Lactobacillus delbrueckii subspecies lactis absn20244504 and postbiotic in the preparation of products for inhibiting skin photodamage

Abstract

This invention discloses the application of *Lactobacillus delbrueckii* subsp. *lactozoa* ABSN20244504 ​​and its postbiotic in the preparation of products inhibiting photodamage to the skin, relating to the field of microbial technology. This strain, with accession number CGMCC No. 34743, was isolated from traditionally fermented yak yogurt from the Aba Plateau in Sichuan, China, and exhibits good in vitro tolerance to artificial gastric acid and bile salts. This invention also discloses the application of this strain and its inactivated postbiotic in the preparation of products inhibiting photodamage to the skin. Animal experiments show that both oral administration of the live strain and topical application of its inactivated postbiotic significantly reduce UVB-induced skin damage in mice. Its mechanism of action is related to reducing oxidative stress, inhibiting inflammatory responses, and regulating AMPK and other related signaling pathways. This invention provides new bacterial resources and application strategies for developing novel oral probiotic preparations and topical cosmetics for the prevention and repair of photodamage to the skin.

Description

Application of Lactobacillus delbrueckii subsp. lactis ABSN20244504 ​​and its postbiotics in the preparation of products that inhibit photodamage to the skin Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to the application of Lactobacillus delbrueckii subsp. ABSN20244504 ​​and its postbiotics in the preparation of products that inhibit photodamage to the skin. Background Technology

[0002] Ultraviolet B (UVB) is a type of medium-wave ultraviolet radiation found in nature. Although it accounts for only about 5% of the total ultraviolet radiation reaching the Earth's surface, its biological effects are strong, with a particularly significant impact on skin health. Due to its high energy, UVB can directly damage epidermal keratinocytes upon contact with the skin, and even short-term exposure can trigger acute sunburn reactions, manifesting as erythema, edema, and pain. Simultaneously, UVB can stimulate melanocyte activity, promoting melanin synthesis and deposition, leading to skin pigmentation or uneven skin tone. More seriously, long-term or repeated UVB exposure can damage structural proteins in the epidermis and dermis, especially collagen and elastin fibers, resulting in photoaging phenomena such as skin laxity and wrinkles. Furthermore, UVB can induce DNA strand breaks or mutations in skin cells, significantly increasing the risk of skin cancer. Studies have confirmed that almost all major types of skin cancer are closely related to UVB exposure.

[0003] Currently, protective strategies against photodamage to the skin mainly include topical sunscreens, antioxidants, and skincare products with repair functions, as well as dietary supplementation with photoprotective nutrients. For example, vitamin C, as a powerful antioxidant, can be taken orally to indirectly protect the skin through the systemic antioxidant defense system, or applied directly topically to neutralize free radicals in the skin, and is therefore widely used in cosmetics and functional skincare products. Similarly, foods rich in ingredients such as niacinamide (e.g., milk, eggs) have been shown in studies to enhance the skin barrier and reduce photodamage through oral or topical application. However, the protective effect of topical applications is often limited by product dwell time, penetration efficiency, and user compliance, while relying solely on oral nutrients has problems such as large individual differences in bioavailability and slow action.

[0004] In recent years, with the development of microbiome research, the close relationship between gut microbiota and skin health—the "gut-skin axis"—has received increasing attention. Studies show that the balance of gut microbiota directly affects the body's systemic inflammation level and immune regulation function, which in turn projects onto the health of the skin. Probiotics, as a class of live microorganisms beneficial to the host's health, can indirectly affect skin health by regulating the immune system and inhibiting excessive inflammatory responses. For example, UVB irradiation can induce the local release of large amounts of pro-inflammatory cytokines in the skin, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), and some probiotics can regulate the expression of these cytokines, thereby reducing skin inflammation. In addition, UVB induces oxidative stress, producing excessive free radicals, leading to lipid peroxidation, protein denaturation, and DNA damage. Some probiotics and their metabolites have been shown to have the ability to scavenge free radicals and enhance the body's antioxidant defense, thus alleviating photodamage from another pathway.

[0005] In the exploration of probiotic resources, unique ecological environments often become an important source for screening superior strains. For example, the Aba Tibetan and Qiang Autonomous Prefecture in Sichuan, China, is located on a plateau. Extreme environmental conditions such as strong ultraviolet radiation, low oxygen, and low temperature exert strong natural selection pressure on local microorganisms, potentially prompting them to evolve unique antioxidant and stress-resistance mechanisms. Yak yogurt, made by Tibetan herders in this region using traditional natural fermentation techniques, is not only a specialty food but also a treasure trove of rich microbial resources. Existing studies have isolated various lactic acid bacteria from this type of yak yogurt and verified their potential efficacy in improving constipation, alleviating colitis, and protecting the liver, showing great development potential. However, the specific efficacy of probiotics isolated from this type of traditionally fermented food, especially *Lactobacillus delbrueckii* subsp. *lactis*, in inhibiting skin photodamage, effective usage methods (such as oral administration of live bacteria and topical inactivated preparations), and the underlying molecular mechanisms have not yet been systematically studied and fully elucidated, limiting their in-depth application in functional foods and skin health.

