A method for preparing salmon DNA sodium raw material and its application

Abstract

This invention discloses a method for preparing sodium DNA from salmon sperm and its application, belonging to the field of biomedical technology. The preparation method includes the following steps: S1. Raw material pretreatment: Frozen salmon or salmon testis tissue is lysed at -30 to -20℃, then a mixed solution containing Tris-HCl buffer, NaOH, and NaCl is added, followed by the addition of a chitosan-embedded complex enzyme with a total enzyme activity ≥350KU / L. The enzyme is then hydrolyzed at 37±1℃ for 26-30 hours, maintaining a pH of 7.8-8.0 to obtain the enzymatic hydrolysate; S2. DNA synergistic lysis: The pH of the enzymatic hydrolysate is adjusted to 7.8, and the mixture is synergistically treated in an ice-water bath at ≤10℃ by alternating cycles of 25-35kHz variable frequency ultrasound and 100-120MPa high-pressure homogenization for 40 minutes to obtain the DNA lysate. This method solves the problems of high protein residue, uneven molecular weight distribution, low transdermal absorption, high cost, and poor environmental friendliness in traditional extraction processes, and has the advantages of high product purity, strong activity, low cost, and green environmental protection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing sodium DNA from salmon extract and its application. Background Technology

[0002] Salmon DNA sodium (DNA-Na), a natural low-molecular-weight DNA derivative, shares 98% base similarity with human DNA in its double-stranded helix structure, exhibiting unique bioactivity in tissue repair and skin rejuvenation. Studies have shown that DNA-Na of different molecular weights has significantly differentiated application value: fragments <60kDa have become core functional ingredients in cosmetics due to their transdermal absorption advantage (epidermal permeability increases 1.8 times for every 10kDa decrease in molecular weight), while fragments of 350–500kDa play a crucial role in tissue regeneration in the pharmaceutical field. However, existing preparation technologies face multiple bottlenecks in meeting these requirements.

[0003] In traditional processes, physical disruption methods (such as ultrasonic and high-pressure homogenization) are low-cost but prone to causing uneven DNA strand breaks, resulting in molecular weight distributions that vary by more than 10 times, failing to meet the narrow molecular weight distribution requirements of cosmetics. While endonuclease methods can achieve a more precise molecular weight distribution, enzyme costs are high (US$8.6 per gram of product) and there is a risk of endotoxin exceeding limits. In the deproteinization step, the use of SDS leads to a wastewater COD value as high as 12000 mg / L, exceeding environmental standards by 6 times. Ethanol precipitation removes less than 30% of <10kDa impurities, and these small molecule proteins are a major trigger for immune responses; clinical data shows that the allergy rate of products containing impurities reaches 12.7%, significantly higher than the pure product group (1.3%). Furthermore, the high proportion of organic solvents used in traditional processes (e.g., ethanol at 73%) not only increases safety risks but also leads to a 15-20% loss of the target product.

[0004] To address the aforementioned issues, this application achieves breakthroughs through the following innovations: It employs a chitosan-encapsulated complex enzyme (containing trypsin, neutral protease, and other multi-enzyme systems) for enzymatic hydrolysis under mild conditions, combined with variable-frequency ultrasound and high-pressure homogenization to achieve precise and controllable DNA fragmentation with a molecular weight distribution range of <3 times. Using a core-shell type phenyl hydrophobic chromatography packing material (with an agarose-silica hybrid matrix) combined with dynamic axial compression column technology, residual protein content is reduced to <0.05%. Simultaneously, a 5kDa ultrafiltration membrane coupled with a nanofiltration system effectively retains target molecular weight fragments. Furthermore, the traditional SDS deproteinization process is eliminated, reducing wastewater COD by 81%; enzyme preparation recycling (reusable ≤5 times) reduces production costs by 42%; and pressure rise testing (PAT) is used in real-time to monitor residual moisture during the freeze-drying process, ensuring product stability and activity.

[0005] Therefore, there is an urgent need for a method for preparing sodium DNA from salmon extract and its application, which would significantly reduce the safety risks of existing products and promote the in-depth application of DNA-Na in precision medicine and functional cosmetics. Summary of the Invention

[0006] Therefore, this invention provides a method for preparing sodium DNA from salmon extract and its application, in order to solve the problems caused by high protein residue, uneven molecular weight distribution, low transdermal absorption rate, high cost and poor environmental performance in the existing technology due to traditional extraction processes.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] According to a first aspect of the present invention, a method for preparing sodium DNA from salmon extract is provided, comprising the following steps:

[0009] S1. Raw material pretreatment: Frozen salmon or salmon testis tissue was lysed at -30 to -20℃, then a mixed solution containing Tris-HCl buffer, NaOH and NaCl was added, followed by the addition of chitosan-embedded complex enzyme with a total enzyme activity ≥350KU / L. The enzyme was then hydrolyzed at 37±1℃ for 26-30h, maintaining a pH of 7.8 to 8.0 to obtain the enzymatic hydrolysate.

[0010] S2. DNA Synergistic Disruption: Adjust the pH of the enzymatic hydrolysate to 7.8, and in an ice-water bath at ≤10℃, synergistically treat it by alternating cycles of 25-35kHz variable frequency ultrasound and 100-120MPa high-pressure homogenization for 40 minutes to obtain DNA disruption solution.

[0011] S3. Deproteinization and fractionation purification: After multiple centrifugations and filtrations, the DNA fragments were separated by nanofiltration, ultrafiltration and core-shell phenyl hydrophobic chromatography packing material, and a specific mobile phase gradient elution was used to collect the target fraction to obtain a high-purity DNA-Na solution.

[0012] S4. Freeze-drying: After pre-freezing the high-purity DNA-Na solution at -45℃, freeze-dry it according to a specific temperature gradient, and then dry it twice while controlling the particle size D90≤40μm to obtain freeze-dried powder.

[0013] S5. Enzyme recovery: Wash the precipitate obtained in S3 to recover chitosan microspheres. If the enzyme activity is ≥70% of the initial value, it can be reused for the next batch of enzymatic hydrolysis.

[0014] Furthermore, the preparation method of the chitosan-encapsulated complex enzyme in S1 is as follows:

[0015] Chitosan was dissolved in 0.5–2% (w / v) acetic acid solution to prepare a 1%–3% (w / v) chitosan solution, and the solution was magnetically stirred until clear. A compound enzyme preparation was added in proportion, and the mixture was sonicated at 50–150 W for 3–10 min to form an enzyme-chitosan mixture. The mixture was then added dropwise to a 0.3% TPP solution, with a chitosan to TPP mass ratio of 3:1–8:1, while simultaneously magnetically stirring at 300–800 rpm. The mixture was cross-linked at room temperature for 20–40 min to form chitosan microspheres with a particle size of 5–10 μm. The microspheres were then collected by centrifugation at 3000–5000 rpm for 5–15 min, washed 2–4 times with 7.5–8.0 Tris-HCl buffer, and vacuum dried at 30–50 °C for 6–15 h.

[0016] Furthermore, the components and weight proportions of the compound enzyme preparation are as follows:

[0017] The weight proportions of the compound enzyme preparation are:

[0018] 40-50 parts trypsin, 10-20 parts neutral protease, 15-25 parts papain, 15-25 parts proteinase K, 12-18 parts NSP enzyme, 7-10 parts cellulase and 3-5 parts β-1,3-glucanase.

[0019] Furthermore, in the mixed solution containing Tris-HCl buffer, NaOH, and NaCl in S1, the concentration of Tris-HCl buffer is 50–100 mmol / L, the concentration of NaOH is 0.1–0.3 mol / L, and the concentration of NaCl is 0.5–1.5 mol / L.

[0020] Furthermore, in S2, the frequency conversion ultrasonic power is 150-300W, the total processing time is 30-60min, and the power is 200-300W for the first 15-30min and 100-200W for the last 15-30min; the pressure of the high-pressure homogenization process is 80-150MPa; the ultrasonic and homogenization processes are alternated 2-6 times, with ultrasonic treatment lasting 3-8min after each homogenization; the ultrasonic working mode is ultrasonic for 3-8s and then paused for 1-3s.

[0021] Further, in step S3, the DNA lysis buffer is centrifuged at pH 8.5-9.2 and 8000-11000 rpm for 15-25 min, and the supernatant is filtered through a 0.5-2 μm ceramic membrane, then the pH is adjusted to 7.5-8.0, allowed to stand for 6-10 h, and centrifuged a second time.

[0022] Furthermore, the nanofiltration system in S3 uses a spiral wound composite membrane made of polyamide, with a molecular weight cutoff of 100–500 Da, an operating pressure of 0.15–0.3 MPa, a temperature of 15–25 °C, a cross-flow filtration membrane surface velocity of 1.5–2.5 m / s, and a 3–10 kDa ultrafiltration membrane after 3–8 h of circulation.

[0023] Further, the freeze-drying process in S4 is as follows: after pre-freezing at -50 to -40℃ for 2 to 3 hours, the temperature is increased to -30 to -20℃ at a rate of 1 to 2℃ / min and maintained for 10 to 14 hours, then increased to -15 to -5℃ and maintained for 10 to 14 hours, then increased to 10 to 20℃ and maintained for 10 to 14 hours, and finally increased to 30 to 40℃ and maintained for 24 to 30 hours; the secondary drying conditions are 40 to 45℃ and vacuum degree ≤10Pa, and the process ends when the pressure increase rate is ≤1Pa / h.

[0024] Furthermore, in step S5, the chitosan microspheres are washed with a 0.05–0.2 mol / L NaCl solution, and the enzyme activity must be tested to be ≥ 70% of the initial value before reuse.

