Pdrn, preparation method and application thereof

Abstract

The application discloses PDRN and a preparation method and application thereof, and the preparation method of the PDRN comprises the following steps: performing fermentation treatment by using lactobacillus plantarum, collecting bacterial bodies after centrifugation of obtained fermentation culture solution; resuspending the obtained bacterial bodies, homogenizing and crushing, centrifuging and removing the precipitate, concentrating by using a 5kDa hollow fiber membrane, purifying the obtained concentrated solution by using a Q column, and then performing concentration and liquid replacement, so that high-purity PDRN is obtained. The PDRN is obtained by fermentation of lactobacillus plantarum for the first time, and compared with the PDRN from current salmon or salmon trout, the production cost of the PDRN is lower, so that a new way is provided for production of the PDRN. Through experimental verification, the PDRN obtained by the method has higher activity in promoting cell proliferation and promoting collagen production.

Description

Technical Field

[0001] This application relates to the field of bio-fermentation technology, and in particular to a PDRN, its preparation method, and its application. Background Technology

[0002] PDRN (Polydeoxyribonucleotides) is a mixture of polynucleotides with a molecular weight between 50 and 1500 kDa. Currently, it is primarily extracted and purified from salmon or trout sperm cells. PDRN has been used in post-skin graft repair, demonstrating excellent biocompatibility and tissue regeneration capabilities. It has been approved by the Korean Food and Drug Administration and is commonly used for tissue repair and wound treatment.

[0003] The core mechanisms of action of PDRN include: Adenosine A2A receptor activation. PDRN breaks down into adenosine, which activates the A2A receptor, triggering signaling pathways, promoting angiogenesis and collagen synthesis, and inhibiting inflammatory responses. Nucleic acid salvage pathway: PDRN degrades into nucleosides and nucleotides, providing DNA repair materials for damaged cells and supporting cell regeneration.

[0004] In recent years, research on PDRN has expanded from medical aesthetics to areas such as skin anti-aging, inflammation repair, and hair regeneration. Research on its combination with ingredients such as hyaluronic acid and collagen, as well as the development of plant-derived and microbial-derived PDRN, has further promoted its application in skincare and medical aesthetic products.

[0005] Although salmon-derived PDRN has considerable activity, its long growth cycle and high breeding costs result in high overall PDRN production costs. Some plant-derived PDRN, such as Chlorella, also have long growth cycles, which limits their industrialization, and their activity is uncertain. Furthermore, the composition of PDRN is complex. Therefore, there is an urgent need to find a method that can produce PDRN with both a short production cycle and good activity. Summary of the Invention

[0006] In view of the above-mentioned technical problems existing in the prior art, the purpose of this invention is to provide a PDRN, its preparation method and application.

[0007] The technical solution adopted in this invention is as follows: A method for preparing PDRN includes the following steps: 1) Fermentation was carried out using Lactobacillus plantarum, and the resulting fermentation broth was centrifuged to collect the bacterial cells; 2) Resuspend the bacterial cells obtained in step 1), homogenize and break them, centrifuge to remove the precipitate, and concentrate them using a 5kDa hollow fiber membrane; 3) The concentrate obtained in step 2) is purified using a Q column, and then concentrated and replaced to obtain PDRN.

[0008] Further, the fermentation medium for step 1) fermentation treatment consists of: 40g glucose, 10g corn steep liquor, 15g fish syrup, 5g diammonium hydrogen phosphate, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1mL Tween 80, 1g defoamer, and water to a final volume of 1L.

[0009] Further, in step 1), during fermentation, Lactobacillus plantarum is inoculated into the fermentation medium at an inoculation rate of 2%-6%, the fermentation temperature is 35-37℃, and the fermentation is carried out for 40-50 hours.

[0010] Further, in step 2), the volume of the original liquid is concentrated to 1 / 8 to 1 / 12 using a 5kDa hollow fiber membrane.