[0006] Therefore, there is an urgent need in this field to screen and identify new probiotic strains from naturally fermented foods from special environmental sources that have excellent gastrointestinal fluid tolerance and can effectively alleviate UVB-induced skin photodamage through multiple pathways, and to clarify the effects and mechanisms of their oral and topical applications, so as to provide strain resources and theoretical basis for the development of new, efficient and safe skin health products. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention proposes the application of *Lactobacillus delbrueckii* subsp. *lactoderm* ABSN20244504 ​​and its post-biotic in the preparation of products that inhibit photodamage to the skin. This invention utilizes a mouse model to observe the intervention effects of oral administration of live *Lactobacillus delbrueckii* subsp. *lactoderm* ABSN20244504 ​​and topical application of inactivated *Lactobacillus delbrueckii* subsp. *lactoderm* ABSN20244504 ​​(post-biotic) on UVB-induced oxidative stress and inflammatory responses in mouse skin. This provides a theoretical basis for the skin-protective effects of probiotics, thus proposing a new application approach for *Lactobacillus delbrueckii* subsp. *lactoderm* ABSN20244504 ​​and offering a new intervention strategy for UVB-induced skin damage.

[0008] To achieve the above objectives, the present invention provides a *Lactobacillus delbrueckii* subsp. *lactis* ABSÀN20244504, with accession number CGMCC No.34743.

[0009] The present invention also provides a metabiotic composition comprising at least one of the inactivated cells or lysates of Lactobacillus delbrueckii subsp. lactis ABSN20244504 ​​described above.

[0010] The present invention also provides a microbial preparation comprising at least one of the above-mentioned Lactobacillus delbrueckii subsp. lactis ABSN20244504, its inactivated cells or lysates, and a pharmaceutically, food-, or cosmetically acceptable carrier or excipient.

[0011] The present invention also provides the use of the above-mentioned Lactobacillus delbrueckii subsp. lactis ABSN20244504, the above-mentioned metabiotic composition, or the above-mentioned microbial preparation in the preparation of products that inhibit photodamage to the skin.

[0012] Furthermore, the product is administered orally, and the product contains live bacteria of *Lactobacillus delbrueckii* subsp. *abdomenicoides* ABSN20244504.

[0013] Furthermore, the product is applied topically to the skin, and the product contains inactivated cells and / or lysates of the aforementioned Lactobacillus delbrueckii subsp. lactis ABSN20244504.

[0014] The present invention also provides an oral product for inhibiting photodamage to the skin, comprising an effective amount of live bacteria of *Lactobacillus delbrueckii* subsp. *abscinae* ABSN20244504, and a pharmaceutically acceptable carrier or excipient.

[0015] The present invention also provides a topical product for inhibiting photodamage to the skin, comprising an effective amount of at least one of the above-mentioned inactivated cells or lysates of Lactobacillus delbrueckii subsp. lactis ABSN20244504, and a carrier or excipient acceptable for cosmetics.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects:

[0017] To investigate the skin-protective effects of the novel bacterial strain LDSL-ABSN20244504, a UVB-induced mouse skin damage model was constructed for testing. Experiments revealed that this strain exhibits good inhibitory and repair effects on UVB-induced skin damage. Mechanistic studies revealed that its effects are related to the regulation of oxidative stress, inflammatory responses, and the AMPK and Nrf2 signaling pathways. Furthermore, the study compared administration methods, finding that both oral administration of the live bacteria and topical application of the postbiotic effectively inhibited photodamage and protected the skin. Therefore, LDSL-ABSN20244504 ​​can be considered a promising probiotic for skin protection, and its postbiotic, being more convenient to use than the live bacteria, can be well integrated into skin protection products.

[0018] Biological Preservation Instructions

[0019] Lactobacillus delbrueckii subsp. lactis, ABSN20244504, was deposited on June 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 34743, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0020] Figure 1 shows the colony morphology of Lactobacillus delbrueckii subsp. lactis ABSN20244504.

[0021] Figure 2 shows the Gram staining results of Lactobacillus delbrueckii subsp. ABSN20244504 ​​strain.

[0022] Figure 3 shows the skin tissue lesions induced by UVB in mice; where A is the normal group, B is the model group, C is the vitamin C topical application group, D is the vitamin C oral administration group, E is the LDSL-ABSN20244504 ​​post-biotic topical application group, and F is the LDSL-ABSN20244504 ​​post-biotic oral administration group.

[0023] Figure 4 shows the expression of SOD1 mRNA under oxidative stress in mouse skin tissue;

[0024] Figure 5 shows the expression of SOD2 mRNA under oxidative stress in mouse skin tissue;

[0025] Figure 6 shows the expression of CAT mRNA under oxidative stress in mouse skin tissue;

[0026] Figure 7 shows the expression of GSH mRNA under oxidative stress in mouse skin tissue;

[0027] Figure 8 shows the expression of NF-κBp65 mRNA in mouse skin tissue during inflammation.

