Preparation method and application of polydeoxyribonucleotide derived from prokaryotes

Abstract

The invention discloses a preparation method and application of polydeoxyribonucleotide derived from prokaryotes. The preparation method comprises the following steps: 1) enriching blue-green algae biomass; 2) ultrasonically crushing; (3) carrying out RNA elution; 4) DNA and protein separation; (5) dissolving the DNA; (6) separating out DNA; 7) gradient cleaning of DNA; (8) dissolving the DNA; and (9) preparing PDRN. The preparation method of the water-blooming cyanobacteria PDRN is developed by taking the prokaryote cyanobacteria as the raw material, the preparation method is simple and easy to implement, the molecular weight of the PDRN can be efficiently controlled, the purity and yield of DNA are ensured by controlling the RNA elution step and the DNA and protein separation step, the antioxidant capacity of the DNA is verified, and the method is suitable for industrial production. And a new way is provided for resource utilization of the water-blooming cyanobacteria biomass.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for preparing polydeoxyribonucleotides derived from prokaryotes and its applications. Background Art

[0002] Water bloom cyanobacteria are a type of photosynthetic prokaryotes that are widely distributed in freshwater and are important primary producers in the aquatic ecosystem. However, when the water body is rich in nutrients such as nitrogen and phosphorus, cyanobacteria will reproduce at an extremely high rate, forming cyanobacterial blooms and accumulating a large amount of biomass in the water body. The unreasonable disposal of this cyanobacterial bloom biomass will cause various harms to the ecological environment and human health.

[0003] It is very necessary to utilize the resources of water bloom cyanobacterial biomass. On the one hand, the large-scale outbreak of cyanobacterial blooms will lead to eutrophication, hypoxia, and a decline in biodiversity of the water body, and release harmful substances such as microcystins, threatening the ecosystem and human health. Through resource utilization, the accumulation of cyanobacteria can be effectively reduced, and the risk of environmental pollution can be lowered. On the other hand, due to the high water content and complex composition of cyanobacterial biomass, traditional treatment methods such as landfilling and incineration have problems such as high costs and potential pollution, while resource utilization provides a more sustainable and efficient alternative. In addition, cyanobacteria are rich in high-value components such as proteins, polysaccharides, lipids, and pigments, which can be converted into organic fertilizers, bioenergy, or industrial raw materials, realizing the recycling of waste and enhancing economic value. Resource utilization also conforms to the concept of circular economy, combining environmental governance with economic benefits, and contributing to promoting sustainable development and alleviating environmental pressure.

[0004] At present, certain progress has been made in the resource utilization of water bloom cyanobacterial biomass, and it shows a diversified development trend. In the agricultural field, cyanobacteria are rich in nutrients such as nitrogen and phosphorus and are used for compost fermentation to produce organic fertilizers. The technology is relatively mature, but the toxin degradation technology still needs to be improved to ensure safety in use. In terms of energy development, carbohydrates and lipids in cyanobacteria can be used to produce biogas, biodiesel, and ethanol fuel, but large-scale application is still limited by technical costs and efficiency, and key bottlenecks need to be broken through. In addition, high-value components such as phycocyanin and polysaccharides in cyanobacteria have been widely used in the fields of food, feed, and cosmetics. Among them, the extraction and application of phycocyanin are the most mature, but extraction costs and toxin control are still restrictive factors. Generally speaking, although the resource utilization of cyanobacteria has great potential, it still faces technical and economic challenges.

[0005] PDRN (Polydeoxyribonucleotide) is a long-chain DNA fragment formed by the linkage of various deoxynucleotides (such as dAMP, dTMP, dGMP, and dCMP) through phosphodiester bonds. The DNA base pairs are between 50 - 2000 bp, and the molecular weight is between 50 - 1500 kDa, with high biocompatibility. PDRN can be degraded by DNA enzymes in the human body into low-molecular-weight nucleotides and nucleosides, participating in various metabolic processes. This property enables it to have significant biological functions in promoting tissue repair and regeneration. At the same time, due to its natural source and the safety of its degradation products, it is widely used in the fields of medical aesthetics and regenerative medicine. Currently, the extraction sources of PDRN include a variety of biological materials, and each source has its unique advantages and limitations. Salmon sperm is the most common extraction source in PDRN research. However, its price is relatively high, and the raw material supply may be limited. In order to expand the commercial application of PDRN, reported extraction sources also include sturgeon sperm, sea cucumber sperm, starfish, and marine red algae. However, all existing PDRNs are derived from eukaryotes, and there has been no research on the extraction and preparation of polydeoxyribonucleotide (PDRN) from prokaryotes (including bloom-forming cyanobacteria). Summary of the Invention

