Preparation method and application of salmon salmon sperm DNA sodium raw material

Abstract

The invention discloses a preparation method and application of a salmon salmon sperm DNA sodium raw material, and belongs to the technical field of biomedicines.The preparation method comprises the following steps that S1, raw material pretreatment is conducted, specifically, after frozen salmon or salmon testis tissue is crushed at the ultralow temperature of-30 DEG C to-20 DEG C, a mixed solution containing a Tris-HCl buffer solution, NaOH and NaCl is added, and a mixed solution is obtained; adding a chitosan embedded compound enzyme with the total enzyme activity of more than or equal to 350KU / L, carrying out enzymolysis at 37 + / -1 DEG C for 26-30 hours, and maintaining the pH value to be 7.8-8.0, so as to prepare an enzymatic hydrolysate; s2, DNA synergistic crushing: adjusting the pH value of the enzymatic hydrolysate to 7.8, and in an ice-water bath of less than or equal to 10 DEG C, carrying out 25-35 kHz variable-frequency ultrasound and 100-120 MPa high-pressure homogenization alternate circulation for 3-5 times to carry out synergistic treatment for 40 minutes, so as to obtain a DNA crushing solution; the method solves the problems of high protein residue, non-uniform molecular weight distribution, low transdermal absorption rate, high cost, poor environmental protection property and the like in the traditional process extraction, and has the advantages of high product purity, high activity, low cost, greenness and environmental protection.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a preparation method of salmon sperm DNA sodium raw material and application thereof. Background Art

[0002] Salmon / Salmon Sperm DNA Sodium (DNA-Na), a natural low-molecular-weight DNA derivative, shares 98% base similarity with human DNA in its double-stranded helix structure, demonstrating unique biological activity in areas such as tissue repair and skin beauty. Research has shown that DNA-Na of varying molecular weights possess significantly differentiated application value: fragments <60 kDa are core functional ingredients in cosmetics due to their superior transdermal absorption (every 10 kDa decrease in molecular weight increases epidermal permeability by 1.8 times), while fragments between 350 and 500 kDa play a key role in tissue regeneration in the pharmaceutical industry. However, existing preparation technologies face multiple bottlenecks in meeting these requirements.

[0003] Traditional processes, while inexpensive, include physical disruption methods (such as ultrasound and high-pressure homogenization), but they can easily lead to uneven DNA strand breakage, resulting in molecular weight distributions that span over 10-fold, failing to meet the narrow molecular weight distribution requirements of cosmetics. Enzymatic methods, while capable of achieving a more precise molecular weight distribution, are expensive (US$8.6 per gram of product) and carry the risk of excessive endotoxin levels. The use of SDS during the deproteinization step results in wastewater COD levels as high as 12,000 mg / L, exceeding environmental standards by six times. Ethanol precipitation removes less than 30% of contaminants <10 kDa. These small molecules are a major cause of immune reactions, with clinical data demonstrating a 12.7% allergy rate in products containing contaminants, significantly higher than the 1.3% rate for pure products. Furthermore, the high proportion of organic solvents used in traditional processes (e.g., 73% of ethanol) not only increases safety risks but also results in a 15-20% loss of target product.

[0004] To address the above issues, this application achieves a breakthrough through the following innovations: using chitosan-encapsulated complex enzymes (including a multi-enzyme system such as trypsin and neutral protease) for enzymatic hydrolysis under mild conditions, combined with variable-frequency ultrasound and high-pressure homogenization for synergistic treatment, to achieve precise and controllable fragmentation of DNA fragments, with a molecular weight distribution span of less than 3 times. By combining a core-shell phenyl hydrophobic chromatography filler (the matrix is ​​an agarose-silica hybrid material) with dynamic axial compression column technology, residual impurities of less than 0.05% are achieved. At the same time, a 5kDa ultrafiltration membrane is combined with a nanofiltration system to effectively intercept the target molecular weight fragments. In addition, by abandoning the traditional SDS deproteinization process, the COD value of the wastewater is reduced by 81%; by recycling and reusing the enzyme preparation (reuse ≤5 times), the production cost is reduced by 42%; and the freeze-drying molding process uses a pressure rise test (PAT) to monitor the residual moisture in real time to ensure product stability and activity.

[0005] Therefore, there is an urgent need for a preparation method and application of salmon sperm DNA sodium raw material, which significantly reduces the safety risks of existing products and promotes the in-depth application of DNA-Na in the fields of precision medicine and functional cosmetics. Summary of the Invention

[0006] To this end, the present invention provides a preparation method of salmon sperm DNA sodium raw material and its application, so as to solve the problems in the prior art caused by high protein residue, uneven molecular weight distribution, low transdermal absorption rate, high cost and poor environmental protection in traditional extraction process.

[0007] In order to achieve the above object, the present invention provides the following technical solutions: According to a first aspect of the present invention, a method for preparing salmon sperm DNA sodium raw material is provided, comprising the following steps: S1. Raw material pretreatment: Frozen salmon or trout testis tissue was ultra-cryogenically crushed at -30 to -20°C, and then added with a mixture of Tris-HCl buffer, NaOH, and NaCl. A chitosan-encapsulated enzyme complex with a total enzyme activity of ≥350 kU / L was then added. The enzyme was hydrolyzed at 37 ± 1°C for 26-30 hours, maintaining a pH of 7.8 to 8.0, to prepare an enzymatic solution. S2. DNA fragmentation: Adjust the pH of the enzymatic solution to 7.8 and perform a synergistic treatment in an ice-water bath at ≤10°C using 25-35 kHz variable frequency ultrasound and 100-120 MPa high pressure homogenization for 40 min, alternating for 3-5 cycles, to obtain a DNA fragmentation solution. S3. Deproteinization and fractional purification: The DNA fragment was centrifuged and filtered multiple times, then separated by nanofiltration, ultrafiltration, and core-shell phenyl hydrophobic chromatography using a specific mobile phase gradient elution. The target fractions were collected to obtain a high-purity DNA-Na solution. S4. Lyophilization: The high-purity DNA-Na solution was pre-frozen at -45°C and lyophilized using a specific temperature gradient. Lyophilized powder was obtained by secondary drying with a controlled particle size (D90) of ≤40 μm. S5. Enzyme preparation recovery: Wash the precipitate obtained in S3 to recover chitosan microspheres. When the enzyme activity is ≥ 70% of the initial value, reuse it for the next batch of enzymatic hydrolysis.

[0008] Furthermore, the preparation method of the chitosan-embedded complex enzyme in S1 is: 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; the complex enzyme preparation was added in proportion, and ultrasonicated at 50-150W for 3-10 minutes to form an enzyme-chitosan mixture; the mixture was dropped into 0.3% TPP solution with a mass ratio of chitosan to TPP of 3:1-8:1, and magnetically stirred at 300-800 rpm. The mixture was cross-linked at room temperature for 20-40 minutes to form chitosan microspheres with a particle size of 5-10 μm, and then the microspheres were collected by centrifugation at 3000-5000 rpm for 5-15 minutes, washed 2-4 times with 7.5-8.0 Tris-HCl buffer, and vacuum dried at 30-50°C for 6-15 hours.

[0009] Furthermore, the components and weight proportions of the complex enzyme preparation are as follows: The weight proportions of the complex enzyme preparation are: 40-50 parts of trypsin, 10-20 parts of neutral protease, 15-25 parts of papain, 15-25 parts of proteinase K, 12-18 parts of NSP enzyme, 7-10 parts of cellulase and 3-5 parts of beta-1,3-glucanase.

[0010] Furthermore, 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.

[0011] Furthermore, the variable frequency ultrasonic power in S2 is 150-300W, the total processing time is 30-60min, and the power is 200-300W for the first 15-30min, and the power is 100-200W for the last 15-30min; the pressure of the high-pressure homogenization treatment is 80-150MPa; the ultrasound and homogenization are alternately cycled 2-6 times, and the ultrasound treatment is 3-8min after each homogenization; the ultrasound working mode is 3-8s ultrasound and 1-3s pause.

[0012] Furthermore, in the S3, the DNA fragmentation solution 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 solution is allowed to stand for 6-10 h and centrifuged a second time.

[0013] Furthermore, the nanofiltration system in S3 uses a polyamide rolled composite membrane with a molecular weight cutoff of 100-500Da, an operating pressure of 0.15-0.3MPa, a temperature of 15-25°C, a cross-flow filtration membrane surface flow rate of 1.5-2.5m / s, and passes through a 3-10kDa ultrafiltration membrane after circulating for 3-8h.

