PDRN, and preparation method therefor and use thereof

Abstract

The present application relates to the field of biotechnology. Provided are a PDRN, and a preparation method therefor and the use thereof. The PDRN has a GC content of 39%-42%. In the PDRN prepared by means of the method recited in the present application, the GC% in the gDNA thereof has a similarity of 98% or more to the GC% of human and is closer to the GC% in a human body than that of a salmon-derived PDRN. The PDRN of the present application has higher safety and efficacy, and compared with the extracting of PDRN from salmon semen in the prior art, the method for preparing the PDRN disclosed in the present application is simpler and more convenient.

Description

A kind of PDRN and its preparation method and application

[0001] This application claims priority to Chinese patent application number 202410195149.3, filed with the Patent Office of China on February 22, 2024, entitled “A PDRN, A Preparation Method, and Application Thereof,” the entire contents of which are incorporated herein by reference.

[0002] This application also claims priority to the Chinese patent application filed with the China Patent Office on February 22, 2024, with application number "202410195108.4" and invention name "Method and Application of Preparing PDRN Using Lactobacillus sakei", the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present application relates to the field of biotechnology, and in particular to a PDRN and a preparation method and application thereof. Background Art

[0004] PDRN (Polydeoxyribonucleotide / Polyribonucleotide) is a biomacromolecule derived from salmon testicular cells, composed of multiple base pairs connected by phosphodiester bonds. Its essence is a mixed product composed of salmon genetic material of different fragment sizes. As early as 1952, the Italian Mastelli company extracted polynucleotide fragments (PDRN) of different sizes from salmon testicular cells based on the phenomenon that fishermen used salmon testicles to treat wounds, and studied their pharmacological activity and other related properties. The experimental results showed that nucleic acid fragments (PDRN) in male salmon testicular cells can significantly promote human cell regeneration, rapidly promote wound healing and reduce scar formation. In subsequent continuous studies, PDRN has been found to have anti-inflammatory, tissue repair, angiogenesis, anti-ischemia and improvement of diabetic foot effects.

[0005] Modern industry mostly extracts PDRN raw materials from the testicular cells of deep-sea salmon, such as the method for isolating 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 external PDRN disclosed in patent CN112315836A, and the preparation method and application of a polydeoxyribonucleotide disclosed in patent CN115074357A. Although the above-mentioned process has always been the main way to obtain PDRN raw materials, with the continuous depletion of marine fishery resources, extracting PDRN from salmon testicular cells has been unable to meet the increasingly strong demand for PDRN raw materials in the new era. In addition, the fuel leaks, nuclear wastewater discharges and the discarding of domestic garbage in coastal areas caused by the constant passing of large ships on ocean routes have all had a severe impact on the marine environment. The resulting problems of heavy metal enrichment, chromosomal aberrations and gene mutations in marine organisms have also greatly affected the safety of salmon sperm PDRN raw materials.

[0006] In summary, there is an urgent need to develop multiple PDRN production pathways to solve the problems of single source and poor safety. Summary of the Invention

[0007] This application prepares PDRN from multiple species (microorganisms, algae, and animals) for the first time, expanding the source of PDRN products while solving the problem of poor PDRN safety caused by marine environmental pollution and better controlling the quality of PDRN extracted raw materials.

[0008] On the one hand, the present application provides a Lactobacillus sakei, wherein the Lactobacillus sakei is Lactobacillus sakei LS-01, which is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 20232285.

[0009] This application has screened a highly active strain of Lactobacillus sakei suitable for preparing PDRN, namely Lactobacillus sakei LS-01, whose 16S rDNA sequence is shown in SEQ ID No. 1. As early as 2014, Lactobacillus sakei was approved by the National Health Commission for inclusion in the "List of New Food Ingredients" as an edible fungus. This application is the first to use Lactobacillus sakei to produce PDRN, providing a new material and new approach for PDRN production.

[0010] On the other hand, the present application also provides a bacterial agent comprising the Lactobacillus sakei.

[0011] Optionally, the Lactobacillus sakei is Lactobacillus sakei LS-01, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20232285.

[0012] Optionally, the bacterial agent contains viable bacteria ≥ 10 6 CFU / g of the above-mentioned sake Lactobacillus dry bacteria and / or wet bacteria.

[0013] The bacterial agent may include various forms of Lactobacillus sake preparations, such as a live Lactobacillus sake suspension, a dead Lactobacillus sake suspension, a Lactobacillus sake metabolite, and a Lactobacillus sake extract.

[0014] Those skilled in the art can culture the Lactobacillus sakei described in this application according to the general culture method of Lactobacillus sakei.

[0015] On the other hand, the present application also provides Bacillus pumilus, which is Bacillus pumilus Bp-1, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025198.

[0016] On the other hand, the present application also provides Bacillus pumilus, which is Bacillus pumilus Bp-2, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025199.

[0017] This application screened Bacillus pumilus, which is highly active and suitable for preparing PDRN.

[0018] On the other hand, the present application also provides a bacterial agent comprising the Bacillus pumilus.

[0019] Those skilled in the art can culture the Bacillus pumilus described in this application according to a general culture method of Bacillus pumilus.

[0020] On the other hand, the present application also provides Komagataella sp. The Komagataella sp. is Komagataella sp. GS115, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025200.

[0021] This application screened Komagataella sp., which is highly active and suitable for preparing PDRN.

[0022] On the other hand, the present application also provides a bacterial agent comprising the Komagataella sp.

[0023] Those skilled in the art can culture the Komagata yeast described in this application according to the general culture method of Komagata yeast.

[0024] Komagataella sp. was formerly known as Pichia pastoris.

[0025] On the other hand, the present application also provides a method for preparing PDRN, characterized in that the method comprises the following steps:

[0026] Step 1: collecting materials and lysing the materials to prepare lysate;

[0027] Step 2: salting out the lysate to prepare a salting-out clear solution;

[0028] Step 3: adding an organic solvent to the salting-out clear solution to precipitate and prepare a crude product;

[0029] Step 4, drying the crude product to obtain a product;

[0030] Optionally, the material includes microorganisms, animals and / or algae;

[0031] More optionally, the animal comprises one or more of guinea pig (Cavia porcellus), Caenorhabditis japonica, Drosophila ananassae, Branchiostoma floridae, Oryzias latipes, and Gallus gallus. More optionally, the animal comprises guinea pig (Cavia porcellus);

[0032] Optionally, the algae comprises freshwater algae, more preferably, the algae comprises Anabaena variabilis;

[0033] More optionally, the microorganism comprises one or more of Lactobacillus, Bacillus, Eubacterium, and yeast. More optionally, the microorganism comprises one or more of Lactobacillus sakei, Eubacterium rectale, Bacillus pumilus, Komagataella sp., and Lactobacillus reuteri.

[0034] Furthermore, the method comprises the following steps:

[0035] Step 1: Collect the material, resuspend it, add enzyme to lyse it, and prepare the lysate;

[0036] Step 2: adding salt and a protein denaturant to the lysate to perform salting out to prepare a salting out clear solution;

[0037] Step 3: adding an organic solvent to the salting-out clear solution to precipitate and prepare a crude product;

[0038] Step 4: drying the crude product to obtain the product.

[0039] Optionally, the enzyme includes one or more of Labiase produced by Streptomyces fusca, mutanolysin, lysostaphin, chromopeptidase, papain, proteinase K, and snailase; optionally, the enzyme is added at a final concentration of 0.2-2 mg / mL.

[0040] The enzyme includes any enzyme that can destroy the cell wall of Gram-positive bacteria or the cell wall of yeast.

[0041] Alternatively, the concentration of the added enzyme may be 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, 2 mg / mL and any value therebetween.

[0042] Optionally, the cleavage step in step 1 includes: adding the enzyme and incubating at a constant temperature of 25° C. to 60° C. for 1 to 5 hours.

[0043] 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 therebetween.

[0044] The constant temperature incubation time can be selected from any value of 1h, 2h, 3h, 4h, and 5h.

[0045] Optionally, the salt includes one or more of sodium chloride, potassium acetate, sodium acetate, ammonium sulfate, and sodium sulfate. Optionally, the added salt is sodium chloride. Optionally, the final concentration of the added salt is 1-5M.

[0046] The final concentration of the salt can be 1 M, 1.5 M, 2 M, 2.5 M, 3 M, 3.5 M, 4 M, 4.5 M, 5 M and any value therebetween.

[0047] Optionally, the protein denaturant includes one or more of guanidine hydrochloride, phenol and a surfactant, wherein the surfactant includes one or more of sodium lauryl sulfate, sodium lauryl sarcosinate, polyethylene glycol octylphenyl ether, polyoxyethylene sorbitan laurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate and polyoxyethylene sorbitan monooleate; optionally, the protein denaturant is sodium lauryl sulfate; and the added concentration of the protein denaturant is 0.5%-2% based on the mass percentage of the bacterial lysate.

[0048] Calculated as a percentage by mass of the 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% and any value therebetween.

[0049] It is understandable that those skilled in the art may choose to use known protein deforming agents to perform this treatment, as long as the final product can achieve salting out. Therefore, the specific method and instrument are not limited here.

[0050] Optionally, the salting-out conditions include: incubation at 40° C.-80° C. for 1-3 hours.

[0051] The constant temperature incubation temperature can be selected from 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C and any value therebetween.

[0052] The constant temperature incubation time can be selected from 1h, 1.5h, 2h, 2.5h, 3h and any value therebetween.

[0053] Optionally, the organic solvent includes one or more of ethanol, methanol, and isopropanol.

[0054] Optionally, the organic solvent is ethanol; more optionally, anhydrous ethanol.

[0055] Optionally, the mass ratio of the salting-out clear liquid to the organic solvent is 1:(1-5); more optionally, 1:2.5.

[0056] The mass ratio of the salting-out clear solution 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 in between.

[0057] Optionally, the precipitation time is 20-60 min; more preferably, 30 min.

[0058] The precipitation time can be selected from 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min, 35min, 36min, 37min, 38min, 39min, 40min, 41min, 42min, 43min, 44min, 45min, 46min, 47min, 48min, 49min, 50min, 51min, 52min, 53min, 54min, 55min, 56min, 57min, 58min, 59min, 60min and any value therebetween.