[0006] Aiming at the problems existing in the prior art, on the one hand, the present invention provides a preparation method of polydeoxyribonucleotide based on prokaryotic sources, which is specifically realized through the following technical solutions:

[0007] 1) Cyanobacteria biomass enrichment: Centrifuge the artificially cultured Microcystis aeruginosa (FACHB 905) and store it in the form of algal sludge for standby.

[0008] 2) Ultrasonic disruption: Add 1 part of algal sludge to 10 parts of 0.14 M sodium chloride / 0.5 M sodium EDTA solution and mix well. Use an ultrasonic disruptor with a power of 400 W and a frequency of 20 kHz to disrupt for 10 minutes (on for 3 s, off for 3 s) to completely disrupt the cyanobacteria cells and release intracellular inclusions.

[0009] 3) RNA elution: Centrifuge the above cell disruption solution at 3000 rpm for 15 min at 25 °C, and discard the supernatant. Re-mix and shake with 10 parts of 0.14 M sodium chloride / 0.5 M sodium EDTA solution, centrifuge and discard the supernatant, repeat 1 - 3 times, and retain the precipitate for standby.

[0010] 4) DNA and protein separation: Mix the above precipitate with 9 parts of 0.14 M sodium chloride / 0.5 M sodium EDTA solution, add 1 part of 20% sodium dodecyl sulfate solution to make the final concentration of sodium dodecyl sulfate 2%, stir at 25, 45, and 65 °C for 30 min respectively, and let it stand to room temperature for standby.

[0011] 5) DNA Dissolution: Add several portions of 10 M sodium chloride solution to the above solution to make the final concentration of sodium chloride reach 2 M, stir at room temperature for 30 min to fully dissolve the DNA. Then, centrifuge at 3000 rpm for 15 min at 25 °C. The supernatant is filtered through a 0.45 μm microporous membrane to fully remove particulate impurities, and the filtrate is reserved for use.

[0012] 6) DNA Precipitation: Add 1.5 times the volume of absolute ethanol to the above filtrate, stir well for 20 min to precipitate, centrifuge at 8000 rpm for 20 min at 25 °C, discard the supernatant, and the precipitate is the crudely extracted DNA;

[0013] 7) DNA Gradient Washing: Add 1 portion of 95% ethanol to the crudely extracted DNA respectively, stir well to mix evenly, centrifuge at 8000 rpm for 20 min at 25 °C, discard the supernatant, and continue to wash once with 80% ethanol and 70% ethanol respectively;

[0014] 8) DNA Dissolution: Add 1 portion of deionized water to the washed DNA precipitate, stir well to mix evenly to fully dissolve the DNA;

[0015] 9) PDRN Preparation: Fragment the dissolved DNA with an ultrasonic crusher under the conditions of ultrasonic power of 300 W, ultrasonic times of 5 min, 10 min, 15 min, 20 min, and 25 min. Finally, the DNA fragment base pairs are within 50 - 2000 bp, and the PDRN preparation is completed. Finally, the PDRN is prepared into a dry powder by vacuum freeze-drying and can be stored at room temperature.

[0016] The second invention of the present invention lies in providing the polydeoxyribonucleotide prepared by the above preparation method.

[0017] The third aspect of the present invention lies in providing the application of the polydeoxyribonucleotide prepared by the above preparation method as an antioxidant.

