Bifidobacterium adolescentis HC2916 for inhibiting melanin synthesis, microbial preparation thereof and application

Abstract

This invention relates to the field of probiotic screening and application technology, specifically to a Bifidobacterium adolescentis HC2916 that inhibits melanin synthesis, its microbial preparations, and applications. Bifidobacterium adolescentis HC2916 was deposited on May 16, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 27352. This strain exhibits excellent antioxidant capacity, capable of scavenging various free radicals and inhibiting lipid peroxidation. Furthermore, its fermentation supernatant and lysate significantly downregulate the protein and mRNA expression of MITF, a core factor in melanin production signaling, and inhibit the expression of its downstream key enzymes TYRP1 and TYRP2. This effectively suppresses UVA-induced increases in melanin content in skin keratinocytes, demonstrating promising application prospects in the field of skin whitening.

Description

Technical Field

[0001] This invention relates to the field of probiotic screening and application technology, specifically to a Bifidobacterium adolescentis HC2916 that inhibits melanin synthesis, its microbial preparations, and applications. Background Technology

[0002] From the perspective of skin pigment composition, human skin color is mainly regulated by carotene and melanin. Melanin, including eumelanin and pheomelanin, plays a dominant role in the formation of skin depth and hue. The maintenance of melanin homeostasis depends on the synergistic effects of multiple stages, including signal transduction regulation, melanin biosynthesis, melanosome transport, and subsequent metabolism, forming a fine and complex molecular regulatory network. In the signal transduction stage, ultraviolet radiation induces keratinocytes to secrete polypeptides and cytokines such as α-melanocyte-stimulating hormone (α-MSH), endothelin-1, and interleukin-2. These substances activate the central regulator of melanin production, MITF, through signaling pathways such as MC1R, cAMP, PKA, and CREB, thereby upregulating the expression of key enzymes such as tyrosinase, tyrosine-associated protein-1 (TYRP1), and tyrosine-associated protein-2 (TYRP2). In the production stage, tyrosine is converted into eumelanin and pheomelanin through multiple enzymatic reactions catalyzed by tyrosinase, ultimately completing the synthesis of melanin. Therefore, regulating the signal transduction and transcription factors related to melanin production becomes a key pathway to achieving skin whitening effects.

[0003] Currently, commercially available skin-whitening products are mainly topical formulations, including tyrosinase inhibitors, melanin transport inhibitors, antioxidant and inflammation-regulating ingredients, and ingredients that promote keratin renewal or metabolism. However, these products typically have limited depth of action and short-lasting effects. Some chemical skin-whitening ingredients may cause skin irritation or allergic reactions with long-term use. Furthermore, topical skin-whitening products cannot improve the body's pigment metabolism at the overall physiological level, and their whitening effect is easily affected by individual skin type and usage methods. Research has found that gut microbiota and their metabolites can significantly impact skin health and pigment metabolism by regulating immune responses, inflammation levels, and oxidative stress. Probiotics, as a relatively safe functional microorganism, show promising application prospects in regulating pigment metabolism and maintaining homeostasis.

[0004] Chinese patent CN119506161B discloses a strain of *Bifidobacterium adolescentis*, a composition for inhibiting the production and accumulation of lipofuscin, its application, and cosmetics. Through nematode lipofuscin testing, it demonstrates that the milk fermentation product obtained by fermenting milk using *Bifidobacterium adolescentis* BF23BA001, along with yeast fermentation products, has a good synergistic effect in reducing lipofuscin. However, this patent relies on the synergistic effect of multiple components of probiotic and yeast fermentation products to be effective; the independent whitening activity of a single strain is weak, increasing the complexity of product formulation and production costs. Furthermore, the aforementioned patent does not explicitly define the regulatory ability of *Bifidobacterium adolescentis* and its milk fermentation products on melanin production, failing to directly meet consumers' core demand for whitening effects that inhibit melanin synthesis. Summary of the Invention

[0005] In view of the technical problems that existing commercially available whitening products have limited depth of action, short duration of effect, and are prone to skin irritation or sensitivity reactions with long-term use, and that probiotic whitening products cannot meet the core requirement of inhibiting melanin synthesis, this invention provides a Bifidobacterium adolescentis HC2916 that inhibits melanin synthesis, its microbial preparation and application.

[0006] In a first aspect, the present invention provides a Bifidobacterium adolescentis HC2916 that inhibits melanin synthesis. Bifidobacterium adolescentis HC2916 was deposited on May 16, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27352, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] Furthermore, the 16S rDNA sequence of Bifidobacterium adolescentis HC2916 is shown in SEQ ID NO:3.

[0008] In a second aspect, the present invention provides a microbial preparation comprising the fermentation supernatant or lysis buffer of the aforementioned Bifidobacterium adolescentis HC2916.

[0009] Furthermore, the preparation method of fermentation supernatant includes: inoculating the activated HC2916 strain into MRS liquid medium, culturing at 37°C for 24-48 h, centrifuging at 6000 rpm for 10 min, and collecting the supernatant, which is the fermentation supernatant.

[0010] Furthermore, the preparation method of the lysate includes: inoculating the activated HC2916 strain into MRS liquid medium, culturing at 37°C for 24-48 h, collecting the fermentation broth along with the bacterial cells and ultrasonically disrupting for 20-30 min to obtain the lysate.

[0011] Thirdly, the present invention provides an application of the above-mentioned Bifidobacterium adolescentis HC2916 or microbial preparation in the preparation of products that inhibit melanin synthesis and have whitening effects.

[0012] Furthermore, the product is designed to inhibit the increase in melanin content caused by UVA.

[0013] Furthermore, the product is related to inhibiting the melanin production process. MITF , TYRP1 and TYRP2 Products for gene mRNA transcription and protein expression.

[0014] Furthermore, the product is a functional food or medicine, and the method of use is oral or topical.

[0015] The beneficial effects of this invention are as follows:

[0016] 1. This invention provides a Bifidobacterium adolescentis HC2916 and its microbial preparation, which possesses excellent antioxidant capabilities, can scavenge various free radicals, and inhibit lipid peroxidation, thereby enhancing the body's antioxidant level. Simultaneously, mechanistic studies have shown that the supernatant and lysate of this strain's bio-fermentation can significantly downregulate the protein and mRNA expression of MITF, a core factor in melanin production signaling, and inhibit the expression of its downstream key enzymes TYRP1 and TYRP2, effectively suppressing UVA-induced increases in melanin content in skin keratinocytes, demonstrating promising application prospects in the field of skin whitening.

[0017] 2. The optimal growth temperature of Bifidobacterium adolescentis HC2916 provided by this invention is 37℃. It does not produce hemolysin, is sensitive to commonly used antibiotics, can survive and colonize intestinal epithelial cells efficiently in a simulated gastrointestinal environment, and has significant cholesterol degradation ability. It also has no obvious toxicity to skin HaCaT cells and melanocyte B16F10 cells, and has good safety. It has high application value in the development of topical and oral beauty, whitening and anti-aging products. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a colony morphology diagram of Bifidobacterium adolescentis HC2916.

[0020] Figure 2 This is an optical microscope image of Bifidobacterium adolescentis HC2916.

[0021] Figure 3 The RAPD fingerprint of Bifidobacterium adolescentis HC2916.

[0022] Figure 4 The rep-PCR fingerprint of Bifidobacterium adolescentis HC2916.

[0023] Figure 5 The graph shows the results of the superoxide anion free radical scavenging rate test of Bifidobacterium adolescentis HC2916; different letters represent significant differences between the two groups (P < 0.05), and the presence of the same letter indicates no significant difference between the two groups.