[0014] Furthermore, the freeze-drying procedure in S4 is as follows: pre-freeze at -50 to -40°C for 2 to 3 hours, then heat to -30 to -20°C at a rate of 1 to 2°C / min and maintain for 10 to 14 hours, then heat to -15 to -5°C and maintain for 10 to 14 hours, continue to heat to 10 to 20°C and maintain for 10 to 14 hours, and finally heat to 30 to 40°C and maintain for 24 to 30 hours; the secondary drying conditions are 40 to 45°C, vacuum degree ≤10Pa, and terminate when the pressure rise rate is ≤1Pa / h.

[0015] Furthermore, in S5, the chitosan microspheres are washed with a 0.05-0.2 mol / L NaCl solution, and the enzyme activity must be detected to be ≥ 70% of the initial value before reuse.

[0016] According to the second aspect of the present invention, there is provided an application of the salmon sperm DNA sodium raw material obtained by the above-mentioned preparation method in the field of cosmetics, for preparing skin care products with anti-wrinkle, skin firming and skin gloss improvement effects.

[0017] The present invention has the following advantages: 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°C. In S2, the enzymatic hydrolyzate is treated by alternating cycles of 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, and chitosan encapsulation improves enzyme stability and recovery rate. Ultrasound and high-pressure homogenization work together to effectively disrupt cells and refine DNA, shortening disruption time, improving DNA yield and purity, and reducing energy consumption.

[0018] 2. In the S3 deproteinization and fractionated purification step, multiple centrifugation and filtration steps are combined with nanofiltration, ultrafiltration, and core-shell phenyl hydrophobic chromatography with 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, and the chromatographic packing further purifies the product. This combination significantly improves the purity of DNA-Na and reduces the impact of impurities on subsequent applications. The resulting high-purity DNA-Na solution can be used in cosmetics, enhancing product stability and efficacy.

[0019] 3. In S5, the chitosan microspheres are washed and recovered. When the enzyme activity is ≥70% of the initial value, they are reused for the next batch of enzymatic hydrolysis. This technology reduces the cost of enzyme preparations. The chitosan-encapsulated complex enzyme can be recycled, reducing raw material waste. Furthermore, stable enzyme activity ensures consistent enzymatic hydrolysis reactions across batches, improving production stability and product quality uniformity, making the preparation process more economical and sustainable. DETAILED DESCRIPTION

[0020] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0021] The present invention provides a method for preparing salmon sperm DNA sodium raw material, comprising the following steps: S1. Raw material pretreatment: Frozen salmon or trout testis tissue was ultra-cryogenically crushed at -30 to -20°C, and then added with a mixture of Tris-HCl buffer, NaOH, and NaCl. A chitosan-encapsulated enzyme complex with a total enzyme activity of ≥350 kU / L was then added. The enzyme was hydrolyzed at 37 ± 1°C for 26-30 hours, maintaining a pH of 7.8 to 8.0, to prepare an enzymatic solution. S2. DNA fragmentation: Adjust the pH of the enzymatic solution to 7.8 and perform a synergistic treatment in an ice-water bath at ≤10°C using 25-35 kHz variable frequency ultrasound and 100-120 MPa high pressure homogenization for 40 min, alternating for 3-5 cycles, to obtain a DNA fragmentation solution. S3. Deproteinization and fractional purification: The DNA fragment was centrifuged and filtered multiple times, then separated by nanofiltration, ultrafiltration, and core-shell phenyl hydrophobic chromatography using a specific mobile phase gradient elution. The target fractions were collected to obtain a high-purity DNA-Na solution. S4. Lyophilization: The high-purity DNA-Na solution was pre-frozen at -45°C and lyophilized using a specific temperature gradient. Lyophilized powder was obtained by secondary drying with a controlled particle size (D90) of ≤40 μm. S5. Enzyme preparation recovery: Wash the precipitate obtained in S3 to recover chitosan microspheres. When the enzyme activity is ≥ 70% of the initial value, reuse it for the next batch of enzymatic hydrolysis.

[0022] The preparation method of the chitosan-embedded 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; the complex enzyme preparation was added in proportion, and ultrasonicated at 50-150W for 3-10 minutes to form an enzyme-chitosan mixture; the mixture was dropped into 0.3% TPP solution with a mass ratio of chitosan to TPP of 3:1-8:1, and magnetically stirred at 300-800 rpm. The mixture was cross-linked at room temperature for 20-40 minutes to form chitosan microspheres with a particle size of 5-10 μm, and then the microspheres were collected by centrifugation at 3000-5000 rpm for 5-15 minutes, washed 2-4 times with 7.5-8.0 Tris-HCl buffer, and vacuum dried at 30-50°C for 6-15 hours.

[0023] Among them, the weight parts of the complex enzyme preparation are: 40-50 parts of trypsin, 10-20 parts of neutral protease, 15-25 parts of papain, 15-25 parts of proteinase K, 12-18 parts of NSP enzyme, 7-10 parts of cellulase and 3-5 parts of beta-1,3-glucanase.

[0024] Wherein, 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.

[0025] Among them, the variable frequency ultrasonic power in S2 is 150~300W, the total processing time is 30~60min, and the power is 200~300W in the first 15~30min, and the power is 100~200W in the last 15~30min; the pressure of high-pressure homogenization treatment is 80~150MPa; ultrasound and homogenization are alternately cycled 2~6 times, and ultrasonic treatment is 3~8min after each homogenization; the ultrasonic working mode is ultrasound for 3~8s and stop for 1~3s.

[0026] In S3, the DNA fragmentation solution is centrifuged at 8000-11000 rpm for 15-25 minutes at pH 8.5-9.2. The supernatant is filtered through a 0.5-2 μm ceramic membrane, adjusted to pH 7.5-8.0, allowed to stand for 6-10 hours, and centrifuged a second time.

[0027] The nanofiltration system in S3 uses a polyamide rolled composite membrane with a molecular weight cutoff of 100-500Da, an operating pressure of 0.15-0.3MPa, a temperature of 15-25°C, a cross-flow filtration membrane surface flow rate of 1.5-2.5m / s, and passes through a 3-10kDa ultrafiltration membrane after circulating for 3-8h.

[0028] The freeze-drying procedure in S4 is as follows: pre-freeze at -50 to -40°C for 2 to 3 hours, then heat to -30 to -20°C at a rate of 1 to 2°C / min and maintain for 10 to 14 hours, then heat to -15 to -5°C and maintain for 10 to 14 hours, continue to heat to 10 to 20°C and maintain for 10 to 14 hours, and finally heat to 30 to 40°C and maintain for 24 to 30 hours; the secondary drying conditions are 40 to 45°C, vacuum degree ≤10Pa, and terminate when the pressure rise rate is ≤1Pa / h.

[0029] In S5, chitosan microspheres were washed with 0.05-0.2 mol / L NaCl solution, and the enzyme activity was tested to be ≥ 70% of the initial value before reuse.

[0030] The present invention provides an application of the salmon sperm DNA sodium raw material obtained by the above-mentioned preparation method in the field of cosmetics, for preparing skin care products with anti-wrinkle, skin firming and skin gloss improvement effects.

[0031] Example 1 Pretreatment of salmon testis raw materials, preparation of chitosan-embedded complex enzyme and enzymatic hydrolysis process 1.1 Raw material pretreatment Deep-sea salmon testis raw materials stored in a -42°C freezer are taken and transferred to an ultra-low temperature crusher at a -28°C environment. They are crushed until they form a slurry, ensuring that the particle size of the crushed material does not exceed 5mm. Subsequently, a specific mixed solution is added to the crushed slurry material in a volume ratio of 1:3 between the testis and the mixed solution. The composition of the mixed solution is: 0.08mol / LTris-HCl buffer (pH 7.8), 0.2mol / LNaOH, and 1mol / L NaCl. After adding the mixed solution, the material is placed in a stirring device and stirred continuously at 300rpm for 30 minutes. At the same time, the temperature is controlled at ≤10°C in an ice bath to complete the pretreatment of the raw materials.

[0032] 1.2 Preparation of chitosan-embedded complex enzyme Preparation of chitosan solution: Weigh chitosan with a degree of deacetylation ≥90% and a viscosity within the range of 300 mPa·s, 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 h until clear, and sterilize through a 0.45 μm filter membrane.

[0033] Preparation of complex enzyme solution: Weigh 45 portions of trypsin (enzyme activity ≥ 250 USPU / mg), purchased from Novozymes (China) Investment Co., Ltd., numbered Trypsin 6.0S; Neutral protease 15 parts (enzyme activity ≥ 10000 U / g), purchased from Novozymes (China) Investment Co., Ltd., model: Neutrase 0.8L; Papain (20 parts) (enzyme activity ≥ 80,000 U / g) was purchased from Nanning Pangbo Bioengineering Co., Ltd., product number PB-Papain-80K; Proteinase K (20 units, activity ≥ 30 U / mg), derived from Tritirachiumalbum, purchased from Sigma-Aldrich, product number V900887; 12 parts of NSP enzyme (β-glucanase activity ≥ 5000 U / g), purchased from Megazyme, product number E-NSPENZ; 8 parts of cellulase (enzyme activity ≥ 1000 U / g), purchased from Novozymes (China) Investment Co., Ltd., model: Celluclast 1.5L; 4 parts of β-1,3-glucanase (enzyme activity ≥500 U / g), purchased from Megazyme, product number E-BGLUC.

