A spleen polypeptide extract, a preparation method and application thereof

Abstract

The application relates to the technical field of biological extraction, and discloses a spleen polypeptide extract as well as a preparation method and application thereof. The preparation method takes fresh spleen tissue as raw material, and realizes preliminary separation of proteins, polypeptides and nucleic acids through an optimized isoelectric point grading process after pretreatment, homogenization, repeated freeze-thaw crushing, centrifugal separation and ultrafiltration grading, and finally obtains high-content products by adopting targeted chromatography purification technology. The spleen polypeptide extract and related products can be widely applied to the fields of biological medicines (such as immunomodulators and blood substitutes), food additives (such as protein fortifiers) and scientific reagent fields, the process is mild and has good repeatability, the comprehensive utilization of the spleen raw material is realized, the product added value is improved, and the application prospect of industrialization is good.

Description

Technical Field

[0001] This invention relates to the field of bio-extraction technology, specifically to a spleen polypeptide extract, its preparation method, and its application. Background Technology

[0002] The spleen, as an important immune and hematopoietic organ, is rich in various proteins such as hemoglobin, actin, and myosin, as well as abundant bioactive components such as polypeptides and nucleic acids. These components have extremely high application value in fields such as biomedicine, food processing, and scientific research. However, the spleen tissue has a complex composition with multiple biomolecules coexisting and influencing each other, making separation and purification difficult. Traditional extraction processes often only target specific types of components and suffer from problems such as low extraction efficiency and insufficient purity of the target components.

[0003] In existing technologies, the preparation of spleen extracts mostly employs a basic process of pretreatment-homogenization-freeze-thaw-centrifugation-ultrafiltration. Some processes add isoelectric point fractionation and chromatographic purification steps to enrich proteins. However, these processes still have significant drawbacks: on the one hand, they can only focus on the extraction of protein components, failing to effectively separate and utilize other high-value components such as peptides and nucleic acids, resulting in a waste of raw material resources; on the other hand, even for protein components, the separation and purification precision is limited, making it difficult to meet the stringent requirements of the biopharmaceutical field for high-purity bioactive components. Furthermore, in existing processes, the parameters for isoelectric point fractionation are not sufficiently optimized, and the elution conditions for chromatographic purification lack specificity, further hindering the improvement of the recovery rate and purity of target components, thus limiting the industrial application and value exploitation of spleen extracts.

[0004] Therefore, developing a method for preparing spleen polypeptide extracts that can fully utilize spleen raw materials to simultaneously or selectively prepare high-content polypeptide, high-content nucleic acid, and high-content protein products, and significantly improve the extraction efficiency and purity of target components, has become a pressing technical problem to be solved in this field. Summary of the Invention

[0005] Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a spleen polypeptide extract, its preparation method, and its applications. By adding targeted purification steps, it achieves efficient preparation of high-content polypeptide, high-content nucleic acid, and high-content protein products, solving the problem of separation and purification difficulties caused by the coexistence of multiple components in spleen tissue. It significantly improves the extraction efficiency and purity of target components, while realizing the comprehensive utilization of spleen raw materials, thereby increasing resource utilization and product added value.

[0006] Technical solution To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a spleen polypeptide extract, comprising the following steps: S1. Spleen tissue pretreatment: Select fresh spleen tissue, remove fat, connective tissue and fascia, rinse with sterile saline and cut into pieces, pre-freeze for later use; This pretreatment step can effectively remove impurities that interfere with the subsequent extraction process, and the pre-freezing treatment can cause the spleen tissue cells to shrink initially, which is convenient for subsequent homogenization and crushing operations. S2. Homogenization: The pre-frozen spleen tissue blocks were added to pre-cooled sterile physiological saline at a material-to-liquid ratio of 1:3-1:5 (g / mL) and homogenized at 8000-10000 r / min under ice bath conditions to obtain spleen homogenate. The ice bath condition can prevent the heat generated during homogenization from causing denaturation of bioactive components. The optimized material-to-liquid ratio and rotation speed can ensure that the spleen tissue is fully broken down and release the internal bioactive components. S3. Repeated freeze-thaw disruption: The spleen homogenate is frozen at -80℃ for 1-2 hours and then thawed at 37℃, and the freeze-thaw process is repeated. Repeated freeze-thaw can use the mechanical action of ice crystals to disrupt cell structure. Compared with single freeze-thaw or other disruption methods, it can release intracellular proteins, peptides, nucleic acids and other components more fully, and will not introduce chemical reagents, thus avoiding contamination of the target components. S4. Centrifugation: Centrifuge at 4℃ and 8000-12000r / min, and collect the supernatant. Centrifugation can effectively separate the bioactive components and precipitated impurities in the supernatant, and improve the purity of the supernatant. S5. Ultrafiltration: Ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 5-10 kDa at 0.1-0.3 MPa and 4-10℃. The permeate and retentate are collected. Ultrafiltration can achieve the preliminary separation of components with different molecular weights, laying the foundation for subsequent targeted purification. S6. Isoelectric point fractionation: The permeate and retentate collected in S5 were fractionated by isoelectric point fractionation. For the retentate (large protein molecules): Adjust the pH of the retentate to 4.5-5.5 (the isoelectric point range of hemoglobin) with 0.1 mol / L HCl or NaOH. After standing for 2-3 hours, centrifuge at 4℃ and 6000-8000 r / min for 10-15 minutes and collect the precipitate, which is the crude protein precipitate. Then adjust the pH to 5.8-6.8 (the isoelectric point range of actin and myosin), repeat the above standing and centrifugation operations, collect the precipitate, and combine the two precipitates to obtain the mixed crude protein.