[0024] Figure 6 Fe for Bifidobacterium adolescentis HC2916 2+ The results of the chelation capacity test are shown in the figure; different letters represent significant differences between the two groups (P < 0.05), while the same letter indicates no significant difference between the two groups.

[0025] Figure 7 The graph shows the total reducing power test results of Bifidobacterium adolescentis HC2916; different letters represent significant differences between the two groups (P < 0.05), and the presence of the same letter indicates no significant difference between the two groups.

[0026] Figure 8 The figure shows the results of the cytotoxicity experiment of Bifidobacterium adolescentis HC2916; where ** indicates P < 0.01.

[0027] Figure 9 The graph shows the results of the test on the ability of Bifidobacterium adolescentis HC2916 to inhibit melanin production; where ** indicates P < 0.01, *** indicates P < 0.001, and ### indicates P < 0.001.

[0028] Figure 10 The figure shows the results of the relative expression level test of melanin synthesis-related proteins; where (a) is the relative expression level of MITF protein; (b) is the relative expression level of TYRP1 protein; and (c) is the relative expression level of TYRP2 protein; * indicates P < 0.05, and ** indicates P < 0.01.

[0029] Figure 11 Figure 1 shows the results of mRNA transcription level tests for melanin synthesis-related proteins; where (a) is the relative expression level of MITF mRNA; (b) is the relative expression level of TYRP1 mRNA; and (c) is the relative expression level of TYRP2 mRNA; * indicates P < 0.05, and ** indicates P < 0.01. Detailed Implementation

[0030] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0031] Example 1: Isolation and Screening of Strains

[0032] 1. Preliminary screening of bacterial strains

[0033] Prepare MRS solid culture medium: 1L pure water, 10g peptone, 10g beef extract, 5g yeast extract, 5g sodium acetate, 5g glucose, 2g KH2PO4, 1mL Tween 80, 2g diamine citrate, 20g CaCO3, 0.58g MgSO4·7H2O, 0.25g MnSO4·7H2O, and 15g agar. Adjust the pH to 6.2-6.5.

[0034] Take 1 mL of breast milk and place it in a sampling bag containing 300 mL of sterile saline. Agitate the sample repeatedly for 5 minutes. The breast milk should be from a healthy lactating woman who has not consumed any probiotic products within the past six months and has signed an informed consent form. Spread 100 μL of the sample solution onto an MRS agar plate. After standing for 10 minutes, invert the plate and place it in an anaerobic bag. Incubate the anaerobic bag at 37°C for 48 hours. Once single colonies have grown on the plate, pick a single colony and streak it. After three purification cultures, pick a single colony for rapid identification using MALDI-TOF-MS.

[0035] Rapid identification of bacterial strains using MALDI-TOF-MS was performed according to the kit instructions, following these steps: Single clones of the bacterial strain were uniformly coated onto a target plate in the form of a thin film. 1 μL of lysis buffer was added to cover the sample, which was then dried. Another 1 μL of matrix solution was added to cover the sample, and the plate was dried again. The sample and target were then placed in the mass spectrometer for identification. The co-crystallized film formed by the sample and matrix was irradiated with a laser, causing the proteins in the sample to ionize. The ions were accelerated through the flight tube under an electric field of 10–20 kV. The molecular weight of the proteins was determined based on their flight time to the detector. Ribosomal protein fingerprints were obtained using Autofms 1000 Analyzer v1.0 software. Based on the MALDI-TOF-MS identification results, 50 strains of Lactobacillus were identified in this study.

[0036] 2. Secondary screening of strains

[0037] The Lactobacillus strains obtained from the initial screening were streaked onto MRS solid medium and anaerobically cultured at 37°C for 16 hours. Single colonies were then picked from the MRS solid medium and transferred to 5 mL of MRS liquid medium, and incubated overnight at 37°C. After incubation, the bacterial suspension was centrifuged at 4000×g for 10 min, the supernatant was discarded, and the precipitate was collected and eluted with 5 mL of PSP buffer (pH 6.8). This process was repeated twice, and the bacterial cells were resuspended in PSP buffer and the OD600 was adjusted to 1 to obtain a bacterial resuspension. The resuspension was incubated statically at 37°C for 6 hours, centrifuged at 4000×g for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter to obtain the fermentation supernatant. The resuspension was then sonicated at 200W for 3 seconds with 10-second intervals for 2 hours using a cell disruptor. The bacterial suspension was then centrifuged again, and the supernatant was filtered through a 0.22 μm filter to obtain the lysis buffer.

[0038] Prepare a 7.4 mM solution of 2,2'-nitro-bis(3-ethylbenzothiazole-6-sulfonic acid) (ABTS) and a 2.6 mM solution of potassium persulfate. Mix 1 mL of ABTS solution with 1 mL of potassium persulfate solution and react in the dark for 24 h to obtain the ABTS working solution. Dilute the ABTS working solution 5 times, and then take 100 μL of sample (fermentation supernatant or lysis buffer) or control (anhydrous ethanol) and mix it with 100 μL of the diluted ABTS solution. React at 37 °C in the dark for 30 min. Calculate the ABTS radical scavenging activity by measuring the absorbance at 734 nm. Perform three replicates for each strain and calculate using the following formula:

[0039]

[0040] In the formula, A1 is the absorbance of 100 μL ABTS diluent + 100 μL sample, A2 is the absorbance of 100 μL ABTS diluent + 100 μL anhydrous ethanol, and A3 is the absorbance of 100 μL anhydrous ethanol + 100 μL sample.

[0041] The results showed that strain H2916 had the highest ABTS radical scavenging rate, with 85.18% ± 4.32% in the fermentation supernatant and 75.29% ± 2.18% in the lysate. Therefore, strain H2916 was selected for further research.

[0042] Example 2 Identification of strain HC2916

[0043] 1. Colony morphology identification

[0044] The HC2916 strain was inoculated onto MRS solid medium and incubated at 37°C for 24 hours. Colony photos are shown below. Figure 1As shown, HC2916 single colonies are off-white, ranging in size from 1.0 to 2.5 mm, with neat edges and a glossy, raised surface. After staining with crystal violet, the image under an optical microscope is as follows. Figure 2 As shown, the bacteria are short rods at both ends, relatively short, and generally uniform in size, with occasional branching.

[0045] 2. Identification of physiological and biochemical characteristics

[0046] Preparation of HC2916 inoculum: The activated HC2916 strain was inoculated into MRS liquid medium at a 2% inoculum size and anaerobically cultured at 37°C for 24 hours until the viable cell count reached 1×10⁻⁶. 9 Centrifuge at CFU / mL for 5000 rpm for 5 min to collect the bacterial cells. Wash the bacterial cells once with PSP solution, then resuspend the bacterial cells with the same volume of PSP solution. Dilute with distilled water 50 times to obtain the inoculum for strain HC2916.

[0047] 2.1 Temperature growth range experiment

[0048] The HC2916 strain inoculum was inoculated into 10 mL of MRS liquid medium at a 10% inoculation rate. 10 mL of uninoculated MRS liquid medium was used as a control. The medium was anaerobically cultured in constant temperature shaking incubators at 0℃, 15℃, 30℃, 37℃, 45℃ and 60℃ for 48 h, respectively, and the culture medium was observed to see if it became turbid.

[0049] The results showed that the culture medium for strain HC2916 remained clear after 4 hours of incubation at 0℃ and 60℃, and became slightly turbid at 15℃ and 45℃. However, strain HC2916 produced a large number of cells after 48 hours of incubation at 30℃ and 37℃, with the highest cell yield and turbidity observed at 37℃. Therefore, the optimal growth temperature for strain HC2916 is 37℃.

