PDRN as well as preparation method and application thereof

Abstract

The invention provides PDRN as well as a preparation method and application thereof, and relates to the technical field of biology. And the GC content of the PDRN is 40.5%-42%. According to the present invention, the similarity between the GC% in the gDNA in the PDRN prepared by using the recorded method and the GC% of the human body is more than 98%, the GC% is closer to the GC% of the human body than the PDRN derived from the salmon, the safety and the efficiency are high, and the PDRN preparation method is simple and convenient compared with the PDRN extracted from the salmon semen in the prior art.

Description

[0001] This application claims the priority of a Chinese patent application titled "A PDRN and Its Preparation Method and Application" with the application number "202410195149.3" filed with the Chinese Patent Office on February 22, 2024, the entire content of which is incorporated herein by reference. Technical Field

[0002] This application relates to the field of biotechnology, and particularly to a PDRN and its preparation method and application. Background Art

[0003] PDRN is a type of biological macromolecular substance derived from salmon sperm cells, which is composed of multiple base pairs connected by phosphodiester bonds. Its essence is a mixed product composed of salmon genetic materials with different fragment sizes. As early as 1952, Italian company Mastelli extracted different-sized polynucleotide fragments (PDRN) from salmon sperm cells based on the phenomenon that fishermen used salmon sperm to treat wounds, and studied its pharmacological activities and other related properties. The experimental results showed that nucleic acid fragments (PDRN) in male salmon sperm cells could significantly promote human cell regeneration, rapidly promote wound healing and reduce scar formation. In subsequent continuous research, PDRN was successively found to have anti-inflammatory, tissue repair, angiogenesis promotion, anti-ischemia, and improvement of diabetic foot effects.

[0004] Modern industry mostly extracts PDRN raw materials from the sperm cells of deep-sea salmon. For example, the method of separating polydeoxyribonucleotides from fish semen disclosed in patent CN107287186A, the preparation and application of a small molecule polydeoxyribonucleotide disclosed in patent CN110747194A, the preparation method and application of an efficient topical PDRN disclosed in patent CN112315836A, and the preparation method and application of a polydeoxyribonucleotide disclosed in patent CN115074357A, etc. Although the above processes have been the main ways to obtain PDRN raw materials, with the continuous depletion of marine fishery resources, it has become difficult to extract PDRN from salmon sperm cells to meet the increasingly strong demand for PDRN raw materials in the new era. In addition, the fuel leakage, nuclear wastewater discharge brought by the passing large ships on the ocean routes, and the disposal of domestic waste in coastal areas have all had an adverse impact on the marine environment. The resulting problems such as heavy metal enrichment, chromosomal aberration, and gene mutation of marine organisms have also had a great impact on the safety of salmon sperm PDRN raw materials.

[0005] In summary, there is an urgent need to develop multiple PDRN production routes to solve the problems of single source and poor safety. Summary of the Invention

[0006] This application first prepares PDRN from multiple species (microorganisms, algae, animals), which not only expands the sources of PDRN products but also solves the problem of poor safety of PDRN caused by marine environmental pollution, and better controls the quality of raw materials for PDRN extraction.

[0007] On the one hand, this application provides a kind of PDRN, and the GC content of the PDRN is 40.5%-42%.

[0008] Preferably, the GC content in the PDRN can be selected from 40.5%, 40.6%, 40.7%, 40.8%, 40.9%, 41%, 41.1%, 41.2%, 41.3%, 41.4%, 41.5%, 41.6%, 41.7%, 41.8%, 41.9%, 42% and any value therebetween.

[0009] Preferably, 40.5-42%

[0010] More preferably, 40.5%-41.5%.

[0011] Furthermore, the molecular weight of the PDRN is 100 kDa - 500 kDa.

[0012] Preferably, the molecular weight of the PDRN is 100 kDa - 300 kDa;

[0013] Preferably, the molecular weight of the PDRN can be selected from 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, 500 kDa and any value therebetween.

[0014] Furthermore, in terms of mass percentage, the protein content of the PDRN is less than or equal to 0.5%.

[0015] Preferably, the protein content in the PDRN is less than or equal to 0.5%, 0.45%, 0.4%, 0.35%, 0.3%, 0.25%, 0.2%, 0.15%, 0.1%, 0.05%, 0.01% and 0% and any value therebetween.

[0016] Preferably, the nucleic acid content in the PDRN is greater than or equal to 96%, and can further be 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100.0% and any value therebetween.

[0017] Preferably, the purity (OD 260 / OD 280 value) of the PDRN is 1.8 - 2.0.

[0018] The purity (OD 260 / OD 280 value) of the PDRN can be 1.8, 1.9, 2.0 and any value therebetween.

[0019] Preferably, the PDRN presents as white or off-white granules or powder.

[0020] Preferably, the PDRN has good solubility, and 1 g of its product can be completely dissolved in 1 L of water within 30 min.

[0021] More preferably, the dissolution time may be 0.5 min, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min, 6 min, 6.5 min, 7 min, 7.5 min, 8 min, 8.5 min, 9 min, 9.5 min, 10 min, 10.5 min, 11 min, 11.5 min, 12 min, 12.5 min, 13 min, 13.5 min, 14 min, 14.5 min, 15 min, 15.5 min, 16 min, 16.5 min, 17 min, 17.5 min, 18 min, 18.5 min, 19 min, 19.5 min, 20 min, 20.5 min, 21 min, 21.5 min, 22 min, 22.5 min, 23 min, 23.5 min, 24 min, 24.5 min, 25 min, 25.5 min, 26 min, 26.5 min, 27 min, 27.5 min, 28 min, 28.5 min, 29 min, 29.5 min, 30 min and any value therebetween.

[0022] On the other hand, the present application also provides a method for preparing PDRN, the method comprising the following steps:

[0023] Step 1, collecting materials and lysing the materials to prepare a lysate;

[0024] Step 2, salting out the lysate to prepare a salted-out supernatant;

[0025] Step 3, adding an organic solvent to the salted-out supernatant for precipitation to prepare a crude product;

[0026] Step 4, drying the crude product to obtain the product.

[0027] Preferably, the method comprises the following steps:

[0028] Step 1, collecting materials and lysing the materials to prepare a lysate;

[0029] Step 2, adding salt and a protein denaturant to the lysate for salting out to prepare a salted-out supernatant;

[0030] Step 3, adding an organic solvent to the salted-out supernatant for precipitation to prepare a crude product;

[0031] Step 4, drying the crude product to obtain the product.

[0032] More preferably, the salt includes one or more of sodium chloride, potassium acetate, sodium acetate, ammonium sulfate, and sodium sulfate. More preferably, the added salt is sodium chloride. More preferably, the final concentration of the added salt is 1-5M.

[0033] The final concentration of the salt can be 1M, 1.5M, 2M, 2.5M, 3M, 3.5M, 4M, 4.5M, 5M, or any value therebetween.

[0034] More preferably, the protein denaturant includes one or more of guanidine hydrochloride, phenol, and surfactants. The surfactants include one or more of sodium dodecyl sulfate, sodium lauroyl sarcosinate, polyoxyethylene octylphenyl ether, polyoxyethylene sorbitan laurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate, and polyoxyethylene sorbitan monooleate. More preferably, the protein denaturant is sodium dodecyl sulfate. Calculated as the mass percentage of the cell lysate, the added concentration of the protein denaturant is 0.5%-2%.

[0035] Calculated as the mass percentage of the cell lysate, the added concentration of the protein denaturant can be 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%, 2.0%, or any value therebetween.

[0036] It can be understood that those skilled in the art can choose to use known protein denaturants for this part of the treatment as long as the final product can achieve salting out. Therefore, the specific methods and instruments are not limited herein.

[0037] Preferably, the salting-out conditions include: incubating at 40°C - 80°C for 1-3h.

[0038] The constant-temperature incubation temperature can be selected from any value among 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C.

[0039] The constant-temperature incubation time can be selected from any value among 1h, 1.5h, 2h, 2.5h, and 3h.

[0040] Preferably, the organic solvent includes one or more of ethanol, methanol, and isopropanol.

[0041] Preferably, the organic solvent is ethanol. More preferably, it is absolute ethanol.

[0042] Preferably, the mass ratio of the salting-out supernatant to the organic solvent is 1:(1-5). More preferably, it is 1:2.5.

[0043] The mass ratio of the salting-out clear liquid to the organic solvent can be selected from 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5 and any value therebetween.

[0044] Preferably, the precipitation time is 20 - 60 min; more preferably, it is 30 min.

[0045] The precipitation time can be selected from 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 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 and any value therebetween.

[0046] Preferably, sodium acetate is further added to the salting-out clear liquid in step three; more preferably, the concentration of the sodium acetate is 0.1 - 0.5 M; more preferably, it is 0.3 M.

[0047] The concentration of the sodium acetate includes 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M and any value therebetween.

[0048] Preferably, the step three further includes a step of rinsing and centrifuging, and the supernatant after centrifugation is discarded to obtain the crude product.

[0049] Preferably, the dehydration and drying in step four includes: steps of dehydration treatment and vacuum drying.

[0050] It can be understood that those skilled in the art can choose to adopt known drying techniques for this part of the treatment, as long as the final product is dehydrated and dried, so the specific method is not limited herein.

[0051] Furthermore, the material includes microorganisms, animals, and / or algae.

[0052] Preferably, the microorganisms include one or more of Lactobacillus, Bacillus, Eubacterium, and yeast. More preferably, the microorganisms include one or more of Lactobacillus sakei, Eubacterium rectale, Bacillus pumilus, Komagataella sp., and Lactobacillus reuteri.

[0053] The Lactobacillus sakei is Lactobacillus sakei LS-01 and / or Lactobacillus sakei CICC21858.

[0054] In a preferred embodiment, the Lactobacillus sakei is Lactobacillus sakei LS-01, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 20232285.

[0055] In a preferred embodiment, the Lactobacillus sakei CICC21858 has the deposit number CICC21858 and is purchased from the China Center for Industrial Culture Collection of Microorganisms.

[0056] In a preferred embodiment, the Lactobacillus rhamnosus has the deposit number CGMCC1.8882 and is purchased from the Institute of Microbiology, Chinese Academy of Sciences.

[0057] The Eubacterium rectale is Eubacterium rectale ATCC 33656 and / or Eubacterium rectale BPB22.

[0058] In a preferred embodiment, the Eubacterium rectale has the deposit number ATCC 33656 and is purchased from the American Type Culture Collection.

[0059] In a preferred embodiment, the Eubacterium rectale BPB22 has the deposit number CCTCC NO: M20232177 and is purchased from the China Center for Type Culture Collection.

[0060] The Bacillus pumilus is Bacillus pumilus Bp-1, Bacillus pumilus Bp-2, and / or Bacillus pumilus ATCC 21143.

[0061] In a preferred embodiment, the Bacillus pumilus Bp-1 is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 2025198.

[0062] In a preferred embodiment, the Bacillus pumilus Bp-2 is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 2025199.

[0063] In a preferred embodiment, the Bacillus pumilus ATCC 21143 with the deposit number ATCC 21143 is purchased from the American Type Culture Collection.

[0064] The Komagataella sp. is Komagataella GS115 and / or Komagataella CICC 33158.

[0065] In a preferred embodiment, the Komagataella GS115 is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 2025200. Komagataella CICC 33158

[0066] In a preferred embodiment, the Komagataella sp. is purchased from the China Center for Industrial Culture Collection of Microorganisms with the deposit number CICC 33158.

[0067] Komagataella sp. was formerly known as Pichia pastoris.

[0068] Those skilled in the art can understand that the above strains can be cultured by conventional methods to obtain the bacterial cells, and no further limitations will be imposed here.

[0069] Preferably, the animal is one or more of guinea pig (Cavia porcellus), Japanese Caenorhabditis elegans (Caenorhabditis japonica), Drosophila ananassae, Florida lancelet (Branchiostoma floridae), medaka (Oryzias latipes), red junglefowl (Gallus gallus), and more preferably guinea pig (Cavia porcellus).

[0070] In a preferred embodiment, the guinea pigs are purchased from Liaoning Changsheng Biotechnology Co., Ltd., ordinary grade.

[0071] Preferably, the algae are freshwater algae, and more preferably, the algae are Anabaena variabilis.

[0072] In a preferred embodiment, the Anabaena variabilis has the deposit number ATCC 29413 and is deposited in the American Type Culture Collection.

[0073] Those skilled in the art can understand that the above-mentioned algae can be obtained by conventional cultivation methods, and no further limitations will be imposed here.

[0074] Furthermore, the step 1 further includes a high-pressure homogenization step.

[0075] Preferably, when the material is a microorganism and / or algae, the step 1 further includes a high-pressure homogenization step.

