Preparation method of bovine spinal cord peptide

Abstract

The invention discloses a preparation method of bovine spinal cord peptide, belongs to the technical field of biology, and is used for solving the technical problems that in the prior art, degreasing agents such as petroleum ether are weak in polar lipid dissolving capacity, and enzymatic hydrolysate is separated by adopting a conventional nanofiltration membrane, so that the manufacturing cost is high, and the service life is short. The preparation method of the bovine spinal cord peptide comprises the following steps: S1, cleaning fresh bovine spinal cords, crushing the cleaned bovine spinal cords into blocks, and pulping the blocks to obtain homogenate tissues; adding a degreasing agent into the homogenized tissue, and performing ultrasonic treatment to obtain degreased spinal cord; s2, adding compound enzyme into the degreased spinal cord for reaction to obtain enzymatic hydrolysate; carrying out enzyme deactivation on the enzymatic hydrolysate in boiling water to prepare a bovine spinal cord crude peptide solution; s3, centrifuging the bovine spinal cord crude peptide solution, and collecting supernate; and passing the supernate through a modified ultrafiltration membrane, and circularly performing ultrafiltration for multiple times to prepare the bovine spinal cord peptide. According to the method, the homogenate tissue is deeply degreased by designing the composite degreasing agent with a surfactant-like structure; and the modified ultrafiltration membrane is used for improving the purity of the filtered bovine spinal cord peptide.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing bovine spinal cord peptides. Background Technology

[0002] Bovine spinal cord peptides are part of the bovine central nervous system and are often consumed as a highly nutritious food. Traditional Chinese medicine believes they have the effects of tonifying the middle energizer, nourishing the spleen and stomach, and strengthening muscles and bones. They are rich in protein, fat, arachidonic acid, palmitic acid, stearic acid, linoleic acid, zinc, calcium, etc., and can replenish blood and qi, and repair tissues. The preparation process of bovine spinal cord peptides typically includes the following steps: high-temperature steaming of bovine spinal cord, tissue homogenization, defatting, enzymatic hydrolysis, ultrafiltration, and freeze-drying / spray drying, thus preparing bovine spinal cord polypeptide powder. Since bovine spinal cord has a high fat content, defatting agents are often used in the defatting process to synthesize defatted spinal cord. Ultrafiltration is one of the commonly used methods for separating polypeptides. Ultrafiltration membranes allow water and small solute particles to pass through under external pressure, while large molecules are retained, thus enabling the separation of polypeptide components with different molecular weights. However, compared to general filtration membranes, nanofiltration membranes have disadvantages such as high manufacturing cost and short service life.

[0003] Patent application CN108949880A discloses a method for preparing bovine spinal cord peptides. In this process, petroleum ether is commonly used as a defatting agent. The prepared enzymatic hydrolysate is separated using ultrafiltration membranes of different molecular weights to obtain different polypeptide solutions. These polypeptide solutions are then spray-dried to prepare bovine spinal cord peptide powder. However, petroleum ether, as a non-polar solvent, has weak solubility for polar lipids such as phospholipids. Furthermore, using ultrafiltration membranes of different molecular weights to separate the enzymatic hydrolysate is cumbersome and increases production costs.

[0004] To address this technical deficiency, a solution is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing bovine spinal cord peptides, which solves the technical problems of high manufacturing cost and short service life in the prior art, where defatting agents such as petroleum ether have weak ability to dissolve polar lipids and enzymatic hydrolysate is separated by nanofiltration membrane.

[0006] The objective of this invention can be achieved through the following technical solutions: A method for preparing bovine spinal cord peptides includes the following steps: S1. Fresh bovine spinal cord is cleaned, broken into pieces, and boiled in distilled water at high temperature to obtain boiled spinal cord tissue; the boiled spinal cord tissue is mixed with distilled water and pulped to obtain homogenized tissue; a compound defatting agent is added to the homogenized tissue and ultrasonically treated to obtain defatted spinal cord. S2. The pH of the defatted spinal cord was adjusted to 7-8 by adding NaOH solution, and a compound enzyme was added to react and obtain an enzymatic hydrolysate. The enzyme hydrolysate was then inactivated in a boiling water bath to prepare a crude bovine spinal cord peptide solution. S3. Centrifuge the crude bovine spinal cord peptide solution and collect the supernatant; pass the supernatant through a modified ultrafiltration membrane and cycle ultrafiltration 2-4 times to obtain the filtrate; freeze-dry the filtrate to prepare bovine spinal cord peptides.