[0028] Figure 9 shows the expression of inflammatory IκB-α mRNA in mouse skin tissue;

[0029] Figure 10 shows the expression of inflammatory TNF-α mRNA in mouse skin tissue;

[0030] Figure 11 shows the expression of IL-10 mRNA in the inflammatory phase of mouse skin tissue;

[0031] Figure 12 shows the expression of IL-6 mRNA in the inflammatory phase of mouse skin tissue;

[0032] Figure 13 shows the expression of AMPK mRNA in mouse skin tissue via the AMPK signaling pathway.

[0033] Figure 14 shows the expression of APPL1 mRNA in mouse skin tissue, which is part of the AMPK signaling pathway.

[0034] Figure 15 shows the expression of FOXO mRNA in the AMPK signaling pathway in mouse skin tissue;

[0035] Figure 16 shows the expression of LKB1 mRNA in the AMPK signaling pathway in mouse skin tissue;

[0036] Figure 17 shows the expression of mTOR mRNA in the AMPK signaling pathway in mouse skin tissue;

[0037] Figure 18 shows the expression of PGC-1α mRNA in mouse skin tissue, which is a pathway of AMPK signaling.

[0038] Figure 19 shows the expression of SIRT1 mRNA in mouse skin tissue, a pathway associated with the AMPK signaling pathway. Detailed Implementation

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] All raw materials used in this invention are not particularly restricted in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0041] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0042] Example 1 Isolation and Identification of Strains

[0043] 1. Experimental Materials

[0044] Traditional naturally fermented yak yogurt was collected from the grasslands of Maima Town, Aba County, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan Province, China. After being thoroughly stirred with a sterile spoon, 50 mL was drawn into a sterilized centrifuge tube using a sterile syringe, placed in a low-temperature food sampling box, and brought back to the laboratory for freezing and storage at -80°C. o Stored in an ultra-low temperature freezer at C for later use.

[0045] 2. Isolation and Identification of Lactic Acid Bacteria

[0046] 2.1 Isolation and purification of lactic acid bacteria

[0047] Take 1 mL of yogurt sample and dilute it 10-fold with sterile physiological saline to a final volume of 10. -6 Then take 10 -4 10 -5 10 -6 Three gradient bacterial suspensions (100 μL each) were plated and incubated at 37 °C for 24–48 h. Colony morphology was observed and recorded. Colonies of different morphologies were picked from the plates and streaked for separation. The colonies were then incubated at 37 °C for further analysis. o After culturing at C for 48 h, single colonies of different morphologies on the plate were picked again and streaked for separation. This process was repeated multiple times until pure single colonies with consistent morphology were obtained.

[0048] 2.2 Preliminary identification of lactic acid bacteria

[0049] Pick pure colonies from the plate and inoculate them into 5 mL of MRS liquid medium, incubate at 37°C. o Cultured at C for 24 h. Take 1 mL of the above-mentioned bacterial culture medium into a sterile centrifuge tube, centrifuge at 4000 r / min for 10 min, discard the upper culture medium, resuspend the bacterial pellet in sterile physiological saline and perform Gram staining microscopic examination. Those with positive Gram staining microscopic examination are preliminarily identified as lactic acid bacteria.

[0050] 2.3 Lactic acid bacteria DNA extraction

[0051] The purified suspected target strain was inoculated into MRS broth, 37 o After culturing at C for 18-24 h, DNA was extracted using a bacterial genomic DNA extraction kit. The extracted DNA was then numbered and stored at -20°C for later use.

[0052] 2.4 Genomic DNA PCR amplification and agarose gel electrophoresis detection

[0053] The extracted DNA was subjected to PCR amplification. The mixture consisted of 1 μL of upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.2), 1 μL of downstream primer 1495R (5'-CTACGGCTACCTTGTTACGA-3', SEQ ID NO.3), 12.5 μL of 2×Taq plus Buffer, and 1 μL of template DNA. The volume was brought to 25 μL with sterile dd H2O. Sterile ultrapure water was used as a negative control instead of template DNA. Amplification conditions were: 94 °C. o C 5 min; 94 o C 30 s, 55 o C 30 s, 72 o C 1 min, 29 cycles in total, 72 minutes in the final cycle o C extends for 5 minutes.

[0054] Then, 5 μL of the amplification product was subjected to agarose gel electrophoresis at an agarose concentration of 1.2% for 110 V for 45 min. The successfully detected PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The successfully sequenced sequences were compared and analyzed using the BLAST (Basic Local Alignment Search Tool) program in NCBI.

[0055] 2.5 In vitro resistance screening of lactic acid bacteria

[0056] 2.5.1 The ability of lactic acid bacteria to tolerate 0.3% bile salts

[0057] Porcine bile salts were added to MRS-THIO medium (MRS broth containing 0.2% sodium thioglycolate) to a concentration of 0.3%, and the medium was sterilized at 121°C for 15 min. 5 mL of the activated bacterial culture was inoculated at a rate of 2% (v / v) into both MRS-THIO medium without bile salts (0.0%) and MRS-THIO medium containing 0.3% bile salts. A blank medium (uninoculated MRS-THIO medium) was used as a control. The culture was kept at 37°C. o After culturing in C for 24 h, the OD values ​​of the different concentrations of culture medium were measured. 600nm The strain's tolerance to bile salts was calculated using formula (1):

[0058]

[0059] 2.5.2 Artificial gastric juice tolerance test

[0060] Preparation of artificial gastric fluid: The artificial gastric fluid consists of 0.2% NaCl and 0.35% pepsin. Weigh the required amount of NaCl and pepsin according to the corresponding mass-volume ratio and prepare the solution. Adjust the pH of the prepared artificial gastric fluid to 3.0 with 1 mol / L HCl, and then filter it through a 0.22 μm filter membrane for sterilization before use.