[0011] Furthermore, in step 3), the purification process using a Q column is as follows: S1: Load the concentrated solution onto the equilibrated Q column. After loading, continue washing with equilibration buffer to reach the baseline. The equilibration buffer contains Tris-HCl buffer with a pH of 7.0 ± 0.2 and a concentration of 17.55 g / L ± 0.5 g / L NaCl. S2: Rinse to baseline with wash buffer containing 29.25 g / L ± 0.5 g / L NaCl in pH 7.0 ± 0.2 Tris-HCl buffer. S3: Load the sample with elution buffer and collect the eluent; the elution buffer contains Tris-HCl buffer with a pH of 7.0±0.2 and a concentration of 40.95 g / L±0.5 g / L NaCl.

[0012] Further, the concentration and liquid exchange process in step 3) is as follows: the purified eluent is concentrated and exchanged using a 5kDa hollow fiber membrane. When the concentration is reduced to 1 / 8 to 1 / 12 of the original solution, 3-5 times the volume of purified water is added. The concentration and liquid exchange are repeated, and the conductivity of the concentrated solution is measured. The liquid exchange operation can be stopped when the conductivity is less than 0.5ms.

[0013] This invention also discloses the application of the aforementioned PDRN in the preparation of products that promote cell proliferation and / or collagen production.

[0014] Compared with the prior art, the beneficial effects achieved by the present invention are: (1) This invention is the first to use Lactobacillus plantarum to ferment PDRN. Compared with PDRN from sources such as salmon or salmon, the production cost of this invention is lower, providing a new way for the production of PDRN.

[0015] (2) This application has been experimentally verified that the PDRN obtained by the method of this invention has higher activity in promoting cell proliferation and collagen production. Attached Figure Description

[0016] Figure 1 These are the gel electrophoresis results of PDRN purification in Example 2; Figure 2 These are the gel electrophoresis results obtained from Example 2 at different storage times; Figure 3 This is a comparison of the PDRN of this invention with the commercial PDRN competing products in terms of HSF cell proliferation ability; Figure 4 This is a comparison of the relative secretion rates of IL-10 in different sample well groups in Example 4 of the present invention; Figure 5 This is a comparison of the relative IL-6 secretion inhibition rates of different sample well groups in Example 4 of the present invention; Figure 6 This is a comparison of the relative secretion rates of Col-III in different sample well groups in Example 5 of the present invention; Figure 7 This is a comparison of the relative secretion rates of Col-I in different sample well groups in Example 5 of the present invention. Detailed Implementation

[0017] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0018] The source information of Lactobacillus plantarum in this embodiment of the invention is as follows: Strain number: BNCC194165; Source strain: CICC22696 (i.e., the original isolate); Manufacturer: Northern Weiye Measurement Group Co., Ltd. (BNCC).

[0019] In this embodiment of the invention, two commercially available PDRN competing products were used as controls. The first was designated as the RH sample, with product batch number RN01-250320. The second was designated as the RGM sample, with product batch number RJMPDRN-M.

[0020] Example 1: Fermentation of Lactobacillus plantarum 1. The culture medium preparation is shown in Table 1.

[0021] Table 1 .

[0022] 2. Fermentation process: Adjust the temperature of 5L fermentation medium to 37℃, adjust the pH of the solution to 6.0 with 25% ammonia water, inoculate with 5% Lactobacillus plantarum, and culture at 37℃ for 48 hours.

[0023] Example 2: PDRN purification 1. The solution preparation is shown in Table 2.

[0024] Table 2 .

[0025] 2. Purification 2.1) Preprocessing (1) Collect the bacterial cells after centrifuging the 5L fermentation broth of Example 1, and resuspend the bacterial cells in 400mL resuspending buffer. (2) The resuspended bacterial solution was crushed in a homogenizer pre-cooled at 4°C, and homogenized 5 to 6 times at a pressure of 1200 to 1300 bar. (3) Centrifuge the homogenized bacterial culture at 12,000 rpm for 20 min, collect the supernatant, and initially remove bacterial fragments; (4) The supernatant was microfiltered with a 0.2 μm hollow fiber membrane to remove the remaining bacterial fragments, and finally microfiltered to obtain 2.5 L of microfiltrate; (5) The microfiltrate was concentrated using a 5kDa hollow fiber membrane to a final concentration of 250mL.