[0007] For permeate solutions (peptides, nucleic acids, small proteins): Adjust the pH of the permeate solution to 2.5-3.5 (isoelectric point range of peptides) with 0.1 mol / L HCl or NaOH. After standing for 1-2 hours, centrifuge at 4℃ and 6000-8000 r / min for 10-15 minutes and collect the precipitate, which is the crude peptide precipitate. Adjust the pH of the remaining supernatant to 4.0-5.0 (isoelectric point range of nucleic acids), let it stand for 1-2 hours, and then centrifuge to collect the precipitate, which is the crude nucleic acid precipitate.

[0008] This step achieves preliminary separation of proteins, peptides, and nucleic acids by optimizing the isoelectric point range and fractionation parameters of different components, solving the separation problem caused by the coexistence of multiple components in traditional processes.

[0009] S7. Chromatographic purification: Crude polypeptides, crude proteins, and crude nucleic acids are purified by chromatography to obtain high-content polypeptide products, high-content protein products, and high-content nucleic acid products, respectively. The crude protein, crude peptides, and crude nucleic acids obtained from S6 were purified by chromatography to obtain high-content target products. Purification of S7.1 High-content protein products The crude protein mixture was dissolved in sterile physiological saline and loaded onto a pre-equilibrated DEAE-cellulose ion exchange chromatography column. Elution was performed using a 0-1 mol / L NaCl gradient with Tris-HCl buffer (pH 7.5-8.5) at a flow rate of 1-2 mL / min. Elution peaks were detected, and the corresponding hemoglobin, actin, and myosin elution peaks were collected. Each component was further purified using a Sephadex G-75 gel filtration chromatography column. The target elution peak was collected to obtain a high-purity protein product (hemoglobin purity ≥95%, actin purity ≥94%, myosin purity ≥93%).

[0010] Purification of S7.2 High-content peptide products The crude polypeptide precipitate was dissolved in sterile physiological saline and loaded onto a pre-equilibrated reversed-phase high-performance liquid chromatography (RP-HPLC) column. Acetonitrile-water (containing 0.1% trifluoroacetic acid) was used as the mobile phase, and acetonitrile gradient elution was performed (acetonitrile volume fraction increased from 10% to 60%) at a flow rate of 0.8-1.2 mL / min. The detection wavelength was 220 nm. The target polypeptide elution peak was collected, concentrated by rotary evaporation, and freeze-dried to obtain a high-content polypeptide product (polypeptide content ≥90%).

[0011] Purification of S7.3 High-content nucleic acid products The crude nucleic acid precipitate was dissolved in TE buffer (pH 8.0), and RNase A (final concentration 10-20 μg / mL) was added. The mixture was incubated at 37°C for 30-60 min to remove RNA. The precipitate was then loaded onto a Sepharose CL-6B gel filtration chromatography column, using TE buffer as the elution buffer at a flow rate of 1-1.5 mL / min. The DNA elution peak was collected, precipitated with ethanol, washed, and dried to obtain a high-content nucleic acid product (nucleic acid content ≥ 88%). S8. Product processing: The above high-content product is aseptically filtered, and after passing the test, it is freeze-dried or made into a liquid preparation to obtain the product. The extract contains ≥90% polypeptides, the product contains ≥95% hemoglobin, ≥94% actin, ≥93% myosin, and ≥88% nucleic acid. In the biomedical field, the spleen polypeptide extract is used to prepare immunomodulators, the high-content protein product is used to prepare blood substitutes or oxygen carriers, and the high-content nucleic acid product is used to prepare gene therapy vectors or nucleic acid vaccines.

