A fibrous structured polynucleotide material and a method for extracting it

Abstract

This invention relates to a fibrous polynucleotide material and its extraction method, belonging to the field of polynucleotide extraction technology. The extraction method includes the following steps: cleaning salmonid testis tissue, homogenizing at low temperature, enzymatically hydrolyzing and centrifuging, purifying the supernatant by salting out and organic solvent extraction, adding an alcohol precipitant and allowing it to stand at low temperature, collecting the precipitate to obtain the fibrous polynucleotide material. The various process parameters of this invention work synergistically to achieve spontaneous, oriented, and ordered self-assembly of PN molecular chains during precipitation. The entire process requires no external templates, electric fields, magnetic fields, or mechanical stretching for orientation, and no post-processing molding steps. It is simple to operate, low in cost, and highly reproducible, applicable to polynucleotide materials from various sources, providing an efficient, controllable, and universal technical solution for preparing biomacromolecules with ordered fibrous microstructures.

Description

Technical Field

[0001] This invention relates to a fibrous polynucleotide material and its extraction method, belonging to the field of polynucleotide extraction technology. Background Technology

[0002] Polynucleotide (PN) materials have broad application prospects in biomedicine, tissue engineering, and cosmetic fillers due to their good biocompatibility, biodegradability, and inherent bioactivity. Currently, most commercially available PN products are in powder or flocculent form. This form lacks a macroscopic physical support structure, resulting in insufficient immediate support when used alone for soft tissue filling. Furthermore, the residence time in the body is difficult to control effectively, limiting their direct application as injectable filler materials.

[0003] To endow PN materials with a fibrous macroscopic morphology, researchers have attempted to prepare nucleic acid fibers through post-processing methods. For example, some studies have used metal ions to induce multi-scale ordered assembly of DNA and combined this with continuous spinning technology to prepare DNA fibers; others have reported the use of DNA-cationic lipid complexes as spinning solutions to produce DNA-based fibers through dry spinning processes. Regarding the assembly mechanism, existing studies have shown that salt ion concentration has a significant impact on the self-assembly behavior and multiphase separation morphology of nucleic acid molecules. Under low ionic strength conditions, the electrostatic repulsion between DNA molecular chains is strong, making it difficult to form ordered aggregates; as the salt concentration increases, the electrostatic shielding effect strengthens, and the molecular chains tend to arrange themselves in an ordered manner and undergo nematic phase separation. In addition, in the preparation of chitosan-DNA complex fibers, it was found that the mechanical properties (strength and flexibility) of the fibers depend on the NaCl concentration in the DNA solution, and the DNA chain length also has a significant impact on phase separation behavior.

[0004] However, all of the existing methods for preparing nucleic acid fibers mentioned above share the following common problems: First, exogenous additives are required. Existing methods typically rely on exogenous additives such as lipids, surfactants, and metal ions to induce the orderly arrangement of nucleic acid molecules. These additives may remain in the final product, leading to reduced biocompatibility and making it difficult to meet the stringent safety requirements for cosmetic injection products.

[0005] Secondly, the process is complex and the secondary processing cost is high. All of the above technical routes require drying the extracted and purified PN raw material into powder first, then redissolving it, adding auxiliaries, and then spinning it into a fiber. The process involves many steps, high energy consumption, and significant material loss due to multiple operations.

[0006] Third, extraction and purification are separated from morphological construction, lacking integrated process design. In existing PN extraction and purification processes, "extraction and purification" and "morphological construction" are usually regarded as two independent steps. The technical possibility of achieving in-situ self-assembly of PN molecules through process parameter control in key steps such as alcohol precipitation, thereby directly obtaining fibrous macroscopic structures, has not been explored.