[0076] Preferably, the high-pressure homogenization includes a pressure of 200 - 3000 bar. More preferably, the high-pressure homogenization includes a pressure of 800 - 1200 bar;

[0077] Preferably, the number of high-pressure homogenization cycles is greater than or equal to 1 time. More preferably, the number of high-pressure homogenization cycles is 1 - 5 times;

[0078] The pressure of the homogenization and lysis process can be selected from 800 bar, 850 bar, 900 bar, 950 bar, 1000 bar, 1500 bar, 1100 bar, 1150 bar, 1200 bar and any value therebetween.

[0079] Among them, the number of high-pressure homogenization cycles can be 1 time, 2 times, 3 times, 4 times, 5 times or more, as long as the final product PDRN with the corresponding molecular weight can be obtained. Therefore, the number of cycles and related equipment and instruments will not be limited here.

[0080] The present invention can also prepare PDRN with different molecular weights by filtration or molecular sieve chromatography according to actual production needs.

[0081] The filtration described above includes but is not limited to pleated filtration, paper chromatography filtration, bag filtration, microfiltration and ultrafiltration.

[0082] The chromatography described above includes but is not limited to adsorption column chromatography, thin-layer chromatography, polyamide film chromatography, ion exchange chromatography, molecular sieve chromatography, affinity chromatography, focusing chromatography and other types;

[0083] Furthermore, the filtration described above can select corresponding membranes such as ultrafiltration membranes for filtration, including but not limited to hollow fiber membranes, spiral wound ultrafiltration membranes, flat membranes, tubular membranes and other types of membranes. Appropriate filter membranes can be selected according to actual needs. Therefore, the types of ultrafiltration membranes and corresponding parameters will not be further limited here.

[0084] Further, the molecular sieve chromatography includes, but is not limited to, agarose-type molecular sieve gel chromatography, dextran-type molecular sieve gel chromatography, pumice-type molecular sieve gel chromatography, agar-type molecular sieve gel chromatography, agarose-type molecular sieve gel chromatography, polyvinyl alcohol-type molecular sieve gel chromatography, and polyacrylamide-type molecular sieve gel chromatography. Appropriate molecular sieves can be selected for filtration according to actual needs, so the types of molecular sieves and corresponding parameters are not further limited here.

[0085] The above filtration method is applicable to the preparation of PDRN with different molecular weights from microorganisms, animals, plants, and algae, and will not be further limited here.

[0086] The present invention can also prepare PDRN with different purities by purification means according to actual production needs.

[0087] The purification means include conventional nucleic acid purification means such as genomic kit purification, magnetic bead purification, lithium chloride precipitation, and TRIzol extraction.

[0088] Further, the lysis method is to add enzymes for lysis.

[0089] Preferably, the enzymes include any enzymes that can break the cell walls of Gram-positive bacteria or yeast cell walls, and can include, but are not limited to, Labiase, mutanolysin, lysostaphin, achromopeptidase and other lysozymes produced by Streptomyces fulvissimus, as well as any one or more of papain, proteinase K, and snailase. The concentration of the added enzyme is 0.2 - 2 mg / mL.

[0090] Preferably, the concentration of the added enzyme can be any value among 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, and 2 mg / mL.

[0091] Preferably, the lysis step in step one includes: incubating at a constant temperature of 25°C - 60°C for 1 - 5 h after adding the enzyme.

[0092] The constant temperature incubation temperature can be selected from 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 50°C, 55°C, 56°C, 60°C, and any value in between.

[0093] The constant temperature incubation time can be selected from 1 h, 2 h, 3 h, 4 h, 5 h, and any value in between.

[0094] Further, when the material is a microorganism or an alga, the method includes the following steps:

[0095] Step 1: Collect materials, resuspend the materials, add enzymes for lysis, and prepare a lysate by high-pressure homogenization.

[0096] Step 2: Add salt and a protein denaturant to the lysate for salting out to prepare a salted-out supernatant.

[0097] Step 3: Add an organic solvent to the salted-out supernatant for precipitation to prepare a crude product.

[0098] Step 4: Dehydrate and dry the crude product to obtain the product.

[0099] In a preferred embodiment, when the material is a microorganism, a method for preparing PDRN, the method comprises the following steps:

[0100] Step 1: Inoculate the bacteria or its bacterial agent into a culture medium, culture at 25°C - 40°C, 100 - 200 rpm for 1 - 12 h to obtain a seed solution; inoculate the seed solution into a culture medium, with an inoculation amount of 1% - 10%, and culture at 25°C - 40°C for 10 - 20 h to obtain a fermentation broth.

[0101] Step 2: Centrifuge to collect the bacteria in the fermentation broth, resuspend the bacteria with a buffer solution, add enzymes for incubation at a constant temperature, the final concentration of the added enzyme is 0.2 - 0.5 mg / mL, incubate at 25°C - 50°C for 1 - 5 h. Then perform a homogenization and lysis process to break the bacteria. The homogenization and lysis process is carried out using a high-pressure homogenizer, the pressure of the homogenization process is 800 - 1200 bar, and cycle 1 - 5 times to prepare a high-pressure homogenate.

[0102] Step 3: Add salt and a surfactant to the prepared high-pressure homogenate for salting out. The salt is sodium chloride, and the final concentration added is 1 - 5 M; the protein denaturant is sodium dodecyl sulfate, and the final concentration of the added sodium dodecyl sulfate is 0.5 - 2%. After adding the salt and the protein denaturant, incubate at 40°C - 80°C for 1 - 3 h, then centrifuge to collect the supernatant to prepare a salted-out supernatant.

[0103] Step 4: Add absolute ethanol and sodium acetate to the salted-out supernatant. The mass ratio of the salted-out supernatant to absolute ethanol is 1:(1 - 5), the concentration of the sodium acetate is 0.1 - 0.5 M, precipitate for 20 - 60 min, rinse and centrifuge to prepare a crude product.

[0104] Step 5: Dehydrate and dry the crude product to obtain the product.

[0105] In a preferred embodiment, when the material is algae, a method for preparing PDRN, the method comprises the following steps:

[0106] Step 1: Inoculate the algae into a culture medium, at 10°C - 50°C, 10 - 50 μE / (m2 · s) Under light illumination conditions, statically culture for 1 - 7 days to obtain the culture solution.

[0107] Step 2: Centrifuge to collect the bacteria in the culture solution. After resuspending the bacteria with buffer, add lysozyme and incubate at a constant temperature. The final concentration of the added lysozyme is 0.2 - 0.5 mg / mL, and incubate at 25°C - 50°C for 1 - 5 h. Then perform a homogenization and lysis process to break the bacteria. The homogenization and lysis process is carried out using a high-pressure homogenizer. The pressure of the homogenization process is 800 - 1200 bar, and cycle 1 - 5 times to prepare a high-pressure homogenate.

[0108] Step 3: Add salt and surfactant to the prepared high-pressure homogenate for salting out. The salt is sodium chloride, and the added final concentration is 1 - 5 M; the protein denaturant is sodium dodecyl sulfate, and the added final concentration of sodium dodecyl sulfate is 0.5 - 2%. After adding salt and protein denaturant, incubate at 40°C - 80°C for 1 - 3 h, then centrifuge to collect the supernatant to prepare a salted-out supernatant.

[0109] Step 4: Add absolute ethanol and sodium acetate to the salted-out supernatant. The mass ratio of the salted-out supernatant to absolute ethanol is 1:(1 - 5), and the concentration of sodium acetate is 0.1 - 0.5 M. Precipitate for 20 - 60 min, wash and centrifuge to prepare a crude product;

[0110] Step 5: Dehydrate and dry the crude product to obtain the product.

[0111] Further, when the material is an animal, the method includes the following steps:

[0112] Step 1: Collect the material, resuspend the material and add enzyme for lysis to prepare a lysate;

[0113] Step 2: Add salt and protein denaturant to the lysate for salting out to prepare a salted-out supernatant;

[0114] Step 3: Add an organic solvent to the salted-out supernatant for precipitation to prepare a crude product;

[0115] Step 4: Dehydrate and dry the crude product to obtain the product.

[0116] In a preferred embodiment, when the material is an animal, a method for preparing PDRN, the method includes the following steps:

[0117] Step 1: Collect animal tissues, digest and prepare tissue lysates;

[0118] Step 2: Add salt and surfactant to the prepared tissue lysate for salting out. The salt is sodium chloride, and the final concentration added is 1 - 5 M; the protein denaturant is sodium dodecyl sulfate, and the final concentration of sodium dodecyl sulfate added is 0.5 - 2%. After adding the salt and protein denaturant, incubate at 40°C - 80°C for 1 - 3 h, then centrifuge to collect the supernatant to prepare the salted-out supernatant.

[0119] Step 3: Add absolute ethanol and sodium acetate to the salted-out supernatant. The mass ratio of the salted-out supernatant to absolute ethanol is 1:(1 - 5), and the concentration of sodium acetate is 0.1 - 0.5 M. Precipitate for 20 - 60 min, then rinse and centrifuge to prepare the crude product;

[0120] Step 4: Dehydrate and dry the crude product to obtain the product.

[0121] Preferably, the animal tissue includes guinea pig liver tissue, kidney tissue, muscle tissue, etc. Grind the guinea pig liver tissue into fine powder at low temperature, resuspend and mix evenly, and add enzyme for lysis.

[0122] On the other hand, the present application also provides a composition, and the composition contains the PDRN as described above. Further, the composition also contains excipients.

[0123] The excipients can be appropriate solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc.

[0124] The composition of the present application can be prepared by a general method, and one or more diluents or carriers can be added, such as aqueous solutions, pills, tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions, etc.

[0125] The composition of the present application can be prepared into any preparation commonly prepared in the art. For example, it can be formulated into preparations such as creams, emulsions, lotions, facial masks, foundations, medical devices, hair cosmetics, etc. Specifically, skin lotions, skin softeners, skin boosters, skin toners, astringents, lotions, moisturizing lotions, nutritional lotions, massage creams, nutritional creams, moisturizing creams, hand creams, foundations, serums, nutritional serums, facial masks, soaps, facial cleansing foams, facial cleansers, facial cleansing creams, body lotions or body washes.

[0126] In the composition of the present application, in addition to PDRN as an essential ingredient, other ingredients commonly formulated in cosmetics can be mixed as needed. For example, oil components, humectants, surfactants, organic pigments, inorganic pigments, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH regulators, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, or pure water can be mixed.

[0127] On the other hand, the present application also provides the use of microorganisms, animals, and / or algae in the preparation of PDRN products.

[0128] Preferably, the microorganism includes one or more of Lactobacillus, Bacillus, Eubacterium, and yeast. More preferably, the microorganism includes one or more of Lactobacillus sakei, Eubacterium rectale, Bacillus pumilus, Komagataella sp., and Lactobacillus reuteri.

[0129] Preferably, the animal includes one or more of guinea pig (Cavia porcellus), Caenorhabditis japonica, Drosophila ananassae, Branchiostoma floridae, Oryzias latipes, and Gallus gallus. More preferably, the animal is guinea pig (Cavia porcellus).

[0130] Preferably, the alga includes freshwater algae. More preferably, the alga includes Anabaena variabilis.

[0131] On the other hand, the present application also provides the use of Lactobacillus sakei in the preparation or production of PDRN.

[0132] On the other hand, the present application also provides the use of Eubacterium rectale in the preparation or production of PDRN.

[0133] On the other hand, the present application also provides the use of Bacillus pumilus in the preparation or production of PDRN.

[0134] On the other hand, the present application also provides the use of Komagataella sp. in the preparation or production of PDRN.

[0135] On the other hand, the present application also provides the use of Anabaena variabilis in the preparation or production of PDRN.

[0136] On the other hand, the present application also provides the use of Cavia porcellus in the preparation or production of PDRN.

[0137] On the other hand, the present application also provides Bacillus pumilus, and the Bacillus pumilus is Bacillus pumilus Bp-1, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M2025198.

[0138] On the other hand, the present application also provides Bacillus pumilus, and the Bacillus pumilus is Bacillus pumilus Bp-2, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M2025199.

[0139] The present application has screened Bacillus pumilus with high activity and suitable for the preparation of PDRN.

[0140] On the other hand, the present application also provides a bacterial agent containing the above-mentioned Bacillus pumilus.

[0141] Preferably, the bacterial agent contains the above-mentioned dry and / or wet bacterial cells of Bacillus pumilus with a viable count ≥ 10 6 CFU / g.

[0142] The bacterial agent may include various forms of Bacillus pumilus preparations such as a viable Bacillus pumilus suspension, a dead Bacillus pumilus suspension, Bacillus pumilus metabolites, and Bacillus pumilus extracts.

[0143] Those skilled in the art can culture the Bacillus pumilus described in the present application according to the general culture method of Bacillus pumilus.

