Halomonas sp. and its application in preparing pdrn and related products

Abstract

The application provides halomonas and application thereof in preparation of PDRN and related products, and relates to the technical field of biotechnology. Halomonas sp. The application extracts natural PDRN from halomonas for the first time, and provides a new way for production of PDRN. The application further provides a method for extracting natural PDRN from halomonas, which is safe and controllable in the whole closed-loop management from culture of the strain to fermentation, and simple in preparation method, free of extreme reaction conditions and easy for large-scale industrial production.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to Halomonas and its application in the preparation of PDRN and related products. Background Technology

[0002] PDRN is a mixture of active polynucleotides, free of antigenic properties and systemic toxicity. Currently, extraction and purification sources include red algae, ginseng, and sperm cells from salmon (golden trout) or salmon (salmon). Derived from nature, PDRN exhibits excellent biocompatibility and safety. Its mechanism of action mimics key aspects of the body's own damage repair process, thus it is considered a highly promising bioactive regenerative therapeutic agent, providing innovative solutions for promoting tissue repair, delaying aging, and improving skin health. It is widely used in fields such as medical aesthetics, wound healing, and tissue regeneration medicine.

[0003] In recent years, there has been increasing research on the application and preparation methods of PDRN. For example, Chinese patent CN118109455A discloses "a method for extracting high-quality polydeoxyribonucleic acid from frozen salmon testis tissue," characterized by homogenizing, lysing, collecting and purifying, precipitating and concentrating, washing, and ultrasonically breaking down DNA in salmon testis, followed by vacuum freeze-drying to obtain a white solid, which is the PDRN sample. However, most of its steps are carried out at low temperatures, resulting in high energy consumption and a complex process. Chinese patent CN11639656A discloses "a method for preparing and applying PDRN derived from Schizochytrium," characterized by aspirating Schizochytrium broth into centrifuge tubes, followed by centrifugation, resuspension, lysis, precipitation, rinsing, resolution, and disruption to obtain a PDRN solution. However, this process is relatively complex and limited by factors such as the adequacy of cell resuspension and dispersion, making large-scale production difficult.

[0004] Modern industry primarily extracts PDRN from the testicular cells of deep-sea salmon. For example, Chinese patent CN119506273A discloses "A method for extracting polydeoxyribonucleotides," CN118207205A discloses "A method for large-scale extraction of small molecule PDRN using marine cryogenic proteases," and CN112315836A discloses "A highly efficient method for preparing topical PDRN and its application," etc. While these processes have long been the main methods for obtaining PDRN, with the continuous depletion of marine fishery resources, extracting PDRN from salmon testicular cells is no longer sufficient to meet the increasingly strong demand for PDRN in the new era. Furthermore, issues such as heavy metal accumulation, chromosomal aberrations, and gene mutations in marine organisms have also significantly impacted the safety of salmon sperm PDRN raw materials.

[0005] In summary, there is an urgent need for a safe and simple PDRN preparation process to meet the needs of PDRN production.

[0006] Halomonas Halomonas sp. It is a type of moderately halophilic Gram-negative bacillus, mainly distributed in salt fields, muddy water, and aquaculture water in China, and has strong environmental adaptability. There are currently no precedents for using *Haloxylon ammodendron* to produce PDRN. Summary of the Invention

[0007] To address the aforementioned problems, the purpose of this invention is to provide a safe and simple PDRN preparation process, and to discover for the first time that *Haloxylon ammodendron* is involved. Halomonas sp. It can be used to prepare and produce PDRN and related products, providing a new raw material for PDRN production.

[0008] On the one hand, this application provides a strain of Halomonas. Halomonas sp. SY-L4 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251411.

[0009] In this invention, a halomonas strain suitable for PDRN preparation was screened. Halomonas sp. SY-L4, whose 16S rDNA sequence is shown in SEQ ID No. 1.

[0010] On the other hand, this application also provides a bacterium containing the aforementioned Halomonas. Halomonas sp. Bacterial agents.

[0011] Preferably, the microbial agent contains ≥10 live bacteria. 6 The above-mentioned dry and / or wet cells of Halomonas bacteria, CFU / g.

[0012] Those skilled in the art can use universal culture methods for halomonas to culture the halomonas described in this application.

[0013] In a preferred embodiment, the method for culturing the *Haloxymonas* includes: inoculating *Haloxymonas* into a seed culture medium and culturing it at 25°C-40°C for 1-2 days.

[0014] The seed culture medium may include: peptone, yeast extract, glucose, and NaCl; preferably, the seed culture medium includes: 10-50 g / L peptone, 10-50 g / L yeast extract, 10-50 g / L glucose, 10-100 g / L NaCl, with the remainder being water. Those skilled in the art may add agar as needed to adjust the form of the seed culture medium.

[0015] In a preferred embodiment, the method for culturing the *Haloxymonas* includes:

[0016] Step 1: Inoculate Halomonas into seed culture medium and incubate at 25℃-40℃ for 1-2 days to obtain seed liquid; preferably, after collecting solid bacteria, the bacterial cells can be resuspended in 10-30 times the amount of water to obtain seed liquid.

[0017] Step 2: Inoculate the seed culture into the fermentation medium at an inoculation rate of 1%-5%, and culture for 30-50 h at 25℃-40℃, stirring at 300-600 rpm, dissolved oxygen 40%-50%, and pH 6.5-7.5.

[0018] The fermentation medium may include glucose, yeast powder, and sodium acetate; preferably, the fermentation medium includes: 400-500 g / L glucose, 10-30 g / L yeast powder, 20-40 g / L sodium acetate, with the remainder being water; more preferably, glucose may be added to the fermentation broth at a constant rate; even more preferably, the glucose addition time is 0-40 h.

[0019] The pH value can be controlled using a pH adjuster. In a preferred embodiment, the pH is controlled using a NaOH solution.

[0020] On the other hand, this application also provides a method for preparing PDRN, the method comprising:

[0021] Step 1: Collecting Halomonas bacteria Halomonas sp. Bacterial cells were resuspended and then lysed to obtain bacterial lysate.

[0022] Step 2: Add a protein denaturant to the bacterial cell lysate and filter to obtain the filtrate;

[0023] Step 3: Add metal salt and organic solvent to the filtrate to precipitate and prepare crude product;

[0024] Step 4: Dry the crude product to obtain PDRN.

[0025] Furthermore, the halomonas Halomonas sp. Including: Halomonas Halomonas sp. SY-L4, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251411, and / or *Halomonas*. Halomonas sp. CGMCC NO.1.8772, purchased from the China General Microbiological Culture Collection Center (CGMCC), with the number CGMCC NO.1.8772.

[0026] Furthermore, the bacterial resuspended to OD 600 The range is 10-1000.

[0027] The bacterial cells are resuspended to OD 600It can be any value from 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or any value in between.

[0028] Preferably, the bacterial resuspended to OD 600 The range is 50-500.

[0029] Furthermore, the fragmentation is ultrasonic fragmentation.

[0030] Preferably, the ultrasonic crushing conditions are a crushing power of 700-900 W.

[0031] Preferably, the ultrasonic fragmentation condition is a fragmentation time of 30-60 min.

[0032] Preferably, the ultrasonic crushing conditions are a crushing temperature of 0℃-10℃.

[0033] The ultrasonic power can be any value among 700 W, 710 W, 720 W, 730 W, 740 W, 750 W, 760 W, 770 W, 780 W, 790 W, 800 W, 810 W, 820 W, 830 W, 840 W, 850 W, 860 W, 870 W, 880 W, 890 W, and 900 W, or any value in between.

[0034] The crushing time can be any value from 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, 60 min, or any value in between.

[0035] The crushing temperature can be any value among 0℃, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, and 10℃, or any value in between.

[0036] More preferably, the ultrasonic crushing conditions are 800 W power, crushing time 45 min, and crushing temperature 6℃.

[0037] Furthermore, step one also includes incubating the bacterial lysate at 30℃-40℃ for 0-24 h.

[0038] The insulation temperature can be selected from any value of 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, or any value in between.

[0039] The heat preservation time can be selected from any value in the range of 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, or any value in between.

[0040] Preferably, step one further includes incubating the bacterial lysis buffer at 37°C for 6-24 hours.

[0041] Those skilled in the art can adjust the incubation temperature and time of the cell lysis buffer according to actual product requirements to obtain products with different DNA contents, and no further limitations are made here.

[0042] Further, the protein denaturant is selected from at least one or more of guanidine hydrochloride, phenol, and surfactants, wherein the surfactants include one or more of sodium dodecyl sulfate, sodium dodecyl sarcosinate, polyethylene glycol octylphenyl ether, polyoxyethylene sorbitan laurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate, and polyoxyethylene sorbitan monooleate; preferably, the protein denaturant is sodium dodecyl sulfate, and the concentration added is 0.5%-2% by weight of the bacterial lysate.

[0043] The concentration can be any value or any value between 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, and 2%.

[0044] Preferably, the concentration of the protein denaturant is 1%.

[0045] Furthermore, the denaturation conditions are a water bath reaction at 40℃-60℃ for 60-120 min.

[0046] The denaturation temperature can be any value among 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, and 60℃, or any value in between.

[0047] The reaction time can be any value among 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, and 120 min, or any value in between.

[0048] Preferably, the denaturation condition is a 55°C water bath reaction for 60 min.