[0007] Therefore, there is an urgent need to provide a fibrous polynucleotide material and its extraction method, which aims to simultaneously achieve in-situ construction of fibrous morphology during PN extraction and purification without the need for external additives and subsequent spinning processes, thereby simplifying the process, reducing costs and improving product safety. Summary of the Invention

[0008] This invention provides a fibrous polynucleotide material and its extraction method. By precisely controlling parameters such as the concentration of PN solution, the type and concentration of salt ions, the type and volume of alcohol precipitant, precipitation temperature, standing time, washing method and drying method in the extraction process, PN molecules can achieve orderly self-assembly and fibrous structure shaping during alcohol precipitation, without the need to introduce any exogenous additives.

[0009] The technical solution provided by this invention is as follows: One objective of this invention is to provide a method for extracting fibrous polynucleotide materials, comprising the following steps: (1) Raw material pretreatment: Take fresh or low-temperature frozen salmonid testis tissue, clean, drain and cut into pieces for later use; (2) Cell disruption and enzymatic hydrolysis: The testis tissue block obtained in step (1) was homogenized and disrupted under low temperature conditions. Proteolytic enzyme was added to the homogenate for enzymatic hydrolysis, and the supernatant was collected by centrifugation. (3) Salting out and purification: Salt out the supernatant obtained in step (2), collect the supernatant by centrifugation, add organic solvent for extraction, and obtain polynucleotide purified solution; (4) One-step alcohol precipitation: Adjust the concentration of the polynucleotide purification solution obtained in step (3) to 1.0~6.0 mg / mL, add alcohol precipitant to the polynucleotide purification solution at a volume ratio of 1:1~2 under low temperature conditions, let stand for 60~180 min, collect the precipitate to obtain fibrous polynucleotide material.

[0010] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, the extraction method further includes the following steps: washing, dehydrating and drying the precipitate obtained in step (4) to obtain a dry fibrous polynucleotide material.

[0011] Further, in step (2), the homogenization and crushing involves transferring testicular tissue blocks to a homogenizer, adding purified water, and performing the process at 4~10℃ and 7000~10000rpm. Further, in step (2), the ratio of the testicular tissue block to purified water is 1g:1~5mL. Further, in step (2), the enzymatic hydrolysis is to add proteolytic enzyme to the homogenate, adjust the pH to 6.5~8.5 with buffer solution, and stir and hydrolyze for 1~5 hours under water bath conditions of 40~55℃; the final concentration of the proteolytic enzyme is 0.1~1g / mL. Further, in step (3), the salting out is to add salt to the supernatant to a final concentration of 0.5~2.0M and let it stand at 4~10℃ for salting out.

[0012] Furthermore, in step (4), the low temperature is 0~10℃.

[0013] Furthermore, the alcohol precipitant is an aqueous solution of ethanol with a volume fraction of 90% to 100%.

[0014] Furthermore, the washing is performed by standing and washing with an ethanol aqueous solution with a volume fraction of 70%~95% for 1~3 times; the dehydration and drying are performed by soaking in anhydrous ethanol, then pre-freezing at -80~-20℃ for 1~4 hours, and then freeze-drying at -20℃ and a vacuum degree ≤10 Pa.

[0015] The second objective of this invention is to provide a fibrous polynucleotide material obtained by the extraction method described above.

[0016] The technical solution provided by this invention has the following advantages compared with the prior art: (1) By controlling the concentration of PN solution, the present invention enables the PN molecular chains to obtain the optimal balance between inter-chain interaction forces and molecular motion capabilities during precipitation, thereby spontaneously inducing the molecular chains to self-assemble in an orderly manner along a specific direction to form a fibrous structure. This avoids the amorphous precipitation of granular or dense blocky particles caused by improper concentration in traditional methods. No post-processing molding process is required, and anisotropic fibrous PN materials can be obtained in one step, which significantly simplifies the preparation process.