[0007] Furthermore, the preparation method of the composite degreasing agent includes the following steps: B1. Mix ethanol and p-aminophenol, then add dichlorodimethylsilane dropwise. After the addition is complete, the reactant is obtained. The reactant is reacted at 45-55℃ for 40-60 min to synthesize the product. The product is filtered and the organic liquid phase is collected. Using ethanol as a solvent, p-aminophenol and dichlorodimethylsilane were reacted to synthesize the product, and the reaction formula is as follows:

[0008] B2. The organic liquid phase and triethylamine are mixed, and then oleyl chloride is added dropwise to obtain the reactant. NaOH solution is added dropwise to adjust the pH of the reactant to 7.5-8. The reaction is continued at 50-60℃ for 4-5 hours. Ethanol is removed by rotary evaporation to prepare the composite degreasing agent.

[0009] A solid degreasing agent is synthesized by the alkaline-catalyzed amidation reaction of oleoyl chloride and triethylamine.

[0010] Further, in step B1, the ratio of ethanol, p-aminophenol, and dichlorodimethylsilane is 50-100 mL: 10-20 g: 5-10 mL.

[0011] Furthermore, in step B2, the ratio of organic liquid phase, triethylamine, and oleoyl chloride is 50-100 mL: 0.3-0.5 g: 15-20 mL.

[0012] Further, in step S3, the method for preparing the modified ultrafiltration membrane includes the following steps: A1. The pretreated microporous membrane is added to the ozone oxidation reactor. Ozone is introduced into the reactor until the ozone concentration is 50-55 mg / L, and the ozone gas flow rate is 0.1-0.2 m³ / L. 3 / hr, the temperature of the ozone oxidation reactor is 75-80℃, and the ozone oxidation time is 1-2h; then the microporous membrane is taken out, which is the oxidized microporous membrane; When the pretreated microporous membrane is exposed to ozone gas, in addition to forming carbonyl and carboxyl groups on the surface, peroxides are generated, as well as surface decomposition free radicals -O- and HO-. -O- has the ability to initiate the polymerization of olefin monomers, thereby initiating a grafting reaction on the surface, while HO- can initiate the homopolymerization of monomers.

[0013] A2, hydroxyethyl methacrylate, isopropanol, hexamethyltrisiloxane and chloroplatinic acid are blended and reacted at 90-100℃ for 5-6 hours. Isopropanol is removed by rotary evaporation to synthesize the modified monomer solution. Using isopropanol as a solvent and chloroplatinic acid as an acidic catalyst, hydroxyethyl methacrylate and hexamethyltrisiloxane undergo an electrophilic addition reaction to synthesize a modified monomeric liquid. The reaction formula is as follows:

[0014] A3. Immerse the oxidized microporous membrane in the modified monomer solution with a solid-liquid volume ratio of 1-2:10, then filter and remove the membrane layer; subject the membrane layer to a crosslinking reaction at 110-120℃ for 30-60 min, then wash with deionized water and dry to constant weight to synthesize the modified ultrafiltration membrane.

[0015] Furthermore, in step A2, the ratio of hydroxyethyl methacrylate, isopropanol, hexamethyltrisiloxane, and chloroplatinic acid is 3-10g:100mL:10-20mL:0.3-0.5g.

[0016] Furthermore, in step S1, the high-temperature cooking temperature is 110-120℃ and the high-temperature cooking time is 15-30min; after pulping, the solid particle size in the homogenized tissue is controlled to be 100-200 mesh; the ratio of degreasing agent to homogenized tissue is 3-5g:50-60mL.

[0017] Further, in step S2, the complex enzyme is obtained by mixing alkaline protease, neutral protease and papain in a mass ratio of 1-2:1:1; the temperature for inactivating the enzyme with boiling water is 95-100℃ and the time for inactivating the enzyme with boiling water is 10-20 min.

[0018] Furthermore, in step S3, the centrifugation speed is 6000-8000 r / min and the centrifugation time is 30-60 min; the temperature of the circulating ultrafiltration is 25-30℃, the ultrafiltration pressure is 1-2 MPa, and the ultrafiltration flow rate is 2-5 mL / min.

[0019] As another aspect of the present invention, bovine spinal cord peptides prepared using the aforementioned method are provided.