[0026] 2.2) Sample purification (1) Q column pretreatment Rinse the Q column (Q column packing volume is 200 mL) with 2 L of equilibration buffer until pH and conductivity are stable.

[0027] (2) Sample loading Load the 250 mL concentrated sample obtained in step 2.1) onto the equilibrated Q column, and monitor the UV. When the UV rises, collect the flow through the column until the UV level is basically reached, then stop collecting.

[0028] (3) Washing off the dregs Rinse with equilibration buffer until UV stabilizes, and collect wash solution 1; Rinse with washing buffer, paying attention to UV and conductivity. When UV and conductivity rise, collect washing buffer 2. Stop collecting when UV drops to the baseline level.

[0029] (4) Washing Rinse with elution buffer, paying attention to UV and conductivity. When UV and conductivity rise, collect the eluent. Stop collecting when UV drops to baseline levels.

[0030] 3. Change the fluid The eluent was concentrated and replaced using a 5kDa hollow fiber optic filter. When the concentration was reduced to 1 / 10 of the original solution, 4 times the volume of purified water was added. The concentration and replacement process was repeated. The conductivity of the concentrate was measured. The replacement process was stopped when the conductivity was less than 0.5ms. Finally, the solution was concentrated to about 50mL to obtain the PDRN purified solution.

[0031] 4. Testing Configure nucleic acid electrophoresis, perform gel electrophoresis, and the results are as follows: Figure 1 As shown, the obtained PDRN purified solution contains components with multiple molecular weights, one of which has a molecular weight around 250 bp.

[0032] The PDRN purified solution obtained in Example 2 was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Sequencing showed that it contained multiple gene fragments, among which the base sequences of five major gene fragments are shown as SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO4 and SEQ ID NO5, respectively.

[0033] Take 0.5 g of the PDRN purified solution obtained in Example 2, dilute it with 1 L of DMEM medium to a PDRN concentration of 0.5 mg / ml, and store the resulting PDRN solution at 4°C for one week. The gel electrophoresis results at different time points are as follows. Figure 2 As shown. Figure 2 The "Marker" in the text refers to an indicator band. "0 weeks" represents the initial state of the PDRN solution when it was first stored at 4°C, and "1 week" represents the state of the PDRN solution after one week of storage at 4°C. According to... Figure 2 The results show that, compared with the original, the PDRN degradation degree after one week is not high, which indicates that the PDNR invented in this patent has strong stability.

[0034] Example 3: Activity Detection - Cell Proliferation The PDRN solution obtained in Example 2 was prepared with serum-free culture medium (purchased from Shanghai Meiji Biomedical Technology Co., Ltd., hereinafter the same) to a concentration of 0.5 mg / ml. The solution was then filtered through a 0.22µm filter membrane for sterilization, and the resulting solution was designated as the JB PDRN sample solution. The commercially available PDRN competitor, RH sample, was also prepared with serum-free culture medium to a concentration of 0.5 mg / ml, and the solution was filtered through a 0.22µm filter membrane for sterilization. The resulting solution was designated as the RH sample solution.

[0035] HSF cells (human skin fibroblasts) in logarithmic growth phase were taken at a concentration of 1×10⁻⁶. 5HSF cells were seeded at a density of 100 µL / mL in 96-well plates, divided into a blank control group, a positive control group, and an experimental group. The plates were incubated in a CO2 cell culture incubator at 37°C and 5% CO2 for 24 h. After 24 h of routine culture, the old culture medium was discarded. The blank control group (containing no serum or PDRN) was treated with 100 µL of serum-free culture medium. The positive control group was treated with 100 µL of the prepared RH sample solution. The experimental group was treated with 100 µL of the prepared JB PDRN sample solution, with three replicates per group. After another 24 h of culture, the culture medium was discarded, and 100 µL of CCK-8 (purchased from Sangon Biotech (Shanghai) Co., Ltd.) diluted 10-fold with serum-free culture medium was added to each well. The plates were then incubated for another 2 h. The relative cell proliferation rate was detected using the CCK-8 assay, with absorbance measured at 450 nm using a microplate reader. The calculation formula is: Relative cell proliferation rate (RGR) % = Absorbance value of experimental group / Absorbance value of blank control group × 100%. Results are as follows: Figure 3 As shown.