[0025] According to a second aspect of the present invention, the application of the salmon extract DNA sodium raw material obtained by the above-described preparation method in the cosmetic field is provided, for use in preparing skin care products with anti-wrinkle, skin-firming, and skin-brightening effects.

[0026] The present invention has the following advantages:

[0027] 1. In the S1 raw material pretreatment, chitosan-encapsulated complex enzyme (total enzyme activity ≥350KU / L) is used to enzymatically hydrolyze the raw material at 37±1℃. In S2, the enzymatic hydrolysate is treated alternately with 25-35kHz variable frequency ultrasound and 100-120MPa high-pressure homogenization. The advantages are that the enzymatic hydrolysis specifically decomposes proteins while gently protecting DNA. Chitosan encapsulation improves enzyme stability and recovery rate. Ultrasound and high-pressure homogenization work together to efficiently break cells and refine DNA, shorten the breaking time, improve DNA yield and purity, and reduce energy consumption.

[0028] 2. In the S3 deproteinization and fractionation purification steps, multiple centrifugation and filtration processes are employed, combined with nanofiltration, ultrafiltration, and core-shell phenyl hydrophobic chromatographic packing for separation, using a specific mobile phase gradient elution. This multi-stage purification method precisely removes proteins and small molecule impurities. Nanofiltration and ultrafiltration membranes separate based on molecular weight differences, and the chromatographic packing further purifies the product. This combination significantly improves DNA-Na purity, reduces the impact of impurities on subsequent applications, and results in a high-purity DNA-Na solution that enhances product stability and efficacy when used in cosmetics.

[0029] In step 3, S5, the chitosan microspheres are washed and recovered. When the enzyme activity is detected to be ≥70% of the initial value, they are reused for the next batch of enzymatic hydrolysis. This technology reduces the cost of enzyme preparations, and the chitosan-embedded composite enzyme can be recycled, reducing raw material waste. Simultaneously, stable enzyme activity ensures the consistency of enzymatic hydrolysis reactions across batches, improving production stability and product quality uniformity, making the preparation process more economical and sustainable. Detailed Implementation

[0030] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0031] This invention provides a method for preparing sodium DNA from salmon extract, comprising the following steps:

[0032] S1. Raw material pretreatment: Frozen salmon or salmon testis tissue was lysed at -30 to -20℃, then a mixed solution containing Tris-HCl buffer, NaOH and NaCl was added, followed by the addition of chitosan-embedded complex enzyme with a total enzyme activity ≥350KU / L. The enzyme was then hydrolyzed at 37±1℃ for 26-30h, maintaining a pH of 7.8 to 8.0 to obtain the enzymatic hydrolysate.

[0033] S2. DNA Synergistic Disruption: Adjust the pH of the enzymatic hydrolysate to 7.8, and in an ice-water bath at ≤10℃, synergistically treat it by alternating cycles of 25-35kHz variable frequency ultrasound and 100-120MPa high-pressure homogenization for 40 minutes to obtain DNA disruption solution.

[0034] S3. Deproteinization and fractionation purification: After multiple centrifugations and filtrations, the DNA fragments were separated by nanofiltration, ultrafiltration and core-shell phenyl hydrophobic chromatography packing material, and a specific mobile phase gradient elution was used to collect the target fraction to obtain a high-purity DNA-Na solution.

[0035] S4. Freeze-drying: After pre-freezing the high-purity DNA-Na solution at -45℃, freeze-dry it according to a specific temperature gradient, and then dry it twice while controlling the particle size D90≤40μm to obtain freeze-dried powder.

[0036] S5. Enzyme recovery: Wash the precipitate obtained in S3 to recover chitosan microspheres. If the enzyme activity is ≥70% of the initial value, it can be reused for the next batch of enzymatic hydrolysis.

[0037] The preparation method of the chitosan-encapsulated complex enzyme in S1 is as follows:

[0038] Chitosan was dissolved in 0.5–2% (w / v) acetic acid solution to prepare a 1%–3% (w / v) chitosan solution, and the solution was magnetically stirred until clear. A compound enzyme preparation was added in proportion, and the mixture was sonicated at 50–150 W for 3–10 min to form an enzyme-chitosan mixture. The mixture was then added dropwise to a 0.3% TPP solution, with a chitosan to TPP mass ratio of 3:1–8:1, while simultaneously magnetically stirring at 300–800 rpm. The mixture was cross-linked at room temperature for 20–40 min to form chitosan microspheres with a particle size of 5–10 μm. The microspheres were then collected by centrifugation at 3000–5000 rpm for 5–15 min, washed 2–4 times with 7.5–8.0 Tris-HCl buffer, and vacuum dried at 30–50 °C for 6–15 h.

[0039] The weight proportions of the compound enzyme preparation are as follows:

[0040] 40-50 parts trypsin, 10-20 parts neutral protease, 15-25 parts papain, 15-25 parts proteinase K, 12-18 parts NSP enzyme, 7-10 parts cellulase and 3-5 parts β-1,3-glucanase.

[0041] In S1, the mixed solution containing Tris-HCl buffer, NaOH, and NaCl has a Tris-HCl buffer concentration of 50–100 mmol / L, a NaOH concentration of 0.1–0.3 mol / L, and a NaCl concentration of 0.5–1.5 mol / L.

[0042] In S2, the frequency conversion ultrasonic power is 150-300W, the total processing time is 30-60min, the power is 200-300W for the first 15-30min and 100-200W for the last 15-30min; the pressure of high-pressure homogenization is 80-150MPa; the ultrasonic and homogenization cycles are repeated 2-6 times, with ultrasonic treatment lasting 3-8min after each homogenization; the ultrasonic working mode is 3-8s ultrasonic treatment followed by 1-3s pause.

[0043] In S3, the DNA lysate is centrifuged at pH 8.5–9.2 and 8000–11000 rpm for 15–25 min. The supernatant is filtered through a 0.5–2 μm ceramic membrane, the pH is adjusted to 7.5–8.0, and the mixture is allowed to stand for 6–10 h before being centrifuged a second time.

[0044] The nanofiltration system in S3 uses a spiral wound composite membrane made of polyamide with a molecular weight cutoff of 100–500 Da, an operating pressure of 0.15–0.3 MPa, a temperature of 15–25 °C, a cross-flow filtration membrane surface velocity of 1.5–2.5 m / s, and a 3–10 kDa ultrafiltration membrane after 3–8 hours of circulation.

[0045] The freeze-drying process in S4 is as follows: after pre-freezing at -50 to -40℃ for 2 to 3 hours, the temperature is increased to -30 to -20℃ at a rate of 1 to 2℃ / min and maintained for 10 to 14 hours, then increased to -15 to -5℃ and maintained for 10 to 14 hours, then increased to 10 to 20℃ and maintained for 10 to 14 hours, and finally increased to 30 to 40℃ and maintained for 24 to 30 hours; the secondary drying conditions are 40 to 45℃ and vacuum degree ≤10Pa, and the process ends when the pressure increase rate is ≤1Pa / h.

[0046] In S5, chitosan microspheres are washed with 0.05–0.2 mol / L NaCl solution. Before reuse, enzyme activity must be tested to be ≥70% of the initial value.

[0047] This invention provides an application of the salmon extract DNA sodium raw material obtained by the above preparation method in the cosmetic field, for the preparation of skin care products with anti-wrinkle, skin-firming and skin-brightening effects.

[0048] Example 1: Pretreatment of salmon testes, preparation of chitosan-encapsulated complex enzymes, and enzymatic hydrolysis process

[0049] 1.1 Raw material pretreatment

[0050] Deep-sea caught salmon testes, stored at -42℃ in a freezer, were transferred to a cryogenic crusher at -28℃ for crushing until a slurry was formed, ensuring the particle size of the crushed material did not exceed 5mm. Subsequently, a specific mixed solution was added to the crushed slurry at a volume ratio of 1:3 (testes to mixed solution). This mixed solution consisted of 0.08 mol / L Tris-HCl buffer (pH 7.8), 0.2 mol / L NaOH, and 1 mol / L NaCl. After adding the mixed solution, the material was placed in a stirring device and continuously stirred at 300 rpm for 30 minutes, while the temperature was controlled to ≤10℃ using an ice bath, thus completing the pretreatment of the raw material.

[0051] 1.2 Preparation of chitosan-encapsulated complex enzyme

[0052] Chitosan solution preparation: Weigh chitosan with a degree of deacetylation ≥90% and a viscosity in the range of 300 mPa·s, and slowly add it to a 1% (w / v) acetic acid solution to prepare a 2% (w / v) chitosan solution. Stir magnetically at 500 rpm for 4 hours until clear, and then sterilize by filtering through a 0.45 μm filter membrane.

[0053] Preparation of compound enzyme solution:

[0054] Weigh out: 45 portions of trypsin (enzyme activity ≥250 USPU / mg), purchased from Novozymes (China) Investment Co., Ltd., product code Trypsin 6.0S;

[0055] 15 samples of neutral protease (enzyme activity ≥10000U / g) were purchased from Novozymes (China) Investment Co., Ltd., model Neutrase 0.8L;

[0056] 20 portions of papain (enzyme activity ≥80000U / g) were purchased from Nanning Pangbo Biotechnology Co., Ltd., product number PB-Papain-80K;

[0057] Proteinase K 20 samples (enzyme activity ≥30U / mg), derived from Candida albicans Tritirachiumalbum, purchased from Sigma-Aldrich, product number V900887;

[0058] 12 samples of NSP enzyme (β-glucanase activity ≥5000U / g), purchased from Megazyme Company, product number E-NSPENZ;

[0059] Eight portions of cellulase (enzyme activity ≥1000U / g) were purchased from Novozymes (China) Investment Co., Ltd., model number Celluclast 1.5L;

[0060] Four portions of β-1,3-glucanase (enzyme activity ≥500U / g), purchased from Megazyme, product number E-BGLUC.

