A pdrn extraction process

Abstract

This invention discloses a PDRN extraction process, relating to the field of cosmetic raw material technology, comprising the following steps: screening for a culture of *Bacillus stearothermophilus* capable of obtaining a specific PDRN molecular weight and degrading fishy odor; culturing *Bacillus stearothermophilus* to a suitable bacterial count in a culture medium containing fish spores to obtain a *Bacillus stearothermophilus* fermentation broth; mixing the fish spore homogenate with the obtained fermentation broth and treating it at multiple temperature stages to obtain an enzymatically hydrolyzed fish spore homogenate; adding disodium EDTA and sodium chloride to the obtained enzymatically hydrolyzed fish spore homogenate to obtain a PDRN extract; filtering and centrifuging the obtained PDRN extract, precipitating the supernatant with ethanol, recovering the precipitate, washing the precipitate, and drying it to obtain pure PDRN. This invention utilizes the synergistic effect of three enzymes secreted by the strain itself, eliminating the need for additional exogenous enzymes, and completing lipid removal, protein removal, and nucleic acid shearing in one step; it domesticates *Bacillus stearothermophilus*, fully degrading the odor from the raw material during the enzymatic hydrolysis of fish spores to prepare PDRN, allowing for processing and utilization without further purification.

Description

Technical Field

[0001] This invention relates to the field of cosmetic raw material technology, specifically a PDRN extraction process. Background Technology

[0002] Polydeoxyribonucleotides (PDRN) are nucleic acid-based active ingredients with tissue regeneration, angiogenesis, and anti-inflammatory repair effects, widely used in medical aesthetics, cosmetics, and wound healing. Due to the high cost of raw materials and extraction processes, PDRN is generally only used in high-end cosmetic products. Fish testes, or milt, are rich in DNA and are commonly used to prepare PDRN. Currently, the mainstream industrial-scale PDRN preparation routes are salmon testes extraction and microbial extraction, both of which have significant cost bottlenecks. Extracting PDRN from milt requires organic solvent extraction or other purification processes to remove the fishy odor. The salmon-derived route suffers from scarce and expensive raw materials. Carp milt typically contains 3-5% DNA, and replacing salmon for PDRN extraction can significantly reduce raw material costs. Currently, salmon-derived PDRN enjoys high market acceptance, while carp-derived PDRN is not yet widely accepted, and its efficacy needs further verification.

[0003] Although microorganisms can secrete various enzymes, current factories in the industry do not use fermentation to extract PDRN, often opting instead for commercially available enzymes. The main reason is that fermentation is difficult to control; the enzyme concentration and optimal conditions are not well-matched to the substrate, making it no more efficient than commercial enzymes. *Geobacillus stearothermophilus*, a Gram-positive aerobic spore-forming bacillus, grows rapidly and can synthesize and secrete various thermostable enzymes. These enzymes can be directly used in key steps of PDRN extraction, such as the degradation of contaminating proteins and the regulation of nucleic acid fragments. They also hold promise as a substitute for alcohol solvents to remove fishy odors and avoid the risk of solvent residue. To date, there are no reports on technologies utilizing enzymes secreted by this strain for PDRN extraction, failing to leverage its advantages to solve the existing challenges of cost, process, and fishy odor removal in large-scale PDRN production. Summary of the Invention

[0004] (a) Technical problems to be solved A novel process for preparing PDRN from carp milt was developed to overcome the limitations of existing extraction processes that rely on exogenous commercial enzymes, require an additional deodorization step during PDRN preparation, and necessitate the use of organic solvents in traditional purification and deodorization methods. This process makes it difficult to achieve integrated impurity removal and targeted regulation of nucleic acid fragments, and the lack of validation for carp-derived PDRN makes it difficult to achieve the same activity as salmon-derived PDRN. This study provides a method for extracting PDRN from carp milt through fermentation with Bacillus thermophilus.