[0034] Dissolve in 50 mmol / L Tris-HCl buffer (pH 7.8) and stir at 200 rpm for 15 min until homogeneous.

[0035] Enzyme-Chitosan Mixing: Slowly add the complex enzyme solution dropwise to the chitosan solution at a volume ratio of 1:2, at a rate of 20 mL / min, while magnetically stirring at 300 rpm. After addition, transfer the mixture to an ultrasonic processor and sonicate at 100 W for 5 minutes. The sonication process uses a pulsed mode (5 seconds of sonication followed by a 1-second pause) in an ice-water bath maintained at ≤25°C.

[0036] Ionic crosslinking: Prepare a 0.3% (w / v) TPP solution as a crosslinker and add 0.15 mol / L NaCl. Add the ultrasonically treated mixture dropwise to the TPP solution at a chitosan:TPP mass ratio of 5:1 using a peristaltic pump at a flow rate of 2 mL / min. During the addition, stir continuously at 500 rpm using a magnetic stirrer for 30 minutes to allow for sufficient ionic crosslinking between the chitosan and TPP, forming 5-10 μm chitosan microspheres.

[0037] Post-processing: After the cross-linking reaction is completed, the mixed solution is transferred to a centrifuge and centrifuged at 4000 rpm for 10 minutes to collect the precipitated microspheres. The microspheres are resuspended and washed with 50 mmol / L Tris-HCl buffer with a pH value of 7.8, and this operation is repeated three times. The washed microspheres are placed in a vacuum drying environment at 40°C and a vacuum degree of -0.09 MPa for 10 hours. After drying, the microspheres are ground and passed through an 80-mesh sieve. Microspheres with a particle size range of 5-10 μm are collected, which are the finished chitosan-encapsulated complex enzymes.

[0038] 1.3 Enzymatic hydrolysis process 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 enzyme complex (total enzyme activity ≥ 350 kU / L) prepared in step 1.2 was added to this solution at a concentration of 0.5% (w / v). The solution was maintained in a 0.08 mol / L Tris-HCl buffer (pH 7.8). The reaction system was maintained at a constant temperature of 37 ± 1°C and magnetic stirring 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 automated titration system to maintain a strict pH control within the range of 7.8 ± 0.1. After the enzymatic hydrolysis reaction, the reaction system was heated to 85°C and maintained for 10 minutes to terminate the reaction. The mixture was then centrifuged at 4000 rpm for 20 minutes, and the supernatant was collected. The DNA yield was measured to be 2.3 mg / g testis, the residual protein content was ≤0.5%, and the DNA purity (A260 / A280) in the enzymatic hydrolyzate was 1.85.

[0039] This example verified that the DNA purity in the enzymatic hydrolyzate reached 1.85 (close to the theoretical value of 1.8), and the residual protein content was ≤0.5%, demonstrating the efficient decomposition ability of the chitosan-encapsulated complex enzyme and the protective effect of the mild enzymatic hydrolysis conditions on DNA, providing high-quality raw materials for subsequent DNA co-fragmentation and purification.

[0040] Example 2 DNA collaborative fragmentation process 2.1 pH adjustment and temperature control The pH of the enzymatic hydrolysate obtained in Example 1 was adjusted to 7.8 using 0.5 mol / L NaOH, and then the solution was placed in an ice water bath to ensure that the temperature was ≤ 8° C., creating suitable environmental conditions for the subsequent collaborative crushing process.

[0041] 2.2 Alternating treatment of variable frequency ultrasound and high pressure homogenization 25-35kHz variable frequency ultrasound and 110MPa high pressure homogenization were used in alternating cycles for 4 times, with a total treatment time of 40 minutes. The specific steps are as follows: Loop 1: First, a high-pressure homogenizer was used to homogenize at a pressure of 110 MPa for 3 min to break up the cell structure by shear force; Then, ultrasonic treatment was performed using 30kHz variable frequency ultrasound, the power was set to 250W, the ultrasonic mode was 5 seconds of work and 1 second of rest, and the treatment lasted for 5 minutes.

[0042] Loop 2-4: The steps of cycle 1 were repeated, i.e., high-pressure homogenization (110 MPa) followed by ultrasonic treatment (250 W, 5 s operation + 1 s rest, 5 min).

[0043] After 20 minutes of treatment: The ultrasonic power was adjusted to 120 W, and the treatment was continued in the same ultrasonic mode (5 s working + 1 s rest) for 20 min.

[0044] 2.3 Crushing effect detection After the crushed liquid was filtered through a 0.45μm filter membrane, the following results were obtained: DNA concentration: 3.2 mg / mL; A260 / A280 ratio: 1.82; Electron microscopy observation: cell fragmentation rate ≥95%, DNA fragment size mainly distributed in the range of 50 to 200 bp.

[0045] Example 3 Deproteinization and fractional purification process 3.1 Centrifugal pretreatment First centrifugation: Adjust the pH of the DNA fragment to 8.8 with 0.5 mol / L NaOH and centrifuge at 9000 rpm for 20 minutes. Discard the precipitate and retain the supernatant. This step primarily removes insoluble impurities and some denatured proteins.

[0046] Second centrifugation: The supernatant was adjusted to pH 7.8 with 0.5 mol / L HCl and allowed to stand at 4°C for 8 h to allow protein precipitation. The supernatant was then centrifuged at 9000 rpm for 20 min, and the supernatant was collected as a pretreatment solution for further removal of protein and other impurities. The results showed an A260 / A280 ratio of 1.75, a DNA concentration of 2.8 mg / mL, and residual protein ≤1.5%.

[0047] 3.2 Membrane separation system Ceramic membrane filtration: The pretreatment liquid is filtered through a 1μm ceramic membrane to remove particulate impurities and macromolecular substances, ensuring the smooth progress of the subsequent nanofiltration process.

[0048] Nanofiltration system treatment: 1. Adjust the pH of the filtrate to 8.8, the membrane inlet pressure to 0.19 MPa, the temperature to 20±1°C, the membrane surface flow rate to 1.8 m / s, and circulate for 5 hours.

[0049] Ultrafiltration and top wash: Filter through a 5 kDa ultrafiltration membrane and top-wash with a 20 mmol / L sodium dihydrogen phosphate solution at pH 7.0 until the conductivity is ≤10 μS / cm; The combined filtrates were adjusted to pH 7.8 to obtain a crude product solution.

[0050] Detection: A260 / A280=1.82, DNA recovery rate 92%, salt content ≤0.1%.

[0051] 3.3 Core-shell chromatographic separation Sterilization treatment: The crude product solution was sterile filtered through a 0.22 μm polyethersulfone membrane to remove microorganisms in the solution and ensure the purity of the subsequent chromatographic separation process.

[0052] Chromatographic separation: The core-shell phenyl hydrophobic chromatography filler (agarose-silica hybrid matrix, specific surface area 350m 2 / g, pore volume 1.2 mL / g), packed in a dynamic axial compression column (DAC column).

[0053] The column pressure was controlled to be 0.28 MPa, the sample loading volume was 0.9 column volumes (BV), and the sample loading linear velocity was 0.5 cm / min.

[0054] Gradient elution program: Balance: mobile phase A:B = 1:1 (A is 100 mmol / L NaCl solution, B is ultrapure water), 2BV; Elution 1: A:B = 1:2, 2BV; Elution 2: A:B:C = 1:2:1 (C is 20 mmol / L potassium hydrogen phosphate solution, pH 8.0), 2 BV; Elution 3: A:B:C=1:1:2, 1BV; Target elution: B:D=5:1 (D is 0.1mol / LNaOH solution), 2BV; During the elution process, the UV260nm absorbance was monitored online. When the absorbance was greater than 0.5, the flow rate was automatically reduced to 6BV / h, and the target fraction was collected to obtain a high-purity DNA-Na solution.

[0055] Target fraction detection: A260 / A280=1.92; DNA concentration 1.5 mg / mL; Protein residue ≤ 0.1%; Endotoxin levels were <0.5 EU / mL.

[0056] Example 4 Freeze-drying molding process 4.1 Pre-freezing treatment Transfer the high-purity DNA-Na solution obtained in Example 3 to the feed tray of the freeze dryer, ensuring a uniform solution thickness of no more than 15 mm. Start the freeze dryer and lower the cold trap (trap) temperature to ≤ -65°C to provide sufficient cooling capacity to capture sublimated water. Simultaneously, lower the feed temperature to -45°C at a rate of 3°C / min and maintain this temperature for 2.5 hours to completely freeze the DNA-Na solution and form a stable ice crystal structure, paving the way for sublimation drying.