[0020] The present invention has the following beneficial effects: 1. This invention involves a series of pretreatments of bovine spinal cord peptides, followed by pulping to form a homogenate with a solid particle size of 100-200 mesh. A composite degreasing agent is added to the homogenate for degreasing, synthesizing degreased spinal cord. The degreased spinal cord is then enzymatically hydrolyzed to obtain a crude bovine spinal cord peptide solution. This crude bovine spinal cord peptide solution is centrifuged and repeatedly filtered through a modified ultrafiltration membrane to obtain a filtrate containing high-purity bovine spinal cord peptides. The filtrate is spray-dried into powder to prepare bovine spinal cord peptides. A nucleophilic substitution reaction is performed between p-aminophenol and dichlorodimethylsilane, followed by an amidation reaction with oleoyl chloride to synthesize a composite degreasing agent. The long carbon chain portion of oleoyl chloride acts as a hydrophobic-lipophilic group, while the dichlorodimethylsilane grafted onto p-aminophenol acts as a hydrophilic group. The resulting surfactant-like agent, through design and control of the molecular weight and length of the grafted chain, yields a composite degreasing agent with a narrow molecular weight distribution. The aforementioned composite degreasing agent possesses excellent wetting, penetrating, emulsifying, and degreasing properties, enabling deep degreasing when added to homogenized tissue. Furthermore, the use of this composite degreasing agent does not require high temperatures or strong acidic / alkaline conditions, which is beneficial for maintaining the tissue structure of bovine spinal cord peptides, and it also exhibits strong degreasing power.

[0021] 2. Defatted spinal cord is enzymatically hydrolyzed using a compound enzyme to obtain an enzymatic hydrolysate. The hydrolysate is a mixture of bovine spinal cord peptides with varying molecular weights, i.e., a crude bovine spinal cord peptide solution. The molecular weight of bovine spinal cord peptides is concentrated between 180-500 Da, corresponding to a pore size cutoff of 1-2 nm, which is much smaller than the pore size of conventional PP microporous membranes. Therefore, conventional PP microporous membranes cannot effectively filter and collect purified bovine spinal cord peptides. However, the PP microporous membrane is chemically modified to prepare a modified ultrafiltration membrane. By grafting modified monomer solutions, the polarity, anti-protein adsorption capacity, and pore size are improved, thereby effectively retaining bovine spinal cord peptides with an average molecular weight in the crude bovine spinal cord peptide solution, thus achieving purification. Compared to conventional nanofiltration membranes, the modified ultrafiltration membrane prepared in this invention has lower raw material costs and a longer service life. The crude bovine spinal cord peptide solution is subjected to multiple ultrafiltrations through the modified ultrafiltration membrane to finally obtain bovine spinal cord peptides. The pretreated microporous membrane is oxidized with ozone to decompose free radicals -O- and HO- on its surface. Then, a modified monomer solution is grafted onto it to obtain a cross-linked microporous membrane, which is the prepared modified ultrafiltration membrane. The reaction of hexamethyltrisiloxane and hydroxyethyl methacrylate introduces silicon into the grafted monomer, which improves the hydrophilicity of the PP microporous membrane, thereby binding a large number of water molecules and forming a physical barrier to further block the approach of residual protein molecules. Grafting 2-hydroxyethyl methacrylate monomer onto the surface of the PP microporous membrane also significantly enhances its water flux and anti-protein adsorption capacity. Detailed Implementation

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] The PP microporous membranes used in Examples 1-3 of this invention were purchased from Nantong Maiborui Biomembrane Technology Co., Ltd., with product code MSPP2801000BO and a pore size of 10μm. The chloroplatinic acid used in Examples 1-3 of this invention was purchased from Jiangsu Jinwo New Materials Co., Ltd. The bovine spinal cord used in Examples 7-9 of this invention was purchased from Putian Hanjiang District Junhan Trading Company. The nanofiltration membrane used in Comparative Example 3 was purchased from Changzhou Hongmo Technology Co., Ltd., with a specification of nanofiltration RS4040-NF500.

[0024] The specific enzyme activity must be specified. Example 1 This embodiment provides a method for preparing a modified ultrafiltration membrane for preparing bovine spinal cord peptides, comprising the following steps: A1. PP microporous membranes were cut into circular samples with a diameter of 5 cm to obtain sample membranes. The sample membranes were then immersed in ethanol for 12 hours to remove surface impurities, and subsequently vacuum dried at 50°C to constant weight to obtain pretreated microporous membranes. The pretreated microporous membranes were then added to an ozone oxidation reactor, with ozone introduced into the reactor at a concentration of 50 mg / L and a flow rate of 0.1 m³ / L. 3 / hr, the temperature of the ozone oxidation reactor is 75℃, the ozone oxidation time is 1h, and then the microporous membrane is taken out to obtain the oxidized microporous membrane.

[0025] A2. Add 3g of hydroxyethyl methacrylate, 100mL of isopropanol, 10mL of hexamethyltrisiloxane and 0.3g of chloroplatinic acid to a 250mL three-necked flask and react at 90℃ for 5h. Remove the isopropanol solvent by vacuum distillation using a rotary evaporator to synthesize the modified monomer solution.