[0005] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a PDRN extraction process, specifically comprising the following steps: S1. Screen for thermophilic Bacillus stearothermophilus cultures that degrade fishy odor, then screen for cultures that can obtain specific PDRN molecular weights, and in the process confirm the appropriate colony count and fermentation extraction conditions. S2. The culture obtained in step S1 is cultured in a medium containing fish milt until it reaches 2 × 10⁻⁶. 6 -5×10 7 The number of bacteria was determined to obtain the fermentation broth of Bacillus stearothermophilus; S3. Mix the fish milt homogenate with the fermentation broth obtained in step S2, adjust the pH to 7-8, keep warm at 58-69℃ for 10-120 min, raise the temperature to 70-75℃, keep warm for 10-60 min, raise the temperature to 76-82℃, keep warm for 10-60 min, and obtain the enzymatically hydrolyzed fish milt homogenate. S4. Add disodium EDTA and sodium chloride to the enzymatically hydrolyzed fish slurry obtained in step S3, adjust the pH to 4-6, keep it at 90-100℃ for 10-60 min, and cool it to room temperature to obtain PDRN extract. S5. After filtering the PDRN extract obtained in step S4 through a filter bag and centrifuging, add 95% ethanol to the supernatant, let it stand for 1-24 hours, centrifuge to recover the precipitate, wash the precipitate with 75% ethanol until the conductivity is less than 100 μS / cm, and dry it to obtain pure PDRN.

[0006] Preferably, the fish milt is commercially available fresh or frozen carp milt, regardless of its origin.

[0007] Preferably, when 95% ethanol is added to the supernatant in step S5, the ratio of supernatant to ethanol is 1:1-3 (v / v).

[0008] Preferably, in step S4, the concentration of disodium ethylenediaminetetraacetate is 0.02% (w / v), and the concentration of sodium chloride is 1.5 mol / L.

[0009] Preferably, in step S3, the ratio of fish slurry to fermentation liquid is 1:5-20 (w / v).

[0010] This invention also provides a fermentation product containing PDRN, the preparation method of which is as follows: T1. The fish paste obtained in step S3 of a PDRN extraction process is adjusted to pH 4-6.5 with citric acid, kept at 90-100℃ for 10-30 minutes, cooled and discharged, filtered through a filter bag and centrifuged to recover the supernatant. T2. After drying the supernatant, a fermentation product containing PDRN was obtained.

[0011] Preferably, the drying of the supernatant in step T2 is spray drying or freeze drying, either alone or after mixing with excipients.

[0012] (III) Beneficial Effects Compared with the prior art, the present invention has the following advantages: (1) This invention fully utilizes the characteristics of Bacillus stearothermophilus itself, and through targeted domestication and regulation of the synergistic expression of multiple intracellular enzyme systems in the strain, it achieves one-step fermentation preparation of PDRN without the need for additional commercial proteases, nucleases and other exogenous enzyme preparations. This effectively simplifies the traditional multi-step enzymatic hydrolysis process, shortens the production process, and significantly improves the preparation efficiency.

[0013] (2) This invention involves physiological domestication and regulation of Bacillus stearothermophilus without any gene modification, mutagenesis, recombination, or other operations that alter the genetic material of the strain, and without artificially creating new strains. The superior functions obtained through domestication are merely environmentally induced physiological adaptive phenotypes and do not possess stable passage characteristics. After being removed from selection pressure, the traits naturally decline, eliminating the need for additional strain preservation. This meets the compliance requirements of microbial fermentation processes, resulting in low barriers to industrialization and high safety.

[0014] (3) This invention uses the degree of reduction in fishy odor and the molecular weight distribution of nucleic acid fragments in the fermentation system as domestication evaluation indicators. Through process domestication, the fermentation system is made capable of efficiently degrading fishy odor substrates, and fishy odor impurities from the raw materials can be removed in situ during PDRN preparation. The fermentation products prepared do not require complex post-processing processes such as organic solvents and physical deodorization, which greatly simplifies the post-processing procedures and significantly reduces the overall production and purification costs.