[0050] 2.2 Salinity tolerance test

[0051] Under aseptic conditions, HC2916 strain inoculum was inoculated at a rate of 10% into 5 mL of MRS liquid medium with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%, respectively. 5 mL of uninoculated MRS liquid medium was used as a control. The medium was incubated at 37°C with shaking for 48 h, and the medium was observed to see if it became turbid.

[0052] The results showed that the HC2916 strain became turbid in the culture medium at salt concentrations of 1%–2%, remained clear in the culture medium at salt concentrations above 3%, and the HC2916 strain exhibited the highest turbidity in the 2% salt medium. Therefore, the maximum salt concentration that the HC2916 strain can tolerate is 2%.

[0053] 2.3 Glucose Acid and Gas Production Test

[0054] The acid-producing culture medium used in this embodiment has the following formulation: 0.5g peptone, 0.3g yeast extract, 0.1mL Tween 80, 0.5mL salt solution A, 0.5mL salt solution B, 0.5g sodium acetate, 2.5g glucose, 0.05mL 2% bromocresol green (w / v), and 100mL distilled water, with a pH of 6.8-7.0. Salt solution A consists of 10.0g KH₂PO₄ and 1.0g K₂HPO₄, dissolved in distilled water and brought to a final volume of 100mL. Salt solution B consists of 11.5g MgSO₄·7H₂O, 2.4g MnSO₄·4H₂O, and 0.68g FeSO₄·7H₂O, dissolved in distilled water and brought to a final volume of 100mL. The prepared acid-producing culture medium was dispensed into large test tubes containing inverted small test tubes, 3mL / tube, and autoclaved at 121℃ for 15min.

[0055] Under aseptic conditions, HC2916 strain inoculum was inoculated into acid-producing medium at a 2% inoculum volume, with uninoculated medium serving as a control. The top was then sealed with 2 mL of sterile liquid paraffin and incubated at 37°C for 48 hours. The color of the medium was then observed. The results showed that after 48 hours of incubation, the acid-producing medium inoculated with HC2916 strain changed from green to yellow, and no gas was produced in the small inverted tubes, indicating that HC2916 strain ferments glucose to produce acid without producing gas.

[0056] 2.4 Antibiotic resistance and hemolytic test

[0057] 2.4.1 Antibiotic tolerance test

[0058] The minimum inhibitory concentration (MIC) of antibiotics against HC2916 strain was determined using the microbroth dilution method. Antibiotic preparation: Ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, and tetracycline were all prepared as stock solutions of 2048 μg / mL and stored at -20℃ for later use. Before use, the stock solutions were serially diluted 2-fold with MRS liquid medium to prepare the working solutions, with concentrations ranging from 1 to 1024 μg / mL.

[0059] Add 190 μL of antibiotic-free MRS liquid medium to each of the eight wells in the first column of a 96-well plate as a negative control. Add 190 μL of MRS liquid medium with serially diluted antibiotic concentrations (1–1024 μg / mL) to each of the eleven wells in columns 2–12, and then inoculate each well with 10 μL of inoculum. Each experiment was repeated four times, with uninoculated wells serving as blank controls. Add 50 μL of autoclaved paraffin oil to each well to prevent moisture evaporation during culture. Incubate the 96-well plate at 37°C, and measure OD every 5 minutes.600 The MIC values ​​for different strains were calculated. Specific results are shown in Table 1.

[0060] Table 1. Antibiotic MIC values ​​of HC2916 strain

[0061]

[0062] Note: MIC is in μg / mL; R indicates resistance, S indicates sensitivity.

[0063] As can be seen from the results in Table 1, the HC2916 strain provided by this invention is sensitive to common antibiotics such as erythromycin, clindamycin and ampicillin, and has inherent resistance to gentamicin, streptomycin and tetracycline, and has good biosafety.

[0064] 2.4.2 Hemolytic test

[0065] Weigh out the following components of TBS basal medium: 1L distilled water, 17.0g tryptone, 5.0g NaCl, 3.0g soybean peptone, 2.5g KH2PO4, and 2.5g glucose. Dissolve the contents and autoclave at 121℃ for 15 minutes. After the medium has cooled to 50℃, add 5% sterile defibrinated sheep blood, mix well, and pour into plates. Streak the HC2916 strain onto the prepared blood cell plates and incubate at 37℃ for 48 hours. Observe whether the HC2916 strain exhibits hemolysis.

[0066] The results showed that strain HC2916 could not grow on hemolysin plates and the hemolysin plates remained unchanged, indicating that strain HC2916 does not produce hemolysin and cannot lyse hemolysin, thus exhibiting good biosafety.

[0067] 2.5 Carbon Source Metabolism Experiment

[0068] Prepare the basic culture medium: 1.5g peptone, 0.6g yeast extract, 0.1g Tween 80, 0.5mL salt solution, 18mg phenol red, and 100mL distilled water. Adjust the pH to 7.4±0.2.

[0069] The salt solution consists of: 11.5g MgSO4·7H2O, 8g MnSO4·4H2O, and 100mL distilled water.

[0070] Prepare a 10 g / mL solution of carbohydrates such as sugars, alcohols, and glycosides, and filter it through a 0.22 μm sterile filter.

[0071] Under aseptic conditions, 20 μL of sterilized carbohydrate solution was added to each well of a 96-well plate, with four replicates for each carbohydrate. Then, 170 μL of sterilized basal medium containing phenol red was added, followed by 10 μL of HC2916 strain inoculation solution. Wells without inoculation served as controls. 50 μL of liquid paraffin was added to each well to prevent moisture evaporation during incubation. The plates were incubated at 37°C for 2 days. Phenol red was used as an indicator to observe the color change of the medium; a yellow color indicated a positive result. The results are shown in Table 2 below. Referring to the "Physiological Characteristics Table - Bifidobacterium," the experimental results were compared with those in the table to determine the genus of the tested bacteria.

[0072] Table 2. Carbon source metabolism results of strain HC2916

[0073]

[0074] Note: + indicates positive, - indicates negative.

[0075] The results of the carbon source metabolism experiment showed that strain HC2916 conforms to the carbon source metabolism pattern of Bifidobacterium adolescentis.

[0076] 3. Molecular biological identification

[0077] Single colonies of strain HC2916 were picked from the plate and placed in MRS liquid medium. After incubation at 37°C for 24 hours, 800 μL of fermentation broth was taken and processed according to TIANGEN. ® The bacterial genomic DNA extraction kit (catalog number: DP302) was used to obtain the genome of this strain for subsequent molecular biological identification.

[0078] 3.1 Identification of 16S rDNA gene sequence

[0079] Using TIANGEN ® The 2×TaqPCR premix kit was used to amplify the 16S rDNA gene of strain HC2916. The reaction system and reaction cycle settings were performed according to the kit instructions.

[0080] The primer sequences are as follows:

[0081] 27F: AGAGTTTGATCCTGGCTCA (SEQ ID NO: 1);

[0082] 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO: 2).

[0083] Electrophoresis confirmed that the PCR amplification product size was approximately 1500 bp, which meets the requirements. The 16S rDNA sequencing results are as follows:

[0084]

[0085] The above sequence was BLAST-aligned on the EzBioCloud website, and it matched that of Bifidobacterium adolescentis (Bifidobacterium adolescentis). Bifidobacterium adolescentis The HC2916 strain showed the highest similarity to Bifidobacterium adolescentis. Therefore, the HC2916 strain was identified as Bifidobacterium adolescentis.

[0086] 3.2 RAPD fingerprint identification

[0087] The HC2916 strain was amplified using the TIANGEN® 2×TaqPCR premix kit. The reaction system and reaction cycle settings were performed according to the kit instructions. The primer sequence was M1: 5'-GAGGGTGGCGGTTCT-3' (SEQ ID NO:4).