[0144] On the other hand, the present application also provides Komagataella sp., and the Komagataella sp. is Komagataella sp. GS115, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 2025200.

[0145] Komagataella sp., formerly known as Pichia pastoris.

[0146] In this application, Komagataella sp. with high activity and suitable for the preparation of PDRN was screened.

[0147] On the other hand, this application also provides a bacterial agent containing the above-mentioned Komagataella sp.

[0148] Optionally, the bacterial agent contains the above-mentioned Komagataella sp. dry cells and / or wet cells with viable cell count ≥ 10 6 CFU / g.

[0149] The bacterial agent can include various forms of Komagataella sp. preparations such as Komagataella sp. live cell suspension, Komagataella sp. dead cell suspension, Komagataella sp. metabolites, Komagataella sp. extracts, etc.

[0150] Those skilled in the art can culture the Komagataella sp. described in this application according to the general culture method of Komagataella sp.

[0151] On the other hand, this application also provides the use of the above-mentioned algae, microorganisms or the bacterial agent of the microorganisms or the above-mentioned PDRN or the composition in the preparation of products for promoting cell proliferation, products for promoting cell migration, products for promoting collagen production, anti-inflammatory and soothing products and / or products for repairing damaged skin barriers.

[0152] The algae include freshwater algae. More preferably, the algae include Anabaena variabilis.

[0153] On the other hand, this application also provides the use of the above-mentioned PDRN or the composition or the PDRN obtained by the preparation method or the Eubacterium rectale or the Bacillus pumilus or the Komagataella sp. or the Anabaena variabilis in the preparation of products for promoting cell proliferation and / or promoting collagen production.

[0154] Optionally, the above-mentioned PDRN includes PDRN from microorganisms, animals and / or algae.

[0155] Furthermore, the above-mentioned PDRN includes PDRN from Lactobacillus sakei.

[0156] Furthermore, the above-mentioned PDRN includes PDRN from Eubacterium rectale.

[0157] Furthermore, the PDRN includes PDRN derived from Komagataella sp.

[0158] Furthermore, the PDRN includes PDRN derived from Anabaena variabilis.

[0159] Furthermore, the PDRN includes PDRN derived from guinea pig.

[0160] The collagen is procollagen Iα-1.

[0161] In an alternative embodiment, the cell is a human skin fibroblast.

[0162] On the other hand, the present application also provides the use of the PDRN, or the PDRN obtained by the composition or the preparation method, or the Eubacterium rectale, or the Bacillus pumilus, or the Komagataella sp., or the Anabaena variabilis in the preparation of an anti-inflammatory and soothing and / or skin barrier repair product.

[0163] Optionally, the PDRN includes PDRN derived from microorganisms, animals, and / or algae.

[0164] Furthermore, the PDRN includes PDRN derived from Lactobacillus sakei.

[0165] Furthermore, the PDRN includes PDRN derived from Eubacterium rectale.

[0166] Furthermore, the PDRN includes PDRN derived from Komagataella sp.

[0167] Furthermore, the PDRN includes PDRN derived from Anabaena variabilis.

[0168] Furthermore, the PDRN includes PDRN derived from guinea pig.

[0169] The anti-inflammatory and soothing is achieved by reducing inflammatory factors; the inflammatory factors include IL-6.

[0170] The repair of damaged skin barrier is achieved by increasing the cell migration rate and / or the cell relative proliferation rate;

[0171] The repair of damaged skin barrier includes repairing the skin barrier damaged by ultraviolet rays and / or repairing the skin barrier damaged by trauma;

[0172] In an alternative embodiment, the cell is a human skin fibroblast.

[0173] The present invention has the following beneficial effects:

[0174] 1. The present invention extracts natural polynucleotide PDRN from Lactobacillus sakei, Eubacterium rectale, Bacillus pumilus, Saccharomyces kloeckeri, Anabaena variabilis, and guinea pigs for the first time, solving the problems of unstable safety, ecological unfriendliness, and poor sustainability of salmon sperm PDRN, expanding the scope of raw materials for PDRN preparation, and providing multiple new ways for the production of PDRN.

[0175] 2. The similarity of GC% between the PDRN prepared by the method described in the present invention and the GC% of the human genome is more than 99%, which is closer to the human GC% than salmon-derived PDRN. Moreover, the PDRN with a GC% content between 40.5% and 42% has better effects in promoting cell proliferation, promoting collagen production, promoting cell migration rate, anti-inflammatory and soothing, and / or repairing damaged skin barriers compared to PDRN outside this range and salmon-derived PDRN. Additionally, compared with the method of extracting PDRN from salmon semen in the prior art, the preparation method is simpler and more convenient.

[0176] 3. The yield of single-source salmon PDRN is easily restricted by raw materials, and the instability of raw materials also limits its application scope. Compared with the growth cycle of salmon, which is usually 3 - 7 years, each species in the present invention can be cultured / raised on a large scale, with a short culture / raising cycle, and has many advantages such as controllable growth environment, pure products, and strong sustainable development ability.

[0177] 4. The present invention also provides a method for naturally extracting PDRN from materials such as Lactobacillus sakei, Eubacterium rectale, Bacillus pumilus, Saccharomyces kloeckeri, Anabaena variabilis, and guinea pigs. This method has a full-process closed-loop management, is safe and controllable, and does not produce pollutants such as waste gas, waste water, and waste residue during the process. Liquid residues and dregs such as fermentation broth and cell debris can be used as animal feed after processing, and the production process is environmentally friendly and has strong sustainable development ability. Description of the Drawings

[0178] The above and / or additional aspects and advantages of the present application will become apparent and easy to understand from the description of the embodiments in conjunction with the following drawings, where:

[0179] Figure 1 is the result graph of cytotoxicity test;

[0180] Figure 2 is the result graph of UV repair efficacy test;

[0181] Figure 3 is the result graph of wound repair efficacy test;

[0182] Figure 4 is the result graph of soothing efficacy test;

[0183] Figure 5 It is a graph showing the test results of collagen-promoting efficacy.

[0184] Biological deposit information:

[0185] A strain of Lactobacillus sakei LS-01 was deposited at the China Center for Type Culture Collection (CCTCC) on November 20, 2023, with the deposit number CCTCC NO: M 20232285, and the deposit address is Wuhan University, Wuhan, China, Zip Code: 430072.

[0186] A strain of Bacillus pumilus Bp-1 was deposited at the China Center for Type Culture Collection (CCTCC) on February 12, 2025, with the deposit number CCTCC NO: M 2025198, and the deposit address is Wuhan University, Wuhan, China, Zip Code: 430072.

[0187] A strain of Bacillus pumilus Bp-2 was deposited at the China Center for Type Culture Collection (CCTCC) on February 12, 2025, with the deposit number CCTCC NO: M 2025199, and the deposit address is Wuhan University, Wuhan, China, Zip Code: 430072.

[0188] A strain of Komagataella sp. GS115 was deposited at the China Center for Type Culture Collection (CCTCC) on February 12, 2025, with the deposit number CCTCC NO: M 2025200, and the deposit address is Wuhan University, Wuhan, China, Zip Code: 430072. Detailed implementation manners

[0189] In order to more clearly illustrate the overall concept of this application, the following will be described in detail by way of examples in combination with the accompanying drawings of the specification. In the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, some technical features well known to the art are not described to avoid confusion with the present invention.

[0190] When numerical ranges are given in the examples, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any one numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art of this technology. In addition to the specific methods, equipment, and materials used in the examples, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, equipment, and materials of the prior art similar to or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0191] For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer.

[0192] Unless otherwise specified, in the following embodiments, for reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained by purchasing in the market.

[0193] The term polydeoxyribonucleotide (PDRN) referred to in the present invention means a high-molecular-weight double-stranded polymer or a low-molecular-weight double-stranded polymer formed by repeating units containing phosphoric acid, deoxyribose, and four bases, including adenine, guanine, thymine, and cytosine.

[0194] Among them, MRS solid medium (02-293) and MRS liquid medium (02-291) were purchased from Beijing Aoboxing; DMEM medium (KGL1206-500) and RPMI-1640 medium (KGL1507-500) were purchased from KeyGen Biotech; Lactobacillus rhamnosus was purchased from the Institute of Microbiology, Chinese Academy of Sciences, with the preservation number CGMCC1.8882; Eubacterium rectale ATCC 33656 was purchased from the American Type Culture Collection, with the number ATCC33656; Eubacterium rectale BPB22 was purchased from the China Center for Type Culture Collection, with the number CCTCC NO: M 20232177; Bacillus pumilus Bp-1 was preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 2025198; Bacillus pumilus Bp-2 was preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 2025199; Bacillus pumilus ATCC 21143 was purchased from the American Type Culture Collection, with the number ATCC 21143; Komagataella sp. GS115 was preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 2025200; Komagataella sp. CICC 33158 was purchased from the China Center for Industrial Culture Collection of Microorganisms, with the number CICC 33158; Anabaena variabilis was purchased from the American Type Culture Collection, with the number ATCC 29413; Cavia porcellus was purchased from Liaoning Changsheng Biotechnology Co., Ltd., of ordinary grade; Lactobacillus sakei CICC21858, with the preservation number CICC21858, was purchased from the China Center for Industrial Culture Collection of Microorganisms; Lysozyme was purchased from Beijing Solarbio Science & Technology Co., Ltd., with the product number L1080; Snailase was purchased from Beijing Solarbio Science & Technology Co., Ltd., with the product number S8280; The brand of the high-pressure homogenizer is ATS, and its model is AH-BASIC 30; The BCA protein quantification kit was purchased from Nanjing Novizan Biotech Co., Ltd., with the product number E112-1.

[0195] In addition, the "water" mentioned in the present invention includes any feasible water that can be used in the cosmetic field, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, purified water, etc.

[0196] Isolation and Identification of Lactobacillus sakei LS-01

[0197] The pickled vegetable samples collected from Meishan, Sichuan were mashed under sterile conditions. After weighing, they were diluted to a 1% (w / v) solution with physiological saline. An appropriate amount of the diluted solution was serially diluted 10-fold with physiological saline. 0.2 mL of each dilution gradient was taken and spread on a solid MRS medium plate, and cultured in an inverted position at 37 °C for 36 - 48 h. Colonies that were milky white, with neat edges, smooth and shiny were selected, and numbered 1 - 10 in descending order of colony size. The above 10 single colonies were separately picked and inoculated into liquid MRS medium, and cultured statically at 37 °C for 48 h. The bacterial cells were collected respectively, and genomic DNA extraction kits were used to extract genomic DNA. Using the genomic DNA as a template, 16S rDNA sequences were obtained through polymerase chain reaction and Sanger sequencing. Homologous sequence alignment was performed by BLAST in the Nucleotide Sequence Database of the National Center for Biotechnology Information in the United States. The results showed that strains 2, 7, and 9 were Lactobacillus sakei. Considering that the colony size reflects the strain activity to a certain extent, strain 2 of Lactobacillus sakei with the largest initial screening colony was selected as the subsequent test strain. The 16S rDNA sequence of strain 2 of Lactobacillus sakei is shown as SEQ ID No.1. Strain 2 of Lactobacillus sakei was named LS-01, and it was deposited in the China Center for Type Culture Collection (CCTCC) on November 20, 2023, with the deposit number CCTCC NO: M 20232285, and the deposit address is Wuhan University, Wuhan, China, Zip Code: 430072.

[0198] Example 1

[0199] Test Example 1

[0200] In this test example, a method for preparing PDRN from Lactobacillus sakei was provided, which is as follows:

[0201] Step 1: Preparation of fermentation broth by Lactobacillus sakei fermentation:

[0202] 1 - 2 loops of bacteria were picked from the MRS solid medium plate of Lactobacillus sakei LS-01 and inoculated into 40 mL of MRS liquid medium, and cultured on a shaker at 37 °C and 160 rpm for 8 h to obtain the seed liquid of Lactobacillus sakei. The seed liquid of Lactobacillus sakei was inoculated into 400 mL of MRS liquid medium at an inoculation amount of 4%, and cultured at 37 °C for 16 h to obtain the fermentation broth of Lactobacillus sakei.

[0203] Step 2: Collection, resuspension of bacterial cells and preparation of bacterial cell lysate (taking 300 mL of fermentation broth as an example):

[0204] Take 300 mL of the bacterial solution (the fermentation broth of Lactobacillus sakei), centrifuge at 9000 rpm for 5 min, discard the supernatant, wash once with 300 mL of TE, centrifuge at 9000 rpm for 5 min, add 20 mL of TE buffer, resuspend and mix well; add lysozyme to the resuspended cells to a final concentration of 0.2 mg / mL, incubate at 37 °C for 2 h, and then perform high-pressure homogenization using a high-pressure homogenizer, maintaining the high-pressure homogenization pressure at 1000 bar and circulating 3 times to prepare the cell lysate.

