A lyophilization protective agent for bioactive peptides, its use and lyophilized powder and method of preparation

Abstract

The application discloses a freeze-drying protective agent for bioactive peptides and application and freeze-dried powder and a preparation method thereof. The application belongs to the technical field of bioactive substance preservation, and particularly relates to a freeze-drying protective agent for bioactive peptides, which is composed of the following components: chitosan oligosaccharide, gamma-polyglutamic acid, trehalose, betaine, glutathione, and the rest is histidine buffer with a concentration of 10-50 mM. A method for preparing freeze-dried powder of bioactive peptides by using the freeze-drying protective agent for bioactive peptides comprises the following steps: pre-freezing, mixing bioactive peptides with the freeze-drying protective agent for bioactive peptides, primary drying, and secondary drying to obtain the freeze-dried powder of bioactive peptides. The freeze-drying protective agent for bioactive peptides and the application and freeze-drying method thereof have the advantages that the freeze-dried powder of bioactive peptides after freeze-drying has high activity retention rate, rapid re-dissolution and excellent long-term stability.

Description

Technical Field

[0001] This invention belongs to the field of bioactive substance preservation technology, and particularly relates to a bioactive peptide freeze-drying protectant, its application, freeze-dried powder, and preparation method. Background Technology

[0002] Bioactive peptides, due to their small molecular weight, high bioavailability, and various biological activities such as antioxidation, blood pressure reduction, and immune regulation, have broad application prospects in functional foods, cosmetics, and pharmaceuticals. However, these peptides have poor stability in aqueous solutions and are prone to deamidation, oxidation, aggregation, and microbial contamination. Therefore, they usually need to be freeze-dried into dry powder formulations to extend shelf life and maintain activity.

[0003] Freeze-drying is one of the most commonly used drying methods for bioactive substances. It primarily involves freezing water at low temperatures and then allowing the ice crystals to sublimate directly under vacuum conditions to achieve dehydration. While this process avoids high-temperature damage, it can still cause various forms of damage to bioactive peptides: Freeze-concentration effect: During ice crystal formation, the solute concentration in the unfrozen aqueous phase increases dramatically, leading to a shortening of the distance between peptide molecules and increasing the risk of aggregation and precipitation. Ice crystal-solution interface-induced denaturation: Peptide molecules may adsorb onto the surface of ice crystals, undergoing conformational changes or irreversible aggregation. pH drift: Certain buffer salts (such as phosphates) cause drastic changes in solution pH during freezing due to selective crystallization, thus affecting peptide stability. Dehydration stress: During the drying stage, the removal of the hydration layer directly disrupts the hydrogen bond network between peptide and water molecules, potentially leading to the loss of secondary structures.

[0004] To overcome the aforementioned problems, extensive research has been conducted on lyophilization protectants. Commonly used protectants include sugars (trehalose, sucrose, lactose), polyols (mannitol, sorbitol), amino acids (arginine, histidine), and polymers (polyethylene glycol, polyvinylpyrrolidone). Among these, trehalose is considered the "gold standard" protectant due to its excellent glass-forming ability and hydrogen bond substitution effect. In addition, some studies have attempted to add surfactants (such as Tween-80) to reduce interfacial adsorption, or add antioxidants (such as vitamin C, glutathione) to inhibit oxidative degradation. In recent years, some studies have reported the use of antifreeze proteins or artificially designed amphiphilic peptides as lyophilization protectants; however, these are costly, have limited availability, and involve the introduction of exogenous proteins or peptides, potentially leading to immunogenicity risks and regulatory compliance issues.

[0005] Therefore, developing a freeze-drying protectant specifically targeting the synergistic protective mechanism of bioactive peptides and its matching freeze-drying method has significant industrial application value and innovative significance. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a lyophilization protectant for bioactive peptides, its application, lyophilized powder, and its preparation method. The lyophilization protectant provided by this invention, combined with the lyophilization method, results in a high activity retention rate, rapid reconstitution, and excellent long-term stability of the lyophilized bioactive peptide powder.

[0007] To achieve the above objectives, the present invention provides a bioactive peptide lyophilization protectant, which is composed of the following components in weight-volume percentages: chitosan oligosaccharide 0.5%–3.0% (w / v), γ-polyglutamic acid 0.1%–1.0% (w / v), trehalose 2%–8% (w / v), betaine 0.5%–2.0% (w / v), glutathione 0.02%–0.1% (w / v), and the balance being a histidine buffer solution with a concentration of 10–50 mM.

[0008] Chitosan oligosaccharides: The abundant hydroxyl and amino functional groups on their molecular chains can form a synergistic hydrogen bond network with peptides. Their low molecular weight allows them to maintain fluidity under freeze-concentration conditions, avoiding structural collapse caused by high viscosity. Chitosan oligosaccharides themselves carry a positive charge, which can interact electrostatically with commonly found negatively charged residues in peptides, stabilizing the peptide chain conformation.