[0049] Furthermore, the filter size is 0.22 μm-1.2 μm.

[0050] The filter size is any value among 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm, 0.29 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.1 μm, and 1.2 μm.

[0051] Preferably, the filtration includes precision filter paperboard filtration and / or polyethersulfone membrane filtration.

[0052] More preferably, the precision filter paperboard is a 1.2 μm precision filter paperboard.

[0053] More preferably, the polyethersulfone filter membrane is a 0.22 μm polyethersulfone filter membrane.

[0054] Furthermore, the metal salt in step three comprises at least one or more of the following: magnesium chloride, sodium chloride, calcium chloride, potassium chloride, zinc chloride, potassium acetate, sodium acetate, ammonium sulfate, and sodium sulfate.

[0055] Preferably, the metal salt is sodium chloride and / or magnesium chloride.

[0056] Furthermore, in step three, the final concentration of the added metal salt is 0.1-5 M, preferably 0.2-2 M.

[0057] The final concentration of the added metal salt can be any value from 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2 M, 3 M, 4 M, 5 M, or any value between these values.

[0058] Preferably, the final concentration of the added metal salt is 0.2-2 M.

[0059] Furthermore, step three also includes a centrifugation step.

[0060] Preferably, the centrifugal speed is 5000-10000 rpm.

[0061] More preferably, the centrifugation time is 1-10 min.

[0062] The centrifugation speed can be any value among 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm, 10000 rpm, and any value in between.

[0063] Centrifugation time can be any value among 1 min, 2 min, 3 min, 4 min, 5 min, or any value in between.

[0064] More preferably, the centrifugation conditions are 6000 rpm for 5 min.

[0065] Furthermore, the organic solvent comprises one or more of ethanol, methanol, and isopropanol.

[0066] Preferably, the organic solvent is at least ethanol; more preferably, the ethanol concentration is greater than or equal to 95%.

[0067] More preferably, the volume ratio of ethanol to filtrate is (1-5):1.

[0068] The volume ratio of ethanol to filtrate can be 1:1, 2:1, 3:1, 4:1, 5:1, or any value between these ratios.

[0069] More preferably, the volume ratio of ethanol to filtrate is 4:1.

[0070] Preferably, the drying process can be vacuum drying.

[0071] More preferably, the vacuum drying conditions are a vacuum degree of -0.1 to -0.5 MPa.

[0072] More preferably, the vacuum drying temperature is 25°C-40°C.

[0073] The vacuum drying temperature can be any value among 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, and 40℃, or any value in between.

[0074] More preferably, the vacuum drying conditions are a vacuum degree of -0.1 MPa and a temperature of 30°C.

[0075] In a preferred embodiment, a method for preparing PDRN includes:

[0076] Step 1: Collecting Halomonas bacteria Halomonas sp. Bacterial cells, resuspended to OD 600 After 10-1000 minutes, ultrasonic disruption was performed. The ultrasonic disruption conditions were: power 700-900 W, disruption time 30-60 min, disruption temperature 0℃-10℃, and holding at 30℃-40℃ for 0-24 h to obtain bacterial cell lysate.

[0077] Step 2: Add 0.5%-2% protein denaturant to the bacterial cell lysate for denaturation. The denaturation conditions are: 40℃-60℃ water bath reaction for 60-120 min, followed by 0.22 μm-1.2 μm filtration to obtain the filtrate.

[0078] Step 3: Add 0.1-5 M of metal salt to the filtrate, centrifuge and resuspend, add organic solvent, the volume ratio of organic solvent to filtrate is (1-5):1, and take the precipitate to obtain crude product;

[0079] Step 4: Dry the crude product to obtain PDRN.

[0080] On the other hand, this application also provides a PDRN or a salt thereof.

[0081] Preferably, the PDRN is a PDRN derived from Halomonas bacteria.

[0082] Preferably, the salt includes one or more of PDRN sodium salt, PDRN magnesium salt, PDRN zinc salt, PDRN potassium salt, and PDRN calcium salt.

[0083] Preferably, the PDRN is a white or off-white powder, granules, or powder or granule product.

[0084] Preferably, the DNA content of the PDRN is greater than or equal to 5%.

[0085] The DNA content of the PDRN can be any value among 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 33%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%, or any value in between.

[0086] Preferably, the pH value of the PDRN is 5-7.

[0087] The pH value of the PDRN can be any value among 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7, or any value in between.

[0088] More preferably, the pH value of the PDRN is 6.1-6.5.

[0089] Preferably, the purity (OD) of the PDRN is... 260 / OD 280 The value is 1.5-2.5.

[0090] The purity (OD) of the PDRN 260 / OD 280 The value can be 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or any value in between.

[0091] Preferably, the total nucleic acid content of the PDRN is greater than or equal to 80%.

[0092] More preferably, the total nucleic acid content of the PDRN is greater than or equal to 90%.

[0093] More preferably, the total nucleic acid content of the PDRN is greater than or equal to 95%.

[0094] The total nucleic acid content of the PDRN can be any value from 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100%, or any value between these values.

[0095] The molecular weight of the PDRN is 50 kDa-1500 kDa.

[0096] The molecular weight of the PDRN can be any value from 50 KDa, 100 KDa, 150 KDa, 200 KDa, 250 KDa, 300 KDa, 350 KDa, 400 KDa, 450 KDa, 500 KDa, 550 KDa, 600 KDa, 650 KDa, 700 KDa, 750 KDa, 800 KDa, 850 KDa, 900 KDa, 950 KDa, 1000 KDa, 1050 KDa, 1100 KDa, 1150 KDa, 1200 KDa, 1250 KDa, 1300 KDa, 1350 KDa, 1400 KDa, 1450 KDa, 1500 KDa, and any value between these values.

[0097] Preferably, the molecular weight of the PDRN is 150 kDa-500 kDa.

[0098] More preferably, the PDRN is prepared according to the above preparation method.

[0099] On the other hand, this application also provides a product comprising the PDRN or a salt thereof.

[0100] The product of this application may also contain excipients, which may include appropriate solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc.

[0101] The product of this application can be prepared by a general method, wherein one or more diluents or carriers may be added, such as aqueous solutions, pills, tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions, etc.

[0102] The product of this application can be prepared into any formulation commonly prepared in this field. For example, it can be formulated into creams, lotions, emulsions, masks, foundations, medical devices, hair cosmetics, pharmaceuticals, etc. Specifically, skin lotions, skin softeners, hyaluronic acid injections, skin toners, astringents, lotions, moisturizing lotions, nourishing lotions, massage creams, nourishing creams, moisturizing creams, hand creams, foundations, serums, nourishing serums, masks, soaps, cleansing foams, cleansing milks, cleansing creams, lotions, or shower gels.

[0103] In the products of this application, in addition to PDRN, which is an essential ingredient, other ingredients commonly formulated in cosmetics may be mixed as needed. These may include, for example, oils, moisturizers, surfactants, organic pigments, inorganic pigments, UV absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation enhancers, cooling agents, antiperspirants, or purified water.

[0104] On the other hand, this application also provides *Haloxylon ammodendron*. Halomonas sp. Applications in the preparation or production of PDRN and related products.

[0105] This application marks the first discovery of Halomonas. Halomonas sp. It can be used to prepare PDRN, and the prepared PDRN has the effects of promoting cell proliferation, promoting skin repair, improving skin inflammation, promoting skin wound healing, promoting angiogenesis, promoting the production of vascular endothelial growth factor, repairing damaged skin barrier, repairing DNA damage, promoting collagen production, anti-oxidation, moisturizing, improving skin elasticity, firming skin and / or anti-wrinkle.

[0106] Furthermore, the halomonas Halomonas sp. Including: Halomonas Halomonas sp. SY-L4, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251411, and / or *Halomonas*. Halomonas sp. CGMCC NO.1.8772, purchased from the China General Microbiological Culture Collection Center (CGMCC), with the number CGMCC NO.1.8772.

[0107] On the other hand, this application also provides the aforementioned halomonas bacteria. Halomonas sp. The bacterial agent, the preparation method, the PDRN or its salt, or the product may contain one or more of the following A1)-A11):

[0108] A1) Prepare products that promote cell proliferation;

[0109] A2) Prepare products for skin repair and / or improvement of skin inflammation and / or skin wound healing and / or promotion of angiogenesis;

[0110] A3) Prepare products that promote the production of vascular endothelial growth factor;

[0111] A4) Prepare products to repair damaged skin barriers;

[0112] A5) Prepare products for repairing DNA damage;

[0113] A6) Prepare products that promote collagen production;

[0114] A7) Preparation of antioxidant products;

[0115] A8) Applications in the preparation of moisturizing products;

[0116] A9) Application in the preparation of products that improve skin elasticity;

[0117] A10) Application in the preparation of skin-firming products;

[0118] A11) Application in the preparation of anti-wrinkle products.

[0119] Preferably, the concentration of PDRN used is 0.001%-20%.

[0120] More preferably, the concentration of PDRN used is 0.001%-10%.

[0121] Those skilled in the art can adjust the concentration of PDRN used according to the actual situation, and no specific limitation is made here.

[0122] Preferably, the *Haloxymonas* Halomonas sp. The application of the bacterial agent, the preparation method, PDRN or its salt, or the product described herein in the preparation of products that promote cell proliferation.

[0123] This application verifies the aforementioned Halomonas. Halomonas sp. The prepared PDRN was non-cytotoxic.

