A lyophilized powder injection of brain protein hydrolysate and a method for preparing the same

Abstract

The application discloses an injection brain protein hydrolysate freeze-dried powder injection and a preparation method thereof, and belongs to the technical field of preparation manufacturing. The preparation method of the injection brain protein hydrolysate freeze-dried powder injection is as follows: (1) taking pig brains, removing impurities and homogenizing; adding pepsin for enzymolysis and adding pancreatin for enzymolysis, and then carrying out enzyme inactivation, cooling, centrifugation, adsorption, filtration and vacuum concentration to obtain a brain protein hydrolysate stock solution; (2) adding an antioxidant stabilizer and mannitol to the brain protein hydrolysate stock solution, adjusting the pH to 6.9-7.5, and then carrying out filtration and freeze-drying to obtain the injection brain protein hydrolysate freeze-dried powder injection. The injection brain protein hydrolysate freeze-dried powder injection prepared in the application has good solubility, clarity, antioxidant property and long-term stability.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical manufacturing technology, specifically to a lyophilized powder injection of hydrolyzed brain protein and its preparation method. Background Technology

[0002] Brain protein hydrolysate is a complex preparation containing various small molecule peptides and free amino acids, obtained from animal brain tissue through enzymatic hydrolysis, separation, and purification processes. Clinically, it is usually formulated as an injection or lyophilized powder for injection. Due to the complex composition of brain protein hydrolysate, some peptides containing oxidatively sensitive amino acid residues such as methionine and tryptophan may undergo varying degrees of oxidation during preparation, lyophilization, and storage, which may affect the peptide composition, aggregation state, color, and solubility.

[0003] Freeze-drying can reduce the impact of moisture on the stability of brain protein hydrolysates to some extent. Existing freeze-dried formulations typically include excipients such as mannitol to improve freeze-drying properties and solubility. However, mannitol mainly serves as a skeletal support and has limited chemical oxidative protection against oxidatively sensitive peptides in brain protein hydrolysates. While conventional antioxidants such as methionine and glutathione can inhibit oxidation reactions to some extent in existing technologies, they still suffer from limited antioxidant protection and insufficient compatibility with complex polypeptide aqueous systems.

[0004] Chinese invention patent CN104096214A discloses a lyophilized powder injection of brain protein hydrolysate and its preparation method. The method is as follows: take pig brain, remove impurities, add purified water and homogenize; the homogenate is hydrolyzed by pepsin and trypsin successively, and the clear liquid is collected; the pH of the clear liquid is adjusted to 1.7-3, heated to 100-110℃, kept at the temperature for 15-45 min, and then the pH is adjusted to 8.0-9.0; ultrafiltration is performed using an 8-10kd membrane, and the filtrate is collected; the filtrate is subjected to column chromatography, the pH of the eluent is adjusted to 8.0-9.0, concentrated using an anion exchange membrane, and filtered through a 0.2μm membrane to obtain the stock solution; the stock solution is frozen, then thawed, excipients are added, the pH is adjusted to 6.9-7.5, ultrafiltration is performed using an 8-10kd membrane, and then filtered through a 0.2μm membrane; the solution is then filled and lyophilized. The obtained lyophilized powder injection contained peptides similar to the reference standard (≥0.90), contained 16 amino acids, and the nitrogen content of the total amino acids accounted for ≥85% of the total nitrogen content. No additional amino acids were required. It was prepared entirely by hydrolyzing pig brain, and was stable and safe. However, the antioxidant properties of the prepared lyophilized powder injection still need to be improved. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing a lyophilized powder injection of brain protein hydrolysate and the preparation method thereof.

[0006] A method for preparing a lyophilized powder injection of hydrolyzed brain protein, comprising the following steps: (1) Take pig brain, remove impurities, homogenize; add pepsin for enzymatic hydrolysis, add pancreatic enzyme for enzymatic hydrolysis, and after enzyme inactivation, cooling, centrifugation, adsorption, filtration, and vacuum concentration, obtain brain protein hydrolysate stock solution; (2) Add antioxidant stabilizer and mannitol to the original brain protein hydrolysate solution, adjust the pH to 6.9-7.5, filter and freeze dry to obtain the final product; The antioxidant stabilizer is obtained by reacting N-acetyl-L-methionine with taurine.