[0205] Step 3 Salting out and centrifugation to prepare the salted-out clear liquid:

[0206] Add NaCl with a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) with a final concentration of 1% to the obtained cell lysate, incubate at 60 °C for 1.5 h, and then collect the supernatant by centrifugation to prepare the salted-out clear liquid.

[0207] Step 4 Ethanol precipitation to prepare the crude product:

[0208] Add sodium acetate with a final concentration of 0.3 M to the salted-out clear liquid obtained in Step 3, stir well and mix evenly; after the sodium acetate is completely dissolved, slowly add 2.5 times the volume of absolute ethanol of the clear liquid to precipitate for 30 min, centrifuge at 9000 rpm for 10 min, and discard the supernatant; wash the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 min, and discard the supernatant to obtain the wet precipitate of the product.

[0209] Step 5 Dehydration and drying to obtain the product:

[0210] Add 20 mL of absolute ethanol to the wet precipitate of the product after washing for dehydration treatment. After the precipitate is thoroughly dispersed, centrifuge at 9000 rpm for 10 min, and discard the supernatant (repeat the operation 3 times). Transfer the obtained precipitate to a disposable petri dish and perform vacuum drying at 40 °C. When the weight of the product no longer changes significantly (the change in the weight of the product is less than 0.001 g), end the drying to obtain the PDRN product.

[0211] Test Example 2

[0212] Step 1 Fermentation of Lactobacillus sakei to prepare the fermentation broth:

[0213] Pick 1 - 2 loops of the strain from the MRS solid medium plate of Lactobacillus sakei LS-01 and inoculate it into 40 mL of MRS liquid medium. Incubate on a shaker at 37 °C and 160 rpm for 8 h to obtain the seed liquid of Lactobacillus sakei. Inoculate the seed liquid of Lactobacillus sakei into 400 mL of MRS liquid medium at an inoculation amount of 4% and incubate at 37 °C for 16 h to obtain the fermentation broth of Lactobacillus sakei.

[0214] Step 2 Cell collection, resuspension and high-pressure homogenization to prepare the cell lysate (taking 300 mL of the fermentation broth as an example):

[0215] Take 300 mL of the bacterial liquid (the fermentation broth of Lactobacillus sakei), centrifuge at 9000 rpm for 5 min, discard the supernatant, wash once with 300 mL of TE, centrifuge at 9000 rpm for 5 min, add 20 mL of TE buffer, resuspend and mix well; add lysozyme to the resuspended cells to a final concentration of 0.2 mg / mL, incubate at 37 °C for 2 h, and perform high-pressure homogenization using a high-pressure homogenizer, maintaining the high-pressure homogenization pressure at 1200 bar and cycling once to prepare the cell lysate.

[0216] Step 3 Salting out and centrifugation to prepare the salted-out clear liquid:

[0217] Add NaCl with a final concentration of 1 M and SDS with a final concentration of 0.5% to the obtained cell lysate, incubate at 80 °C for 1 h, and then collect the supernatant by centrifugation to prepare the salted-out clear liquid.

[0218] Step 4 Ethanol precipitation to prepare the crude product:

[0219] Add sodium acetate with a final concentration of 0.3 M to the salted-out clear liquid obtained in Step 3, stir well and mix evenly; after the sodium acetate is completely dissolved, slowly add 2.5 times the volume of absolute ethanol of the clear liquid to precipitate for 30 min, centrifuge at 9000 rpm for 10 min, and discard the supernatant; wash the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 min, and discard the supernatant to obtain the wet precipitate of the product.

[0220] Step 5 Dehydration and drying to obtain the product:

[0221] Add 20 mL of absolute ethanol to the wet precipitate of the product after washing for dehydration treatment. After the precipitate is thoroughly dispersed, centrifuge at 9000 rpm for 10 min, and discard the supernatant (repeat the operation 3 times). Transfer the obtained precipitate to a disposable petri dish and perform vacuum drying at 40 °C. When the weight of the product no longer changes significantly (the change in the weight of the product is less than 0.001 g), stop drying to obtain the PDRN product.

[0222] Experimental Example 3

[0223] Step 1 Fermentation of Lactobacillus sakei to prepare the fermentation broth:

[0224] Pick 1 - 2 loops of the strain from the MRS solid medium plate of Lactobacillus sakei LS-01, inoculate it into 40 mL of MRS liquid medium, and culture it on a shaker at 37 °C and 160 rpm for 8 h to obtain the seed liquid of Lactobacillus sakei. Inoculate the seed liquid of Lactobacillus sakei into 400 mL of MRS liquid medium at an inoculation amount of 4%, and culture it at 37 °C for 16 h to obtain the fermentation broth of Lactobacillus sakei.

[0225] Step 2: Collection, resuspension of bacteria cells and preparation of bacterial cell lysate by high-pressure homogenization (taking 300 mL of fermentation broth as an example):

[0226] Take 300 mL of bacterial liquid (fermentation broth of Lactobacillus sakei), centrifuge at 9000 rpm for 5 min, discard the supernatant, wash once with 300 mL of TE, centrifuge at 9000 rpm for 5 min, add 20 mL of TE buffer, resuspend and mix well; to the bacteria cells resuspended in TE, add lysozyme with a final concentration of 0.2 mg / mL, incubate at 37 °C for 2 h, then perform high-pressure homogenization using a high-pressure homogenizer, maintain the high-pressure homogenization pressure at 900 bar, circulate 4 times to prepare the bacterial cell lysate.

[0227] Step 3: Salting out and centrifugation to prepare the salted-out clear liquid:

[0228] Add NaCl with a final concentration of 4 M and SDS with a final concentration of 1.5% to the obtained bacterial cell lysate, incubate at 50 °C for 2.5 h. Then collect the supernatant by centrifugation to prepare the salted-out clear liquid.

[0229] Step 4: Ethanol precipitation to prepare the crude product:

[0230] Add sodium acetate with a final concentration of 0.3 M to the salted-out clear liquid obtained in Step 3, stir and mix well; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of absolute ethanol of the clear liquid to precipitate for 30 min, centrifuge at 9000 rpm for 10 min, discard the supernatant; wash the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 min, discard the supernatant to obtain the wet precipitate of the product.

[0231] Step 5: Dehydration and drying to obtain the product:

[0232] Add 20 mL of absolute ethanol to the wet precipitate of the product after washing for dehydration treatment. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min, discard the supernatant (repeat the operation 3 times). Transfer the obtained precipitate to a disposable petri dish, perform vacuum drying at 40 °C, and end the drying when the weight of the product no longer changes significantly (the change in the weight of the product is less than 0.001 g) to obtain the PDRN product.

[0233] Experimental Example 4

[0234] Step 1: Fermentation of Lactobacillus sakei to prepare the fermentation broth:

[0235] Pick 1 - 2 loops of bacterial strains from the MRS solid medium plate of Lactobacillus sakei LS-01, inoculate them into 40 mL of MRS liquid medium, and culture them on a shaker at 37 °C and 160 rpm for 8 h to obtain the seed liquid of Lactobacillus sakei. Inoculate the seed liquid of Lactobacillus sakei into 400 mL of MRS liquid medium at an inoculation amount of 4%, and culture at 37 °C for 16 h to obtain the fermentation broth of Lactobacillus sakei.

[0236] Step 2 Bacterial cell collection, resuspension and high-pressure homogenization to prepare bacterial cell lysate (taking 300 mL of fermentation broth as an example):

[0237] Take 300 mL of bacterial liquid (fermentation broth of Lactobacillus sakei), centrifuge at 9000 rpm for 5 min, discard the supernatant, wash once with 300 mL of TE, centrifuge at 9000 rpm for 5 min, add 20 mL of TE buffer, resuspend and mix well; add lysozyme to the bacteria resuspended in TE to a final concentration of 0.4 mg / mL, incubate at 37 °C for 2 h, then perform high-pressure homogenization using a high-pressure homogenizer, maintain the high-pressure homogenization pressure at 800 bar, cycle 2 times to prepare the bacterial cell lysate.

[0238] Step 3 Salting out and centrifugation to prepare salted-out clear liquid:

[0239] Add NaCl with a final concentration of 2.5 M and SDS with a final concentration of 1% to the obtained bacterial cell lysate, incubate at 60 °C for 1.5 h. Then collect the supernatant by centrifugation to prepare the salted-out clear liquid.

[0240] Step 4 Ethanol precipitation to prepare the crude product:

[0241] Add sodium acetate with a final concentration of 0.3 M to the salted-out clear liquid obtained in Step 3, stir well and mix evenly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of absolute ethanol of the clear liquid to the clear liquid to precipitate for 30 min, centrifuge at 9000 rpm for 10 min, discard the supernatant; wash the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 min, discard the supernatant to obtain the wet precipitate of the product.

[0242] Step 5 Dehydration and drying to obtain the product:

[0243] Add 20 mL of absolute ethanol to the wet precipitate of the product after washing to perform dehydration treatment. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min, discard the supernatant (repeat the operation 3 times). Transfer the obtained precipitate to a disposable petri dish and perform vacuum drying at 40 °C. When the weight of the product no longer changes significantly (the change in the weight of the product is less than 0.001 g), end the drying to obtain the PDRN product.

[0244] Test Example 5

[0245] Step 1 Fermentation of Lactobacillus sakei to prepare the fermentation broth:

[0246] Pick 1-2 loops of bacterial strains from the MRS solid medium plate of Lactobacillus sakei LS-01, inoculate them into 40 mL of MRS liquid medium, and culture them on a shaker at 37 °C and 160 rpm for 8 h to obtain the seed liquid of Lactobacillus sakei. Inoculate the seed liquid of Lactobacillus sakei into 400 mL of MRS liquid medium at an inoculation amount of 4%, and culture it at 37 °C for 16 h to obtain the fermentation broth of Lactobacillus sakei.

[0247] Step 2 Bacterial cell collection, resuspension and high-pressure homogenization to prepare bacterial cell lysate (taking 300 mL of fermentation broth as an example):

[0248] Take 300 mL of bacterial liquid (fermentation broth of Lactobacillus sakei), centrifuge at 9000 rpm for 5 min, discard the supernatant, wash once with 300 mL of TE, centrifuge at 9000 rpm for 5 min, add 20 mL of TE buffer, resuspend and mix well; add lysozyme to the bacteria resuspended in TE to a final concentration of 0.4 mg / mL, incubate at 37 °C for 4 h, and then perform high-pressure homogenization using a high-pressure homogenizer, maintaining the high-pressure homogenization pressure at 800 bar and circulating once to prepare the bacterial cell lysate.

[0249] Step 3 Salting out and centrifugation to prepare salted-out clear liquid:

[0250] Add NaCl with a final concentration of 2.5 M and SDS with a final concentration of 1% to the obtained bacterial cell lysate, and incubate at 60 °C for 1.5 h. Then collect the supernatant by centrifugation to prepare the salted-out clear liquid.

[0251] Step 4 Ethanol precipitation to prepare the crude product:

[0252] Add sodium acetate with a final concentration of 0.3 M to the salted-out clear liquid obtained in Step 3, and stir well; after the sodium acetate is completely dissolved, slowly add 2.5 times the volume of absolute ethanol of the clear liquid to precipitate for 30 min, centrifuge at 9000 rpm for 10 min, and discard the supernatant; wash the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 min, and discard the supernatant to obtain the wet precipitate of the product.

[0253] Step 5 Dehydration and drying to obtain the product:

[0254] Add 20 mL of absolute ethanol to the wet precipitate of the product after washing for dehydration treatment. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min, and discard the supernatant (repeat the operation 3 times). Transfer the obtained precipitate to a disposable petri dish and perform vacuum drying at 40 °C. When the weight of the product no longer changes significantly (the change in the weight of the product is less than 0.001 g), end the drying to obtain the PDRN product.

[0255] Test Example 6

[0256] The difference between this test example and Test Example 1 is only that the fermentation bacterial strain used is Lactobacillus sakei CICC21858, with the preservation number of CICC 21858, purchased from the China Center for Industrial Culture Collection.

[0257] Step 1 Fermentation of Lactobacillus sakei to prepare the fermentation broth:

[0258] Pick 1 - 2 loops of bacteria from the MRS solid medium plate of Lactobacillus sakei CICC21858 and inoculate them into 40 mL of MRS liquid medium. Incubate in a shaker at 37°C and 160 rpm for 8 h to obtain the seed liquid of Lactobacillus sakei. Inoculate the seed liquid of Lactobacillus sakei into 400 mL of MRS liquid medium at an inoculation amount of 4%, and incubate at 37°C for 16 h to obtain the fermentation liquid of Lactobacillus sakei.