[0026] A3. Immerse the oxidized microporous membrane in the modified monomer solution and react for 1 hour, with a solid-liquid volume ratio of 1:10. After filtration, remove the membrane layer. Transfer the membrane layer to an oven at 110°C and heat for a crosslinking reaction for 30 minutes. Then wash with deionized water and dry at 50°C to constant weight to synthesize the modified ultrafiltration membrane.

[0027] Example 2 This embodiment provides a method for preparing a modified ultrafiltration membrane for preparing bovine spinal cord peptides, comprising the following steps: A1. PP microporous membranes were cut into circular samples with a diameter of 5 cm to obtain sample membranes. The sample membranes were then immersed in ethanol for 12 hours to remove surface impurities, and subsequently vacuum dried at 55°C to constant weight to obtain pretreated microporous membranes. The pretreated microporous membranes were then added to an ozone oxidation reactor, with ozone concentrations of 52 mg / L and a flow rate of 0.15 m³ / L. 3 / hr, the temperature of the ozone oxidation reactor is 78℃, the ozone oxidation time is 1.5h, and then the microporous membrane is taken out to obtain the oxidized microporous membrane.

[0028] A2. Add 6g of hydroxyethyl methacrylate, 100mL of isopropanol, 15mL of hexamethyltrisiloxane and 0.4g of chloroplatinic acid to a 250mL three-necked flask and react at 95℃ for 5.5h. Remove the isopropanol solvent by vacuum distillation using a rotary evaporator to synthesize the modified monomer solution.

[0029] A3. The oxidized microporous membrane was immersed in the modified monomer solution and reacted for 1.5 h with a solid-liquid volume ratio of 1.5:10. After filtration, the membrane layer was removed. The membrane layer was transferred to an oven at 115 °C and heated for 50 min for crosslinking reaction. Then it was washed with deionized water and dried at 55 °C to constant weight to synthesize the modified ultrafiltration membrane.

[0030] Example 3 This embodiment provides a method for preparing a modified ultrafiltration membrane for preparing bovine spinal cord peptides, comprising the following steps: A1. PP microporous membranes were cut into circular samples with a diameter of 5 cm to obtain sample membranes. The sample membranes were then immersed in ethanol for 12 hours to remove surface impurities, and subsequently vacuum dried at 60°C to constant weight to obtain pretreated microporous membranes. The pretreated microporous membranes were then added to an ozone oxidation reactor, with ozone introduced into the reactor at a concentration of 55 mg / L and a flow rate of 0.2 m³ / L. 3 / hr, the temperature of the ozone oxidation reactor is 80℃, the ozone oxidation time is 1-2h, and then the microporous membrane is taken out to obtain the oxidized microporous membrane.

[0031] A2. Add 10g of hydroxyethyl methacrylate, 100mL of isopropanol, 20mL of hexamethyltrisiloxane and 0.5g of chloroplatinic acid to a 250mL three-necked flask and react at 100℃ for 6h to synthesize the modified monomer solution.

[0032] A3. The oxidized microporous membrane was immersed in the modified monomer solution and reacted for 2 hours at a solid-liquid volume ratio of 2:10. After filtration, the membrane layer was removed. The membrane layer was transferred to an oven at 120°C and heated for 60 minutes for crosslinking reaction. Then, it was washed with deionized water and dried at 60°C to constant weight to synthesize the modified ultrafiltration membrane.

[0033] Example 4 This embodiment provides a method for preparing a compound defatting agent for preparing bovine spinal cord peptides, comprising the following steps: B1. A 250 mL four-necked flask is fitted with an external heating mantle. The flask is connected to a thermometer, condenser, stirrer, and constant-pressure funnel. 50 mL of ethanol and 10 g of p-aminophenol are added to the flask and mixed thoroughly. 5 mL of dichlorodimethylsilane is then added dropwise through the constant-pressure funnel over 6 minutes, yielding the reactants. The flask is heated to 50 °C and reacted at this temperature for 55 minutes to synthesize the product. The product is filtered, and the organic liquid phase is collected.

[0034] B2. Measure 70 mL of the organic liquid phase and 0.4 g of the acid-binding agent triethylamine into a 250 mL flask. Add 15 mL of oleoyl chloride dropwise to the flask at room temperature to obtain the reactant. Add 0.1 mol / L NaOH solution dropwise to the reactant to adjust the pH of the system to 7.8. After the addition is complete, continue the reaction at 55 °C for 4.6 h. Then, remove the solvent ethanol from the organic liquid phase by rotary evaporation at 55 °C to prepare the composite degreasing agent.