[0009] γ-Polyglutamic acid: a natural biodegradable polymer, rich in carboxyl groups, forms a hydration layer network in aqueous solution. Its "molecular fence" effect can confine peptide molecules in a hydration microenvironment during freeze-drying, reducing direct contact at the ice crystal interface, inhibiting interface-induced aggregation and denaturation, and forming a polysaccharide-peptide synergistic dual network with chitosan oligosaccharide.

[0010] Trehalose: a glassy protective agent that forms an amorphous matrix, replaces hydrogen bonds in water molecules, and increases the glass transition temperature of the system.

[0011] Betaine: Its zwitterionic properties allow it to stabilize both the hydrophilic and hydrophobic regions of peptide molecules. Under freeze-concentration conditions, it can preferentially hydrate, locking water molecules around the peptide molecules.

[0012] Glutathione: Prevents easily oxidized methionine and cysteine ​​residues in peptides from being oxidized during freeze-drying and storage.

[0013] Histidine buffer: to prevent pH drift caused by freeze concentration and maintain the optimal stable pH range of peptides.

[0014] Preferably, the chitosan oligosaccharide has a molecular weight ≤1500 Da and a degree of deacetylation ≥90%.

[0015] Preferably, the pH of the histidine buffer solution is 6.0 to 7.5.

[0016] The present invention also provides the application of the bioactive peptide freeze-drying protectant in the preparation of bioactive peptide freeze-dried powder.

[0017] The present invention also provides a method for preparing lyophilized bioactive peptide powder using the aforementioned lyophilization protectant, comprising the following steps: 1) Pre-freezing: Mix bioactive peptides with bioactive peptide freeze-drying protectant, cool to -10℃ and hold for 20~40min to induce uniform ice nucleus formation and generate a primary ice crystal distribution network. Quickly cool to -45℃ and hold for 80~100min to form a fine and dense ice crystal structure, reducing mechanical damage to peptide molecules. Heat to -15℃ and hold for 50~70min to promote controlled crystallization of chitosan oligosaccharide and some trehalose, forming a through-hole microporous network, providing a smooth channel for water sublimation, and releasing the internal stress generated by freeze concentration, reducing compression damage to peptide molecules. Cool to -50℃ and hold for 20~40min to solidify the pore structure and stabilize the spatial configuration of the protective matrix. 2) Single drying: Under a vacuum of 10~15Pa, heat to 0℃ at a heating rate of 0.2~0.5℃ / min, and hold for 20~40min for every 5℃ increase; 3) Secondary drying: Under vacuum of ≤0.1Pa, the temperature is increased to 35℃ at a rate of 0.1~0.3℃ / min to obtain lyophilized bioactive peptide powder.

[0018] Preferably, in step 1), the ratio of bioactive peptide to bioactive peptide freeze-drying protectant is 0.5~5 mg: 1 mL.

[0019] Preferably, in step 1), the cooling rate to -10℃ is 0.4~0.6℃ / min, the cooling rate to -45℃ is 2~4℃ / min, the heating rate to -15℃ is 0.8~1.2℃ / min, and the cooling rate to -50℃ is 1~3℃ / min.

[0020] Preferably, the vacuum degree during the heating process in step 2) is 12~13 Pa.

[0021] Preferably, the vacuum degree during the heating process in step 3) is ≤0.05Pa.

[0022] The present invention also provides a method for preparing lyophilized bioactive peptide powder to obtain the lyophilized bioactive peptide powder.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides a bioactive peptide lyophilization protectant, its application, and a lyophilization method. The bioactive peptide lyophilization protectant of this invention is composed of chitosan oligosaccharide, γ-polyglutamic acid, trehalose, betaine, glutathione, and histidine buffer. The lyophilization protection effect of chitosan oligosaccharide stems from its unique multiple synergistic mechanisms: interfacial exclusion effect: the high hydration capacity of chitosan oligosaccharide allows it to be excluded from the unfrozen aqueous phase during ice crystal formation, forming a protective molecular brush layer that prevents peptide molecules from directly adsorbing onto the ice crystal-solution interface, reducing interface-induced peptide aggregation and conformational changes; electrostatic anchoring effect: the -NH3 group of chitosan oligosaccharide... + The -COO group of common Asp and Glu residues in peptides - Electrostatic interactions anchor peptide molecules within the protective matrix, preventing migration and collision aggregation during freeze-drying. A hydrogen bond substitution network is also employed: the polyhydroxy structure of chitosan oligosaccharides forms a substitute hydrogen bond network with peptide molecules during dehydration, stabilizing the peptide's secondary structure. This invention is the first to combine chitosan oligosaccharides with γ-polyglutamic acid (PGA) to form a polysaccharide-polymer synergistic dual-network protection system. Electrostatic attraction and hydrogen bonding exist between the carboxyl groups of γ-PGA and the amino groups of chitosan oligosaccharides, forming a physical cross-linked network. This network is compressed and enriched during ice crystal formation, forming a high-concentration protective concentrated shell that encapsulates the oligopeptide molecules, achieving dual isolation protection. γ-PGA provides a molecular fence confining hydration microenvironment, while chitosan oligosaccharides provide interfacial exclusion and hydrogen bond substitution. Their synergistic effect significantly reduces structural damage during freeze-drying. The formulation of this invention simultaneously achieves the synergistic effect of three protection mechanisms: chitosan oligosaccharide interfacial protection, γ-PGA and betaine hydration layer protection, and trehalose glassy state protection, constituting a "triple insurance" freeze-drying protection system. The freeze-drying method described in this invention employs a two-step annealing process. The first annealing is carried out at -10°C, which allows some of the water to form an oriented ice crystal framework. The second annealing is carried out at -15°C, with the temperature precisely set within the crystallization temperature range of chitosan oligosaccharides, achieving "designable micropores." This two-step annealing strategy, combined with the freeze-drying protectant formulation, creates a synergistic effect. The three-dimensional network structure formed by the chitosan oligosaccharides during annealing provides a preferential pathway for subsequent water sublimation, significantly shortening the drying time by approximately 30% while ensuring the mechanical strength of the cake structure. The freeze-drying protectant provided by this invention, combined with the freeze-drying method, results in a high activity retention rate, rapid reconstitution, and excellent long-term stability of the freeze-dried bioactive peptide powder. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The DPPH clearance rate of tuna oligopeptides in each group; Figure 2 The ACE inhibition rate of tuna oligopeptides in each group; Figure 3 The hydroxyl radical scavenging rate of each group of tuna oligopeptides is represented. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] The materials used in this invention were sourced as follows: histidine buffer was purchased from Merck; tuna oligopeptides, anserine, and fish bone collagen oligopeptides were obtained from Hainan Yutai Biopharmaceutical Co., Ltd.; chitosan was purchased from Qingdao Bozhi Huifeng Biotechnology; γ-polyglutamic acid was purchased from Nanjing Xuankai Biotechnology; trehalose was purchased from Hayashihara, Japan; betaine was purchased from Sigma-Aldrich; and glutathione was purchased from Aladdin.