[0124] In a preferred embodiment, the cell is a HaCaT cell (human immortalized keratinocyte).

[0125] Preferably, the *Haloxymonas* Halomonas sp. The application of the bacterial agent, the preparation method, the PDRN or its salt, or the product described herein in the preparation of products for skin repair and / or improvement of skin inflammation and / or skin wound healing and / or promotion of angiogenesis.

[0126] The promotion of angiogenesis is achieved by promoting the production of vascular endothelial growth factor.

[0127] Preferably, the *Haloxymonas* Halomonas sp. The application of the bacterial agent, the preparation method, PDRN or its salt, or the product described herein in the preparation of products that promote the production of vascular endothelial growth factor.

[0128] This application verifies the aforementioned Halomonas. Halomonas sp. The prepared PDRN significantly promoted the production of vascular endothelial growth factor in cells compared to PDRN from other sources.

[0129] In a preferred embodiment, the cell is a HaCaT cell (human immortalized keratinocyte).

[0130] Preferably, the *Haloxymonas* Halomonas sp. The application of the bacterial agent, the preparation method, the PDRN or its salt, or the product in the preparation of products for repairing damaged skin barriers.

[0131] Preferably, the *Haloxymonas* Halomonas sp. The application of the bacterial agent, the preparation method, the PDRN or its salt, or the product in the preparation of DNA damage repair products.

[0132] Preferably, the DNA damage is at least DNA damage caused by ultraviolet light; more preferably, the ultraviolet light includes UVB and / or UVA.

[0133] This application verifies the aforementioned Halomonas. Halomonas sp. The prepared PDRN has the function of repairing DNA damage, specifically repairing DNA damage caused by ultraviolet radiation (UVB and / or UVA), which is manifested by inhibiting the production of CPD-DNA in damaged cells.

[0134] In a preferred embodiment, the cell is a HaCaT cell (human immortalized keratinocyte).

[0135] Preferably, the *Haloxymonas* Halomonas sp. The bacterial agent, the preparation method, PDRN or its salt, or the product may be used in the preparation of a product that promotes collagen production.

[0136] More preferably, the collagen includes type I collagen and / or type III collagen.

[0137] This application verifies the aforementioned Halomonas. Halomonas sp. The prepared PDRN has the function of promoting the production of type I collagen and / or type III collagen, while PDRN prepared by existing methods does not have this effect.

[0138] In a preferred embodiment, the cells are HSF cells (human skin fibroblasts).

[0139] Preferably, the *Haloxymonas* Halomonas sp. The application of the bacterial agent, the preparation method, PDRN or its salt, or the product in the preparation of antioxidant products.

[0140] More preferably, the application includes inhibiting cellular ROS generation.

[0141] In a preferred embodiment, the cell is a HaCaT cell (human immortalized keratinocyte).

[0142] Preferably, the *Haloxymonas* Halomonas sp. The application of the aforementioned microbial agent, the aforementioned preparation method, the aforementioned PDRN or its salt, or the aforementioned product in the preparation of moisturizing products.

[0143] Preferably, the *Haloxymonas* Halomonas sp. The application of the aforementioned bacterial agent, the aforementioned preparation method, the aforementioned PDRN or its salt, or the aforementioned product in the preparation of products that improve skin elasticity.

[0144] Preferably, the *Haloxymonas* Halomonas sp. The application of the bacterial agent, the preparation method, the PDRN or its salt, or the product in the preparation of firm skin.

[0145] Preferably, the *Haloxymonas* Halomonas sp. The application of the bacterial agent, the preparation method, PDRN or its salt, or the product in the preparation of anti-wrinkle products.

[0146] The present invention has the following beneficial effects:

[0147] This invention is the first to obtain from Halomonas bacteria Halomonas sp. Extracting natural PDRN provides a new approach for PDRN production.

[0148] This invention also provides a method for extracting natural PDRN from Halomonas bacteria. This method features closed-loop management from strain cultivation to fermentation in tanks, ensuring safety and controllability. Furthermore, the preparation method is simple, has no extreme reaction conditions, and is easily mass-produced industrially.

[0149] The fermentation cycle of Halomonas in this invention is extremely short, requiring only 30-48 hours from inoculation to the end of fermentation. It also has many advantages such as controllable growth environment, pure product, low fermentation cost, and strong sustainable development capability, making the future application scenarios of Halomonas PDRN more extensive. Attached Figure Description

[0150] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0151] Figure 1 It is Halomonas Halomonas sp. Photographs of the colony morphology of SY-L4 CCTCC NO: M 20251411;

[0152] Figure 2It is Halomonas Halomonas sp. Micrograph of SY-L4 CCTCC NO: M 20251411 bacterial cells, magnified 1000 times;

[0153] Figure 3 This is a standard curve of DNA concentration.

[0154] Information on the preservation of biological materials:

[0155] The Halomonas bacterium described in this invention Halomonas sp. SY-L4 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251411, deposited on June 17, 2025, at Wuhan University, Luojia Mountain, Wuchang District, Wuhan, Hubei Province, 430072, China. Detailed Implementation

[0156] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0157] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0158] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0159] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0160] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0161] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related areas.

[0162] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0163] In this invention, the terms "nucleic acid," "nucleic acid molecule," "nucleotide," "nucleotide sequence," and "polynucleotide" are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule"), or any phosphate ester analogue thereof, such as single-stranded or double-stranded helical thiophosphates and thioesters. A single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The term nucleic acid molecule, particularly DNA or RNA molecule, refers only to the primary and secondary structures of the molecule and is not limited to any particular tertiary form. A "recombinant DNA molecule" is a DNA molecule that has undergone molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. The "product" disclosed in this invention comprises one or more nucleic acids as described herein.

[0164] Polydeoxyribonucleotides are single or double-stranded polymers consisting of phosphate, deoxyribose, and four bases (adenine, guanine, thymine, and cytosine).

[0165] Polyribonucleotides are single-chain polymers consisting of phosphate, ribose, and four bases (including adenine, guanine, cytosine, and uracil).

[0166] In this article, PDRN refers to polydeoxyribonucleotides and / or polyribonucleotides and their associated salts.

[0167] In this article, PDRN refers to polydeoxyribonucleotides and / or polyribonucleotides and their associated salts with a molecular weight of less than or equal to 1500 kDa.

[0168] In addition, those skilled in the art will recognize that PDRN often exists as a water-soluble salt formed with metal ions, and the PDRN prepared by this invention also covers the case of metal salts of nucleic acid molecules.

[0169] As used herein, the term "physiologically acceptable" means a molecular entity and composition that is physiologically tolerable and, when administered to humans, generally does not produce toxicity or sensitization or similar adverse reactions (such as stomach upset, dizziness, etc.). Optionally, as used herein, the term "physiologically acceptable" means approved by a government regulatory agency or approved in a pharmacopoeia or other generally recognized manner for use in animals or humans.

[0170] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0171] Example 1: Halomonas Halomonas sp. SY-L4 screening and identification process

[0172] In this embodiment, silt samples collected from Chaka Salt Lake in Qinghai Province were dispersed in 100 times their mass of sterile water. The solutions were then evenly spread onto high-salt solid culture medium plates using a spreader and incubated at 33°C for 1-2 days. Single colonies with round and smooth morphology were selected and inoculated onto solid culture medium slant tubes, incubated at 33°C for 1-2 days, and then stored at 4°C after the cells had grown sufficiently. The solid culture medium consisted of: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, 100 g / L NaCl, 20 g / L agar, and the remainder was water.

[0173] The selected bacterial strain was named SY-L4 and sent to Beijing Liuhe BGI Genomics Co., Ltd. for genome sequencing identification. The results showed that the strain was *Haliotis diversicolor*. Halomonas sp. The 16S rDNA gene sequence of this strain is shown in SEQ ID NO.1.

[0174] Halomonas Halomonas sp. SY-L4 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251411, deposited on June 17, 2025, at Wuhan University, Luojia Mountain, Wuchang District, Wuhan, Hubei Province, 430072, China.

[0175] This Halomonas Halomonas sp. The morphological characteristics of SY-L4 are as follows:

[0176] Halomonas Halomonas sp. SY-L4 colonies on solid culture plates have a uniform texture, neat edges, and appear milky tea-colored to the naked eye. The color is uniform, and the surface is smooth and moist. Figure 1 As shown; under a microscope at 1000x magnification, the bacteria are rod-shaped with a simple morphology, as... Figure 2 As shown.

[0177] Example 2: Preparation of Halomonas by fermentation Halomonas sp. Bacterial cells

[0178] In this embodiment, the Halomonas bacteria from the solid test tube in Example 1 are used. Halomonas sp. SY-L4 was washed with 20 times its weight of sterile physiological saline and inoculated into the fermentation medium at a rate of 1%. After fermentation, the cells were collected by centrifugation at 6000 rpm for 5 min. The fermentation medium consisted of 450.0 g / L glucose, 20.0 g / L yeast extract, and 30.0 g / L sodium acetate, with the remainder being water. Glucose was added to the fermentation broth at a constant flow rate from inoculation until 38 h. The culture conditions were: 33℃, stirring at 500 rpm, dissolved oxygen 40%-50%, and pH 7.0, for 40 h. The pH was controlled using NaOH solution.