[0017] (2) By systematically optimizing the types and concentrations of salt ions, this invention utilizes the electrostatic repulsion effect of salt ions shielding the negative charges between PN molecular chains, achieving a precise match between the inter-chain repulsion force and the aggregation driving force, effectively promoting the parallel orientation of molecular chains during alcohol precipitation, successfully suppressing the formation of amorphous flocculent precipitates, and endowing the resulting fibrous material with higher structural regularity and mechanical property consistency.

[0018] (3) The addition volume of alcohol precipitant in this invention allows the precipitation process to proceed at a moderate rate, providing sufficient time window for the PN molecular chains to complete the ordered arrangement and fibrous growth; at the same time, it avoids disordered stacking of molecular chains caused by excessively rapid precipitation, thereby ensuring the uniformity of morphology and structural integrity of the fibrous product.

[0019] (4) The present invention controls the precipitation temperature in the low temperature range. By reducing the rate of thermal motion of molecular chains, the orientation order of molecular chains during precipitation is greatly improved, so that the fibrous structure grows preferentially in a specific direction. At the same time, it avoids the problem of incomplete precipitation or increased fiber brittleness caused by excessively low temperature, and also avoids the drawback of thermal motion interfering with the orderly arrangement when the temperature is too high. The resulting fibrous PN material has good mechanical toughness and structural stability.

[0020] (5) The present invention uses a static tilting washing method to wash the fibrous PN precipitate after alcohol precipitation, and combines it with a gentle drying treatment to avoid the damage to the fiber morphology caused by mechanical stress such as stirring and oscillation. This preserves the fibrous ordered structure formed during the precipitation process to the greatest extent, ensuring the high fidelity of the fiber morphology of the final product throughout the entire process from precipitation to drying. This solves the problem of the fibrous structure being easily broken and deformed in the post-processing stage in the prior art.

[0021] (6) The various process parameters of the present invention work together to achieve spontaneous, oriented, and ordered self-assembly of PN molecular chains during precipitation. The entire process does not require external templates, electric fields, magnetic fields or mechanical stretching or other auxiliary orientation methods, nor does it require any post-processing molding steps. It is simple to operate, low in cost and has good reproducibility. It is applicable to polynucleotide materials from various sources and provides an efficient, controllable and universal technical solution for preparing biomacromolecule materials with ordered fiber microstructures. Attached Figure Description

[0022] Figure 1 A photograph of the fibrous polynucleotide material prepared in Example 1 of this invention; Figure 2 This is a SEM image of the fibrous polynucleotide material prepared in Example 1 of the present invention; Figure 3 This is an electrophoretic image of the fibrous polynucleotide material prepared in Example 1 of the present invention. Detailed Implementation

[0023] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0024] Example 1 A method for extracting fibrous polynucleotide materials includes the following steps: Step 1: Pretreatment of fish testes Take 200 g of fresh, frozen Atlantic salmon testis tissue, thaw it slowly in a 4°C freezer, remove any remaining fat, connective tissue, and blood, rinse repeatedly with purified water three times until no blood remains, drain the surface moisture, and cut into approximately 1 cm pieces. 3 Small pieces are kept for later use.

[0025] Step 2: Cell disruption and enzymatic deproteinization Transfer 500g of the testicular tissue block obtained in step 1 to a homogenizer, add 1000 mL of pre-cooled purified water, and homogenize at 8000 rpm for 5 minutes at 4°C to obtain a uniform milky white homogenate. Keep the temperature low during homogenization to avoid nucleic acid degradation.

[0026] Add proteinase K (final concentration approximately 0.2 g / mL) to the homogenate, adjust the pH to 7.5-8.0 with 1 M Tris-HCl buffer, and gently stir in a 50°C water bath for 3 hours for enzymatic hydrolysis. After hydrolysis, cool the hydrolysate to room temperature, centrifuge at 12000 rpm for 15 minutes, collect the supernatant (containing nucleic acid), and discard the precipitate.