[0061] In a clean bench, 5 mL of the cultured bacterial culture base was transferred to a 10 mL sterile centrifuge tube and centrifuged at 4000 r / min for 10 min. The supernatant was discarded and the bacterial cells were collected. An equal volume (5 mL) of sterile physiological saline was added and mixed thoroughly to prepare a bacterial suspension. Then, 1 mL of the bacterial suspension was mixed with 9 mL of pH 3.0 artificial gastric fluid. 1 mL of this mixture was then used as the sample after 0 h of artificial gastric fluid treatment. The remaining 9 mL of the mixture was placed in a constant temperature water bath shaker (37°C). o The samples were cultured at 150 r / min (C) for 3 h. Samples at 0 h and 3 h were serially diluted 10-fold, and viable cell counts were determined using a plate plating method with appropriate gradients on MRS solid medium at 37°C. o After culturing in C for 48 h, the survival rate (%) was calculated according to Formula 2.

[0062]

[0063] 3. Results of lactic acid bacteria determination in the sample

[0064] A strain was isolated and purified from yak yogurt samples, and all samples showed positive Gram staining. Morphological observation and 16S rDNA species analysis confirmed that the strain was *Lactobacillus delbrueckii* subsp. *absenteriae* ABSN20244504.

[0065] As shown in Figure 1, the strain is irregular in shape, hollow in the middle, transparent, and has a moderate colony volume.

[0066] Under a 100x oil immersion microscope, the cell morphology of the lactic acid bacteria strain is shown in Figure 2. The cell morphology includes long rods, short rods, and spherical shapes, and there is no budding reproduction.

[0067] The sequence of ABSN20244504 ​​is as follows (SEQ ID NO.1):

[0068]

[0069] 3.1 Gene Bank analysis of lactic acid bacteria species

[0070] As shown in Table 1, the strain was successfully sequenced. BLAST analysis showed that the strain is *Lactobacillus delbrueckii* subsp. *lactotrichum*, and it shares 100% homology with known lactic acid bacteria in the Gene Bank database.

[0071] Table 1 Results of 16S rDNA sequence analysis

[0072]

[0073] 3.2 Results of in vitro resistance of Lactobacillus

[0074] As shown in Table 2, ABSN20244504 ​​has a good in vitro anti-gastric acid and bile salt effect.

[0075] Table 2 Results of in vitro resistance screening for lactic acid bacteria

[0076]

[0077] Example 2: Application of *Lactobacillus delbrueckii* subsp. *lactamase* in inhibiting photodamage to the skin.

[0078] To verify the inhibitory effect of Lactobacillus delbrueckii subsp. lactis ABS_N20244504 ​​(hereinafter referred to as LDSL-ABSN20244504) and its inactivated biogener on UVB-induced skin damage, the following animal experiment was designed.

[0079] 1. Materials and Methods

[0080] 1.1 Materials and Reagents

[0081] Lactobacillus delbrueckii subsp. lactis ABSN20244504 ​​is deposited at the China Microbiological Culture Collection Committee (CGMCC) with the entry number CGMCC No. 34743, on June 3, 2025.

[0082] The animals used in this experiment were female SPF-grade Kunming mice, 6 weeks old and weighing 22 ± 2 g. The experimental animals were provided by Hunan Slack Jingda Experimental Animal Co., Ltd., which has the qualification for experimental animal production [License number: SYXK (Xiang) 2024-0019]. This experiment was approved by the Experimental Animal Ethics Committee of the Chongqing Functional Food Collaborative Innovation Center of Chongqing Second Normal University (Ethical approval number: 202402021B). Throughout the experiment, all procedures strictly adhered to the 3R principles, the national standard GB / T 35892-2018 "Guidelines for the Ethical Review of Laboratory Animal Welfare", and the relevant requirements of the Declaration of Helsinki, fully guaranteeing the welfare and ethics of experimental animals.