[0179] Example 3: Purification and preparation of Halomonas bacillus Halomonas sp. PDRN products

[0180] This embodiment provides a *Haloxymonas* species. Halomonas sp. Preparation method of SY-L4 PDRN product:

[0181] The Halomonas obtained in Example 2 Halomonas sp. SY-L4 cells were resuspended in water to OD. 600 After 300 minutes, the cells were ultrasonically disrupted under the following conditions: power 800 W, disruption time 45 min, and disruption temperature 6℃. 1% sodium dodecyl sulfate was added to the bacterial lysate, and the mixture was reacted in a 55℃ water bath for 60 min. The denatured solution was then filtered sequentially through a 1.2 μm precision filter paper and a 0.22 μm polyethersulfone filter membrane to obtain a clear filtrate. 1.2 M sodium chloride solution was added to the filtrate to further precipitate the nucleic acids. The mixture was centrifuged at 6000 rpm for 5 min to obtain a crude product precipitate. The crude product was dissolved in purified water and then precipitated with 4 times its volume of ethanol. The precipitate was then vacuum dried at -0.1 MPa and 30℃ to obtain the PDRN product S1.

[0182] Example 4: Purification and preparation of Halomonas bacillus Halomonas sp. PDRN products

[0183] This embodiment provides a *Haloxymonas* species. Halomonas sp. Preparation method of SY-L4 PDRN product:

[0184] The Halomonas obtained in Example 2 Halomonas sp. SY-L4 cells were resuspended in water to OD. 600 After 300 minutes, the cells were ultrasonically disrupted under the following conditions: power 800 W, disruption time 45 min, disruption temperature 6℃. After disruption, the cells were incubated at 37℃ for 6 h. Then, 1% sodium dodecyl sulfate was added to the bacterial lysate, and the mixture was reacted in a water bath at 55℃ for 60 min. The denatured solution was then filtered sequentially through a 1.2 μm precision filter paper and a 0.22 μm polyethersulfone filter membrane to obtain a clear filtrate. 1.2 M sodium chloride solution was added to the filtrate to further precipitate the nucleic acids. The mixture was centrifuged at 6000 rpm for 5 min to obtain a crude product precipitate. The crude product was dissolved in purified water and then precipitated with 4 volumes of ethanol. The precipitate was then vacuum dried at -0.1 MPa and 30℃ to obtain the PDRN product S2.

[0185] Example 5: Purification and preparation of Halomonas bacillus Halomonas sp. PDRN products

[0186] This embodiment provides a *Haloxymonas* species. Halomonas sp. Preparation method of SY-L4 PDRN product:

[0187] The Halomonas obtained in Example 2 Halomonas sp. SY-L4 cells were resuspended in water to OD. 600 After 300 minutes, the cells were ultrasonically disrupted under the following conditions: power 800 W, disruption time 45 min, disruption temperature 6℃. After disruption, the cells were incubated at 37℃ for 24 h. Then, 1% sodium dodecyl sulfate was added to the lysate, and the mixture was reacted in a water bath at 55℃ for 60 min. The denatured solution was then filtered sequentially through a 1.2 μm precision filter paper and a 0.22 μm polyethersulfone filter membrane to obtain a clear filtrate. 1.2 M sodium chloride solution was added to the filtrate to further precipitate the nucleic acids. The mixture was centrifuged at 6000 rpm for 5 min to obtain a crude product precipitate. The crude product was dissolved in purified water and then precipitated with 4 volumes of ethanol. The precipitate was then vacuum dried at -0.1 MPa and 30℃ to obtain the PDRN product S3.

[0188] Example 6: Purification and preparation of Halomonas bacillus Halomonas sp. PDRN products

[0189] This embodiment provides a *Haloxymonas* species. Halomonas sp.Preparation method of SY-L4 PDRN product:

[0190] The Halomonas obtained in Example 2 Halomonas sp. SY-L4 cells were resuspended in water to OD. 600 After 50 minutes, the cells were ultrasonically disrupted under the following conditions: power 800 W, disruption time 45 min, disruption temperature 6℃. After disruption, the cells were incubated at 37℃ for 6 h. Then, 1% sodium dodecyl sulfate was added to the lysate, and the mixture was reacted in a water bath at 55℃ for 60 min. The denatured solution was then filtered sequentially through a 1.2 μm precision filter paper and a 0.22 μm polyethersulfone filter membrane to obtain a clear filtrate. 1.2 M magnesium chloride solution was added to the filtrate to further precipitate the nucleic acids. The mixture was centrifuged at 6000 rpm for 5 min to obtain a crude product precipitate. The crude product was dissolved in purified water and then precipitated with 4 times its volume of ethanol. The precipitate was then vacuum dried at -0.1 MPa and 30℃ to obtain PDRN product S4.

[0191] Example 7: Purification and Preparation of Halomonas Halomonas sp. PDRN products

[0192] This embodiment provides a *Haloxymonas* species. Halomonas sp. Preparation method of SY-L4 PDRN product:

[0193] The Halomonas obtained in Example 2 Halomonas sp. SY-L4 cells were resuspended in water to OD. 600 After 500 mmol / L, the cells were ultrasonically disrupted under the following conditions: power 800 W, disruption time 45 min, disruption temperature 6℃. After disruption, the cells were incubated at 37℃ for 6 h. Then, 1% sodium dodecyl sulfate was added to the lysate, and the mixture was reacted in a water bath at 55℃ for 60 min. The denatured solution was then filtered sequentially through a 1.2 μm precision filter paper and a 0.22 μm polyethersulfone filter membrane to obtain a clear filtrate. 1.2 M sodium chloride solution was added to the filtrate to further precipitate the nucleic acids. The mixture was centrifuged at 6000 rpm for 5 min to obtain a crude product precipitate. The crude product was dissolved in purified water and then precipitated with 4 volumes of ethanol. The precipitate was then vacuum dried at -0.1 MPa and 30℃ to obtain the PDRN product S5.

[0194] Example 8: Purification and Preparation of Halomonas Halomonas sp. PDRN products

[0195] This embodiment provides a *Haloxymonas* species. Halomonas sp. Preparation method of SY-L4 PDRN product:

[0196] The Halomonas obtained in Example 2 Halomonas sp. SY-L4 cells were resuspended in water to OD. 600After 300 minutes, the cells were ultrasonically disrupted under the following conditions: power 800 W, disruption time 45 min, disruption temperature 6℃. After disruption, the cells were incubated at 37℃ for 6 h. Then, 1% sodium dodecyl sulfate was added to the lysate, and the mixture was reacted in a water bath at 55℃ for 60 min. The denatured solution was then filtered sequentially through a 1.2 μm precision filter paper and a 0.22 μm polyethersulfone filter membrane to obtain a clear filtrate. 0.2 M sodium chloride solution was added to the filtrate to further precipitate the nucleic acids. The mixture was centrifuged at 6000 rpm for 5 min to obtain a crude product precipitate. The crude product was dissolved in purified water and then precipitated with 4 volumes of ethanol. The precipitate was then vacuum dried at -0.1 MPa and 30℃ to obtain the PDRN product S6.

[0197] Example 9: Purification and Preparation of Halomonas Halomonas sp. PDRN products

[0198] This embodiment provides a *Haloxymonas* species. Halomonas sp. Preparation method of SY-L4 PDRN product:

[0199] The Halomonas obtained in Example 2 Halomonas sp. SY-L4 cells were resuspended in water to OD. 600 After 300 minutes, the cells were ultrasonically disrupted under the following conditions: power 800 W, disruption time 45 min, disruption temperature 6℃. After disruption, the cells were incubated at 37℃ for 6 h. Then, 1% sodium dodecyl sulfate was added to the lysate, and the mixture was reacted in a water bath at 55℃ for 60 min. The denatured solution was then filtered sequentially through a 1.2 μm precision filter paper and a 0.22 μm polyethersulfone filter membrane to obtain a clear filtrate. 2 M sodium chloride solution was added to the filtrate to further precipitate the nucleic acids. The mixture was centrifuged at 6000 rpm for 5 min to obtain a crude product precipitate. The crude product was dissolved in purified water and then precipitated with 4 volumes of ethanol. The precipitate was then vacuum dried at -0.1 MPa and 30℃ to obtain the PDRN product S7.

[0200] Example 10: Purification and Preparation of Halomonas Halomonas sp. PDRN products

[0201] This embodiment provides a *Haloxymonas* species. Halomonas sp. Preparation method of SY-L4 PDRN product:

[0202] The Halomonas obtained in Example 2 Halomonas sp. SY-L4 cells were resuspended in water to OD. 600After 300 minutes, the cells were ultrasonically disrupted under the following conditions: power 800 W, disruption time 45 min, disruption temperature 6℃. After disruption, the cells were incubated at 37℃ for 6 h. Then, 1% sodium dodecyl sulfate was added to the lysate, and the mixture was reacted in a water bath at 55℃ for 60 min. The denatured solution was then filtered sequentially through a 1.2 μm precision filter paper and a 0.22 μm polyethersulfone filter membrane to obtain a clear filtrate. 1.2 M magnesium chloride solution was added to the filtrate to further precipitate the nucleic acids. The mixture was centrifuged at 6000 rpm for 5 min to obtain a crude product precipitate. The crude product was dissolved in purified water and then precipitated with 4 volumes of ethanol. The precipitate was then vacuum dried at -0.1 MPa and 30℃ to obtain the PDRN product S8.

