Bromelain freeze-drying protective agent and preparation method thereof

Abstract

The invention belongs to the technical field of biological enzyme preservation, and particularly relates to a bromelain freeze-drying protective agent and a preparation method thereof. The trehalose and pulullan compound freeze-dried powder injection is prepared from the following components in parts by weight: 30 to 50 parts of trehalose, 8 to 10 parts of pulullan, 3 to 10 parts of maltodextrin, 12 to 22 parts of mannitol, 1 to 4 parts of sorbitol, 4 to 12 parts of glycine, 0.05 to 0.2 part of polysorbate-20, 1.5 to 4.5 parts of a buffering agent, 0.2 to 0.8 part of N-acetylcysteine, 0.05 to 0.3 part of oxidized glutathione, 0.8 to 2.5 parts of PEG (Polyethylene Glycol) and 0.05 to 0.3 part of an antioxidant. According to the invention, trehalose-pullulan is taken as a main skeleton, so that the growth of ice crystals is inhibited, and the water activity is reduced; mannitol constructs a crystal phase support network, and polysorbate-20 stabilizes an interface; sulfydryl is temporarily sealed through oxidized glutathione, and controllable reduction during redissolution is realized through microencapsulated N-acetylcysteine; and a sodium phosphate buffer system keeps the pH stable.

Description

Technical Field

[0001] The invention belongs to the technical field of biological enzyme preservation, and particularly relates to a bromelain freeze-drying protective agent and a preparation method thereof. Background Art

[0002] Bromelain is a sulfur-rich cysteine ​​protease with an optimal reaction environment in a neutral environment. Its efficient hydrolysis of a variety of protein substrates has led to its widespread application in the pharmaceutical, food, and biocatalysis industries. However, its conformation relies on disulfide bonds, free sulfhydryl groups, and a surrounding hydration layer. Therefore, it is rapidly inactivated by exposure to high temperatures, extreme pH, metal ions, or oxidative stress. Therefore, industrial production requires gentle and precise drying and storage processes. Freeze-drying significantly delays degradation by freezing at low temperatures followed by sublimation of the water under reduced pressure. However, during the freezing, primary drying, and secondary drying stages, the enzyme molecules are simultaneously subjected to ice crystal shearing, solute concentration, interfacial exposure, and residual water interference. To mitigate these stresses, a pre-freeze-drying excipient—a lyophilized enzyme protectant—is added before lyophilization. This preservative forms a glass or crystalline scaffold to restrict molecular motion in the dry state, replaces water molecules with polyols or non-reducing sugars to maintain the hydrogen bond network, utilizes surfactants to mitigate conformational disruption at the air-liquid and ice-liquid interfaces, and employs antioxidants, sulfhydryl protectants, and buffer salt systems to maintain the chemical environment, synergistically enhancing stability. However, traditional formulations typically rely on single sugar or protein fillers, which offer insufficient protection against the dual stresses of ice crystal growth and freeze concentration, resulting in a 30% to 40% loss of activity after freeze-drying. If residual moisture levels are high, active sulfhydryl groups can easily trigger self-cleavage and disulfide bond rearrangement, making it difficult for the enzyme to last more than a year at room temperature. Reducing sugars can undergo the Maillard reaction during secondary drying, causing browning and further weakening enzyme activity. If the crystallinity or glass transition temperature of the support is improperly controlled, the freeze-dried cake can easily collapse and pulverize, resulting in slow reconstitution and incomplete activity recovery. The lack of an interfacial stabilizer can also induce adsorption inactivation and foaming during filling and transportation. Given these shortcomings, it is crucial to develop a highly stable lyoprotectant that can provide effective protection throughout the freeze-drying process and rapidly restore bromelain activity upon reconstitution. Summary of the Invention

[0003] In response to the shortcomings of the prior art, the present invention aims to provide a bromelain freeze-drying protective agent and a preparation method thereof. Traditional freeze-drying protective systems based on trehalose and mannitol often form dense amorphous glass with limited interconnected capillaries, resulting in slow wetting of the freeze-dried cake, long re-dissolution time, and a large number of suspended particles. The present invention addresses this problem by introducing a dual module of oxidized glutathione reversible thiol temporary sealing / N-acetylcysteine ​​microcapsule delayed reduction, combined with microenvironmental regulation of low ionic strength sodium phosphate buffering, to address the risk of pH drift caused by freeze fractionation and achieve a comprehensive improvement in the clarity, activity retention, and pH stability of freeze-dried bromelain.