[0059] Optionally, sodium acetate is further added to the salting-out clear solution in step three; more optionally, the concentration of sodium acetate is 0.1-0.5M; more optionally, 0.3M.

[0060] The concentration of sodium acetate can be selected from 0.1M, 0.2M, 0.3M, 0.4M, 0.5M and any value therebetween.

[0061] Optionally, step three further includes a rinsing and centrifuging step, and the supernatant after centrifugation is discarded to obtain a crude product.

[0062] Optionally, the drying in step 4 includes: dehydration treatment and vacuum drying steps.

[0063] It is understandable that those skilled in the art may choose to use known drying technology to carry out this part of the treatment, as long as the final product is dehydrated and dried, so the specific method is not limited here.

[0064] Optionally, the material includes microorganisms, animals and / or algae;

[0065] More optionally, the animal comprises one or more of guinea pig (Cavia porcellus), Caenorhabditis japonica, Drosophila ananassae, Branchiostoma floridae, Oryzias latipes, and Gallus gallus. More optionally, the animal comprises guinea pig (Cavia porcellus);

[0066] More optionally, the algae comprises freshwater algae, more optionally, the algae comprises Anabaena variabilis;

[0067] More optionally, the microorganism is one or more of Lactobacillus, Bacillus, Eubacterium, and yeast, and more optionally one or more of Lactobacillus sakei, Eubacterium rectale, Bacillus pumilus, Komagataella sp., and Lactobacillus reuteri.

[0068] The Lactobacillus sakei is Lactobacillus sakei LS-01 and / or Lactobacillus sakei CICC 21858.

[0069] In an optional embodiment, the Lactobacillus sakei is Lactobacillus sakei LS-01, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20232285.

[0070] In an optional embodiment, the Lactobacillus sakei CICC 21858 has a preservation number of CICC 21858 and is purchased from the China Industrial Microorganism Culture Collection Center.

[0071] In an optional embodiment, the Lactobacillus rhamnosus has a preservation number of CGMCC 1.8882 and is purchased from the Institute of Microbiology, Chinese Academy of Sciences.

[0072] The Eubacterium rectum is Eubacterium rectum ATCC 33656 and / or Eubacterium rectum BPB22.

[0073] In an optional embodiment, the Eubacterium rectale has a deposit number of ATCC 33656, which is purchased from the American Type Culture Collection.

[0074] In an optional embodiment, the Eubacterium rectum BPB22, with a preservation number of CCTCC NO: M 20232177, was purchased from China Center for Type Culture Collection.

[0075] The Bacillus pumilus is Bacillus pumilus Bp-1, Bacillus pumilus Bp-2 and / or Bacillus pumilus ATCC 21143.

[0076] In an optional embodiment, the Bacillus pumilus Bp-1 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025198.

[0077] In an optional embodiment, the Bacillus pumilus Bp-2 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025199.

[0078] In an optional embodiment, the Bacillus pumilus ATCC 21143 has a deposit number of ATCC 21143, which is deposited in the American Type Culture Collection.

[0079] The Komagataella sp. is Komagataella sp. GS115 and / or Komagataella sp. CICC 33158;

[0080] Among them, Komagataella sp. was formerly known as Pichia pastoris.

[0081] In an optional embodiment, the Komagata yeast GS115 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025200.

[0082] In an optional embodiment, the accession number of the Komagata yeast CICC 33158 is CICC 33158, which is deposited in the China Industrial Microorganism Culture Collection Center.

[0083] Those skilled in the art will appreciate that the bacterial cells can be obtained by culturing the above-mentioned strains through conventional methods, and no further restrictions will be imposed here.

[0084] In an optional embodiment, the guinea pig is purchased from Liaoning Changsheng Biotechnology Co., Ltd. and is of ordinary grade.

[0085] In an optional embodiment, the Houttuynia cordata Thunb. has an accession number of ATCC 29413, which is deposited in the American Type Culture Collection.

[0086] Furthermore, when the material is microorganisms and / or algae, step one further includes a high-pressure homogenization step, the high-pressure homogenization pressure is 200-3000 bar, and the number of cycles is greater than or equal to 1.

[0087] Optionally, more optionally, the high-pressure homogenization includes a pressure of 800-1200 bar;

[0088] Optionally, the high pressure homogenization cycle is 1-5 times;

[0089] The homogenization cracking process pressure 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.

[0090] Among them, the number of cycles of high-pressure homogenization can be 1, 2, 3, 4, 5 or more times, as long as the final product PDRN of the corresponding molecular weight is obtained. Therefore, the number of cycles and related equipment and instruments are no longer limited here.

[0091] This application can also prepare PDRN of different molecular weights by filtration or chromatography in step 1 according to actual production needs;

[0092] The filtration includes but is not limited to pleated filtration, paper filtration, bag filtration, microfiltration, ultrafiltration and nanofiltration;

[0093] The chromatography 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 the like;

[0094] Furthermore, the filtration can be performed using corresponding membranes such as ultrafiltration, including but not limited to hollow fiber membranes, rolled ultrafiltration membranes, flat membranes, tubular membranes, etc., and appropriate membranes can be selected for filtration according to actual needs. Therefore, the types of ultrafiltration membranes and corresponding parameters are not further limited here.

[0095] Furthermore, the filtration can be performed using corresponding membranes such as nanofiltration, including but not limited to porous nanofiltration membranes, jet nanofiltration membranes, hollow fiber nanofiltration membranes, multilayer nanofiltration membranes, and nanofiltration composite membranes. Suitable filter membranes can be selected for filtration according to actual needs, so the nanofiltration membrane types and corresponding parameters are not further limited here.

[0096] Furthermore, 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. Suitable molecular sieves can be selected for filtration according to actual needs, so the molecular sieve type and corresponding parameters are not further limited here.

[0097] The above-described filtration method is applicable to the preparation of PDRN of different molecular weights from microorganisms, animals, plants, and algae, and is not further limited here.

[0098] Furthermore, when the material is a microorganism or algae, the method comprises the following steps:

[0099] Step 1: Collect the material, resuspend the material, add enzyme lysis, and prepare lysate by high-pressure homogenization;

[0100] Step 2: adding salt and a protein denaturant to the lysate to perform salting out to prepare a salting out clear solution;

[0101] Step 3: adding an organic solvent to the salting-out clear solution to precipitate and prepare a crude product;

[0102] Step 4: dehydrate and dry the crude product to obtain the product.

[0103] In an optional embodiment, when the material is a microorganism, a method for preparing PDRN comprises the following steps:

[0104] Step 1: inoculate the bacteria or its inoculum into the culture medium, culture at 25°C-40°C, 100-200 rpm for 1-12 hours to obtain a seed solution; inoculate the seed solution into the culture medium at an inoculum amount of 1%-10%, and culture at 25°C-40°C for 10-20 hours to obtain a fermentation liquid;

[0105] Step 2: Collect the cells from the fermentation broth by centrifugation, resuspend the cells in buffer, add enzyme, and incubate at a constant temperature. The final concentration of the enzyme is 0.2-0.5 mg / mL, and the temperature is 25°C-50°C for 1-5 hours. Then, a homogenization lysis process is performed to disrupt the cells. The homogenization lysis process is performed using a high-pressure homogenizer at a pressure of 800-1200 bar, and the homogenization process is repeated 1-5 times to prepare a high-pressure homogenized solution.

[0106] Step 3: Add salt and a surfactant to the prepared high-pressure homogenized solution for salting out. The salt is sodium chloride, added to a final concentration of 1-5 M; the protein denaturant is sodium dodecyl sulfate, added to a final concentration of 0.5%-2%. After adding the salt and protein denaturant, incubate at 40°C-80°C for 1-3 hours, and then centrifuge to collect the supernatant to prepare a salting-out supernatant.

[0107] Step 4: adding anhydrous ethanol and sodium acetate to the salting-out supernatant, wherein the mass ratio of the salting-out supernatant to anhydrous ethanol is 1:(1-5), the concentration of the sodium acetate is 0.1-0.5M, settling for 20-60 minutes, rinsing and centrifuging to prepare a crude product;

[0108] Step 5: dehydrate and dry the crude product to obtain the product.

[0109] Furthermore, the microorganisms include one or more of Lactobacillus, Bacillus, Eubacterium, and yeast;

[0110] Furthermore, the microorganisms include one or more of Lactobacillus sakei, Eubacterium rectale, Bacillus pumilus, Komagataella sp., and Lactobacillus reuteri;

[0111] Among them, Komagataella sp. was formerly known as Pichia pastoris.

[0112] In an optional embodiment, when the material is algae, a method for preparing PDRN comprises the following steps:

[0113] Step 1: Inoculate algae into the culture medium at 10-50°C and 10-50 μE / (m 2 ·s) Incubate the culture under light conditions for 1-7 days to obtain the culture solution.

[0114] Step 2: Collect the cells from the culture medium by centrifugation, resuspend the cells in buffer, add enzyme, and incubate at a constant temperature. The final concentration of the enzyme is 0.2-0.5 mg / mL, and the temperature is 25-50°C for 1-5 hours. Then, a homogenization lysis process is performed to disrupt the cells. The homogenization lysis process is performed using a high-pressure homogenizer at a pressure of 800-1200 bar, and the homogenization process is repeated 1-5 times to prepare a high-pressure homogenized solution.

[0115] Step 3: Add salt and a surfactant to the prepared high-pressure homogenized solution for salting out. The salt is sodium chloride, added to a final concentration of 1-5 M; the protein denaturant is sodium dodecyl sulfate, added to a final concentration of 0.5-2%. After adding the salt and protein denaturant, incubate at 40°C-80°C for 1-3 hours, and then centrifuge to collect the supernatant to prepare a salting-out supernatant.

[0116] Step 4: adding anhydrous ethanol and sodium acetate to the salting-out supernatant, wherein the mass ratio of the salting-out supernatant to anhydrous ethanol is 1:(1-5), the concentration of the sodium acetate is 0.1-0.5M, settling for 20-60 minutes, rinsing and centrifuging to prepare a crude product;

[0117] Step 5: dehydrate and dry the crude product to obtain the product.