[0036] Figure 3 In this context, DMEM represents the blank control group, JB PDRN represents the experimental group, and RH represents the positive control group. Figure 3 The results showed that, within the selected concentration range, the PDRN of the present invention had a superior ability to promote HSF cell proliferation compared to commercial PDRN competitors.

[0037] Example 4: Activity Detection - Anti-wrinkle and Soothing Effects (1) Effects of PDRN anti-inflammatory factor IL-10 1. Add 100 μL of DMEM complete medium to each well of a 96-well plate, and add 5000 mouse macrophage leukemia cells (RAW264.7) to each well of the medium. Incubate in a CO2 incubator at 37°C and 5% CO2 for 24 hours.

[0038] 2. After culturing for 24 hours, the medium was replaced with serum-free medium and then placed in a CO2 incubator at 37°C and 5% CO2 for 2 hours.

[0039] 3. After culturing for 2 hours, add LPS to each well at a final concentration of 10 ug / ml, and place in a CO2 incubator at 37℃ and 5% CO2 for another 6 hours.

[0040] Centrifuge at 3000 rpm for 20 min and collect the supernatant.

[0041] 5. The mouse interleukin-10 enzyme-linked immunosorbent assay kit from Jiangsu Enzyme Immunoassay Co., Ltd. was selected.

[0042] 6. Sample Addition: Set up blank wells (blank control wells do not contain sample or enzyme-labeled reagent; all other steps are the same) and sample wells. Add 40 μL of sample diluent to the sample wells, then add 10 μL of the sample solution (sample dilution is 5-fold, resulting in a concentration of 0.5 mg / mL). Add the sample to the bottom of the wells, avoiding contact with the well walls, and gently shake to mix.

[0043] 7. Incubation: After sealing the plate with sealing film, incubate at 37°C for 30 minutes.

[0044] 8. Solution preparation: Dilute the 20-fold concentrated washing solution with distilled water 20 times and set aside.

[0045] 9. Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let stand for 30 seconds, then discard. Repeat 5 times, then pat dry.

[0046] 10. Add enzyme: Add 50 μL of enzyme-labeled reagent to the sample wells to be tested, and add 50 μL of DMEM complete culture medium to the blank wells.

[0047] 11. Incubation: The procedure is the same as step 7.

[0048] 12. Washing: Same as step 9.

[0049] 13. Color development: Add 50 μL of developer A to each well, then add 50 μL of developer B, gently shake to mix, and develop at 37°C in the dark for 10 minutes.

[0050] 14. Termination: Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).

[0051] 15. Measurement: Measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0052] Relative secretion rate (%) = OD of the sample well group 450nm Value / OD of blank hole group 450nm Value * 100%.

[0053] With the relative IL-10 secretion rate of the blank well group being 100%, the comparison results of the relative IL-10 secretion rates of different test sample well groups are shown in the figure. Figure 4 . Figure 4 In the diagram, PDRN without PDRN represents a blank well group, JB PDRN represents a well group of test samples with PDRN obtained in Example 2, and RH represents a well group of test samples with commercial PDRN competitors.

[0054] according to Figure 4 Conclusion: OD was measured using an ELISA reader. 450The data show that the group that added JI PDRN had a IL-10 secretion level of 121.7%, while the competing product had a IL-10 secretion level of 99.7%, indicating that JB PDRN has a significant anti-inflammatory effect.

[0055] (2) Effect of PDRN on the pro-inflammatory factor IL-6 1. Add 100 μL of DMEM complete medium to each well of a 96-well plate, and add 5000 mouse macrophage leukemia cells (RAW264.7) to each well of the medium. Incubate in a CO2 incubator at 37°C and 5% CO2 for 24 hours.