[0007] The molar ratio of N-acetyl-L-methionine to taurine is 1:1.05.

[0008] The reaction of N-acetyl-L-methionine with taurine was carried out at 25°C for 8 hours.

[0009] The amount of pepsin and pancreatin used is 0.35-0.45% of the weight of the pig brain.

[0010] The temperature for adding pepsin for enzymatic hydrolysis is 35-40℃, and the time is 2.5-3.5h.

[0011] The temperature for adding pancreatic enzyme for enzymatic hydrolysis is 35-40℃, and the time is 1.5-2.5h.

[0012] The enzyme inactivation temperature is 85-90℃, and the time is 10-15 minutes.

[0013] The mass ratio of the brain protein hydrolysate stock solution, antioxidant stabilizer, and mannitol is (0.9-1.1):(0.04-0.06):5, wherein the brain protein hydrolysate stock solution is calculated based on total nitrogen content.

[0014] The freeze-drying process is as follows: pre-freeze at -45℃ to -40℃ for 2-3 hours; evacuate to an absolute pressure of 20Pa, freeze-dry at -30℃ to -25℃ for 3-4 hours, then freeze-dry at -25℃ to -22℃ for 10-12 hours; reduce the absolute pressure to 15Pa, and raise the temperature to 25-30℃ for vacuum drying for 4-5 hours.

[0015] A lyophilized powder injection of brain protein hydrolysate is prepared by the above-described preparation method.

[0016] Due to the adoption of the above technical solutions, the beneficial effects of the present invention include: This invention improves the overall performance of lyophilized brain protein hydrolysate injections by introducing an antioxidant stabilizer and combining it with the supporting effect of mannitol on the lyophilized structure. Mannitol forms a loose, stable porous framework during lyophilization, which helps maintain the structural integrity of the lyophilized powder and promotes rapid water penetration during reconstitution, thereby improving solubility. The thioether group in the antioxidant stabilizer molecule can act as a preferential oxidation site to participate in the consumption of reactive oxygen species, reducing the degree of oxidation of oxidation-sensitive amino acid residues and small peptides in the brain protein hydrolysate. Its sulfonic acid group helps improve the dispersibility of the antioxidant stabilizer in the aqueous phase and its compatibility with the peptide system. Mannitol works synergistically with antioxidant stabilizers to reduce peptide oxidation, aggregation, and fine particle formation during freeze-drying and storage by stabilizing the physical structure of the freeze-dried product and providing chemical antioxidant protection. This improves the clarity of the reconstituted solution and delays the decay of antioxidant properties, thereby giving the prepared lyophilized powder of brain protein hydrolysate for injection good solubility, clarity, antioxidant properties, and long-term stability. Attached Figure Description

[0017] Figure 1 Comparison of infrared spectra of N-acetyl-L-methionine, taurine, and antioxidant stabilizers. Detailed Implementation

[0018] Example 1: Preparation of antioxidant stabilizers Under nitrogen protection, 150 mL of purified water, 0.1 mol N-acetyl-L-methionine, and 0.1 mol N-hydroxysuccinimide were mixed thoroughly. The pH was adjusted to 5.2 using a 1 mol / L sodium hydroxide aqueous solution. Under ice bath conditions, 120 mL of purified aqueous solution containing 0.11 mol 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added dropwise over 30 min. The mixture was then stirred and activated for 45 min. Next, 180 mL of purified aqueous solution containing 0.105 mol taurine was slowly added dropwise (the pH was adjusted and maintained at 7.8 ± 0.2 using a 1 mol / L sodium hydroxide aqueous solution during the addition), over 50 min. The reaction was carried out at 25 °C for 8 h. Finally, the pH was adjusted to 2.8 using a 1 mol / L hydrochloric acid aqueous solution, and then added to Cl... - In a strong-base quaternary ammonium type I anion exchange resin, adsorption was carried out by stirring at 20℃ and 150 rpm for 1 h. After filtration, the resin was collected and washed with purified water (2 × 350 mL). 400 mL of 0.4 mol / L ammonium bicarbonate aqueous solution was added, and the mixture was stirred at 20℃ and 150 rpm for 1 h for desorption. After filtration, the filtrate was added to H₂O. +In a strong acid cation exchange resin, the mixture was stirred at 20°C and 150 rpm for 30 min, filtered, and the filtrate was concentrated under reduced pressure at 40°C and -0.095 MPa for 1 h. The filtrate was then filtered through a 0.22 μm filter membrane, pre-frozen at -45°C for 2 h, evacuated to an absolute pressure of 20 Pa, freeze-dried at -25°C for 10 h, and then heated to 25°C and vacuum-dried at an absolute pressure of 15 Pa for 4 h to obtain an antioxidant stabilizer. The reaction equation is shown below:

[0019] Its proton NMR spectrum data are as follows: 1 H NMR (400 MHz, DMSO- d 6 ) δ 10.40 (s, 1H), 8.13-8.05 (m, 2H), 4.27-4.18 (m, 1H), 3.51-3.37 (m, 2H), 2.79-2.68 (m, 2H), 2.64-2.57 (m, 2H), 2.06 (s, 3H), 1.91 (s, 3H), 1.88-1.82 (m, 2H); HRMS (m / z):299.0731[M+H] + .

[0020] Figure 1 This is a comparison of the infrared spectra of N-acetyl-L-methionine, taurine, and antioxidant stabilizers. As shown in the figure, N-acetyl-L-methionine has a peak density at 1723 cm⁻¹. -1 A carboxyl-related C=O stretching vibration absorption peak appeared nearby, at 1658 cm⁻¹. -1 and 1540cm -1 Characteristic absorption peaks of amide I and amide II bands appeared nearby. Taurine showed a peak at 1202 cm⁻¹. -1 and 1042cm -1 The strong absorption peaks observed nearby are mainly attributed to SO3-related vibrations in the sulfonic acid groups. Compared to the two raw materials, the antioxidant stabilizer exhibits a higher absorption peak at 1654 cm⁻¹. -1 and 1544cm -1 The characteristic absorption peaks of amide I and amide II bands are retained nearby, and their peak intensities are significantly enhanced relative to N-acetyl-L-methionine, with a peak intensity of 1723 cm⁻¹. -1 The disappearance of the characteristic absorption indicates that the carboxyl group of N-acetyl-L-methionine has been transformed into an amide structure. Furthermore, the antioxidant stabilizer at 1192 cm⁻¹... -1 and 1046cm -1 The area still retains a clear characteristic absorption of SO3, and at 759 cm⁻¹ -1 and 706cm -1The observation of absorptions related to CS bonds and molecular skeleton vibrations indicates that both the sulfonic acid group derived from taurine and the thioether structure derived from N-acetyl-L-methionine are present in the obtained product. These infrared spectral results are consistent with the expected structure of an amide bond formed by the condensation of N-acetyl-L-methionine and taurine.

[0021] Example 2: Preparation of Lyophilized Powder of Brain Protein Hydrolysate for Injection (1) Take 10 kg of fresh pig brain, remove the meninges, obvious blood vessels, blood clots and adipose tissue, rinse with 5℃ purified water (2×10L), and drain the surface water; cut the processed pig brain into 2 cm pieces, add 25L of 5℃ purified water, and homogenize twice at 10℃ using a colloid mill (gap controlled at 0.2 mm); transfer to a reactor, heat to 35℃ at 150 rpm, adjust the pH to 2.0 with 1 mol / L hydrochloric acid aqueous solution, add 35 g of pepsin (enzyme activity of 3000 U / g), and enzymatically hydrolyze at 35℃ and 150 rpm for 3.5 h; adjust the pH to 7.8 with 1 mol / L sodium hydroxide aqueous solution, add 35 g of trypsin (enzyme activity of 5000 U / g), and continue enzymatic hydrolysis at 35℃ and 150 rpm for 2.5 h; heat to 85℃ and keep warm for 1 hour. Enzyme inactivation was performed for 5 minutes; after cooling to room temperature, the mixture was centrifuged at 5000 rpm for 20 minutes, and the supernatant was collected. 40 g of pharmaceutical activated carbon was added to the supernatant, and the mixture was stirred and adsorbed at 25℃ and 200 rpm for 20 minutes. The mixture was then filtered through 5 μm and 0.45 μm filter membranes, and ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa (ultrafiltration temperature: 15℃, transmembrane pressure: 0.15 MPa). The permeate was collected and concentrated under reduced pressure at 40℃ and a gauge pressure of -0.095 MPa for 1 hour to obtain the brain protein hydrolysate stock solution (total nitrogen concentration: 11.25 mg / mL). (2) Take 800 mL of the brain protein hydrolysate stock solution prepared in step (1) (total nitrogen content is 9 g), add 20 mL of water for injection containing 0.4 g of antioxidant stabilizer (prepared in Example 1), stir at 10°C and 150 rpm for 20 min, add 180 mL of water for injection containing 50 g of mannitol, stir at 15°C and 150 rpm for 15 min, adjust the pH to 6.9 with 0.1 mol / L sodium hydroxide aqueous solution, and continue stirring for 10 min; after 0. The sample was filtered through a 22μm sterile filter membrane and aseptically filled into vials, 2mL per vial. After half-stopping, the vials were placed in a freeze dryer and pre-frozen at -45℃ for 2h. The vacuum was then evacuated to an absolute pressure of 20Pa, and freeze-dried at -30℃ for 3h, followed by freeze-drying at -25℃ for 10h. The absolute pressure was reduced to 15Pa, and the temperature was raised to 25℃ for vacuum drying for 5h. Sterile nitrogen was introduced to restore atmospheric pressure, and the vials were stoppered in the freeze dryer. After removal, the vials were capped to obtain the lyophilized powder injection of brain protein hydrolysate.