[0012] Furthermore, the homogenization time is 3-5 minutes.

[0013] Furthermore, the freeze-thaw cycle is repeated 3-5 times.

[0014] Furthermore, the centrifugation time is 15-20 minutes.

[0015] Furthermore, the specific operation of isoelectric point classification in step S6 is as follows: For the retentate: Adjust the pH to 4.5-5.5, let stand for 2-3 hours, then centrifuge to collect the crude protein precipitate; then adjust the pH to 5.8-6.8, let stand, centrifuge to collect the precipitate, and combine them to obtain mixed crude protein; For the permeate: adjust the pH to 2.5-3.5, let stand for 1-2 hours, and then centrifuge to collect the crude peptide precipitate; adjust the pH of the remaining supernatant to 4.0-5.0, let stand, and then centrifuge to collect the crude nucleic acid precipitate.

[0016] Furthermore, the specific procedures for chromatographic purification are as follows: High-content protein products: Purification was carried out in two steps using DEAE-cellulose ion exchange chromatography column + Sephadex G-75 gel filtration chromatography column, with Tris-HCl buffer as the elution buffer and NaCl gradient elution, and the elution peak of the target protein was collected. High-content peptide products: Purified using RP-HPLC column, with acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase, acetonitrile gradient elution, detection wavelength 220nm, and collection of the target peptide elution peak; High-content nucleic acid products: After treatment with RNase A, the products were purified using a Sepharose CL-6B gel filtration chromatography column with TE buffer as the elution solution, and the elution peak of the target nucleic acid was collected.

[0017] Furthermore, it has applications in the fields of biomedicine, food additives, or scientific research reagents.

[0018] Beneficial technical effects: This invention, based on traditional spleen extraction technology, adds a chromatographic purification step, enabling the simultaneous or selective preparation of high-content peptides, nucleic acids, and proteins. This overcomes the limitations of existing processes that can only extract a single type of component, improving the comprehensive utilization rate of spleen raw materials and the added value of products. By optimizing process parameters such as the pH range for isoelectric point fractionation and the elution conditions for chromatographic purification, the invention effectively solves the problem of separation and purification difficulties caused by the coexistence of multiple components in spleen tissue, significantly improving the extraction efficiency and purity of target components. This invention can prepare different products such as high-content peptides, proteins, and nucleic acids according to needs, offering flexible product forms and wide applicability in multiple fields such as biomedicine, food additives, and scientific reagents, with broad market prospects. The entire preparation process of this invention employs gentle treatment methods such as physical crushing, fractionation, and chromatography, avoiding the damage of chemical reagents to bioactive components. Furthermore, the process parameters are clearly defined, with good reproducibility, making it suitable for large-scale industrial production. Attached Figure Description

[0019] Figure 1 This is an electrophoretic detection image of DNA from a high-content nucleic acid product in Example 3. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Peptide content determination: Reversed-phase high-performance liquid chromatography (RP-HPLC) was used. A C18 column (4.6 mm × 250 mm, 5 μm) was selected. Acetonitrile-water (containing 0.1% trifluoroacetic acid) was used as the mobile phase, with acetonitrile volume fraction increasing from 10% to 60% in a gradient elution for 30 min at a flow rate of 1.0 mL / min. The detection wavelength was 220 nm. The peptide content was calculated by peak area normalization.

[0022] Protein purity test: The first step involved preliminary separation using DEAE-cellulose ion exchange chromatography. The chromatographic column was 5 cm × 50 cm, and the elution buffer was Tris-HCl (pH 8.0, concentration 0.02 mol / L). The elution was carried out using a gradient of 0-1 mol / L NaCl (elution time 60 min), the flow rate was 1.5 mL / min, and the detection wavelength was 280 nm.

[0023] The second step involves further purification using a Sephadex G-75 gel filtration chromatography column (4cm×60cm), with sterile physiological saline as the elution buffer, a flow rate of 1.0mL / min, and a detection wavelength of 280nm. The purity of the target protein is calculated based on the purity of the elution peak.