[0056] 2. After culturing for 24 hours, the medium was replaced with serum-free medium and then placed in a CO2 incubator at 37°C and 5% CO2 for 2 hours.

[0057] 3. After culturing for 2 hours, add LPS to each well at a final concentration of 10 ug / ml, and place in a CO2 incubator at 37℃ and 5% CO2 for another 6 hours.

[0058] 4. Centrifuge at 3000 rpm for 20 min and collect the supernatant.

[0059] 5. The mouse interleukin-6 enzyme-linked immunosorbent assay kit from Jiangsu Enzyme Immunoassay Co., Ltd. was selected.

[0060] 6. Sample Addition: Separate blank wells (blank control wells do not contain sample or enzyme-labeled reagent; all other steps are the same) and sample wells. The sample wells are divided into three groups: ① JB PDRN-0.25% group: Add 50 μL of the sample solution (sample concentration is 2.5 mg / mL) to the sample wells; ② JB PDRN-0.1% group: Add 30 μL of sample diluent to the sample wells, then add 20 μL of the sample solution (diluted sample concentration is 1.0 mg / mL); ③ JB PDRN-0.05% group: Add 40 μL of sample diluent to the sample wells, then add 10 μL of the sample solution (final sample dilution is 5-fold, diluted sample concentration is 0.5 mg / mL). Add the sample to the bottom of the wells, avoiding contact with the well walls, and gently shake to mix.

[0061] 7. Incubation: After sealing the plate with sealing film, incubate at 37°C for 30 minutes.

[0062] 8. Solution preparation: Dilute the 20-fold concentrated washing solution with distilled water 20 times and set aside.

[0063] 9. Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let stand for 30 seconds, then discard. Repeat 5 times, then pat dry.

[0064] 10. Add enzyme: Add 50 μL of enzyme-labeled reagent to the sample wells to be tested, and add 50 μL of DMEM complete culture medium to the blank wells.

[0065] 11. Incubation: The procedure is the same as step 7.

[0066] 12. Washing: Same as step 9.

[0067] 13. Color development: Add 50 μL of developer A to each well, then add 50 μL of developer B, gently shake to mix, and develop at 37°C in the dark for 10 minutes.

[0068] 14. Termination: Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).

[0069] 15. Measurement: Measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0070] Relative secretion rate (%) = (OD of blank wells) 450nm Value - OD of the well group of the sample to be tested 450nm (value) / OD of blank well group 450nm Value * 100%.

[0071] Assuming a relative inhibition rate of IL-6 secretion of 0% in the blank well group, the comparison results of the relative inhibition rates of IL-6 secretion in different sample well groups are shown in the figure. Figure 5 . Figure 5 In the diagram, "no protein added" represents the blank well group, and "JB PDRN" represents the well group of test samples containing PDRN obtained in Example 2.

[0072] Conclusion: OD450 values ​​measured by an ELISA reader indicate that JI PDRN inhibits the inflammatory cytokine IL-6 at different concentrations. Specifically, JB PDRN at a concentration of 0.05% inhibits IL-6 secretion by 32% (see results below). Figure 5 (As shown).

[0073] In conclusion, JB PDRN can not only promote the secretion of anti-inflammatory IL-10, but also inhibit the secretion of inflammatory factor IL-6, indicating that it has a good anti-inflammatory and soothing effect.

[0074] Example 5: Activity Detection - Promoting Collagen Regeneration (1) PDRN promotes type III collagen regeneration 1. Add 100 μL of DMEM complete medium to each well of a 96-well plate, and add 5000 human skin fibroblasts (HSF) to each well of the medium. Incubate in a CO2 incubator at 37°C and 5% CO2 for 24 hours.

[0075] 2. After culturing for 24 hours, wash once with PBS, then replace with sample culture medium, and incubate in a CO2 incubator at 37℃ and 5% CO2 for 48 hours.