[0022] Example 3: Preparation of Lyophilized Powder of Brain Protein Hydrolysate for Injection (1) Take 10 kg of fresh pig brain, remove the meninges, obvious blood vessels, blood clots and adipose tissue, rinse with 5℃ purified water (2×10L), and drain the surface water; cut the processed pig brain into 2 cm pieces, add 25 L of 5℃ purified water, and homogenize twice at 10℃ using a colloid mill (gap controlled at 0.2 mm); transfer to a reactor, heat to 38℃ at 150 rpm, adjust the pH to 2.0 with 1 mol / L hydrochloric acid aqueous solution, add 40 g of pepsin (enzyme activity of 3000 U / g), and enzymatically hydrolyze for 3 h at 38℃ and 150 rpm; adjust the pH to 7.8 with 1 mol / L sodium hydroxide aqueous solution, add 40 g of trypsin (enzyme activity of 5000 U / g), and continue enzymatic hydrolysis for 2 h at 38℃ and 150 rpm; heat to 88℃ and keep warm for 12 m The enzyme was inactivated by induction; after cooling to room temperature, the mixture was centrifuged at 5000 rpm for 20 min, and the supernatant was collected. 40 g of pharmaceutical activated carbon was added to the supernatant, and the mixture was stirred and adsorbed at 25 °C and 200 rpm for 20 min. The mixture was then filtered through 5 μm and 0.45 μm filter membranes, and ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa (ultrafiltration temperature: 15 °C, transmembrane pressure: 0.15 MPa). The permeate was collected and concentrated under reduced pressure at 40 °C and a gauge pressure of -0.095 MPa for 1 h to obtain the brain protein hydrolysate stock solution (total nitrogen concentration: 12.51 mg / mL). (2) Take 800 mL of the brain protein hydrolysate stock solution prepared in step (1) (total nitrogen content is 10 g), add 20 mL of water for injection containing 0.5 g of antioxidant stabilizer (prepared in Example 1), stir at 10°C and 150 rpm for 20 min, add 180 mL of water for injection containing 50 g of mannitol, stir at 15°C and 150 rpm for 15 min, adjust the pH to 7.2 using 0.1 mol / L sodium hydroxide aqueous solution, and continue stirring for 10 min; after 0.22 The sample was filtered through a μm sterile membrane and aseptically filled into vials, 2 mL per vial. After partially capping, the vials were placed in a freeze dryer and pre-frozen at -42°C for 2.5 h. The vacuum was then reduced to an absolute pressure of 20 Pa, and the vials were freeze-dried at -28°C for 3.5 h, followed by freeze-drying at -24°C for 11 h. The absolute pressure was then reduced to 15 Pa, and the vials were heated to 28°C for vacuum drying for 4.5 h. Sterile nitrogen was introduced to restore atmospheric pressure, and the vials were capped in the freeze dryer to obtain a lyophilized powder injection of hydrolyzed brain protein.