[0118] Furthermore, when the material is an animal, the method comprises the following steps:

[0119] Step 1: Collect the material, resuspend it, add enzyme to lyse it, and prepare the lysate;

[0120] Step 2: adding salt and a protein denaturant to the lysate to perform salting out to prepare a salting out clear solution;

[0121] Step 3: adding an organic solvent to the salting-out clear solution to precipitate and prepare a crude product;

[0122] Step 4: dehydrate and dry the crude product to obtain the product.

[0123] In an optional embodiment, when the material is an animal, a method for preparing PDRN comprises the following steps:

[0124] Step 1: Collect animal tissue, digest and prepare tissue lysate;

[0125] Step 2: Add salt and a surfactant to the prepared tissue lysate to perform salting-out. The salt is sodium chloride, added to a final concentration of 1-5 M; the protein denaturant is sodium dodecyl sulfate, added to a final concentration of 0.5-2%. After adding the salt and protein denaturant, incubate at 40°C-80°C for 1-3 hours, and then centrifuge to collect the supernatant to prepare the salting-out supernatant.

[0126] Step 3: adding anhydrous ethanol and sodium acetate to the salting-out supernatant, wherein the mass ratio of the salting-out supernatant to anhydrous ethanol is 1:(1-5), the concentration of the sodium acetate is 0.1-0.5M, settling for 20-60 minutes, rinsing and centrifuging to prepare a crude product;

[0127] Step 4: dehydrate and dry the crude product to obtain the product.

[0128] Optionally, the animal tissue comprises guinea pig liver tissue, muscle tissue, and kidney tissue. The guinea pig tissue is ground into fine powder at low temperature, resuspended and mixed, and then enzyme is added for lysis.

[0129] On the other hand, the present application also provides the use of microorganisms, animals and / or algae in the preparation of PDRN products;

[0130] Optionally, 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.

[0131] Optionally, the algae comprises freshwater algae, more preferably, the algae comprises Anabaena variabilis;

[0132] Optionally, the microorganism comprises one or more of lactobacillus, bacillus, eubacterium, and yeast. More optionally, the microorganism comprises one or more of Lactobacillus sakei, Eubacterium rectale, Bacillus pumilus, Komagataella sp., and Lactobacillus reuteri.

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

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

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

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

[0137] On the other hand, the present application also provides the use of Anabaena variabilis in the preparation or production of PDRN.

[0138] On the other hand, the present application also provides the use of guinea pigs (Cavia porcellus) in the preparation or production of PDRN.

[0139] On the other hand, the present application also provides a PDRN, wherein the GC content of the PDRN is 40.5%-42%.

[0140] The GC content of the PDRN prepared by the method is 40.5%-42%.

[0141] Optionally, 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 in between.

[0142] Optional, 40.5%-41.5%.

[0143] Furthermore, the weight average molecular weight of the PDRN is 100 kDa-500 kDa;

[0144] And / or, in terms of mass percentage, the protein content in the PDRN is less than or equal to 0.5%; and / or, the nucleic acid content in the PDRN is greater than or equal to 96.3%.

[0145] Alternatively, the weight average molecular weight of the PDRN may be selected from 100kDa, 110kDa, 120kDa, 130kDa, 140kDa, 150kDa, 160kDa, 170kDa, 180kDa, 190kDa, 200kDa, 210kDa, 220kDa, 230kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa kDa, 470 kDa, 480 kDa, 490 kDa, 500 kDa and any value therebetween.

[0146] Optionally, 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.

[0147] Optionally, 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 in between.

[0148] Optionally, the purity of the PDRN (OD 260 / OD 280 value) is 1.8-2.0.

[0149] The purity of the PDRN (OD 260 / OD 280 value) can be 1.8, 1.9, 2.0 and any value in between.

[0150] Optionally, the PDRN is in the form of white or off-white granules or powder.

[0151] Optionally, the PDRN has good solubility, and 1g of the product is completely dissolved in 1L of water within 30min.

[0152] More optionally, the dissolution time can 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 in, 16min, 16.5min, 17min, 17.5min, 18min, 18.5min, 19min, 19.5min, 20min, 20.5min, 21min, 21.5min, 22min, 22.5min, 23min, 23.5min, 24min, 24.5min, 25min, 25.5min, 26min, 26.5min, 27min, 27.5min, 28min, 28.5min, 29min, 29.5min, 30min and any value in between.

[0153] Optionally, the PDRN comprises PDRN of microbial, animal and / or algae origin.

[0154] Furthermore, the PDRN contains Lactobacillus sakei-derived PDRN.

[0155] Furthermore, the PDRN comprises a PDRN derived from Eubacterium rectum.

[0156] Furthermore, the PDRN comprises Bacillus pumilus-derived PDRN.

[0157] Furthermore, the PDRN comprises a Pseudomonas aeruginosa-derived PDRN.

[0158] Furthermore, the PDRN comprises Lactobacillus reuteri-derived PDRN.

[0159] Furthermore, the PDRN comprises a variable Houttuynia cordata source PDRN.

[0160] Furthermore, the PDRN comprises guinea pig-derived PDRN.

[0161] On the other hand, the present application also provides a composition comprising the PDRN.

[0162] Optionally, the PDRN comprises PDRN of microbial, animal and / or algae origin.

[0163] Furthermore, the PDRN contains Lactobacillus sakei-derived PDRN.

[0164] Furthermore, the PDRN comprises Bacillus pumilus-derived PDRN.

[0165] Furthermore, the PDRN comprises a PDRN derived from Eubacterium rectum.

[0166] Furthermore, the PDRN comprises a Pseudomonas aeruginosa-derived PDRN.

[0167] Furthermore, the PDRN comprises Lactobacillus reuteri-derived PDRN.

[0168] Furthermore, the PDRN comprises a variable Houttuynia cordata source PDRN.

[0169] Furthermore, the PDRN comprises guinea pig-derived PDRN.

[0170] Furthermore, the composition further comprises an auxiliary material.

[0171] The excipients may be appropriate solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc.

[0172] The composition of the present application can be prepared by general methods, wherein one or more diluents or carriers can be added, such as water, pills, tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions, etc.

[0173] The composition of the present application can be prepared into any formulation commonly prepared in the art. For example, it can be formulated into a cream, lotion, emulsion, facial mask, foundation, medical device, hair cosmetics, and other formulations. Furthermore, it can be formulated into a skin lotion, skin softener, water-lightening injection, skin toner, astringent, toner, lotion, moisturizing lotion, nourishing lotion, massage cream, nourishing cream, moisturizing cream, hand cream, foundation, essence, nourishing essence, facial mask, soap, cleansing foam, cleansing milk, cleansing cream, body lotion, or shower gel.

[0174] In the composition of the present application, in addition to the PDRN as an essential ingredient, other ingredients commonly formulated in cosmetics can be mixed as needed. For example, oil components, moisturizers, surfactants, organic pigments, inorganic pigments, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH regulators, alcohols, pigments, spices, blood circulation promoters, cooling agents, antiperspirants or purified water can be mixed.

[0175] On the other hand, the present application also provides the use of the algae, microorganisms, or the bacterial agent described by the microorganisms, or the PDRN, or the composition in the preparation of products that promote cell proliferation, promote cell migration, promote collagen production, anti-inflammatory and soothing products, and / or repair products for damaged skin barriers.

[0176] The algae comprises freshwater algae, more preferably, the algae comprises Anabaena variabilis.

[0177] 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.

[0178] Optionally, the PDRN comprises PDRN of microbial, animal and / or algae origin.

[0179] Furthermore, the PDRN contains Lactobacillus sakei-derived PDRN.

[0180] Furthermore, the PDRN comprises Bacillus pumilus-derived PDRN.

[0181] Furthermore, the PDRN comprises a PDRN derived from Eubacterium rectum.

[0182] Furthermore, the PDRN comprises a Pseudomonas aeruginosa-derived PDRN.

[0183] Furthermore, the PDRN comprises Lactobacillus reuteri-derived PDRN.

[0184] Furthermore, the PDRN comprises a variable Houttuynia cordata source PDRN.

[0185] Furthermore, the PDRN comprises guinea pig-derived PDRN.

[0186] The collagen is procollagen I α-1.

[0187] The anti-inflammatory relief is achieved by reducing inflammatory factors; the inflammatory factors include IL-6.

[0188] The repair of damaged skin barrier is achieved by increasing cell migration rate and / or relative cell proliferation rate;

[0189] Repairing the damaged skin barrier includes repairing the damaged skin barrier caused by ultraviolet rays and / or repairing the damaged skin barrier caused by trauma;

[0190] In an alternative embodiment, the cells are human dermal fibroblasts.

[0191] This application has the following beneficial effects:

[0192] This application is the first to extract PDRN from materials such as Lactobacillus sakei, Eubacterium rectale, Bacillus pumilus, Saccharomyces cerevisiae, Anabaena variabilis, and guinea pigs, 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.

[0193] The GC% in the gDNA of the PDRN prepared by the method described in this application is more than 99% similar to the GC% of humans, which is closer to the human GC% than the PDRN derived from salmon, and the PDRN whose GC% content is between 40.5% and 42% has better effects on promoting cell proliferation, promoting collagen production, promoting cell migration, anti-inflammatory and soothing, and / or repairing damaged skin barriers than PDRN outside this range and PDRN derived from salmon. Compared with the prior art of extracting PDRN from salmon semen, the preparation method is simpler and more convenient.

[0194] The present application also provides a method for naturally extracting PDRN from materials such as Lactobacillus sakei, Eubacterium rectum, Bacillus pumilus, Saccharomyces cerevisiae, Anabaena variabilis, and guinea pigs. The method is fully closed-loop managed, safe and controllable, and does not produce pollutants such as waste gas, wastewater, and waste residue during the process. Liquid residues and residues such as fermentation liquid and bacterial fragments can also be used as animal feed after processing. The production process is eco-friendly and has strong sustainable development capabilities.

[0195] The production of salmon PDRN from a single source is easily restricted by raw materials, and the instability of raw materials also limits its application range. Compared with the salmon's growth cycle of 3-7 years, the fermentation cycle of the various microorganisms (Lactobacillus sakei, Eubacterium rectum, Bacillus pumilus, and Saccharomyces cerevisiae) materials in this application is extremely short, and the culture cycle of materials such as Anabaena variabilis and guinea pigs is short. It has many advantages such as controllable growth environment, pure products, low cost, and strong sustainable development capabilities. The source of edible materials makes the future application scenarios of Lactobacillus sake PDRN broader. BRIEF DESCRIPTION OF THE DRAWINGS

[0196] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0197] FIG1 is a diagram showing the results of a cytotoxicity test;

[0198] FIG2 is a graph showing the results of UV repair efficacy testing;

[0199] FIG3 is a diagram showing the results of a wound repair efficacy test;

[0200] FIG4 is a diagram showing the soothing efficacy test results;

[0201] FIG5 is a graph showing the results of the collagen-promoting efficacy test.