[0259] Step 2 Bacterial cell collection, resuspension and preparation of cell lysate (taking 300 mL of fermentation liquid as an example):

[0260] Take 300 mL of bacterial liquid (fermentation liquid of Lactobacillus sakei), centrifuge at 9000 rpm for 5 min, discard the supernatant, wash once with 300 mL of TE, centrifuge at 9000 rpm for 5 min, add 20 mL of TE buffer, resuspend and mix well; to the cells resuspended in TE, add lysozyme with a final concentration of 0.2 mg / mL, incubate at 37°C for 2 h, and then perform high - pressure homogenization using a high - pressure homogenizer, maintaining the high - pressure homogenization pressure at 1000 bar and circulating 3 times to prepare the cell lysate.

[0261] Step 3 Salting - out, centrifugation to prepare the salted - out clear liquid:

[0262] Add NaCl with a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) with a final concentration of 1% to the obtained cell lysate, and incubate at 60°C for 1.5 h. Then collect the supernatant by centrifugation to prepare the salted - out clear liquid.

[0263] Step 4 Ethanol precipitation to prepare the crude product:

[0264] Add sodium acetate with a final concentration of 0.3 M to the salted - out clear liquid obtained in Step 3, stir well; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of absolute ethanol of the clear liquid to precipitate for 30 min, centrifuge at 9000 rpm for 10 min, and discard the supernatant; wash the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 min, and discard the supernatant to obtain the wet precipitate of the product.

[0265] Step 5 Dehydration and drying to obtain the product:

[0266] Add 20 mL of absolute ethanol to the wet precipitate of the product after washing for dehydration treatment. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min, and discard the supernatant (repeat the operation 3 times). Transfer the obtained precipitate to a disposable petri dish and perform vacuum drying at 40°C. End the drying when the weight of the product no longer changes significantly (the change in the weight of the product is less than 0.001 g) to obtain the PDRN product.

[0267] Experimental example 7

[0268] This test example provides a method for preparing PDRN from Lactobacillus rhamnosus (deposit number: CGMCC1.8882), which is as follows:

[0269] Step 1: Ferment Lactobacillus rhamnosus to prepare a fermentation broth:

[0270] Pick 1 - 2 loops of bacteria from the MRS solid medium plate for culturing Lactobacillus rhamnosus, inoculate them into 40 mL of MRS liquid medium, and culture anaerobically at 37°C for 8 h to obtain the seed liquid of Lactobacillus rhamnosus. Inoculate the seed liquid of Lactobacillus rhamnosus into 400 mL of MRS liquid medium at an inoculation amount of 4%, and culture at 37°C for 16 h to obtain the fermentation broth of Lactobacillus rhamnosus.

[0271] Step 2: Collect, resuspend the bacteria, and perform high - pressure homogenization to prepare a bacterial cell lysate (taking 300 mL of fermentation broth as an example):

[0272] Take 300 mL of the bacterial liquid (fermentation broth of Lactobacillus rhamnosus), centrifuge at 9000 rpm for 5 min, discard the supernatant, wash once with 300 mL of TE, centrifuge at 9000 rpm for 5 min, add 20 mL of TE buffer, resuspend and mix well; add lysozyme to the bacteria resuspended in TE to a final concentration of 0.2 mg / mL, incubate at 37°C for 2 h, and then perform high - pressure homogenization using a high - pressure homogenizer, maintaining the high - pressure homogenization pressure at 1000 bar and cycling 3 times to prepare the bacterial cell lysate.

[0273] Step 3: Perform salting - out and centrifugation to prepare a salting - out supernatant:

[0274] Add NaCl with a final concentration of 2.5 M and SDS with a final concentration of 1% to the obtained bacterial cell lysate, and incubate at 60°C for 1.5 h. Then collect the supernatant by centrifugation to prepare the salting - out supernatant.

[0275] Step 4: Perform ethanol precipitation to prepare a crude product:

[0276] Add sodium acetate with a final concentration of 0.3 M to the salting - out supernatant obtained in Step 3, stir well; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of absolute ethanol of the supernatant to precipitate for 30 min, centrifuge at 9000 rpm for 10 min, and discard the supernatant; wash the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 min, and discard the supernatant to obtain the wet precipitate of the product.

[0277] Step 5: Dehydrate and dry to obtain the product:

[0278] Add 20 mL of absolute ethanol to the wet precipitate after rinsing for dehydration treatment. After the precipitate is fully dispersed by stirring, centrifuge at 9000 rpm for 10 min, and discard the supernatant (repeat the operation 3 times). Transfer the obtained precipitate to a disposable petri dish and perform vacuum drying at 40 °C. When the weight of the product no longer changes significantly (the change in the weight of the product is less than 0.001 g), end the drying to obtain the PDRN product.

[0279] Test Example 8

[0280] This test example provides a method for preparing PDRN from Eubacterium rectale ATCC 33656 (purchased from the American Type Culture Collection, numbered ATCC 33656), which is as follows:

[0281] Step 1: Ferment Eubacterium rectale to prepare a fermentation broth:

[0282] Pick 1-2 loops of bacteria from the PYG (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number LA5570) solid medium plate of Eubacterium rectale ATCC 33656 and inoculate it into 40 mL of PYG liquid medium. Anaerobically culture at 37 °C for 16 h to obtain the seed liquid of Eubacterium rectale. Inoculate the seed liquid of Eubacterium rectale into 400 mL of PYG liquid medium at an inoculation amount of 4%, and anaerobically culture at 37 °C for 48 h to obtain the fermentation broth of Eubacterium rectale.

[0283] Step 2: Collect, resuspend the bacteria and prepare a bacterial cell lysate (taking 300 mL of fermentation broth as an example):

[0284] Take 300 mL of bacterial liquid (the fermentation broth of Eubacterium rectale), centrifuge at 9000 rpm for 5 min, discard the supernatant, rinse once with 300 mL of TE, centrifuge at 9000 rpm for 5 min, add 20 mL of TE buffer, resuspend and mix well; add lysozyme to the bacteria resuspended in TE to a final concentration of 0.2 mg / mL, incubate at 37 °C for 2 h. Then, through a high-pressure homogenizer, maintain the high-pressure homogenization pressure at 1000 bar and cycle 3 times to prepare a bacterial cell lysate.

[0285] Step 3: Salting out, centrifuging to prepare a salting-out supernatant:

[0286] Add NaCl with a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) with a final concentration of 1% to the obtained bacterial cell lysate, and incubate at 60 °C for 1.5 h. Then, collect the supernatant by centrifugation to prepare a salting-out supernatant.

[0287] Step 4: Ethanol precipitation to prepare a crude product:

[0288] To the salting-out supernatant obtained in step 3, add sodium acetate with a final concentration of 0.3 M, and stir well to mix evenly. After the sodium acetate is completely dissolved, slowly add 2.5 times the volume of absolute ethanol to the supernatant to precipitate for 30 min, centrifuge at 9000 rpm for 10 min, and discard the supernatant. The precipitate is rinsed once with 75% ethanol, centrifuged at 9000 rpm for 10 min, and the supernatant is discarded to obtain a wet precipitate of the product.

[0289] Step 5 Dehydration and drying to obtain the product:

[0290] Add 20 mL of absolute ethanol to the rinsed precipitate for dehydration treatment. After the precipitate is thoroughly dispersed, centrifuge at 9000 rpm for 10 min, and discard the supernatant (repeat the operation 3 times). The obtained precipitate is transferred to a disposable petri dish and dried under vacuum at 40 °C. When the weight of the product no longer changes significantly (the change in the weight of the product is less than 0.001 g), stop drying to obtain the PDRN product.

[0291] Test Example 9

[0292] This test example provides a method for preparing PDRN from Eubacterium rectale BPB22 (purchased from the China Center for Type Culture Collection, with the accession number CCTCC NO: M 20232177), which is as follows:

[0293] Step 1 Fermentation of Eubacterium rectale to prepare fermentation broth:

[0294] Pick 1-2 loops of bacteria from the PYG (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number LA5570) solid medium plate of Eubacterium rectale BPB22, inoculate it into 40 mL of PYG liquid medium, and anaerobically culture at 37 °C for 16 h to obtain the seed liquid of Eubacterium rectale. Inoculate the seed liquid of Eubacterium rectale into 400 mL of PYG liquid medium at an inoculation amount of 4%, and anaerobically culture at 37 °C for 48 h to obtain the fermentation broth of Eubacterium rectale.

[0295] Step 2 Bacterial cell collection, resuspension and preparation of bacterial cell lysate (taking 300 mL of fermentation broth as an example):

[0296] Take 300 mL of bacterial liquid (fermentation broth of Eubacterium rectale), centrifuge at 9000 rpm for 5 min, discard the supernatant, rinse once with 300 mL of TE, centrifuge at 9000 rpm for 5 min, add 20 mL of TE buffer, and resuspend and mix evenly. To the bacteria resuspended in TE, add lysozyme with a final concentration of 0.2 mg / mL, incubate at 37 °C for 2 h. Then, through a high-pressure homogenizer, maintain the high-pressure homogenization pressure at 1000 bar and cycle 3 times to prepare the bacterial cell lysate.

[0297] Step 3 Salting out and centrifugation to prepare salting-out supernatant:

[0298] Add NaCl with a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) with a final concentration of 1% to the obtained bacterial cell lysate, and incubate at 60 °C for 1.5 h. Then collect the supernatant by centrifugation to prepare the salting-out supernatant.

[0299] Step 4 Preparation of crude product by ethanol precipitation:

[0300] Add sodium acetate with a final concentration of 0.3 M to the salting-out supernatant obtained in Step 3, and stir well; after the sodium acetate is completely dissolved, slowly add 2.5 times the volume of absolute ethanol of the supernatant to precipitate for 30 min, centrifuge at 9000 rpm for 10 min, and discard the supernatant; wash the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 min, and discard the supernatant to obtain the wet precipitate of the product.

[0301] Step 5 Obtaining the product by dehydration and drying:

[0302] Add 20 mL of absolute ethanol to the washed precipitate for dehydration treatment. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min, and discard the supernatant (repeat the operation 3 times). Transfer the obtained precipitate to a disposable petri dish and perform vacuum drying at 40 °C. When the weight of the product no longer changes significantly (the change in the weight of the product is less than 0.001 g), end the drying to obtain the PDRN product.

[0303] Test Example 10

[0304] This test example provides a method for preparing PDRN from Bacillus pumilus Bp-1 (deposited in the China Center for Type Culture Collection, deposit number CCTCC NO: M 2025198), which is as follows:

[0305] Step 1 Preparation of fermentation broth by fermentation of Bacillus pumilus:

[0306] Pick 1-2 loops of bacteria from the LB (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number L1010) solid medium plate of Bacillus pumilus Bp-1 strain, inoculate it into 40 mL of LB liquid medium, and culture at 37 °C and 150 rpm for 12 h to obtain the seed liquid of Bacillus pumilus. Inoculate the seed liquid of Bacillus pumilus into 400 mL of LB liquid medium at an inoculation amount of 4%, and culture at 37 °C and 150 rpm for 24 h to obtain the fermentation broth of Bacillus pumilus.

[0307] Step 2 Collection, resuspension and preparation of bacterial cell lysate (taking 300 mL of fermentation broth as an example):

[0308] Take 300 mL of the bacterial solution (the fermentation broth of Bacillus pumilus), centrifuge at 9000 rpm for 5 min, discard the supernatant, wash once with 300 mL of TE, centrifuge at 9000 rpm for 5 min, add 20 mL of TE buffer, resuspend and mix well; add lysozyme to the resuspended cells to a final concentration of 0.2 mg / mL, incubate at 37 °C for 2 h. Then, use a high-pressure homogenizer to maintain a high-pressure homogenization pressure of 1000 bar and circulate 3 times to prepare the cell lysate.

[0309] Step 3 Salting out and centrifugation to prepare the salted-out supernatant:

[0310] Add NaCl with a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) with a final concentration of 1% to the obtained cell lysate, incubate at 60 °C for 1.5 h. Then, collect the supernatant by centrifugation to prepare the salted-out supernatant.

[0311] Step 4 Ethanol precipitation to prepare the crude product:

[0312] Add sodium acetate with a final concentration of 0.3 M to the salted-out supernatant obtained in Step 3, stir well and mix evenly; after the sodium acetate is completely dissolved, slowly add 2.5 times the volume of absolute ethanol of the supernatant to precipitate for 30 min, centrifuge at 9000 rpm for 10 min, discard the supernatant; wash the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 min, discard the supernatant to obtain the wet precipitate of the product.

[0313] Step 5 Dehydration and drying to obtain the product:

[0314] Add 20 mL of absolute ethanol to the washed precipitate for dehydration treatment. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min, discard the supernatant (repeat the operation 3 times). Transfer the obtained precipitate to a disposable petri dish and perform vacuum drying at 40 °C. When the weight of the product no longer changes significantly (the change in the weight of the product is less than 0.001 g), end the drying to obtain the PDRN product.