[0088] A 1.5% agarose gel plate was prepared, with a DL2000 DNA Marker used as a result control. Electrophoresis was performed at a constant voltage of 100V for 80 minutes. Finally, the electrophoresis pattern was detected using a gel imaging system. The RAPD fingerprint of strain HC2916 is shown below. Figure 3 As shown. A comparison revealed no similarity in existing publicly available reports. Figure 3 The matching RAPD fingerprint pattern indicates that strain HC2916 is a novel Bifidobacterium adolescentis strain.

[0089] 3.3 Rep-PCR fingerprint identification

[0090] Using TIANGEN ® The 2×TaqPCR premix kit was used to amplify strain HC2916. The reaction system and reaction cycle settings were performed according to the kit instructions. The rep-PCR primer was 5'-GTGGTGGTGGTGGTG-3' (SEQ ID NO:5).

[0091] Prepare 1.5% agarose gel plates, using a DL2000 DNA Marker as a result control. Electrophoresis was performed at 100V for 80 minutes to detect the amplification results. The rep-PCR fingerprint of strain HC2916 is shown below. Figure 4 As shown. A comparison revealed no similarity in existing publicly available reports. Figure 4 The matching rep-PCR fingerprint pattern indicates that the HC2916 strain screened in this invention is a new Bifidobacterium adolescentis strain.

[0092] Based on the combined results of MALDI-TOF-MS identification, molecular biology experiments, carbon source metabolism experiments, and glucose acid and gas production experiments, strain HC2916 is identified as Bifidobacterium adolescentis. Bifidobacterium adolescentis The new strain was named Bifidobacterium adolescentis HC2916.

[0093] Bifidobacterium adolescentis HC2916 was deposited on May 16, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 27352, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as Bifidobacterium adolescentis. Bifidobacterium adolescentis .

[0094] Example 3: Gastrointestinal Fluid Tolerance Test

[0095] (1) Preparation of artificial simulated gastrointestinal fluid: Add 5g peptone, 2.5g yeast extract, 1g glucose and 2g NaCl to 1L of distilled water in sequence. After thorough mixing, adjust the pH of the system to 3.0 and sterilize at 115℃ for 20 minutes. After sterilization, wait for the solution to cool to a suitable temperature. Before the experiment, add 3.2g porcine mucosal pepsin powder and gently shake the container to mix the powder thoroughly to obtain artificial simulated gastric fluid. Add 5g peptone, 2.5g yeast extract, 1g glucose, 6.8g potassium dihydrogen phosphate and 3.0g bile salt to 1L of distilled water in sequence. Stir to dissolve and add 77mL NaOH solution (0.2N). Adjust the pH of the system to 6.8±0.1 and then sterilize at 115℃ for 20 minutes. Before the experiment, add 1.0g pancreatic enzyme powder to the cooled sterilized solution and gently shake to mix to obtain artificial simulated intestinal fluid.

[0096] (2) The absorbance OD of the activated Bifidobacterium adolescentis HC2916 bacterial solution 600 Adjust to 1.5. Centrifuge 1 mL of bacterial culture at 6000 × g for 10 min at 4℃, discard the supernatant, resuspend the bacterial culture in 1 mL of simulated gastric fluid, and incubate in an anaerobic environment at 37℃ for 3 h. Perform plate colony counting on the sample at the beginning (0 h) and end (3 h) of the incubation. Subsequently, centrifuge the bacterial culture in the simulated gastric fluid for 3 h again at 6000 × g for 10 min at 4℃, discard the supernatant, and resuspend the bacterial culture in an equal volume of simulated intestinal fluid. Continue incubation at 37℃ for 2 h, followed by plate colony counting. Use commercially available Bifidobacterium adolescentis BA-3 as the control strain. The survival rate of the strains was calculated according to the following formula, and the results are shown in Table 2.

[0097]

[0098] In the formula, N0 and N1 are the number of surviving colonies (CFU / mL) of the strain before and after treatment with simulated gastrointestinal fluid, respectively.

[0099] Table 3. Tolerance of HC2916 to simulated gastrointestinal fluid

[0100]

[0101] Table 3 shows that *Bifidobacterium adolescentis* HC2916 exhibits high tolerance to simulated gastric and intestinal fluids. After digestion with simulated gastric fluid, the viable bacterial survival rate was 97.89% ± 1.65%, while the survival rate of the control strain *Bifidobacterium adolescentis* BA-3 was 71.78% ± 6.12%. This indicates that *Bifidobacterium adolescentis* HC2916 can withstand the harsh acidic environment of the stomach and successfully enter the small intestine, while the control strain *Bifidobacterium adolescentis* BA-3 suffered a loss of over 29% after gastric acid digestion. After further digestion with simulated intestinal fluid, the viable bacterial survival rate of *Bifidobacterium adolescentis* HC2916 remained at 95.77% ± 2.12%, while the survival rate of the control strain *Bifidobacterium adolescentis* BA-3 was only 41.91% ± 2.52%, with a viable bacterial loss rate exceeding 59%. This demonstrates that *Bifidobacterium adolescentis* HC2916 can not only withstand the harsh acidic environment of the stomach but also the bile salt environment of the intestine, which is conducive to its survival and arrival in the gastrointestinal tract.

[0102] Example 4: Cell Surface Hydrophobicity Test

[0103] Activated Bifidobacterium adolescentis HC2916 colonies were picked and inoculated into freshly prepared MRS liquid medium. The culture was incubated at 37°C with shaking for 24 h. Then, 1% of the culture was added to the MRS liquid medium and cultured at 37°C with shaking for another 24 h. After centrifugation at 6000×g for 10 min, the bacterial cells were collected and washed twice with sterile physiological saline. The bacterial cells were then resuspended in 1 mL of sterile 0.1M KNO3 solution to obtain the bacterial suspension for testing.

[0104] Add 50 μL of the above bacterial suspension to 2450 μL of 0.1 M KNO3 solution and record the OD. 600 Let A0 be the concentration. Mix 1.5 mL of bacterial suspension with 500 μL of xylene and let stand at room temperature for 10 min. Vortex the two-phase system for 2 min and then let it stand for 20 min to reform the aqueous and organic phases. Pipette the aqueous phase and measure the absorbance A1 at 600 nm. Cell hydrophobicity is calculated using the following formula:

[0105]

[0106] Commercially available *Bifidobacterium adolescentis* BA-3 was used as a control strain. Both groups were measured three times in parallel, and the average value was taken. The results showed that the surface hydrophobicity of *Bifidobacterium adolescentis* HC2916 provided by this invention was 79.43% ± 11.88%, which was superior to the control strain *Bifidobacterium adolescentis* BA-3 (28.63% ± 7.35%). The hydrophobicity of lactic acid bacteria surfaces is positively correlated with their adhesiveness; higher hydrophobicity indicates stronger adhesive properties. *Bifidobacterium adolescentis* HC2916 has significant adhesion potential, which is beneficial for its adhesion to intestinal epithelial cells, thereby colonizing the intestinal environment and exerting its probiotic properties.

[0107] Example 5: Intestinal Epithelial Cell Adhesion Test

[0108] HT-29 cells were removed from the liquid nitrogen tank, revived, and passaged. Once the cell number reached the required level, and observation under an inverted microscope showed that cell confluence was approximately 80%, subsequent experiments could proceed. The original culture medium in the cell culture flask was discarded, and the cells were rinsed twice with PBS solution. An appropriate amount of trypsin was added to digest the cells. After adding trypsin, the cells were returned to the CO2 incubator. Once complete cell detachment was observed visually, 2-3 times the volume of trypsin culture medium was added to stop digestion. The cells were repeatedly pipetted approximately 10 times, and under a microscope, the cells were observed to be as single-celled as possible. The single-cell suspension was aspirated into 15mL centrifuge tubes, centrifuged at 1000rpm for 5 minutes, and the supernatant was discarded. The cell pellet was gently dispersed, and an appropriate amount of fresh culture medium was added for resuspending. Cell counting was performed using a hemocytometer. The cell suspension was diluted with PBS solution, and the number of cells in each well of a 6-well plate was 2 × 10⁶. 6 Add 2 mL of culture medium to each well. After placing the 6-well plate in a CO2 incubator for 24 hours, subsequent cell adhesion experiments can be performed.