[0061] Dissolve in 50 mmol / L Tris-HCl buffer (pH 7.8) and stir at 200 rpm for 15 min until homogeneous.

[0062] Enzyme-Chitosan Mixing: The complex enzyme solution was slowly added dropwise to the chitosan solution at a volume ratio of 1:2 (enzyme solution to chitosan solution), with a dropping rate controlled at 20 mL / min, while simultaneously stirring magnetically at 300 rpm. After addition, the mixture was transferred to an ultrasonic processor and ultrasonicated for 5 minutes at 100 W. The ultrasonic process used a pulse mode, i.e., 5 seconds of ultrasonication followed by a 1-second pause, with the temperature controlled in an ice-water bath ≤25℃.

[0063] Ionic crosslinking: A 0.3% (w / v) TPP solution was prepared as a crosslinking agent, and 0.15 mol / L NaCl was added to it. The ultrasonically treated mixture was added dropwise to the TPP solution using a peristaltic pump at a flow rate of 2 mL / min, according to a chitosan to TPP mass ratio of 5:1. During the addition process, a magnetic stirrer was used to continuously stir at 500 rpm for 30 min to ensure sufficient ionic crosslinking between chitosan and TPP, forming 5–10 μm chitosan microspheres.

[0064] Post-processing: After the cross-linking reaction, the mixture was transferred to a centrifuge and centrifuged at 4000 rpm for 10 min. The precipitated microspheres were collected. The microspheres were resuspended and washed with 50 mmol / L Tris-HCl buffer (pH 7.8), and this operation was repeated 3 times. The washed microspheres were then vacuum-dried at 40℃ and -0.09 MPa for 10 h. After drying, the microspheres were ground and passed through an 80-mesh sieve. Microspheres with a particle size in the range of 5-10 μm were collected, which were the chitosan-encapsulated complex enzyme product.

[0065] 1.3 Enzymatic hydrolysis process

[0066] The pretreated material was used as a substrate and prepared into a 10% (w / v) solution based on the dry weight of the testes. The chitosan-encapsulated complex enzyme (total enzyme activity ≥350 KU / L) prepared in step 1.2 was added to this solution at a concentration of 0.5% (w / v), while maintaining a buffer system of 0.08 mol / L Tris-HCl (pH 7.8). The reaction system was placed in a constant temperature environment of 37±1℃ and subjected to enzymatic hydrolysis using a magnetic stirrer at 300 rpm for 28 hours. During the enzymatic hydrolysis, 0.5 mol / L NaOH or HCl solution was added in real-time using an automatic titration system to strictly control the pH of the reaction system within the range of 7.8±0.1. After the enzymatic hydrolysis reaction was completed, the reaction system was heated to 85℃ and maintained for 10 min to terminate the reaction. Then, it was centrifuged at 4000 rpm for 20 min, and the supernatant was collected. The DNA yield was 2.3 mg / g testes, the protein residue was ≤0.5%, and the DNA purity (A260 / A280) in the enzymatic hydrolysate was 1.85.

[0067] This embodiment verifies that the DNA purity in the enzymatic hydrolysate reaches 1.85 (close to the theoretical value of 1.8), and the protein residue is ≤0.5%, demonstrating the high efficiency of the chitosan-encapsulated complex enzyme in decomposition and the protective effect of the mild enzymatic hydrolysis conditions on DNA, providing high-quality raw materials for subsequent DNA synergistic fragmentation and purification.

[0068] Example 2 DNA Co-disruption Process

[0069] 2.1 pH Adjustment and Temperature Control

[0070] The enzymatic hydrolysate obtained in Example 1 was adjusted to pH 7.8 using 0.5 mol / L NaOH. The solution was then placed in an ice-water bath to ensure the temperature was ≤8°C, creating suitable environmental conditions for subsequent synergistic crushing.

[0071] 2.2 Alternating treatment with variable frequency ultrasound and high-pressure homogenization

[0072] The treatment involved alternating cycles of 25–35 kHz variable frequency ultrasound and 110 MPa high-pressure homogenization four times, with a total processing time of 40 minutes. The specific steps are as follows:

[0073] Loop 1:

[0074] First, the cells were homogenized at 110 MPa for 3 minutes using a high-pressure homogenizer to break down the cell structure through shear force.

[0075] Subsequently, ultrasonic treatment was performed using 30kHz variable frequency ultrasound with a power setting of 250W. The ultrasound mode was set to work for 5 seconds, then pause for 1 second, and the treatment lasted for 5 minutes.

[0076] Cycle 2-4:

[0077] Repeat the steps of cycle 1, namely, first high-pressure homogenization (110MPa), then ultrasonic treatment (250W, 5s working + 1s rest, 5min).

[0078] Processing in the last 20 minutes:

[0079] Adjust the ultrasonic power to 120W and continue treatment for 20 minutes using the same ultrasonic mode (5s working + 1s interval).

[0080] 2.3 Crushing effect test

[0081] After the rupture fluid was filtered through a 0.45 μm filter membrane, the following results were obtained:

[0082] DNA concentration: 3.2 mg / mL;

[0083] A260 / A280 ratio: 1.82;

[0084] Electron microscopy revealed that the cell fragmentation rate was ≥95%, and the DNA fragment sizes were mainly distributed between 50 and 200 bp.

[0085] Example 3: Deproteinization and Fractionation Purification Process

[0086] 3.1 Centrifugation Pretreatment

[0087] First centrifugation:

[0088] The pH of the DNA lysis buffer was adjusted to 8.8 with 0.5 mol / L NaOH, and then centrifuged at 9000 rpm for 20 min. After centrifugation, the precipitate was discarded, and the supernatant was retained. This step mainly removes insoluble impurities and some denatured proteins.

[0089] Second centrifugation:

[0090] The supernatant was adjusted to pH 7.8 with 0.5 mol / L HCl and allowed to stand at 4°C for 8 hours to allow for complete protein precipitation. Afterwards, it was centrifuged at 9000 rpm for 20 minutes, and the supernatant was collected as a pretreatment solution to further remove proteins and other impurities. The A260 / A280 ratio was 1.75, the DNA concentration was 2.8 mg / mL, and the residual protein content was ≤1.5%.

[0091] 3.2 Membrane Separation System

[0092] Ceramic membrane filtration:

[0093] The pretreated solution is filtered through a 1μm ceramic membrane to remove particulate impurities and macromolecules, ensuring the smooth progress of the subsequent nanofiltration process.

[0094] Nanofiltration system processing:

[0095] 1. Adjust the pH of the filtrate to 8.8, set the inlet pressure to 0.19 MPa, the temperature to 20 ± 1℃, the membrane flow rate to 1.8 m / s, and circulate for 5 hours.

[0096] Ultrafiltration and top washing:

[0097] Filtration was performed using a 5 kDa ultrafiltration membrane, followed by headwashing with a 20 mmol / L sodium dihydrogen phosphate solution at pH 7.0 until the conductivity was ≤10 μS / cm.

[0098] Combine the filtrates and adjust the pH to 7.8 to obtain the crude product solution.

[0099] Detection results: A260 / A280 = 1.82, DNA recovery rate 92%, salt content ≤0.1%.

[0100] 3.3 Core-shell chromatographic separation

[0101] Sterilization treatment:

[0102] The crude solution was sterilized by filtration through a 0.22 μm polyethersulfone membrane to remove microorganisms and ensure the purity of subsequent chromatographic separation processes.

[0103] Chromatographic separation:

[0104] The core-shell type phenyl hydrophobic chromatographic packing material (agarose-silica hybrid matrix, specific surface area 350 m²) was used. 2 / g, pore volume 1.2mL / g), packed into a dynamic axial compression column (DAC column).

[0105] The column pressure was controlled at 0.28 MPa, the sample loading volume was 0.9 column volumes (BV), and the sample loading speed was 0.5 cm / min.

[0106] Gradient elution procedure:

[0107] Equilibrium: Mobile phase A:B = 1:1 (A is 100 mmol / L NaCl solution, B is ultrapure water), 2 BV;

[0108] Elution 1: A:B = 1:2, 2 BV;

[0109] Elution 2: A:B:C = 1:2:1 (C is 20 mmol / L dipotassium hydrogen phosphate solution, pH 8.0), 2 BV;

[0110] Elution 3: A:B:C = 1:1:2, 1 BV;

[0111] Target elution: B:D = 5:1 (D is 0.1 mol / L NaOH solution), 2 BV;

[0112] During the elution process, the absorbance at UV260nm is monitored online. When the absorbance is >0.5, the flow rate is automatically reduced to 6BV / h, and the target fraction is collected to obtain a high-purity DNA-Na solution.

[0113] Target flow segmentation detection:

[0114] A260 / A280=1.92;

[0115] DNA concentration 1.5 mg / mL;

[0116] Protein residue ≤0.1%;

[0117] Endotoxin level <0.5 EU / mL.

[0118] Example 4: Freeze-drying molding process

[0119] 4.1 Pre-freezing treatment

[0120] The high-purity DNA-Na solution obtained in Example 3 was transferred to the material tray of the freeze dryer, ensuring a uniform solution thickness not exceeding 15 mm. The freeze dryer was started, and the temperature of the cold trap (collector) was lowered to ≤-65°C to provide sufficient cooling to capture sublimated water. Simultaneously, the material temperature was lowered to -45°C at a rate of 3°C / min and maintained at this temperature for 2.5 hours to completely freeze the DNA-Na solution, forming a stable ice crystal structure, laying the foundation for sublimation drying.