[0315] Test Example 11

[0316] This test example provides a method for preparing PDRN from Bacillus pumilus Bp-2 (preserved in the China Center for Type Culture Collection, preservation number CCTCC NO: M 2025199), which is as follows:

[0317] Step 1 Fermentation of Bacillus pumilus to prepare the fermentation broth:

[0318] Pick 1-2 loops of bacteria from the LB solid medium plate of Bacillus pumilus strain Bp-2 (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number L1010), inoculate them into 40 mL of LB liquid medium, and culture at 37°C and 150 rpm for 12 h to obtain the seed liquid of Bacillus pumilus. Inoculate the seed liquid of Bacillus pumilus into 400 mL of LB liquid medium at an inoculation amount of 4%, and culture at 37°C and 150 rpm for 24 h to obtain the fermentation liquid of Bacillus pumilus.

[0319] Step 2 Bacterial cell collection, resuspension and preparation of cell lysate (taking 300 mL of fermentation liquid as an example):

[0320] Take 300 mL of bacterial liquid (fermentation liquid of Bacillus pumilus), centrifuge at 9000 rpm for 5 min, discard the supernatant, wash once with 300 mL of TE, centrifuge at 9000 rpm for 5 min, add 20 mL of TE buffer, resuspend and mix well; add lysozyme to the cells resuspended in TE to a final concentration of 0.2 mg / mL, incubate at 37°C for 2 h. Then, use a high-pressure homogenizer to maintain a high-pressure homogenization pressure of 1000 bar and circulate 3 times to prepare the cell lysate.

[0321] Step 3 Salting out and centrifugation to prepare the salting-out supernatant:

[0322] Add NaCl with a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) with a final concentration of 1% to the obtained cell lysate, and incubate at 60°C for 1.5 h. Then, collect the supernatant by centrifugation to prepare the salting-out supernatant.

[0323] Step 4 Ethanol precipitation to prepare the crude product:

[0324] Add sodium acetate with a final concentration of 0.3 M to the salting-out supernatant obtained in Step 3, and stir well; after the sodium acetate is completely dissolved, slowly add 2.5 times the volume of absolute ethanol of the supernatant to precipitate for 30 min, centrifuge at 9000 rpm for 10 min, and discard the supernatant; wash the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 min, and discard the supernatant to obtain the wet precipitate of the product.

[0325] Step 5 Dehydration and drying to obtain the product:

[0326] Add 20 mL of absolute ethanol to the washed precipitate for dehydration treatment. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min, and discard the supernatant (repeat the operation 3 times). Transfer the obtained precipitate to a disposable petri dish and perform vacuum drying at 40°C until the weight of the product no longer changes significantly (the weight change of the product is less than 0.001 g), then end the drying to obtain the PDRN product.

[0327] Test Example 12

[0328] This test example provides a method for preparing PDRN from Bacillus pumilus ATCC 21143 (purchased from the American Type Culture Collection, numbered ATCC 21143), which is as follows:

[0329] Step 1: Ferment Bacillus pumilus to prepare a fermentation broth:

[0330] Pick 1 - 2 loops of bacteria from the LB (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number L1010) solid medium plate of Bacillus pumilus ATCC 21143, inoculate it into 40 mL of LB liquid medium, and culture it at 37 °C and 150 rpm for 12 h to obtain the seed liquid of Bacillus pumilus. Inoculate the seed liquid of Bacillus pumilus into 400 mL of LB liquid medium at an inoculation amount of 4%, and culture it at 37 °C and 150 rpm for 24 h to obtain the fermentation broth of Bacillus pumilus.

[0331] Step 2: Collect, resuspend the bacteria and prepare a bacterial cell lysate (taking 300 mL of fermentation broth as an example):

[0332] Take 300 mL of the bacterial liquid (fermentation broth of Bacillus pumilus), centrifuge it at 9000 rpm for 5 min, discard the supernatant, wash it once with 300 mL of TE, centrifuge it at 9000 rpm for 5 min, add 20 mL of TE buffer, resuspend and mix well; add lysozyme to the bacteria resuspended in TE to a final concentration of 0.2 mg / mL, incubate at 37 °C for 2 h. Then, use a high - pressure homogenizer to maintain a high - pressure homogenization pressure of 1000 bar and cycle 3 times to prepare the bacterial cell lysate.

[0333] Step 3: Prepare a salting - out supernatant by salting - out and centrifugation:

[0334] Add NaCl with a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) with a final concentration of 1% to the obtained bacterial cell lysate, and incubate at 60 °C for 1.5 h. Then, collect the supernatant by centrifugation to prepare the salting - out supernatant.

[0335] Step 4: Prepare a crude product by ethanol precipitation:

[0336] Add sodium acetate with a final concentration of 0.3 M to the salting - out supernatant obtained in Step 3, and stir well; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of absolute ethanol of the supernatant to the supernatant and precipitate for 30 min, centrifuge at 9000 rpm for 10 min, and discard the supernatant; wash the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 min, and discard the supernatant to obtain the wet precipitate of the product.

[0337] Step 5: Dehydrate and dry to obtain the product:

[0338] Add 20 mL of absolute ethanol to the washed precipitate for dehydration treatment. After thoroughly dispersing the precipitate, centrifuge at 9000 rpm for 10 min, and discard the supernatant (repeat the operation 3 times). Transfer the obtained precipitate to a disposable petri dish and perform vacuum drying at 40 °C. When the weight of the product no longer changes significantly (the change in the weight of the product is less than 0.001 g), end the drying to obtain the PDRN product.

[0339] Test Example 13

[0340] This test example provides a method for preparing PDRN from Komagataella sp. GS115 (deposited in the China Center for Type Culture Collection, deposit number CCTCC NO: M 2025200), which is as follows:

[0341] Step 1: Ferment Komagataella sp. to prepare a fermentation broth:

[0342] Pick 1-2 loops of bacteria from the YPD (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number LA0220) solid medium plate of Komagataella sp. GS115 and inoculate them into 40 mL of YPD liquid medium. Culture on a shaker at 30 °C and 220 rpm for 16 h to obtain the seed liquid of Komagataella sp. Inoculate the seed liquid of Komagataella sp. into 400 mL of YPD liquid medium at an inoculation amount of 4%, and culture at 30 °C and 220 rpm for 30 h to obtain the fermentation broth of Komagataella sp.

[0343] Step 2: Collect, resuspend the cells and prepare a cell lysate (taking 300 mL of fermentation broth as an example):

[0344] Take 300 mL of the bacterial solution (the fermentation broth of Komagataella sp.), centrifuge at 9000 rpm for 5 min, discard the supernatant, wash it once with 300 mL of TE, centrifuge at 9000 rpm for 5 min, add 20 mL of TE buffer, and resuspend and mix well; add snailase (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number S8280) to the cells resuspended in TE, with a final concentration of 0.2 mg / mL, and incubate at 45 °C for 2 h. Then, use a high-pressure homogenizer to maintain a high-pressure homogenization pressure of 1000 bar and circulate 3 times to prepare a cell lysate.

[0345] Step 3: Salt out, centrifuge to prepare a salted-out supernatant:

[0346] Add NaCl with a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) with a final concentration of 1% to the obtained cell lysate, and incubate at 60 °C for 1.5 h. Then, collect the supernatant by centrifugation to prepare a salted-out supernatant.

[0347] Step 4: Ethanol precipitation to prepare a crude product:

[0348] To the salting-out supernatant obtained in step 3, add sodium acetate with a final concentration of 0.3 M, and stir well to mix evenly. After the sodium acetate is completely dissolved, slowly add 2.5 times the volume of absolute ethanol of the supernatant to precipitate for 30 min, centrifuge at 9000 rpm for 10 min, and discard the supernatant. The precipitate is rinsed once with 75% ethanol, centrifuged at 9000 rpm for 10 min, and the supernatant is discarded to obtain the wet precipitate of the product.

[0349] Step 5 Dehydration and drying to obtain the product:

[0350] Add 20 mL of absolute ethanol to the rinsed precipitate for dehydration treatment. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min, and discard the supernatant (repeat the operation 3 times). The obtained precipitate is transferred to a disposable petri dish and vacuum dried at 40 °C. When the weight of the product no longer changes significantly (the change in the weight of the product is less than 0.001 g), the drying is terminated to obtain the PDRN product.

[0351] Test Example 14

[0352] This test example provides a method for preparing PDRN from Komagataella sp. CICC 33158 (purchased from China Center for Industrial Culture Collection, with the preservation number CICC 33158), which is as follows:

[0353] Step 1 Fermentation of Komagataella sp. to prepare the fermentation broth:

[0354] Pick 1-2 loops of bacteria from the YPD (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number LA0220) solid medium plate of Komagataella sp. CICC 33158, inoculate it into 40 mL of YPD liquid medium, and culture it on a shaker at 30 °C and 220 rpm for 16 h to obtain the seed liquid of Komagataella sp. The seed liquid of Komagataella sp. is inoculated into 400 mL of YPD liquid medium at an inoculation amount of 4%, and cultured at 30 °C and 220 rpm for 30 h to obtain the fermentation broth of Komagataella sp.

[0355] Step 2 Bacterial cell collection, resuspension and preparation of bacterial cell lysate (taking 300 mL of fermentation broth as an example):

[0356] Take 300 mL of bacterial liquid (the fermentation broth of Komagataella sp.), centrifuge at 9000 rpm for 5 min, discard the supernatant, rinse it once with 300 mL of TE, centrifuge at 9000 rpm for 5 min, add 20 mL of TE buffer, and resuspend and mix evenly. To the bacteria resuspended in TE, add snailase (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number S8280) with a final concentration of 0.2 mg / mL, and incubate at 45 °C for 2 h. Then, through a high-pressure homogenizer, maintain the high-pressure homogenization pressure at 1000 bar and circulate 3 times to prepare the bacterial cell lysate.

[0357] Step 3: Salting out and centrifugation to prepare the salting-out supernatant:

[0358] Add NaCl with a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) with a final concentration of 1% to the obtained cell lysate, and incubate at 60 °C for 1.5 h. Then collect the supernatant by centrifugation to prepare the salting-out supernatant.

[0359] Step 4: Ethanol precipitation to prepare the crude product:

[0360] Add sodium acetate with a final concentration of 0.3 M to the salting-out supernatant obtained in Step 3, and stir well; after the sodium acetate is completely dissolved, slowly add 2.5 times the volume of absolute ethanol of the supernatant to precipitate for 30 min, centrifuge at 9000 rpm for 10 min, and discard the supernatant; wash the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 min, and discard the supernatant to obtain the wet precipitate of the product.

[0361] Step 5: Dehydration and drying to obtain the product:

[0362] Add 20 mL of absolute ethanol to the washed precipitate for dehydration treatment. After the precipitate is thoroughly dispersed, centrifuge at 9000 rpm for 10 min, and discard the supernatant (repeat the operation 3 times). Transfer the obtained precipitate to a disposable petri dish and perform vacuum drying at 40 °C. When the weight of the product no longer changes significantly (the change in the weight of the product is less than 0.001 g), end the drying to obtain the PDRN product.

[0363] Test Example 15

[0364] This test provides a method for preparing PDRN from Anabaena variabilis (purchased from the American Type Culture Collection, numbered ATCC 29413), which is as follows:

[0365] Step 1: Culturing Anabaena variabilis to prepare the culture medium:

[0366] Inoculate the Anabaena variabilis strain into the AA liquid medium diluted 8 times (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number LA8520), and statically culture at 30 °C under the light condition of 30 μE / (m 2 ·s) for 5 days to obtain the Anabaena variabilis culture medium.

[0367] Step 2: Collection, resuspension, and preparation of the cell lysate of the algae (taking 300 mL of the culture medium as an example):

[0368] Take 300 mL of the culture medium (the culture medium of Anabaena variabilis), centrifuge at 9000 rpm for 5 min, discard the supernatant, wash it once with 300 mL of TE, centrifuge at 9000 rpm for 5 min, add 20 mL of TE buffer, resuspend and mix well; to the cells resuspended in TE, add lysozyme with a final concentration of 0.2 mg / mL, incubate at 37 °C for 2 h. Then, use a high-pressure homogenizer to maintain a high-pressure homogenization pressure of 1000 bar and circulate 3 times to prepare the algal cell lysate.

[0369] Step 3 Salting out and centrifugation to prepare the salted-out supernatant:

[0370] To the obtained algal cell lysate, add NaCl with a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) with a final concentration of 1%, incubate at 60 °C for 1.5 h. Then, collect the supernatant by centrifugation to prepare the salted-out supernatant.