[0109] Bifidobacterium adolescentis HC2916 and control strain Bifidobacterium adolescentis BA-3 were cultured to the stationary phase, washed twice with PBS, and resuspended in MRS liquid medium to a concentration of 5 × 10⁻⁶. 7 CFU / mL for later use (OD) 600 The value is around 0.4. The HT-29 monolayer that has adhered to the wells of a 6-well plate was washed twice with PBS, and 1 mL of antibiotic-free cell culture medium and 1 mL of the above-mentioned 5×10⁻⁶ cells were added. 7 CFU / mL bacterial suspension was incubated in a CO2 incubator for 2 hours. After incubation, HT-29 cells were washed three times with PBS to remove unattached bacteria. PBS buffer was slowly added along the cell wall to avoid upsetting the cell layer. 500 μL of trypsin was added for 3 min of digestion, followed by 1.5 mL of cell culture medium to terminate the digestion. The cells were repeatedly pipetted and the resulting solution was collected into sterile EP tubes. The collected solution was serially diluted 10-fold, 100-fold, 1000-fold, and 10000-fold, and the number of attached bacteria was counted using the plate count method.

[0110] The adhesion ability of the tested strains was calculated using the following formula:

[0111]

[0112] The results showed that the adhesion ability of Bifidobacterium adolescentis HC2916 to HT-29 cells was 64.12±5.35. The adhesion ability of Bifidobacterium adolescentis BA-3 was 20.34±4.36. This indicates that Bifidobacterium adolescentis HC2916 has excellent adhesion ability to intestinal epithelial cells HT-29, which is conducive to its colonization in the intestine and exerting its probiotic properties.

[0113] Example 6: In vitro cholesterol degradation test of Bifidobacterium adolescentis HC2916

[0114] Weigh 1g of cholesterol, dissolve it in anhydrous ethanol, and bring the volume to 100mL. Filter the solution under sterile conditions through a 0.22µm microporous membrane to obtain a cholesterol solution. Weigh 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium citrate, 20.0g of glucose, 1.0mL of Tween 80, 5.0g of CH3COONa, 0.1g of MgSO4, 0.05g of MnSO4, 2.0g of K2HPO4, and 1000mL of distilled water. After dissolving, adjust the pH to 7.3, sterilize at 115℃ for 30min, and after the culture medium cools, add the cholesterol solution to bring the final cholesterol concentration to 0.1%, obtaining a cholesterol-containing liquid culture medium.

[0115] The Bifidobacterium adolescentis HC2916 inoculum was inoculated at 0.1% ( v / v The bacterial culture was inoculated into a cholesterol-containing liquid culture medium at an inoculation rate of 0.2 mL and cultured statically at 37°C for 48 h. Then, 0.2 mL of the bacterial culture was added to 1.8 mL of anhydrous ethanol, mixed, and allowed to stand for 10 min. The mixture was then centrifuged at 3000 rpm for 5 min, and the supernatant was collected. The cholesterol content was determined according to the method specified in GB 5009.128-2016 "Determination of Cholesterol in Food," and the cholesterol degradation rate was calculated. *Bifidobacterium adolescentis* BA-3 was used as a control strain. Cholesterol is an essential nutrient for the human body, and excessive cholesterol in the blood can easily induce cardiovascular and cerebrovascular diseases. The results showed that the cholesterol degradation rate of *Bifidobacterium adolescentis* HC2916 was 73.57% ± 3.28%, while the cholesterol degradation rate of the control strain *Bifidobacterium adolescentis* BA-3 was only 10.29% ± 2.26%. This indicates that *Bifidobacterium adolescentis* HC2916 has a stronger ability to degrade cholesterol, helping to lower blood cholesterol levels and promote cardiovascular and cerebrovascular health.

[0116] Example 7: Determination of the antioxidant function of Bifidobacterium adolescentis HC2916

[0117] 1. Determination of DPPH and hydroxyl radical scavenging capacity

[0118] The strain culture and preparation of fermentation supernatant and lysis buffer in this embodiment are as follows: After activation for 3 generations, the HC2916 strain was cultured in MRS liquid medium at 37°C for 24 hours, then centrifuged at 6000 rpm for 10 minutes, and the supernatant was collected as the fermentation supernatant. After culturing the HC2916 strain in MRS liquid medium at 37°C for 24 hours, the fermentation broth, along with the bacterial cells, was collected and ultrasonically disrupted for 20 minutes to obtain the HC2916 strain lysis buffer. Commercially available *Bifidobacterium adolescentis* BA-3 was used as a control strain, and its fermentation supernatant and lysis buffer were prepared using the same methods as those for the HC2916 strain.

[0119] 1.1 Determination of DPPH scavenging ability

[0120] Take 1 mL of fermentation supernatant or lysis buffer of the test strain, add 1 mL of 0.4 mM freshly prepared DPPH radical solution, mix well, and incubate at room temperature in the dark for 30 min. Then measure the absorbance A of the fermentation supernatant and lysis buffer samples at a wavelength of 517 nm. 样品 The measurements were performed in triplicate. The control group samples were prepared with an equal volume of PBS solution and a DPPH-ethanol mixture, and a blank was zeroed using an equal volume of fermentation supernatant or lysis buffer sample mixed with ethanol. The DPPH scavenging rate was calculated using the following formula, and the results are shown in Table 4.

[0121]

[0122] Table 4 DPPH free radical scavenging rate of strain HC2916

[0123]

[0124] As shown in Table 4, the DPPH removal rate of fermentation supernatant and lysate of Bifidobacterium adolescentis HC2916 was significantly better than that of the control strain Bifidobacterium adolescentis BA-3.

[0125] 1.2 Determination of hydroxyl radical (HRS) scavenging ability

[0126] Mix 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate, 500 μL of deionized water, and 200 μL of Bifidobacterium adolescentis HC2916 fermentation supernatant or lysis buffer. Then add 100 μL of 3 mM hydrogen peroxide solution. Incubate at 37°C for 15 min. Measure the absorbance of sample A at 510 nm. Calculate the HRS scavenging rate using the following formula:

[0127]

[0128] Among them, A 控制 A represents the absorbance of the sample reaction, where deionized water is used instead. 空白The absorbance is calculated by replacing the sample with deionized water and reacting it with H2O2. Bifidobacterium adolescentis BA-3, a commercially available product, was used as a control strain. The results are shown in Table 5.

[0129] Table 5 HRS free radical scavenging rate of HC2916 strain

[0130]

[0131] As shown in Table 5, the fermentation supernatant and lysate of Bifidobacterium adolescentis HC2916 showed significantly better HRS clearance rates than the control strain BA-3.

[0132] 2. Determination of anti-lipid peroxidation capacity

[0133] Preparation of linoleic acid emulsion: Mix 0.1 mL linoleic acid, 0.2 mL Tween 20, and 19.7 mL deionized water thoroughly. Add 1 mL of the linoleic acid emulsion and 1 mL of 1% FeSO4 solution to 0.5 mL of PBS solution, then add 0.5 mL of the fermentation supernatant or lysis buffer of the test strain. Incubate at 37°C for 1.5 h. Add 0.2 mL of TCA solution (4 wt%) and 2 mL of LTBA solution (8 wt%) to the mixture, incubate at 100°C for 30 min, cool rapidly, centrifuge at 4000 rpm for 15 min, collect the supernatant, and measure the absorbance A at 532 nm. Replace the sample reaction with 0.5 mL of distilled water and measure the absorbance A0 at 532 nm. Calculate the inhibition rate using the following formula:

[0134]

[0135] The commercially available Bifidobacterium adolescentis BA-3 strain was used as a control strain, and the results are shown in Table 6.