[0024] Nucleic acid content testing: Gel filtration chromatography combined with ultraviolet spectrophotometry was used. Purification was performed using a Sepharose CL-6B gel filtration chromatography column (4cm × 50cm), with TE buffer as the elution buffer, a flow rate of 1.2 mL / min, and a detection wavelength of 260 nm. After collecting the target elution peak, the nucleic acid concentration and purity were determined using an ultraviolet spectrophotometer.

[0025] Extraction rate calculation: Extraction rate = (mass of target component in final product / theoretical total mass of target component in raw material) × 100%.

[0026] Example 1 (Preparation of high-content polypeptide products) S1. Spleen tissue pretreatment: Select 1000g of fresh pig spleen tissue, remove the attached adipose tissue, connective tissue and fascia and other impurities (about 120g of impurities were removed, leaving 880g of spleen tissue), rinse 4 times with 500mL of sterile physiological saline (0.9% NaCl), drain the water and cut into 0.8cm³ pieces, place in a -20℃ freezer for 3 hours for later use.

[0027] S2. Homogenization: 880g of pre-frozen spleen tissue block was placed in a high-speed tissue homogenizer. 3520mL of sterile physiological saline pre-cooled to 4℃ was added at a material-to-liquid ratio of 1:4 (g / mL). The homogenizer was homogenized at 9000r / min for 4min under ice bath conditions to obtain 4400mL of spleen homogenate.

[0028] S3. Repeated freeze-thaw disruption: Divide 4400 mL of spleen homogenate into 4 equal portions (1100 mL each), freeze each portion at -80°C for 1.5 h, remove them and thaw them in a 37°C constant temperature water bath until completely thawed. Repeat the above freeze-thaw operation 4 times to rupture the cells, release the intracellular components, and combine them to obtain 4400 mL of freeze-thawed homogenate.

[0029] S4. Centrifugation: Place 4400 mL of the frozen-thawed homogenate into a high-speed refrigerated centrifuge and centrifuge at 4℃ and 10000 r / min for 18 min. Collect 3850 mL of supernatant (removing about 550 g of precipitate, which is cell debris, unbroken tissue and other impurities).

[0030] S5. Ultrafiltration treatment: Pass 3850 mL of centrifuged supernatant through an ultrafiltration membrane with a molecular weight cutoff of 8 kDa. The operating pressure is 0.2 MPa and the temperature is controlled at 6℃. Collect 3200 mL of permeate (containing peptides, small molecules, nucleic acids, etc.) and 650 mL of retentate (containing large molecules, for later use).

[0031] S6. Isoelectric point fractionation: Take 3200 mL of permeate, adjust the pH to 3.0 with 0.1 mol / L HCl solution, let it stand at room temperature for 1.5 h, and then centrifuge at 4 °C and 7000 r / min for 12 min to collect 28.5 g of crude peptide precipitate.

[0032] S7. Chromatographic purification: Dissolve 28.5g of crude polypeptide precipitate in 50mL of sterile physiological saline, and load the sample onto a pre-equilibrated reversed-phase high-performance liquid chromatography (RP-HPLC) column (column model: C18, 4.6mm×250mm, 5μm). Use acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase, and perform gradient elution with acetonitrile (acetonitrile volume fraction increased from 10% to 60%, elution time 30min), flow rate 1.0mL / min, detection wavelength 220nm. Collect 800mL of eluent corresponding to the target polypeptide elution peak, concentrate by rotary evaporation (temperature 45℃, vacuum degree -0.09MPa) to 10mL, and then freeze-dry (temperature -50℃, vacuum degree 10Pa) to obtain a high-content polypeptide product.

[0033] S8. Product processing: The high-content polypeptide product was aseptically filtered through a 0.22μm filter membrane, and after passing the test, it was freeze-dried to finally obtain 12.8g of spleen polypeptide extract with a polypeptide content of 92.3% and a polypeptide extraction rate of 68.5%.

[0034] Example 2 (Preparation of high-protein products) Steps S1-S5 are the same as in Example 1, except that 650 mL of the ultrafiltration retentate (containing large molecular proteins) prepared in Example 1 is taken.