[0035] Example 5 This embodiment provides a method for preparing a compound defatting agent for preparing bovine spinal cord peptides, comprising the following steps: B1. A 250 mL four-necked flask is fitted with an external heating mantle. The flask is connected to a thermometer, condenser, stirrer, and constant-pressure funnel. 70 mL of ethanol and 15 g of p-aminophenol are added to the flask and mixed thoroughly. 8 mL of dichlorodimethylsilane is then added dropwise through the constant-pressure funnel over 6 minutes, yielding the reactants. The flask is heated to 50 °C and reacted at this temperature for 50 minutes to synthesize the product. The product is filtered, and the organic liquid phase is collected.

[0036] B2. Measure 80 mL of the organic liquid phase and 0.4 g of the acid-binding agent triethylamine into a 250 mL flask. Add 15 mL of oleyl chloride dropwise to the flask at room temperature to obtain the reactant. Add 0.1 mol / L NaOH solution dropwise to the reactant to adjust the pH of the system to 7.8. After the addition is complete, continue the reaction at 55 °C for 4.4 h. Then, remove the solvent ethanol from the organic liquid phase by rotary evaporation at 58 °C to prepare the composite degreasing agent.

[0037] Example 6 This embodiment provides a method for preparing a compound defatting agent for preparing bovine spinal cord peptides, comprising the following steps: B1. A 250 mL four-necked flask is fitted with an external heating mantle. The flask is connected to a thermometer, condenser, stirrer, and constant-pressure funnel. 100 mL of ethanol and 20 g of p-aminophenol are added to the flask and mixed thoroughly. 10 mL of dichlorodimethylsilane is then added dropwise through the constant-pressure funnel over 10 minutes, yielding the reactants. The flask is heated to 55 °C and reacted at this temperature for 60 minutes to synthesize the product. The product is filtered, and the organic liquid phase is collected.

[0038] B2. Measure 100 mL of organic liquid phase and 0.5 g of triethylamine, an acid-binding agent, into a 250 mL flask. Add 20 mL of oleoyl chloride dropwise to the flask at room temperature to obtain the reactants. Add 0.1 mol / L NaOH solution dropwise to the reactants to adjust the pH of the system to 8. After the addition is complete, continue the reaction at 60 °C for 5 h. Then, remove the solvent ethanol from the organic liquid phase by rotary evaporation at 60 °C to prepare the composite degreasing agent.

[0039] Example 7 This embodiment provides a method for preparing bovine spinal cord peptides, including the following steps: S1. Select fresh bovine spinal cord, rinse with running water to remove impurities, and obtain cleaned bovine spinal cord. Then, break the cleaned bovine spinal cord into pieces, add distilled water, and steam at high temperature. The volume ratio of cleaned bovine spinal cord to distilled water is 1:3. The steaming temperature is 70℃, and the steaming time is 15 minutes, resulting in steamed spinal cord tissue. Mix the steamed spinal cord tissue with distilled water and homogenize. The volume ratio of steamed spinal cord tissue to distilled water is 1:5, resulting in a homogenized tissue. Control the solid particle size in the homogenized tissue to be 200 mesh. Add 3g of the defatting agent prepared in Example 4 to 50mL of the homogenized tissue, and sonicate for 20 minutes at a frequency of 20kHz to synthesize defatted spinal cord.

[0040] S2, alkaline protease, neutral protease, and papain were mixed in a mass ratio of 1:1:1 to obtain a compound enzyme. Defatted spinal cord was added to an enzymatic hydrolysis flask, and the pH of the defatted spinal cord was adjusted to 7 by adding 0.5 mol / L NaOH solution. The compound enzyme was then added to initiate the reaction. The amount of compound enzyme added was 2% of the mass of the defatted spinal cord substrate. The enzymatic hydrolysis temperature was 45℃, and the hydrolysis time was 2 hours, yielding an enzymatic hydrolysate. The hydrolysate was then inactivated in a boiling water bath at 95℃ for 10 minutes to prepare a crude bovine spinal cord peptide solution.

[0041] S3. The crude bovine spinal cord peptide solution was centrifuged at 6000 r / min for 30 min to remove precipitates and macromolecular impurities, and the supernatant was collected. The supernatant was filtered twice through the modified ultrafiltration membrane prepared in Example 1, and the filtrate was collected. During ultrafiltration, the pH was 7, the ultrafiltration temperature was 25℃, the ultrafiltration pressure was 1 MPa, and the ultrafiltration flow rate was adjusted to 2 mL / min. The filtrate was spray-dried at an inlet air temperature of 110℃ for 15 h to finally obtain bovine spinal cord peptides.