[0121] 4.2 Gradient Temperature Freeze-drying

[0122] After pre-freezing, the main drying stage begins, and freeze-drying is carried out according to the set temperature gradient:

[0123] First stage: The material temperature is raised from -45℃ to -25℃ at a rate of 1.5℃ / min and maintained at this temperature for 12 hours. During this time, the system vacuum is maintained at 10-15 Pa, which promotes the direct sublimation of ice crystals into water vapor at low temperature, removing most of the free water.

[0124] Second stage: Continue to raise the temperature to -10℃ at a rate of 1.5℃ / min and maintain it for 12 hours. In this stage, further remove tightly bound moisture, and control the vacuum degree at 8-10 Pa to ensure the stable progress of the sublimation process.

[0125] Third stage: Raise the temperature to 15℃ and maintain it for 12 hours. At this time, the ice crystals in the material have basically sublimated, and some of the adsorbed water begins to be removed. The vacuum degree is adjusted to 5-8 Pa.

[0126] Fourth stage: Finally, raise the temperature to 35℃ and maintain it for 26 hours. This stage mainly removes residual adsorbed water, and the vacuum degree is maintained at ≤8Pa to achieve a low moisture content in the DNA-Na product.

[0127] 4.3 Secondary Drying and Quality Control

[0128] Pressure Rise Test (PAT): After maintaining a temperature of 35℃ for 26 hours, the secondary drying stage begins. The temperature is then further increased to 42℃, with the vacuum level controlled at ≤8Pa, and the pressure rise test is initiated. By shutting down the vacuum pump, the rate of change of system pressure over time (pressure rise rate) is monitored to track the residual moisture content in the material in real time.

[0129] Online particle size monitoring: During the secondary drying process, a laser particle size analyzer is used to monitor the particle size of the freeze-dried product online to ensure that the D90 particle size (i.e., the diameter of 90% of the particles) is ≤36μm, which meets the process requirements.

[0130] Drying endpoint determination: Continuously monitor the pressure rise rate. When the pressure rise rate is ≤0.4Pa / h, it is determined that the residual moisture in the material has reached the specified standard (≤3%), and the drying process is terminated at this time.

[0131] Example 5 Enzyme Recycling and Reuse

[0132] 5.1 Sedimentation Collection and Washing

[0133] Precipitation source: The precipitate obtained after the first centrifugation (pH 8.8, 9000 rpm, 20 min) in Example 3.1 mainly contains chitosan-embedded complex enzyme microspheres and some co-precipitated impurities.

[0134] Washing procedure:

[0135] Add 0.1 mol / L NaCl solution to the precipitate, the amount added being 3 times the volume of the precipitate (v).

[0136] Stir with a magnetic stirrer at 200 rpm for 15 minutes to fully suspend the precipitate.

[0137] Centrifuge at 4000 rpm for 10 min, discard the supernatant, and collect the washed microspheres.

[0138] Repeat the washing steps above once to thoroughly remove impurities and residual protein.

[0139] 5.2 Microsphere Recovery and Enzyme Activity Detection

[0140] Microsphere recycling:

[0141] The washed chitosan microspheres were transferred to a sterile container, weighed, and the recovery volume was recorded. Microscopic observation (400x magnification) confirmed that the microspheres were intact and showed no obvious damage or aggregation.

[0142] Enzyme activity assay:

[0143] Take an appropriate amount of recovered microspheres, add Tris-HCl buffer (pH 7.8, 50 mmol / L), and shake on a shaker at 150 rpm for 30 min to fully release the enzyme.

[0144] The total activity of the protease was determined by the Folin-phenol method (using bovine serum albumin as a standard, measured at a wavelength of 750 nm), and the recovery rate was calculated based on the initial enzyme activity (380 KU / L).

[0145] When the enzyme activity drops to 70% of the initial value (i.e., 266 KU / L), the replenishment process begins.

[0146] 5.3 Reuse and Enzyme Activity Supplementation

[0147] Reusable:

[0148] The recovered chitosan microspheres were directly used in the next batch of enzymatic hydrolysis reaction (Step 3 of Example 1), with the same amount added as the fresh enzyme preparation (0.5% w / v).

[0149] Enzyme activity supplementation:

[0150] Enzyme activity is tested after each cycle of 5 uses, and a replenishment process is initiated when it drops to 70% of the initial value.

[0151] Calculate the replenishment amount using the following formula:

[0152] Supplemented enzyme amount (g) = (Initial enzyme activity - Current enzyme activity) × Reaction system volume × Dilution factor

[0153] (Note: "Initial enzyme activity" refers to the total enzyme activity of the fresh enzyme preparation (380 KU / L), and "current enzyme activity" is the measured value of the recovered enzyme; the dilution factor of 1.2 is the enzyme activity loss compensation factor.)

[0154] Use after thoroughly mixing the replenished fresh enzyme preparation with the recycled microspheres.

[0155] Example 6 Product Performance Verification Experiment

[0156] 6.1 Purity and Molecular Weight Detection

[0157] Protein residue determination

[0158] Detection method: The BCA (diquinoline carboxylic acid) method was used, with bovine serum albumin (BSA) as the standard.

[0159] Sample processing:

[0160] Weigh 10 mg of the lyophilized product precisely and dissolve it in 1 mL of ultrapure water. Shake at room temperature for 30 min until completely dissolved. Take 20 μL of the sample solution, add 200 μL of LCA working reagent (solution A:solution B = 50:1), mix thoroughly, and incubate at 37 °C for 30 min.

[0161] Testing conditions:

[0162] The absorbance was measured at 562 nm using a spectrophotometer, and the protein content was calculated using a standard curve (0-1000 μg / mL BSA).

[0163] result:

[0164] The measured residual protein content was 0.026%, which is 48% lower than that of the existing technology (0.05%), indicating that the deproteinization effect of this process is significantly better than that of the traditional method.

[0165] Molecular weight distribution determination

[0166] Detection method: High performance liquid size exclusion chromatography (SEC-HPLC).

[0167] Chromatographic conditions:

[0168] Chromatographic column: TSKgel G4000PWXL (7.8mm×30cm, Tosoh, Japan).

[0169] Mobile phase: 0.1 mol / L phosphate buffer (pH 7.0) containing 0.1 mol / L NaCl.

[0170] Flow rate: 0.5 mL / min, column temperature: 25℃, injection volume: 20 μL, UV detection: 260 nm.

[0171] Standard products:

[0172] A calibration curve was established using DNA molecular weight standards (100bp-100kb, Sigma-Aldrich).

[0173] result:

[0174] The product contains 92.5% DNA fragments below 20kDa, indicating that it is mainly composed of small molecules and is suitable for transdermal absorption.

[0175] The fact that the percentage of high molecular weight components (above 50 kDa) is less than 1.5% indicates that the process effectively avoids excessive DNA polymerization.

[0176] The molecular weight distribution span (D90 / D10) was 2.7 times, indicating good fragment homogeneity.

[0177] Transdermal absorption rate determination

[0178] Experimental model: Franz diffusion cell (effective diffusion area 1.77 cm²) 2 (Receiver chamber volume 5mL), using abdominal skin of nude mice (thickness 0.2-0.3mm).

[0179] Experimental conditions:

[0180] Supply chamber: 1 mL of PBS solution containing 1 mg / mL DNA (pH 7.4).

[0181] Receiving chamber: PBS solution (containing 0.1% BSA), constant temperature at 37℃, magnetic stirring at 600 rpm.

[0182] Sampling method:

[0183] Take 0.5 mL of the receiving solution at 2, 4, 6, 8, 12 and 24 h respectively, and replenish with an equal amount of fresh solution.

[0184] Detection method:

[0185] After sampling, Hoechst 33258 fluorescent dye was added, and the DNA content was determined using a fluorescence spectrophotometer (excitation 350 nm, emission 460 nm).

[0186] result:

[0187] The 24-hour transdermal absorption rate was 22.9%, which is 25% higher than the existing technology (18.3%), indicating that the DNA prepared by this process has better transdermal performance.

[0188] 6.2 Yield and Activity Assay

[0189] Yield determination

[0190] Experimental scale:

[0191] Feed 50kg of frozen salmon testes (stored at -42℃, frozen within 2 hours of catching).

[0192] Calculation method:

[0193] The theoretical yield is calculated based on an average salmon testis DNA content of 1.07% (i.e., 50kg × 1.07% = 535g).

[0194] Actual yield = (weight of lyophilized product / weight of theoretical DNA) × 100%.

[0195] result:

[0196] The final yield was 23.8g of freeze-dried product;

[0197] Theoretical yield: DNA content of 50kg of testes = 50000g × 1.07% = 535g;

[0198] Actual yield: 23.8g / 535g×100%=4.45%, indicating that process loss is controllable.

[0199] Bioactivity assay

[0200] Cell model: Human skin fibroblasts (HSF, ATCCCRL-2097).

[0201] Experimental Groups:

[0202] Blank group: DMEM medium without DNA.

[0203] Experimental group: Culture medium containing 0.1 mg / mL DNA from this process.

[0204] Control group: Culture medium containing 0.1 mg / mL of existing technology DNA.

[0205] The existing methods for preparing DNA are as follows:

[0206] 1. Raw material pretreatment

[0207] Take 50 kg of frozen salmon testes (stored at -20℃, frozen within 24 hours of catching), thaw at room temperature, and then homogenize into a paste using a tissue homogenizer.

[0208] Add a mixture of 0.1 mol / L Tris-HCl buffer (pH 8.0) and 0.5 mol / L NaCl at a ratio of 1:5 (w / v), and stir to mix thoroughly.

[0209] 2. Enzymatic hydrolysis

[0210] Add a single proteinase K (enzyme activity ≥30U / mg) at an amount of 0.1% (w / v) of the testis weight and enzymatically hydrolyze at 50℃ for 12h.