[0004] The technical effect of the present invention is achieved through the following technical scheme: a bromelain freeze-drying protective agent, which comprises the following components in parts by weight: 30-50 parts of trehalose, 8-10 parts of pullulan, 3-10 parts of maltodextrin, 12-22 parts of mannitol, 1-4 parts of sorbitol, 4-12 parts of glycine, 0.05-0.2 parts of polysorbate-20, 1.5-4.5 parts of a buffer, 0.2-0.8 parts of N-acetylcysteine, 0.05-0.3 parts of oxidized glutathione, 0.8-2.5 parts of PEG and 0.05-0.3 parts of an antioxidant.

[0005] Preferably, the DE value of the maltodextrin is 5 to 10, preferably 6 to 8; Preferably, the PEG is PEG-4000; Preferably, the buffer is any one of a sodium citrate buffer system and a sodium phosphate buffer system, preferably a sodium phosphate buffer system; Preferably, the antioxidant is any one of α-tocopheryl acetate and ascorbyl palmitate; Preferably, another aspect of the present invention is to provide a method for preparing a bromelain lyoprotectant, comprising the following steps: S1: Cool pure water to 5-10°C, dissolve trehalose, pullulan and maltodextrin in sequence, add buffer to adjust the pH to 4.8-5.2, add oxidized glutathione, filter through 0.22 μm, concentrate by vacuum thin film evaporation to a solid content of 45-55%, and spray dry to obtain a primary powder; S2: First, dissolve 99% of the weight of mannitol and glycine and sorbitol in pure water in proportion, add water to adjust the solid content to 25-30%, heat in a water bath to 60°C, stir until completely clear, filter, and slowly cool until crystals begin to precipitate, then begin to add the remaining weight of mannitol, continue stirring, cool to room temperature and age for 30-60 minutes, centrifuge at 1000-2000g for 5-10 minutes, remove the supernatant, take the precipitate and spray freeze it with liquid nitrogen, vacuum freeze-dry, and sieve to obtain 50-200µm free-flowing crystals; S3: Under nitrogen protection, PEG is heated to 60°C to obtain a clear melt, an antioxidant is added, and when cooled to 35-40°C, N-acetylcysteine ​​is added and treated at 2000 rpm for 30-60 seconds to form a semi-solid suspension; the semi-solid suspension is added to 0.5% polysorbate-20 buffer cooled to 5-10°C, treated at 10000 rpm for 60-120 seconds, emulsified into a fine emulsion, spray-dried at low temperature, and film-coated with a trehalose / pullulan aqueous solution in a fluidized bed, and screened through a 40-mesh screen to obtain microcapsules; S4: In a clean environment, with the humidity controlled at 15%, under a nitrogen atmosphere, add the primary powder of step S1 to a mixer and premix for 2-3 minutes, then slowly add the free-flowing crystals of step S2 and mix at a low speed for 5-15 minutes, then add the microcapsules of step S3 by continuous spraying within 15-30 minutes, mix at a low speed for 5-8 minutes, sieve through 20 mesh, package and store to obtain a lyoprotectant; Preferably, in step S1, the spray drying parameters are: inlet 100-120°C, outlet 60-70°C, and atomization pressure 0.6-0.8 MPa; Preferably, in step S2, the vacuum freeze-drying parameters are: sublimation treatment at -20°C, followed by heating to 25°C and secondary drying until the water content is less than 2%; Preferably, in step S3, the 0.5% polysorbate 20 buffer is prepared by adding polysorbate 20 to a buffer; Preferably, in step S3, the low-temperature spray drying parameters are: nozzle 0.7 mm, inlet 70-90° C., outlet 40-50° C., atomization pressure 0.6-0.8 MPa; Preferably, in step S3, the specific operation of the fluidized bed film coating is: using 5-8% trehalose / pullulan aqueous solution, an air inlet temperature of 32-35°C, a bed temperature of 28-32°C, a spray rate of 0.5-1 g / min•kg, and an atomization pressure of 1-1.5 bar; Preferably, in step S4, the parameters of the continuous spraying are: carrier gas pressure 0.1-0.2 MPa, and the spray gun is 10-20 cm away from the material surface.