[0057] 4.2 Gradient temperature increase freeze-drying After the pre-freezing is completed, the main drying stage begins, and freeze-drying is performed according to the set temperature gradient: Phase 1: The material temperature is raised from -45°C to -25°C at a rate of 1.5°C / min and maintained at this temperature for 12 hours. During this time, the system vacuum is maintained at 10-15 Pa, which allows ice crystals to sublime directly into water vapor at low temperatures, removing most of the free water.

[0058] Stage 2: Continue to raise the temperature to -10°C at a rate of 1.5°C / min and maintain it for 12 hours. This stage further removes tightly bound water, and the vacuum is controlled at 8-10 Pa to ensure a stable sublimation process.

[0059] The third stage: the temperature is raised to 15 ° C and maintained for 12 hours. At this time, the ice crystals in the material have basically sublimated and some adsorbed water has begun to be removed. The vacuum degree is adjusted to 5-8 Pa.

[0060] Stage 4: Finally, the temperature is raised to 35°C and maintained for 26 hours. This stage mainly removes residual adsorbed water. The vacuum degree is maintained at ≤8Pa to achieve a low moisture content in the DNA-Na product.

[0061] 4.3 Secondary drying and quality control Pressure Rise Test (PAT): After the temperature is raised to 35°C and maintained for 26 hours, the secondary drying phase begins. At this point, the temperature is further raised to 42°C, the vacuum level is controlled at ≤8 Pa, and the pressure rise test is initiated. By shutting off the vacuum pump and monitoring the rate of change of system pressure over time (pressure rise rate), the residual moisture content in the material is monitored in real time.

[0062] 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) of the product is ≤36μm, which meets the process requirements.

[0063] Determination of drying end point: 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.

[0064] Example 5 Enzyme Preparation Recovery and Reuse 5.1 Precipitate collection and washing Source of precipitate: The precipitate obtained after the first centrifugation (pH 8.8, 9000 rpm, 20 min) in Example 3.1 was taken. The precipitate mainly contained chitosan-embedded complex enzyme microspheres and some co-precipitated impurities.

[0065] Washing operation: Add 0.1 mol / L NaCl solution to the precipitate in an amount 3 times the volume of the precipitate (v).

[0066] The mixture was stirred at 200 rpm with a magnetic stirrer for 15 min to fully suspend the precipitate.

[0067] The microspheres were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the washed microspheres were collected.

[0068] Repeat the above washing steps once to completely remove impurities and residual proteins.

[0069] 5.2 Microsphere recovery and enzyme activity detection Microsphere recovery: Transfer the washed chitosan microspheres to a sterile container, weigh them, and record the recovered amount. Observe them under a microscope (400x magnification) to ensure that the microspheres are intact and free of obvious damage or agglomeration.

[0070] Enzyme activity detection: Take an appropriate amount of recovered microspheres, add Tris-HCl buffer (50 mmol / L) at pH 7.8, and shake on a shaker at 150 rpm for 30 min to fully release the enzyme.

[0071] The total protease activity was determined by the Folin-phenol method (using bovine serum albumin as the standard and measuring at a wavelength of 750 nm), and the recovery was calculated based on the initial enzyme activity (380 KU / L).

[0072] When the enzyme activity drops to 70% of the initial value (i.e. 266KU / L), the supplementation process begins.

[0073] 5.3 Reuse and enzyme activity supplementation Recycling: The recovered chitosan microspheres were directly used in the next batch of enzymatic hydrolysis reaction (step 3 of Example 1) with the same addition amount as the fresh enzyme preparation (0.5% w / v).

[0074] Enzyme supplementation: The enzyme activity was tested after 5 cycles of use, and the replenishment process was started when it dropped to 70% of the initial value.

[0075] The supplement amount is calculated according to the following formula: Supplementary enzyme amount (g) = (initial enzyme activity - current enzyme activity) × reaction system volume × dilution factor (Note: "Initial enzyme activity" refers to the total enzyme activity of the fresh enzyme preparation (380 KU / L); "Current enzyme activity" refers to the actual measured value of the recovered enzyme; the dilution factor of 1.2 is the compensation factor for enzyme activity loss.) The fresh enzyme preparation was mixed thoroughly with the recovered microspheres before use.

[0076] Example 6 Product Performance Verification Experiment 6.1 Purity and molecular weight detection Protein residue determination Detection method: BCA (bicinchoninic acid) method was used with bovine serum albumin (BSA) as the standard.

[0077] Sample processing: Accurately weigh 10 mg of the lyophilized product and dissolve it in 1 mL of ultrapure water. Oscillate at room temperature for 30 minutes until completely dissolved. Add 20 μL of the sample solution to 200 μL of BCA working reagent (Solution A:Solution B = 50:1), mix thoroughly, and incubate at 37°C for 30 minutes.

[0078] Testing conditions: The absorbance was measured at a wavelength of 562 nm using a spectrophotometer, and the protein content was calculated using a standard curve (0-1000 μg / mL BSA).

[0079] result: The measured protein residue was 0.026%, which was 48% lower than the existing technology (0.05%), indicating that the deproteinization effect of this process is significantly better than the traditional method.

[0080] Molecular weight distribution determination Detection method: High performance liquid exclusion chromatography (SEC-HPLC).

[0081] Chromatographic conditions: Chromatographic column: TSKgelG4000PWXL (7.8 mm × 30 cm, Tosoh, Japan).

[0082] Mobile phase: 0.1 mol / L phosphate buffer (pH 7.0), containing 0.1 mol / L NaCl.

[0083] Flow rate: 0.5 mL / min, column temperature 25°C, injection volume 20 μL, UV 260 nm detection.

[0084] Standards: A calibration curve was established using DNA molecular weight standards (100 bp–100 kb, Sigma-Aldrich).

[0085] result: DNA fragments below 20 kDa account for 92.5%, indicating that the product is mainly composed of small molecules and is suitable for transdermal absorption.

[0086] The high molecular weight components above 50 kDa were less than 1.5%, indicating that the process effectively avoided excessive polymerization of DNA.

[0087] The molecular weight distribution span (D90 / D10) is 2.7 times, indicating good fragment uniformity.

[0088] Transdermal absorption rate determination Experimental model: Franz diffusion cell (effective diffusion area 1.77cm 2 , receiving chamber volume 5 mL), and nude mouse abdominal skin (thickness 0.2-0.3 mm) was used.

[0089] Experimental conditions: Donor chamber: 1 mL of PBS solution (pH 7.4) containing 1 mg / mL DNA.

[0090] Receiving chamber: PBS solution (containing 0.1% BSA), constant temperature at 37°C, magnetic stirring at 600 rpm.

[0091] Sampling method: 0.5 mL of the receiving solution was sampled at 2, 4, 6, 8, 12, and 24 h, and an equal amount of fresh solution was added.

[0092] Detection method: After sampling, Hoechst 33258 fluorescent dye was added, and the DNA content was measured using a fluorescence spectrophotometer (excitation 350 nm, emission 460 nm).

[0093] result: The 24-hour transdermal absorption rate was 22.9%, a 25% increase compared to the existing technology (18.3%), indicating that the DNA prepared by this process has better transdermal performance.

[0094] 6.2 Yield and activity detection Yield determination Experimental scale: 50 kg of frozen salmon testicles (stored at -42°C and frozen within 2 hours after catching) were fed.

[0095] Calculation method: The theoretical yield was calculated based on the average salmon testis DNA content of 1.07% (i.e. 50 kg × 1.07% = 535 g).

[0096] Actual yield = (freeze-dried product weight / theoretical DNA weight) × 100%.

[0097] result: Finally, 23.8 g of freeze-dried product was obtained; Theoretical yield: 50 kg testis contains DNA = 50,000 g × 1.07% = 535 g; The actual yield was 23.8 g / 535 g × 100% = 4.45%, indicating that the process loss was controllable.

[0098] Biological activity detection Cell model: human skin fibroblasts (HSF, ATCC CRL-2097).

[0099] Experimental groups: Blank group: DMEM medium without DNA.

[0100] Experimental group: culture medium containing 0.1 mg / mL DNA of this process.

[0101] Control group: culture medium containing 0.1 mg / mL prior art DNA.

[0102] The preparation method of prior art DNA is as follows: 1. Raw material pretreatment 50 kg of frozen salmon testes (stored at -20°C and frozen within 24 h after capture) were taken and thawed at room temperature and then crushed into a homogenate using a tissue crusher.