[0027] Step 3: Salting out and purification Add sodium chloride to the supernatant to a final concentration of 1.0 M, allow to stand at 4°C for 30 minutes to salt out, centrifuge at 8000 rpm for 10 minutes, collect the supernatant, add an equal volume of chloroform-isoamyl alcohol mixture (chloroform to isoamyl alcohol volume ratio of 24:1) to the supernatant, shake vigorously for 5 minutes, centrifuge at 8000 rpm for 10 minutes, collect the upper aqueous phase, which is the PN purification solution.

[0028] The concentration of PN purification solution was determined by ultraviolet spectrophotometry. The A260 absorbance was measured, and the concentration of PN purification solution was calculated according to the standard curve. The concentration of PN purification solution was found to be approximately 3.2 mg / mL, A260 / A280 = 1.91, and the protein residue was <2%.

[0029] Step 4: One-step alcohol precipitation of fibrous PN The PN purified solution obtained in step 3 was placed in an ice bath and cooled to 4°C. Under stirring conditions, pre-cooled anhydrous ethanol (4°C) was slowly added at a volume ratio of 1:1.5 while stirring gently. When about half of the ethanol was added, white fibrous substances began to appear in the solution. At this time, the remaining ethanol was added slowly while keeping the stirring speed stable. After all the ethanol was added, stirring was stopped. The precipitation system was allowed to stand at 4°C for 90 minutes to allow the PN molecular chains to fully self-assemble along the orientation direction during the precipitation process, and the fibrous precipitate was completely formed.

[0030] The precipitate was collected to obtain fibrous PN with a fiber diameter of approximately 0.01–1 mm and a fiber length of 4–12 cm.

[0031] Step 5: Washing after sedimentation Transfer the fibrous PN precipitate obtained in step 4 to a clean petri dish, gently pour a 75% ethanol aqueous solution at room temperature onto the surface of the precipitate, let it stand for 15 minutes, gently pour off the washing solution, and repeat the washing once. During the washing process, maintain the integrity of the fibrous precipitate and do not stir or shake violently.

[0032] Step 6: Dehydration and Drying The fibrous PN precipitate obtained from step 5 was soaked in anhydrous ethanol at room temperature (25°C) for 15 minutes. The washing liquid was gently poured off, and then the precipitate was transferred to a freeze dryer. It was pre-frozen at -40°C for 2 hours and freeze-dried at -20°C and a vacuum degree ≤10 Pa for 48 hours to obtain the dried fibrous polynucleotide material, denoted as PN-F-1.

[0033] The morphology of PN-F-1 is as follows Figure 1 As shown, the SEM image is as follows Figure 2 As shown. From Figure 1 As can be seen, this fibrous polynucleotide material exhibits a macroscopic filamentous morphology. Measurements show that the dried diameter of PN-F-1 fibers ranges from 0.01 to 1.0 mm, and the length ranges from 3 to 11 cm. Individual fibers maintain a stable fibrous morphology in the dry state, exhibiting good flexibility, slight elasticity, and resistance to breakage. Upon immersion in physiological saline, they spontaneously swell to form a hydrophilic gel. Figure 2 As can be seen, the PN-F-1 fiber has a directionally arranged three-dimensional porous network structure, which appears as a honeycomb or sponge, with a pore size distribution of 10~100μm.

[0034] The PN-F-1 sample obtained in Example 1 was tested according to the general methods of the 2025 edition of the Chinese Pharmacopoeia. The purity was determined to be 98.3% by ultraviolet spectrophotometry, and A260 / A280 = 1.82.

[0035] The molecular weight of the PN-F-1 samples was analyzed by agarose gel electrophoresis (1.2% agarose, TAE buffer, 100 V for 45 minutes, ethidium bromide staining). The results are as follows: Figure 3 As shown, the PN-F-1 sample exhibits a clear diffuse band, with a molecular weight range of 100~5000 bp. The main band is concentrated in the 200~3000 bp range, which is consistent with the molecular weight distribution characteristics of PN. This indicates that the fibrous self-assembly during alcohol precipitation did not significantly degrade the molecular weight of PN.