[0202] Biodeposit Information:

[0203] A strain of Lactobacillus sakei LS-01 was deposited in the China Center for Type Culture Collection (CCTCC) on November 20, 2023, with the deposit number CCTCC NO: M 20232285. The deposit address is Wuhan University, Wuhan, China, Postal Code: 430072.

[0204] A strain of Bacillus pumilus Bp-1 was deposited in the China Center for Type Culture Collection (CCTCC) on February 12, 2025, with the deposit number CCTCC NO: M 2025198. The deposit address is Wuhan University, Wuhan, China, Postal Code: 430072.

[0205] A strain of Bacillus pumilus Bp-2 was deposited in the China Center for Type Culture Collection (CCTCC) on February 12, 2025, with the deposit number CCTCC NO: M 2025199. The deposit address is Wuhan University, Wuhan, China, Postal Code: 430072.

[0206] A strain of Komagataella sp. GS115 was deposited in the China Center for Type Culture Collection (CCTCC) on February 13, 2025, with the deposit number CCTCC NO: M 2025200. The deposit address is Wuhan University, Wuhan, China, Postal Code: 430072. DETAILED DESCRIPTION

[0207] In order to more clearly illustrate the overall concept of the present application, the following is a detailed description of the embodiments in conjunction with the accompanying drawings. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present application. However, it is obvious to those skilled in the art that the present application can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present application, some technical features well known in the art are not described.

[0208] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified herein, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of this application, any method, equipment, and material of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present application can also be used to realize this application.

[0209] The term polydeoxyribonucleotide (PDRN) referred to in this application refers to a high molecular weight double-stranded polymer or a low molecular weight double-stranded polymer composed of phosphate, deoxyribose and four bases, including adenine, guanine, thymine and cytosine, which constitute repeating units.

[0210] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0211] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market.

[0212] 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 Keygene Biotechnology; Lactobacillus rhamnosus was purchased from the Institute of Microbiology, Chinese Academy of Sciences, with the accession number CGMCC1.8882; Eubacterium rectale ATCC 33656 was purchased from the American Type Culture Collection, with the accession number ATCC 33656; Eubacterium rectale BPB22 was purchased from the China Center for Type Culture Collection, with the accession number CCTCC NO: M 20232177; Bacillus pumilus Bp-1 was deposited in the China Center for Type Culture Collection, with the accession number CCTCC NO: M 2025198; Bacillus pumilus Bp-2, which was deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025199; Bacillus pumilus ATCC 21143, purchased from the American Type Culture Collection with a deposit number of ATCC 21143; Komagataella sp. GS115, which was deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025200; Komagataella sp. CICC 33158, purchased from the China Center for Industrial Microbiological Culture Collection with a deposit number of CICC 33158; Anabaena variabilis, purchased from the American Type Culture Collection with a deposit number of ATCC 29413; Cavia pumilus porcellus, purchased from Liaoning Changsheng Biotechnology Co., Ltd., ordinary grade; Lactobacillus sakei CICC21858, with the accession number CICC21858, purchased from China Industrial Microbiological Culture Collection Administration Center; lysozyme was purchased from Beijing Solebold Technology Co., Ltd., item number L1080; snailase was purchased from Beijing Solebold Technology Co., Ltd., item number S8280; the brand of high-pressure homogenizer was ATS, and its model was AH-BASIC 30; BCA protein quantification kit was purchased from Nanjing Novezan Biotechnology Co., Ltd., item number E112-1.

[0213] In addition, the "water" mentioned in this application 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, and purified water.

[0214] In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight.

[0215] Example 1 Isolation and Identification of Lactobacillus sakei

[0216] Kimchi samples collected in Meishan, Sichuan, were aseptically crushed, weighed, and diluted with physiological saline to a 1% (w / v) solution. Appropriate amounts of the diluted solution were diluted with physiological saline in 10-fold gradients. 0.2 mL of each dilution gradient was spread onto MRS medium plates and incubated inverted at 37°C for 36-48 hours. Milky white colonies with neat, smooth, and shiny edges were selected and numbered 1-10 from largest to smallest according to size. Ten individual colonies were inoculated into liquid MRS medium and incubated at 37°C for 48 hours. The strains were collected and their genomes extracted using a genomic DNA extraction kit. Using the genomic DNA as a template, 16S rDNA sequences were obtained by polymerase chain reaction and Sanger sequencing. Sequence alignment using BLAST against the National Center for Biotechnology Information (NCBI) Nucleic Acid Database revealed that strains 2, 7, and 9 were Lactobacillus sakei. Considering that the colony size reflects the activity of the strain to a certain extent, the sake Lactobacillus No. 2 with the largest colony in the initial screening was selected as the subsequent test strain.

[0217] The 16S rDNA sequence of Lactobacillus sakei No. 2 is shown in SEQ ID No. 1. Lactobacillus sakei No. 2 was named LS-01 and was deposited in the China Center for Type Culture Collection (CCTCC) on November 20, 2023, with the deposit number CCTCCNO: M 20232285. The deposit address is Wuhan University, Wuhan, China, Postal Code: 430072.

[0218] Example 2

[0219] Test Example 1

[0220] In the present embodiment, a method for preparing PDRN derived from Lactobacillus sakei is provided as follows:

[0221] Step 1: Fermentation of Lactobacillus sakei to prepare fermentation broth:

[0222] Adopt above-described embodiment 1 method to cultivate lactobacillus sake LS-01, from the MRS solid culture medium flat board of lactobacillus sake LS-01, picking 1-2 ringlet bacterial classification, be inoculated in 40mL MRS liquid nutrient medium, 37 ℃, 160rpm shaking table are cultivated 8h, promptly get the seed liquor of lactobacillus sake.The seed liquor of lactobacillus sake is inoculated in 400mL MRS liquid nutrient medium by 4% inoculum size, 37 ℃ of cultivations 16h, promptly get the fermented liquid of lactobacillus sake.

[0223] Step 2: Collect, resuspend and prepare bacterial lysate (taking 300 mL fermentation broth as an example):

[0224] Take 300 mL of bacterial liquid (fermentation broth of Lactobacillus sakei), 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; 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 repeat 3 cycles to prepare a bacterial lysate.

[0225] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0226] To the obtained bacterial lysate, NaCl at a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) at a final concentration of 1% were added, and the mixture was incubated at 60° C. for 1.5 h. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0227] Step 4: Ethanol precipitation to prepare crude product:

[0228] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0229] Step 5: Dehydration and drying to obtain the product:

[0230] After rinsing, add 20 mL of anhydrous ethanol to the wet precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40°C until the product weight no longer changes significantly (the product weight change is less than 0.001 g). Drying is then completed to obtain the PDRN product.

[0231] Test Example 2

[0232] Step 1: Fermentation of Lactobacillus sakei to prepare fermentation broth:

[0233] Adopt above-described embodiment 1 method to cultivate lactobacillus sake LS-01, from the MRS solid culture medium flat board of lactobacillus sake LS-01, picking 1-2 ringlet bacterial classification, be inoculated in 40mL MRS liquid nutrient medium, 37 ℃, 160rpm shaking table are cultivated 8h, promptly get the seed liquor of lactobacillus sake.The seed liquor of lactobacillus sake is inoculated in 400mL MRS liquid nutrient medium by 4% inoculum size, 37 ℃ of cultivations 16h, promptly get the fermented liquid of lactobacillus sake.

[0234] Step 2: Collect, resuspend and homogenize the cells to prepare cell lysate (taking 300 mL of fermentation broth as an example):

[0235] Take 300 mL of bacterial liquid (fermentation broth of Lactobacillus sakei), 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; 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 1200 bar, and repeat once to prepare a bacterial lysate.

[0236] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0237] To the obtained bacterial lysate, NaCl at a final concentration of 1 M and SDS at a final concentration of 0.5% were added, and the mixture was incubated at 80° C. for 1 hour. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0238] Step 4: Ethanol precipitation to prepare crude product:

[0239] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0240] Step 5: Dehydration and drying to obtain the product:

[0241] After rinsing, add 20 mL of anhydrous ethanol to the wet precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40°C until the product weight no longer changes significantly (the product weight change is less than 0.001 g). Drying is then completed to obtain the PDRN product.

[0242] Test Example 3

[0243] Step 1: Fermentation of Lactobacillus sakei to prepare fermentation broth:

[0244] Adopt above-described embodiment 1 method to cultivate lactobacillus sake LS-01, from the MRS solid culture medium flat board of lactobacillus sake LS-01, picking 1-2 ringlet bacterial classification, be inoculated in 40mL MRS liquid nutrient medium, 37 ℃, 160rpm shaking table are cultivated 8h, promptly get the seed liquor of lactobacillus sake.The seed liquor of lactobacillus sake is inoculated in 400mL MRS liquid nutrient medium by 4% inoculum size, 37 ℃ of cultivations 16h, promptly get the fermented liquid of lactobacillus sake.

[0245] Step 2: Collect, resuspend and homogenize the cells to prepare cell lysate (taking 300 mL of fermentation broth as an example):

[0246] Take 300 mL of bacterial liquid (fermentation broth of Lactobacillus sakei), 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; 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 900 bar, and repeat 4 times to prepare a bacterial lysate.

[0247] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0248] To the obtained bacterial lysate, NaCl at a final concentration of 4 M and SDS at a final concentration of 1.5% were added, and the mixture was incubated at 50° C. for 2.5 h. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0249] Step 4: Ethanol precipitation to prepare crude product:

[0250] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0251] Step 5: Dehydration and drying to obtain the product:

[0252] After rinsing, add 20 mL of anhydrous ethanol to the wet precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40°C until the product weight no longer changes significantly (the product weight change is less than 0.001 g). Drying is then completed to obtain the PDRN product.