[0015] (4) Compared with commercially available salmon-derived PDRN products, the PDRN product prepared by this invention can effectively promote the proliferation and growth of human keratinocytes, and significantly increase the expression level of human type I collagen, exhibiting outstanding biological activity. Furthermore, the fermentation product retains the active functional components produced by the strain's metabolism, possessing excellent overall biological activity and skincare application potential, with functionality superior to single purified PDRN raw materials. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the molecular weight of PDRN obtained in this invention. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] The strain used in the following examples is ATCC7953. All raw materials, reagents, and equipment used can be obtained through conventional commercial channels.

[0019] Please see Figure 1 This invention provides ten technical solutions, specifically including the following embodiments: Example 1: Single colonies of *Bacillus steatophilus* on agar plates were inoculated into liquid culture medium. Liquid culture medium: 1 part by weight of carp milt and 99 parts by weight of water, sterilized at 115°C for 30 min. Culture conditions: 60°C, shaker at 200 rpm for 24 h to obtain a culture solution. The culture solution was inoculated into a new liquid culture medium at an inoculation volume of 0.1% (v / v), and cultured under the same conditions. Using the same inoculation volume, the culture solution was inoculated into a new liquid culture medium again, repeated three times to obtain culture solutions for odor screening. Multiple single colonies were inoculated each time to obtain multiple culture solution samples for odor screening, and each sample was evaluated for odor. Samples without a significant fishy odor were selected, and the corresponding culture solutions were mixed with 20% glycerol to a final concentration and then frozen for preservation to obtain cultures that could degrade fishy odor. A certain number of cultures were accumulated for further screening.

[0020] Example 2: The culture obtained in Example 1 was inoculated into fermentation medium at a ratio of 1:1000 (v / v) and cultured at 60°C on a shaker at 200 rpm, with multiple parallel studies. Fermentation medium consisted of 2 wt% glucose, 1 wt% carp milt, 0.01 wt% dipotassium hydrogen phosphate, 0.07 wt% magnesium sulfate heptahydrate, 0.01 wt% calcium chloride, with the remainder being water. The pH was adjusted to 7.5 with sodium hydroxide, and the medium was sterilized at 115°C for 30 min. Samples of the culture medium were taken at different time points to detect bacterial counts. Half of the culture medium was refrigerated and stored, while the remaining medium was used for further fermentation. Carp milt was soaked in 95°C water for 3 min, drained, and ground into a homogenate. The homogenate was added to the fermentation medium after sampling at a ratio of 1:7 (w / v), and multiple temperature and time settings were set for continued culturing. Nucleic acid electrophoresis was performed on samples of the culture medium at intervals. Based on the electrophoresis results, samples with a molecular weight around 100 bp and no fishy odor were selected, and the fermentation parameters were confirmed. The corresponding refrigerated culture medium was mixed with 20% glycerol and then frozen for preservation, yielding cultures exhibiting two different metabolic characteristics.

[0021] Example 3: The culture obtained in Example 2 was inoculated into fermentation medium at a ratio of 1:1000 (v / v) and cultured in a fermenter at 60°C until it reached 5 × 10⁵ cells / year. 6The bacterial count was determined to yield the fermentation broth of *Bacillus stearothermophilus*. The fermentation medium consisted of 2 wt% glucose, 1 wt% carp milt, 0.01 wt% dipotassium hydrogen phosphate, 0.07 wt% magnesium sulfate heptahydrate, and 0.01 wt% calcium chloride, with the remainder being water. The pH was adjusted to 7.5 with sodium hydroxide, and the mixture was sterilized at 115°C for 30 min. The carp milt was soaked in 95°C water for 3 min, drained, and then ground into a homogenate. This homogenate was added to the fermentation broth at a ratio of 1:7 (w / v). The mixture was stirred at 100 rpm, and the pH was controlled at 7.50 using phosphoric acid and sodium hydroxide solution. The mixture was incubated at 62°C for 60 min, then heated to 71°C for 60 min, and finally heated to 78°C for 30 min. The resulting enzymatically hydrolyzed carp milt homogenate was denoted as X1.