[0032] The preparation method of the bioactive peptide lyophilization protectant of this invention is as follows: Take approximately 80 mL of histidine buffer solution in a 100 mL beaker, place it on a magnetic stirrer, and start stirring at 300-500 rpm. Add the components in the following order (adding the next component only after complete dissolution): trehalose, betaine, chitosan oligosaccharide, γ-polyglutamic acid, and glutathione. After all components are completely dissolved, transfer the solution to a 100 mL volumetric flask. Wash the beaker several times with histidine buffer solution and transfer the solution to the volumetric flask, finally bringing the volume to the 100 mL mark. Take the diluted solution and measure the pH value using a precision pH meter. Fine-tune the pH to 6.8 ± 0.1 using 1M NaOH or 1M HCl (ensuring the final pH is within the range of 6.0-7.5). Transfer the diluted solution back to the beaker and continue stirring for 5 minutes to ensure uniform mixing. Use a 0.22 μm PES (polyethersulfone) or PVDF (polyvinylidene fluoride) sterile filter membrane, with the diameter selected according to the filtration volume (47 mm or 50 mm recommended). In a biosafety cabinet or clean bench, use a sterile syringe or peristaltic pump to push the cryoprotectant solution through a 0.22 μm filter membrane into a sterile container. Aliquot the single-use amount into sterile lyophilized vials or centrifuge tubes and seal tightly. Store at 2–8°C protected from light. For long-term storage, freeze at -20°C.

[0033] Example 1 The bioactive peptide lyophilization protectant consists of the following components by weight / volume percentage: chitosan oligosaccharide 1.5% (w / v), γ-polyglutamic acid 0.5% (w / v), trehalose 5% (w / v), betaine 1.0% (w / v), glutathione 0.05% (w / v), with the balance being a 20 mM histidine buffer at pH 6.8. The chitosan oligosaccharide has a molecular weight ≤1500 Da and a degree of deacetylation ≥90%.

[0034] 1) Pre-freezing: Mix tuna oligopeptides and bioactive peptide freeze-drying protectant at a ratio of 2.5 mg: 1 mL, cool to -10 °C at a rate of 0.5 °C / min and hold for 30 min, then rapidly cool to -45 °C at a rate of 3 °C / min and hold for 90 min, then heat to -15 °C at a rate of 1 °C / min and hold for 60 min, then cool to -50 °C at a rate of 2 °C / min and hold for 30 min.

[0035] 2) Single drying: Heat to 0℃ at a rate of 0.3℃ / min and a vacuum of 12~13Pa, and hold for 30min for every 5℃ increase.

[0036] 3) Secondary drying: The temperature is increased to 35℃ at a heating rate of 0.2℃ / min and a vacuum degree of ≤0.05Pa to obtain tuna oligopeptide freeze-dried powder.

[0037] Example 2 The bioactive peptide lyophilization protectant consists of the following components by weight / volume percentage: chitosan oligosaccharide 0.5% (w / v), γ-polyglutamic acid 0.1% (w / v), trehalose 2% (w / v), betaine 0.5% (w / v), glutathione 0.02% (w / v), with the balance being a 10 mM histidine buffer at pH 6.0. The chitosan oligosaccharide has a molecular weight ≤1500 Da and a degree of deacetylation ≥90%.