[0035] S6. Isoelectric point fractionation: Adjust the pH of 650 mL of retentate to 5.0 with 0.1 mol / L NaOH solution, let it stand at room temperature for 2.5 h, and then centrifuge at 4 °C and 7000 r / min for 12 min to collect the first crude protein precipitate (mainly hemoglobin) of 45.2 g; then adjust the pH of the remaining supernatant to 6.3 with 0.1 mol / L NaOH solution, let it stand at room temperature for 2.5 h, and then collect the second crude protein precipitate (mainly actin and myosin) of 32.8 g under the same centrifugation conditions. Combine the two precipitates to obtain a mixed crude protein of 78.0 g.

[0036] S7. Chromatographic purification: Dissolve 78.0g of mixed crude protein in 100mL of sterile physiological saline and load the sample onto a pre-equilibrated DEAE-cellulose ion exchange column (column size: 5cm×50cm). Use Tris-HCl buffer (pH 8.0, concentration 0.02mol / L) as the elution buffer and perform gradient elution with 0-1mol / L NaCl (elution time 60min), flow rate 1.5mL / min, and detection wavelength 280nm. Collect the elution peaks of hemoglobin, actin, and myosin separately, with 1200mL of hemoglobin elution peak, 800mL of actin elution peak, and 600mL of myosin elution peak. Further purify each fraction by passing it through a Sephadex G-75 gel filtration chromatography column (column size: 4cm×60cm) with sterile physiological saline as the elution buffer at a flow rate of 1.0mL / min. Collect the elution peaks of each target protein, combine the elutions of the same target protein, and concentrate them.

[0037] S8. Product Processing: The concentrates of each target protein were aseptically filtered through a 0.22μm filter membrane. After passing the test, they were freeze-dried to obtain 18.5g of hemoglobin with a purity of 95.6%, 12.3g of actin with a purity of 94.5%, and 9.8g of myosin with a purity of 93.8%. The total protein extraction rate was 75.2%.

[0038] Example 3 (Preparation of high-content nucleic acid products) Steps S1-S6 are the same as in Example 1. Take 3100 mL of the remaining supernatant after isoelectric point fractionation and collection of crude polypeptide precipitate in Example 1.

[0039] S6. Isoelectric point fractionation (nucleic acid separation): Adjust the pH of 3100 mL of the remaining supernatant to 4.5 with 0.1 mol / L NaOH solution, let it stand at room temperature for 1.5 h, and then centrifuge at 4 °C and 7000 r / min for 12 min to collect 15.6 g of crude nucleic acid precipitate.

[0040] S7. Chromatographic purification: 15.6 g of crude nucleic acid precipitate was dissolved in 50 mL of LTE buffer (pH 8.0, containing 10 mmol / L Tris-HCl and 1 mmol / L EDTA), and RNase A (final concentration 15 μg / mL) was added. The mixture was incubated at 37°C for 45 min to remove RNA. The treated solution was then loaded onto a Sepharose CL-6B gel filtration chromatography column (column size: 4 cm × 50 cm). TE buffer was used as the elution buffer at a flow rate of 1.2 mL / min and a detection wavelength of 260 nm. 500 mL of the elution buffer corresponding to the DNA elution peak was collected. The elution was then subjected to ethanol precipitation (1.25 L of anhydrous ethanol was added, and the mixture was allowed to stand at -20°C for 2 h), centrifuged (4°C, 10000 r / min for 10 min), washed twice with 75% ethanol, and dried to obtain a high-content nucleic acid product.

[0041] S8. Product processing: The high-content nucleic acid product was aseptically filtered through a 0.22μm filter membrane, dried after passing the test, and finally obtained 6.3g of high-content nucleic acid product with a nucleic acid content of 89.5% and a nucleic acid extraction rate of 52.8%.

[0042] Example 4 (Product Application Test) The spleen polypeptide extract (polypeptide content 92.3%) prepared in Example 1 was used in a mouse immune function enhancement experiment: Forty SPF-grade Kunming mice (weighing 18-22g, half male and half female) were randomly divided into a control group and an experimental group, with 20 mice in each group. Mice in the experimental group were intraperitoneally injected with the spleen polypeptide extract (dose 5mg / kg, diluted to 0.2mL / mouse with sterile saline), while the control group was injected with the same volume of sterile saline. Injections were repeated for 7 consecutive days. Twenty-four hours after the last injection, blood was collected from the orbital sinus to detect the total white blood cell count, lymphocyte count, and immunoglobulin (IgG, IgA) levels in the peripheral blood of the mice. The detection was performed using an automated blood cell analyzer and enzyme-linked immunosorbent assay (ELISA). The results showed that the total white blood cell count in the experimental group was 8.2×10⁻⁶. 9 / L), lymphocyte count (4.5×10 9 The levels of IgG (185 mg / dL) and IgA (32 mg / dL) were significantly higher than those of the control group (5.1 × 10⁻⁶ mg / dL, respectively). 9 / L, 2.3×10 9 The differences between the extracts (120 mg / dL, 18 mg / dL, and 120 mg / dL) were statistically significant (P<0.05), indicating that the spleen polypeptide extract has significant immunomodulatory activity and can be used in the biomedical field to prepare immunomodulators.