[0211] After enzymatic hydrolysis, the enzyme was inactivated at 95°C for 10 min, centrifuged at 4000 rpm for 15 min, and the supernatant was collected.

[0212] 3. Protein removal and precipitation

[0213] Add the supernatant to a phenol-chloroform-isoamyl alcohol (25:24:1) mixture at a volume ratio of 1:1, vortex for 10 min, centrifuge at 12000 rpm for 10 min, and collect the aqueous phase.

[0214] Repeat the extraction twice until there is no protein layer at the interface.

[0215] Add 0.1 volume of 3 mol / L sodium acetate (pH 5.2) and 2 volumes of anhydrous ethanol to the aqueous phase, precipitate at -20℃ for 2 h, centrifuge at 12000 rpm for 10 min, and discard the supernatant.

[0216] The precipitate was washed twice with 70% ethanol, dried under vacuum, and then dissolved in 10 mmol / L Tris-HCl buffer (pH 8.0).

[0217] 4. Preliminary purification

[0218] The solution was filtered through a 0.45 μm filter membrane and purified by DEAE-cellulose column chromatography (stationary phase: DEAE-52; mobile phase: 0.1–1.0 mol / L NaCl gradient elution). The elution peak containing DNA was collected.

[0219] The eluent was concentrated by rotary evaporation, precipitated again with ethanol, dried, dissolved in sterile water, and stored at -20°C.

[0220] Bioactivity assay procedure:

[0221] Cells were seeded in 24-well plates at a density of 5 × 10⁶ cells / well. 4 Cells / well were cultured for 24 hours until adherence.

[0222] Replace the culture medium with one containing DNA and continue culturing for 48 hours.

[0223] Total RNA was extracted (Trizol method) and reverse transcribed into cDNA (PrimeScriptRT kit).

[0224] The expression level of collagen I (COL1A1) mRNA was detected by qPCR, with GAPDH as an internal control.

[0225] Primer sequences:

[0226] COL1A1-F: 5'-CTGGCAAGGTGGACATCG-3' (amplified fragment 120bp).

[0227] COL1A1-R: 5'-TGGGGCACACACCTTCT-3'.

[0228] GAPDH-F: 5'-GAGTCAACGGATTTGGTCGT-3'.

[0229] GAPDH-R: 5'-TTGATTTTGGAGGGATCTCG-3'.

[0230] qPCR conditions:

[0231] Pre-denaturation at 95℃ for 30 seconds, followed by 40 cycles (5 seconds at 95℃, 30 seconds at 60℃).

[0232] Result calculation:

[0233] The relative expression level was calculated using the 2^(-ΔΔCt) method, and the experiment was repeated three times and the average value was taken.

[0234] result:

[0235] The expression level of COL1A1 mRNA in the experimental group increased by 41% compared with the blank group and by 32% compared with the control group, indicating that the product of this process can stimulate collagen synthesis by 28%.

[0236] Example 7: Cosmetic Application Efficacy Test

[0237] 7.1 Preparation of the serum

[0238] Formula composition:

[0239] Active ingredient: Salmon DNA-Na (prepared in Example 4), content 0.6% (w / v).

[0240] Solvent system: glycerol (10% w / v), deionized water (79.4% w / v), PEG-400 (10% w / v).

[0241] Additives: Sodium hyaluronate (0.5% w / v), disodium EDTA (0.1% w / v), phenoxyethanol (0.4% w / v).

[0242] Preparation process:

[0243] Mix glycerin with PEG-400 and stir at 60°C until homogeneous.

[0244] Add DNA-Na powder and continue stirring for 30 minutes until completely dissolved.

[0245] Slowly add deionized water (containing pre-dissolved sodium hyaluronate and disodium EDTA) while stirring.

[0246] After cooling to room temperature, add phenoxyethanol as a preservative and stir for 10 minutes.

[0247] Sterilize by filtration through a 0.22μm filter membrane, dispense into brown glass bottles, and store away from light.

[0248] 7.2 Human Efficacy Testing

[0249] Subject screening:

[0250] Female subjects aged 35-50 (n=60) with facial wrinkles classified as Fitzpatrick grade II-III were recruited.

[0251] Exclusion criteria: allergic constitution, history of skin disease, and use of anti-wrinkle products within 3 months prior to the test.

[0252] Students were randomly divided into two groups: the experimental group (n=30, using DNA-Na-containing essence) and the control group (n=30, using blank matrix without DNA-Na).

[0253] How to use:

[0254] Take 2mg / cm after cleansing your face every morning and evening. 2 Apply the serum evenly to the face and gently massage until absorbed.

[0255] Use continuously for 8 weeks, avoiding the use of other anti-wrinkle products during this period.

[0256] Detection indicators and methods:

[0257] Collagen content:

[0258] Detection method: ELISA (Human Collagen Type I ELISA Kit, Abcam).

[0259] Sampling: 3 mm skin biopsy samples were taken from the preauricular region of the subjects at week 0 and week 8.

[0260] Procedure: After digestion, the content of type I collagen in the sample was measured, and the results were expressed as μg / mg tissue.

[0261] Skin elasticity:

[0262] Testing instrument: Cutometer MPA580 (Courage+Khazaka).

[0263] Parameter settings: Test probe diameter 2mm, suction force 500mbar, measurement time 1s.

[0264] Evaluation metric: R2 value (elastic recovery rate), taken as the average value of both cheeks.

[0265] Wrinkle depth:

[0266] Testing instrument: Visioscan VC98 (Courage+Khazaka).

[0267] Detection area: frown lines (deepest vertical wrinkles).

[0268] Analysis method: Wrinkle depth (μm) was measured using SEDERM software.

[0269] Test Results

[0270] Changes in collagen content:

[0271] Experimental group: Collagen content increased from (25.4±3.1) μg / mg at baseline to (35.3±4.2) μg / mg, an increase of 39% (p<0.01).

[0272] Control group: The concentration increased from (24.9±2.8) μg / mg to (27.1±3.0) μg / mg, an increase of 8.8% (not statistically significant).

[0273] Improved skin elasticity:

[0274] Experimental group: R2 value increased from 0.42±0.05 at baseline to 0.55±0.06, an increase of 31%.

[0275] Control group: R2 value increased from 0.41±0.04 to 0.44±0.05, an increase of 7.3%.

[0276] Wrinkle depth reduced:

[0277] Experimental group: The depth of glabellar lines decreased from (48.6±7.2) μm to (36.9±5.8) μm, a reduction of 24%.

[0278] Control group: decreased from (47.9±6.5) μm to (45.2±6.1) μm, a reduction of 5.6%.

[0279] Statistical analysis:

[0280] Paired t-tests were used to compare differences within groups, and independent samples t-tests were used to compare differences between groups.

[0281] All p-values ​​were <0.01, indicating that the differences between the experimental group and the control group were highly statistically significant.

[0282] Key conclusions

[0283] Significant anti-wrinkle effects:

[0284] The serum containing 0.6% salmon DNA-Na significantly increased skin collagen content (+39%) and elasticity (+31%) within 8 weeks, and reduced wrinkle depth (-24%), with better results than the blank matrix.

[0285] Security verification:

[0286] No allergic or irritant reactions were observed in any of the subjects during the testing period, indicating that this concentration of DNA-Na has good safety in cosmetics.

[0287] Mechanism of action support:

[0288] The experimental results were highly consistent with the in vitro activity data of Example 6 (a 41% increase in collagen mRNA expression), further verifying that DNA-Na exerts its anti-wrinkle effect by stimulating fibroblasts to synthesize collagen.

[0289] This efficacy test shows that the salmon extract DNA-Na prepared by this process has a significant anti-wrinkle effect as an active ingredient in cosmetics, providing clinical evidence for its application in high-end skincare products.

[0290] Comparative Example 1: Traditional enzymatic hydrolysis process (without chitosan-encapsulated complex enzyme)

[0291] Step 1: Raw material pretreatment

[0292] Raw material preparation:

[0293] Take 20 kg of frozen rainbow trout testes (stored at -20℃ and frozen within 48 hours after catch), thaw them, add 2 times the volume (40 L) of 40 mmol / L Tris-water solution (pH 7.5), and use a high-speed tissue homogenizer (10000 rpm) to homogenize for 10 min.

[0294] Cell lysis:

[0295] Add 0.2% (w / v) Triton X-100 (80g), adjust the pH to 7.5 with 0.5mol / L NaOH, and stir at 25℃ for 8 hours to promote cell lysis and release DNA.

[0296] Step 2: Enzymatic hydrolysis

[0297] Enzymatic hydrolysis conditions:

[0298] After adjusting the homogenate to pH 7.5, add free trypsin (250 KU / L, 20 L volume, 5000 KU total activity) and digest at 37°C with stirring for 24 h.

[0299] Process monitoring:

[0300] Samples were taken every 4 hours, and enzyme activity was detected using the Folin-Ciocalteu method. It was observed that after 24 hours, the enzyme activity was only 45% of the initial value (112.5 KU / L).

[0301] Test results

[0302] Protein residue:

[0303] The protein content of the enzymatic hydrolysate determined by BCA method was 4.2 ± 0.3% (n=3), which was significantly higher than 0.87% in Example 1.

[0304] DNA fragment distribution:

[0305] Gel electrophoresis showed that the main fragments were in the range of 50-100 kDa, with only 65% ​​below 20 kDa, indicating a wide molecular weight distribution.

[0306] Enzyme activity attenuation:

[0307] The enzyme activity residual rate was 45% after 24 hours, indicating that the free enzyme has poor stability and requires frequent enzyme replenishment to maintain reaction efficiency.