[0038] 1) Pre-freezing: Mix goose muscle peptide and bioactive peptide freeze-drying protectant at a ratio of 0.5 mg: 1 mL, cool to -10 °C at a rate of 0.4 °C / min, hold for 20 min, rapidly cool to -45 °C at a rate of 2 °C / min, hold for 80 min, heat to -15 °C at a rate of 0.8 °C / min, hold for 50 min, and cool to -50 °C at a rate of 1 °C / min, hold for 20 min.

[0039] 2) Single drying: Heat to 0℃ at a rate of 0.2℃ / min and a vacuum of 10~15Pa, and hold for 20 min for every 5℃ increase.

[0040] 3) Secondary drying: The temperature is raised to 35℃ at a heating rate of 0.1℃ / min and a vacuum degree of ≤0.1Pa to obtain goose muscle peptide lyophilized powder.

[0041] Example 3 The bioactive peptide lyophilization protectant consists of the following components by weight / volume percentage: chitosan oligosaccharide 3% (w / v), γ-polyglutamic acid 1.0% (w / v), trehalose 8% (w / v), betaine 2.0% (w / v), glutathione 0.1% (w / v), with the balance being a 50 mM histidine buffer at pH 7.5. The chitosan oligosaccharide has a molecular weight ≤1500 Da and a degree of deacetylation ≥90%.

[0042] 1) Pre-freezing: Mix fish bone collagen oligopeptides with bioactive peptide freeze-drying protectant at a ratio of 5 mg: 1 mL, cool to -10°C at a rate of 0.6°C / min and hold for 40 min, then rapidly cool to -45°C at a rate of 4°C / min and hold for 100 min, then heat to -15°C at a rate of 1.2°C / min and hold for 70 min, then cool to -50°C at a rate of 3°C / min and hold for 40 min.

[0043] 2) Single drying: Heat to 0℃ at a rate of 0.5℃ / min and a vacuum of 10~15Pa, and hold for 40 min for every 5℃ increase.

[0044] 3) Secondary drying: The temperature is raised to 35℃ at a heating rate of 0.3℃ / min and a vacuum degree of ≤0.1Pa to obtain fish bone collagen oligopeptide freeze-dried powder.

[0045] Comparative Example 1 Tuna oligopeptides were dissolved in a 5% (w / v) trehalose aqueous solution to prepare a solution system with a final concentration of 1.5 mg / mL. A conventional linear temperature-increase freeze-drying process was adopted. In the pre-freezing stage, the temperature was decreased to -45℃ at 1℃ / min and held directly for 3 hours. In the first drying stage, the temperature was linearly increased from -45℃ to 0℃ at 0.5℃ / min, and the vacuum degree was maintained at 15 Pa. In the second drying stage, the temperature was increased to 35℃ at 0.5℃ / min, and the vacuum degree was 0.2 Pa, to obtain tuna oligopeptide freeze-dried powder.

[0046] Comparative Example 2 Tuna oligopeptides were dissolved in a 5% (w / v) trehalose and 2% (w / v) mannitol aqueous solution (currently a commonly used freeze-drying protectant in the industry) to prepare a solution system with a final concentration of 1.5 mg / mL. A conventional linear temperature-increase freeze-drying process was adopted. In the pre-freezing stage, the temperature was decreased to -45℃ at 1℃ / min and held directly for 3 hours. In the first drying stage, the temperature was linearly increased from -45℃ to 0℃ at 0.5℃ / min, and the vacuum degree was maintained at 15 Pa. In the second drying stage, the temperature was increased to 35℃ at 0.5℃ / min, and the vacuum degree was 0.2 Pa to obtain tuna oligopeptide freeze-dried powder.

[0047] Comparative Example 3 Goose muscle peptide was dissolved in a 5% (w / v) trehalose aqueous solution to prepare a solution system with a final concentration of 1.5 mg / mL. A conventional linear temperature-increase freeze-drying process was adopted. In the pre-freezing stage, the temperature was decreased to -45℃ at 1℃ / min and held directly for 3 hours. In the first drying stage, the temperature was linearly increased from -45℃ to 0℃ at 0.5℃ / min, and the vacuum degree was maintained at 15 Pa. In the second drying stage, the temperature was increased to 35℃ at 0.5℃ / min, and the vacuum degree was 0.2 Pa to obtain goose muscle peptide freeze-dried powder.

[0048] Comparative Example 4 Fish bone collagen oligopeptides were dissolved in a 5% (w / v) trehalose aqueous solution to prepare a solution system with a final concentration of 1.5 mg / mL. A conventional linear temperature-increase freeze-drying process was adopted. In the pre-freezing stage, the temperature was decreased to -45℃ at 1℃ / min and held directly for 3 hours. In the first drying stage, the temperature was linearly increased from -45℃ to 0℃ at 0.5℃ / min, and the vacuum degree was maintained at 15 Pa. In the second drying stage, the temperature was increased to 35℃ at 0.5℃ / min, and the vacuum degree was 0.2 Pa, to obtain fish bone collagen oligopeptide freeze-dried powder.