[0203] Example 11 Preparation of *Halomycinobacterium salina* Halomonas sp. CGMCC NO. 1.8772 PDRN product

[0204] This embodiment provides a *Halomonas hydrophila*. Halomonas sp. Preparation method of CGMCC NO.1.8772 PDRN product:

[0205] sea ​​salt monocytogenes Halomonas sp. CGMCC NO.1.8772 was purchased from the China General Microbiological Culture Collection Center (CGMCC), with the catalog number CGMCC NO.1.8772.

[0206] (1) Fermentation preparation of cell culture

[0207] Seawater halomonas on solid test tubes Halomonas sp. CGMCC NO.1.8772 was washed with 20 times its weight of sterile physiological saline and inoculated into the fermentation medium at an inoculum volume of 1%. After fermentation, the cells were collected by centrifugation at 6000 rpm for 5 min. The fermentation medium consisted of 450.0 g / L glucose, 20.0 g / L yeast extract, and 30.0 g / L sodium acetate, with the remainder being water. Glucose was added to the fermentation broth at a constant flow rate from the start of inoculation until 38 h. The culture conditions were: 33℃, stirring at 500 rpm, dissolved oxygen 40%-50%, and pH 7.0, for 40 h. The pH was controlled using NaOH solution.

[0208] (2) Purification and preparation of PDRN products

[0209] sea ​​salt monocytogenes Halomonas sp. CGMCC NO.1.8772 bacterial cells were resuspended in water to OD. 600After 300 minutes, the cells were ultrasonically disrupted under the following conditions: power 800 W, disruption time 45 min, disruption temperature 6℃. After disruption, the cells were incubated at 37℃ for 6 h. Then, 1% sodium dodecyl sulfate was added to the lysate, and the mixture was reacted in a water bath at 55℃ for 60 min. The denatured solution was then filtered sequentially through a 1.2 μm precision filter paper and a 0.22 μm polyethersulfone filter membrane to obtain a clear filtrate. 1.2M sodium chloride solution was added to the filtrate to further precipitate the nucleic acids. The mixture was centrifuged at 6000 rpm for 5 min to obtain a crude product precipitate. The crude product was dissolved in purified water and then precipitated with 4 volumes of ethanol. The precipitate was then vacuum dried at -0.1 MPa and 30℃ to obtain the PDRN product S9.

[0210] Comparative Example 1: Saccharomyces cerevisiae Saccharomyces cerevisiae Preparation of PDRN

[0211] This comparative example provides a brewing yeast. Saccharomyces cerevisiae Preparation method of PDRN products:

[0212] brewing yeast Saccharomyces cerevisiae Purchased from China General Microbiological Culture Collection Center (CGMCC), with the number CGMCC NO.2.3854.

[0213] (1) Fermentation preparation of cell culture

[0214] The brewer's yeast on the solid test tube Saccharomyces cerevisiae CGMCC NO.2.3854 was washed with 20 times its weight of sterile physiological saline and inoculated into the fermentation medium at an inoculum volume of 1%. After fermentation, the cells were collected by centrifugation at 6000 rpm for 5 min. The fermentation medium consisted of 450.0 g / L glucose, 20.0 g / L yeast extract, and 40.0 g / L peptone, with the remainder being water. Glucose was added to the fermentation broth at a constant flow rate from the start of inoculation until 38 h. The culture conditions were: 28℃, stirring at 300 rpm, dissolved oxygen 20%-30%, and pH 7.0, for 40 h. The pH was controlled using NaOH solution.

[0215] (2) Purification and preparation of PDRN products

[0216] brewer's yeast Saccharomyces cerevisiae CGMCC NO.2.3854 cells were resuspended in water to OD. 600After 300 minutes, the cells were ultrasonically disrupted under the following conditions: power 800 W, disruption time 45 min, disruption temperature 6℃. After disruption, the cells were incubated at 37℃ for 6 h. Then, 1% sodium dodecyl sulfate was added to the bacterial lysate, and the mixture was reacted in a water bath at 55℃ for 60 min. The denatured solution was then filtered sequentially through a 1.2 μm precision filter paper and a 0.22 μm polyethersulfone filter membrane to obtain a clear filtrate. 1.2 M sodium chloride solution was added to the filtrate to further precipitate the nucleic acids. The mixture was centrifuged at 6000 rpm for 5 min to obtain a crude product precipitate. The crude product was dissolved in purified water and then precipitated with 4 volumes of ethanol. The precipitate was then vacuum dried at -0.1 MPa and 30℃ to obtain the PDRN product C1.

[0217] Comparative Example 2: Lactobacillus plantarum Lactobacillus plantarum Preparation of PDRN

[0218] This embodiment provides a *Lactobacillus plantarum*. Lactobacillus plantarum Preparation method of PDRN products:

[0219] Lactobacillus plantarum Lactobacillus plantarum Purchased from China General Microbiological Culture Collection Center (CGMCC), with the number CGMCC NO.1.3921.

[0220] (1) Fermentation preparation of cell culture

[0221] Lactobacillus plantarum on solid test tubes Lactobacillus plantarum CGMCC NO.1.3921 was washed with 20 times its weight of sterile physiological saline and inoculated into the fermentation medium at an inoculum volume of 1%. After fermentation, the cells were collected by centrifugation at 6000 rpm for 5 min. The fermentation medium consisted of: glucose 200.0 g / L, peptone 30 g / L, yeast extract 20.0 g / L, sodium acetate 30.0 g / L, diammonium hydrogen citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.1 g / L, with the remainder being water. Glucose was added to the fermentation broth at a constant flow rate from the start of inoculation until 38 h. The culture conditions were: 37℃, stirring at 200 rpm, dissolved oxygen 20%-30%, and pH 5.0, for 40 h. The pH was controlled using NaOH solution.

[0222] (2) Purification and preparation of PDRN products

[0223] Lactobacillus plantarum Lactobacillus plantarum CGMCC NO.1.3921 bacterial cells were resuspended in water to OD. 600After 300 minutes, the cells were ultrasonically disrupted under the following conditions: power 800 W, disruption time 45 min, disruption temperature 6℃. After disruption, the cells were incubated at 37℃ for 6 h. Then, 1% sodium dodecyl sulfate was added to the lysate, and the mixture was reacted in a water bath at 55℃ for 60 min. The denatured solution was then filtered sequentially through a 1.2 μm precision filter paper and a 0.22 μm polyethersulfone filter membrane to obtain a clear filtrate. 1.2 M sodium chloride solution was added to the filtrate to further precipitate the nucleic acids. The mixture was centrifuged at 6000 rpm for 5 min to obtain a crude product precipitate. The crude product was dissolved in purified water and then precipitated with 4 volumes of ethanol. The precipitate was then vacuum dried at -0.1 MPa and 30℃ to obtain the PDRN product C2.

[0224] Test case

[0225] The PDRN products S1-S9 and C1-C2 prepared in Examples 3-11 and Comparative Examples 1 and 2 were used as samples for the following experiments.

[0226] I. Testing of Physicochemical Properties

[0227] 1. Appearance

[0228] By visual inspection, the PDRN product should appear as white or off-white granules or powder. As shown in Table 1, the PDRN prepared by the above methods all meet the appearance requirements.

[0229] Table 1 Appearance characteristics of different samples

[0230]

[0231] 2. DNA content

[0232] Accurately weigh 1.0 g of crystalline diphenylamine, dissolve it in 100 mL of analytical grade glacial acetic acid, and add 10 mL of 60% perchloric acid and mix well. Add 1 mL of 1.6% acetaldehyde solution immediately before use. This solvent should be colorless. Accurately weigh 100 mg of calf thymus DNA, dissolve it in 0.1 M KOH and bring the volume to 500 mL, resulting in a final concentration of 200 mg / L. Prepare 8 stoppered test tubes. Add the volumes of DNA standard solution, purified water, and diphenylamine reagent shown in Table 2 to each tube sequentially. After shaking and mixing, heat at 60°C for 1 h. After cooling, measure the absorbance at 595 nm using a UV spectrophotometer. Plot a standard curve based on the absorbance and the DNA concentration in the standard. Figure 3 As shown.

[0233] Table 2 Reagent Addition Ratio

[0234]

[0235] Accurately weigh 100 mg of sample, dissolve it in purified water and bring the volume to 200 mL to obtain the sample solution.

[0236] Each group consisted of three test tubes. The first group served as a blank control, labeled tube 0, while the others were experimental groups. 1 mL of sample solution (an equal volume of water was added to the blank control) and 2 mL of diphenylamine reagent were added to each group. The mixture was incubated at 60°C for 1 h. After cooling, the absorbance of the DNA was measured at 595 nm, with the blank control group used to adjust the zero point. Based on the measured absorbance, the corresponding nucleic acid / DNA content was determined from the standard curve. The formula for calculating the DNA content is as follows, and the results are shown in Table 3.

[0237] DNA content (%) = ;

[0238] Loss on drying: Take about 1.0 g of this product and place it in the tray of a halogen moisture analyzer. Measure at 110℃ for 15 min, and record the result as h (%) as automatically printed by the instrument. Perform two parallel determinations and report the average of the two parallel determinations (deviation requirement: RD% = |(X1-X2) / (X1+X2)| × 100%, the relative deviation between the two parallel determinations should not exceed 5%).