[0023] Example 4: Preparation of Lyophilized Powder of Brain Protein Hydrolysate for Injection (1) Take 10 kg of fresh pig brain, remove the meninges, obvious blood vessels, blood clots and adipose tissue, rinse with 5℃ purified water (2×10L), and drain the surface water; cut the processed pig brain into 2 cm pieces, add 25 L of 5℃ purified water, and homogenize twice at 10℃ using a colloid mill (gap controlled at 0.2 mm); transfer to a reactor, heat to 40℃ at 150 rpm, adjust the pH to 2.0 with 1 mol / L hydrochloric acid aqueous solution, add 45 g of pepsin (enzyme activity of 3000 U / g), and enzymatically hydrolyze at 40℃ and 150 rpm for 2.5 h; adjust the pH to 7.8 with 1 mol / L sodium hydroxide aqueous solution, add 45 g of trypsin (enzyme activity of 5000 U / g), and continue enzymatic hydrolysis at 40℃ and 150 rpm for 1.5 h; heat to 90℃ and keep warm for 1 hour. Enzyme inactivation was performed at 0 min; after cooling to room temperature, the mixture was centrifuged at 5000 rpm for 20 min, and the supernatant was collected. 40 g of pharmaceutical activated carbon was added to the supernatant, and the mixture was stirred and adsorbed at 25 °C and 200 rpm for 20 min. The mixture was then filtered sequentially through 5 μm and 0.45 μm filter membranes, and ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa (ultrafiltration temperature: 15 °C, transmembrane pressure: 0.15 MPa). The permeate was collected and concentrated under reduced pressure at 40 °C and a gauge pressure of -0.095 MPa for 1 h to obtain the original brain protein hydrolysate solution (total nitrogen concentration: 13.78 mg / mL). (2) Take 800 mL of the brain protein hydrolysate stock solution prepared in step (1) (total nitrogen content is 11 g), add 20 mL of water for injection containing 0.6 g of antioxidant stabilizer (prepared in Example 1), stir at 10°C and 150 rpm for 20 min, add 180 mL of water for injection containing 50 g of mannitol, stir at 15°C and 150 rpm for 15 min, adjust the pH to 7.5 with 0.1 mol / L sodium hydroxide aqueous solution, and continue stirring for 10 min; after 0 The sample was filtered through a .22μm sterile filter membrane and aseptically filled into vials, 2mL per vial. After half-stopping, the vials were placed in a lyophilizer and pre-frozen at -40℃ for 3 hours. The pressure was then evacuated to 20Pa and freeze-dried at -25℃ for 4 hours, followed by sublimation drying at -22℃ for 12 hours. The pressure was then reduced to 15Pa, and the sample was heated to 30℃ for vacuum drying for 4 hours. Sterile nitrogen was introduced to restore atmospheric pressure, and the vials were sealed in the lyophilizer. The vials were then removed and capped to obtain the lyophilized powder of brain protein hydrolysate for injection.

[0024] Comparative Example 1: The preparation method of the lyophilized powder injection of brain protein hydrolysate is basically the same as that of Example 3, except that the antioxidant stabilizer is replaced with a physical mixture of 0.3g N-acetyl-L-methionine and 0.2g taurine.

[0025] Comparative Example 2: The preparation method of the lyophilized powder of brain protein hydrolysate for injection is basically the same as that of Example 3, except that the antioxidant stabilizer is replaced with an equal weight of antioxidant stabilizer prepared by the following method: The preparation method of the antioxidant stabilizer is basically the same as that in Example 1, except that N-acetyl-L-methionine is replaced with an equimolar amount of N-acetyl-L-leucine.

[0026] Comparative Example 3: The preparation method of the lyophilized powder injection of brain protein hydrolysate is basically the same as that of Example 3, except that the antioxidant stabilizer is replaced with an equal weight of antioxidant stabilizer prepared by the following method: The preparation method of the antioxidant stabilizer is basically the same as that in Example 1, except that taurine is replaced with an equimolar amount of 4-aminobutane-1-sulfonic acid.

[0027] The preparation method of the lyophilized powder of brain protein hydrolysate for injection in Comparative Example 4 is basically the same as that in Example 3, except that the amount of antioxidant stabilizer is replaced with 0.25g.