[0371] Step 4 Ethanol precipitation to prepare the crude product:

[0372] To the salted-out supernatant obtained in Step 3, add sodium acetate with a final concentration of 0.3 M, stir well and mix evenly; after the sodium acetate is completely dissolved, slowly add 2.5 times the volume of absolute ethanol of the supernatant to precipitate for 30 min, centrifuge at 9000 rpm for 10 min, and discard the supernatant; wash the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 min, and discard the supernatant to obtain the wet precipitate of the product.

[0373] Step 5 Dehydration and drying to obtain the product:

[0374] Add 20 mL of absolute ethanol to the washed precipitate for dehydration treatment. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat the operation 3 times). Transfer the obtained precipitate to a disposable petri dish and perform vacuum drying at 40 °C. When the weight of the product no longer changes significantly (the change in the weight of the product is less than 0.001 g), end the drying to obtain the PDRN product.

[0375] Test Example 16

[0376] This test provides a method for preparing PDRN from guinea pigs (Cavia porcellus) (purchased from Liaoning Changsheng Biotechnology Co., Ltd., ordinary grade), which is as follows:

[0377] Step 1 Guinea pig feeding:

[0378] Normally feed the 3-week-old ordinary-grade guinea pigs purchased from Liaoning Changsheng Biotechnology Co., Ltd. for 3 months.

[0379] Step 2 Collection, digestion and preparation of tissue lysate from guinea pig liver tissue (taking 1 g of liver tissue as an example):

[0380] The guinea pigs were sacrificed by cervical dislocation, and the liver tissues were collected by routine dissection of the guinea pig samples and frozen in liquid nitrogen for 10 min. The frozen liver tissues were taken out and ground into fine powder in liquid nitrogen. Take 1 g, add 20 mL of TE buffer, resuspend and mix well; add proteinase K (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number P9460) to the TE resuspension at a final concentration of 2 mg / mL, incubate at 56 °C for 2 h to prepare tissue lysate.

[0381] Step 3 Salting out and centrifugation to prepare salting-out supernatant:

[0382] Add NaCl with a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) with a final concentration of 1% to the obtained tissue lysate, and incubate at 60 °C for 1.5 h. Then collect the supernatant by centrifugation to prepare the salting-out supernatant.

[0383] Step 4 Ethanol precipitation to prepare crude product:

[0384] Add sodium acetate with a final concentration of 0.3 M to the salting-out supernatant obtained in Step 3, stir well and mix evenly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of absolute ethanol of the supernatant to precipitate for 30 min, centrifuge at 9000 rpm for 10 min, and discard the supernatant; wash the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 min, and discard the supernatant to obtain the wet precipitate of the product.

[0385] Step 5 Dehydration and drying to obtain the product:

[0386] Add 20 mL of absolute ethanol to the washed precipitate for dehydration treatment. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min, and discard the supernatant (repeat the operation 3 times). Transfer the obtained precipitate to a disposable culture dish and perform vacuum drying at 40 °C. When the weight of the product no longer changes significantly (the change in the weight of the product is less than 0.001 g), end the drying to obtain the PDRN product.

[0387] In Comparative Example 1, the PDRN product was prepared by the method of CN107287186A in the background technology.

[0388] In Comparative Example 2, the PDRN product was prepared by the method of CN112315836A in the background technology.

[0389] In Comparative Example 3, the PDRN product was prepared by the method of CN115074357A in the background technology.

[0390] The PDRN products obtained in the above Test Examples 1-16 and Comparative Examples 1-3 were subjected to the following tests.

[0391] Purity and nucleic acid content detection:

[0392] Accurately weigh an appropriate amount of the above-mentioned dried product, reconstitute it with water at a concentration of 5 mg / mL, invert the solution up and down after incubating at 40 °C for 30 min (repeat the operation 2 - 3 times until the product is completely dissolved), and use this solution for the purity detection of polynucleotides. After the above-mentioned dried product is dissolved in water, use an ultra-micro ultraviolet spectrophotometer to measure the absorbance of the reconstituted sample at 260 nm and 280 nm, and determine the purity and nucleic acid content of PDRN in the product according to the results feedback by the instrument. The purity calculation formula is: Product purity = OD 260 / OD 280 ; Nucleic acid content = Detected sample concentration (ng / μL) / (5 mg / mL) / 1000 × 100%.

[0393] Protein detection:

[0394] In this example, a BCA protein quantification kit was used to determine the protein content in the product.

[0395] First, prepare the BCA working solution by mixing 50 volumes of BCAReagent A with 1 volume of BCAReagent B (50:1) (both BCAReagent A and B are included in the kit), and mix well.

[0396] Take an enzyme-linked immunosorbent assay (ELISA) plate, add reagents according to the data in Table 1, mix well by shaking, and place it at 37 °C for 30 min. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at A562 nm. Take the protein content (μg) as the abscissa and the absorbance as the ordinate to draw a standard curve.

[0397] Table 1

[0398]

[0399] *: Standard protein is included in the kit

[0400] Sample detection: Accurately weigh 100 mg of the PDRN sample, dissolve it in 200 mL of deionized water to prepare a 0.5 g / L sample solution. Take 20 μL and add 200 μL of the BCA working solution. Mix well by shaking and place it at 37 °C for 30 min. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at A562 nm, with the absorbance without standard protein as the blank control. According to the measured absorbance, substitute it into the standard curve to calculate the protein content in the test sample on the standard curve.

[0401] Product protein% = Protein content in the test sample / 0.5 * 100%.

[0402] Molecular weight detection:

[0403] In this example, a fully automatic nucleic acid analyzer was used to determine the molecular weight of the product by capillary electrophoresis.

[0404] Among them, instruments and equipment: fully automatic nucleic acid analyzer Qsep100, electronic balance, vortex mixer, ultra-micro ultraviolet spectrophotometer, etc.

[0405] Test solution: Take 0.01 g of the sample, dissolve it in water and dilute it to make a solution containing about 5 mg per 1 mL (0.5%). Then continue to dilute the sample solution 100 times until the final concentration of the sample is 50 ng / μL.

[0406] Testing on the machine: Turn on the power of the instrument and the air compressor switch, double-click the software icon; Add a new New Project; Select "Connect" to establish system connection; Configure the buffer and Marker; Select "Change buffer", put in the Marker and buffer; Load the sample tray; Select "Change Sample" to open the transparent door cover and put in the sample (sample volume ≥ 10 μL); Punch holes in the new cartridge; Open the cartridge door, put in the cartridge, and then close the cartridge door tightly; Select "Latch"; Calibrate the cartridge; Select the blank space and select in turn: (1) sample position, (2) test scheme, injection time, number of tests, (3) result file naming, (4) calculation.

[0407] Result processing: After the program runs to completion, manually eliminate some short fragments and overly long fragments with relatively low content, and calculate the weight-average molecular weight (the product molecular weight is automatically calculated by the fully automatic nucleic acid analyzer).

[0408] Detection of bases and nucleotides:

[0409] In this example, the perchloric acid hydrolysis method is used to detect the base and nucleotide content in the product.

[0410] Among them, weigh 5 mg of the sample to be tested and place it in 6 test tubes respectively. Take every 3 test tubes as a group, add 300 μL of 70% perchloric acid. The control group adds an equal amount of water, and then seal the test tubes into ampoules. Heat in boiling water at 100 °C for 1 h. After cooling, open the ampoule and transfer it completely into a 25 mL volumetric flask. Adjust the pH to 5.3 with 6M potassium hydroxide and make up the volume with water. Filter with a 0.22 μm MCE filter membrane to obtain the sample for determination.

[0411] Preparation method of standard solution: Adenine and uracil are dissolved in 0.1M KOH solution respectively, and cytosine nucleotide and uracil nucleotide are dissolved in ultrapure water respectively, with a final concentration of 1 mg / mL. Gradient dilute with ultrapure water to concentrations of 0.1 μg / mL, 1 μg / mL, 10 μg / mL, 0.1 mg / mL and 1 mg / mL respectively. Filter through a 0.22 μm MCE water-based filter membrane, and then perform liquid phase detection.

[0412] Base and nucleotide analysis method: Agilent ZORBAX EC-C18 chromatographic column (4μm, 4.6mm×150mm), mobile phase A: 5mM ammonium acetate solution; mobile phase B: methanol; gradient elution program: 0-10min 0%-5% methanol; 10-30min 5-20% methanol. The flow rate is set at 0.45mL / min. The column temperature is maintained at 25°C. The DAD detection wavelength is set at 254nm, and the injection volume is 5μL for collecting chromatograms and quantitative analysis. GC% is calculated by high performance liquid chromatography integration.

[0413] Detection of appearance and solubility:

[0414] In this example, the appearance of the product was observed and recorded, and the dissolution performance of the product was also detected. The specific method is as follows:

[0415] Observation of appearance characteristics:

[0416] By visual observation, the appearance characteristics of the sample to be tested should be white or off-white granules or powder.

[0417] Solubility test:

[0418] Weigh 1g of the sample to be tested, add 1L of pure water to make up the volume, mix gently, and observe the solubility after standing for 30min. If the resulting solution is clear, transparent, free of impurities and turbidity, the sample to be tested passes the solubility test; otherwise, it fails the solubility test.

[0419] The test results of the above test examples and comparative examples and their comparisons are shown in Table 2.

[0420] Table 2

[0421] Sample Properties <![CDATA[Solubility * > Purity Nucleic acid content Protein content Molecular weight GC% Test Example 1 White powder Completely soluble 1.95 99.50％ 0.19％ 249.5 kda 40.91％ Test Example 2 White powder Completely soluble 1.84 97.10％ 0.21％ 430.3 kda 40.94％ Test Example 3 White powder Completely soluble 1.82 96.30％ 0.43％ 186.6 kda 40.98％ Test Example 4 White powder Completely soluble 1.93 98.70％ 0.23％ 373.8 kda 40.83％ Test Example 5 White powder Completely soluble 1.87 99.10％ 0.37％ 493.9 kda 41.06％ Test Example 6 White powder Completely soluble 1.84 96.01％ 0.41％ 253.7 kda 40.93％ Test Example 7 White powder Completely soluble 1.83 95.10％ 0.35％ 237.2 kda 46.72％ Test Example 8 White powder Completely soluble 1.92 98.01％ 0.39％ 223.4 kda 41.49％ Test Example 9 White powder Completely soluble 1.93 99.31％ 0.19％ 249.1 kda 41.49％ Test Example 10 White powder Completely soluble 1.85 99.10％ 0.24％ 237.6 kda 41.33％ Test Example 11 White powder Completely soluble 1.84 98.73％ 0.21％ 241.8 kda 41.41％ Test Example 12 White powder Completely soluble 1.90 99.28％ 0.25％ 231.9 kda 41.25％ Test Example 13 White powder Completely soluble 1.88 99.01％ 0.18％ 271.7 kda 41.09％ Test Example 14 White powder Completely soluble 1.93 98.10％ 0.45％ 288.2 kda 41.16％ Test Example 15 White powder Completely soluble 1.97 97.76％ 0.33％ 437.7 kda 41.50％ Test Example 16 White powder Completely soluble 1.93 98.10％ 0.45％ 513.9 kda 39.89％ Comparative Example 1 Light yellow powder There are a few insoluble substances 1.73 90.10％ 1.81％ 255.6 kda 43.24％ Comparative Example 2 White powder Completely soluble 1.85 98.60％ 0.79％ 215.9 kda 43.41％ Comparative Example 3 White powder Completely soluble 1.84 98.30％ 0.31％ 239.8 kda 43.32％

[0422] *: 1g of the product dissolves in 1L of water within 30min

[0423] As can be seen from the content of Table 2, the PDRN product prepared by the method recorded in the test example has a high purity and nucleic acid content, and a low protein content. The GC content of the PDRN product is between 39.5% and 41.5%, which is very close to the human GC content of 40.91%, with more than 98% homology, and has a high nucleic acid content, molecular weight and yield, and is suitable for industrial production.

[0424] Example 2 Efficacy test

[0425] In this example, the PDRN products prepared in Test Examples 1 and / or 2 and / or 3 and / or 4 and / or 5 and / or 6 and / or 7 and / or 8 and / or 9 and / or 10 and / or 11 and / or 12 and / or 13 and / or 14 and / or 15 and / or 16 and Comparative Examples 1-3 in Example 1 were used as samples for efficacy testing.

[0426] Cytotoxicity test

[0427] Specifically, the above samples, Test Examples 1, 7-16 and Comparative Examples 1-3 were used for cytotoxicity testing. Each sample was tested at three concentrations of 0.01%, 0.05% and 0.1% respectively. The test sample information is shown in Table 3. Using HS27 cells as the experimental object, the culture system was DMEM medium (supplemented with 10% fetal bovine serum), and the culture conditions were 37 °C and 5% CO 2 in a carbon dioxide incubator. After culturing the cells for 24 h, the original medium was discarded and the cells were continued to be cultured for 24 h in serum-free medium mixed with the sample. The control group was water, and the CCK-8 method was used to detect the relative cell proliferation rate. The relative proliferation rate data are shown in Table 4. Based on the data in Table 4, Figure 1 .