[0308] Comparative Example 2: Single ultrasonic fragmentation process (without high-pressure homogenization synergy)

[0309] Step 1: Pretreatment of enzymatic hydrolysate

[0310] Dilution and Adjustment:

[0311] Take 20 L of the enzyme hydrolysate from Comparative Example 1, add an equal volume of 40 mmol / L Tris-water solution (pH 7.5), add 0.1% Triton X-100 (20 g), and adjust the pH to 7.5 with 0.5 mol / L HCl.

[0312] Step 2: Ultrasonic fragmentation

[0313] Equipment and parameters:

[0314] A 20kHz constant frequency ultrasonic disruptor (500W power) was used, with an ultrasonic mode of 5 seconds of operation followed by 1 second of pause. The total processing time was 40 minutes, and the temperature was controlled at 20℃±5℃ using circulating cooling water.

[0315] Step 3: Separation and Purification

[0316] Subsequent processes:

[0317] The treatment was carried out according to the steps in Example 3, including centrifugation (9000 rpm, 20 min), ceramic membrane filtration (1 μm), nanofiltration (200 Da molecular weight cutoff), and ultrafiltration (5 kDa).

[0318] Test results

[0319] Purity index:

[0320] OD260 / OD280 = 1.65 ± 0.03 (n = 3), indicating protein contamination; BCA method protein residue was 0.075 ± 0.005%, higher than 0.026% in Example 3.

[0321] Molecular weight distribution:

[0322] Size exclusion chromatography showed that DNA with a content below 20 kDa accounted for 82.3 ± 1.2%, and DNA with a content above 50 kDa accounted for 4.8 ± 0.5%, which is significantly different from 92.5% and <1.5% in Example 2.

[0323] Crushing efficiency:

[0324] The average DNA fragment length was 120 bp, which is 50% longer than the 80 bp in Example 2, indicating that the fragmentation effect was insufficient.

[0325] Comparative Example 3: Traditional column chromatography purification (butyl hydrophobic packing material)

[0326] Step 1: Sample Pretreatment

[0327] Solution preparation:

[0328] Take 10 L of the crude DNA solution obtained in Comparative Example 2 (concentration approximately 0.5 g / L), add an equal volume of 20 mmol / L NaCl solution (10 L), and dilute the final NaCl concentration to 10 mmol / L.

[0329] Filtration and impurity removal:

[0330] The mixed solution was filtered under reduced pressure through a 0.45 μm cellulose acetate membrane (142 mm in diameter) to remove insoluble impurities and obtain a clear filtrate.

[0331] Step 2: Hydrophobic chromatographic separation

[0332] Column packing:

[0333] A stainless steel column (5 cm inner diameter, 20 cm column height, 500 mL column volume) was packed with Proteomix® HICButyl hydrophobic chromatographic packing material (30 μm particle size, 100 Å pore size) at a packing pressure of 1.5 MPa. The measured column efficiency was 6000 theoretical plates / m.

[0334] Balancing and Sample Loading:

[0335] Equilibration buffer: 20 mmol / L NaCl (pH 7.8), equilibrate for 5 column volumes (CV) at a linear velocity of 2 cm / min.

[0336] Sample loading conditions: Load the pretreated sample at a linear velocity of 0.5 cm / min, with a sample loading volume of 1.5 column volume (750 mL).

[0337] Step 3: Gradient elution

[0338] Washing procedure:

[0339] The same gradient elution program as S34 was used, but the elution linear velocity was fixed at 1 cm / min (not dynamically adjusted based on UV monitoring values):

[0340] 0-10CV: 20 mmol / L NaCl (pH 7.8) → 20 mmol / L NaCl + 1.5 mol / L (NH4)2SO4 (pH 7.8) linear gradient.

[0341] 10-15CV: Isocratic elution with 20 mmol / L NaCl + 1.5 mol / L (NH4)2SO4 (pH 7.8).

[0342] 15-20CV: Rinse with pure water.

[0343] Monitoring and collection:

[0344] The elution curve was monitored by an ultraviolet detector (260 nm), and the main peak portion (retention time approximately 8-12 CV) was collected.

[0345] Step 4: Freeze-drying and shaping

[0346] Concentration and Dialysis:

[0347] The collected eluent was concentrated to 500 mL using a 10 kDa molecular weight cutoff ultrafiltration membrane (GE Healthcare), and then desalted by dialyzing with 10 mmol / L Tris-HCl buffer (pH 7.5).

[0348] Freeze-drying process:

[0349] The process was carried out according to the freeze-drying conditions of Example 4:

[0350] Pre-freezing: -45℃, 2.5h, collector temperature ≤-65℃.

[0351] Sublimation: -25℃ for 12 hours → -10℃ for 12 hours → 15℃ for 12 hours → 35℃ for 26 hours.

[0352] Secondary drying: 42℃, vacuum degree ≤8Pa, drying for 3h, PAT monitoring until the pressure rise rate is ≤0.4Pa / h.

[0353] Test results

[0354] Separation efficiency:

[0355] Column efficiency change: When the sample loading volume is 1.5 BV, the column efficiency decreases from the initial 6000 / m to 4800 / m (a decrease of 20%).

[0356] Peak shape parameters: The target peak tailing factor (As) is 1.8 (1.2 in the core-shell packing in Example 3), and the half-peak width (W1 / 2) is increased by 35%.

[0357] Yield and purity:

[0358] Overall yield: 40 kg of rainbow trout testes were fed, and 16.2 g of freeze-dried product was obtained, with a yield of 40.5% (43.27% in Example 3).

[0359] Purity index:

[0360] Protein residue: 0.048 ± 0.002% (BCA method), higher than 0.026% in Example 3.

[0361] DNA molecular weight distribution: 85.7 ± 0.9% of DNA molecules are below 20 kDa, and 3.2 ± 0.4% of DNA molecules are above 50 kDa (the corresponding values ​​in Example 3 are 92.5% and < 1.5%).

[0362] Process stability:

[0363] Continuous purification test: After five consecutive purifications, the sample loading capacity of the packing material decreased from 1.5 BV to 1.0 BV (a decrease of 33%), requiring regeneration with 2M NaCl + 20% ethanol solution.

[0364] Service life: After 10 repeated uses, the column efficiency dropped to 65% of the initial value (3900 / m), while the core-shell packing of Example 3 still maintained a column efficiency of over 90%.

[0365] Key Difference Analysis

[0366] index Comparative Example 3 (Butyl Hydrophobic Filler) Example 3 (Core-shell packing material) Explanation of differences Sample volume (BV) 1.5 (decreased to 1.0) 3.0 (Stable) Core-shell structure improves mass transfer efficiency Column efficiency (theoretical plates / m) 4800 (after sample loading) 8500 (stable) Core-shell packing materials have stronger resistance to contamination. Target peak tailing factor 1.8 1.2 Butyl packing material exhibits non-specific adsorption. Percentage of DNA with a DNA level below 20kDa (%) 85.7±0.9 92.5±0.7 Higher separation selectivity Sample load reduction after 5 cycles 33% ＜5% Core-shell packing has a longer service life

[0367] in conclusion:

[0368] Traditional butyl hydrophobic packing material is significantly inferior to the core-shell chromatographic packing material in Example 3 in terms of sample loading capacity, separation efficiency, and service life. The fixed flow rate elution strategy failed to dynamically adjust according to actual separation conditions, further exacerbating peak broadening and impurity residue problems. This indicates that the core-shell packing material and its accompanying intelligent elution strategy employed in this invention play a crucial role in improving DNA purification efficiency and process stability.

[0369] Comparative Example 4: Unoptimized freeze-drying process (without pressure rise test)

[0370] Step 1: Pre-freezing treatment

[0371] Sample preparation:

[0372] Take the high-purity DNA-Na solution (concentration 10 mg / mL, OD260 / OD280=1.90) prepared in Example 3 and dispense it into 50 lyophilized bottles (10 mL per bottle, 500 mL in total).

[0373] Pre-freezing conditions:

[0374] Place the freeze-drying flasks in the drying chamber of the Labconco FreeZone 2.5L freeze dryer, set the cold trap temperature to -55°C, and cool down to -40°C at a rate of 5°C / min, maintaining this temperature for 2 hours. Set the collector temperature to -55°C and the vacuum degree to ≤10Pa.

[0375] Step 2: Sublimation drying

[0376] Heating program:

[0377] The temperature should be increased according to the following gradient, while maintaining a vacuum level of ≤10 Pa throughout the process:

[0378] Phase 1: Increase the temperature from -40℃ to -25℃ at a rate of 0.5℃ / min and maintain the temperature for 10 hours.

[0379] Phase 2: Increase the temperature to -10℃ at a rate of 0.3℃ / min and maintain for 10 hours.

[0380] Phase 3: Increase the temperature to 15℃ at a rate of 0.5℃ / min and maintain for 10 hours.

[0381] Phase 4: Increase the temperature to 35℃ at a rate of 1℃ / min and maintain for 20 hours.

[0382] Step 3: Secondary drying

[0383] Final dehydration:

[0384] Raise the temperature to 40°C, maintain the vacuum at 10 Pa, and dry for 2 hours.

[0385] Termination condition:

[0386] Pressure rise test (PAT) was not used to monitor residual moisture; the drying endpoint was controlled solely by time. After drying, nitrogen gas was introduced, and the container was sealed with a stopper.

[0387] Test results

[0388] Moisture content determination:

[0389] Detection method: Karl Fischer volumetric method (Metrohm 870KFTitrino).

[0390] Results: The residual moisture content of the freeze-dried product was 1.3±0.1% (n=5), which was significantly higher than that of Example 4 (0.8±0.05%).

[0391] Reconstitution assessment:

[0392] Procedure: Take 100 mg of lyophilized product, add 10 mL of ultrapure water, and shake to dissolve at room temperature for 10 min.

[0393] Changes in purity:

[0394] Before freeze drying, OD260 / OD280 = 1.90 ± 0.01.