[0028] Comparative Example 5: The preparation method of the lyophilized powder of brain protein hydrolysate for injection is basically the same as that of Example 3, except that no antioxidant stabilizer is added, and 200 ml of water for injection containing 50.5 g mannitol is added.

[0029] The embodiments and comparative examples in this application use Cl - The strong basic quaternary ammonium type I anion exchange resin, model number 201×7, is manufactured by Zhejiang Zhengguang Industrial Co., Ltd.; H + The type of strong acid cation exchange resin is Purolite. TM C100H.

[0030] The lyophilized powder of brain protein hydrolysate prepared in the examples and comparative examples was tested for solubility, clarity, antioxidant properties and long-term stability. The test results are shown in Table 1.

[0031] Solubility test: Randomly take 6 vials of lyophilized powder for injection from each group, and equilibrate at 25°C for 30 min; place the vials on a shaker, use a syringe to add 2 mL of water for injection at 25°C to each vial at a constant speed, with the water adding duration being 5 s. Immediately start the shaker and a stopwatch upon completion of water addition, and perform shaking reconstitution at 25°C and 100 rpm; observe the dissolution state of the lyophilized powder for injection, and take the point at which the lyophilized powder for injection is completely dissolved with no undissolved solid visible to the naked eye in the solution as the reconstitution endpoint. Bubbles generated during the shaking process shall not be judged as undissolved solids; record the time required from the completion of water addition to reaching the reconstitution endpoint. Test 6 vials for each group, calculate the average reconstitution time, and the result is expressed in seconds (s). A shorter reconstitution time indicates better solubility of the lyophilized powder for injection.

[0032] Clarity test: The clarity test is carried out with reference to the first method (visual method) of the 0902 Clarity Inspection Method in the 2025 Edition Volume IV of the Chinese Pharmacopoeia. Randomly take 6 vials of lyophilized powder for injection from each group, fully reconstitute the samples according to the solubility test method, and let the samples stand at 25°C for 10 min after reconstitution is completed to obtain the reconstituted solution. Take 1 mL of the reconstituted solution and an equal volume of No. 0.5 turbidity standard solution, place them respectively in glass tubes for turbidity measurement of the same specification (colorless and transparent, inner diameter 16 mm, flat bottom), place the tubes on a clarity tester (model HN200A, manufacturer Shanghai Yinhuang Technology Co., Ltd.) with an illuminance of 1000 LX, and observe the turbidity degree of the reconstituted solution from the horizontal direction; if the turbidity degree of the reconstituted solution does not exceed that of the No. 0.5 turbidity standard solution, it is judged as qualified.

[0033] Antioxidant performance test: Randomly take 6 vials of lyophilized powder for injection from each group, fully reconstitute the samples according to the solubility test method, and let the samples stand at 25°C for 10 min after reconstitution is completed to obtain the reconstituted solution. Mix 1 mL of the reconstituted solution with 1 mL of 0.1 mmol / L DPPH ethanol solution as Group A1; mix 1 mL of water for injection with 1 mL of 0.1 mmol / L DPPH ethanol solution as Group A0; mix 1 mL of the reconstituted solution with 1 mL of absolute ethanol solution as Group A2. Let the solutions of the above groups react in the dark at 25°C for 10 min, measure the absorbance at 517 nm, and calculate the DPPH radical scavenging rate according to the formula: DPPH radical scavenging rate = [A0-(A1-A2)] / A0×100%. Test 6 parallel samples for each group, and take the average value of the results.

[0034] Long-term stability test: Randomly take 6 vials of lyophilized powder for injection from each group, store them at 5°C under light-shielded conditions for 6 months, and then perform the antioxidant performance test again according to the test method for antioxidant performance.

[0035] Table 1 Performance test data sheet

[0036] As shown in Table 1, the lyophilized powder for injection of brain protein hydrolysate prepared in this application has good solubility, clarity, antioxidant properties and long-term stability.

[0037] In Comparative Example 1, N-acetyl-L-methionine and taurine were only physically mixed, and the two did not form a unified molecular structure through amide bonds. The thioether group and the hydrophilic sulfonic acid group could not work synergistically, so its antioxidant properties and long-term stability were reduced.