[0076] 3. After culturing for 48 hours, centrifuge at 3000 rpm for 20 minutes and collect the supernatant.

[0077] 4. The human type III collagen protein-linked immunosorbent assay kit from Jiangsu Enzyme Immunoassay Co., Ltd. was selected.

[0078] 5. Sample Addition: Set up blank wells (blank control wells do not contain sample or enzyme-labeled reagent; all other steps are the same) and sample wells. Add 40 μL of sample diluent to the sample wells, then add 10 μL of the sample to be tested (the final sample dilution is 5-fold, and the concentration after dilution is 0.5 mg / mL). Add the sample to the bottom of the wells, avoiding contact with the well walls, and gently shake to mix.

[0079] 6. Incubation: After sealing the plate with sealing film, incubate at 37°C for 30 minutes.

[0080] 7. Solution preparation: Dilute the 20-fold concentrated washing solution with distilled water 20 times and set aside.

[0081] 8. Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let stand for 30 seconds, then discard. Repeat 5 times, then pat dry.

[0082] 9. Add enzyme: Add 50 μL of enzyme-labeled reagent to the sample wells to be tested, and add 50 μL of DMEM complete culture medium to the blank wells.

[0083] 10. Incubation: The procedure is the same as step 6.

[0084] 11. Washing: Same as step 8.

[0085] 12. Color development: Add 50 μL of developer A to each well, then add 50 μL of developer B, gently shake to mix, and develop at 37°C in the dark for 10 minutes.

[0086] 13. Termination: Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).

[0087] 14. Measurement: Measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0088] Relative secretion rate (%) = OD of the sample well group 450nm Value / OD of blank hole group 450nm Value * 100%.

[0089] With the relative secretion rate of Col-III in the blank well group being 100%, the comparison results of the relative secretion rates of Col-III in different sample well groups are shown in the figure. Figure 6 OD was measured using an ELISA reader. 450 The data shows that adding JI PDRN to the group promoted the secretion of type III collagen by 112.8%, while the competing product RH promoted the secretion of collagen by 100.2% and the competing product RJM promoted the secretion of type III collagen by 103%. This indicates that JI PDRN is superior to the competing products RH and RJM in promoting the regeneration of type III collagen and can beautify and care for the skin.

[0090] (2) PDRN promotes type I collagen regeneration 1. Add 100 μL of DMEM complete medium to each well of a 96-well plate, and add 5000 human skin fibroblasts (HSF) to each well of the medium. Incubate in a CO2 incubator at 37°C and 5% CO2 for 24 hours.

[0091] 2. After culturing for 24 hours, wash once with PBS, then replace with sample culture medium, and incubate in a CO2 incubator at 37℃ and 5% CO2 for 48 hours.

[0092] 3. After culturing for 48 hours, centrifuge at 3000 rpm for 20 minutes and collect the supernatant.

[0093] 4. The human type I collagen protein-linked immunosorbent assay kit from Jiangsu Enzyme Immunoassay Co., Ltd. was selected.

[0094] 5. Sample Addition: Set up blank wells (blank control wells do not contain sample or enzyme-labeled reagent; all other steps are the same) and sample wells. Add 40 μL of sample diluent to the sample wells, then add 10 μL of the sample to be tested (the final sample dilution is 5-fold, and the concentration after dilution is 0.5 mg / mL). Add the sample to the bottom of the wells, avoiding contact with the well walls, and gently shake to mix.

[0095] 6. Incubation: After sealing the plate with sealing film, incubate at 37°C for 30 minutes.

[0096] 7. Solution preparation: Dilute the 20-fold concentrated washing solution with distilled water 20 times and set aside.

[0097] 8. Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let stand for 30 seconds, then discard. Repeat 5 times, then pat dry.

[0098] 9. Add enzyme: Add 50 μL of enzyme-labeled reagent to the sample wells to be tested, and add 50 μL of DMEM complete culture medium to the blank wells.

[0099] 10. Incubation: The procedure is the same as step 6.

[0100] 11. Washing: Same as step 8.