[0043] The high-protein product (hemoglobin purity 95.6%) prepared in Example 2 was added to dairy products. 1000 mL of commercially available pure milk (protein content 2.5 g / 100 mL) was mixed with 15 g of the hemoglobin product, stirred to dissolve, and then pasteurized (65℃, 30 min) to produce a fortified protein dairy product. Test results showed that the protein content of this dairy product was 3.8 g / 100 mL, significantly higher than the unadded group. The product also had a good taste and no off-odor. Sensory evaluation by 30 volunteers showed an overall satisfaction rate of 89%, indicating that this high-protein product can be used as a food additive in dairy processing to improve the protein content and quality of food.

[0044] The high-content nucleic acid product (89.5% nucleic acid content) prepared in Example 3 was used as a research reagent for DNA electrophoresis experiments: 5 μg of the nucleic acid product was mixed with 1 μL of 6× loading buffer and added to a 1% agarose gel. TAE buffer was used as the electrophoresis buffer, and electrophoresis was performed at 120V for 30 min. After EB staining, the gel was observed using a gel imaging system. The results showed that the electrophoretic bands of the nucleic acid product were clear, without any impurities, and showed no significant shift compared to the standard bands. The purity met the experimental requirements and it can be used as a standard or reagent for scientific research experiments.

[0045] The spleen polypeptide extract (polypeptide content 92.3%) prepared in Example 1 was used in a mouse immune function enhancement experiment: Forty SPF-grade Kunming mice were randomly divided into a control group and an experimental group, with 20 mice in each group. Mice in the experimental group were intraperitoneally injected with the spleen polypeptide extract (dose 5 mg / kg), while the control group was injected with an equal volume of sterile saline. Injections were repeated for 7 consecutive days. Twenty-four hours after the last injection, the total white blood cell count, lymphocyte count, and immunoglobulin (IgG, IgA) levels in the peripheral blood of the mice were measured. The results showed that the total white blood cell count, lymphocyte count, and IgG and IgA levels in the experimental group were significantly higher than those in the control group (P<0.05), indicating that the spleen polypeptide extract has significant immune-enhancing activity and can be used in the biomedical field to prepare immunomodulators.

[0046] The high-protein product (hemoglobin purity 95.6%) prepared in Example 2 was added to dairy products to make fortified protein dairy products. The protein content of the product was tested to be 3.8g / 100mL, which was significantly higher than that of the unadded group (protein content 2.5g / 100mL). The product also had a good taste and no off-odor, indicating that the high-protein product can be used as a food additive in dairy product processing to improve the protein content and quality of food.

[0047] The high-content nucleic acid product (89.5% nucleic acid content) prepared in Example 3 was used as a scientific research reagent for DNA electrophoresis experiments. The results showed that the nucleic acid product had clear electrophoretic bands with no impurities, and the purity met the experimental requirements. It can be used as a standard or reagent for scientific research experiments.

[0048] Comparative Example 1 The spleen extract was prepared using existing traditional processes, and the specific steps are as follows. The raw material amounts and basic process parameters are the same as in Example 1: S1. Spleen tissue pretreatment: Same as in Example 1, 1000g of fresh pig spleen tissue was selected, and after removing impurities, 880g of spleen tissue was obtained. The tissue was cut into pieces and pre-frozen for later use.

[0049] S2. Homogenization: Same as in Example 1, 880g spleen tissue block was added to 3520mL of pre-cooled sterile saline, homogenized at 9000r / min for 4min in an ice bath, and 4400mL of spleen homogenate was obtained.

[0050] S3. Repeated freeze-thaw disruption: Same as in Example 1, the homogenate was repeatedly frozen and thawed 4 times to obtain 4400 mL of the frozen and thawed homogenate.

[0051] S4. Centrifugation: Same as in Example 1, centrifuge at 4℃ and 10000r / min for 18min, and collect 3850mL of supernatant.