[0006] The beneficial effects of the present invention are as follows: The freeze-drying protection system used in the present invention uses non-reducing, high-glass-transition-temperature trehalose as its main framework, combined with the high-molecular-weight pullulan polysaccharide. Together, they form a dense hydrogen-bonded network and a viscous unfrozen phase, effectively restricting free water migration and buffering initial ice crystal growth during the early freezing phase. In the dry state, a glass matrix with restricted molecular motion is formed. To improve dissolution and processability and dry powder formation, a small proportion of low-reducing-end maltodextrin is added to adjust rheological properties. Because the trehalose / pullulan combination is dominant and significantly reduces water activity in the dry state, it can effectively inhibit the potential Maillard reaction rate of maltodextrin, thereby balancing process adaptability and color / activity stability. Mannitol, as the primary crystalline filler, readily crystallizes spontaneously under conventional freeze-drying conditions, forming discrete support domains to improve the mechanical integrity of the freeze-dried cake and provide a less dense flow path for aqueous infiltration during reconstitution. By controlling the amount of sorbitol incorporated, it is only used to adjust the pre-freeze solution viscosity and freeze-concentrate fluidity, avoiding any impact on the overall dry-state glass transition temperature. In this system, glycine primarily assists in forming and reduces the risk of collapse. The interfacial inhibition module utilizes polysorbate 20, which acts by reducing the interfacial tension between the nascent gas-liquid / ice-liquid interface and competitively adsorbing it, thereby mitigating interface-induced enzyme conformational unfolding and aggregation.

[0007] While the polysorbate 20 used in this invention does not itself drive the active directional migration of hydrophobic antioxidants, it can help the pre-dispersed lipophilic antioxidants rapidly wet and redisperse during reconstitution. These lipophilic antioxidants, due to their hydrophobic affinity, spontaneously partition into hydrophobic microdomains or interfaces, thereby more effectively intercepting free radicals and synergistically protecting the active cysteine ​​residues of bromelain. PEG-4000 is added at low levels primarily to adjust the rheology of the pre-freeze solution and aid in the dispersion of the lipophilic antioxidant microparticles. Its interfacial protective effect is limited, and because polyether peroxide impurities can damage sulfhydryl groups, its dosage is controlled and synergistically combined with the polysorbate 20 / lipophilic antioxidant to reduce the overall oxidative load. The sodium phosphate buffer system, by adjusting the initial pH to the 5-6 range and limiting the ionic strength, preserves enzyme activity while mitigating the dramatic pH excursions caused by freeze-induced fractionation. The buffer system works synergistically with the trehalose / pullulan glass matrix to maintain the reconstitution pH close to the initial filling value after freeze-concentration and drying, protecting the enzyme from adverse pH extremes. To address the inherent vulnerability of active cysteine ​​sites to oxidation, this system incorporates a low equivalent of oxidized glutathione (GSSG) during liquid phase preparation, forming a partially reversible S-glutathionylation seal in a slightly acidic environment to reduce the risk of sulfhydryl oxidation and self-cleavage during storage. Simultaneously, N-acetylcysteine ​​(NAC) is physically isolated from the main glass phase in the form of dry microcapsules, allowing it to be gradually released only after reconstitution and water absorption. By establishing a mild reducing potential, NAC and GSSG undergo a controlled sulfur-disulfide exchange, removing the sulfhydryl mask, restoring catalytic activity, and capturing residual oxides, thereby preventing premature reaction between the two during liquid phase preparation and freeze-drying, which could weaken the protective efficacy. After these multi-modules pre-encode the restriction, support, interface stabilization, antioxidant-reduction, and pH microenvironmental adjustments at the formulation level, a structurally intact, color-stable, controlled residual water content, and rapid reconstitution bromelain freeze-dried formulation can be obtained using a conventional freeze-drying process. Both its dry-state activity retention and post-reconstitution activity recovery significantly outperform traditional systems that rely on monosaccharide alcohols or simple excipient stacking. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0009] Figure 1 1. It is a graph showing the freeze-drying reconstitution time and reconstitution activity change results of the freeze-dried protective agents of Examples 1 to 3 of the present invention, Comparative Examples 1 to 3, and a control group; Figure 2 1. It is a graph showing the freeze-dried and reconstituted clarity and pH change results of the freeze-dried and reconstituted freeze-dried protective agents of Examples 1 to 3 of the present invention, Comparative Examples 1 to 3, and a control group; Figure 3 This is a graph showing the results of the accelerated test of protease activity changes of Examples 1 to 3 of the present invention, Comparative Examples 1 to 3, and the lyophilized protective agent of the control group; Figure 4 This is a graph showing the change in residual water rate of the lyophilized protective agents of Examples 1 to 3 of the present invention, Comparative Examples 1 to 3, and a control group in an accelerated test; Figure 5 1 is a graph showing the change in protease activity of the freeze-thaw cycle test results of Examples 1 to 3 of the present invention, Comparative Examples 1 to 3, and the lyophilized protective agent of the control group; Figure 6 3. It is a graph showing the change in residual water rate of freeze-thaw cycle test results of the freeze-dried protective agents of Examples 1 to 3 of the present invention, Comparative Examples 1 to 3 and the control group. DETAILED DESCRIPTION