[0042] Example 8 This embodiment provides a method for preparing bovine spinal cord peptides, including the following steps: S1. Select fresh bovine spinal cord and rinse it with running water to remove impurities, obtaining cleaned bovine spinal cord. Then, break the cleaned bovine spinal cord into pieces, add distilled water, and steam at high temperature. The volume ratio of cleaned bovine spinal cord to distilled water is 1:4. The steaming temperature is 115℃, and the steaming time is 25 minutes, obtaining steamed spinal cord tissue. Mix the steamed spinal cord tissue with distilled water and homogenize. The volume ratio of steamed spinal cord tissue to distilled water is 1:8, obtaining a homogenized tissue. Control the solid particle size in the homogenized tissue to be 120 mesh. Add 4g of the defatting agent prepared in Example 5 to 55mL of the homogenized tissue and sonicate for 25 minutes at a frequency of 25kHz to synthesize defatted spinal cord.

[0043] S2, alkaline protease, neutral protease, and papain were mixed in a mass ratio of 1:1:1 to obtain a compound enzyme. Defatted spinal cord was added to an enzymatic hydrolysis flask, and the pH of the defatted spinal cord was adjusted to 7.7 by adding 0.5 mol / L NaOH solution. The compound enzyme was then added to initiate the reaction. The amount of compound enzyme added was 3% of the mass of the defatted spinal cord substrate. The enzymatic hydrolysis temperature was 55℃, and the hydrolysis time was 3 hours to obtain the enzymatic hydrolysate. The enzymatic hydrolysate was then inactivated in a boiling water bath at 98℃ for 15 minutes to prepare a crude bovine spinal cord peptide solution.

[0044] S3. Centrifuge the crude bovine spinal cord peptide solution at 7000 r / min for 50 min to remove precipitates and macromolecular impurities, and collect the supernatant. Pass the supernatant through the modified ultrafiltration membrane prepared in Example 2, circulating and filtering three times. Collect the filtrate. During ultrafiltration, the ultrafiltration temperature is 25℃, the ultrafiltration pressure is 1.5 MPa, and the ultrafiltration flow rate is adjusted to 3 mL / min. Spray dry the filtrate at an inlet air temperature of 115℃ for 18 h to finally obtain bovine spinal cord peptides.

[0045] Example 9 This embodiment provides a method for preparing bovine spinal cord peptides, including the following steps: S1. Select fresh bovine spinal cord, rinse with running water to remove impurities, and obtain cleaned bovine spinal cord. Then, break the cleaned bovine spinal cord into pieces, add distilled water, and steam at high temperature. The volume ratio of cleaned bovine spinal cord to distilled water is 1:5. The steaming temperature is 80℃, and the steaming time is 30 minutes, resulting in steamed spinal cord tissue. Mix the steamed spinal cord tissue with distilled water and homogenize. The volume ratio of steamed spinal cord tissue to distilled water is 1:10, resulting in a homogenized tissue. Control the solid particle size in the homogenized tissue to be 100 mesh. Add 5g of the defatting agent prepared in Example 6 to 60mL of the homogenized tissue, and sonicate for 30 minutes at a frequency of 30kHz to synthesize defatted spinal cord.

[0046] S2, alkaline protease, neutral protease, and papain were mixed in a mass ratio of 2:1:1 to obtain a compound enzyme. Defatted spinal cord was added to an enzymatic hydrolysis flask, and the pH of the defatted spinal cord was adjusted to 8 by adding 0.5 mol / L NaOH solution. The compound enzyme was then added to initiate the reaction. The amount of compound enzyme added was 5% of the mass of the defatted spinal cord substrate. The enzymatic hydrolysis temperature was 65℃, and the hydrolysis time was 5 hours, yielding an enzymatic hydrolysate. The hydrolysate was then inactivated in a boiling water bath at 100℃ for 20 minutes to prepare a crude bovine spinal cord peptide solution.

[0047] S3. Centrifuge the crude bovine spinal cord peptide solution at 8000 r / min for 60 min to remove precipitates and macromolecular impurities, and collect the supernatant. Pass the supernatant through the modified ultrafiltration membrane prepared in Example 3, circulating and filtering four times. Collect the filtrate. During ultrafiltration, the pH value is 7, the ultrafiltration temperature is 25℃, the ultrafiltration pressure is 2 MPa, and the ultrafiltration flow rate is adjusted to 5 mL / min. Spray dry the filtrate at an inlet air temperature of 120℃ for 20 h to finally obtain bovine spinal cord peptides.

[0048] Comparative Example 1 The difference between this comparative example and Example 9 lies in the steps for synthesizing the modified monomer solution during the preparation of the modified ultrafiltration membrane. Specifically: Mix 10g of hydroxyethyl methacrylate and 100mL of isopropanol to obtain the synthesized modified monomer solution.

[0049] Comparative Example 2 The difference between this comparative example and Example 9 is that the method of synthesizing the organic liquid phase is different when preparing the composite degreasing agent. The specific steps are as follows: 100 mL of solvent ethanol and 20 g of p-aminophenol are added to a four-necked flask, mixed well, the four-necked flask is heated to 55°C, and stirred until the p-aminophenol is completely dissolved in ethanol. The product is the synthesized organic liquid phase.