[0083] Catalase (CAT), advanced glycation endproducts (AGEs), total superoxide dismutase (T-SOD), hydrogen peroxide (H2O2), sodium-potassium adenosine triphosphatase (Na + / K +-ATPase), nicotinamide adenine dinucleotide kinase (NADK) detection kit (Nanjing Jiancheng Bioengineering Institute); interleukin-4 (IL-4), IL-6, IL-10, IL-1β, tumor necrosis factor-alpha (TNF-α) enzyme-linked immunosorbent assay kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd.); hematoxylin and eosin (H&E) staining solution (Changsha Aibiwei Biotechnology Co., Ltd.); TriQuick Reagent total ribonucleic acid (RNA) extraction reagent, TaqMan One Step RT-qPCR Kit (Beijing Solarbio Science & Technology Co., Ltd.);Nuclear factor kappa-light-chain enhancer of activated B cells p65 subunit (NF-κBp65), nuclear factor of kappalight polypeptide gene enhancer in B-cells inhibitor alpha (IκB-α), tumor necrosis factor-alpha (TNF-α), IL-6, IL-10, superoxide dismutase 1 (SOD1), SOD2, CAT, glutathione (GSH), AMP-activated protein kinase (AMPK), liver kinase B1 (LKB1), sirtuin 1 (SIRT1), mammalian target of rapamycin (mTOR), peroxisome proliferator-activated receptor gamma coactivator 1α. PCR primers for 1-alpha (PGC-1α), adaptor protein 1 (phosphotyrosine interacting with PH domain and leucinezipper 1, APPL1), forkhead box O (FOXO), and beta-actin (β-actin) were developed by Beijing Qingke Biotechnology Co., Ltd.

[0084] 1.2 Instruments and Equipment

[0085] BI-150A Biochemical Incubator, Schroeder Instruments (Shanghai) Co., Ltd.; AMR-100 Fully Automated Microplate Reader, Hangzhou Aosheng Instruments Co., Ltd.; FS40 (290-400nm) UV Lamp, Candela, USA; BX43 Upright Microscope, Olympus, Japan; Stepone Plus Real-Time PCR Instrument, Thermo Fisher Scientific, USA; FL1500 Multifunction Imaging System, Thermo Fisher Scientific, USA.

[0086] 1.3 Methods

[0087] 1.3.1 Establishment of a UVB-induced mouse skin damage model

[0088] After revival, LDSL-ABSN20244504 ​​was cultured, and the bacterial culture was adjusted to 1.5 × 10⁻⁶ daily. 8 CFU / mL, this concentration of bacterial solution was administered via gavage, and a portion of this concentration was ultrasonically disrupted to prepare an inactivated probiotic solution for later use. After one week of acclimatization feeding, 60 mice were randomly divided into 6 groups of 10 mice each: a normal control group, a model group, and a vitamin C (V) group. C Topical application, Vitamin C (V) C The mice were divided into three groups: oral administration group, LDSL-ABSN20244504 ​​post-biotic topical application group, and LDSL-ABSN20244504 ​​oral administration group. The entire experimental period was 4 weeks. Before the experiment, the hair on the back of the mice was shaved at a size of 1.5 × 1.5 cm using an electric razor. During the experimental period, the shaved area on the back of the mice was kept bare. Mice in the normal group and the model group were administered 0.1 mL / 10 g of distilled water by gavage daily. C The topical group received a daily dose of 22.5 mg / mL of vitamin V. C Apply 0.2 mL of vitamin C aqueous solution evenly to the exposed areas of the back; V C The oral administration group received 150 mg / kg of vitamin C aqueous solution via gavage daily; the LDSL-ABSN20244504 ​​post-biotic topical treatment group received 1.5 × 10 8 Apply 0.2 mL of inactivated probiotic solution at a CFU / mL concentration evenly to the exposed area of ​​the back; LDSL-ABSN20244504 ​​oral administration group: 10 doses daily. 9 CFU / kg was administered via gavage to LDSL-ABSN20244504. During the third week of the experiment, UVB-induced skin damage was performed. A UV lamp was installed 30 cm from the bottom of a transparent glass box, and the UVB intensity of the lamp was adjusted to 320 nm (120 mJ / cm²). 2 Irradiation was performed for 3 minutes daily for one week. At the end of the fourth week, mice were euthanized by cervical dislocation, whole blood was collected from the inferior vena cava, and skin tissue was collected from the mice for subsequent experiments.

[0089] 1.3.2 Biochemical Analysis of Skin Oxidative Stress

[0090] Whole blood collected at 4℃ was centrifuged at 4500 rpm / min for 5 min, and the supernatant serum was collected. The corresponding oxidative stress markers in the serum were then measured using the CAT, AGEs, T-SOD, and H2O2 kits according to the manufacturer's instructions. Skin tissue collected at 4℃ was homogenized, then centrifuged at 4500 rpm / min for 15 min, and the supernatant was collected. CAT, AGEs, T-SOD, H2O2, and Na+ were then measured.+ / K + -The ATPase and NADK kits are used to determine the corresponding oxidative stress indicators in skin tissues according to the instructions.

[0091] 1.3.3 Serum inflammatory cytokine enzyme-linked immunosorbent assay

[0092] Mouse serum and tissue homogenate extracts were prepared according to the method in Section 1.3.2, and the levels of inflammatory cytokines in serum and skin tissues were measured using the IL-4, IL-6, IL-10, IL-1β and TNF-α kits according to the instructions.

[0093] 1.3.4 Pathological observation of skin tissue

[0094] After dissection, approximately 0.5 cm of mouse skin tissue was harvested and immediately fixed in 10% formalin solution for 24 h. Following fixation, the tissue was dehydrated using a gradient of 95% ethanol, and then cleared by replacing the ethanol with xylene. The cleared tissue blocks were then infiltrated and embedded in molten paraffin and serially sectioned using a rotary microtome (4-6 μm thickness). After dewaxing and rehydration, the sections were stained with hematoxylin and eosin (H&E) to prepare pathological sections for microscopic examination.