[0018] The present invention develops a preparation method step of bloom cyanobacteria PDRN using prokaryotic cyanobacteria as a raw material. The preparation method is simple and easy to implement and can efficiently control the molecular weight of PDRN. By controlling the RNA elution step and the DNA and protein separation steps, the DNA purity and yield are ensured, and its antioxidant ability is verified, providing a new way for the resource utilization of bloom cyanobacteria biomass. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the process flow chart of the present invention;

[0020] Figure 2 is the influence of different numbers of RNA on the purity and concentration of PDRN;

[0021] Figure 3 Effect of different temperatures on the protein residue amount in PDRN products;

[0022] Figure 4 For gel electrophoresis and DNA fragment distribution (A: Gel electrophoresis result after DNA dissolution; B: Distribution of DNA fragments after different ultrasonic treatment times);

[0023] Figure 5 For the antioxidant capacity evaluation of PDRN. Specific implementation mode

[0024] The process flow of the preparation method of polydeoxyribonucleotide derived from prokaryotes according to the present invention is as Figure 1 shown, successively including cyanobacteria biomass enrichment, ultrasonic disruption, RNA elution, DNA and protein separation, DNA dissolution, DNA precipitation, DNA gradient washing, DNA dissolution, RDRN preparation, and PDRN antioxidant application.

[0025] Example 1

[0026] The preparation method of polydeoxyribonucleotide derived from prokaryotes (the number of RNA elution times is 1), and the specific steps are as follows:

[0027] Artificially cultured Microcystis aeruginosa (FACHB 905) was collected by centrifugation and stored in the form of algal sludge for later use; 1 part of the algal sludge was added to 10 parts of 0.14 M sodium chloride / 0.5 M sodium EDTA solution and mixed well. It was then broken by an ultrasonic crusher with a power of 400 W and a frequency of 20 kHz for 10 minutes (on for 3 s, off for 3 s) to completely break the cyanobacterial cells and release the intracellular inclusions. The above cell lysate was centrifuged at 3000 rpm for 15 min at 25 °C, and the supernatant was discarded. It was remixed and shaken with 10 parts of 0.14 M sodium chloride / 0.5 M sodium EDTA solution, centrifuged, and the supernatant was discarded, leaving the precipitate for later use; the above precipitate was mixed with 9 parts of 0.14 M sodium chloride / 0.5 M sodium EDTA solution, and 1 part of 20% sodium dodecyl sulfate solution was added to make the final concentration of sodium dodecyl sulfate 2%. It was stirred at 65 °C for 30 min and left to stand at room temperature for later use; several parts of 10 M sodium chloride solution were added to the above solution to make the final concentration of sodium chloride reach 2 M, and it was stirred at room temperature for 30 min to fully dissolve the DNA. Then, it was centrifuged at 3000 rpm for 15 min at 25 °C, and the supernatant was filtered through a 0.45 μm microporous membrane to completely remove particulate impurities; 1.5 times the volume of absolute ethanol was added to the above filtrate, stirred well for 20 min to precipitate, centrifuged at 8000 rpm for 20 min at 25 °C, and the supernatant was discarded. The precipitate was the crude DNA; 1 part of 95% ethanol was added to the crude DNA respectively, stirred well, centrifuged at 8000 rpm for 20 min at 25 °C, and the supernatant was discarded. It was then washed once with 80% ethanol and 70% ethanol respectively; 1 part of deionized water was added to the washed DNA precipitate, stirred well to fully dissolve the DNA; the dissolved DNA was fragmented by an ultrasonic crusher under the conditions of an ultrasonic power of 300 W and an ultrasonic time of 10 min. Finally, the DNA fragment base pairs were between 50 and 2000 bp, and the preparation of PDRN was completed. The content was 227.6 μg / mL, and A260 / A280 was 1.69. Finally, the PDRN was prepared into a dry powder by vacuum freeze-drying and could be stored at room temperature.

[0028] Example 2

[0029] Preparation method of polydeoxyribonucleotide derived from prokaryotes (the number of RNA elution times is 2 times), the specific steps are as follows:

[0030] Artificially cultured Microcystis aeruginosa (FACHB 905) was collected by centrifugation and stored in the form of algal sludge for later use; 1 part of algal sludge was added to 10 parts of 0.14 M sodium chloride / 0.5 M sodium EDTA solution and mixed well. It was then broken with a ultrasonic crusher with a power of 400 W and a frequency of 20 kHz for 10 minutes (on for 3 s, off for 3 s) to completely break the cyanobacterial cells and release intracellular inclusions; the above cell lysate was centrifuged at 3000 rpm, 15 min, 25 °C, and the supernatant was discarded. It was remixed and shaken with 10 parts of 0.14 M sodium chloride / 0.5 M sodium EDTA solution, centrifuged, and the supernatant was discarded. This washing step was repeated 2 times, and the precipitate was reserved for later use; the above precipitate was mixed with 9 parts of 0.14 M sodium chloride / 0.5 M sodium EDTA solution, and 1 part of 20% sodium dodecyl sulfate solution was added to make the final concentration of sodium dodecyl sulfate 2%. It was stirred at 65 °C for 30 min and left to stand at room temperature for later use; several parts of 10 M sodium chloride solution were added to the above solution to make the final concentration of sodium chloride reach 2 M, and it was stirred at room temperature for 30 min to fully dissolve the DNA. Then, it was centrifuged at 3000 rpm, 15 min, 25 °C, and the supernatant was filtered through a 0.45 μm microporous filter membrane to fully remove particulate impurities; 1.5 times the volume of absolute ethanol was added to the above filtrate, and it was stirred well for 20 min to precipitate. It was centrifuged at 8000 rpm, 20 min, 25 °C, and the supernatant was discarded. The precipitate was the crude DNA; 1 part of 95% ethanol was added to the crude DNA respectively, stirred well, centrifuged at 8000 rpm, 20 min, 25 °C, and the supernatant was discarded. It was then washed once with 80% ethanol and 70% ethanol respectively; 1 part of deionized water was added to the washed DNA precipitate, stirred well to fully dissolve the DNA; the dissolved DNA was fragmented with a ultrasonic crusher under the conditions of ultrasonic power of 300 W and ultrasonic time of 10 min. Finally, the DNA fragment base pairs were between 50 - 2000 bp, and the preparation of PDRN was completed. The content was 220.3 μg / mL, and A260 / A280 was 1.85. Finally, PDRN was prepared into a dry powder by vacuum freeze-drying and could be stored at room temperature.

[0031] The effects of different numbers of RNA elutions on the purity and concentration of PDRN are shown in Table 1 and Figure 2 as follows.

[0032] Table 1

[0033] Number of RNA elution times PDRN content (μg / ml) A260 / A280 1 227.6 1.69 2 220.3 1.85 3 167.2 1.84

[0034] The results showed that in the technological process, performing two RNA elutions could maximize the yield of PDRN while ensuring its purity (A260 / A280 between 1.80 - 2.0).

[0035] Example 3

[0036] Preparation method of polydeoxyribonucleotide derived from prokaryotes (temperature condition during DNA and protein separation process: 25°C), and the specific steps are as follows:

[0037] Centrifuge to collect artificially cultured Microcystis aeruginosa (FACHB 905) and store it in the form of algal sludge for later use; add 1 part of algal sludge to 10 parts of 0.14M sodium chloride / 0.5M sodium EDTA solution and mix well, then use an ultrasonic crusher with a power of 400W and a frequency of 20kHz to crush for 10 minutes (on for 3s, off for 3s) to completely break the cyanobacterial cells and release intracellular inclusions; centrifuge the above cell lysate at 3000rpm, 15min, 25°C, and discard the supernatant. Re-mix and shake with 10 parts of 0.14M sodium chloride / 0.5M sodium EDTA solution, centrifuge and discard the supernatant, repeat this washing step 2 times, and keep the precipitate for later use; mix the above precipitate with 9 parts of 0.14M sodium chloride / 0.5M sodium EDTA solution, add 1 part of 20% sodium dodecyl sulfate solution to make the final concentration of sodium dodecyl sulfate 2%, stir at 25°C for 30min, and let it stand at room temperature for later use; add several parts of 10M sodium chloride solution to the above solution to make the final concentration of sodium chloride reach 2M, stir at room temperature for 30min to fully dissolve the DNA. Then, centrifuge at 3000rpm, 15min, 25°C, filter the supernatant through a 0.45μm microporous filter membrane to fully remove particulate impurities; add 1.5 times the volume of absolute ethanol to the above filtrate, stir well for 20min to precipitate, centrifuge at 8000rpm, 20min, 25°C, and discard the supernatant, and the precipitate is the crude DNA; add 1 part of 95% ethanol to the crude DNA respectively, stir well, centrifuge at 8000rpm, 20min, 25°C, discard the supernatant, and continue to wash with 80% ethanol and 70% ethanol once each; add 1 part of deionized water to the washed DNA precipitate, stir well to fully dissolve the DNA; fragment the dissolved DNA with an ultrasonic crusher, with the conditions of ultrasonic power of 300W and ultrasonic time of 10min, and the final DNA fragment base pairs are between 50 - 2000bp, that is, the preparation of PDRN is completed, and the protein residue is 0.2745mg / mL. Finally, the PDRN is prepared into dry powder by vacuum freeze-drying and can be stored at room temperature.