[0428] Table 3

[0429] Sample name Sample concentration % Test Example 1 0.01、0.05、0.1 Test Example 7 0.01、0.05、0.1 Test Example 8 0.01、0.05、0.1 Test Example 9 0.01、0.05、0.1 Test Example 10 0.01、0.05、0.1 Test Example 11 0.01、0.05、0.1 Test Example 12 0.01、0.05、0.1 Test Example 13 0.01、0.05、0.1 Test Example 14 0.01、0.05、0.1 Test Example 15 0.01、0.05、0.1 Test Example 16 0.01、0.05、0.1 Comparative Example 1 0.01、0.05、0.1 Comparative Example 2 0.01、0.05、0.1 Comparative Example 3 0.01、0.05、0.1

[0430] Table 4

[0431]

[0432]

[0433] The cytotoxicity test results in Table 4 and Figure 1 showed that the products prepared in Test Examples 1, 8-15 of the present invention had no cytotoxicity (cell proliferation rate > 80%). The overall cell proliferation promoting effects of PDRN derived from Lactobacillus sakei, Eubacterium rectale, Bacillus pumilus, Saccharomyces kloeckeri and Anabaena variabilis were better than those of Test Example 7 and Comparative Examples 1-3, and were slightly better than that of PDRN derived from guinea pigs.

[0434] UV repair efficacy test

[0435] Continuing to use the above samples, Test Examples 1, 7-16 and Comparative Examples 1-3 (the sample concentration was 0.01%) for ultraviolet repair efficacy testing. Specifically, using HS27 cells as the experimental object, the culture system was DMEM medium (supplemented with 10% fetal bovine serum), and the culture conditions were 37 °C and 5% CO 2 in a carbon dioxide incubator. After the cells were irradiated with ultraviolet light (2000 μW / cm 2, irradiated for 80 min), discard the original culture medium, add serum-free medium mixed with the sample, and continue culturing for 24 h. The CCK-8 method was used to detect the relative cell proliferation rate. The control group was not irradiated with ultraviolet light. The ultraviolet group only added an equal amount of serum-free medium without adding the sample after ultraviolet irradiation. The relative proliferation rate data are shown in Table 5. Based on the data in Table 5, Figure 2 .

[0436] Table 5

[0437] Sample name Relative cell proliferation rate % Variance Control 100.00 1.63 UV 64.32 6.50 Test Example 1 95.08 6.51 Test Example 7 65.93 4.15 Test Example 8 91.89 2.74 Test Example 9 92.34 3.71 Test Example 10 94.38 4.02 Test Example 11 91.23 6.15 Test Example 12 89.76 3.86 Test Example 13 93.57 5.76 Test Example 14 91.18 2.76 Test Example 15 92.08 3.45 Test Example 16 88.01 2.92 Comparative Example 1 81.17 2.07 Comparative Example 2 88.05 3.33 Comparative Example 3 87.37 7.04

[0438] From Table 5 and Figure 2 The results of the ultraviolet repair efficacy test showed that the ultraviolet repair effects of the products prepared in Test Examples 1, 8-16 of the present invention were better than those of the PDRN products prepared in Test Example 7 and Comparative Examples 1-3. Moreover, the ultraviolet repair effects of PDRN derived from Lactobacillus sakei, Eubacterium rectale, Bacillus pumilus, Saccharomyces kloeckeri, and Anabaena variabilis were better than those of PDRN derived from guinea pigs.

[0439] Wound repair efficacy test

[0440] Continue to use the above-mentioned Sample Test Examples 1, 7-16 and Comparative Examples 1-3 (the sample concentration is 0.01%) for the wound repair efficacy test.

[0441] Specifically, HS27 cells were used as the experimental object, the culture system was DMEM medium (added with 10% fetal bovine serum), and the culture conditions were 37 °C and 5% CO 2 cultivation in an incubator. The sample treatment process was as follows: Place an ibidi chamber in a 24-well culture plate. After culturing the cells for 24 h, remove the ibidi chamber. Scratch and take pictures, discard the original culture medium, add serum-free medium mixed with the sample, and continue culturing for 24 h, 36 h, and 48 h (observe and take pictures). The control group only added an equal amount of serum-free medium without adding the sample. The area of the scratched area in the photo was analyzed using ImageJ software. The cell migration rate (%) = (1 - the area of the scratched area after adding the drug / the area of the initial scratched area) × 100. The cell migration rate data are shown in Table 6. Based on the data in Table 6, Figure 3 .

[0442] Table 6

[0443] Sample name Cell migration rate % 24h Variance Cell migration rate % 36h Variance Control 60.80 2.11 98.13 2.10 Test Example 1 66.85 2.04 99.51 0.99 Test Example 7 42.67 1.04 79.98 1.28 Test Example 8 61.45 2.46 98.75 2.48 Test Example 9 63.08 1.43 99.24 1.37 Test Example 10 59.83 1.43 99.67 2.34 Test Example 11 62.71 2.19 98.91 3.19 Test Example 12 57.19 1.46 97.57 3.27 Test Example 13 70.35 2.59 99.26 2.07 Test Example 14 65.43 1.88 98.74 3.52 Test Example 15 65.78 2.63 99.47 2.67 Test Example 16 56.35 0.18 93.79 1.39 Comparative Example 1 59.23 1.72 90.76 2.44 Comparative Example 2 65.10 1.64 97.22 2.18 Comparative Example 3 66.86 3.19 98.32 1.88

[0444] From Table 6 and Figure 3The results show that after 24 hours of treatment with PDRN from different sources, the PDRN prepared in Test Examples 1, 8 - 15 of the present invention is more remarkable in promoting cell migration compared with PDRN from other processes and sources, indicating that the PDRN from Lactobacillus kefiri, Eubacterium rectale, Bacillus pumilus, Saccharomyces kloeckeri and Anabaena variabilis prepared by the process of the present invention has good wound repair efficacy.

[0445] Soothing efficacy test

[0446] Continue to use the above Sample Test Examples 1, 7 - 16 and Comparative Examples 1 - 3 (sample concentration is 0.01%) for soothing efficacy testing.

[0447] Specifically, using Raw264.7 cells as the experimental object, the culture system is RPMI1640 medium (added with 10% fetal bovine serum), and the culture conditions are 37°C and 5% CO 2 cultivation in a carbon dioxide incubator. The sample treatment process is as follows: after culturing the cells for 24 hours, discard the original medium and add serum-free medium mixed with the sample and continue to culture for 24 hours (using lipopolysaccharide (LPS) as a model control). Use the CCK-8 method to detect the relative proliferation rate of the cells, centrifuge to collect the cell supernatant, and detect the inflammatory factors according to the instructions of the ELISA kit. The relevant data are shown in Table 7, and the data in Table 7 are used to draw Figure 4 .

[0448] Table 7

[0449]

[0450]

[0451] From Table 7 and Figure 4 the results show that after treating the LPS-induced cell line with the products prepared in Test Examples 1, 8 - 15 of the present invention, the content of inflammatory factor IL-6 is significantly reduced, indicating that the PDRN from Lactobacillus kefiri, Eubacterium rectale, Bacillus pumilus, Saccharomyces kloeckeri and Anabaena variabilis prepared by the process of the present invention has good soothing efficacy.

[0452] Collagen promotion efficacy test

[0453] Continue to use the above Sample Test Examples 1, 7 - 16 and Comparative Examples 1 - 3 (sample concentration is 0.01%) for collagen promotion efficacy testing.

[0454] Specifically, using HS27 cells as the experimental object, the culture system is DMEM medium (added with 10% fetal bovine serum), and the culture conditions are 37°C and 5% CO 2Cultivation. The sample treatment process is as follows: After culturing the cells for 24 h, discard the original culture medium and add serum-free culture medium mixed with the sample and continue culturing for 24 h. Collect the cell supernatant. The control group only adds an equal amount of serum-free culture medium without adding the sample. Refer to the Pro-Collagen Iα-1 ELISA kit to determine the content of Pro-Collagen Iα-1 (procollagen Iα-1). The relevant data are shown in Table 8, and the following is drawn based on the data in Table 8 Figure 5 .

[0455] Table 8

[0456]

[0457]

[0458] From Table 8 and Figure 5 The results show that after the products prepared in Test Examples 1, 8-15 of the present invention are treated for 24 h, the content of procollagen Iα-1 in the cells increases significantly. It shows that the PDRN derived from Lactobacillus kefiri, Eubacterium rectale, Bacillus pumilus, Saccharomyces kloeckeri and Anabaena variabilis prepared by the process of the present invention can significantly promote the synthesis of collagen in cells, and the effect is better than that of PDRN derived from guinea pigs.

[0459] After verification, the PDRN products derived from Lactobacillus kefiri, Eubacterium rectale, Bacillus pumilus, Saccharomyces kloeckeri and Anabaena variabilis prepared by the method described in the present invention have no cytotoxicity, and have ultraviolet repair efficacy, wound repair efficacy and collagen-promoting efficacy.

[0460] The above are only the embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A PDRN, characterized in that: The GC content of the PDRN is 40.5%-42%.

2. The PDRN according to claim 1, characterized in that The weight average molecular weight of the PDRN comprises 100kDa-500kDa.

3. A method for preparing PDRN, characterized in that: The method comprises the following steps: Step 1, collecting materials, and lysing the materials to prepare lysate; Step 2, salting out the lysate to prepare a salting-out clear solution; Step 3, adding an organic solvent to the salting-out clear solution to precipitate and prepare a crude product; Step 4: Dry the crude product to obtain the product.

4. The preparation method according to claim 3, characterized in that: The material includes microorganisms, animals and / or algae; Preferably, the microorganism comprises one or more of lactobacillus, bacillus, eubacterium, and yeast, and more preferably, the microorganism comprises one or more of Eubacterium rectale, Bacillus pumilus, Komagataella sp., and Lactobacillus reuteri; Preferably, the animal comprises one or more of Cavia porcellus, Caenorhabditis japonica, Drosophila ananassae, Branchiostoma floridae, Oryzias latipes, and Gallus gallus. More preferably, the animal comprises Cavia porcellus. Preferably, the algae comprises freshwater algae, more preferably, the algae comprises Anabaenavariabilis; More preferably, the method comprises the steps of: Step 1: Collect the material, resuspend the material, add enzyme to lyse it, and prepare lysate by high pressure homogenization; Step 2, adding salt and a protein denaturant to the lysate to perform salting out to prepare a salting out clear solution; Step 3, adding an organic solvent to the salting-out clear solution to precipitate and prepare a crude product; Step 4, drying the crude product to obtain a product; The enzymes include one or more of Labiase produced by Streptomyces fusca, mutanolysin, lysostaphin, achromopeptidase, papain, proteinase K, and snailase; The salt includes one or more of sodium chloride, potassium acetate, sodium acetate, ammonium sulfate, and sodium sulfate; The protein denaturant includes one or more of guanidine hydrochloride, phenol and a surfactant; The organic solvent includes one or more of ethanol, methanol and isopropanol.

5. A composition, characterized in that The composition comprises the PDRN described in claim 1 or 2 or the PDRN prepared according to claim 3 or 4.

6. Applications, including at least any one of A1) to A6) and above: A1) Application of Eubacterium rectale in the preparation of PDRN products; A2) Application of Bacillus pumilus in the preparation of PDRN products; A3) Application of Komagataella sp. in the preparation of PDRN products; A4) Application of Lactobacillus reuteri in the preparation of PDRN products; A5) Use of guinea pigs (Caviaporcellus) in the preparation of PDRN products; A6) Application of Anabaena variabilis in the preparation of PDRN products.

7. Bacillus pumilus, characterized in that The Bacillus pumilus is Bacillus pumilus Bp-1, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025198.

8. Bacillus pumilus, characterized in that The Bacillus pumilus is Bacillus pumilus Bp-2, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025199.

9. Komagataella sp. is characterized by: The Komagata yeast GS115 is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025200.

10. The PDRN prepared by the preparation method as claimed in claim 1 or 2 or claim 3 or 4 or the composition as claimed in claim 5 or Bacillus pumilus as claimed in claim 7 or Bacillus pumilus as claimed in claim 8 or Komagataella sp. as claimed in claim 9 or the application of Anabaena variabilis described in claim 4 in preparing a product that promotes cell proliferation and / or cell migration and / or promotes collagen production and / or anti-inflammatory relief and / or repairs skin barrier damage.

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