[0049] Example 4 I. Testing the freeze-drying effect of tuna oligopeptides in Example 1, Comparative Example 1, and Comparative Example 2: (1) Determination of DPPH free radical scavenging activity: 1,1-Diphenyl-2-picrylhydrazine (DPPH) free radical scavenging rate was used as an indicator of antioxidant activity. A precise 0.2 mM DPPH ethanol solution was prepared (stored protected from light, freshly prepared). Tuna oligopeptide samples were diluted with deionized water to a series of concentration gradients (0.05, 0.1, 0.2, 0.5, 1.0 mg / mL). 2 mL of sample solution was mixed with 2 mL of DPPH solution, shaken thoroughly, and reacted at room temperature in the dark for 30 minutes. The absorbance at 517 nm was measured (A1). Deionized water was used as a blank control (A0), and a mixture of the corresponding concentration of sample solution and anhydrous ethanol was used as a background control (A2) to eliminate interference from the sample's own color. The DPPH free radical scavenging rate was calculated as follows: DPPH scavenging rate (%) = [1 – (A1 – A2) / A0] × 100%.

[0050] Plot a curve of sample concentration against clearance rate and calculate the half-inhibitory concentration (IC50). 50 IC 50 The smaller the value, the stronger the antioxidant activity.

[0051] (2) Determination of angiotensin-converting enzyme inhibitory activity: ACE inhibitory activity was determined by high-performance liquid chromatography (HPLC). Hippuric acid was generated by adding ACE enzyme to a substrate, and the peak area of ​​hippuric acid was determined using a C18 reversed-phase column. Tuna oligopeptide samples, before or after lyophilization, were dissolved and diluted with deionized water to a series of concentration gradients (0.5, 1.0, 2.0, 3.0, 5.0 mg / mL) to determine the ACE inhibition rate at different concentrations, and the IC50 was calculated. 50 The reaction was performed by adding 20 μL of ACE solution (0.1 U / mL), 20 μL of sample solution, and 200 μL of HHL substrate (5 mmol / L, dissolved in 0.1 mol / L borate buffer containing 0.3 mol / L NaCl, pH 8.3) to the sample tube and incubating at 37 °C for 30 minutes. The reaction was then terminated by adding 250 μL of 1 mol / L HCl. The control tube was treated with buffer solution instead of the sample. The reaction solution was filtered through a 0.22 μm filter and 20 μL was injected. The peak area of ​​hippuric acid was measured at 228 nm. The ACE inhibition rate was calculated as follows: ACE inhibition rate (%) = (A control – A sample) / (A control – A blank) × 100%, where A control is the peak area of ​​hippuric acid in the control tube, A sample is the peak area of ​​hippuric acid in the sample tube, and A blank is the peak area of ​​the blank tube.

[0052] Plot a curve of sample concentration versus inhibition rate and calculate IC. 50 IC 50 The smaller the value, the stronger the ACE inhibitory activity.

[0053] (3) Determination of hydroxyl radical scavenging activity: The hydroxyl radical scavenging rate was determined using the Fenton reaction system. 1 mL each of 9 mmol / L FeSO4 solution and 9 mmol / L salicylic acid-ethanol solution were added sequentially to test tubes, followed by 1 mL of sample solutions of different concentrations (0.2, 0.5, 1.0, 2.0, and 5.0 mg / mL). Finally, 1 mL of 6 mmol / L H2O2 solution was added to initiate the reaction. The reaction was carried out in a 37°C water bath for 30 minutes, and the absorbance at 510 nm was measured. Deionized water was used as a blank control instead of the sample, and deionized water was used as a background control instead of H2O2 to eliminate interference from the sample's own color.

[0054] (4) Determination of total peptide content: Biuret reagent: Weigh 1.5g of copper sulfate (CuSO4·5H2O) and 6.0g of potassium sodium tartrate (C4H4O6KNa·4H2O), dissolve them in 500mL of deionized water, add 300mL of 10% sodium hydroxide solution while stirring, and make up to 1000mL with deionized water. Mix well and store at room temperature for later use.

[0055] Standard peptide solution: Accurately weigh glycyl-glycyl-glycine (Gly-Gly-Gly, molecular weight 189.2) standard dried to constant weight at 110℃, prepare a stock solution of 1.0 mg / mL with deionized water, and then dilute to prepare standard working solutions of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL.

[0056] Accurately weigh approximately 10 mg of the lyophilized powder sample to be tested (before or after lyophilization), dissolve it in deionized water, and bring the volume to 10 mL (final concentration approximately 1.0 mg / mL). Take 1.0 mL of the above sample solution, add 4.0 mL of biuret reagent, mix immediately, and let stand at room temperature for 30 minutes. Use 1.0 mL of deionized water and 4.0 mL of biuret reagent as blank controls, and measure the absorbance at 540 nm. Similarly, take 1.0 mL of each concentration of standard working solution, add 4.0 mL of biuret reagent, and measure the absorbance using the same method. Plot a standard curve (absorbance A versus concentration C, mg / mL). Calculate the total peptide concentration in the sample solution according to the standard curve equation, and then calculate the total peptide content (mg / g) in the lyophilized powder according to the dilution factor. Total peptide content retention rate (%) = (total peptide content of the sample after lyophilization / total peptide content of the original solution before lyophilization) × 100%. For lyophilized powders containing multiple excipients, the biuret method may be interfered with; therefore, high-performance liquid chromatography (HPLC) with external standard method is used for verification. HPLC conditions: C18 column (4.6 × 250 mm, 5 μm), mobile phase A: 0.1% trifluoroacetic acid-water, mobile phase B: 0.1% trifluoroacetic acid-acetonitrile, gradient elution (0 → 10 min, 5% B → 40% B), detection wavelength 214 nm, flow rate 1.0 mL / min, injection volume 20 μL. Glycyl-glycyl-glycine is used as the external standard for quantification.

