability of cells.

[0140] Example 8: Changes in the content of cellular inflammatory factors

[0141] UVB can induce the production of IL-1β, IL-6, and TNF-α in the stratum corneum of the skin, causing skin inflammation. These inflammatory cytokines can promote the production of large amounts of ROS, exacerbating oxidative stress in skin cells. ROS can further mediate skin inflammation and accelerate photoaging of the skin.

[0142] Photodamaged HaCaT cells treated with bacterial fermentation products were cultured and collected according to method 6.1.1. The supernatant was taken and the contents of inflammatory factors tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6) were detected according to the instructions of Jiangsu Enzyme Immunosorbent Assay Kit (catalog numbers: MM-0122H2, MM-0181H2 and MM-0049H2).

[0143] like Figures 12-14 As shown, after UVB irradiation treatment, the levels of cellular inflammatory factors TNF-α and IL-6 significantly increased, while the level of IL-1β did not change significantly, indicating that the immune system can sense UVB damage and exhibit an immune response. Treatment with selenium-enriched compound bacteria fermentation products reduced the levels of cellular inflammatory factors TNF-α and IL-6, with the effect of treatment reaching a highly significant level.

[0144] Example 9: Repair of photodamaged mouse skin by fermentation products of selenium-enriched complex bacteria

[0145] according to Figure 15The experiment will be conducted according to the procedure shown below.

[0146] 9.1 Laboratory Animals: After obtaining approval from the Laboratory Animal Welfare and Animal Experimentation Ethics Review Committee of China Agricultural University, 40 6-8 week old female Balb / c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were purchased. After professional clean-grade packaging, they were sent to the specific pathogen-free (SPF) animal facility at the West Campus of China Agricultural University. The relative humidity of the rearing environment was 55±10%, and the temperature was 22±2℃. A strict 12-hour light-dark cycle was maintained, and the mice had free access to food and water. Mice were anesthetized by intraperitoneal injection of 3% chloral hydrate solution. The fur on the back of the mice was shaved off using a razor, covering an area of ​​approximately 3 cm × 3 cm.

[0147] 9.2 Animal Grouping: Mice were randomly divided into 5 groups of 8 mice each and underwent a one-week acclimatization period. After the acclimatization period, except for the blank control group, the other groups of mice were treated with UVB irradiation daily. The mice were placed in a dark room under a UVB lamp with the light source fixed at a distance of approximately 10 cm from the mouse's back. A broadband UVB phototherapy device (emission spectrum 290-320 nm, peak 310 nm) was used to irradiate the mice every 24 hours for 3 consecutive days (the endpoint of modeling was determined based on the state of skin damage in the mice) to establish a skin photodamage model. Before the start of irradiation, the irradiation power was measured using an irradiation device, and the irradiation dose was 1000 mJ / cm2 per day. The UVB group mice were irradiated for the corresponding time according to the measured power. During the UVB modeling period, each treatment group was administered medication transdermally 30 minutes before UVB irradiation. After the experiment, mice were euthanized by cervical dislocation, and back skin tissue was collected. One portion was transferred to 1.5 mL centrifuge tubes containing 4% neutral formaldehyde and fixed at 4°C, while the other portion was stored at -80°C. The specific groupings are as follows:

[0148] Blank control group (NC): 200 μL of physiological saline was applied percutaneously every day, without UVB irradiation;

[0149] UVB group (UVB): UVB irradiation was performed daily after applying 200 μL of physiological saline to the skin.

[0150] Selenium-enriched H15 group (Se-H): 200 μL of fermentation product filtrate containing 10% selenium-enriched Bifidobacterium H15 was applied transdermally every day and UVB irradiation was performed;

[0151] Selenium-enriched ES23 group (Se-E): 200 μL of filtrate containing 10% selenium-enriched Lactobacillus coccidioides fermentation product was applied transdermally daily and UVB irradiation was performed;

[0152] Selenium-enriched compound bacteria group (Se-F): 200μL of fermentation product filtrate containing 10% selenium-enriched compound bacteria (H15+ES23) was applied percutaneously daily and irradiated with UVB.

[0153] 9.3 Degree of skin damage: After the experiment, the degree of skin damage in mice was determined by observing the erythema and the extent of skin damage on the back of the mice.

[0154] 9.4 Skin histopathological analysis

[0155] 9.4.1 HE staining: Mice were euthanized after the experiment, and their skin tissue was embedded in paraffin and sectioned. The tissue was then dewaxed with xylene, dexylene was removed using a gradient of ethanol, and the sections were washed with water to obtain mouse skin tissue sections. After completion, the sections were placed in hematoxylin staining solution for 5 minutes, then washed twice with water and placed in eosin staining solution for 30 seconds. The sections were observed under a light microscope and photographed using a photomicroscope to observe histological changes.

[0156] 9.4.2 Masson staining:

[0157] (1) Sampling and preparation of paraffin sections: Skin tissue samples were collected and fixed with 4% paraformaldehyde solution. After fixation, the tissue was dehydrated, cleared with xylene, and impregnated with paraffin. The tissue was then placed in a mold and molten paraffin was poured in. The mold was allowed to solidify to form a paraffin block. The fixed paraffin-embedded tissue block was cut on a microtome, and the section thickness was 6-8 μm.