[0038] The antioxidant stabilizer prepared in Comparative Example 2 lacks thioether groups that can participate in preferential oxidation, which weakens its ability to consume reactive oxygen species, resulting in a decrease in the initial DPPH radical scavenging rate.

[0039] Comparative Example 3 used 4-aminobutane-1-sulfonic acid instead of taurine. The added methylene group increased the hydrophobic segment of the antioxidant stabilizer, which changed the spatial distance between the thioether group and the sulfonic acid group and the hydrophilic / hydrophobic balance of the molecule. This was not conducive to the uniform distribution of the antioxidant stabilizer in the aqueous phase of brain protein hydrolysate. Therefore, the reconstitution time was slightly prolonged and the long-term stability was reduced.

[0040] In Comparative Example 4, after reducing the amount of antioxidant stabilizer, the number of effective thioether antioxidant sites in the system was insufficient, the ability to consume free radicals and reactive oxygen species decreased, and the antioxidant components that could continuously play a protective role during storage were insufficient, thus reducing antioxidant properties and long-term stability.

[0041] Comparative Example 5 did not contain any antioxidant stabilizers, and the system lacked a thioether antioxidant structure, relying mainly on the antioxidant capacity of the brain protein hydrolysate itself, resulting in a significant reduction in antioxidant properties and long-term stability.

[0042] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. However, any modifications, alterations, and variations made by those skilled in the art without departing from the scope of the present invention based on the disclosed technical content are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and variations made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a lyophilized powder injection of hydrolyzed brain protein, characterized in that, Includes the following steps: (1) Take pig brain, remove impurities, homogenize; add pepsin for enzymatic hydrolysis, add pancreatic enzyme for enzymatic hydrolysis, and after enzyme inactivation, cooling, centrifugation, adsorption, filtration, and vacuum concentration, obtain brain protein hydrolysate stock solution; (2) Add antioxidant stabilizer and mannitol to the original brain protein hydrolysate solution, adjust the pH to 6.9-7.5, filter and freeze dry to obtain the final product; The antioxidant stabilizer is obtained by reacting N-acetyl-L-methionine with taurine.

2. The method for preparing a lyophilized powder injection of hydrolyzed brain protein as described in claim 1, characterized in that, The molar ratio of N-acetyl-L-methionine to taurine is 1:1.

05.

3. The method for preparing a lyophilized powder injection of hydrolyzed brain protein as described in claim 1, characterized in that, The reaction of N-acetyl-L-methionine with taurine was carried out at 25°C for 8 hours.

4. The method for preparing a lyophilized powder injection of hydrolyzed brain protein as described in claim 1, characterized in that, The amount of pepsin and pancreatin used is 0.35-0.45% of the weight of the pig brain.

5. The method for preparing a lyophilized powder injection of hydrolyzed brain protein as described in claim 1, characterized in that, The temperature for adding pepsin for enzymatic hydrolysis is 35-40℃, and the time is 2.5-3.5h.

6. The method for preparing a lyophilized powder injection of hydrolyzed brain protein as described in claim 1, characterized in that, The temperature for adding pancreatic enzyme for enzymatic hydrolysis is 35-40℃, and the time is 1.5-2.5h.

7. The method for preparing a lyophilized powder injection of hydrolyzed brain protein as described in claim 1, characterized in that, The enzyme inactivation temperature is 85-90℃, and the time is 10-15 minutes.

8. The method for preparing a lyophilized powder injection of hydrolyzed brain protein as described in claim 1, characterized in that, The mass ratio of the brain protein hydrolysate stock solution, antioxidant stabilizer, and mannitol is (0.9-1.1):(0.04-0.06):5, wherein the brain protein hydrolysate stock solution is calculated based on total nitrogen content.

9. The method for preparing a lyophilized powder injection of hydrolyzed brain protein as described in claim 1, characterized in that, The freeze-drying process is as follows: pre-freeze at -45℃ to -40℃ for 2-3 hours; evacuate to an absolute pressure of 20Pa, freeze-dry at -30℃ to -25℃ for 3-4 hours, then freeze-dry at -25℃ to -22℃ for 10-12 hours; reduce the absolute pressure to 15Pa, and raise the temperature to 25-30℃ for vacuum drying for 4-5 hours.

10. A lyophilized powder injection of hydrolyzed brain protein, characterized in that, It is prepared by the preparation method according to any one of claims 1-9.

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