[0136] Table 6. Lipid peroxidation resistance rate of HC2916 strain

[0137]

[0138] As shown in Table 6, the fermentation supernatant and lysate of Bifidobacterium adolescentis HC2916 provided by the present invention have strong anti-lipid peroxidation ability, and the anti-lipid peroxidation effect is better than that of the control strain BA-3.

[0139] Based on the combined performance of Bifidobacterium adolescentis HC2916 in DPPH scavenging rate, HRS scavenging rate and anti-lipid peroxidation, it can be concluded that the Bifidobacterium adolescentis HC2916 provided by the present invention has excellent antioxidant capacity.

[0140] 3. Determination of superoxide anion free radical scavenging capacity

[0141] Add 2.8 mL of Tris-HCl buffer (0.05 mol / L, pH 8.2), 0.1 mL of pyrogallol (0.05 mol / L), and 0.1 mL of the sample to a test tube sequentially. After mixing, react at 25°C in the dark for 4 min. Stop the reaction by adding 1 mL of HCl solution (8 mol / L), and measure the OD value at 320 nm to obtain A1. Use a test tube without the sample as a blank control and measure the absorbance A0. Calculate the superoxide anion radical scavenging rate of the sample using the following formula:

[0142]

[0143] The results are as follows Figure 5 As shown, the supernatant and lysate of *Bifidobacterium adolescentis* HC2916 exhibited superoxide anion radical scavenging rates of 17.76%±1.88% and 15.65%±2.04%, respectively, while the control strain *Bifidobacterium adolescentis* BA-3 showed superoxide anion radical scavenging rates of 6.45%±0.72% and 3.89%±1.59%, respectively. This indicates that *Bifidobacterium adolescentis* HC2916 demonstrates superior superoxide anion radical scavenging ability compared to the control strain.

[0144] 4. Fe 2+ Chelation capacity determination

[0145] Add 0.1 mL of ascorbic acid (1%), 0.1 mL of FeSO4 (0.4%), and 1 mL of NaOH (0.2 mol / L) sequentially to a test tube, mix well, and then add 0.5 mL of the sample to be tested. Place the mixture in a 37℃ water bath for 20 min, then add 0.2 mL of TCA (10%) and react at room temperature for 20 min to precipitate the protein. After the reaction is complete, centrifuge the mixture at 3000×g and 4℃ for 10 min. Take 0.2 mL of the supernatant and mix it with 2 mL of o-phenanthroline (0.1%). After standing for 10 min, measure the OD value at 510 nm, which is the absorbance A1. Use PBS as a blank control to measure the absorbance A0. The sample's effect on Fe... 2+ Chelating capacity is calculated using the following formula:

[0146]

[0147] The results are as follows Figure 6 As shown, the fermentation supernatant and lysis buffer of Bifidobacterium adolescentis HC2916 are Fe 2+ The chelation abilities were 84.21%±11.87% and 87.28%±6.55%, respectively, compared with the fermentation supernatant and lysate of the control strain Bifidobacterium adolescentis BA-3. Fe2+The chelating capacities were 71.09%±9.80% and 65.30%±8.57%, respectively. This indicates that *Bifidobacterium adolescentis* HC2916 has a strong chelating capacity for Fe... 2+ The chelating ability was superior to that of the control strain.

[0148] 4. Total reducing power determination

[0149] Take 0.5 mL of the sample to be tested, add 0.5 mL of potassium ferricyanide (1 wt%) and 0.5 mL of PBS, mix well, incubate at 50 °C for 20 min, then rapidly cool. Add 0.5 mL of 10 g / 100 mL trichloroacetic acid, centrifuge at 7000 × g for 10 min, and collect the supernatant. Take 1 mL of the supernatant, add 1 mL of 0.1 g / 100 mL ferric chloride and 1 mL of water, mix again, and let stand for 10 min. Measure the absorbance A1 at 700 nm. Measure the absorbance A0 when an equal volume of water is used instead of the sample to be tested. Calculate using the following formula:

[0150]

[0151] like Figure 7 As shown, the total reducing power of the fermentation supernatant and lysis buffer of *Bifidobacterium adolescentis* HC2916 was 94.66%±2.32% and 92.11%±1.92%, respectively, while that of the control strain *Bifidobacterium adolescentis* BA-3 was 90.10%±4.89% and 87.35%±2.30%, respectively. The total reducing power of *Bifidobacterium adolescentis* HC2916 was comparable to that of the control strain.

[0152] The comprehensive analysis of Bifidobacterium adolescentis HC2916 showed improvements in DPPH clearance rate, HRS clearance rate, anti-lipid peroxidation, superoxide anion radical scavenging ability, and Fe... 2+ The performance of this strain in multiple antioxidant indicators, such as chelation capacity and total reducing power, demonstrates its excellent antioxidant capacity through multiple pathways and synergistic effects. It can directly scavenge various reactive oxygen species, inhibit lipid peroxidation, and reduce metal ion-mediated free radical generation, thereby comprehensively enhancing the body's antioxidant defense level. Excessive accumulation of free radicals in the body can trigger oxidative stress, damaging nucleic acids, proteins, and lipids, thereby interfering with normal skin cell function, promoting abnormal melanin production and deposition, and causing dullness, age spots, and accelerated aging. Bifidobacterium adolescentis HC2916 helps maintain skin cell homeostasis and melanin metabolism balance by reducing free radical levels and alleviating oxidative stress, inhibiting oxidative stress-induced pigmentation abnormalities at the source, thus maintaining even and bright skin tone and exerting a comprehensive anti-aging and whitening effect.

[0153] Example 8: Cytotoxicity test of Bifidobacterium adolescentis HC2916 on skin cells

[0154] The preparation methods of the fermentation supernatant and lysis buffer of Bifidobacterium adolescentis HC2916 used in this embodiment are the same as those in Example 7.

[0155] The MTT assay was used to detect the cytotoxic effects of *Bifidobacterium adolescentis* HC2916 on skin epithelial cells. Immortalized human keratinocytes (HaCaT cells) or mouse skin melanoma cells (B16F10 cells) were resuscitated from liquid nitrogen and cultured to the required quantity. When the cell density reached approximately 80%, the cells were digested with trypsin into a single-cell suspension, counted using a hemocytometer, and seeded into 96-well plates at a seeding density of 2 × 10⁶ cells / well. 5 Cells / well, with 150 μL of culture medium added to each well. Subsequent experiments were conducted after 24 hours of cell culture.

[0156] Filter 50 mL of fetal bovine serum and 5 mL of penicillin-streptomycin mixture separately into 500 mL of 1640 medium using a 0.22 µm PES disposable syringe filter to prepare 1640 complete medium. Weigh 0.2 mg of 5-fluorouracil and dissolve it in 1 mL of 1640 complete medium to prepare a medium containing 0.2 mg / mL 5-fluorouracil as a positive control. Dilute the fermentation supernatant or lysis buffer of strain HC2916 to a concentration of 5% (v / v) using 1640 complete medium.