[0101] 12. Color development: Add 50 μL of developer A to each well, then add 50 μL of developer B, gently shake to mix, and develop at 37°C in the dark for 10 minutes.

[0102] 13. Termination: Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).

[0103] 14. Measurement: Measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0104] Relative secretion rate (%) = OD of the sample well group 450nm Value / OD of blank hole group 450nm Value * 100%.

[0105] With the relative secretion rate of Col-I in the blank well group being 100%, the comparison results of the relative secretion rates of Col-I in different sample well groups are shown in the figure. Figure 7 .

[0106] Conclusion: OD was measured using an ELISA reader. 450 The data shows that adding JB PDRN to the group promoted the secretion of type I collagen by 108.30%, while the competing product RH promoted the secretion of collagen by 106%, and the competing product RJM promoted the secretion of type III collagen by 104.7%. This indicates that JB PDRN is superior to the competing products RH and RJM in promoting the regeneration of type I collagen, and can have anti-wrinkle and skin-firming effects.

[0107] In conclusion, JB PDRN not only significantly promotes the regeneration of type III collagen, but also the regeneration of type I collagen, indicating that it not only has skincare benefits, but also has the potential for medical aesthetic treatments.

[0108] The contents described in this specification are merely an enumeration of the implementation forms of the inventive concept, and the scope of protection of this invention should not be regarded as limited to the specific forms described in the embodiments.

Claims

1. A method for preparing PDRN, characterized in that, Includes the following steps: 1) Fermentation was carried out using Lactobacillus plantarum, and the resulting fermentation broth was centrifuged to collect the bacterial cells; 2) Resuspend the bacterial cells obtained in step 1), homogenize and break them, centrifuge to remove the precipitate, and concentrate them using a 5kDa hollow fiber membrane; 3) The concentrate obtained in step 2) is purified using a Q column, and then concentrated and replaced to obtain PDRN.

2. The method for preparing PDRN as described in claim 1, characterized in that, Step 1) The fermentation medium for fermentation treatment consists of: 40g glucose, 10g corn steep liquor, 15g fish broth, 5g diammonium hydrogen phosphate, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1mL Tween 80, 1g defoamer, and water to a final volume of 1L.

3. The method for preparing PDRN as described in claim 1, characterized in that, Step 1) During fermentation, inoculate Lactobacillus plantarum into the fermentation medium at an inoculation rate of 2%-6%, and ferment at a temperature of 35-37℃ for 40-50 hours.

4. The method for preparing PDRN as described in claim 1, characterized in that, In step 2), the volume of the original liquid is concentrated to 1 / 8 to 1 / 12 using a 5kDa hollow fiber membrane.

5. The method for preparing PDRN as described in claim 1, characterized in that, In step 3), the purification process using a Q column is as follows: S1: Load the concentrated solution onto the equilibrated Q column. After loading, continue washing with equilibration buffer to reach the baseline. The equilibration buffer contains Tris-HCl buffer with a pH of 7.0 ± 0.2 and a concentration of 17.55 g / L ± 0.5 g / L NaCl. S2: Rinse to baseline with wash buffer containing 29.25 g / L ± 0.5 g / L NaCl in pH 7.0 ± 0.2 Tris-HCl buffer. S3: Load the sample with elution buffer and collect the eluent; the elution buffer contains a pH=7.0±0.2 Tris-HCl buffer with a concentration of 40.95 g / L±0.5 g / L NaCl.

6. The method for preparing PDRN as described in claim 1, characterized in that, Step 3) involves the following process for concentration and liquid exchange: The purified eluent is concentrated and exchanged using a 5kDa hollow fiber membrane. When the concentration reaches 1 / 8 to 1 / 12 of the original solution, 3-5 times the volume of purified water is added. The concentration and liquid exchange are repeated, and the conductivity of the concentrated solution is measured. The liquid exchange operation can be stopped when the conductivity is less than 0.5ms.

7. A PDRN prepared by the method described in any one of claims 1-7.

8. The use of the PDRN as described in claim 7 in the preparation of products that promote cell proliferation and / or collagen production.

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