[0038] Example 4

[0039] Preparation method of polydeoxyribonucleotide derived from prokaryotes (temperature condition during DNA and protein separation process: 65°C), and the specific steps are as follows:

[0040] Artificially cultured Microcystis aeruginosa (FACHB 905) was collected by centrifugation and stored in the form of algal sludge for later use. One part of the algal sludge was added to 10 parts of 0.14M sodium chloride / 0.5M sodium EDTA solution and mixed well. It was then broken by an ultrasonic crusher with a power of 400W and a frequency of 20kHz for 10 minutes (on for 3s, off for 3s) to completely break the cyanobacterial cells and release the intracellular contents. The above cell lysate was centrifuged at 3000rpm, 15min, 25°C, and the supernatant was discarded. It was remixed and shaken with 10 parts of 0.14M sodium chloride / 0.5M sodium EDTA solution, centrifuged, and the supernatant was discarded. This washing step was repeated twice, and the precipitate was reserved for later use. The above precipitate was mixed with 9 parts of 0.14M sodium chloride / 0.5M sodium EDTA solution, and 1 part of 20% sodium dodecyl sulfate solution was added to make the final concentration of sodium dodecyl sulfate 2%. It was stirred at 25°C for 30min and left to stand at room temperature for later use. Several parts of 10M sodium chloride solution were added to the above solution to make the final concentration of sodium chloride reach 2M, and it was stirred at room temperature for 30min to fully dissolve the DNA. Then, it was centrifuged at 3000rpm, 15min, 65°C, and the supernatant was filtered through a 0.45μm microporous filter membrane to fully remove particulate impurities. 1.5 times the volume of absolute ethanol was added to the above filtrate, and it was stirred well for 20min to precipitate. It was centrifuged at 8000rpm, 20min, 25°C, and the supernatant was discarded. The precipitate was the crude DNA. 1 part of 95% ethanol was added to the crude DNA respectively, stirred well and mixed, centrifuged at 8000rpm, 20min, 25°C, and the supernatant was discarded. It was then washed once with 80% ethanol and 70% ethanol respectively. 1 part of deionized water was added to the washed DNA precipitate, stirred well and mixed to fully dissolve the DNA. The dissolved DNA was fragmented by an ultrasonic crusher under the conditions of an ultrasonic power of 300W and an ultrasonic time of 10min. Finally, the DNA fragment base pairs were in the range of 50 - 2000bp, and the preparation of PDRN was completed, with the protein residue content being 0.1426mg / mL. Finally, the PDRN was prepared into a dry powder by vacuum freeze-drying and could be stored at room temperature.

[0041] The gel electrophoresis result after DNA dissolution is shown in Figure 4 A in Figure 4 As can be seen from A, the DNA base pairs are mainly distributed above 5000bp; after different ultrasonic treatment times, the distribution of DNA fragments is as shown in Figure 4 B in Figure 4 As can be seen from B, after 10min of ultrasonic fragmentation, all DNA fragments can reach the PDRN base pair range (between 50 - 2000bp), realizing efficient preparation.

[0042] The effects of different temperatures on the protein residue content in the PDRN product are shown in Table 2 andFigure 3 As shown. The results show that during the DNA and protein separation process, increasing the reaction temperature can improve the separation efficiency of DNA and proteins, thereby reducing the protein residue in PDRN and increasing the purity of the PDRN product.