[0253] Test Example 4

[0254] Step 1: Fermentation of Lactobacillus sakei to prepare fermentation broth:

[0255] Adopt above-described embodiment 1 method to cultivate lactobacillus sake LS-01, from the MRS solid culture medium flat board of lactobacillus sake LS-01, picking 1-2 ringlet bacterial classification, be inoculated in 40mL MRS liquid nutrient medium, 37 ℃, 160rpm shaking table are cultivated 8h, promptly get the seed liquor of lactobacillus sake.The seed liquor of lactobacillus sake is inoculated in 400mL MRS liquid nutrient medium by 4% inoculum size, 37 ℃ of cultivations 16h, promptly get the fermented liquid of lactobacillus sake.

[0256] Step 2: Collect, resuspend and homogenize the cells to prepare cell lysate (taking 300 mL of fermentation broth as an example):

[0257] Take 300 mL of bacterial liquid (fermentation broth of Lactobacillus sakei), 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; add lysozyme to the bacteria resuspended in TE to a final concentration of 0.4 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 800 bar, and repeat twice to prepare a bacterial lysate.

[0258] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0259] To the obtained bacterial lysate, NaCl at a final concentration of 2.5 M and SDS at a final concentration of 1% were added, and the mixture was incubated at 60° C. for 1.5 h. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0260] Step 4: Ethanol precipitation to prepare crude product:

[0261] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0262] Step 5: Dehydration and drying to obtain the product:

[0263] After rinsing, add 20 mL of anhydrous ethanol to the wet precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40°C until the product weight no longer changes significantly (the product weight change is less than 0.001 g). Drying is then completed to obtain the PDRN product.

[0264] Test Example 5

[0265] Step 1: Fermentation of Lactobacillus sakei to prepare fermentation broth:

[0266] Adopt above-described embodiment 1 method to cultivate lactobacillus sake LS-01, from the MRS solid culture medium flat board of lactobacillus sake LS-01, picking 1-2 ringlet bacterial classification, be inoculated in 40mL MRS liquid nutrient medium, 37 ℃, 160rpm shaking table are cultivated 8h, promptly get the seed liquor of lactobacillus sake.The seed liquor of lactobacillus sake is inoculated in 400mL MRS liquid nutrient medium by 4% inoculum size, 37 ℃ of cultivations 16h, promptly get the fermented liquid of lactobacillus sake.

[0267] Step 2: Collect, resuspend and homogenize the cells to prepare cell lysate (taking 300 mL of fermentation broth as an example):

[0268] Take 300 mL of bacterial liquid (fermentation broth of Lactobacillus sakei), 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; 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 repeat once to prepare a bacterial lysate.

[0269] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0270] To the obtained bacterial lysate, NaCl at a final concentration of 2.5 M and SDS at a final concentration of 1% were added, and the mixture was incubated at 60° C. for 1.5 h. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0271] Step 4: Ethanol precipitation to prepare crude product:

[0272] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0273] Step 5: Dehydration and drying to obtain the product:

[0274] After rinsing, add 20 mL of anhydrous ethanol to the wet precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40°C until the product weight no longer changes significantly (the product weight change is less than 0.001 g). Drying is then completed to obtain the PDRN product.

[0275] Test Example 6

[0276] The only difference between this test example and test example 1 is that the fermentation strain used is Lactobacillus sakei CICC21858, with a preservation number of CICC21858, purchased from the China Industrial Microbiological Culture Collection Center.

[0277] Step 1: Fermentation of Lactobacillus sakei to prepare fermentation broth:

[0278] Lactobacillus sakei CICC21858 was cultured according to the method of Example 1. 1-2 loops of strains were picked from the MRS solid culture medium plate of Lactobacillus sakei CICC21858 and inoculated into 40 mL of MRS liquid culture medium. The culture was shaken at 37° C. and 160 rpm for 8 h to obtain a seed solution of Lactobacillus sakei. The seed solution of Lactobacillus sakei was inoculated into 400 mL of MRS liquid culture medium at a 4% inoculum size and incubated at 37° C. for 16 h to obtain a fermentation liquid of Lactobacillus sakei.

[0279] Step 2: Collect, resuspend and prepare bacterial lysate (taking 300 mL fermentation broth as an example):

[0280] Take 300 mL of bacterial liquid (fermentation broth of Lactobacillus sakei), 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; 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 repeat 3 cycles to prepare a bacterial lysate.

[0281] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0282] To the obtained bacterial lysate, NaCl at a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) at a final concentration of 1% were added, and the mixture was incubated at 60° C. for 1.5 h. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0283] Step 4: Ethanol precipitation to prepare crude product:

[0284] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0285] Step 5: Dehydration and drying to obtain the product:

[0286] After rinsing, add 20 mL of anhydrous ethanol to the wet precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40°C until the product weight no longer changes significantly (the product weight change is less than 0.001 g). Drying is then completed to obtain the PDRN product.

[0287] Test Example 7

[0288] This test example provides a method for preparing PDRN derived from Lactobacillus rhamnosus (Accession No. CGMCC 1.8882), as follows:

[0289] Step 1: Fermentation of Lactobacillus rhamnosus to prepare fermentation broth:

[0290] Pick 1-2 loops of bacteria from an MRS solid medium plate cultured with Lactobacillus rhamnosus and inoculate them into 40 mL of MRS liquid medium. Incubate anaerobically at 37°C for 8 hours to obtain a Lactobacillus rhamnosus seed solution. Inoculate 400 mL of MRS liquid medium at a 4% inoculum of the Lactobacillus rhamnosus seed solution and incubate at 37°C for 16 hours to obtain a Lactobacillus rhamnosus fermentation broth.

[0291] Step 2: Collect, resuspend and homogenize the cells to prepare cell lysate (taking 300 mL of fermentation broth as an example):

[0292] Take 300 mL of bacterial liquid (fermentation broth of Lactobacillus rhamnosus), 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; 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 repeat 3 cycles to prepare a bacterial lysate.

[0293] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0294] To the obtained bacterial lysate, NaCl at a final concentration of 2.5 M and SDS at a final concentration of 1% were added, and the mixture was incubated at 60° C. for 1.5 h. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0295] Step 4: Ethanol precipitation to prepare crude product:

[0296] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0297] Step 5: Dehydration and drying to obtain the product:

[0298] After rinsing, add 20 mL of anhydrous ethanol to the wet precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40°C until the product weight no longer changes significantly (the product weight change is less than 0.001 g). Drying is then completed to obtain the PDRN product.

[0299] Test Example 8

[0300] This test example provides a method for preparing PDRN derived from Eubacterium rectale ATCC 33656 (purchased from the American Type Culture Collection, numbered ATCC 33656) as follows:

[0301] Step 1: Fermentation of Eubacterium rectum to prepare fermentation broth:

[0302] One to two loopfuls of Eubacterium rectum ATCC 33656 (PYG) solid culture medium (purchased from Beijing Solaibao Technology Co., Ltd., catalog number LA5570) were inoculated into 40 mL of PYG liquid culture medium. The culture was then anaerobically cultured at 37°C for 16 hours to obtain a seed solution of Eubacterium rectum. A 4% inoculum of the seed solution was then inoculated into 400 mL of PYG liquid culture medium. The fermentation broth was then anaerobically cultured at 37°C for 48 hours to obtain a fermentation solution of Eubacterium rectum.

[0303] Step 2: Collect, resuspend and prepare bacterial lysate (taking 300 mL fermentation broth as an example):

[0304] Centrifuge 300 mL of bacterial culture (fermentation broth of Eubacterium rectum) at 9000 rpm for 5 minutes, discard the supernatant, rinse once with 300 mL of TE, centrifuge at 9000 rpm for 5 minutes, resuspend in 20 mL of TE buffer, and mix thoroughly. Add lysozyme to the resuspended cells at a final concentration of 0.2 mg / mL and incubate at 37°C for 2 hours. Then, pass the cells through a high-pressure homogenizer, maintaining a homogenization pressure of 1000 bar, for three cycles to prepare a bacterial lysate.

[0305] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0306] To the obtained bacterial lysate, NaCl at a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) at a final concentration of 1% were added, and the mixture was incubated at 60° C. for 1.5 h. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0307] Step 4: Ethanol precipitation to prepare crude product:

[0308] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0309] Step 5: Dehydration and drying to obtain the product:

[0310] After rinsing, add 20 mL of anhydrous ethanol to the precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40 ° C. When the product weight no longer changes significantly (the product weight change is less than 0.001 g), the drying is completed to obtain the PDRN product.

[0311] Test Example 9

[0312] This test example provides a method for preparing PDRN derived from Eubacterium rectale BPB22 (purchased from the China Center for Type Culture Collection, numbered CCTCC NO: M 20232177), as follows:

[0313] Step 1: Fermentation of Eubacterium rectum to prepare fermentation broth:

[0314] One to two loopfuls of Eubacterium rectum BPB22 culture medium (PYG, purchased from Beijing Solaibao Technology Co., Ltd., catalog number LA5570) were inoculated into 40 mL of PYG liquid medium. The culture was then anaerobically cultured at 37°C for 16 hours to obtain a seed solution of Eubacterium rectum. A 4% inoculum of the seed solution was then inoculated into 400 mL of PYG liquid medium. The culture was then anaerobically cultured at 37°C for 48 hours to obtain a fermentation broth of Eubacterium rectum.

[0315] Step 2: Collect, resuspend and prepare bacterial lysate (taking 300 mL fermentation broth as an example):

[0316] Centrifuge 300 mL of bacterial culture (fermentation broth of Eubacterium rectum) at 9000 rpm for 5 minutes, discard the supernatant, rinse once with 300 mL of TE, centrifuge at 9000 rpm for 5 minutes, resuspend in 20 mL of TE buffer, and mix thoroughly. Add lysozyme to the resuspended cells at a final concentration of 0.2 mg / mL and incubate at 37°C for 2 hours. Then, pass the cells through a high-pressure homogenizer, maintaining a homogenization pressure of 1000 bar, for three cycles to prepare a bacterial lysate.

[0317] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0318] To the obtained bacterial lysate, NaCl at a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) at a final concentration of 1% were added, and the mixture was incubated at 60° C. for 1.5 h. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0319] Step 4: Ethanol precipitation to prepare crude product:

[0320] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0321] Step 5: Dehydration and drying to obtain the product:

[0322] After rinsing, add 20 mL of anhydrous ethanol to the precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40 ° C. When the product weight no longer changes significantly (the product weight change is less than 0.001 g), the drying is completed to obtain the PDRN product.