[0022] Example 4: Sodium chloride (1.5 mol / L) and disodium ethylenediaminetetraacetate (0.02% w / v) were added to the enzymatically hydrolyzed fish milt homogenate X1 obtained in Example 3. The pH was then adjusted to 4.50 with citric acid, and the mixture was incubated at 95°C for 15 min. After cooling, PDRN extract was obtained. The extract was filtered through a 1 μm filter bag and centrifuged at 9000 rpm for 5 min, and the supernatant was collected. The supernatant was mixed with 95% ethanol at a volume ratio of 1:2, allowed to stand for 2 h, and the precipitate was collected by centrifugation. The precipitate was washed with 75% ethanol at a ratio of 1:10 (w / v) multiple times until the conductivity was below 100 μS / cm. PDRN was obtained by vacuum drying at 45°C to constant weight, denoted as P1.

[0023] Example 5: The enzymatically hydrolyzed fish milt homogenate X1 obtained in Example 3 was adjusted to pH 5.5 with citric acid, incubated at 95℃ for 15 min, cooled, and discharged. After filtration through a 1 μm filter bag, it was centrifuged at 9000 rpm for 10 min, and the supernatant was recovered. The supernatant was mixed with maltodextrin at a ratio of 50:1 (w / w), and spray-dried to obtain a fermentation product containing PDRN, denoted as H1.

[0024] Example 6: Following the fermentation product obtained in Example 5, 10 wt% of the product was mixed with 0.02 wt% disodium ethylenediaminetetraacetate (EDTA), 3 wt% sodium chloride, and the remainder water, and dissolved. The dissolved solution was mixed with 95% ethanol at a ratio of 1:2 (v / v), allowed to stand for 2 hours, and centrifuged at 9000 rpm for 3 minutes to recover the precipitate. The precipitate was washed with 75% ethanol at a ratio of 1:10 (w / v) multiple times until the conductivity was below 100 μS / cm. The precipitate was then vacuum dried at 45°C to constant weight to obtain PDRN, denoted as G1. The PDRN content of the fermentation product = (mass of G1 / mass of H1) × 100%.

[0025] Comparative Example 1: The culture used in Example 3 was replaced with an unselected culture cultured in LB medium, with all other conditions remaining unchanged, to obtain an enzymatically hydrolyzed fish milt homogenate. The resulting sample was denoted as X2. X2 replaced X1 in Example 4, with all other conditions remaining unchanged, and the resulting sample was denoted as P2. X2 replaced X1 in Example 5, with all other conditions remaining unchanged, and the resulting sample was denoted as H2.

[0026] Comparative Example 2: The culture used in Example 3 was replaced with a culture medium that had a fishy smell after screening, while keeping all other conditions unchanged. The resulting sample was denoted as X3. X3 replaced X1 in Example 4, while keeping all other conditions unchanged. The resulting sample was denoted as P3. X3 replaced X1 in Example 5, while keeping all other conditions unchanged. The resulting sample was denoted as H3.

[0027] Example 7: The basic indicators of the product were tested as follows: The samples prepared in the above examples and comparative examples were weighed and diluted with purified water to a concentration of 1 mg / mL, i.e., 1000 ng / μL. The nucleic acid content was detected using the DNA concentration measurement mode of an ultra-micro UV spectrophotometer. DNA content = (sample concentration obtained (ng / μL) / (1000 ng / μL)) × 100%. The DNA content of sample H1 was calculated based on the PDRN content in sample G1 obtained in Example 5. Based on the measured DNA content, a 1 mg / mL solution was prepared with purified water, and the sample was detected by nucleic acid electrophoresis. The molecular weight distribution range of PDRN was observed by comparing it with a marker in the range of 100-5000 bp. Based on the measured DNA content, a 0.2% solution (w / v) was prepared with purified water, dissolved at room temperature, and allowed to stand for 30 min before observing the appearance.