[0043] Table 2

[0044] Temperature during DNA and protein separation process (℃) Residual amount of PDRN protein (mg / mL) 25 0.2745 45 0.2146 65 0.1426

[0045] This example

[0046] Verification example: Antioxidant verification

[0047] The test process is as follows: Prepare an ABTS solution with a concentration of 7 mM and a potassium persulfate solution with a concentration of 2.45 mM, mix them in equal amounts to form a working solution, and react in the dark for 12 h. Before use, dilute the working solution with PBS (10 mM, pH 7.4) to an absorbance value of 0.7 ± 0.02 (734 nm). When measuring the antioxidant capacity, the blank group is a mixture of 150 μL of ABTS working solution and 10 μL of deionized water, and the sample group is a mixture of 150 μL of ABTS working solution and 10 μL of 10 mg / mL PDRN sample (PDRN is prepared in Example 4), react in the dark for 6 min, and measure the absorbance value at 734 nm. The antioxidant capacity is calculated using the following index: ABTS radical scavenging rate (%) = (Ablank - Asample) / Ablank × 100%. The radical scavenging rate is as Figure 5 shown. The results show that the ABTS radical scavenging rate of 10 mg / mL PDRN is 77.7%.

Claims

1. A method for preparing a polydeoxyribonucleotide from a prokaryotic organism, characterized in that: The preparation method comprises the following steps: 1) Enrichment of cyanobacterial biomass: Artificially cultured Microcystis aeruginosa (FACHB 905) was collected by centrifugation and stored in the form of algae mud for later use; 2) Ultrasonic disruption: Add 0.14M sodium chloride / 0.5M sodium EDTA solution to the algae mud and mix thoroughly. Use an ultrasonic disruptor to completely disrupt the cyanobacterial cells and release the intracellular contents. 3) RNA elution: centrifuge the cell disruption solution at 3000 rpm, 15 min, 25°C, and discard the supernatant; remix and shake with the same amount of 0.14 M sodium chloride / 0.5 M EDTA sodium salt solution, centrifuge and discard the supernatant, repeat 1-3 times, and keep the precipitate for later use; 4) DNA and protein separation: Mix the precipitate obtained in step 3) with 0.14M sodium chloride / 0.5M sodium EDTA solution, add 20% sodium dodecyl sulfate solution to make the final concentration of sodium dodecyl sulfate 2%, stir at 25-65°C for 30 minutes, and let stand at room temperature for use; 5) DNA dissolution: Add 10M sodium chloride solution to the solution in step 4) to make the final concentration of sodium chloride reach 2M, stir at room temperature for 30 minutes to fully dissolve the DNA, then centrifuge, filter the supernatant with a 0.45μm microporous filter membrane to fully remove particulate impurities, and use the filtrate for later use; 6) DNA precipitation: add 1.5 times the volume of anhydrous ethanol to the filtrate of step 5), stir thoroughly to precipitate for 20 minutes, centrifuge, discard the supernatant, and the precipitate is the crude DNA; 7) DNA gradient cleaning: add 1 part of 95% ethanol to the crude DNA, stir and mix thoroughly, centrifuge, discard the supernatant, and continue to wash with 80% ethanol and 70% ethanol once each; 8) DNA dissolution: Add 1 part of deionized water to the washed DNA precipitate and stir thoroughly to dissolve the DNA completely; 9) Preparation of PDRN: The dissolved DNA was fragmented using an ultrasonic disruptor at an ultrasonic power of 300 W and an ultrasonic time of 5-25 min to complete the preparation of PDRN.

2. The method for preparing polydeoxyribonucleotides from prokaryotes as claimed in claim 1, characterized in that In step 2), the ratio of algae mud to sodium chloride / EDTA sodium salt solution is 1:10; the conditions for ultrasonic crushing are: 400W, 20kHz, crushing for 10min, and the crushing period is on for 3s and off for 3s.

3. The method for preparing polydeoxyribonucleotides from prokaryotes as claimed in claim 1, characterized in that In step 4), the sodium chloride / EDTA sodium salt solution is used in 9 parts and the sodium dodecyl sulfate solution is used in 1 part.

4. The method for preparing polydeoxyribonucleotides from prokaryotes as claimed in claim 1, characterized in that In step 5), the centrifugation conditions are: 25° C., 3000 rpm, and 15 min.

5. The method for preparing polydeoxyribonucleotides from prokaryotes as claimed in claim 1, characterized in that In step 6) and step 7), the centrifugation conditions are: 8000 rpm, 20 min, 25°C.

6. The polydeoxyribonucleotide obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the polydeoxyribonucleotide according to claim 6 as an antioxidant.

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