[0239] Table 3 DNA content of different samples

[0240]

[0241] 3. pH value

[0242] Weigh 0.1 g of the sample (accurate to 0.01 g) precisely into a beaker, add freshly boiled and cooled water to a final volume of 100 g, seal the beaker, and stir magnetically until dissolved. This is the test solution. Wash the electrode with water, blot dry with filter paper, insert the electrode into the test solution, start the stirrer, and wait for the pH meter reading to stabilize for 1 minute. Then stop the stirrer and read the pH value directly from the instrument. The pH values ​​of different samples are shown in Table 4.

[0243] Table 4 pH values ​​of different samples

[0244]

[0245] 4. Total nucleic acid content detection

[0246] Accurately weigh the sample, dissolve it in purified water, and quantitatively dilute it to prepare a test solution containing approximately 0.04 mg per 1 mL.

[0247] Take the test solution and use an ultra-micro UV spectrophotometer to measure the absorbance of the reconstituted sample at 260 nm. Determine the PDRN nucleic acid content in the product based on the results fed back by the instrument.

[0248] For standard samples, when A 260 When =1: the total nucleic acid concentration is approximately 50 µg / mL.

[0249] Total nucleic acid content (%) = ;

[0250] Loss on drying: Take about 1.0 g of this product and place it in the tray of a halogen moisture analyzer. Measure at 110℃ for 15 min, and record the result as h (%) as automatically printed by the instrument. Perform two parallel determinations and report the average of the two parallel determinations (deviation requirement: RD% = |(X1-X2) / (X1+X2)| × 100%, the relative deviation between the two parallel determinations should not exceed 5%).

[0251] Take the test solution and measure the absorbance at 260 nm.

[0252] Table 5 Total nucleic acid content of different samples

[0253]

[0254] 5. Molecular weight

[0255] (1) Instruments

[0256] Electronic balance (accuracy 0.01 g and 0.1 mg), DAWN multi-angle laser light scattering instrument-high performance liquid chromatography system.

[0257] (2) Chromatographic conditions

[0258] Chromatographic column: SRT SEC-500 liquid chromatography column, 7.8 × 300 mm 5 μm;

[0259] Column temperature: 35℃;

[0260] Mobile phase: 0.05 mol / L sodium chloride solution (containing 0.2% ProClin 200);

[0261] Flow rate: 0.5 mL / min.

[0262] (3) Reagents

[0263] Sodium chloride (analytical grade), ProClin 200 (preservative).

[0264] (4) Solution preparation

[0265] Mobile phase: Weigh 2.92 g of sodium chloride, measure 0.2 mL of ProClin 200, add water to dissolve, and dilute to 1000 mL. Filter through a 0.22 μm filter membrane to obtain a 0.05 mol / L sodium chloride solution.

[0266] Test solution: Weigh 5 mg of sample and dissolve it in 10 mL of pure water to prepare a concentrated solution. Dilute the concentrated solution with the mobile phase to a sample content of 0.1 mg / mL, shake well, and prepare two parallel solutions as test solutions.

[0267] (5) Measurement

[0268] Determination of test solutions: Take 500 μL of each of the two test solutions, inject each once, and determine using the same method. Record the chromatogram.

[0269] (6) Data processing

[0270] The weight-average molecular weight and molecular weight distribution of the test sample were calculated using specialized software. The refractive index increment (dn / dc) of the polydeoxyribonucleotide was calculated to be 0.158, and the results are shown in Table 6.

[0271] Table 6. Molecular weight of different samples

[0272]

[0273] II. Effect Evaluation

[0274] 1. Cytotoxicity evaluation

[0275] Plating: HaCaT cells in logarithmic growth phase were used, at a concentration of 1×10⁻⁶ cells / cells. 5 Cells were seeded at a density of 100 μL / mL in 96-well plates. The culture medium was DMEM high glucose medium with 10% serum added. The seeded cells were incubated overnight in a CO2 incubator at 37°C with 5% CO2.

[0276] Sample solution preparation: Dissolve the sample in serum-free DMEM high-glucose medium to prepare sample solutions of different concentrations. Filter the solutions through a 0.22 μm filter membrane for sterilization and use immediately.

[0277] Drug administration: After 24 h of routine culture, the old culture medium was discarded. The experimental group was replaced with 100 μL of sample solution, and the negative control group was given an equal volume of serum-free DMEM high glucose medium. There were 6 parallel wells in each level.

[0278] Assay: After culturing for another 24 h, the relative cell proliferation rate was detected using the CCK-8 assay. The culture medium was discarded, and 100 μL of CCK-8 diluted 10-fold with serum-free DMEM high-glucose medium was added to each well. The cells were then incubated in a cell culture incubator for another 2 h, and the absorbance was measured at 450 nm using a microplate reader. The relative proliferation rate (RGR) was the ratio of the absorbance of the experimental group to that of the negative control group (NC). The results are shown in Table 7.

[0279] Table 7. Relative cell proliferation rate (%)

[0280]

[0281] Note: * indicates p < 0.05 compared to the NC group; ** indicates p < 0.01 compared to the NC group; *** indicates p < 0.001 compared to the NC group.

[0282] According to the standard of a relative proliferation rate of 70% or higher, the cells were considered to be non-cytotoxic. The results are shown in Table 7. The cell proliferation rate of the negative control group (NC) was set at 100%. All samples in each group showed no potential cytotoxicity within the experimental concentration range. The concentrations of 0.01% and 0.05% were selected for subsequent experiments.

[0283] 2. Evaluation of the efficacy in promoting VEGF production

[0284] Plating: HaCaT cells in logarithmic growth phase were plated at a density of 2 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of 1 / mL in 12-well plates, with 1 mL per well. The culture medium was DMEM high-glucose medium supplemented with 10% fetal bovine serum. The seeded cells were incubated overnight in a CO2 incubator at 37°C with 5% CO2.

[0285] Sample solution preparation: The sample was prepared into a 0.2% stock solution using serum-free DMEM high glucose medium, and diluted to 0.05% before use. It should be prepared and used immediately.

[0286] Drug administration: After 24 h of routine culture, the old culture medium was discarded. 1 mL of sample solution was added to the experimental group, an equal volume of serum-free DMEM high glucose medium was added to the negative control group (NC), and 100 ng / mL of TGF-β1 prepared with serum-free high glucose DMEM medium was added to the positive control group (PC). Culture was continued for 24 h.

[0287] ELISA assay: After incubation, the cell culture supernatant was collected, and the VEGF content was determined according to the human VEGF ELISA kit. The results are shown in Table 8.

[0288] Table 8. Effects of samples on VEGF secretion in HaCaT cells

[0289]

[0290] Note: ** indicates p < 0.01 compared to NC; *** indicates p < 0.001 compared to NC; & indicates p < 0.05 compared to S2; && indicates p < 0.01 compared to S2; &&& indicates p < 0.001 compared to S2.

[0291] The active expression of cytokines in wound repair makes them a new reference parameter for inferring damage levels, serving as a molecular-level "living response" indicator in in vivo injuries. VEGF is a key factor in the late inflammatory and proliferative phases of wound healing, playing a crucial role in granulation tissue formation, wound healing, angiogenesis, collagen fiber synthesis, and the repair of hair follicles and skin appendages. Therefore, detecting VEGF expression levels can effectively reflect the progress of cellular repair.

[0292] The effect of PDRN on VEGF secretion in HaCaT cells was investigated. The samples were applied to HaCaT cells, and the results are shown in Table 8. TGF-β1 significantly increased VEGF secretion. All samples within the experimental concentration range significantly increased VEGF secretion. Samples S2 and S9 showed significantly better VEGF-promoting effects than the control group samples. Therefore, the PDRN of this invention has a good effect on promoting VEGF production.

[0293] 3. Evaluation of DNA damage repair efficacy

[0294] Plating: HaCaT cells in logarithmic growth phase were plated at a concentration of 2.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 / mL in 12-well plates, with 1 mL per well. The culture medium was DMEM with 10% fetal bovine serum added. The seeded cells were incubated in a CO2 incubator at 37°C and 5% CO2 for 24 h.

[0295] Irradiation: Remove the lid of the culture plate, cover with plastic wrap, and irradiate the model group (MC) and sample group with UVB at 700 µw / cm². 2 The subjects were irradiated with the specified intensity for 6 minutes, while the negative control group (NC) was covered with aluminum foil and not irradiated.

[0296] Sample solution preparation: Prepare a 0.2% stock solution of the sample using serum-free DMEM medium, and dilute to 0.05% before use. Prepare fresh solution immediately before use.

[0297] Drug administration: After irradiation, discard the old culture medium, replace the experimental group with 1 mL of sample solution, and add an equal volume of serum-free DMEM culture medium to the negative control group. Place in an incubator and continue culturing for 24 h.

[0298] Detection: Cells were collected, and total DNA was extracted using a universal column-based genomic DNA extraction kit. The CPD-DNA content was determined using a human CPD ELISA kit. The results are shown in Table 9.

[0299] Table 9. Effects of samples on CPD-DNA generation

[0300]

[0301] Note: ### indicates p < 0.001 compared to the NC group; * indicates p < 0.05 compared to the model group; ** indicates p < 0.01 compared to the model group; *** indicates p < 0.001 compared to the model group; & indicates p < 0.05 compared to S2; && indicates p < 0.01 compared to S2; &&& indicates p < 0.001 compared to S2.