[0395] After reconstitution, OD260 / OD280 = 1.82 ± 0.02, and the OD value decreased by 4% from 1.90 before lyophilization to 1.82, indicating slight degradation of nucleic acid or increased protein contamination.

[0396] Accelerated stability testing:

[0397] Conditions: Place the freeze-dried product in an environment of 40℃ and 75% relative humidity (RH75%) for 1 month for aging.

[0398] Molecular weight change:

[0399] Initially, DNA with a content below 20 kDa accounted for 91.5 ± 0.6% (data from Example 3).

[0400] After aging, the degradation rate decreased to 87.2 ± 0.8%, and the degradation rate was 4.7% ((91.5-87.2) / 91.5×100%), which was higher than the 3.1% in Example 4.

[0401] Comparison of key process parameters

[0402] index Comparative Example 4 (Unoptimized freeze-drying) Example 4 (Optimized freeze-drying) Difference analysis Pre-freezing rate (°C / min) 5 3 Rapid cooling may form large ice crystals. Total sublimation time (h) 50 48 The time was similar but the temperature gradient was different. Secondary drying temperature (°C) 40 42 Temperature slightly lower but lack of PAT monitoring Secondary drying time (h) 2 3 Insufficient time leads to high moisture residue Pressure rise test none Yes (≤0.4Pa / h) Unable to determine the drying endpoint in real time Final moisture content (%) 1.3±0.1 0.8±0.05 Residual moisture affects stability Degradation rate at 40℃ (%) 4.3 3.1 Moisture promotes nucleic acid hydrolysis and oxidation

[0403] in conclusion:

[0404] Unoptimized freeze-drying processes, lacking pressure rise testing (PAT) monitoring, lead to inaccurate determination of the drying endpoint and higher residual moisture content. Higher moisture content accelerates DNA degradation during storage, manifested as a decrease in the OD260 / OD280 ratio and changes in molecular weight distribution. Example 4, by optimizing the pre-freezing rate, adjusting the temperature gradient, and combining PAT monitoring, effectively reduced residual moisture and improved product stability. This demonstrates that PAT technology plays a crucial role in controlling the freeze-drying process endpoint and ensuring consistent product quality.

[0405] Comparative Example 5: Traditional process without enzyme recovery

[0406] Step 1: Enzymatic hydrolysis (for unrecovered chitosan microspheres)

[0407] Raw material processing:

[0408] Take 50 kg of fresh rainbow trout testes and treat them according to steps 1.1 and 1.2 in Example 1. After obtaining a homogenate, adjust the pH to 7.5.

[0409] Enzymatic hydrolysis reaction:

[0410] Add chitosan-embedded complex enzymes (trypsin:papain = 3:1, total activity 1000 KU), and stir at 37°C for 12 h.

[0411] Centrifugal separation:

[0412] After enzymatic hydrolysis, centrifuge directly (9000 rpm, 20 min), discard the precipitate (containing unrecovered chitosan microspheres), and retain the supernatant for subsequent purification.

[0413] Steps 2 to 4: Subsequent Processes

[0414] Crushing and Purification:

[0415] Following the steps in Example 2, ultrasonic-high pressure homogenization was performed, followed by centrifugation, ceramic membrane filtration, and hydrophobic chromatography purification following the steps in Example 3.

[0416] Freeze-drying:

[0417] The DNA-Na lyophilized product was obtained by performing freeze-drying according to the steps in Example 4.

[0418] Test results

[0419] Cost analysis:

[0420] Enzyme consumption:

[0421] The cost of directly adding fresh enzyme preparations per batch is 2,850 yuan (the cost drops to 1,965 yuan after recycling in Example 5), an increase of 45%.

[0422] Total cost comparison:

[0423] Single batch production cost (enzyme + consumables):

[0424] Comparative Example 5: Product priced at 4230 yuan / kg.

[0425] Example 5: Product priced at 2910 yuan / kg, saving 31.2%.

[0426] Environmental indicators:

[0427] Solid waste generation:

[0428] Solid waste generated per 1 kg of DNA-Na produced:

[0429] Comparative Example 5: 5.2 kg (including unrecovered chitosan microspheres).

[0430] Example 5: 2.9 kg (chitosan recovery rate 85%), a reduction of 44.2%.

[0431] Wastewater discharge:

[0432] Wastewater COD value: Comparative Example 5 was 12800 mg / L, Example 5 was 8600 mg / L, a reduction of 32.8%.

[0433] Long-term stability:

[0434] Continuous batch testing:

[0435] Five consecutive production batches were conducted, with 50 kg of testes fed into each batch.

[0436] batch Yield (%) Protein residue (%) Batch 1 42.8 0.031 Batch 5 38.7 0.042

[0437] Performance changes:

[0438] Compared with Example 5, the decrease in yield (9.6% vs 2.1%) and the increase in protein residue (35.5% vs 11.5%) were both significantly greater.

[0439] Key Difference Analysis

[0440] index Comparative Example 5 (unrecovered enzyme) Example 5 (Enzyme Recovery) Explanation of differences Enzyme preparation cost (RMB / kg) 2850 1965 Chitosan microspheres can be recycled 5 times. Solid waste generation (kg / kg) 5.2 2.9 Recycling chitosan reduces waste Yield decreased after 5 batches 9.6% 2.1% Loss of enzyme activity leads to incomplete degradation Increase in protein residue after 5 batches 35.5% 11.5% Decreased enzyme activity affects protein removal efficiency Wastewater COD (mg / L) 12800 8600 Reduce enzyme protein residue contamination

[0441] in conclusion:

[0442] Traditional processes that do not recover chitosan-encapsulated enzymes are inferior to the enzyme recovery process in Example 5 in terms of cost, environmental impact, and process stability. Recycling chitosan microspheres can reduce enzyme consumption costs, decrease solid waste and wastewater discharge, and ensure stable enzyme activity, thereby guaranteeing production efficiency and product purity. This application constructs a green and efficient preparation system through chitosan encapsulation, ultrasonic-homogenization, and PAT monitoring, achieving breakthroughs in molecular weight control, purity, cost, and environmental protection, providing a new path for the industrialization of natural nucleic acid raw materials.

[0443] Comparative Example 6: Validation of the synergistic effect of complex enzyme encapsulation vs. single enzyme encapsulation

[0444] Step 1: Raw material processing

[0445] Take 20 kg of frozen salmon testes, crush them at -25℃, add 40 L of mixed solution (containing 0.08 mol / L Tris-HCl, 0.2 mol / L NaOH, and 1.0 mol / L NaCl), stir magnetically at 300 rpm for 30 min, and stir evenly in an ice-water bath at a temperature controlled at ≤10℃.

[0446] Step 2: Enzyme preparation

[0447] 1. Complex enzyme encapsulation group: Complex enzyme encapsulation group:

[0448] Chitosan solution preparation: Dissolve 20g of chitosan in 1% (w / v) acetic acid solution to prepare 1000mL of 2% (w / v) chitosan solution. Stir magnetically at 500rpm for 4h until clear, and sterilize with a 0.45μm filter membrane.

[0449] Mixing of the complex enzymes: Weigh out 45g of trypsin, 15g of neutral protease, 20g of papain, 20g of proteinase K, 15g of NSP enzyme, 8g of cellulase, and 4g of β-1,3-glucanase according to the weight proportions, dissolve them in 500mL of pH7.8 Tris-HCl buffer, and sonicate at 100W for 5min (sonicate for 5s, pause for 1s) to mix well.

[0450] Ionic crosslinking: The enzyme-chitosan mixture (enzyme solution: chitosan solution = 1:2 volume ratio) was added dropwise to 0.3% (w / v) TPP solution (containing 0.15 mol / L NaCl) at a flow rate of 2 mL / min. The mass ratio of chitosan to TPP was 5:1. The mixture was magnetically stirred at 500 rpm for 30 min to form microspheres of 5-10 μm.

[0451] Microsphere collection: Centrifuge at 4000 rpm for 10 min, wash 3 times with pH 7.8 Tris-HCl buffer, and vacuum dry at 40℃ for 10 h (vacuum degree ≤ 10 Pa).

[0452] 2. Single enzyme encapsulation group: only 45g of trypsin was added, and the remaining encapsulation process was the same as that of the complex enzyme group (the chitosan solution concentration and cross-linking conditions were the same).

[0453] Step 3: Enzymatic hydrolysis reaction

[0454] Two groups were respectively added with enzyme-encapsulated microspheres (total enzyme activity 350 KU / L) to the raw material slurry, enzymatically hydrolyzed at 37±1℃ for 28h, and the pH was maintained at 7.8±0.1 by an automatic titration system and magnetically stirred at 300rpm.

[0455] After enzymatic hydrolysis, the enzyme was inactivated at 85℃ for 10 min, centrifuged at 4000 rpm for 20 min, and the supernatant was collected.

[0456] Step 4: Detection and Analysis

[0457] DNA yield determination:

[0458] Ultraviolet spectrophotometry (A260): Dilute the supernatant 100 times, measure the absorbance at 260 nm, and calculate the yield (mg / g raw material) according to DNA concentration (mg / mL) = 50 × A260 × dilution factor.

[0459] Complex enzyme group: 5.8±0.5mg / g, single enzyme group: 4.2±0.3mg / g (n=3, p<0.01).

[0460] Purity assessment:

[0461] The A260 / A280 ratio was determined to be 1.92±0.02 for the complex enzyme group and 1.75±0.03 for the single enzyme group (n=3, p<0.05).

[0462] Cell fragmentation rate detection:

[0463] Trypan blue staining method: Take the enzyme digest smear, count 100 cells under an optical microscope (1000×), and calculate the percentage of ruptured cells.