[0010] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention. It should be noted that unless otherwise specified, the raw materials involved in the present invention were purchased through conventional commercial channels.

[0011] Example 1: A bromelain freeze-drying protectant, comprising the following components in parts by weight: 30 parts of trehalose, 8 parts of pullulan, 3 parts of maltodextrin, 12 parts of mannitol, 1 part of sorbitol, 4 parts of glycine, 0.05 parts of polysorbate 20, 1.5 parts of buffer, 0.2 parts of N-acetylcysteine, 0.05 parts of oxidized glutathione, 0.8 parts of PEG and 0.05 parts of antioxidant.

[0012] The DE value of the maltodextrin is 5 to 10; The preparation of the bromelain freeze-drying protectant comprises the following steps: S1: Cool pure water to 10°C, dissolve trehalose, pullulan and maltodextrin in sequence, add sodium phosphate buffer to adjust the pH to 4.8, add oxidized glutathione, filter through 0.22 μm, and concentrate by vacuum thin film evaporation to a solid content of 55%. Spray dry at an inlet of 120°C, an outlet of 60°C, and an atomizing pressure of 0.8 MPa to obtain a primary powder; S2: First, dissolve 99% of the weight of mannitol and glycine and sorbitol in pure water in proportion, add water to adjust the solid content to 30%, heat in a water bath to 60°C, stir until completely clear, filter, and slowly cool until crystals begin to precipitate, then add the remaining weight of mannitol, continue stirring, cool to room temperature and age for 60 minutes, centrifuge at 2000g for 5 minutes, remove the supernatant, take the precipitate and quick-freeze it by liquid nitrogen spray, sublime it at -20°C, then heat to 25°C and dry it again until the water content is less than 2%, and sieve to obtain free-flowing crystals of 50 to 200µm; S3: Under nitrogen protection, PEG-4000 was heated to 60°C to obtain a clear melt, and ascorbyl palmitate was added. When cooled to 40°C, N-acetylcysteine ​​was added and treated at 2000 rpm for 60 seconds to form a semisolid suspension. The semisolid suspension was added to a sodium phosphate buffer solution containing 0.5% polysorbate 20 cooled to 10°C, and treated at 10000 rpm for 120 seconds to emulsify into a fine emulsion. The solution was spray-dried at a nozzle of 0.7 mm, an inlet of 90°C, an outlet of 40°C, and an atomization pressure of 0.8 MPa. The solution was then film-coated in a fluidized bed using an 8% trehalose / pullulan aqueous solution at an inlet air temperature of 35°C, a bed temperature of 32°C, a spray rate of 1 g / min•kg, and an atomization pressure of 1.5 bar. The solution was then screened through a 40-mesh filter to obtain microcapsules. S4: In a clean environment, with the humidity controlled at 15%, under a nitrogen atmosphere, the primary powder of step S1 was added to the mixer and premixed for 3 minutes. Then, the free-flowing crystals of step S2 were slowly added and mixed at a low speed for 15 minutes. The carrier gas pressure was 0.2 MPa, and the spray gun was 20 cm away from the material surface. The microcapsules of step S3 were continuously added within 15 minutes. After mixing at a low speed for 8 minutes, the mixture was sieved through a 20-mesh screen, packaged, and stored to obtain a lyoprotectant.