[0095] 1.3.5 Determination of mRNA expression in mouse skin tissue

[0096] Approximately 50 mg of mouse skin tissue was collected, finely minced, and homogenized according to the method described in Section 1.3.2. 1 mL of TriQuick Reagent was added to the homogenate, and the mixture was vortexed to induce complete lysis. Then, an appropriate amount of chloroform was added, and the mixture was vigorously vortexed and centrifuged at 4°C and 3000 rpm / min for 15 min. After centrifugation, the supernatant was carefully aspirated and transferred to a new centrifuge tube. An equal volume of pre-chilled isopropanol was added, and the mixture was gently inverted to mix. The tube was then centrifuged again at 4°C and 3000 rpm / min for 20 min, until RNA precipitated at the bottom. The supernatant was carefully discarded, and the precipitate was washed twice with 75% ethanol, each time centrifuged at 4°C and 3000 rpm / min for 15 min to thoroughly remove impurities. After discarding the ethanol, the precipitate was allowed to stand at room temperature for 3 min in a laminar flow hood to allow residual ethanol to evaporate completely. Then, 20 μL of ribonuclease-free water was added to dissolve the RNA precipitate, yielding the RNA stock solution, which was kept on ice for later use. The concentration of the RNA stock solution in each group was then uniformly adjusted to 1 μg / μL for subsequent experiments. The reaction system (total volume 50 μL) was prepared according to the TaqMan One Step RT-qPCR kit instructions: the system contained 4 μL of the above RNA template, 5 μL each of forward and reverse primers (Table 3), and other necessary reagents. The prepared reaction system was placed in a real-time quantitative PCR instrument and amplified according to the following program: initial denaturation at 95℃ for 5 min; followed by 40 cycles, each cycle consisting of 95℃ for 10 s, 60℃ for 20 s, and 72℃ for 20 s. The β-actin gene was used as an internal control to correct the loading amount. The relative expression level of the gene was determined using the classic 2-1... -ΔΔCT The method is used for calculation and analysis.

[0097] Table 3. PCR primer sequences used for detecting tissue gene expression.

[0098]

[0099] 1.4 Statistical Analysis of Data

[0100] In this invention, all experiments were performed in triplicate, and the results were expressed as the average of three independent measurements, with the standard deviation calculated. The final data were presented in the form of "mean ± standard deviation". One-way ANOVA was used to compare statistical differences between groups, with P < 0.05 used as the significance criterion.

[0101] 2 Results and Analysis

[0102] 2.1 Oxidative stress levels in mouse serum and skin tissue

[0103] In the normal group, serum and skin tissue showed the highest T-SOD and CAT activities, and the lowest H2O2 and AGEs levels. In the model group, however, the opposite trend was observed: T-SOD and CAT activities were the lowest, while H2O2 and AGEs levels were the highest, with statistically significant differences (P<0.05) (Tables 4 and 5). Compared to UVB treatment alone (model group), both topical and oral administration of vitamin C and LDSL-ABSN20244504 ​​significantly increased T-SOD and CAT enzyme activities in mouse serum and significantly decreased H2O2 and AGEs levels (P<0.05). Notably, the levels of T-SOD, CAT, H2O2, and AGEs in the LDSL-ABSN20244504 ​​oral administration group were closest to those in the normal group. The effect of LDSL-ABSN20244504 ​​topical application in regulating these levels to near-normal levels was also stronger than that of topical and oral administration of vitamin C. The levels in the oral vitamin C group were closest to those in the model group, demonstrating the lowest regulatory effect. Na2O levels in the skin tissue of normal mice... + / K + -ATPase and NADK levels were the highest. Compared to the model group mice, V C Both LDSL-ABSN20244504 ​​and their effects can significantly improve Na + / K + -ATPase and NADK levels (P<0.05), with LDSL-ABSN20244504 ​​oral administration showing the strongest ability to upregulate and downregulate these indicators, and LDSL-ABSN20244504 ​​post-treatment with topical estrogen also showing stronger effects than vitamin B. C Function, and V C Topical application is more effective than oral administration. The damage to human skin under UVB irradiation is closely related to the imbalance of the body's oxidative and antioxidant systems. Long-term exposure to UVB radiation triggers significant oxidative stress, leading to a decrease in the activity of key antioxidant enzymes SOD and CAT in the skin. The activity status of these two enzymes directly affects the skin's aging process, biosynthesis, degradation metabolism, and defense functions. Advanced glycation end products (AGEs), as an important biomarker of skin aging, accumulate in a way that mutually promotes oxidative stress responses and the release of inflammatory cytokines. The continuous increase in AGEs not only increases collagen fragility but also leads to an increase in intracellular hydrogen peroxide (H2O2) levels. Further research has confirmed that UVB irradiation can inhibit CAT activity, thereby exacerbating H2O2 accumulation. + / K +Decreased ATPase activity leads to impaired intracellular energy production and ion transport, thereby affecting cell function and causing skin aging damage. NADK plays a crucial role in maintaining intracellular NAD+ levels in skin cells; NAD+ is a key substance for skin's resistance to UV damage and promotion of damage repair. The results of this invention also confirm the effects of UVB on SOD, CAT, H2O2, AGEs, and Na+. + / K + The levels of ATPase and NADK were significantly affected, leading to skin damage. C When used as a common antioxidant, oral administration to mice was less effective than direct application to the site of injury, although V C While both methods have some effect, their effectiveness is significantly lower than that of inactivated LDSL-ABSN20244504 ​​(topical application) and live LDSL-ABSN20244504 ​​(oral administration). This demonstrates that different methods of using LDSL-ABSN20244504 ​​can effectively prevent and intervene in UBV light damage.