[0057] (5) Determination of aggregate content: The content of aggregates was determined by size exclusion high-performance liquid chromatography (SEC-HPLC). A TSK gel G2000SWXL gel column was used, with an acetonitrile-water solution containing 0.1% trifluoroacetic acid (30:70, v / v) as the mobile phase, a flow rate of 0.5 mL / min, and a detection wavelength of 214 nm. The retention time was used to distinguish between monomeric peptides and aggregate peaks, and the aggregate content was calculated as a percentage of peak area.

[0058] (6) Determination of reconstitution time: Take one bottle of lyophilized sample, add purified water at 25°C to the indicated volume, shake gently, and record the time required from the addition of water until the cake is completely dispersed and dissolved.

[0059] (7) Calculation method for activity retention rate: The activity retention rate of lyophilized bioactive peptides was calculated using the following formula: Relative activity retention rate (%) = (IC50 of the lyophilized sample) 50 -1 IC of the stock solution before lyophilization 50 -1 ) × 100%; where IC 50 The half-maximal inhibitory concentration (or half-maximal effective concentration) is the IC50. 50 -1It is directly proportional to the activity intensity. For experiments that directly measure the scavenging rate or inhibition rate (such as hydroxyl radical scavenging rate, total peptide content), the following formula is used: Relative activity retention rate (%) = (measured value of the lyophilized sample / measured value of the original solution before lyophilization) × 100%.

[0060] Taking DPPH removal rate as an example: IC50 of tuna oligopeptide stock solution before freeze-drying 50 The concentration was 0.204 mg / mL, and its reciprocal (1 / 0.204) was 4.902; the IC50 of the lyophilized sample from Example 1 was... 50 The concentration is 0.213 mg / mL, and its reciprocal is 4.694; therefore, the relative activity retention rate = (4.694 / 4.902) × 100% = 95.8% (consistent with 95.4% in Table 1, the difference is due to rounding). When the measured value is a percentage value (such as hydroxyl radical scavenging rate), the retention rate is calculated directly using the ratio of the measured values.

[0061] (8) Moisture content determination: The moisture content of lyophilized powder was determined using the Karl Fischer coulometric titration method. Specific procedures: Accurately weigh approximately 10-20 mg of the lyophilized powder sample and quickly transfer it to the titration cell of the Karl Fischer moisture analyzer. Perform the determination according to the instrument's operating procedures and record the moisture content (%). Each sample was measured in triplicate, and the average value was taken. Instrument parameters: electrolysis current 400 mA, endpoint potential 50 mV, moderate stirring speed. A blank vial (without sample) was used as a background control to subtract background moisture.

[0062] II. Results: Table 1. Effects of different preservatives and freeze-drying methods on the freeze-drying effect of tuna oligopeptides

[0063] Combining Table 1 above and Figures 1-3 This demonstrates that the freeze-drying protectant and freeze-drying method of the present invention have excellent protective effects on tuna oligopeptides: the activity retention rate is significantly improved: after freeze-drying, the DPPH free radical scavenging activity retention rate of the present invention reached 95.4%, the ACE inhibition activity retention rate reached 96.0%, the hydroxyl free radical scavenging activity retention rate reached 94.9%, and the total peptide content retention rate reached 96.8%. In contrast, the activity retention rates of Comparative Example 1 and Comparative Example 2 were only about 63%~72%, indicating that the conventional protectant system has limited protective effect on bioactive peptides.

[0064] In summary, using tuna oligopeptides as bioactive peptides, and employing the protective agent provided by this invention in combination with the freeze-drying method of this invention, the activity retention rate of the freeze-dried bioactive peptide powder obtained after freeze-drying is stable at over 95%, the aggregate content is effectively controlled within 1.5%, and reconstitution is rapid.

[0065] Example 5 Verification of the freeze-drying effect of goose muscle peptide: Using the same detection method as in Example 4, various indicators of goose muscle peptide before freeze-drying, goose muscle peptide freeze-dried powder prepared in Example 2, and goose muscle peptide freeze-dried powder prepared by conventional freeze-drying process in Comparative Example 3 (control) were measured. The activity of goose muscle peptide was evaluated using xanthine oxidase inhibition rate, which was determined by high performance liquid chromatography (HPLC). Allopurinol was used as a positive control, and IC50 was calculated. 50 value.