[0323] Test Example 10

[0324] This test example provides a method for preparing PDRN derived from Bacillus pumilus Bp-1 (deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 2025198), as follows:

[0325] Step 1: Fermentation with Bacillus pumilus to prepare fermentation broth:

[0326] Take one to two loops of Bacillus pumilus Bp-1 from an LB solid culture medium plate (purchased from Beijing Solaibao Technology Co., Ltd., Cat. No. L1010) and inoculate into 40 mL of LB liquid culture medium. Incubate at 37°C, 150 rpm for 12 hours to obtain a Bacillus pumilus seed solution. Inoculate 400 mL of LB liquid culture medium at a 4% inoculum of the Bacillus pumilus seed solution and incubate at 37°C, 150 rpm for 24 hours to obtain a Bacillus pumilus fermentation broth.

[0327] Step 2: Collect, resuspend and prepare bacterial lysate (taking 300 mL fermentation broth as an example):

[0328] Centrifuge 300 mL of Bacillus pumilus fermentation broth at 9000 rpm for 5 minutes, discard the supernatant, rinse once with 300 mL of TE, centrifuge at 9000 rpm for 5 minutes, resuspend in 20 mL of TE buffer, and mix thoroughly. Add lysozyme to the resuspended bacteria at a final concentration of 0.2 mg / mL and incubate at 37°C for 2 hours. Then, pass the mixture through a high-pressure homogenizer, maintaining the homogenization pressure at 1000 bar, for three cycles to prepare a bacterial lysate.

[0329] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0330] To the obtained bacterial lysate, NaCl at a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) at a final concentration of 1% were added, and the mixture was incubated at 60° C. for 1.5 h. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0331] Step 4: Ethanol precipitation to prepare crude product:

[0332] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0333] Step 5: Dehydration and drying to obtain the product:

[0334] After rinsing, add 20 mL of anhydrous ethanol to the precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40 ° C. When the product weight no longer changes significantly (the product weight change is less than 0.001 g), the drying is completed to obtain the PDRN product.

[0335] Test Example 11

[0336] This test example provides a method for preparing PDRN derived from Bacillus pumilus Bp-2 (deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 2025199), as follows:

[0337] Step 1: Fermentation with Bacillus pumilus to prepare fermentation broth:

[0338] Take one to two loops of Bacillus pumilus Bp-2 from an LB solid culture medium plate (purchased from Beijing Solaibao Technology Co., Ltd., Cat. No. L1010) and inoculate into 40 mL of LB liquid culture medium. Incubate at 37°C, 150 rpm for 12 hours to obtain a Bacillus pumilus seed solution. Inoculate 400 mL of LB liquid culture medium at a 4% inoculum of the Bacillus pumilus seed solution and incubate at 37°C, 150 rpm for 24 hours to obtain a Bacillus pumilus fermentation broth.

[0339] Step 2: Collect, resuspend and prepare bacterial lysate (taking 300 mL fermentation broth as an example):

[0340] Centrifuge 300 mL of Bacillus pumilus fermentation broth at 9000 rpm for 5 minutes, discard the supernatant, rinse once with 300 mL of TE, centrifuge at 9000 rpm for 5 minutes, resuspend in 20 mL of TE buffer, and mix thoroughly. Add lysozyme to the resuspended bacteria at a final concentration of 0.2 mg / mL and incubate at 37°C for 2 hours. Then, pass the mixture through a high-pressure homogenizer, maintaining the homogenization pressure at 1000 bar, for three cycles to prepare a bacterial lysate.

[0341] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0342] To the obtained bacterial lysate, NaCl at a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) at a final concentration of 1% were added, and the mixture was incubated at 60° C. for 1.5 h. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0343] Step 4: Ethanol precipitation to prepare crude product:

[0344] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0345] Step 5: Dehydration and drying to obtain the product:

[0346] After rinsing, add 20 mL of anhydrous ethanol to the precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40 ° C. When the product weight no longer changes significantly (the product weight change is less than 0.001 g), the drying is completed to obtain the PDRN product.

[0347] Test Example 12

[0348] This test example provides a method for preparing PDRN derived from Bacillus pumilus ATCC 21143 (purchased from the American Type Culture Collection, numbered ATCC 21143), as follows:

[0349] Step 1: Fermentation with Bacillus pumilus to prepare fermentation broth:

[0350] Take one to two loops of Bacillus pumilus ATCC 21143 from a LB solid culture plate (purchased from Beijing Solaibao Technology Co., Ltd., Cat. No. L1010) and inoculate them into 40 mL of LB liquid culture medium. Incubate at 37°C, 150 rpm, for 12 hours to obtain a Bacillus pumilus seed solution. Inoculate 400 mL of LB liquid culture medium at a 4% inoculum of the Bacillus pumilus seed solution and incubate at 37°C, 150 rpm, for 24 hours to obtain a Bacillus pumilus fermentation broth.

[0351] Step 2: Collect, resuspend and prepare bacterial lysate (taking 300 mL fermentation broth as an example):

[0352] Centrifuge 300 mL of Bacillus pumilus fermentation broth at 9000 rpm for 5 minutes, discard the supernatant, rinse once with 300 mL of TE, centrifuge at 9000 rpm for 5 minutes, resuspend in 20 mL of TE buffer, and mix thoroughly. Add lysozyme to the resuspended bacteria at a final concentration of 0.2 mg / mL and incubate at 37°C for 2 hours. Then, pass the mixture through a high-pressure homogenizer, maintaining the homogenization pressure at 1000 bar, for three cycles to prepare a bacterial lysate.

[0353] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0354] To the obtained bacterial lysate, NaCl at a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) at a final concentration of 1% were added, and the mixture was incubated at 60° C. for 1.5 h. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0355] Step 4: Ethanol precipitation to prepare crude product:

[0356] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0357] Step 5: Dehydration and drying to obtain the product:

[0358] After rinsing, add 20 mL of anhydrous ethanol to the precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40 ° C. When the product weight no longer changes significantly (the product weight change is less than 0.001 g), the drying is completed to obtain the PDRN product.

[0359] Test Example 13

[0360] This test example provides a method for preparing PDRN derived from Komagataella sp. GS115 (deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 2025200), as follows:

[0361] Step 1: Fermentation of yeast to prepare fermentation broth:

[0362] Take one to two loopfuls of yeast from a YPD solid culture plate of Saccharomyces komagata GS115 (purchased from Beijing Solaibao Technology Co., Ltd., Catalog No. LA0220) and inoculate into 40 mL of YPD liquid culture. Incubate at 30°C, 220 rpm, and shake for 16 hours to obtain a Saccharomyces komagata seed solution. Inoculate 400 mL of YPD liquid culture medium at a 4% inoculum rate and incubate at 30°C, 220 rpm, for 30 hours to obtain a Saccharomyces komagata fermentation broth.

[0363] Step 2: Collect, resuspend and prepare bacterial lysate (taking 300 mL fermentation broth as an example):

[0364] Take 300 mL of bacterial broth (fermentation broth of Saccharomyces komagata), centrifuge at 9000 rpm for 5 minutes, discard the supernatant, rinse once with 300 mL of TE, centrifuge at 9000 rpm for 5 minutes, add 20 mL of TE buffer, and resuspend and mix thoroughly. Add snail enzyme (purchased from Beijing Solebow Technology Co., Ltd., Cat. No. S8280) to the resuspended bacteria in TE to a final concentration of 0.2 mg / mL and incubate at 45°C for 2 hours. Then pass through a high-pressure homogenizer, maintaining the high-pressure homogenization pressure at 1000 bar, and repeat three cycles to prepare a bacterial lysate.

[0365] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0366] To the obtained bacterial lysate, NaCl at a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) at a final concentration of 1% were added, and the mixture was incubated at 60° C. for 1.5 h. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0367] Step 4: Ethanol precipitation to prepare crude product:

[0368] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0369] Step 5: Dehydration and drying to obtain the product:

[0370] After rinsing, add 20 mL of anhydrous ethanol to the precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40 ° C. When the product weight no longer changes significantly (the product weight change is less than 0.001 g), the drying is completed to obtain the PDRN product.

[0371] Test Example 14

[0372] This test example provides a method for preparing PDRN derived from Komagataella sp. CICC 33158 (purchased from the China Industrial Microbiological Culture Collection Center, with the collection number CICC 33158), as follows:

[0373] Step 1: Fermentation of yeast to prepare fermentation broth:

[0374] Take one to two loops of yeast from a YPD solid medium plate (purchased from Beijing Solebao Technology Co., Ltd., Catalog No. LA0220) and inoculate them into 40 mL of YPD liquid medium. Incubate at 30°C, 220 rpm, and shake for 16 hours to obtain a Y. komagata seed solution. Inoculate 400 mL of YPD liquid medium with a 4% inoculum of the Y. komagata seed solution and incubate at 30°C, 220 rpm, for 30 hours to obtain a Y. komagata fermentation broth.

[0375] Step 2: Collect, resuspend and prepare bacterial lysate (taking 300 mL fermentation broth as an example):

[0376] Take 300 mL of bacterial broth (fermentation broth of Saccharomyces komagata), centrifuge at 9000 rpm for 5 minutes, discard the supernatant, rinse once with 300 mL of TE, centrifuge at 9000 rpm for 5 minutes, add 20 mL of TE buffer, and resuspend and mix thoroughly. Add snail enzyme (purchased from Beijing Solebow Technology Co., Ltd., Cat. No. S8280) to the resuspended bacteria in TE to a final concentration of 0.2 mg / mL and incubate at 45°C for 2 hours. Then pass through a high-pressure homogenizer, maintaining the high-pressure homogenization pressure at 1000 bar, and repeat three cycles to prepare a bacterial lysate.

[0377] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0378] To the obtained bacterial lysate, NaCl at a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) at a final concentration of 1% were added, and the mixture was incubated at 60° C. for 1.5 h. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0379] Step 4: Ethanol precipitation to prepare crude product:

[0380] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0381] Step 5: Dehydration and drying to obtain the product:

[0382] After rinsing, add 20 mL of anhydrous ethanol to the precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40 ° C. When the product weight no longer changes significantly (the product weight change is less than 0.001 g), the drying is completed to obtain the PDRN product.