[0028] The results are shown in Table 1. The PDRN prepared using this invention has a high content and concentrated distribution. After dissolving at a concentration of 0.2%, it is clear and has minimal impact on cosmetic formulations. Cultures not screened using this invention produce PDRN with a wider distribution range and lower content.

[0029] Table 1 First Data Table Example 8: Based on the DNA content measured in the samples, each sample was prepared with serum-free medium to a concentration of 250 μL / mL. The PDRN content in sample H1 was calculated based on the PDRN content in sample G1 obtained in Example 7. The final PDRN concentration after sample addition was 50 μL / mL. The positive control was 25 ng / mL EGF. At this concentration, the ability of the samples to promote cell proliferation was evaluated using the following method: Human keratinocytes (HaCaT) in the logarithmic growth phase were used for biological activity assays. The culture medium in the culture flasks was discarded, the cells were digested and collected, and the cells were reconstituted with culture medium containing 10% fetal bovine serum to a density of 7.0 × 10⁻⁶ cells / mL. 4Cell suspension of 100 μL / mL was seeded into 96-well cell culture plates and cultured at 37°C and 5% CO2 for 8 h to allow cell adhesion. The serum-containing medium was then discarded. 80 μL of serum-free medium was added to each well, and the cells were cultured for approximately 16 h. Then, 20 μL of positive control or sample solution was added to each well, and 20 μL of serum-free medium was added to each well for the blank control. Three replicates were made for each group, and the cells were cultured at 37°C and 5% CO2 for 48 h. 10 μL of CCK8 was added to each well, mixed, and the cells were cultured for another 2 h. The absorbance was measured at 450 nm, and the results were recorded. Relative cell proliferation rate (RGR) % = (Experimental group absorbance value / Blank control absorbance value) × 100%.

[0030] The results are shown in Table 2. Compared with competing products, the PDRN obtained by this invention also has the effect of promoting cell proliferation. The fermentation product obtained by this invention has better activity than purified PDRN, and with the same amount of PDRN added, it has more advantages in cosmetic formulation applications.

[0031] Table 2 Second Data Table Example 9: Based on the DNA content measured in Example 6, samples with a concentration of 1000 μg / mL were uniformly prepared using serum-free culture medium. The PDRN content of sample H1 was calculated based on the PDRN content in sample G1 obtained in Example 5. The DNA content of all samples was diluted with serum-free culture medium to 50 μg / mL and 100 μL / mL as detection concentrations. The positive control was 100 ng / mL of TGF-β1. The upregulation rate of type I collagen after sample addition was used to assess whether it could promote type I collagen synthesis.

[0032] Human fibroblasts (HFF-1) were cultured in complete medium at 37°C and 5% CO2. Cells in the logarithmic growth phase were harvested, the culture medium was discarded, and the cells were enzymatically digested, resuspended in fresh 1640 medium containing 10% FBS, and cell counts were performed to prepare 1×10⁻⁶ cells / cells. 5 Cell suspension at a concentration of [number] cells / mL was seeded into 96-well plates, with 200 μL of cell suspension added to each well. The plates were incubated at 37°C and 5% CO2 for 24 h. The culture medium was removed, and 200 μL of positive control or sample solution was added to each well. For the blank control, 200 μL of serum-free culture medium was added to each well. Three replicates were created for each group, and the plates were incubated at 37°C and 5% CO2 for 24 h. After incubation, 200 μL of cell culture supernatant was collected from each well and analyzed using a human type I collagen ELISA kit. The average of the three replicates from each group was taken as the final type I collagen result. The type I collagen upregulation rate was calculated as: (Sample group content - Blank group content) / Blank group content × 100%.