[0066] Phosphate buffer (PBS, pH 7.5): Weigh 1.36 g of potassium dihydrogen phosphate (KH2PO4) and 4.46 g of disodium hydrogen phosphate (Na2HPO4·12H2O), dissolve them in about 800 mL of deionized water, adjust the pH to 7.5 with 1 M NaOH or 1 M HCl, bring the volume to 1000 mL, filter through a 0.22 μm filter membrane, and store at 4 °C for later use.

[0067] Xanthine substrate solution: Accurately weigh 15.2 mg of xanthine standard (purity ≥99%), dissolve it with a small amount of 0.1 M NaOH, and then make up to 100 mL with PBS (pH 7.5) to prepare a xanthine solution with a final concentration of 1.0 mmol / L. Store at 4°C protected from light and use immediately.

[0068] Xanthine oxidase solution: Take xanthine oxidase (derived from milk, specific activity ≥0.4U / mg protein, Sigma-Aldrich), dilute with PBS (pH 7.5) to 0.1U / mL, aliquot and store at -20℃. Thaw and place on ice before use.

[0069] Positive control (allopurinol) solution: Accurately weigh 1.36 mg of allopurinol standard (purity ≥99%), dissolve it in 0.1 M NaOH, dilute to volume with PBS (pH 7.5) to prepare a stock solution with a concentration of 10 μmol / L, and then serially dilute to prepare standard working solutions of 0.1, 0.5, 1.0, 2.0, 5.0, and 10.0 μmol / L.

[0070] Sample solution preparation: Accurately weigh the lyophilized goose muscle peptide powder (before or after lyophilization), dissolve and dilute it with PBS (pH 7.5) to create a series of concentration gradients, typically set as follows: 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 mg / mL (adjust according to preliminary experimental results). Filter all sample solutions through a 0.22 μm filter membrane before use.

[0071] The reaction was carried out using 96-well plates or 2 mL centrifuge tubes, with a total reaction volume of 200 μL. The reaction system is shown in Table 2 below: Table 2 Reaction System

[0072] Reaction Procedure: Mix all components except the xanthine substrate (PBS, enzyme solution, sample or positive control) and pre-incubate at 37°C for 10 min. Add the xanthine substrate to start the reaction and react at 37°C for a precise 30 min. Add 50 μL of 1M HCl to terminate the reaction, mix well, and cool in an ice bath. Blank group: Replace the sample solution with 50 μL of PBS, otherwise the same. Sample background group: Replace the enzyme solution with 50 μL of PBS to subtract interference from the sample itself at the detection wavelength. Blank background group: Replace the sample with 50 μL of PBS and the enzyme solution with 50 μL of PBS.

[0073] High-performance liquid chromatography (HPLC) conditions: Column: C18 reversed-phase column (4.6 × 250 mm, 5 μm), such as Agilent ZORBAX SB-C18. Mobile phase: methanol: 0.01% phosphoric acid aqueous solution = 5:95 (v / v), isocratic elution. Flow rate: 1.0 mL / min. Column temperature: 30℃. Detection wavelength: 290 nm (maximum absorption wavelength of uric acid). Injection volume: 20 μL. Run time: 10 min (uric acid retention time approximately 3.5–4.5 min, xanthine approximately 2.5–3.0 min, complete separation between the two).

[0074] Data processing and IC 50 Calculation: Record the peak area of ​​uric acid in each group (subtracting the peak area of ​​the blank background group). The formula for calculating the xanthine oxidase inhibition rate is: Inhibition rate (%) = (A blank - A sample) / (A blank - A background) × 100%, where, A blank: the peak area of ​​uric acid measured after the reaction in the blank group (no sample, with enzyme); A sample: the peak area of ​​uric acid measured after the reaction in the sample group (with sample, with enzyme); A background: the peak area of ​​uric acid measured after the reaction in the sample background group (with sample, without enzyme), used to subtract the absorption interference of the sample itself at 290nm.

[0075] Positive control verification: Allopurinol working solutions of various concentrations were used instead of samples, and the inhibition rate was determined using the same method. A standard curve was constructed, and the IC50 of allopurinol was calculated. 50 The value (should be in the range of 0.5~2.0 μmol / L) is used to verify the reliability of the system.

[0076] IC 50 Calculation: Using the logarithm of sample concentration (mg / mL) as the x-axis and inhibition rate (%) as the y-axis, a dose-response curve was fitted using nonlinear regression (four-parameter logistic equation) in GraphPad Prism or similar software to calculate the half-inhibitory concentration (IC50). 50 IC 50 The smaller the value, the stronger the inhibitory activity of the sample on xanthine oxidase.

[0077] Activity retention rate calculation: Relative activity retention rate (%) = (IC50 of the lyophilized sample) 50 -1 IC of the stock solution before lyophilization 50 -1 () × 100%. The anserine sample in Example 2 was determined using the above method, IC50 before lyophilization. 50 =125.3±5.2μg / mL, IC of sample in Example 2 after lyophilization 50 =131.6±6.1μg / mL, relative activity retention rate = (1 / 131.6) / (1 / 125.3)×100%=95.0% (as shown in Table 3).

[0078] Table 3. Effects of different preservatives and freeze-drying methods on the freeze-drying effect of goose muscle peptides.