[0464] Complex enzyme group: 96±2%, single enzyme group: 70±3% (n=3, p<0.01).

[0465] Polysaccharide residue (DNS method):

[0466] Complex enzyme group: 0.12±0.01 mg / mL, single enzyme group: 0.35±0.02 mg / mL (n=3, p<0.01).

[0467] Comparative Example 7: Stability Comparison of Encapsulated vs. Free Complex Enzymes

[0468] Step 1: Enzyme preparation

[0469] 1. Composite enzyme encapsulation group: Encapsulated microspheres were prepared in the same manner as in comparative example 6;

[0470] 2. Free complex enzyme group: Prepare free complex enzyme solution with the same components and enzyme activity, and dissolve in pH 7.8 Tris-HCl buffer (free state).

[0471] Step 2: Enzymatic hydrolysis and recovery

[0472] Single enzymatic hydrolysis:

[0473] Two groups of 20 kg salmon testes were treated and enzymatically hydrolyzed at 37℃ for 28 h while maintaining pH 7.8. The residual enzyme activity and DNA yield were recorded after hydrolysis.

[0474] Continuous batch recycling:

[0475] Encapsulation group: After each enzymatic digestion, the microspheres were recovered by centrifugation at 4000 rpm, washed three times with 0.1 mol / L NaCl, and the enzyme activity was tested (if ≥ 70% of the initial value, the microspheres were reused). This process was repeated for 5 consecutive batches.

[0476] Free enzyme group: After each enzymatic digestion, the free enzyme was recovered through an ultrafiltration membrane (30kDa), and the activity of the recovered enzyme was measured. This was repeated for 5 batches.

[0477] Step 3: Detection and Analysis

[0478] Enzyme activity residual rate (Folin-Ciocalteu method):

[0479] After a single enzymatic digestion: 87±3% in the encapsulated group and 52±4% in the free group (n=3, p<0.01).

[0480] After 5 batches: the enzyme activity in the embedded group was 75±5%, while that in the free group was only 32±3% (n=3, p<0.01).

[0481] DNA yield:

[0482] Single enzymatic digestion: 5.6±0.4 mg / g in the encapsulated group and 4.1±0.3 mg / g in the free group (n=3, p<0.01).

[0483] After 5 batches: the yield of the embedded group decreased by 2.1%, and that of the free group decreased by 37.6% (n=3, p<0.01).

[0484] Protein residue (BCA method):

[0485] Embedded group: 0.031±0.002%, free group: 0.075±0.005% (n=3, p<0.01).

[0486] Microsphere size stability:

[0487] Laser particle size analyzer test: After 5 repetitions, the D90 of the embedded group was 39±2μm, which was not significantly different from the initial value (38±2μm) (p>0.05).

[0488] in conclusion

[0489] 1. Comparative Example 6 data showed that the DNA yield of the complex enzyme-encapsulated group (5.8 mg / g) was 38% higher than that of the single enzyme-encapsulated group (4.2 mg / g), the cell disruption rate increased from 70% to 96%, and the polysaccharide residue decreased by 66%. The synergistic effect of multiple enzymes (such as protease degradation of proteins and cellulase decomposition of cell walls) significantly improved DNA release efficiency, while the effect of single enzymes was poor due to substrate specificity limitations.

[0490] 2. Comparative Example 7 data showed that after a single enzymatic hydrolysis, the residual enzyme activity rate in the encapsulated group (87%) was 67% higher than that in the free group (52%); after 5 batches of reuse, the yield in the encapsulated group decreased by only 2.1%, while that in the free group decreased by 37.6%. Chitosan microspheres effectively delayed enzyme inactivation and degradation through physical isolation and structural protection, thus improving the efficiency of enzyme reuse.

[0491] Combined with Comparative Examples 6 and 7, the composite enzyme encapsulation technology simultaneously achieved high DNA yield (5.8 mg / g vs 4.2 mg / g), high purity (A260 / A280 = 1.92 vs 1.75), low impurity residue (polysaccharide 0.12 mg / mL vs 0.35 mg / mL) and long shelf life (yield decay of 2.1% vs 37.6% after 5 batches).

[0492] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing sodium DNA from salmon extract, characterized in that, Includes the following steps: S1. Raw material pretreatment: Frozen salmon or salmon testis tissue was lysed at -30 to -20℃, then a mixed solution containing Tris-HCl buffer, NaOH and NaCl was added, followed by the addition of chitosan-embedded complex enzyme with a total enzyme activity ≥350KU / L. The enzyme was then hydrolyzed at 37±1℃ for 26-30h, maintaining a pH of 7.8 to 8.0 to obtain the enzymatic hydrolysate. S2. DNA Synergistic Disruption: Adjust the pH of the enzymatic hydrolysate to 7.8, and in an ice-water bath at ≤10℃, synergistically treat it by alternating cycles of 25-35kHz variable frequency ultrasound and 100-120MPa high-pressure homogenization for 40 minutes to obtain DNA disruption solution. S3. Deproteinization and fractionation purification: After multiple centrifugations and filtrations, the DNA fragments were separated by nanofiltration, ultrafiltration and core-shell phenyl hydrophobic chromatography packing material, and a specific mobile phase gradient elution was used to collect the target fraction to obtain a high-purity DNA-Na solution. S4. Freeze-drying: After pre-freezing the high-purity DNA-Na solution at -45℃, freeze-dry it according to a specific temperature gradient, and then dry it twice while controlling the particle size D90≤40μm to obtain freeze-dried powder. S5. Enzyme recovery: Wash the precipitate obtained in S3 to recover chitosan microspheres. If the enzyme activity is ≥70% of the initial value, it can be reused for the next batch of enzymatic hydrolysis. The weight proportions of the compound enzyme preparation are: 40-50 parts trypsin, 10-20 parts neutral protease, 15-25 parts papain, 15-25 parts proteinase K, 12-18 parts NSP enzyme, 7-10 parts cellulase and 3-5 parts β-1,3-glucanase.

2. The method for preparing salmon DNA sodium raw material as described in claim 1, characterized in that, The preparation method of the chitosan-encapsulated complex enzyme in S1 is as follows: Chitosan was dissolved in 0.5–2% w / v acetic acid solution to prepare a 1%–3% w / v chitosan solution, and magnetically stirred until clear. A compound enzyme preparation was added in proportion, and the mixture was sonicated at 50–150 W for 3–10 min to form an enzyme-chitosan mixture. The mixture was then added dropwise to a 0.3% TPP solution, with a chitosan to TPP mass ratio of 3:1–8:1, while magnetically stirring at 300–800 rpm. The mixture was cross-linked at room temperature for 20–40 min to form chitosan microspheres with a particle size of 5–10 μm. The microspheres were then collected by centrifugation at 3000–5000 rpm for 5–15 min, washed 2–4 times with 7.5–8.0 Tris-HCl buffer, and vacuum dried at 30–50 °C for 6–15 h.

3. The method for preparing salmon DNA sodium raw material as described in claim 1, characterized in that, In the mixed solution of Tris-HCl buffer, NaOH and NaCl in S1, the concentration of Tris-HCl buffer is 50-100 mmol / L, the concentration of NaOH is 0.1-0.3 mol / L, and the concentration of NaCl is 0.5-1.5 mol / L.

4. The method for preparing salmon DNA sodium raw material as described in claim 1, characterized in that, The variable frequency ultrasonic power in S2 is 150-300W, the total processing time is 30-60min, the power is 200-300W for the first 15-30min and 100-200W for the last 15-30min; the pressure of high-pressure homogenization is 80-150MPa; the ultrasonic and homogenization cycles are repeated 2-6 times, with ultrasonic treatment lasting 3-8min after each homogenization; the ultrasonic working mode is ultrasonic for 3-8s and then paused for 1-3s.

5. The method for preparing salmon DNA sodium raw material as described in claim 1, characterized in that, In step S3, the DNA lysis buffer is centrifuged at pH 8.5-9.2 and 8000-11000 rpm for 15-25 min. The supernatant is filtered through a 0.5-2 μm ceramic membrane, the pH is adjusted to 7.5-8.0, and the mixture is allowed to stand for 6-10 h before being centrifuged a second time.

6. The method for preparing salmon DNA sodium raw material as described in claim 5, characterized in that, The nanofiltration system in S3 uses a spiral wound composite membrane made of polyamide with a molecular weight cutoff of 100–500 Da, an operating pressure of 0.15–0.3 MPa, a temperature of 15–25 °C, a cross-flow filtration membrane surface velocity of 1.5–2.5 m / s, and a 3–10 kDa ultrafiltration membrane after 3–8 hours of circulation.

7. The method for preparing salmon DNA sodium raw material as described in claim 1, characterized in that, The freeze-drying process in S4 is as follows: after pre-freezing at -50 to -40℃ for 2 to 3 hours, the temperature is increased to -30 to -20℃ at a rate of 1 to 2℃ / min and maintained for 10 to 14 hours, then increased to -15 to -5℃ and maintained for 10 to 14 hours, then increased to 10 to 20℃ and maintained for 10 to 14 hours, and finally increased to 30 to 40℃ and maintained for 24 to 30 hours; the secondary drying conditions are 40 to 45℃ and vacuum degree ≤10Pa, and the process ends when the pressure increase rate is ≤1Pa / h.

8. The method for preparing salmon DNA sodium raw material as described in claim 1, characterized in that, In step S5, the chitosan microspheres are washed with 0.05–0.2 mol / L NaCl solution, and the enzyme activity must be tested to be ≥ 70% of the initial value before reuse.

9. The application of a salmon extract DNA sodium raw material obtained by the preparation method according to any one of claims 1-8 in the cosmetic field, characterized in that, Used to prepare skin care products with anti-wrinkle, skin-firming, and skin-brightening effects.