[0302] Cell proliferation and survival depend on the stable transmission of genetic information, but DNA damage factors prevalent in the intracellular and external environments can disrupt this stability. One mechanism by which ultraviolet radiation damages skin is by damaging cellular DNA, forming "sunburned cells," and inducing a variety of variations in intracellular DNA. The most significant variation is the production of cyclobutane pyrimidine dimers (CPD). Therefore, testing changes in CPD-DNA levels in cells can directly observe the progress of cellular UV damage repair.

[0303] The results are shown in Table 9. After UVB irradiation, the CPD-DNA content in the model group of HaCaT cells was significantly higher than that in the negative control group, indicating that the DNA damage model was successfully established. Compared with the model group, samples S2, S3, and S9 could inhibit CPD-DNA production within the experimental concentration range, demonstrating a repair effect on cellular DNA damage; samples C1 and C2 could not inhibit CPD-DNA production and had no DNA damage repair effect. Therefore, the PDRN of this invention has good DNA loss repair efficacy.

[0304] 4. Evaluation of the efficacy in promoting type I collagen

[0305] Plating: Take HSF cells in logarithmic growth phase, digest them with trypsin to make the cells round, add an appropriate amount of serum-containing DMEM medium to stop digestion, and adjust the cell density to 1×10⁶. 5 Cells were seeded at a density of 1 mL / well in 24-well plates, ensuring even distribution at the bottom of the plates. The seeded cells were then incubated at 37°C with 5% CO2 for 24 hours.

[0306] Sample solution preparation: Prepare a 0.2% stock solution of the sample using serum-free DMEM medium, and dilute to 0.05% before use. Prepare fresh solution immediately before use.

[0307] Drug administration: After 24 h of routine culture, the culture medium in the well plate was discarded. The experimental group was given sample solution, and the negative control group (NC) was given an equal volume of serum-free DMEM medium. The plates were then incubated in a CO2 incubator for another 24 h. A positive control group (PC) was set up, which was given 100 ng / mL of TGF prepared with serum-free DMEM medium.

[0308] Supernatant collection: After the incubation and culture are completed, collect the cell culture supernatant for later use.

[0309] ELISA assay: The content of Pro-Collagen I alpha 1 (procollagen I α-1) in the supernatant was determined using the human Pro-Collagen I alpha 1 ELISA kit. The results are shown in Table 10.

[0310] Table 10 Effects of the test samples on type I collagen production

[0311]

[0312] Note: * indicates p < 0.05 compared to the NC group; ** indicates p < 0.01 compared to the NC group; & indicates p < 0.05 compared to S2; && indicates p < 0.01 compared to S2; &&& indicates p < 0.001 compared to S2.

[0313] Pro-Collagen I alpha 1 is the precursor form of type I collagen. Both are different stages of the same substance. Therefore, by measuring the content of Pro-Collagen I alpha 1, the secretion of type I collagen can be determined.

[0314] The results are shown in Table 10. Experimental results showed that samples S2 and S9 at a concentration of 0.05% significantly promoted the secretion of type I collagen; samples C1 and C2 did not significantly promote type I collagen secretion. Therefore, the PDRN of this invention has a significant effect on promoting type I collagen production.

[0315] 5. Evaluation of the efficacy in promoting type III collagen

[0316] Spreading: Take the logarithmic growth phase HSF, with 1×10 5 Cells were seeded at a density of 1 mL / well in 24-well plates. The culture medium was DMEM with 15% fetal bovine serum added. The seeded cells were incubated in a CO2 incubator at 37°C and 5% CO2 for 24 hours.

[0317] Sample solution preparation: Prepare a 0.2% stock solution of the sample using serum-free DMEM medium, and dilute to 0.05% before use. Prepare fresh solution immediately before use.

[0318] Drug addition: After 24 h of routine culture, the old culture medium was discarded, and the experimental group was replaced with 1 mL of sample solution. The negative control group (NC) was given an equal volume of serum-free DMEM medium, and 100 ng / mL TGF prepared with serum-free DMEM medium was added as a positive control (PC). The culture was continued for 24 h.

[0319] Supernatant collection: After the incubation and culture are completed, collect the cell culture supernatant for later use.

[0320] ELISA assay: The COL3A1 content in the supernatant was determined using the human type III collagen (Col III) enzyme-linked immunosorbent assay kit.

[0321] Table 11 Effects of the test samples on type III collagen formation

[0322]

[0323] Note: * indicates p < 0.05 compared to the NC group; ** indicates p < 0.01 compared to the NC group; *** indicates p < 0.001 compared to the NC group; & indicates p < 0.05 compared to S2; && indicates p < 0.01 compared to S2; &&& indicates p < 0.001 compared to S2.

[0324] The results are shown in Table 11. The S2 sample showed significantly higher efficacy in promoting type III collagen production compared to the comparative sample. Therefore, the PDRN of this invention has a better effect on promoting type III collagen production.

[0325] 6. Evaluation of antioxidant efficacy

[0326] Spreading: Take HaCaT in the logarithmic growth phase, at 2.5 × 10⁻⁶ 5 Cells were seeded at a density of 1 / mL in 12-well plates, with 1 mL per well. The culture medium was DMEM with 10% fetal bovine serum added. The seeded cells were incubated in a CO2 incubator at 37°C and 5% CO2 for 24 h.

[0327] Sample solution preparation: The sample was prepared into a 0.2% stock solution using serum-free DMEM medium, and diluted to 0.01% or 0.05% before use. The solution was prepared and used immediately.

[0328] Preparation of positive control solution: Prepare a 1% VC solution using serum-free DMEM cell culture medium, and sterilize by filtration through a 0.22 µm filter membrane. Prepare fresh and use immediately, diluting before use.

[0329] Preparation of dichlorofluorescein diacetate (DCFH-DA) probe solution: dilute at a ratio of 0.375 µL DCFH-DA: 1 mL PBS.

[0330] Drug addition: After 24 h of routine culture, the old culture medium was discarded, and 1 mL of sample solution was added to each experimental group to culture the cells. The oxidative model group and the negative control group (NC) were added with an equal volume of serum-free DMEM medium, and the positive control group (PC) was added with VC solution (0.01%). The cells were cultured for another 24 h.

[0331] Oxidation model establishment: 24 h later, both the oxidation model group (MC) and the drug-treated group (positive control group and experimental group) were given H2O2 solution with a final concentration of 600 μmol / L and oxidized for 4 h.

[0332] Flow cytometry analysis: After culture, discard all culture medium and wash twice with PBS. Add 1.5 mL of DCFH-DA to each well and incubate for 30 min in a cell culture incubator, mixing every 5 min to ensure probe binding. Discard the probe, wash twice with preheated serum-free DMEM medium, add 1 mL of serum-free DMEM medium to each well, and incubate at 37°C for 10 min. After washing once with PBS, trypsin digest the cells, wash twice with PBS, resuspend in 300 μL PBS, and analyze using flow cytometry (FITC-A channel). Collect 10,000 cells per sample. Calculate the average fluorescence intensity and ROS inhibition rate. The ROS inhibition rate is calculated using the following formula, and the results are shown in Table 12.

[0333] ROS inhibition rate = (1 - (average fluorescence intensity of experimental group / average fluorescence intensity of model group)) × 100%.

[0334] Table 12 Effect of samples on reactive oxygen species (ROS) content

[0335]

[0336] Note: ### indicates p<0.001 compared to NC; * indicates p<0.05 compared to the model group; ** indicates p<0.01 compared to the model group; *** indicates p<0.001 compared to the model group.

[0337] The results are shown in Table 12. The average fluorescence intensity of ROS in the model group was significantly higher than that in the negative control group, indicating that the H2O2-induced ROS model was successfully established. Compared with the model group (MC), the positive control group (VC) significantly inhibited ROS generation. Samples S2 and S9 significantly inhibited ROS generation within the experimental concentration range, demonstrating antioxidant effects. Therefore, the PDRN of this invention has good antioxidant effects.

[0338] 7. Evaluation of UV damage repair efficacy

[0339] Plating: HaCaT cells in the logarithmic growth phase were harvested, digested with trypsin, and then the cell density was adjusted to 1×10⁻⁶. 5 / mL, seeded into 96-well cell culture plates, 100 μL of cell suspension per well, cultured in DMEM medium supplemented with 15% fetal bovine serum, and incubated overnight at 37°C with 5% CO2.

[0340] Irradiation: Remove the lid from the 96-well cell culture plate, wrap it with plastic wrap, and cover the negative control group (NC) with aluminum foil to avoid irradiation. Place the plate under a UV lamp at 2000 μW / cm². 2 30 min of UVA irradiation at 700 μW / cm 2 Cells were irradiated with UVB at high intensity for 6 minutes to obtain a cell model.

[0341] Sample solution preparation: The sample is prepared into a 0.2% stock solution using serum-free DMEM medium, and diluted to 0.01% or 0.05% before use. Prepare fresh solution immediately before use.

[0342] Drug addition: After the irradiation of the experimental group, the old culture medium was discarded and 100 μL of sample solution was added to each well. The old culture medium of the negative group and the model group (MC) was discarded and 100 μL of fresh serum-free DMEM medium was added. The wells were then placed in an incubator and cultured for another 24 h.