[0036] Example 2: Effect of different PN purification solution concentrations on the formation of fibrous structures Following the preparation method of Example 1, after step 3, the concentration of the PN purification solution was adjusted to 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.2 mg / mL (concentration of Example 1), 4.0 mg / mL, 6.0 mg / mL, 7.0 mg / mL, and 8.0 mg / mL, respectively, by dilution or concentration, while keeping other conditions unchanged. The effect of different PN purification solution concentrations on the formation of fibrous structures was investigated. Each concentration was measured in triplicate. The results are shown in Table 1.

[0037] Table 1. Effect of different PN purification solution concentrations on the formation of fibrous structures.

[0038] The results showed that PN purification solution concentrations within the range of 1.0–6.0 mg / mL could form fibrous structures, with the most uniform fiber morphology and highest yield observed at concentrations of 2.0–4.0 mg / mL. When the PN purification solution concentration was below 1.0 mg / mL, the intermolecular chain interactions were weak, resulting in the precipitate being dispersed into fine particles and difficult to form continuous fibers. When the PN purification solution concentration was above 6.0 mg / mL, the solution viscosity was too high, restricting molecular chain movement, and the precipitate was mainly amorphous lumps or flocs.

[0039] Example 3: Effect of different salt ion types on the formation of fibrous structures Following the preparation method of Example 1, sodium chloride (NaCl, Example 1), potassium chloride (KCl), sodium acetate (CH3COONa), and potassium acetate (CH3COOK) were added to a final concentration of 1.0 M in step 3, while keeping other conditions unchanged, to investigate the effect of different salt ion types on the formation of fibrous structures. The results are shown in Table 2.

[0040] Table 2. Effects of different salt ion types on the formation of fibrous structures

[0041] The results showed that the addition of salt was the key factor in the formation of fibrous precipitates. Without salt, the alcohol precipitate was a white flocculent or powdery substance, without the formation of fibrous structures. All four salt ions could effectively induce the formation of fibrous structures, with sodium chloride and sodium acetate showing slightly better effects and producing the best uniformity of fibers.

[0042] Example 4: Effect of different salt concentrations on the formation of fibrous structures Following the preparation method of Example 1, sodium chloride was added in step 3 and the final concentrations were adjusted to 0.2 M, 0.5 M, 1.0 M (Example 1), 1.5 M, 2.0 M, 2.5 M, and 3.0 M, respectively, with other conditions remaining unchanged. The effect of salt concentration on the formation of fibrous structures was investigated. The results are shown in Table 3.

[0043] Table 3. Effects of different salt concentrations on the formation of fibrous structures

[0044] The results showed that fibrous structures could be formed in NaCl concentrations ranging from 0.5 to 2.0 M, with the most regular fiber morphology observed at concentrations of 1.0 to 1.5 M. When the NaCl concentration was below 0.5 M, electrostatic repulsion was insufficient to shield the structure, making it difficult for molecular chains to aggregate in an orderly manner, resulting in an indistinct fibrous structure. When the concentration was above 2.0 M, the precipitation rate was too fast, and the fibers were coarse and prone to sticking together.

[0045] Example 5: Effect of different types of alcohol precipitants on the formation of fibrous structures Following the preparation method of Example 1, in step 4, anhydrous ethanol (Example 1), 95% ethanol, 90% ethanol, 85% ethanol, 80% ethanol, 75% ethanol, and isopropanol were used as alcohol precipitants, with other conditions remaining unchanged, to investigate the effect of different alcohol precipitants on the formation of fibrous structures. The results are shown in Table 4.