[0103] Add a mixed solution of 0.1 mol / L Tris-HCl buffer (pH 8.0) and 0.5 mol / L NaCl at a material-liquid ratio of 1:5 (w / v) and stir to mix.

[0104] 2. Enzymatic fragmentation Add single proteinase K (enzyme activity ≥ 30 U / mg) at an amount of 0.1% (w / v) of the testis weight and enzymolyze at 50°C for 12 h.

[0105] After the enzymatic hydrolysis, the cells were inactivated at 95°C for 10 min, centrifuged at 4000 rpm for 15 min, and the supernatant was collected.

[0106] 3. Deproteinization and precipitation The supernatant was added with a mixture of phenol-chloroform-isoamyl alcohol (25:24:1) at a volume ratio of 1:1, vortexed for 10 min, centrifuged at 12000 rpm for 10 min, and the aqueous phase was collected.

[0107] Repeat the extraction twice until there is no protein layer at the interface.

[0108] Add 0.1 times the volume of 3 mol / L sodium acetate (pH 5.2) and 2 times the volume of anhydrous ethanol to the aqueous phase, precipitate at -20°C for 2 hours, centrifuge at 12000 rpm for 10 minutes, and discard the supernatant.

[0109] The precipitate was washed twice with 70% ethanol, dried in vacuo, and then dissolved in 10 mmol / L Tris-HCl buffer (pH 8.0).

[0110] 4. Preliminary Purification The solution was filtered through a 0.45 μm filter membrane and purified by DEAE-cellulose column chromatography (the stationary phase was DEAE-52 and the mobile phase was 0.1-1.0 mol / L NaCl gradient elution), and the elution peak containing DNA was collected.

[0111] The eluate was concentrated by rotary evaporation, precipitated again with ethanol, dried, dissolved in sterile water, and stored at -20°C.

[0112] Biological activity detection steps: Cells were seeded in 24-well plates at a density of 5 × 10 4 cells / well and cultured for 24 h until adherence.

[0113] The culture medium containing DNA was replaced and the culture was continued for 48 h.

[0114] Total RNA was extracted (Trizol method) and reverse transcribed into cDNA (PrimeScript RT kit).

[0115] Collagen I (COL1A1) mRNA expression was detected by qPCR, with GAPDH as the internal reference.

[0116] Primer sequences: COL1A1-F: 5′-CTGGCAAGGTGGACATCG-3′ (amplified fragment 120 bp).

[0117] COL1A1-R: 5'-TGGGGCACACACCTTCT-3'.

[0118] GAPDH-F: 5'-GAGTCAACGGATTTGGTCGT-3'.

[0119] GAPDH-R: 5'-TTGATTTTGGAGGGATCTCG-3'.

[0120] qPCR conditions: Initial denaturation was performed at 95°C for 30 s, followed by 40 cycles (95°C for 5 s, 60°C for 30 s).

[0121] Result calculation: The relative expression level was calculated using the 2^(-ΔΔCt) method, and the experiment was repeated three times to obtain the average value.

[0122] result: The expression of COL1A1 mRNA in the experimental group increased by 41% compared with the blank group, and that in the control group increased by 32%, indicating that the ability of this process product to stimulate collagen synthesis increased by 28%.

[0123] Example 7 Cosmetic Application Efficacy Test 7.1 Essence Preparation Recipe composition: Active ingredient: salmon sperm DNA-Na (prepared in Example 4), content 0.6% (w / v).

[0124] Solvent system: glycerol (10% w / v), deionized water (79.4% w / v), PEG-400 (10% w / v).

[0125] Additives: Sodium hyaluronate (0.5% w / v), disodium EDTA (0.1% w / v), phenoxyethanol (0.4% w / v).

[0126] Preparation process: Mix glycerol and PEG-400 and stir at 60℃ until homogeneous.

[0127] Add DNA-Na powder and continue stirring for 30 min until it is completely dissolved.

[0128] Slowly add deionized water (containing pre-dissolved sodium hyaluronate and disodium EDTA) while stirring.

[0129] After cooling to room temperature, phenoxyethanol was added as a preservative and stirred for 10 min.

[0130] Filter through a 0.22 μm filter membrane for sterilization, dispense into brown glass bottles, and store away from light.

[0131] 7.2 Human efficacy testing Subject screening: Female subjects aged 35-50 years (n=60) with facial wrinkles grade II-III according to Fitzpatrick classification were recruited.

[0132] Exclusion criteria: allergic constitution, history of skin disease, and use of anti-wrinkle products within 3 months before the test.

[0133] The mice were randomly divided into two groups: experimental group (n=30, using DNA-Na serum) and control group (n=30, using blank matrix without DNA-Na).

[0134] Directions: After cleansing your face every morning and evening, take 2mg / cm 2 Apply the essence evenly on the face and massage gently until absorbed.

[0135] Continue using for 8 weeks and avoid using other anti-wrinkle products during this period.

[0136] Testing indicators and methods: Collagen content: Detection method: ELISA (Human Collagen Type I ELISA Kit, Abcam).

[0137] Sampling: 3 mm skin biopsy samples were obtained from the preauricular area of ​​the subjects at week 0 and week 8.

[0138] Procedure: After digestion, the sample was assayed for type I collagen content, and the results were expressed as μg / mg tissue.

[0139] Skin elasticity: Detection instrument: CutometerMPA580 (Courage+Khazaka).

[0140] Parameter settings: test probe diameter 2mm, suction force 500mbar, measurement time 1s.

[0141] Evaluation index: R2 value (elastic recovery rate), taking the average value of both cheeks.

[0142] Wrinkle depth: Testing instrument: VisioscanVC98 (Courage+Khazaka).

[0143] Testing area: Glabellar lines (deepest vertical wrinkles).

[0144] Analysis method: Wrinkle depth (μm) was measured using SEDERM software.

[0145] Test results Changes in collagen content: Experimental group: Collagen content increased from baseline (25.4±3.1) μg / mg to (35.3±4.2) μg / mg, an increase of 39% (p<0.01).

[0146] Control group: increased from (24.9±2.8) μg / mg to (27.1±3.0) μg / mg, an increase of 8.8% (not statistically significant).

[0147] Improved skin elasticity: Experimental group: The R2 value increased from 0.42±0.05 at baseline to 0.55±0.06, an increase of 31%.

[0148] Control group: R2 value increased from 0.41±0.04 to 0.44±0.05, an increase of 7.3%.

[0149] Wrinkle Depth Reduction: Experimental group: The depth of glabellar wrinkles decreased from (48.6±7.2) μm to (36.9±5.8) μm, a decrease of 24%.

[0150] Control group: decreased from (47.9±6.5) μm to (45.2±6.1) μm, a decrease of 5.6%.

[0151] Statistical analysis: Paired t-test was used to compare intra-group differences, and independent sample t-test was used to compare inter-group differences.

[0152] All p values ​​were < 0.01, indicating that the differences between the experimental and control groups were highly statistically significant.

[0153] Key conclusions Significant anti-wrinkle effect: The serum containing 0.6% salmon sperm DNA-Na significantly increased skin collagen content (+39%) and elasticity (+31%), and reduced wrinkle depth (-24%) within 8 weeks, with better results than the blank matrix.

[0154] Security Verification: All subjects did not experience allergic or irritation reactions during the test period, indicating that this concentration of DNA-Na has good safety in cosmetics.

[0155] Mechanism of action supports: The experimental results were highly consistent with the in vitro activity data of Example 6 (collagen mRNA expression increased by 41%), further verifying that DNA-Na exerts its anti-wrinkle effect by stimulating fibroblasts to synthesize collagen.

[0156] This efficacy test shows that the salmon sperm DNA-Na prepared by this process has a significant anti-wrinkle effect as a cosmetic active ingredient, providing a clinical basis for its application in high-end skin care products.

[0157] Comparative Example 1: Traditional enzymatic hydrolysis process (without chitosan-encapsulated complex enzyme) Step 1: Raw material pretreatment Raw materials preparation: Take 20 kg of frozen rainbow trout testes (stored at -20℃ and frozen within 48 hours after capture), thaw them, add 2 times the volume (40 L) of 40 mmol / L Tris-water solution (pH 7.5), and break them up with a high-speed tissue grinder (10000 rpm) for 10 minutes.

[0158] Cell lysis: 0.2% (w / v) Triton X-100 (80 g) was added, and the pH was adjusted to 7.5 with 0.5 mol / L NaOH. The mixture was stirred at a constant temperature of 25°C for 8 h to promote cell lysis and release DNA.

[0159] Step 2: Enzymatic treatment Enzymatic hydrolysis conditions: After adjusting the homogenate to pH 7.5, free trypsin (250 KU / L, volume 20 L, total activity 5000 KU) was added and digested at 37°C with stirring for 24 h.