[0013] Example 2: A bromelain freeze-drying protectant, comprising the following components in parts by weight: 50 parts of trehalose, 10 parts of pullulan, 10 parts of maltodextrin, 22 parts of mannitol, 4 parts of sorbitol, 12 parts of glycine, 0.2 parts of polysorbate 20, 4.5 parts of buffer, 0.8 parts of N-acetylcysteine, 0.3 parts of oxidized glutathione, 2.5 parts of PEG and 0.3 parts of antioxidant.

[0014] The DE value of the maltodextrin is 5 to 10; The preparation of the bromelain freeze-drying protectant comprises the following steps: S1: Cool pure water to 5°C, dissolve trehalose, pullulan and maltodextrin in sequence, add sodium citrate buffer to adjust the pH to 4.8, add oxidized glutathione, filter through 0.22 μm, and concentrate by vacuum thin film evaporation to a solid content of 45%. Spray dry at an inlet of 100°C, an outlet of 70°C, and an atomizing pressure of 0.6 MPa to obtain a primary powder; S2: First, dissolve 99% of the weight of mannitol and glycine and sorbitol in pure water in proportion, add water to adjust the solid content to 25%, heat in a water bath to 60°C, stir until completely clear, filter, and slowly cool until crystals begin to precipitate, then add the remaining weight of mannitol, continue stirring, cool to room temperature and age for 30 minutes, centrifuge at 1000g for 10 minutes, remove the supernatant, take the precipitate and quick-freeze it by liquid nitrogen spray, sublime it at -20°C, then heat it to 25°C and dry it again until the water content is less than 2%, and sieve it to obtain free-flowing crystals of 50 to 200µm; S3: Under nitrogen protection, PEG-4000 was heated to 60°C to obtain a clear melt, α-tocopheryl acetate was added, and when cooled to 35°C, N-acetylcysteine ​​was added and treated at 2000 rpm for 30 seconds to form a semisolid suspension. The semisolid suspension was added to a sodium citrate buffer solution containing 0.5% polysorbate 20 cooled to 5°C, and treated at 10000 rpm for 60 seconds to emulsify into a fine emulsion. The solution was spray-dried at a nozzle of 0.7 mm, an inlet of 70°C, an outlet of 50°C, and an atomization pressure of 0.6 MPa. The solution was then film-coated in a fluidized bed using a 5% trehalose / pullulan aqueous solution at an inlet air temperature of 32°C, a bed temperature of 28°C, a spray rate of 0.5 g / min•kg, and an atomization pressure of 1 bar. The solution was then screened through a 40-mesh screen to obtain microcapsules. S4: In a clean environment, control the humidity at 15%, and under a nitrogen atmosphere, add the primary powder of step S1 to the mixer, premix for 2 minutes, then slowly add the free-flowing crystals of step S2, and mix at a low speed for 5 minutes; the carrier gas pressure is 0.1 MPa, the spray gun is 10 cm away from the material surface, and the microcapsules of step S3 are continuously added within 30 minutes. After mixing at a low speed for 5 minutes, the mixture is sieved through 20 mesh to control the moisture content to less than 3%, and the mixture is packaged and stored to obtain a freeze-dried protective agent.

[0015] Example 3: A bromelain freeze-drying protectant, comprising the following components in parts by weight: 45 parts of trehalose, 9 parts of pullulan, 6 parts of maltodextrin, 20 parts of mannitol, 2 parts of sorbitol, 8 parts of glycine, 0.12 parts of polysorbate 20, 3 parts of buffer, 0.6 parts of N-acetylcysteine, 0.15 parts of oxidized glutathione, 1.5 parts of PEG and 0.18 parts of antioxidant.