[0158] (2) Dewaxing and hydration of paraffin sections: The sections were placed in xylene I and xylene II for 15 min each to remove paraffin. Then, the sections were transferred to anhydrous ethanol for 15 min to degrease, and then placed in 90% ethanol, 80% ethanol and 70% ethanol for 5 min each to hydrate. Finally, the sections were rinsed with distilled water for 2 min.

[0159] (3) Weigert iron hematoxylin staining of collagen fibers: Weigert iron hematoxylin staining for 5 min, wash off excess staining solution with distilled water, differentiate with 1% hydrochloric acid alcohol for 5-15 s, and rinse with distilled water for 30 s.

[0160] (4) Ponceau Acid Fuchsia Dye on muscle fibers: Treat with Ponceau Acid Fuchsia Dye for 5 min, wash away excess dye with distilled water, treat with phosphomolybdic acid solution for 5 min, then counter-dye with aniline blue dye for 1-2 min, and wash with 1% glacial acetic acid aqueous solution for 30 s;

[0161] (5) Dehydration and mounting: The sections were quickly immersed in 95% ethanol for 3 s and then in anhydrous ethanol twice for 10 s each time. Subsequently, the sections were cleared in xylene III, xylene II and xylene I for 2 min each, and then mounted with neutral resin.

[0162] (6) Microscopic examination of slides: Collagen fibers appear in blue, while muscle fibers and cellulose appear in red. Analysis and calculations were performed using ImageJ software.

[0163] 9.5 Detection of malondialdehyde (MDA) and superoxide dismutase (SOD) antioxidant markers in mouse skin:

[0164] The detection and calculation were performed according to the detection methods in the instructions of Beyotime Lipid Oxidation (MDA) Assay Kit (Catalog No.: S0131M) and Total SOD Activity Assay Kit (WST-8 Method) (Catalog No.: S0101M).

[0165] like Figure 16 As shown, the skin morphology of mice at the experimental endpoint was observed ( Figure 16 A) and B) showed that the area of ​​skin crusting in mice in the Se-F group was significantly lower than that in the model group, the degree of skin damage was reduced, and the redness, swelling, and wrinkling were all alleviated. Both the Se-F and Se-H groups had significant repair effects on UVB-induced skin damage in mice, and the difference between the Se-F and Se-H groups was significant, indicating that both Se-H and Se-F fermentation products inhibited UVB-induced skin damage in mice to some extent, with the compound bacterial fermentation product (Se-F) group showing the most significant effect.

[0166] UVB irradiation increases ROS in mouse skin tissue, leading to alterations in protein and gene structure and photodamage. Epidermal thickness, a quantitative parameter for evaluating the degree of photodamage, increases due to skin edema and inflammation accumulation. After photodamage, epidermal thickness increases by approximately 200%, with inflammatory infiltration in the dermis and disruption of the connection between the epidermis and dermis. In normal skin, the skin layers are evenly distributed, the epidermis is thinner, the connection between the epidermis and dermis is wavy, and inflammatory cell infiltration in the dermis is minimal. (Based on HE staining results...) Figure 16 As can be seen from C and 14E, after treatment with Se-H and Se-F, the thickness of the mouse epidermis was significantly reduced and the infiltration of inflammatory cells in the dermis was reduced, but the changes in the Se-E group were not obvious.

[0167] Changes in collagen fibers, particularly CVF (capillary fiber flow), are also key indicators for assessing photodamage to the skin. CVF reflects the percentage of collagen in the tissue and the degree of skin fibrosis. Masson staining shows (…). Figure 16Following UVB irradiation (D, 14F), the collagen fibers in the dermal layer of mouse skin exhibited a relatively disordered and intertwined arrangement, making it difficult to discern a clear structural pattern. The CVF of photodamaged skin decreased by approximately 50%. Mice treated with Se-H and Se-F via transdermal application showed more orderly collagen deposition and fiber arrangement. After transdermal treatment, the CVF of the Se-F and Se-H groups significantly increased, but the Se-E group showed no significant change. Compared to the UVB model group, the CVF improvement in the Se-F group (P<0.01) was more significant than that in the Se-H group (P<0.05), indicating that the fermentation products of selenium-enriched complex bacteria exert a more significant protective effect on collagen than those of single-bacterial fermentation products.

[0168] MDA and SOD can directly reflect the state of redox reactions in the body. MDA is one of the main products of lipid peroxidation. By detecting changes in MDA levels, the degree of oxygen free radical attack and lipid peroxidation can be reflected. Therefore, MDA is one of the commonly used indicators for evaluating the degree of oxidative stress. SOD is an important antioxidant enzyme in the human body, with the ability to scavenge free radicals and prevent oxygen free radicals from damaging body cells. In addition, antioxidant enzymes play an important role in the process of clearing ROS in the body. The activity of antioxidant enzymes is related to the body's ability to scavenge oxygen free radicals; the higher the activity of antioxidant enzymes, the stronger the body's ability to scavenge oxygen free radicals. Therefore, detecting MDA levels and SOD enzyme activity can indirectly reflect the level of oxidative stress in the body.