[0160] Process monitoring: Samples were taken every 4 h and the enzyme activity was tested using the Folin-phenol method. It was observed that after 24 h, the enzyme activity was only 45% of the initial value (112.5 KU / L).

[0161] Test results Protein residue: The protein content of the enzymatic hydrolysate was determined by BCA method to be 4.2±0.3% (n=3), which was significantly higher than 0.87% in Example 1.

[0162] DNA fragment distribution: Gel electrophoresis showed that the main fragments were between 50 and 100 kDa, with fragments below 20 kDa accounting for only 65%, and the molecular weight distribution was relatively wide.

[0163] Enzyme activity decay: The 24-hour enzyme activity residual rate was 45%, indicating that the free enzyme had poor stability and required frequent enzyme addition to maintain reaction efficiency.

[0164] Comparative Example 2: Single ultrasonic crushing process (without high-pressure homogenization) Step 1: Enzyme pretreatment Dilution and adjustment: Take 20 L of the enzymatic hydrolyzate of 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.

[0165] Step 2: Ultrasonic fragmentation Equipment and parameters: A 20 kHz constant frequency ultrasonic disruptor (power 500 W) was used, the ultrasonic mode was 5 s working and 1 s pause, the total processing time was 40 min, and the temperature was controlled at 20 °C ± 5 °C by circulating cooling water.

[0166] Step 3: Separation and purification Subsequent process: The steps in Example 3 were followed by centrifugation (9000 rpm, 20 min), ceramic membrane filtration (1 μm), nanofiltration (200 Da molecular weight cut-off), and ultrafiltration (5 kDa).

[0167] Test results Purity index: OD260 / OD280=1.65±0.03 (n=3), indicating protein contamination; the protein residual amount by BCA method was 0.075±0.005%, which was higher than 0.026% in Example 3.

[0168] Molecular weight distribution: Size exclusion chromatography showed that DNA below 20 kDa accounted for 82.3±1.2%, and DNA above 50 kDa accounted for 4.8±0.5%, which was significantly lower than 92.5% and <1.5% in Example 2.

[0169] Crushing efficiency: The average DNA fragment length was 120 bp, 50% longer than the 80 bp in Example 2, indicating that the fragmentation effect was insufficient.

[0170] Comparative Example 3: Traditional column chromatography purification (butyl hydrophobic filler) Step 1: Sample pretreatment Solution preparation: Take 10 L of the crude DNA solution obtained in Comparative Example 2 (concentration is about 0.5 g / L), add an equal volume of 20 mmol / L NaCl solution (10 L) to dilute the final NaCl concentration to 10 mmol / L.

[0171] Filtering and removing impurities: The mixed solution was filtered through a 0.45 μm cellulose acetate filter membrane (142 mm in diameter) under reduced pressure to remove insoluble impurities and obtain a clear filtrate.

[0172] Step 2: Hydrophobic chromatography separation Column packing: A stainless steel column (inner diameter 5 cm, column height 20 cm, column volume 500 mL) was packed with Proteomix® HICButyl hydrophobic chromatography packing (particle size 30 μm, pore size 100 Å) at a packing pressure of 1.5 MPa. The column efficiency was measured to be 6000 theoretical plates / meter.

[0173] Balancing and loading: Equilibration buffer: 20 mmol / L NaCl (pH 7.8), equilibration for 5 column volumes (CV) at a linear velocity of 2 cm / min.

[0174] Loading conditions: The pretreated sample was loaded at a linear velocity of 0.5 cm / min and the loading volume was 1.5 column volumes (750 mL).

[0175] Step 3: Gradient elution Elution procedure: The same gradient elution program as S34 was used, but the elution linear velocity was fixed at 1 cm / min (not dynamically adjusted according to UV monitoring values): 0-10CV: 20mmol / LNaCl (pH 7.8) → 20mmol / LNaCl + 1.5mol / L(NH4)2SO4 (pH 7.8), linear gradient.

[0176] 10-15CV: Isocratic elution with 20mmol / LNaCl + 1.5mol / L(NH4)2SO4 (pH7.8).

[0177] 15-20CV: Rinse with pure water.

[0178] Monitoring and collection: The elution curve was monitored by a UV detector (260 nm) and the main peak fraction (retention time was approximately 8-12 CV) was collected.

[0179] Step 4: Freeze-drying Concentration and dialysis: The collected eluate was concentrated to 500 mL through a 10 kDa molecular weight cutoff ultrafiltration membrane (GE Healthcare) and then dialyzed against 10 mmol / L Tris-HCl buffer (pH 7.5) for desalting.

[0180] Freeze-drying process: The freeze-drying conditions of Example 4 were followed: Pre-freezing: -45℃, 2.5h, trap temperature ≤-65℃.

[0181] Sublimation: -25℃ for 12h → -10℃ for 12h → 15℃ for 12h → 35℃ for 26h.

[0182] Secondary drying: 42°C, vacuum degree ≤8 Pa, drying for 3 h, PAT monitoring to a pressure rise rate ≤0.4 Pa / h.

[0183] Test results Separation efficiency: Column efficiency change: When the sample volume was 1.5 BV, the column efficiency dropped from the initial 6000 / m to 4800 / m (a decrease of 20%).

[0184] Peak shape parameters: the target peak tailing factor (As) is 1.8 (1.2 for the core-shell filler in Example 3), and the half-peak width (W1 / 2) is increased by 35%.

[0185] Yield and purity: Total yield: 40 kg of rainbow trout testis was fed, and 16.2 g of freeze-dried product was finally obtained, with a yield of 40.5% (43.27% in Example 3).

[0186] Purity index: Residual protein: 0.048±0.002% (BCA method), higher than 0.026% in Example 3.

[0187] DNA molecular weight distribution: 85.7±0.9% of the DNA was below 20 kDa, and 3.2±0.4% of the DNA was above 50 kDa (the corresponding values ​​in Example 3 were 92.5% and <1.5%).

[0188] Process stability: Continuous purification test: After 5 consecutive purifications, the filler loading capacity decreased from 1.5 BV to 1.0 BV (a decrease of 33%) and required regeneration with 2M NaCl + 20% ethanol solution.

[0189] Service life: After repeated use 10 times, 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 more than 90%.

[0190] Key Difference Analysis index Comparative Example 3 (Butyl Hydrophobic Filler) Example 3 (core-shell filler) Explanation of the differences Loading volume (BV) 1.5 (down to 1.0) 3.0 (stable) Core-shell structure improves mass transfer efficiency Column efficiency (theoretical plates / m) 4800 (after loading) 8500 (stable) Core-shell fillers have stronger anti-pollution ability Target peak tailing factor 1.8 1.2 Butyl fillers have nonspecific adsorption Proportion of DNA below 20 kDa (%) 85.7±0.9 92.5±0.7 Higher separation selectivity The decrease in sample loading after 5 cycles 33% ＜5% Core-shell packing has a longer service life in conclusion: The traditional butyl hydrophobic filler significantly outperformed the core-shell chromatographic filler of Example 3 in terms of sample loading capacity, separation efficiency, and service life. The fixed flow rate elution strategy failed to dynamically adjust to the actual separation conditions, further exacerbating peak broadening and impurity residue issues. This demonstrates that the core-shell filler and its accompanying intelligent elution strategy employed in the present invention play a key role in improving DNA purification efficiency and process stability.

[0191] Comparative Example 4: Unoptimized freeze-drying process (no pressure rise test) Step 1: Pre-freezing Sample preparation: The high-purity DNA-Na solution prepared in Example 3 (concentration 10 mg / mL, OD260 / OD280=1.90) was dispensed into 50 lyophilized vials (10 mL per vial, 500 mL in total).

[0192] Pre-freezing conditions: The lyophilized vial was placed in the drying chamber of a freeze dryer (Labconco Free Zone 2.5 L). The cold trap temperature was set to -55 °C and then cooled to -40 °C at a rate of 5 °C / min for 2 h. The trap temperature was set to -55 °C and the vacuum degree was ≤10 Pa.

[0193] Step 2: Sublimation drying Heating program: Raise the temperature according to the following gradient, maintaining the vacuum degree ≤10Pa throughout the process: Stage 1: Raise the temperature from -40°C to -25°C at 0.5°C / min and maintain for 10 h.

[0194] Stage 2: Raise the temperature to -10°C at 0.3°C / min and maintain for 10 h.

[0195] Stage 3: Raise the temperature to 15°C at 0.5°C / min and maintain for 10 h.

[0196] Stage 4: Raise the temperature to 35°C at 1°C / min and maintain for 20 h.

[0197] Step 3: Secondary drying Final Dehydration: The temperature was raised to 40°C, the vacuum was maintained at 10 Pa, and the mixture was dried for 2 h.