[0046] Table 4. Effects of different alcohol precipitants on the formation of fibrous structures

[0047] The results showed that anhydrous ethanol and 95% ethanol produced the most complete fibrous structures when used as precipitants, while the fiber formation effect decreased significantly when the ethanol concentration was below 85%. This result is consistent with the basic principle of PN precipitation: the final concentration of ethanol in the aqueous solution is the key factor for PN precipitation. When the final ethanol concentration is below about 50%, PN is difficult to precipitate effectively; when the final concentration is in the range of 50% to 60%, the precipitation rate is moderate, and the PN molecular chains can obtain sufficient time to arrange themselves in an orderly manner to form a fibrous structure; when the final concentration is too high (such as when a high proportion of anhydrous ethanol is added directly, the local concentration is too high), precipitation is too fast. However, this invention maintains the final concentration within a suitable range by controlling the proportion of ethanol added, thus ensuring the formation of the fibrous structure.

[0048] Example 6 Effect of different alcohol addition volume ratios on the formation of fibrous structures Following the preparation method of Example 1, in step 4, pre-cooled anhydrous ethanol was added at volume ratios of PN purification solution to anhydrous ethanol of 1:0.5, 1:0.8, 1:1, 1:1.5 (Example 1), 1:2, 1:2.5, and 1:3, respectively, with other conditions remaining unchanged, to investigate the effect of the alcohol addition volume ratio on the formation of fibrous structures. The results are shown in Table 5.

[0049] Table 5. Effect of different alcohol addition volume ratios on the formation of fibrous structures.

[0050] The results showed that when the volume ratio of PN purification solution to anhydrous ethanol was 1:1 to 1:2 (final ethanol concentration 50%~67%), obvious fibrous precipitates could be formed, with the most intact fiber morphology observed at a ratio of 1:1.5 (final ethanol concentration approximately 60%). When the amount of ethanol added was too high (final ethanol concentration ≥71%), both the local and overall ethanol concentrations were too high, causing the PN molecular chains to desolvate abruptly and aggregate before they could arrange themselves in an orderly manner. The excessively rapid precipitation rate made it difficult for fibrous structures to form, and the product was mainly a flocculent precipitate.

[0051] Example 7: Effect of different alcohol precipitation temperatures on the formation of fibrous structures Following the preparation method of Example 1, in step 4, the alcohol precipitation temperature was set to -10℃, 0℃, 4℃ (Example 1), 10℃, 15℃, and 25℃ (room temperature), respectively, with other conditions remaining unchanged, to investigate the effect of alcohol precipitation temperature on the formation of fibrous structures. The results are shown in Table 6.

[0052] Table 6. Effect of different alcohol precipitation temperatures on the formation of fibrous structures.

[0053] The results showed that the fibrous structure formed best at a precipitation temperature of 0-4℃, exhibiting complete morphology and good flexibility. At excessively low temperatures (-10℃), PN precipitation was incomplete and the fibers became brittle; at excessively high temperatures (above 10℃), the thermal motion of the PN molecular chains was intense, making it difficult for them to arrange themselves in an orderly manner, and the precipitation was mainly flocculent.

[0054] Example 8: Effect of different precipitation and settling times on the formation of fibrous structures Following the preparation method of Example 1, in step 4, the settling time after alcohol precipitation was set to 10 min, 30 min, 60 min, 90 min (Example 1), 120 min, and 180 min, respectively, with other conditions remaining unchanged, to investigate the effect of settling time on the formation of fibrous structures. The results are shown in Table 7.

[0055] Table 7. Effects of different settling times on the formation of fibrous structures.

[0056] The results showed that precipitation was complete when the settling time reached 60 minutes, and further extending the settling time did not significantly improve the fiber morphology. When the settling time was too short (less than 30 minutes), precipitation was incomplete, and the fiber length was too short.

[0057] Example 9: Effect of different washing methods on the retention of fibrous structure Following the preparation method of Example 1, in step 5, the washing methods were set as follows: static rinsing (the washing liquid was gently poured onto the surface of the precipitate, and after standing for 15 minutes, it was gently poured off, Example 1), stirring rinsing (the precipitate was stirred with a glass rod and mixed with the ethanol solution, and then allowed to stand), and rinsing by pouring (the washing liquid was directly poured into the container and then immediately poured out). Other conditions remained unchanged, and the effect of different washing methods on the retention of the fibrous structure was investigated. The results are shown in Table 8.