[0157] After cell attachment, the culture medium was aspirated, and 150 μL of culture medium containing either 5-fluorouracil or fermentation supernatant or lysis buffer of strain HC2916 was added to each well. Blank control wells and zeroing wells were also included. Cells were cultured for another 24 hours, with five replicates per group. After culture, the supernatant was carefully aspirated, and 90 μL of fresh culture medium (serum-free, antibiotic-free) was added, followed by 10 μL of MTT solution (Solepro MTT cytotoxicity assay kit). Cells were cultured for another 4 hours. The supernatant was then aspirated, and 110 μL of Formazan lysis buffer was added to each well. The cells were shaken on a shaker at low speed for 10 min to fully dissolve any crystals. The absorbance of each well was measured at 490 nm using an ELISA reader. The cytotoxicity inhibition rate was calculated using the following formula:

[0158]

[0159] Where: As, absorbance of the experimental well; Ac, absorbance of the blank control; Ab, absorbance of the zeroing well.

[0160] like Figure 8As shown, the fermentation supernatant and lysis buffer of *Bifidobacterium adolescentis* HC2916 inhibited HaCaT cells by 6.01%±3.68% and 2.83%±0.76%, respectively, while 5-fluorouracil inhibited HaCaT cells by 97.09%±2.02%. Similarly, the fermentation supernatant and lysis buffer of *Bifidobacterium adolescentis* HC2916 inhibited B16F10 cells by 4.81%±1.48% and 3.09%±1.04%, respectively, while 5-fluorouracil inhibited B16F10 cells by 96.85%±2.63%. These results indicate that *Bifidobacterium adolescentis* HC2916 inhibited the activity of both skin HaCaT cells and B16F10 cells by less than 6.00%, demonstrating no cytotoxicity and good biocompatibility.

[0161] Example 9: Effect of Bifidobacterium adolescentis HC2916 on intracellular melanin content

[0162] The preparation methods of the HC2916 fermentation supernatant and lysis buffer used in this embodiment are the same as in Example 7.

[0163] Arbutin was prepared into a cell culture medium containing 500 μM arbutin using DMEM high-glucose medium as a positive control. The fermentation supernatant and lysis buffer of HC2916 were diluted to a concentration of 5% (v / v) using DMEM high-glucose medium.

[0164] HaCaT cells were resuscitated from liquid nitrogen and cultured to the required quantity. When the cell density reached approximately 80%, they were digested with trypsin into a single-cell suspension, counted using a hemocytometer, and seeded into 96-well plates at a density of 2 × 10⁶ cells / well. 5 Cells / well, with 150 μL of culture medium added to each well. After 24 h of cell culture, subsequent experiments were performed. HaCaT cells in good growth condition and in the logarithmic growth phase were used for the next experiment. After trypsin digestion, the cells were seeded into 96-well plates. After observing that the adherent cells grew to about 80%, they were randomly divided into: blank group, UVA group, UVA + arbutin (arbutin group), UVA + HC2916 fermentation supernatant (supernatant group), and UVA + HC2916 lysis buffer (lysis buffer group), with 5 replicates in each group.

[0165] On day 1, HaCaT cells were placed in DMEM high-glucose medium containing different components and cultured in a 37°C, 5% CO2 incubator. Cells that reached the logarithmic growth phase were harvested, digested with trypsin, and then cultured in 3cm culture dishes. On day 2, cell density was observed to reach 60%–70%. Cells were then irradiated with UVA for three consecutive days. Except for the control group, cells in other groups received UVA irradiation at a dose of 15 J / cm². 2 The irradiation time (s) is determined based on the UVA irradiation dose and UV irradiation intensity (W / cm²).2 The ratio was calculated. Before each illumination, the UV lamp was turned on and stabilized for 0.5 hours. Once the UV irradiator display stabilized, illumination began. The culture medium for cell groups requiring UVA irradiation was aspirated, and 1 mL of PBS solution was added. The cells were then irradiated according to the designed dosage and method. Cell groups not requiring illumination were covered with aluminum foil to protect them from light. After illumination, the cells were replaced with the appropriate culture medium and cultured in the incubator for another 24 hours.

[0166] On the third day after UVA irradiation, the cell culture medium for each group was aspirated, and the cells were washed once with PBS solution. The PBS was then removed, and 1 mL of 0.4% trypsin was added to the cells for digestion for 2 min. The cells were then centrifuged at 3000 rpm for 2 min, and the supernatant was discarded. 1 mL of PBS buffer was added to the pellet, and the cells were washed once. Most of the PBS buffer was removed, leaving a small amount of supernatant. Finally, the cell pellet was broken up. 100 μL of 1M NaOH solution containing 10% DMSO was added to the UVA-irradiated HaCaT cells, and the mixture was thoroughly mixed by pipetting. The cells were then incubated in an 80°C metal bath for 2 h. After incubation, the mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. The relative melanin content in the samples was determined using the BCA method. The absorbance A1 of the treatment group was measured at 562 nm and calculated using the following formula:

[0167]

[0168] The results are as follows Figure 9 As shown, compared with the control group, the relative melanin content of cells in the UVA group increased significantly after UVA irradiation, with a 94.20% increase (P<0.001). Compared with the UVA group, the relative melanin content in the arbutin group decreased by 5.77% (P<0.01). Compared with the UVA group, the relative melanin content in the supernatant group decreased by 24.41% (P<0.001). Compared with the UVA group, the relative melanin content in the lysate group decreased by 28.01% (P<0.001). The relative melanin content decreased significantly between the lysate group and the supernatant group (P<0.01).

[0169] HaCat cells showed a significant increase in melanin content after UVA irradiation. Arbutin can inhibit melanin production, thereby reducing skin pigmentation and removing age spots and freckles. When used as a positive control, it significantly reduced the increase in melanin content induced by UVA. Compared to arbutin, both the fermentation supernatant and lysate of *Bifidobacterium adolescentis* HC2916 reduced the increase in melanin content induced by UVA, with better results than arbutin. However, there was no difference in the reduction of melanin content between the fermentation supernatant and lysate of *Bifidobacterium adolescentis* HC2916; their effects were comparable.

[0170] Example 10: Effects of Bifidobacterium adolescentis HC2916 on the transcription and expression levels of melanin production-related genes.

[0171] The preparation of fermentation supernatant and lysis buffer of Bifidobacterium adolescentis HC2916 was as described in Example 7, and the cell culture method and grouping were as described in Example 9, except that the arbutin group was not included in this example. The experimental protocol was as described in Example 9, except that in this example, total RNA and protein were extracted from HaCat cells on day 3 after all UVA irradiation was completed, and the mRNA transcription levels and protein expression levels of MITF, TYRP1, and TYRP2 were detected.

[0172] 1. Detect protein expression levels

[0173] Take 4 mL of RIPA lysis buffer (Beyotime). ® Add 40 μL of PMSF solution (Solepro) ® Mix well and place on ice. Add 250 μL of lysis buffer to each well of cells and lyse on ice. After 30 min, centrifuge at 14000 × g for 5 min at 4 °C. Determine the protein concentration from the supernatant and store at -80 °C for later use. Perform 10% SDS-PAGE electrophoresis on the extracted proteins and transfer them to a PVDF membrane. Block the PVDF membrane with TBST blocking buffer containing 5% skim milk at room temperature. Incubate with mouse anti-MITF, rabbit anti-TYRP1, rabbit anti-TYRP2, and mouse anti-β-actin monoclonal antibodies, respectively. Wash the membrane with TBST blocking buffer and react with secondary antibodies. Immunoassay bands were analyzed using Amersham ECL Plus. TM Chemiluminescence staining was performed using the Western Blotting Detection System kit, and the signal was exposed onto X-ray film. The grayscale values ​​of the protein bands were read using QuantityOne 4.3.0 software, and the grayscale values ​​were analyzed using QuantityOne 4.3.0 software. The ratio of the grayscale value of the target protein to that of the internal control protein β-actin was used as the relative expression level of the target protein. The results of the relative protein expression levels for each group are shown below. Figure 10As shown, the specific analysis is as follows:

[0174] like Figure 10 As shown in (a), compared with the control group, the relative expression level of MITF protein in the UVA group increased by 1.46-fold, with a highly significant difference (P<0.01). Compared with the UVA group, the relative expression levels of MITF protein in the supernatant group and the lysis buffer group decreased by 27.44% and 49.15%, respectively, with highly significant differences (2.46±0.20 vs 1.79±0.09, P<0.01; 2.46±0.20 vs 1.25±0.10, P<0.01). The relative expression level of MITF protein in the lysis buffer group decreased compared with the supernatant group, and the difference was significant (P<0.05).