[0383] Test Example 15

[0384] This experiment provides a method for preparing polydeoxyribonucleotide (PDRN) derived from Anabaena variabilis ATCC 29413 (purchased from the American Type Culture Collection, numbered ATCC 29413) as follows:

[0385] Step 1: Cultivate Anabaena variabilis and prepare culture medium:

[0386] The Anabaena variabilis strain was inoculated into AA liquid medium diluted 8 times (purchased from Beijing Solebeau Technology Co., Ltd., product number LA8520) at 30°C and 30 μE / (m 2 s) under light conditions, and culture the mixture statically for 5 days to obtain the culture solution of Anabaena variabilis.

[0387] Step 2: Collect, resuspend and prepare algal lysate (taking 300 mL of culture medium as an example):

[0388] Take 300 mL of culture medium (Anabaena variabilis), centrifuge at 9000 rpm for 5 minutes, discard the supernatant, rinse once with 300 mL of TE, centrifuge at 9000 rpm for 5 minutes, add 20 mL of TE buffer, and resuspend and mix thoroughly. Add lysozyme to the resuspended cells in TE to a final concentration of 0.2 mg / mL and incubate at 37°C for 2 hours. Then, pass the cells through a high-pressure homogenizer, maintaining a high-pressure homogenization pressure of 1000 bar, for three cycles to prepare the algal lysate.

[0389] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0390] To the obtained algal lysate, NaCl at a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) at a final concentration of 1% were added, and the mixture was incubated at 60° C. for 1.5 h. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0391] Step 4: Ethanol precipitation to prepare crude product:

[0392] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0393] Step 5: Dehydration and drying to obtain the product:

[0394] After rinsing, add 20 mL of anhydrous ethanol to the precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40 ° C. When the product weight no longer changes significantly (the product weight change is less than 0.001 g), the drying is completed to obtain the PDRN product.

[0395] Test Example 16

[0396] This study provides a method for preparing polydeoxyribonucleotide (PDRN) from guinea pigs (Cavia porcellus) (purchased from Liaoning Changsheng Biotechnology Co., Ltd., ordinary grade) as follows:

[0397] Step 1: Guinea Pig Breeding:

[0398] Three-week-old ordinary-grade guinea pigs purchased from Liaoning Changsheng Biotechnology Co., Ltd. were raised normally for three months.

[0399] Step 2: Collect, digest and prepare tissue lysate from guinea pig liver tissue (taking 1g of liver tissue as an example):

[0400] Guinea pigs were sacrificed by cervical dislocation. Liver tissue was collected by conventional dissection and frozen in liquid nitrogen for 10 minutes. The frozen liver tissue was removed and ground into a fine powder in liquid nitrogen. One gram of the liver was resuspended in 20 mL of TE buffer and mixed thoroughly. Proteinase K (purchased from Beijing Solebold Technology Co., Ltd., Cat. No. P9460) was added to the TE suspension to a final concentration of 2 mg / mL and incubated at 56°C for 2 hours to prepare tissue lysate.

[0401] Step 3: salting out and centrifuging to prepare salting out clear solution:

[0402] To the obtained tissue lysate, NaCl at a final concentration of 2.5 M and SDS (sodium dodecyl sulfate) at a final concentration of 1% were added, and the mixture was incubated at 60° C. for 1.5 h. The supernatant was then collected by centrifugation to prepare a salting-out supernatant.

[0403] Step 4: Ethanol precipitation to prepare crude product:

[0404] To the salting-out clear solution obtained in step 3, add sodium acetate at a final concentration of 0.3 M and stir thoroughly; after the sodium acetate is fully dissolved, slowly add 2.5 times the volume of anhydrous ethanol to the clear solution and precipitate for 30 minutes, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant; rinse the precipitate once with 75% ethanol, centrifuge at 9000 rpm for 10 minutes, and discard the supernatant to obtain the product wet precipitate.

[0405] Step 5: Dehydration and drying to obtain the product:

[0406] After rinsing, add 20 mL of anhydrous ethanol to the precipitate for dehydration. After the precipitate is fully dispersed, centrifuge at 9000 rpm for 10 min and discard the supernatant (repeat this operation 3 times). The resulting precipitate is transferred to a disposable petri dish and vacuum-dried at 40 ° C. When the product weight no longer changes significantly (the product weight change is less than 0.001 g), the drying is completed to obtain the PDRN product.

[0407] Comparative Example 1 uses the method of CN107287186A in the background technology to prepare the PDRN product.

[0408] Comparative Example 2 uses the method of CN112315836A in the background technology to prepare the PDRN product.

[0409] Comparative Example 3 uses the method of CN115074357A in the background technology to prepare the PDRN product.

[0410] The PDRN products obtained in the above-mentioned Test Examples 1-16 and Comparative Examples 1-3 were subjected to the following tests.

[0411] Purity and nucleic acid content testing:

[0412] Accurately weigh the above-mentioned dry product, re-dissolve it with water at a concentration of 5 mg / mL, incubate at 40°C for 30 minutes, and then turn the solution upside down to mix (repeat the operation 2-3 times until the product is completely dissolved). Use this solution to detect the purity of polynucleotides. After the above-mentioned dry product is dissolved in water, use an ultra-micro UV spectrophotometer to measure the absorbance of the re-dissolved sample at 260nm and 280nm. Determine the purity and nucleic acid content of the PDRN in the product based on the results fed back 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%.

[0413] Protein detection:

[0414] In this example, the protein content in the product was determined using a BCA protein quantification kit.

[0415] First, prepare BCA working solution by adding 1 volume of BCA Reagent B to 50 volumes of BCA Reagent A (50:1) (BCA Reagent A & B are included in the kit) and mix thoroughly.

[0416] Take a microplate and add the reagents according to the data in Table 1. After vortexing and mixing, incubate at 37°C for 30 minutes. Measure the absorbance at A562 nm using a microplate reader. Draw a standard curve with protein content (μg) as the horizontal axis and absorbance as the vertical axis.

[0417] Table 1

[0418] *: Standard protein included in the kit

[0419] Sample testing: Accurately weigh 100 mg of PDRN sample and dissolve it in 200 mL of deionized water to prepare a 0.5 g / L sample solution. Take 20 μL of the sample and add 200 μL of BCA working solution. After vortexing and mixing, incubate at 37°C for 30 min. Measure the absorbance at 562 nm using a microplate reader. Use the absorbance of a sample without standard protein as a blank control. Substitute the measured absorbance value into the standard curve to calculate the protein content in the test sample.

[0420] Product protein % = protein content in test sample / 0.5 * 100%.

[0421] Molecular weight detection:

[0422] In this embodiment, a fully automatic nucleic acid analyzer was used to determine the molecular weight of the product by capillary electrophoresis.

[0423] Among them, the instruments and equipment include: fully automatic nucleic acid analyzer Qsep100, electronic balance, vortex mixer, ultra-micro UV spectrophotometer, etc.

[0424] Test solution: Take 0.01 g of sample, dissolve it in water and dilute it to make a solution containing about 5 mg per 1 mL (0.5%). Continue to dilute the sample solution 100 times to a final sample concentration of 50 ng / μL.

[0425] On-machine testing: Turn on the instrument power and air compressor switch, double-click the software icon; add a new project; click "Connect" to establish system connection; configure the buffer and marker; click "Change buffer" to add the marker and buffer; load the sample tray; click "Change Sample" to open the transparent door and add the sample (sample volume ≥ 10μL); pierce the new card clip; open the card clip door, insert the card clip, and then close the card clip door; click "Latch"; card clip calibration; click the blank space to select: (1) sample position, (2) test plan, injection time, number of tests, (3) result file name, (4) calculation.

[0426] Result processing: After the program is completed, some short fragments with low content and excessively long fragments are manually removed, and the weight-average molecular weight is calculated (the product molecular weight is automatically calculated by the fully automatic nucleic acid analyzer).

[0427] Detection of bases and nucleotides:

[0428] In this example, the perchloric acid hydrolysis method was used to detect the base and nucleotide content in the product.

[0429] 5 mg of the sample to be tested was weighed and placed into six test tubes, with three test tubes in each group. 300 μL of 70% perchloric acid was added, while an equal amount of water was added to the control group. The test tubes were then sealed into ampoules. The samples were heated in boiling water at 100°C for 1 hour. After cooling, the ampoule was opened and completely transferred to a 25 mL volumetric flask. The pH was adjusted to 5.3 with 6 M potassium hydroxide and the volume was brought to volume with water. The samples were filtered through a 0.22 μm MCE filter membrane for analysis.

[0430] Standard solution preparation: Dissolve adenine and uracil in 0.1 M KOH, and cytosine and uracil in ultrapure water, respectively, to a final concentration of 1 mg / mL. Gradual dilutions were performed using 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 filter membrane before liquid chromatography detection.

[0431] Base and nucleotide analysis methods: An Agilent ZORBAX EC-C18 column (4 μm, 4.6 mm × 150 mm) was used. Mobile phase A: 5 mM ammonium acetate; mobile phase B: methanol. Gradient elution program: 0% to 5% methanol (0–10 min); 5–20% methanol (10–30 min). The flow rate was set at 0.45 mL / min. The column temperature was maintained at 25°C. The DAD detection wavelength was set at 254 nm, and a 5 μL injection volume was used for chromatographic acquisition and quantitative analysis. GC% was calculated by HPLC integration.

[0432] Appearance and solubility testing:

[0433] In this embodiment, the appearance of the product was observed and recorded, and the solubility of the product was tested.

[0434] Observation of appearance characteristics:

[0435] When observed with the naked eye, the sample to be tested should appear as white or off-white granules or powder.

[0436] Solubility test:

[0437] Weigh 1g of the sample to be tested, add 1L of pure water to the volume, mix gently, let it stand for 30 minutes, and then observe the solubility. If the resulting solution is clear, free of impurities and turbidity, the sample has passed the solubility test; otherwise, it has failed the solubility test.

[0438] The test results of the above examples and comparative examples and their comparison are shown in Table 2.