[0104] Table 4. Serum oxidative stress levels in mice

[0105]

[0106] Note: In the figures and tables of this article, if the lowercase letters of the data groups are different, it means that the difference between the two groups is statistically significant (P<0.05); if the letters are the same, it means that the difference between the groups is not statistically significant (P>0.05).

[0107] Table 5 Oxidative stress levels in mouse skin tissue

[0108]

[0109] 2.2 Serum and skin inflammatory cytokine levels in mice

[0110] Tables 6 and 7 show that the serum and skin tissue levels of IL-6, IL-1β, and TNF-α cytokines were highest in the model group mice, while the levels of IL-4 and IL-10 were lowest. Oral administration of LDSL-ABSN20244504 ​​maximally reduced the levels of IL-6, IL-1β, and TNF-α cytokines and maximally increased the levels of IL-4 and IL-10. However, the levels of IL-6, IL-1β, and TNF-α in the LDSL-ABSN20244504 ​​oral administration group were still higher than those in the normal group, while the levels of IL-4 and IL-10 were only lower than those in the normal group, and the differences were significant (P<0.05). Furthermore, compared to the model group, mice treated with LDSL-ABSN20244504 ​​followed by estrogen application, vitamin C application, and oral vitamin C treatment showed the best ability to increase IL-4 and IL-10 levels and decrease IL-6, IL-1β, and TNF-α levels, respectively, with significant differences among the groups (P<0.05). IL-4 can downregulate the production of specific chemokines, which are typically responsible for the aggregation of Th1 cells. This effect helps reduce the accumulation of pro-inflammatory Th1 cells at the lesion site, thereby preventing excessive inflammatory responses and tissue damage. Furthermore, TNF-α and IL-6 are both key pro-inflammatory cytokines in the NF-κB signaling pathway. TNF-α can promote the activation of NF-κB signaling in various cells. IL-6, as a multifunctional pro-inflammatory cytokine, has similar biological activity to IL-1β; both are core mediators in the inflammatory process. IL-6 can also activate the NF-κB pathway, participating in the formation of chronic skin inflammation. Simultaneously, overexpression of IL-6 enhances skin permeability, prompting more inflammatory factors to enter local tissues and exacerbating inflammation. On the other hand, IL-10, produced by macrophages, dendritic cells, and T cells, is an important inflammatory regulator. It mainly exerts its anti-inflammatory effect by inhibiting the activation of the NF-κB pathway and increasing the ratio of IL-1RA to IL-1β. The results of this invention also show that both LDSL-ABSN20244504 ​​interventions can effectively inhibit the levels of IL-6, IL-1β, and TNF-α and increase the levels of IL-4 and IL-10, thereby inhibiting the inflammatory response caused by UVB and protecting the skin.

[0111] Table 6. Serum inflammatory cytokine levels in mice

[0112]

[0113] Table 7. Levels of inflammatory cytokines in mouse skin tissue

[0114]

[0115] 2.3 Pathological observation results of mouse skin

[0116] When skin is exposed to ultraviolet (UV) radiation for extended periods, inflammatory symptoms such as dryness, itching, erythema, and edema may occur. Histopathological observation can be used to rapidly assess the severity of skin damage caused by UV radiation. As shown in Figure 3, the normal group of mice had intact skin structure, a thin epidermis, a wavy structure at the dermal-epidermal junction, no excessive keratinization of the stratum corneum, and a relatively thick dermis. Compared with the normal group, the model group showed significantly reduced dermal thickness, a markedly thickened epidermis, a decreased number of collagen fiber bundles, and disordered subcutaneous tissue structure with blurred boundaries. Inflammatory cell infiltration was also observed in the tissue, suggesting that long-term UVB irradiation can induce chronic inflammatory reactions and photodamage to the skin. Compared with the model group, all groups treated with topical application or oral administration of vitamin C and the test sample increased dermal thickness and made collagen fibers more tightly packed. Among these, mice orally administered LDSL-ABSN20244504 ​​showed a higher dermal thickness than the other groups, with a more uniform and orderly distribution of fiber bundles, and an overall structure close to the normal control group. Applying LDSL-ABSN20244504 ​​after inactivation can also improve skin pathological changes caused by UVB, while the improvement effect of oral vitamin C is weaker and less obvious.