[0058] Table 8. Effects of different washing methods on the retention of fibrous structure

[0059] The results showed that static tilting washing is the washing method that causes the least damage to the fiber structure. After washing, the fiber morphology remains intact, and the fiber breakage rate is less than 5%. Agitation washing will severely damage the fiber structure and should be strictly avoided.

[0060] Example 10: Effects of different drying methods on the morphology of fibrous structures The fibrous PN precipitate after washing in step 6 of Example 1 was processed using the following drying methods: Vacuum drying at room temperature: 25℃, vacuum degree ≤0.08 MPa, drying for 24 hours.

[0061] Freeze-drying (Example 1): Pre-freeze at -40℃ for 2 hours, then freeze-dry at -20℃ and vacuum degree ≤10 Pa for 48 hours.

[0062] Low-temperature drying: 35℃ oven, normal pressure, dry for 12 hours.

[0063] Air dry at room temperature: 25℃, normal pressure, and ventilation, for 48 hours.

[0064] The results are shown in Table 9.

[0065] Table 9. Effects of different drying methods on the morphology of fibrous structures.

[0066] The results show that the fibrous PN material obtained by freeze-drying has a more obvious porous microstructure and the best expansion performance after rehydration, which is the preferred drying method of this invention.

[0067] Comparative Example 1: Comparison of Conventional PN Powder PN was extracted using the same process as in Example 1, but the precipitate was vigorously stirred during the alcohol precipitation process in step 4 to disperse it in a flocculent manner. No special protection was provided during the washing and drying processes, and conventional PN powder was finally obtained, which is referred to as Comparative Example 1.

[0068] Water absorption swelling rate: The gravimetric method was used. The initial sample was dried to constant weight and recorded as W1. The sample was then immersed in PBS solution for 1 hour. After being taken out, the surface moisture was absorbed with absorbent paper and weighed again and recorded as W2. Water absorption swelling rate = [(W2-W1) / W1]×100%.

[0069] The water absorption swelling rate of the conventional PN powder obtained in Comparative Example 1 was 310±30%, which was significantly lower than the water absorption swelling rate of PN-F-1 obtained in Example 1 (920±70%).

[0070] The results show that the PN powder extracted by conventional processes lacks a macroscopic physical support structure and is significantly inferior to the PN-F-1 fibrous material of this invention in terms of water absorption and swelling performance.

[0071] Comparative Example 2: Comparison of PN powder preparation followed by dissolution and alcohol precipitation (simulating existing post-processing routes) Take the PN powder obtained from Comparative Example 1, redissolve it in purified water to prepare a 3.2 mg / mL PN solution, and carry out alcohol precipitation, washing, dehydration and drying according to the conditions of steps 4 to 6 of Example 1 to prepare fibrous PN material, which is referred to as Comparative Example 2.

[0072] The diameter of the fibers obtained in Comparative Example 2 was 0.7–1.3 mm. The reason for this is that during the redissolution of the PN powder, some PN molecular chains underwent irreversible aggregation and conformational changes. This resulted in the redissolved PN solution failing to achieve the same uniform dispersion and molecular chain extension as the original purified solution, affecting the fiber formation effect during subsequent alcohol precipitation. This comparative example demonstrates that the "one-step method" of directly forming a fibrous structure during the extraction stage is superior in terms of material properties to the post-processing route of "preparing powder first and then reshaping."

[0073] Comparative Example 3: Alcohol precipitant added in reverse order (fibers could not form). The preparation method of Example 1 was followed, but step 4 was adjusted as follows: pre-cooled anhydrous ethanol was placed in a container first, and then the PN purification solution obtained in step 3 was slowly added to the anhydrous ethanol (i.e., the PN purification solution was added to the ethanol, rather than the ethanol being added to the PN purification solution). Other conditions remained unchanged, and PN material was prepared, which was recorded as Comparative Example 3.