[0050] Comparative Example 3 The difference between this comparative example and Example 9 is that, in the preparation of bovine spinal cord peptides, a nanofiltration membrane is used instead of a modified ultrafiltration membrane in step S3.

[0051] Performance testing: 1. The degree of hydrolysis of the bovine spinal cord crude peptide solutions in Examples 7-9 and Comparative Example 2 was determined sequentially using the trichloroacetic acid method. 10% wt trichloroacetic acid solution was added sequentially to the bovine spinal cord crude peptide solutions in Examples 7-9 and Comparative Example 2. After thorough mixing, the solutions were centrifuged at high speed (3000 rpm) for 5 minutes. The supernatant contained small peptides and amino acids, while the precipitate contained unhydrolyzed proteins and larger peptides. The mass of the precipitate was weighed, and the mass of the acid-soluble protein was obtained by subtracting the mass of the precipitate from the total unhydrolyzed protein mass. The change in the ratio of acid-soluble protein to total protein was calculated to determine the degree of hydrolysis.

[0052] Degree of hydrolysis = 1 - acid-soluble protein / total protein. See Table 1 for specific data. Table 1. Sample performance test data

[0053] Data Analysis: In Examples 7-9 of this invention, defatted tissue was used to obtain defatted spinal cord; after enzymatic hydrolysis of the defatted spinal cord, a crude bovine spinal cord peptide solution was synthesized. The degree of defatting of bovine spinal cord affects its hydrolysis process; defatting treatment can destroy cell membrane structure and lipoprotein complexes, thereby accelerating the hydrolysis reaction and increasing the concentration of hydrolysis products. The defatted spinal cord synthesized in Examples 7-9 of this invention all exhibited a high degree of hydrolysis after enzymatic hydrolysis. In the defatting agent, increasing the molecular weight, length, and number of polar functional groups of the grafted chains improves emulsification efficiency and defatting efficacy, thereby accelerating hydrolysis and increasing the degree of hydrolysis. However, in Comparative Example 2, when preparing the composite defatting agent, dichlorodimethylsilane was not used to graft p-aminophenol, resulting in a decreased degree of hydrolysis in the synthesized crude bovine spinal cord peptide solution after enzymatic hydrolysis in Comparative Example 2.

[0054] 2. The protein concentration was calculated by measuring the absorbance of the filtrates prepared in Examples 7-9 and Comparative Examples 1-3 at 562 nm using the BCA protein quantification method.

[0055] 3. Configure ABTS + Mother liquor, ABTS + The stock solution was obtained by redox reaction of 7 mmol / L ABTS stock solution with 2.45 mM potassium persulfate solution. Before use, ABTS was diluted with PBS solution. + The absorbance of the mother liquor was diluted to 0.7 and used as the working solution. 20 μL of the filtrates prepared in Examples 7-9, Comparative Examples 1, and Comparative Example 3, and 180 μL of ABTS were added sequentially to a 96-well plate. +The mother liquor was reacted at room temperature in the dark for 5 minutes, with distilled water as a blank control group. The ABTS free radical scavenging rate was calculated by measuring the absorbance at 734 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0056] ABTS radical scavenging rate (%) = (A0 - A1) / A0 × 100% A0 represents the absorbance of the blank control group; A1 represents the absorbance values ​​of different sample solutions.

[0057] Table 2. Sample performance test data

[0058] Data Analysis: Table 2 shows that the filtrate after three ultrafiltrations has low protein and impurity concentrations, indicating a low protein concentration. However, in Comparative Example 1, the oxidized PP microporous membrane was not grafted with hexamethyltrisiloxane, resulting in decreased protein filtration performance and increased protein concentration in the filtrate. Since the filtration performance of the modified ultrafiltration membranes prepared in Comparative Example 1 is inferior to that of the modified ultrafiltration membranes prepared in Examples 7-9, the ABTS free radical scavenging rate of the filtrate after filtration in Comparative Example 1 is lower.

[0059] In Comparative Example 3, a conventional nanofiltration membrane was used instead of the modified ultrafiltration membrane. Its protein filtration capacity and ABTS free radical scavenging rate were similar to those in Examples 7-9. This indicates that the modified microporous membrane can achieve similar filtration capacity to the nanofiltration membrane, or even better performance. Therefore, the modified ultrafiltration membrane prepared using this invention has advantages over conventional nanofiltration membranes in terms of better purification performance of bovine spinal cord peptides, lower raw material costs, and longer service life.