[0033] The experimental results are shown in Table 3. The PDRN and PDRN complex obtained in this invention can increase type I collagen at concentrations of 50 μg / mL and 100 μg / mL, demonstrating a collagen-promoting effect similar to competing products. Among them, the fermentation product obtained in this invention showed the best effect, outperforming other samples at the same PDRN content.

[0034] Table 3 Third Data Table Example 10: Fermentation products of equal mass obtained from the examples and comparative examples were diluted directly with the original product or purified water and then subjected to olfactory sensory evaluation. The fish shavings homogenate ground in Example 3 was diluted with purified water to 0.05% as a control (w / v). Twenty adults were selected, half male and half female. If one or more individuals detected a similar odor to the control, it was recorded as "+"; if none of the 20 individuals detected a similar odor, it was recorded as "-".

[0035] The results are shown in Table 4. The fermentation product obtained by this invention has no obvious characteristic odor and will not affect subsequent applications due to odor. PDRN usage costs are also lower. Even when diluted 100 times, the fermentation product obtained without this invention still retains its characteristic odor.

[0036] Table 4 Fourth Data Table Furthermore, any content not described in detail in this specification is existing technology known to those skilled in the art.

[0037] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0038] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A PDRN extraction process, characterized in that: Specifically, the following steps are included: S1. Screen for thermophilic Bacillus stearothermophilus cultures that degrade fishy odor, then screen for cultures that can obtain specific PDRN molecular weights, and in the process confirm the appropriate colony count and fermentation extraction conditions. S2. The culture obtained in step S1 is cultured in a medium containing fish milt until it reaches 2 × 10⁻⁶. 6 -5×10 7 The number of bacteria was determined to obtain the fermentation broth of Bacillus stearothermophilus; S3. Mix the fish milt homogenate with the fermentation broth obtained in step S2, adjust the pH to 7-8, keep warm at 58-69℃ for 10-120 min, raise the temperature to 70-75℃, keep warm for 10-60 min, raise the temperature to 76-82℃, keep warm for 10-60 min, and obtain the enzymatically hydrolyzed fish milt homogenate. S4. Add disodium EDTA and sodium chloride to the enzymatically hydrolyzed fish slurry obtained in step S3, adjust the pH to 4-6, keep it at 90-100℃ for 10-60 min, and cool it to room temperature to obtain PDRN extract. S5. After filtering the PDRN extract obtained in step S4 through a filter bag and centrifuging, add 95% ethanol to the supernatant, let it stand for 1-24 hours, centrifuge to recover the precipitate, wash the precipitate with 75% ethanol until the conductivity is less than 100 μS / cm, and dry it to obtain pure PDRN.

2. The PDRN extraction process according to claim 1, characterized in that: In step S5, when 95% ethanol is added to the supernatant, the ratio of supernatant to ethanol is 1:1-3.

3. The PDRN extraction process according to claim 1, characterized in that: In step S4, the concentration of disodium ethylenediaminetetraacetate is 0.02%, and the concentration of sodium chloride is 1.5 mol / L.

4. The PDRN extraction process according to claim 1, characterized in that: In step S3, the ratio of fish slurry to fermentation liquid is 1:5-20.

5. A fermentation product containing PDRN according to claim 1, characterized in that: The fish milt mentioned is commercially available fresh or frozen carp milt.

6. A fermentation product containing PDRN, characterized in that: Its preparation method is as follows: T1. The fish paste obtained in step S3 of the PDRN extraction process according to any one of claims 1-5 is adjusted to pH 4-6.5 with citric acid, kept at 90-100℃ for 10-30 min, cooled and discharged, filtered through a filter bag and centrifuged to recover the supernatant. T2. After drying the supernatant, a fermentation product containing PDRN was obtained.

7. A fermentation product containing PDRN according to claim 6, characterized in that: In step T2, the supernatant drying is performed by spray drying or freeze drying, either alone or after mixing with excipients.

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