[0439] Table 2

[0440] *: 1g of product dissolves in 1L of water within 30 minutes

[0441] As can be seen from the contents of Table 2, the PDRN product prepared by the method described in the experimental example has high purity and nucleic acid content, and has a low protein content. The GC content of the PDRN product is 39.5%-41.5%, which is very close to the human GC content of 40%, and is more than 98% homologous. It has a high nucleic acid content, molecular weight and yield, and is suitable for industrial production.

[0442] Example 3 Efficacy Test

[0443] In this embodiment, 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 2 were used as samples for efficacy testing.

[0444] Cytotoxicity test:

[0445] Specifically, cytotoxicity tests were performed using the above-mentioned samples in Test Examples 1, 7-16, and Comparative Examples 1-3. Each sample was tested at three concentrations, 0.01%, 0.05%, and 0.1%. The test sample information is shown in Table 3. HS27 cells were used as experimental subjects, and the culture system was DMEM medium (supplemented with 10% fetal bovine serum) in a carbon dioxide incubator at 37°C and 5% CO2. After 24 hours of cell culture, the original culture medium was discarded and serum-free medium mixed with the sample was added to continue culturing for 24 hours. The sample concentrations are shown in Table 3. The control group was water. The relative cell proliferation rate was detected by CCK-8 assay. The relative proliferation rate data are shown in Table 4. Figure 1 was plotted based on the data in Table 4.

[0446] Table 3

[0447] Table 4

[0448] The cytotoxicity test results in Table 4 and Figure 1 show that the products prepared in Experimental Examples 1 and 8-15 of the present application are not cytotoxic (cell proliferation rate>80%), and the overall cell proliferation promoting effect of PDRN derived from Lactobacillus sakei, Eubacterium rectum, Bacillus pumilus, Saccharomyces cerevisiae and Anabaena variabilis is better than that of Experimental Example 7 and Comparative Examples 1-3, and is better than that of PDRN derived from guinea pigs.

[0449] UV repair efficacy test:

[0450] The above-mentioned sample test examples 1, 7-16 and comparative examples 1-3 (sample concentrations are all 0.01%) were used to test the UV repair efficacy. Specifically, HS27 cells were used as experimental subjects, the culture system was DMEM medium (supplemented with 10% fetal bovine serum), and the culture conditions were a carbon dioxide incubator at 37°C and 5% CO2. After the cells were irradiated with UV light (2000 μW / cm 2 The cells were then irradiated for 80 minutes (80 minutes). The original culture medium was discarded and serum-free culture medium mixed with the sample was added for another 24 hours. The relative cell proliferation rate was measured using the CCK-8 assay. A control group was not exposed to UV irradiation, while the UV group was exposed to UV irradiation followed by an equal amount of serum-free culture medium without sample. The relative proliferation rate data are shown in Table 5, and Figure 2 was plotted based on the data in Table 5.

[0451] Table 5

[0452] The UV repair efficacy test results in Table 5 and Figure 2 show that the UV repair effect of the products prepared in Experimental Examples 1 and 8-16 of the present application is better than the UV repair effect of the PDRN products prepared in Experimental Examples 7 and Comparative Examples 1-3. In addition, the UV repair effect of PDRN derived from Lactobacillus sakei, Eubacterium rectum, Bacillus pumilus, Saccharomyces cerevisiae, and Anabaena variabilis is better than that of PDRN derived from guinea pigs.

[0453] Wound repair efficacy test

[0454] The wound repair efficacy test was continued using the above sample test examples 1, 7-16 and comparative examples 1-3 (the sample concentration was all 0.01%).

[0455] Specifically, using HS27 cell as experimental subject, culture system is DMEM culture medium (adding 10% fetal calf serum), and culture condition is 37 ℃, 5% CO2 of carbon dioxide incubator to cultivate.The sample treatment process is: in 24-well culture plates, place the ibidi chamber, after cell culture 24h, take off the ibidi chamber.The line is taken pictures, and discards original culture medium and adds the serum-free medium that is mixed with sample and continues to cultivate 24h, 36h and 48h (observe and take pictures), and the matched group only adds equal amount of serum-free medium and does not add sample, and the area of ​​the scratched area is analyzed with ImageJ software in the photo, cell migration rate (%)=(scratched area area / initial scratched area area after 1-dosing) × 100.Cell migration rate data are as shown in Table 6, and according to Table 6 data drawing, Fig. 3 is obtained.

[0456] Table 6

[0457] The results in Table 6 and Figure 3 show that after 24 hours of treatment with PDRN from different sources, the PDRN prepared in Experimental Examples 1 and 8-15 of the present application is more effective in promoting cell migration than PDRN from other processes and sources, indicating that the PDRN derived from Lactobacillus vinifera, Eubacterium rectum, Bacillus pumilus, Saccharomyces cerevisiae and Anabaena variabilis prepared by the process of this application has good wound repair effect.

[0458] Soothing efficacy test

[0459] The soothing efficacy test was continued using the above sample test examples 1, 7-16 and comparative examples 1-3 (the sample concentration was all 0.01%).

[0460] Specifically, Raw264.7 cells were cultured in RPMI1640 medium (supplemented with 10% fetal bovine serum) in a CO2 incubator at 37°C and 5% CO2. Sample processing was as follows: After 24 hours of cell culture, the original culture medium was discarded and serum-free medium mixed with the sample was added for another 24 hours (lipopolysaccharide (LPS) was used as a model control). The relative cell proliferation rate was measured using the CCK-8 assay, and the supernatant was collected by centrifugation and assayed for inflammatory factors according to the ELISA kit instructions. The relevant data are shown in Table 7, and Figure 4 was plotted based on the data in Table 7.

[0461] Table 7

[0462] The results in Table 7 and Figure 4 show that after the products prepared in Experimental Examples 1 and 8-15 of the present application were used to treat the LPS model cell line, the content of the inflammatory factor IL-6 was significantly reduced, indicating that the PDRN derived from Lactobacillus vinifera, Eubacterium rectum, Bacillus pumilus, Saccharomyces cerevisiae and Anabaena variabilis prepared by the process of the present application has good soothing effect.

[0463] Collagen-promoting efficacy test

[0464] The above-mentioned sample test examples 1, 7-16 and comparative examples 1-3 (all sample concentrations were 0.01%) were used to carry out collagen-promoting efficacy tests.

[0465] Specifically, HS27 cells were cultured in DMEM (supplemented with 10% fetal bovine serum) in a CO2 incubator at 37°C and 5% CO2. Sample processing was as follows: After 24 hours of cell culture, the original culture medium was discarded and replaced with serum-free culture medium mixed with the sample for another 24 hours. The cell supernatant was collected. A control group received an equal amount of serum-free culture medium without the sample, and procollagen Iα-1 (procollagen Iα-1) content was measured using a Pro-Collagen Iα-1 ELISA kit. The relevant data are shown in Table 8, and Figure 5 was plotted based on the data in Table 8.

[0466] Table 8

[0467] The results in Table 8 and Figure 5 show that after 24 hours of treatment with the products prepared in Experimental Examples 1 and 8-15 of this application, the content of procollagen I α-1 in cells was significantly increased. This indicates that the PDRN derived from Lactobacillus vinifera, Eubacterium rectum, Bacillus pumilus, Saccharomyces cerevisiae, and Anabaena variabilis prepared using the process of this application can significantly promote collagen synthesis in cells, and the effect is better than that of PDRN derived from guinea pigs.

[0468] It has been verified that the PDRN product derived from Lactobacillus brevicornis, Eubacterium rectale, Bacillus pumilus, Saccharomyces cerevisiae and Anabaena variabilis prepared by the method described in this application is non-cytotoxic and has ultraviolet repair effect, wound repair effect and collagen-promoting effect.

[0469] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all 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, wherein The weight average molecular weight of the PDRN is 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, drying the crude product to obtain a product; Optionally, the material includes microorganisms, animals and / or algae; More optionally, the animal comprises one or more of guinea pig (Cavia porcellus), Caenorhabditis japonica, Drosophila ananassae, Branchiostoma floridae, Oryzias latipes, and Gallus gallus. More optionally, the animal comprises guinea pig (Cavia porcellus); Optionally, the algae comprises freshwater algae, more preferably, the algae comprises Anabaena variabilis; More optionally, the microorganism comprises one or more of Lactobacillus, Bacillus, Eubacterium, and yeast. More optionally, the microorganism comprises one or more of Lactobacillus sakei, Eubacterium rectale, Bacillus pumilus, Komagataella sp., and Lactobacillus reuteri.

4. The method according to claim 3, characterized in that The method comprises the following steps: Step 1: Collect the material, resuspend it, add enzyme to lyse it, and prepare the lysate; 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; Optionally, the enzyme includes one or more of Labiase, mutanolysin, lysostaphin, chromopeptidase, papain, proteinase K, and snailase produced by Streptomyces fusca; optionally, the enzyme is added at a final concentration of 0.2-2 mg / mL; Optionally, the salt includes one or more of sodium chloride, potassium acetate, sodium acetate, ammonium sulfate, and sodium sulfate; optionally, the final concentration of the salt added is 1-5M; Optionally, the protein denaturant includes one or more of guanidine hydrochloride, phenol and a surfactant; Optionally, 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 A7) or more: A1) Application of Lactobacillus sakei in preparing PDRN products; A2) Use of Eubacterium rectale in the preparation of PDRN products; A3) Use of Bacillus pumilus in the preparation of PDRN products; A4) Use of Komagataella sp. in the preparation of PDRN products; A5) Use of Lactobacillus reuteri in the preparation of PDRN products; A6) Use of guinea pigs (Cavia porcellus) in the preparation of PDRN products; A7) Use 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 the 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 the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025199.

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

10. Lactobacillus sakei, characterized in that The Lactobacillus sakei is Lactobacillus sakei LS-01, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20232285.

11. the PDRN as claimed in claim 1 or 2 or the PDRN obtained by the preparation method as claimed in 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 Lactobacillus sakei as claimed in claim 10 or the application of Anabaena variabilis described in claim 3 in the product of promoting cell proliferation and / or cell migration and / or promoting collagen production.

12. The use of the PDRN as claimed in claim 1 or 2, or the PDRN obtained by the preparation method as claimed in 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 Lactobacillus sakei as claimed in claim 10, or Anabaena variabilis as described in claim 3 in the preparation of anti-inflammatory, soothing and / or repairing skin barrier damaged products.

Images