[0169] like Figure 16 As shown in Figure G, compared with the control group (NC), the MDA content in the skin tissue of mice in the UVB model group increased by 1.76 times, which was highly significant (P<0.01). Compared with the UVB model group, percutaneous application of Se-H and Se-F significantly decreased the MDA level in the tissue, but the change was not significant in the Se-E group. The MDA decrease in the Se-F group reached a highly significant level, and the MDA decrease in the Se-H group reached a significant level.

[0170] like Figure 16 As shown in Figure H, compared with the control group (NC), the activity of the antioxidant enzyme SOD in the skin tissue of mice in the UVB model group decreased by 44.67%, which was highly significant (P<0.01). Compared with the UVB model group, the SOD level in the tissue significantly increased after percutaneous application of Se-H and Se-F, but the change was not obvious in the Se-E group.

[0171] The above results indicate that the fermentation products of selenium-enriched compound bacteria (Se-F) and the fermentation product of selenium-enriched bacterium H15 (Se-H) have a certain repair effect on UVB-induced photodamage to mouse skin, while the effect of selenium-enriched bacterium ES23 alone is not significant. The repair effect of the Se-F group is significantly better than that of the Se-H group, indicating that the fermentation products of selenium-enriched compound bacteria exhibit stronger antioxidant activity and collagen protection function through synergistic enhancement, and have potential application value in the repair of photodamage to the skin.

[0172] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A compound bacteria, characterized in that, The compound bacteria include Bifidobacterium animalis and Lactobacillus corynebacterium; The animal bifidobacteria is animal bifidobacteria ( Bifidobacterium animals H15 was deposited on June 16, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.27654. The *Lactobacillus* species is *Lactobacillus* (…). Lactobacillus coryniformiss ES23 was deposited on June 16, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.27653. The volume ratio of Bifidobacterium animalis H15 to Lactobacillus corynebacterium ES23 is 1~5:1~5, the effective viable count of Bifidobacterium animalis H15 is 9.46 lg CFU / mL, and the effective viable count of Lactobacillus corynebacterium ES23 is 8.48 lg CFU / mL.

2. A fermentation product of selenium-enriched compound bacteria, characterized in that, The preparation method of the selenium-enriched compound bacteria fermentation product includes the following steps: (1) The animal bifidobacterium H15 and the rod-shaped lactobacillus ES23 described in claim 1 were respectively inoculated into MRSc medium and anaerobic cultured to obtain animal bifidobacterium H15 seed solution and rod-shaped lactobacillus ES23 seed solution; (2) The Bifidobacterium animalis H15 seed liquid and the Lactobacillus ES23 seed liquid were mixed and inoculated into MRSc medium containing sodium selenite, and anaerobic culture was carried out to obtain selenium-enriched compound fermentation liquid. (3) Adjust the pH of the selenium-enriched compound fermentation liquid, break the cell wall, and filter to remove bacteria to obtain the selenium-enriched compound fermentation product.

3. The selenium-enriched compound bacteria fermentation product according to claim 2, characterized in that, In step (1), the inoculation amount of Bifidobacterium animalis H15 and Lactobacillus ES23 is 1-5%.

4. The selenium-enriched compound bacteria fermentation product according to claim 2, characterized in that, The mixing volume ratio of the H15 seed liquid and the ES23 seed liquid in step (2) is 1~5:1~5.

5. The selenium-enriched compound bacteria fermentation product according to claim 2, characterized in that, The sodium selenite content in the MRSc medium described in step (2) is 50~150 μg / mL.

6. The selenium-enriched compound bacteria fermentation product according to claim 2, characterized in that, The selenium content in the fermentation product of the selenium-enriched compound bacteria is 15~25 μg / mL.

7. A composition, characterized in that, The composition comprises the fermentation product of the compound bacteria as described in claim 1 or any of the selenium-enriched compound bacteria as described in claims 2-6.

8. The application of the fermentation product of the compound bacteria according to claim 1 or any one of claims 2 to 6 in the preparation of antioxidant products.

9. The application according to claim 8, characterized in that, The products mentioned are health foods, medicines, cosmetics, or animal feed.

10. The application of the fermentation product of the compound bacteria according to claim 1 or any of the selenium-enriched compound bacteria according to claims 2 to 6 in the preparation of products with photodamage repair function.

11. The application according to claim 10, characterized in that, The application includes at least one of the following: (1) Application in the preparation of products that can improve cell viability; (2) Application in the preparation of products that can enhance the activity of cellular antioxidant enzymes; (3) Application in the preparation of products that can increase the hydroxyproline content of cells; (4) Application in the preparation of products that can reduce cellular inflammation levels; (5) Application in the preparation of products that can inhibit skin damage caused by UVB; (6) Application in the preparation of products with collagen protection function.

12. The application according to any one of claims 10-11, characterized in that, The product is a pharmaceutical, cosmetic, or animal feed.

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