[0060] The above description is merely an example and illustration of the structure of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the structure of the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

[0061] In the description of this specification, references to terms such as "an embodiment," "example," and "specific example" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0062] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to specific implementations. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for preparing bovine spinal cord peptides, characterized in that, Includes the following steps: S1. Fresh bovine spinal cord is cleaned, broken into pieces, and boiled in distilled water at high temperature to obtain boiled spinal cord tissue. The boiled spinal cord tissue is mixed with distilled water and homogenized to obtain a homogenate. A compound defatting agent is added to the homogenate, and the tissue is ultrasonically treated to obtain defatted spinal cord. S2. The pH of the defatted spinal cord is adjusted to 7-8, and a compound enzyme is added to react and obtain an enzymatic hydrolysate. The enzyme in the hydrolysate is inactivated in a boiling water bath to prepare a crude bovine spinal cord peptide solution. S3. The crude bovine spinal cord peptide solution is centrifuged, and the supernatant is collected. The supernatant is passed through a modified ultrafiltration membrane and ultrafiltration is repeated 2-4 times to obtain a filtrate. The filtrate is freeze-dried to prepare bovine spinal cord peptides.

2. The method for preparing bovine spinal cord peptide according to claim 1, characterized in that, In step S1, the preparation method of the composite degreasing agent includes the following steps: B1. Mix ethanol and p-aminophenol, then add dichlorodimethylsilane dropwise. After the addition is complete, the reactant is obtained. The reactant is reacted at 45-55℃ for 40-60 min to synthesize the product. The product is filtered and the organic liquid phase is collected. B2. The organic liquid phase and triethylamine are mixed, and then oleyl chloride is added dropwise to obtain the reactant; the pH of the reactant is adjusted to 7.5-8, and the reaction is continued at 50-60℃ for 4-5 hours. The ethanol is removed by rotary evaporation to prepare the composite degreasing agent.

3. The method for preparing bovine spinal cord peptide according to claim 2, characterized in that, In step B1, the ratio of ethanol, p-aminophenol, and dichlorodimethylsilane is 50-100 mL: 10-20 g: 5-10 mL.

4. The method for preparing bovine spinal cord peptide according to claim 2, characterized in that, In step B2, the ratio of organic liquid phase, triethylamine and oleoyl chloride is 50-100mL: 0.3-0.5g: 15-20mL.

5. The method for preparing bovine spinal cord peptide according to claim 1, characterized in that, In step S3, the method for preparing the modified ultrafiltration membrane includes the following steps: A1. The pretreated microporous membrane is added to the ozone oxidation reactor. Ozone is introduced into the reactor until the ozone concentration is 50-55 mg / L, and the ozone gas flow rate is 0.1-0.2 m³ / L. 3 / hr, the temperature of the ozone oxidation reactor is 75-80℃, and the ozone oxidation time is 1-2h; then the microporous membrane is taken out, which is the oxidized microporous membrane; A2, hydroxyethyl methacrylate, isopropanol, hexamethyltrisiloxane and chloroplatinic acid are blended and reacted at 90-100℃ for 5-6 hours. Isopropanol is removed by rotary evaporation to synthesize the modified monomer solution. A3. Immerse the oxidized microporous membrane in the modified monomer solution with a solid-liquid volume ratio of 1-2:10, then filter and remove the membrane layer; subject the membrane layer to a crosslinking reaction at 110-120℃ for 30-60 min, then wash with deionized water and dry to constant weight to synthesize the modified ultrafiltration membrane.

6. The method for preparing bovine spinal cord peptide according to claim 5, characterized in that, In step A2, the ratio of hydroxyethyl methacrylate, isopropanol, hexamethyltrisiloxane and chloroplatinic acid is 3-10g:100mL:10-20mL:0.3-0.5g.

7. The method for preparing bovine spinal cord peptide according to claim 1, characterized in that, In step S1, the high-temperature cooking temperature is 110-120℃ and the high-temperature cooking time is 15-30min; after pulping, the solid particle size in the homogenized tissue is controlled to be 100-200 mesh; the ratio of degreasing agent to homogenized tissue is 3-5g:50-60mL.

8. The method for preparing bovine spinal cord peptide according to claim 1, characterized in that, In step S2, the complex enzyme is obtained by mixing alkaline protease, neutral protease and papain in a mass ratio of 1-2:1:1; the enzyme is inactivated by boiling water at a temperature of 95-100℃ for 10-20 minutes.

9. The method for preparing bovine spinal cord peptide according to claim 1, characterized in that, In step S3, the centrifugation speed is 6000-8000 r / min and the centrifugation time is 30-60 min; the temperature of the circulating ultrafiltration is 25-30℃, the ultrafiltration pressure is 1-2 MPa, and the ultrafiltration flow rate is 2-5 mL / min.

Images