[0052] S5. Ultrafiltration treatment: Same as in Example 1, 3850 mL of supernatant was ultrafiltered through an 8 kDa ultrafiltration membrane, and 3200 mL of permeate and 650 mL of retentate were collected.

[0053] S6. Isoelectric point fractionation: The pH of 650 mL of retentate was adjusted to 5.0-6.8 with 0.1 mol / L NaOH, allowed to stand for 2.5 h, and then centrifuged to collect 68.5 g of crude protein precipitate; 3200 mL of permeate was not fractionated and was used directly.

[0054] S7. Purification: Only 68.5g of crude protein was purified by single DEAE-cellulose ion exchange chromatography (under the same chromatography conditions as in Example 2). The protein eluent was collected and concentrated. 3200mL of permeate was directly concentrated to 50mL by rotary evaporation without further purification.

[0055] S8. Product Processing: The purified protein concentrate and permeate concentrate were aseptically filtered and freeze-dried to obtain 25.3g of conventional process protein product (mixed protein) and conventional process peptide concentrate (peptide content 58.6%). Examples 1-3, by optimizing isoelectric point fractionation parameters and designing targeted chromatographic purification processes, solved the core problems of difficult separation and purification, low resource utilization, and insufficient product purity in traditional processes. They achieved efficient separation and high-purity preparation of three high-value components—peptides, proteins, and nucleic acids—from spleen raw materials. The products can be widely used in multiple fields, and the process has the potential for industrial-scale promotion. Comparative Example 1 highlights the limitations of traditional processes. A direct comparison with the examples fully verifies the advantages of the technical solution of this invention.

[0056] 1. Nucleic acid extraction rate calculation: step: Take a certain mass of spleen tissue (e.g., 100g), break it up, extract it, purify it, and then bring it to a certain volume (e.g., 100mL).

[0057] The absorbance of the extract at 260 nm was measured using a UV spectrophotometer (A). 260 Nucleic acid concentration is calculated using the formula: Nucleic acid concentration (μg / mL) = A 260 ×50×Dilution factor; Calculate the total mass of nucleic acids in the extract: Total nucleic acid mass = Nucleic acid concentration × Fixed volume Assuming the theoretical nucleic acid content in spleen tissue is C 理论 (e.g., 0.5g / 100g tissue), then the extraction rate is: Nucleic acid extraction rate (%) = (total nucleic acid mass / spleen tissue mass × C theoretical) × 100%. Protein extraction rate (taking hemoglobin as an example) calculation: Protein extraction rate (%) = (total protein mass / spleen tissue mass × C theoretical) × 100%. Total protein extraction rate: = (total protein mass / spleen tissue mass × C theoretical) × 100%.

[0058] 2. Experimental Design Subjects: 30 healthy volunteers (half male and half female, aged 18–45 years) with no history of taste disorders or allergies.

[0059] Samples: Spleen polypeptide oral liquid (experimental group) from Example 1, and similar commercially available products (control group).

[0060] Evaluation criteria: taste (sweetness, bitterness, astringency, smoothness), flavor (aftertaste, irritation), using a 10-point scoring system (0=none, 10=extremely strong).

[0061] Statistical method: Independent samples t-test, P<0.05 was considered statistically significant.

[0062] Table 1: Taste Test project Experimental group (Example 1) control group p-value Sweetness 3.2±0.8 4.5±1.1 0.002 bitterness 1.5±0.6 3.1±0.9 <0.001 astringency 1.2±0.4 2.8±0.7 <0.001 Smoothness 7.8±1.0 5.6±1.2 <0.001 Aftertaste comfort 8.1±0.9 5.3±1.3 <0.001 Oral irritation 0.9±0.3 2.4±0.8 <0.001 As shown in Table 1, the spleen polypeptide oral liquid of Example 1 has significantly lower bitterness, astringency and oral irritation, and significantly higher smoothness and aftertaste comfort (P<0.001), resulting in a better taste and flavor experience.

[0063] Experimental Design Animal model: Immunosuppressed mice (cyclophosphamide model) were randomly divided into a control group (physiological saline) and an experimental group (Example 1: spleen polypeptide injection, 20 mg / kg / d, for 7 consecutive days).

[0064] Detection methods: Fully automated blood cell analyzer (white blood cells, lymphocytes); immunoturbidimetric assay (IgG, IgA).

[0065] Statistical method: Independent samples t-test, P<0.05 was considered statistically significant.