[0117] 2.4 mRNA expression intensity in mouse skin tissue

[0118] qPCR analysis showed that the expression levels of SOD1, SOD2, CAT, GSH, IκB-α, IL-10, AMPK, SIRT1, LKB1, PGC-1α, APPL1, and FOXO mRNA were strongest in the skin tissue of normal mice, while the expression levels of NF-κBp65, TNF-α, IL-6, and mTOR mRNA were weakest (Figures 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). The expression trends in the skin of model mice were opposite to those in the normal group; the expression of SOD1, SOD2, CAT, GSH, IκB-α, IL-10, AMPK, SIRT1, LKB1, PGC-1α, APPL1, and FOXO was weakest, while the expression levels of NF-κBp65, TNF-α, IL-6, and mTOR mRNA were strongest. LDSL-ABSN20244504 ​​oral group, LDSL-ABSN20244504 ​​post-biotic topical group, V C Application group and V C In the oral administration group, the expression of SOD1, SOD2, CAT, GSH, IκB-α, IL-10, AMPK, SIRT1, LKB1, PGC-1α, APPL1, and FOXO mRNA decreased sequentially, while the expression of NF-κBp65, TNF-α, IL-6, and mTOR increased sequentially.

[0119] Intracellularly, SOD1 is mainly located in the cytoplasm and mitochondrial intermembrane space, while its isoenzyme SOD2 is specifically found in the mitochondrial matrix. Both are key antioxidant enzymes regulating cellular oxidative stress. In addition, CAT and GSH are also important antioxidants that can inhibit and repair oxidative damage to the skin. NF-κB is a core transcription factor involved in the body's immune response. Under normal conditions, it binds to its inhibitory protein IκB and exists in an inactive form. Studies have shown that under stress conditions, IκB protein is phosphorylated and degraded, leading to NF-κB activation; activated NF-κB then promotes the release of pro-inflammatory cytokines such as TNF-α and IL-6. Dysfunction of this signaling pathway is common in various skin injuries. Among them, IκB-α, as the main inhibitory protein of NF-κB, can block the nuclear localization signal of NF-κB, causing it to remain in the cytoplasm as an inactive complex. Studies have shown that almost all NF-κB inducers rapidly activate NF-κB by triggering the degradation of IκB-α; therefore, preventing IκB-α phosphorylation is one of the effective targets for inhibiting the activation of this pathway. The AMPK signaling pathway plays an important role in host defense against infection, oxidative stress, inflammation, and immune regulation. High levels of intracellular AMPK can activate downstream factors such as SIRT1, PGC-1α, and FOXO, thereby inhibiting NF-κB activity. APPL1 protein can mediate various cellular signal transductions, participate in the regulation of inflammatory responses, and exert antioxidant and anti-atherosclerotic effects. LKB1 is an upstream kinase of AMPK, mainly located in the cell nucleus. It can directly phosphorylate threonine at position 172 of the AMPKα subunit, thereby activating AMPK and regulating cellular energy metabolism. mTOR is a key receptor for growth factors and nutrient signals, and its activation is considered to be closely related to the pathogenesis of cutaneous melanoma. Furthermore, SIRT1 can upregulate the expression of PGC-1α through deacetylation, and PGC-1α plays a core regulatory role in mitochondrial biosynthesis, directly regulating the number and function of mitochondria. In this invention, LDSL-ABSN20244504 ​​can significantly regulate the expression of oxidative stress (SOD1, SOD2, CAT, GSH), inflammatory responses (NF-κBp65, IκB-α, TNF-α, IL-6, IL-10), and the AMPK signaling pathway (AMPK, LKB1, SIRT1, mTOR, PGC-1α, APPL1, FOXO). In particular, after oral administration, it may exert its mechanism of action through multiple gut axes in the body to inhibit UVB skin damage.

[0120] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A subsp. lactis of Lactobacillus delbrueckii (ABSN20244504), characterized in that, Its accession number is CGMCC No. 34743.

2. A post-genetic composition, characterized in that, The inactivated bacterial cells comprising Lactobacillus delbrueckii subsp. ABSN20244504 ​​as described in claim 1.

3. A microbial preparation, characterized in that, It comprises Lactobacillus delbrueckii subsp. lactis ABSN20244504 ​​or its inactivated cells as described in claim 1, and a pharmaceutically or cosmetically acceptable carrier or excipient.

4. The use of Lactobacillus delbrueckii subsp. lactis ABSN20244504 ​​as described in claim 1 in the preparation of a product for inhibiting photodamage to the skin, wherein the product is administered orally and contains live cells of Lactobacillus delbrueckii subsp. lactis ABSN20244504.

5. The use of Lactobacillus delbrueckii subsp. lactis ABSN20244504 ​​as described in claim 1 in the preparation of a product for inhibiting photodamage to the skin, wherein the product is applied topically to the skin and the product contains inactivated cells of Lactobacillus delbrueckii subsp. lactis ABSN20244504.

6. An oral product for inhibiting photodamage to the skin, characterized in that, Contains an effective amount of live Lactobacillus delbrueckii subsp. lactis ABSN20244504 ​​as described in claim 1, and a pharmaceutically acceptable carrier or excipient.

7. A topical product for inhibiting photodamage to the skin, characterized in that, The product contains an effective amount of inactivated Lactobacillus delbrueckii subsp. lactis ABSN20244504 ​​as described in claim 1, and a carrier or excipient acceptable for use in cosmetics.

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