[0343] Detection: Discard the old culture medium, add 100 μL of CCK-8 diluted 10-fold with serum-free DMEM medium to each well, and incubate in a cell culture incubator for 2 h. Measure the absorbance at 450 nm using a microplate reader. The relative cell proliferation rate is the ratio of the absorbance of the experimental group to that of the negative control group (NC). Calculate the relative cell proliferation rate. The results are shown in Table 13.

[0344] Table 13. Repair effect of test samples on HaCaT cells after UV irradiation

[0345]

[0346] Note: ### indicates p < 0.001 compared to the NC group; * indicates p < 0.05 compared to the MC group; ** indicates p < 0.01 compared to the MC group; *** indicates p < 0.001 compared to the MC group.

[0347] After combined UVA / UVB irradiation, the cell proliferation rate in the model group decreased by 22.82% compared to the NC group, a significant difference, indicating successful damage modeling. The results are shown in Table 13. Compared to the UV model group, samples S2 and S9 significantly promoted cell proliferation within the experimental concentration range, demonstrating UV damage repair efficacy, with lower concentrations showing better repair effects. Therefore, the PDRN of this invention exhibits good UV damage repair efficacy even at low concentrations.

[0348] 8. Evaluation of Human Efficacy

[0349] Mix the S2 sample evenly according to the formula in Table 14 below to prepare an essence for human efficacy testing.

[0350] Table 14 Serum Formula

[0351]

[0352] Sample usage instructions:

[0353] Sample group: After cleansing, take an appropriate amount of product in the palm of your hand, apply it evenly to the face (half face), and massage gently until fully absorbed;

[0354] Placebo: After cleansing, take an appropriate amount of product in the palm of your hand, apply it evenly to the face (half face), and massage gently until fully absorbed.

[0355] Detection method:

[0356] (1) Moisturizing effect: The moisture content of the stratum corneum of the skin was measured using an indirect measurement method with a Corneometer CM825. Data were collected three times and the average value was taken.

[0357] (2) Repair efficacy: The transepidermal water loss rate of the skin was measured using an indirect measurement method with a Tewameter™ Hex meter. Data were collected three times and the average value was taken.

[0358] (3) Firming effect: The indirect measurement method was used, and the skin elasticity parameter R2 value / skin elasticity parameter R5 value was measured using a skin elasticity tester (Cutometer®MPA580Probe). Data were collected twice and the average value was taken.

[0359] (4) Anti-wrinkle effect: The indirect measurement method was used to collect facial images using the Antera3D®CS3.0 skin analysis system and analyze the maximum depth of the nasolabial folds.

[0360] Test results:

[0361] (1) Changes in skin moisturizing parameters

[0362] The results are shown in Table 15. After 30 min of sample use, compared with baseline (skin condition before sample use), the stratum corneum moisture content significantly increased by 37.76% (p<0.001); and compared with placebo (difference), the stratum corneum moisture content significantly increased by 15.54% (p<0.001). After 2 h of sample use, compared with baseline, the stratum corneum moisture content significantly increased by 33.04% (p<0.001); and compared with placebo (difference), the stratum corneum moisture content significantly increased by 14.14% (p<0.001). After 4 h of sample use, compared with baseline, the stratum corneum moisture content significantly increased by 32.02% (p<0.001); and compared with placebo (difference), the stratum corneum moisture content significantly increased by 14.45% (p<0.001). After 6 h of sample use… Compared with baseline, the stratum corneum moisture content increased significantly by 29.48% (p<0.001); and compared with placebo (difference), the stratum corneum moisture content increased significantly by 13.93% (p<0.001). Therefore, under the study conditions, product S2 is considered to have moisturizing effects.

[0363] Table 15 Changes in stratum corneum moisture content before and after sample use

[0364]

[0365] (2) Changes in skin repair parameters

[0366] The results are shown in Table 16: After 30 min of sample use, compared with baseline (skin condition before sample use), the transepidermal water loss rate was significantly reduced by 8.35% (p<0.001); and compared with placebo (difference), the transepidermal water loss rate was significantly reduced by 8.55% (p<0.001); after 2 h of sample use, compared with baseline, the transepidermal water loss rate was significantly reduced by 9.19% (p<0.001); and compared with placebo (difference), the transepidermal water loss rate was significantly reduced by 8.90% (p<0.001); after 4 h of sample use, compared with baseline, the transepidermal water loss rate was significantly reduced by 11.03% (p<0.001); and compared with placebo (difference), the transepidermal water loss rate was significantly reduced by 6.56% (p<0.001); after 6 h of sample use… Compared with baseline, the transepidermal water loss rate was significantly reduced by 14.30% (p<0.001); and compared with placebo (difference), the transepidermal water loss rate was significantly reduced by 9.25% (p<0.001). Therefore, under the study conditions, product S2 is considered to have repair efficacy.

[0367] Table 16 Changes in transepidermal water loss rate before and after sample use.

[0368]

[0369] (3) Skin elasticity parameter R5 value

[0370] The results are shown in Table 17: Compared with baseline (skin condition before sample use) 2 hours after sample use, the skin elasticity parameter R5 value increased significantly by 5.76% ( p <0.050); and compared with placebo (difference), the skin elasticity parameter R5 value increased significantly by 9.81% ( p <0.050); After 4 hours of sample use compared with baseline, the R5 value of the skin elasticity parameter increased significantly by 7.97% ( p <0.050); and compared with placebo (difference), the skin elasticity parameter R5 value increased significantly by 11.59% ( p <0.050); Compared with baseline after 6 hours of sample use, the R5 value of skin elasticity parameter increased significantly by 7.47% ( p <0.050). Therefore, under the study conditions, it is concluded that the tested product S2 has a firming effect.

[0371] Table 17 Changes in the R5 value of skin elasticity parameter before and after using the sample.

[0372]

[0373] (4) Changes in skin anti-wrinkle parameters

[0374] The results are shown in Table 18: After 30 min of sample use, compared with baseline (skin condition before sample use), the maximum depth of nasolabial folds significantly decreased by 20.24% (p<0.050); and compared with placebo (difference), the maximum depth of nasolabial folds significantly decreased by 28.84% (p<0.050); after 2 h of sample use, compared with baseline, the maximum depth of nasolabial folds significantly decreased by 27.54% (p<0.001); and compared with placebo (difference), the maximum depth of nasolabial folds significantly decreased by 34.53% (p<0.001); after 4 h of sample use, compared with baseline, the maximum depth of nasolabial folds significantly decreased by 28.50% (p<0.001); and compared with placebo (difference), the maximum depth of nasolabial folds significantly decreased by 30.12% (p<0.010); after 6 h of sample use… Compared with baseline, the maximum depth of nasolabial folds was significantly reduced by 35.27% (p<0.001); and compared with placebo (difference), the maximum depth of nasolabial folds was significantly reduced by 45.48% (p<0.001). Therefore, under the study conditions, product S2 is considered to have anti-wrinkle efficacy.

[0375] Table 18. Maximum change in nasolabial fold depth before and after using the sample.

[0376]

[0377] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A strain of Halomonas Halomonas sp. SY-L4 is characterized by, It is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20251411.

2. Containing the *Halomonas* strain of claim 1 Halomonas sp. Bacterial agents.

3. A method for preparing PDRN, characterized in that, The method includes: Step 1: Collecting Halomonas bacteria Halomonas sp. Bacterial cells were resuspended and then disrupted to obtain a bacterial lysate; the disruption was performed by ultrasonic disruption. Step 2: Add a protein denaturant to the bacterial cell lysate and filter to obtain the filtrate; the protein denaturant is sodium dodecyl sulfate. Step 3: Add a metal salt or ammonium sulfate and an organic solvent to the filtrate to precipitate and prepare a crude product; the final concentration of the metal salt or ammonium sulfate added is 0.1-5 M; the metal salt includes one or more of magnesium chloride, sodium chloride, calcium chloride, potassium chloride, zinc chloride, potassium acetate, sodium acetate, and sodium sulfate; Step 4: Dry the crude product to obtain PDRN; The Halomonas Halomonas sp. Including: Halomonas Halomonas sp. SY-L4, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251411, and / or *Halomonas*. Halomonas sp. CGMCC NO.1.8772, purchased from the China General Microbiological Culture Collection Center, with the number CGMCC NO.1.8772.

4. The method according to claim 3, characterized in that, Step one also includes incubating the bacterial lysis buffer at 30℃-40℃ for 0-24 h.

5. PDRN prepared by the method according to any one of claims 3-4.

6. A product characterized in that, The product includes the PDRN as described in claim 5.

7. Halomonas Halomonas sp. Its application in the preparation or production of PDRN is characterized by, The Halomonas Halomonas sp. Including: Halomonas Halomonas sp. SY-L4, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251411, and / or *Halomonas*. Halomonas sp. CGMCC NO.1.8772, purchased from the China General Microbiological Culture Collection Center, with the number CGMCC NO.1.8772.

8. The use of the Halomonas bacillus as described in claim 1, the bacterial agent as described in claim 2, the PDRN as described in claim 5, or the product as described in claim 6 in any one or more of the following A1)-A7): A1) Application in the preparation of products for skin repair and / or improvement of skin inflammation and / or skin wound healing; A2) Application in the preparation of products for repairing damaged skin barriers; A3) Applications in the preparation of antioxidant products; A4) Applications in the preparation of moisturizing products; A5) Application in the preparation of products that improve skin elasticity; A6) Application in the preparation of skin-firming products; A7) Application in the preparation of anti-wrinkle products.

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