[0079] Verification of the freeze-drying effect of fish bone collagen oligopeptides: Using the same detection method as in Example 4, the various indicators of fish bone collagen oligopeptides before freeze-drying, the freeze-dried fish bone collagen oligopeptides prepared in Example 3, and the freeze-dried fish bone collagen oligopeptides prepared by conventional freeze-drying process in Comparative Example 4 (control) were measured.

[0080] Table 4. Effects of different preservatives and freeze-drying methods on the freeze-drying effect of fish bone collagen oligopeptides.

[0081] The above results show that the protective agent and freeze-drying method proposed in this invention also have excellent protective effects on goose muscle peptides and fish bone collagen oligopeptides. The activity retention rate after freeze-drying is stable at over 95%, indicating that the technical solution of this invention has broad-spectrum adaptability to bioactive peptides of different sources and structures.

[0082] Existing research indicates that while freeze-drying is superior to spray-drying, it still carries the risk of peptide aggregation due to the freeze-concentration effect and the challenge of dehydration stress disrupting hydrogen bond networks. This invention demonstrates that the synergistic protection system of chitosan oligosaccharide and γ-PGA significantly alleviates these problems through interfacial exclusion and electrostatic anchoring effects. Aggregation was effectively inhibited: the aggregate content in the freeze-dried group of this invention increased by only 0.7% (from 0.6% to 1.3%), while the aggregate content in Comparative Example 1 increased by 8.8%, and in Comparative Example 2 by 8.1%. This difference stems from the triple synergistic mechanism of the protective agents in this invention—chitosan oligosaccharide provides interfacial exclusion and electrostatic anchoring effects, γ-PGA provides a hydration layer "molecular fence" effect, and trehalose provides glassy protection, collectively inhibiting peptide aggregation behavior during freeze-drying. It is noteworthy that tuna oligopeptides are rich in hydrophobic amino acids, which are more prone to hydrophobic interactions driving aggregation under freeze-concentration conditions. In the protective agent system of this invention, the high hydration capacity of γ-PGA and the electrostatic covering effect of chitosan oligosaccharide can effectively shield hydrophobic regions and reduce hydrophobically driven aggregation. Excellent physical properties: The freeze-dried cakes of this invention are white, loose, and intact, without collapse, and the reconstitution time is only ≤18s, much faster than Comparative Example 1 and Comparative Example 2.

[0083] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A lyophilization protectant for biologically active peptides, characterized in that, It consists of the following components by weight-volume percentage: chitosan oligosaccharide 0.5%–3.0% (w / v), γ-polyglutamic acid 0.1%–1.0% (w / v), trehalose 2%–8% (w / v), betaine 0.5%–2.0% (w / v), glutathione 0.02%–0.1% (w / v), with the balance being histidine buffer at a concentration of 10–50 mM.

2. The bioactive peptide lyophilization protectant according to claim 1, characterized in that, The molecular weight of the chitosan oligosaccharide is ≤1500 Da, and the degree of deacetylation is ≥90%.

3. The bioactive peptide lyophilization protectant according to claim 1, characterized in that, The pH of the histidine buffer solution is 6.0~7.

5.

4. The use of the bioactive peptide freeze-drying protectant as described in any one of claims 1 to 3 in the preparation of bioactive peptide freeze-dried powder.

5. A method for preparing lyophilized bioactive peptide powder using the lyophilization protectant of any one of claims 1 to 3, characterized in that, Includes the following steps: 1) Pre-freezing: Mix bioactive peptides with bioactive peptide freeze-drying protectant, cool to -10℃, hold for 20~40min, rapidly cool to -45℃, hold for 80~100min, heat to -15℃, hold for 50~70min, cool to -50℃, hold for 20~40min; 2) Single drying: Under a vacuum of 10~15Pa, heat to 0℃ at a heating rate of 0.2~0.5℃ / min, and hold for 20~40min for every 5℃ increase; 3) Secondary drying: Under vacuum conditions of ≤0.1Pa, the temperature is increased to 35℃ at a heating rate of 0.1~0.3℃ / min to obtain lyophilized bioactive peptide powder.

6. The method for preparing lyophilized bioactive peptide powder according to claim 5, characterized in that, In step 1), the cooling rate to -10℃ is 0.4~0.6℃ / min, the cooling rate to -45℃ is 2~4℃ / min, the heating rate to -15℃ is 0.8~1.2℃ / min, and the cooling rate to -50℃ is 1~3℃ / min.

7. The method for preparing lyophilized bioactive peptide powder according to claim 5, characterized in that, In step 1), the ratio of bioactive peptide to bioactive peptide freeze-drying protectant is 0.5~5mg:1mL.

8. The method for preparing lyophilized bioactive peptide powder according to claim 5, characterized in that, The vacuum level during the heating process in step 2) is 12~13 Pa.

9. The method for preparing lyophilized bioactive peptide powder according to claim 5, characterized in that, The vacuum degree during the heating process in step 3) is ≤0.05Pa.

10. The bioactive peptide lyophilized powder prepared by the method for preparing bioactive peptide lyophilized powder according to any one of claims 5 to 9.

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