[0066] Test data (n=10, mean ± standard deviation).

[0067] Table 2: Data on total white blood cell count, lymphocyte count, and immunoglobulin (IgG, IgA) levels Group <![CDATA[Total white blood cell count (×10 9 / L)]]> <![CDATA[Number of lymphocytes (×10 9 / L)]]> control group 5.1±0.8 2.3±0.5 experimental group 8.2±1.1* 4.5±0.7* As shown in Table 2, the total number of white blood cells, lymphocytes, and levels of IgG and IgA in the experimental group were significantly higher than those in the control group (P<0.001), indicating that the spleen polypeptide in Example 1 can significantly enhance the body's immune function.

[0068] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0069] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0070] Those skilled in the art should understand that the above descriptions are merely several specific embodiments of the present invention, and not all embodiments.

Claims

1. A method for preparing a spleen polypeptide extract, characterized in that, Includes the following steps: S1. Spleen tissue pretreatment: Select fresh pig spleen tissue, remove fat, connective tissue and fascia, rinse with sterile saline, cut into pieces, and pre-freeze for later use; S2. Homogenization: Add pre-frozen spleen tissue blocks to pre-cooled sterile physiological saline at a material-to-liquid ratio of 1:3-1:5 (g / mL), and homogenize at 8000-10000 r / min under ice bath conditions to obtain spleen homogenate. S3. Repeated freeze-thaw disruption: Freeze the spleen homogenate at -80℃ for 1-2 hours and then thaw it at 37℃, repeating the freeze-thaw process. S4. Centrifugation: Centrifuge at 4℃ and 8000-12000 r / min, and collect the supernatant; S5. Ultrafiltration treatment: Ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 5-10 kDa at 0.1-0.3 MPa and 4-10℃, and the permeate and retentate are collected. S6. Isoelectric point fractionation: Adjust the pH values ​​of the permeate and retentate respectively, and perform fractionation centrifugation for different target components to collect crude peptide precipitates, crude protein precipitates, and crude nucleic acid precipitates; S7. Chromatographic purification: Crude polypeptides, crude proteins, and crude nucleic acids are purified by chromatography to obtain high-content polypeptide products, high-content protein products, and high-content nucleic acid products, respectively. S8. Product processing: The above high-content product is aseptically filtered, and after passing the test, it is freeze-dried or made into a liquid preparation.

2. The method for preparing the spleen polypeptide extract according to claim 1, characterized in that, In step S2, the homogenization time is 3-5 minutes.

3. The method for preparing the spleen polypeptide extract according to claim 1, characterized in that, In step S3, the freeze-thaw cycle is repeated 3-5 times.

4. The method for preparing the spleen polypeptide extract according to claim 1, characterized in that, In step S4, the centrifugation time is 15-20 minutes.

5. The method for preparing the spleen polypeptide extract according to claim 1, characterized in that, In step S6, the specific operation of isoelectric point classification is as follows: For the retentate: Adjust the pH to 4.5-5.5, let stand for 2-3 hours, then centrifuge to collect the crude protein precipitate; then adjust the pH to 5.8-6.8, let stand, centrifuge to collect the precipitate, and combine them to obtain mixed crude protein; For the permeate: adjust the pH to 2.5-3.5, let stand for 1-2 hours, and then centrifuge to collect the crude peptide precipitate; adjust the pH of the remaining supernatant to 4.0-5.0, let stand, and then centrifuge to collect the crude nucleic acid precipitate.

6. The method for preparing the spleen polypeptide extract according to claim 1, characterized in that, In step S7, the specific operations of chromatographic purification are as follows: High-content protein products: Purification was carried out in two steps using DEAE-cellulose ion exchange chromatography column + Sephadex G-75 gel filtration chromatography column, with Tris-HCl buffer as the elution buffer and NaCl gradient elution, and the elution peak of the target protein was collected. High-content peptide products: Purified using RP-HPLC column, with acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase, acetonitrile gradient elution, detection wavelength 220nm, and collection of the target peptide elution peak; High-content nucleic acid products: After treatment with RNase A, the products were purified using a Sepharose CL-6B gel filtration chromatography column with TE buffer as the elution solution, and the elution peak of the target nucleic acid was collected.

7. A spleen polypeptide extract, characterized in that, It is prepared using any one of claims 1-6.

8. The spleen polypeptide extract according to claim 7, characterized in that, Applications in the fields of biomedicine, food additives, or scientific research reagents.

Images