[0198] End conditions: No pressure rise test (PAT) was used to monitor residual moisture; the drying endpoint was determined solely by time. After drying, the container was filled with nitrogen and sealed with a stopper.

[0199] Test results Moisture content determination: Detection method: Karl Fischer volumetry (Metrohm 870KF Titrino).

[0200] Results: The residual moisture content of the freeze-dried product was 1.3±0.1% (n=5), which was significantly higher than 0.8±0.05% in Example 4.

[0201] Resolubility assessment: Procedure: Take 100 mg of lyophilized product, add 10 mL of ultrapure water, and dissolve by shaking at room temperature for 10 min.

[0202] Purity changes: OD260 / OD280 before lyophilization=1.90±0.01.

[0203] After reconstitution, OD260 / OD280 was 1.82±0.02, and the OD value decreased by 4% from 1.90 before freeze-drying to 1.82, indicating slight degradation of nucleic acid or increased protein contamination.

[0204] Accelerated stability testing: Conditions: The freeze-dried product was placed in an environment of 40°C and 75% relative humidity (RH75%) for aging for 1 month.

[0205] Molecular weight change: Initially, DNA below 20 kDa accounted for 91.5 ± 0.6% (data from Example 3).

[0206] After aging, it dropped to 87.2±0.8%, and the degradation rate was 4.7% ((91.5-87.2) / 91.5×100%), which was higher than 3.1% in Example 4.

[0207] Comparison of key process parameters index Comparative Example 4 (freeze-drying not optimized) Example 4 (Optimization of freeze-drying) Gap Analysis Prefreezing rate (℃ / min) 5 3 Rapid cooling may form large ice crystals Total sublimation time (h) 50 48 Similar time but different temperature gradients Secondary drying temperature (℃) 40 42 Slightly lower temperature but lack of PAT monitoring Secondary drying time (h) 2 3 Insufficient time leads to high residual moisture 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 40℃ aging degradation rate (%) 4.3 3.1 Water promotes hydrolysis and oxidation of nucleic acids in conclusion: The lack of pressure rise testing (PAT) monitoring in unoptimized freeze-drying processes leads to inaccurate determination of the drying endpoint and high residual moisture. High moisture content accelerates DNA degradation during storage, manifesting as a decrease in the OD260 / OD280 ratio and changes in molecular weight distribution. Example 4 effectively reduces residual moisture and improves product stability by optimizing the prefreezing rate, adjusting the heating gradient, and incorporating PAT monitoring. This demonstrates the critical role of PAT technology in controlling the freeze-drying endpoint and ensuring consistent product quality.

[0208] Comparative Example 5: Traditional process without enzyme recovery Step 1: Enzymatic hydrolysis (chitosan microspheres not recovered) Raw material processing: 50 kg of fresh rainbow trout testes were taken and processed according to 1.1 and 1.2 of Example 1 to obtain a homogenate, and the pH was adjusted to 7.5.

[0209] Enzymatic hydrolysis reaction: Chitosan-encapsulated complex enzyme (trypsin: papain = 3:1, total activity 1000 KU) was added and stirred at 37°C for 12 h.

[0210] Centrifugal separation: After the enzymatic hydrolysis, centrifugation was performed directly (9000 rpm, 20 min), the precipitate (including unrecovered chitosan microspheres) was discarded, and the supernatant was retained for subsequent purification.

[0211] Step 2 to Step 4: Subsequent Process Crushing and purification: Ultrasonic-high-pressure homogenization synergistic disruption was performed according to the steps of Example 2, and then centrifugation, ceramic membrane filtration, and hydrophobic chromatography purification were performed according to the steps of Example 3.

[0212] Freeze-dried molding: Lyophilization was performed according to the steps in Example 4 to obtain a DNA-Na lyophilized product.

[0213] Test results Cost Analysis: Enzyme consumption: The cost of directly investing in fresh enzyme preparation per batch is RMB 2,850 (the cost is reduced to RMB 1,965 after recycling in Example 5), which is a 45% increase in cost.

[0214] Total cost comparison: Single batch production cost (enzyme + consumables): Comparative Example 5: 4230 yuan / kg product.

[0215] Example 5: 2910 yuan / kg product, saving 31.2%.

[0216] Environmental indicators: Amount of solid waste generated: Amount of solid waste generated per kg DNA-Na production: Comparative Example 5: 5.2 kg (containing unrecovered chitosan microspheres).

[0217] Example 5: 2.9 kg (chitosan recovery rate 85%), a reduction of 44.2%.

[0218] Wastewater Discharge: Wastewater COD value: Comparative Example 5 is 12800 mg / L, while Example 5 is 8600 mg / L, a decrease of 32.8%.

[0219] Long-term stability: Continuous batch testing: 5 batches of production were carried out continuously, with 50 kg of testes in each batch: batch Yield (%) Residual protein (%) Batch 1 42.8 0.031 Batch 5 38.7 0.042 Performance changes: 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 significantly higher.

[0220] Key Difference Analysis index Comparative Example 5 (no enzyme recovery) Example 5 (Enzyme recovery) Explanation of the differences Enzyme preparation cost (yuan / kg) 2850 1965 Chitosan microspheres can be recycled 5 times Amount of solid waste generated (kg / kg) 5.2 2.9 Recycling chitosan to reduce waste Yield decreased after 5 batches 9.6% 2.1% Loss of enzyme activity leads to incomplete degradation Protein residue increase after 5 batches 35.5% 11.5% Decreased enzyme activity affects protein removal efficiency Wastewater COD (mg / L) 12800 8600 Reduce enzyme protein residual contamination in conclusion: The traditional process of not recovering chitosan-encapsulated enzymes is inferior to the enzyme recovery process of Example 5 in terms of cost, environmental protection, and process stability. The recycling of chitosan microspheres can reduce enzyme consumption costs, reduce solid waste and wastewater discharge, and stabilize enzyme activity to ensure production efficiency and product purity. This application constructs a green and efficient preparation system through chitosan encapsulation, ultrasound-homogenization coupling, 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.

[0221] Comparative Example 6: Verification of the synergistic effect of complex enzyme encapsulation vs. single enzyme encapsulation Step 1: Raw material processing Take 20 kg of frozen salmon testicles, crush them at -25℃ ultra-low temperature, add 40 L of mixed solution (containing 0.08 mol / L Tris-HCl, 0.2 mol / L NaOH, 1.0 mol / L NaCl), stir magnetically at 300 rpm for 30 min, and stir evenly in an ice-water bath controlled at ≤10℃.

[0222] Step 2: Enzyme preparation 1. Complex enzyme embedding group: Complex enzyme embedding group: Preparation of chitosan solution: Dissolve 20 g of chitosan in 1% (w / v) acetic acid solution to prepare 1000 mL of 2% (w / v) chitosan solution. Stir magnetically at 500 rpm for 4 h until clear, and sterilize with a 0.45 μm filter membrane.

[0223] Complex enzyme mixture: Weigh 45 g of trypsin, 15 g of neutral protease, 20 g of papain, 20 g of proteinase K, 15 g of NSP enzyme, 8 g of cellulase, and 4 g of β-1,3-glucanase according to weight, dissolve in 500 mL of pH 7.8 Tris-HCl buffer, and mix under 100 W ultrasonication for 5 min (5 s on, 1 s off).

[0224] 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 chitosan:TPP mass ratio was 5:1. The mixture was magnetically stirred at 500 rpm for 30 min to form 5-10 μm microspheres.

[0225] Microsphere collection: centrifugation at 4000 rpm for 10 min, washing three times with pH 7.8 Tris-HCl buffer, and vacuum drying at 40°C for 10 h (vacuum degree ≤ 10 Pa).

[0226] 2. Single enzyme embedding group: Only 45g trypsin was added, and the rest of the embedding process was the same as that of the complex enzyme group (chitosan solution concentration and cross-linking conditions were the same).

[0227] Step 3: Enzymatic hydrolysis Enzyme-encapsulated microspheres (total enzyme activity 350 KU / L) were added to the raw material slurry in both groups and enzymatically hydrolyzed at 37 ± 1 °C for 28 h. The pH was maintained at 7.8 ± 0.1 by an automatic titration system and magnetic stirring was performed at 300 rpm. After the enzymatic hydrolysis, the cells were inactivated at 85°C for 10 min, centrifuged at 4000 rpm for 20 min, and the supernatant was collected.

[0228] Step 4: Detection and Analysis DNA yield determination: UV spectrophotometry (A260): Dilute the supernatant 100-fold, measure the absorbance at 260 nm, and calculate the yield (mg / g raw material) as follows: DNA concentration (mg / mL) = 50 × A260 × dilution factor.

[0229] Complex enzyme group: 5.8±0.5 mg / g, single enzyme group: 4.2±0.3 mg / g (n=3, p<0.01).