[0074] Results: A large amount of precipitate formed instantly, appearing as white flocculent material that settled rapidly without forming a fibrous structure. The final product was a white flocculent powder. Analysis of the cause: When the PN purification solution was added to a large amount of alcohol, the local alcohol concentration was too high, causing the PN molecular chains to desolvate abruptly and aggregate and precipitate before any orderly arrangement could occur, thus preventing the formation of a fibrous structure. This comparative example demonstrates that the order of alcohol addition (addition of alcohol to the aqueous phase) is one of the key process parameters for achieving a fibrous structure.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting fibrous polynucleotide materials, characterized in that, Includes the following steps: (1) Raw material pretreatment: Take fresh or frozen salmonid testis tissue, clean, drain and cut into pieces for later use; (2) Cell disruption and enzymatic hydrolysis: The testis tissue block obtained in step (1) was homogenized and disrupted under low temperature conditions. Proteolytic enzyme was added to the homogenate for enzymatic hydrolysis, and the supernatant was collected by centrifugation. (3) Salting out and purification: Salt out the supernatant obtained in step (2), collect the supernatant by centrifugation, add organic solvent for extraction, and obtain polynucleotide purified solution; (4) One-step alcohol precipitation: Adjust the concentration of the polynucleotide purification solution obtained in step (3) to 1.0~6.0 mg / mL, add alcohol precipitant to the polynucleotide purification solution at a volume ratio of 1:1~2 under low temperature conditions, let stand for 60~180 min, collect the precipitate to obtain fibrous polynucleotide material.

2. The method for extracting fibrous polynucleotide materials according to claim 1, characterized in that, It also includes the following steps: The precipitate obtained in step (4) is washed, dehydrated and dried to obtain a dry fibrous polynucleotide material.

3. The method for extracting fibrous polynucleotide materials according to claim 1, characterized in that, In step (2), the homogenization and crushing involves transferring testicular tissue blocks to a homogenizer, adding purified water, and performing the homogenization at 4~10℃ and 7000~10000rpm.

4. The method for extracting fibrous polynucleotide materials according to claim 3, characterized in that, In step (2), the ratio of testicular tissue block to purified water is 1g:1~5mL.

5. The method for extracting fibrous polynucleotide materials according to claim 1, characterized in that, In step (2), the enzymatic hydrolysis involves adding proteolytic enzyme to the homogenate, adjusting the pH to 6.5-8.5 with buffer solution, and stirring the homogenate in a water bath at 40-55°C for 1-5 hours; the final concentration of the proteolytic enzyme is 0.1-1 g / mL.

6. The method for extracting fibrous polynucleotide materials according to claim 1, characterized in that, In step (3), the salting out involves adding salt to the supernatant to a final concentration of 0.5~2.0M and allowing it to stand at 4~10℃ for salting out.

7. The method for extracting fibrous polynucleotide materials according to claim 1, characterized in that, In step (4), the low temperature is 0~10℃.

8. The method for extracting fibrous polynucleotide materials according to claim 1 or 7, characterized in that, The alcohol precipitant is an aqueous solution of ethanol with a volume fraction of 90% to 100%.

9. The method for extracting fibrous polynucleotide materials according to claim 2, characterized in that, The washing process involves washing the food 1 to 3 times with a 70% to 95% ethanol aqueous solution. The dehydration and drying process involves soaking the food in anhydrous ethanol, pre-freezing it at -80 to -20°C for 1 to 4 hours, and then freeze-drying it at -20°C under a vacuum of ≤10 Pa.

10. A fibrous polynucleotide material, characterized in that, It is obtained by the extraction method described in any one of claims 1 to 9.

Images