[0175] like Figure 10 As shown in (b), compared with the control group, the relative expression level of TYRP1 protein in the UVA group increased by 1.56-fold, with a highly significant difference (P<0.01). Compared with the UVA group, the relative expression levels of TYRP1 protein in the supernatant group and the lysis buffer group decreased by 32.14% and 52.43%, respectively, with highly significant differences (2.56±0.10 vs 1.73±0.17, P<0.01; 2.56±0.10 vs 1.22±0.06, P<0.01). The relative expression level of TYRP1 protein in the lysis buffer group decreased compared with the supernatant group, and the difference was significant (P<0.05).

[0176] like Figure 10 As shown in (c), compared with the control group, the relative expression level of TYRP2 protein in the UVA group increased by 1.11-fold, which was statistically significant (P<0.01). Compared with the UVA group, the relative expression levels of TYRP2 protein in the supernatant group and the lysis buffer group decreased by 21.83% and 50.83%, respectively, which were also statistically significant (2.12±0.23 vs 1.65±0.16, P<0.05; 2.12±0.23 vs 1.04±0.25, P<0.01). The relative expression level of TYRP2 protein in the lysis buffer group was significantly lower than that in the supernatant group (P<0.05).

[0177] 2. Detecting mRNA transcription levels

[0178] TRNzol Universal Total RNA Extraction Kit (Tiangen) was used ® Total RNA was extracted using DP424, and then the results were analyzed using the FastKing one-step RT-PCR kit (Tiangen). ®The target genes MITF, TYRP1, TYRP2, and the internal reference gene β-actin were reverse transcribed and PCR was performed using the KR123 kit. The reaction system and PCR procedure were performed according to the kit instructions, and the primers are shown in Table 10.

[0179] Table 10 Primer sequences for target and internal reference genes in quantitative PCR

[0180]

[0181] The relative expression level of the target gene was calculated using the ΔΔCt method, as follows:

[0182] First, using β-actin as an internal reference gene, the Ct values ​​of the target gene in the samples were standardized, and ΔCt_experimental group (ΔCt_experimental group = Ct_target gene) was calculated. Ct (internal reference gene) and ΔCt (blank group) were used to obtain ΔΔCt (ΔΔCt = ΔCt_experimental group). ΔCt_blank group), and finally expressed using the formula "relative expression level = 2^ "ΔΔCt" is used to calculate the fold change in the expression of the target gene in the experimental group relative to the control group. The results are as follows: Figure 11 As shown.

[0183] like Figure 11 As shown in (a), compared with the blank group, the UVA group MITF The relative expression level of mRNA increased by 2.46-fold, a highly significant difference (P<0.01). Compared with the UVA group, the supernatant group and the lysis buffer group showed significantly higher mRNA expression levels. MITF The relative expression levels of mRNA decreased by 22.40% and 55.88%, respectively, with highly significant differences (3.46±0.20 vs 2.69±0.13, P<0.01; 3.46±0.20 vs 1.53±0.21, P<0.01). Lysis buffer group MITF The relative expression level of mRNA decreased compared with that of the supernatant group, and the difference was highly significant (P<0.01).

[0184] like Figure 11 As shown in (b) above, compared to the blank group, the UVA group... TYRP1 The relative expression level of mRNA increased by 1.22-fold, which was statistically significant (P<0.01). Compared with the UVA group, the supernatant group and the lysis buffer group showed significantly higher mRNA expression levels. TYRP1The relative expression levels of mRNA decreased by 23.75% and 44.24%, respectively, and the differences were statistically significant (2.23±0.35 vs 1.70±0.09, P<0.05; 2.23±0.35 vs 1.24±0.07, P<0.01). Lysis buffer group TYRP1 The relative expression level of mRNA decreased significantly compared with the supernatant group (P<0.05).

[0185] like Figure 11 As shown in (c), compared with the blank group, the UVA group TYRP2 The relative expression level of mRNA increased by 1.38-fold, a highly significant difference (P<0.01). Compared with the UVA group, the supernatant group and the lysis buffer group showed significantly higher mRNA expression levels. TYRP2 The relative expression levels of mRNA decreased by 31.16% and 53.69%, respectively, with highly significant differences (2.38±0.11 vs 1.64±0.18, P<0.01; 2.38±0.11 vs 1.10±0.20, P<0.01). Lysis buffer group TYRP2 The relative expression level of mRNA was significantly different from that of the supernatant group (P<0.05).

[0186] The results showed that *Bifidobacterium adolescentis* HC2916 significantly downregulated the expression level of MITF, a central regulatory factor in the signal transduction stage of melanin production, and downregulated the expression levels of TYRP1 and TYRP2, two key protein factors in the melanin production stage of the downstream signaling pathway of MITF. This indicates that *Bifidobacterium adolescentis* HC2916 can intervene in the melanin biosynthesis pathway at the transcriptional regulatory level by inhibiting... MITF The expression of this core transcription factor, in turn, synergistically reduces the expression of its downstream melanin synthesis-related genes. TYRP1 and TYRP2 The expression level of melanin can be reduced by blocking the transmission of melanin production signals from upstream to downstream, thereby effectively reducing the synthesis and accumulation of melanin.

[0187] Compared to the fermentation supernatant, the lysate of *Bifidobacterium adolescentis* HC2916 showed a more significant effect in downregulating the expression of the aforementioned key factors, suggesting that the main active components of HC2916 may exist in intracellular components or in forms related to cell structure, which is more conducive to exerting a regulatory effect on the melanin production pathway. This result further demonstrates that *Bifidobacterium adolescentis* HC2916 has the potential to inhibit melanin production at the source of signal regulation, providing experimental evidence at the molecular level for its application in skin whitening and skin health products.

[0188] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A Bifidobacterium adolescentis HC2916 that inhibits melanin synthesis, characterized in that, Bifidobacterium adolescentis ( Bifidobacterium adolescentis HC2916 was deposited on May 16, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27352, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. A microbial preparation, characterized in that, The lysate includes the fermentation supernatant or lysis buffer of Bifidobacterium adolescentis HC2916 as described in claim 1. The preparation method of the lysate includes: inoculating the activated HC2916 strain into MRS liquid medium, culturing at 37°C for 24-48 h, collecting the fermentation broth along with the bacterial cells and sonicating for 20-30 min to obtain the lysate.

3. The microbial preparation according to claim 2, characterized in that, The preparation method of fermentation supernatant includes: inoculating the activated HC2916 strain into MRS liquid medium, culturing at 37℃ for 24~48h, centrifuging at 6000rpm for 10min, and collecting the supernatant, which is the fermentation supernatant.

4. The use of the Bifidobacterium adolescentis HC2916 as described in claim 1 or the microbial preparation as described in any one of claims 2-3 in the preparation of a pharmaceutical product that inhibits melanin synthesis and has whitening effects.

5. The application as described in claim 4, characterized in that, The drug is designed to inhibit the increase in melanin content caused by UVA.

6. The application as described in claim 5, characterized in that, The drug is related to the process of inhibiting melanin production. MITF , TYRP1 and TYRP2 Drugs that involve gene mRNA transcription and protein expression.

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