[0230] Purity assessment: The A260 / A280 ratio was measured and was 1.92±0.02 in the combined enzyme group and 1.75±0.03 in the single enzyme group (n=3, p<0.05).

[0231] Cell disruption rate detection: Trypan blue staining method: Take a smear of the enzymatic solution, count 100 cells under an optical microscope (1000×), and calculate the proportion of ruptured cells.

[0232] Combined enzyme group: 96±2%, single enzyme group: 70±3% (n=3, p<0.01).

[0233] Residual polysaccharide content (DNS method): Combined enzyme group: 0.12±0.01 mg / mL, single enzyme group: 0.35±0.02 mg / mL (n=3, p<0.01).

[0234] Comparative Example 7: Stability comparison of encapsulated complex enzyme vs. free complex enzyme Step 1: Enzyme preparation 1. Complex enzyme embedding group: embedding microspheres were prepared in the same manner as in Comparative Example 6; 2. Free complex enzyme group: Prepare free complex enzyme solution according to the same components and enzyme activity, and dissolve in pH 7.8 Tris-HCl buffer (free state).

[0235] Step 2: Enzymatic hydrolysis and recovery Single enzymatic hydrolysis: The two groups treated 20 kg of salmon testes respectively, enzymatically hydrolyzed them at 37°C for 28 hours, maintaining the pH at 7.8, and recorded the residual enzyme activity and DNA yield after enzymatic hydrolysis.

[0236] Continuous batch recovery: Embedding group: After each enzymatic hydrolysis, the microspheres were recovered by centrifugation at 4000 rpm, washed three times with 0.1 mol / L NaCl, and the enzyme activity was tested (reused when ≥70% of the initial value). This was repeated for five batches.

[0237] Free group: After each enzymatic hydrolysis, the free enzyme was recovered by ultrafiltration membrane (30 kDa), and the recovered enzyme activity was measured. This was repeated for 5 batches.

[0238] Step 3: Detection and Analysis Residual enzyme activity (Folin-phenol method): After a single enzymatic hydrolysis: 87±3% in the embedded group and 52±4% in the free group (n=3, p<0.01).

[0239] 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).

[0240] DNA yield: Single enzymatic hydrolysis: 5.6±0.4 mg / g in the embedded group and 4.1±0.3 mg / g in the free group (n=3, p<0.01).

[0241] 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).

[0242] Residual protein (BCA method): Embedded group: 0.031±0.002%, free group: 0.075±0.005% (n=3, p<0.01).

[0243] Microsphere particle size stability: Laser particle size analyzer detection: D90 of the embedding group after 5 repetitions was 39±2μm, which was not significantly different from the initial value (38±2μm) (p>0.05).

[0244] in conclusion 1. Data from Comparative Example 6 show that the DNA yield (5.8 mg / g) of the complex enzyme-encapsulated group increased by 38% compared to the single enzyme-encapsulated group (4.2 mg / g), the cell disruption rate increased from 70% to 96%, and the residual polysaccharide content decreased by 66%. The synergistic action of multiple enzymes (e.g., protease degradation of proteins and cellulase decomposition of cell walls) significantly improved DNA release efficiency, whereas the effectiveness of single enzymes was less effective due to limited substrate specificity.

[0245] 2. Data from Comparative Example 7 show that after a single enzymatic hydrolysis, the residual enzyme activity in the encapsulated group (87%) was 67% higher than that in the free group (52%). After five batches of repeated use, the yield in the encapsulated group decreased by only 2.1%, while that in the free group decreased by 37.6%. Chitosan microspheres effectively delay enzyme inactivation and degradation through physical isolation and structural protection, enhancing the efficiency of enzyme reuse.

[0246] Combined with Comparative Examples 6 and 7, the complex 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 life (yield attenuation 2.1% vs 37.6% after 5 batches).

[0247] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for preparing salmon sperm DNA sodium raw material, characterized in that: The following steps are involved: S1. Raw material pretreatment: Frozen salmon or trout testis tissue was ultra-cryogenically crushed at -30 to -20°C, and then added with a mixture of Tris-HCl buffer, NaOH, and NaCl. A chitosan-encapsulated enzyme complex with a total enzyme activity of ≥350 kU / L was then added. The enzyme was hydrolyzed at 37 ± 1°C for 26-30 hours, maintaining a pH of 7.8 to 8.0, to prepare an enzymatic solution. S2. DNA fragmentation: Adjust the pH of the enzymatic solution to 7.8 and perform a synergistic treatment in an ice-water bath at ≤10°C using 25-35 kHz variable frequency ultrasound and 100-120 MPa high pressure homogenization for 40 min, alternating for 3-5 cycles, to obtain a DNA fragmentation solution. S3. Deproteinization and fractional purification: The DNA fragment was centrifuged and filtered multiple times, then separated by nanofiltration, ultrafiltration, and core-shell phenyl hydrophobic chromatography using a specific mobile phase gradient elution. The target fractions were collected to obtain a high-purity DNA-Na solution. S4. Lyophilization: The high-purity DNA-Na solution was pre-frozen at -45°C and lyophilized using a specific temperature gradient. Lyophilized powder was obtained by secondary drying with a controlled particle size (D90) of ≤40 μm. S5. Enzyme preparation recovery: Wash the precipitate obtained in S3 to recover chitosan microspheres. When the enzyme activity is ≥ 70% of the initial value, reuse it for the next batch of enzymatic hydrolysis.

2. The method for preparing salmon sperm DNA sodium raw material according to claim 1, wherein The preparation method of the chitosan-embedded complex enzyme in S1 is: 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; the complex enzyme preparation was added in proportion, and ultrasonicated at 50-150W for 3-10 minutes to form an enzyme-chitosan mixture; the mixture was dropped into 0.3% TPP solution with a mass ratio of chitosan to TPP of 3:1-8:1, and magnetically stirred at 300-800 rpm. The mixture was cross-linked at room temperature for 20-40 minutes to form chitosan microspheres with a particle size of 5-10 μm, and then the microspheres were collected by centrifugation at 3000-5000 rpm for 5-15 minutes, washed 2-4 times with 7.5-8.0 Tris-HCl buffer, and vacuum dried at 30-50°C for 6-15 hours.

3. The method for preparing salmon sperm DNA sodium raw material according to claim 2, wherein The weight proportions of the complex enzyme preparation are: 40-50 parts of trypsin, 10-20 parts of neutral protease, 15-25 parts of papain, 15-25 parts of proteinase K, 12-18 parts of NSP enzyme, 7-10 parts of cellulase and 3-5 parts of beta-1,3-glucanase.

4. The method for preparing salmon sperm DNA sodium raw material according to claim 1, wherein The mixed solution of Tris-HCl buffer, NaOH and NaCl in S1 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.

5. The method for preparing salmon sperm DNA sodium raw material according to claim 1, wherein The variable frequency ultrasonic power in S2 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 treatment is 80-150MPa; ultrasound and homogenization are alternately cycled 2-6 times, and ultrasonic treatment is performed for 3-8min after each homogenization; the ultrasonic working mode is ultrasound for 3-8s and rest for 1-3s.

6. The method for preparing salmon sperm DNA sodium raw material according to claim 1, wherein: In the S3, the DNA fragmentation solution 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, adjusted to pH 7.5-8.0, allowed to stand for 6-10 h, and centrifuged a second time.

7. The method for preparing salmon sperm DNA sodium raw material according to claim 6, characterized in that: The nanofiltration system in S3 uses a polyamide rolled composite membrane with a molecular weight cutoff of 100-500Da, an operating pressure of 0.15-0.3MPa, a temperature of 15-25°C, a cross-flow filtration membrane surface flow rate of 1.5-2.5m / s, and passes through a 3-10kDa ultrafiltration membrane after circulating for 3-8h.

8. The method for preparing salmon sperm DNA sodium raw material according to claim 1, wherein: The freeze-drying procedure in S4 is as follows: pre-freeze at -50 to -40°C for 2 to 3 hours, then heat to -30 to -20°C at a rate of 1 to 2°C / min and maintain for 10 to 14 hours, then heat to -15 to -5°C and maintain for 10 to 14 hours, then heat to 10 to 20°C and maintain for 10 to 14 hours, and finally heat to 30 to 40°C and maintain for 24 to 30 hours; the secondary drying conditions are 40 to 45°C, vacuum degree ≤10Pa, and the temperature is terminated when the pressure rise rate is ≤1Pa / h.

9. The method for preparing salmon sperm DNA sodium raw material according to claim 1, wherein: In the 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.

10. An application of the salmon sperm DNA sodium raw material obtained by the preparation method according to any one of claims 1 to 9 in the field of cosmetics, characterized in that: Used to prepare skin care products with anti-wrinkle, skin firming and skin gloss improvement effects.