[0016] The DE value of the maltodextrin is 6 to 8; The preparation of the bromelain freeze-drying protectant comprises the following steps: S1: Cool pure water to 8°C, dissolve trehalose, pullulan and maltodextrin in sequence, add sodium phosphate buffer to adjust the pH to 5, add oxidized glutathione, filter through 0.22 μm, and concentrate by vacuum thin film evaporation to a solid content of 50%. Spray dry at an inlet of 110°C, an outlet of 65°C, and an atomizing pressure of 0.7 MPa to obtain a primary powder; S2: First, dissolve 99% of the weight of mannitol and glycine and sorbitol in pure water in proportion, add water to adjust the solid content to 28%, heat in a water bath to 60°C, stir until completely clear, filter, and slowly cool until crystals begin to precipitate, then add the remaining weight of mannitol, continue stirring, cool to room temperature and age for 45 minutes, centrifuge at 1500g for 8 minutes, remove the supernatant, take the precipitate and quick-freeze it by liquid nitrogen spray, sublime it at -20°C, then heat it to 25°C and dry it again until the water content is less than 2%, and sieve it to obtain free-flowing crystals of 50 to 200µm; S3: Under nitrogen protection, PEG-4000 was heated to 60°C to obtain a clear melt, and ascorbyl palmitate was added. When cooled to 38°C, N-acetylcysteine ​​was added and treated at 2000 rpm for 50 seconds to form a semisolid suspension. The semisolid suspension was added to a sodium phosphate buffer solution containing 0.5% polysorbate 20 cooled to 8°C, and treated at 10000 rpm for 100 seconds to emulsify into a fine emulsion. The solution was spray-dried at a nozzle of 0.7 mm, an inlet of 80°C, an outlet of 45°C, and an atomization pressure of 0.7 MPa. The solution was then film-coated in a fluidized bed using a 6% trehalose / pullulan aqueous solution at an inlet air temperature of 34°C, a bed temperature of 30°C, a spray rate of 0.8 g / min•kg, and an atomization pressure of 1.2 bar. The solution was then screened through a 40-mesh screen to obtain microcapsules. S4: In a clean environment, with the humidity controlled at 15%, under a nitrogen atmosphere, the primary powder of step S1 was added to the mixer and premixed for 2.5 minutes. The free-flowing crystals of step S2 were then slowly added and mixed at a low speed for 10 minutes. The carrier gas pressure was 0.15 MPa, the spray gun was 15 cm away from the material surface, and the microcapsules of step S3 were continuously added within 25 minutes. After mixing at a low speed for 6 minutes, the mixture was sieved through a 20-mesh screen, packaged, and stored to obtain a lyoprotectant.

[0017] Comparative Example 1: The operating process parameters of Comparative Example 1 and Example 3 are basically the same. The main difference is that in Comparative Example 1, N-acetylcysteine ​​and oxidized glutathione are removed, and their missing proportions are filled with trehalose, and their related operating processes are removed at the same time.

[0018] Comparative Example 2: The operating process parameters of Comparative Example 2 and Example 3 are basically the same. The main difference is that in Comparative Example 2, mannitol, sorbitol and glycine are removed and their missing proportions are filled with trehalose, that is, the free-flowing grains prepared in step S2 are removed.

[0019] Comparative Example 3: The operating process parameters of Comparative Example 3 and Example 3 are basically the same. The main difference is that in Comparative Example 3, N-acetylcysteine ​​is not embedded, but PEG, antioxidant and N-acetylcysteine ​​are directly added to the polysorbate 20 buffer.

[0020] Performance testing: Lyophilization and reconstitution test: 6 g of the lyophilized protective agent sample prepared in Examples 1 to 3, Comparative Examples 1 to 3, and the control group (conventional lyophilized protective agent trehalose + mannitol + buffer + polysorbate-20) was added to 100 mL of pure water, stirred and dissolved evenly, and the buffer was slightly adjusted to pH 5, 0.45 μm PES filter was used to remove particles to obtain protective agent solution. 8 mL of 20 mg / mL bromelain solution was added to 92 mL of protective agent solution and gently shaken to mix evenly. Then, the product was allowed to stand at 5°C for 30 min and pre-frozen at -40°C for 60 min. The product was placed at -25°C and heated to -15°C at a rate of 1°C / h under vacuum of 10 Pa and maintained for 2 h. Then, the temperature was increased to 25°C at a rate of 0.2°C / min and maintained for 6 h to obtain freeze-dried samples. 10 mL of pure water was added to each sample and gently shaken once every 5 s. A stopwatch was started to record the reconstitution time and the clarity (NTU) and pH fluctuation after reconstitution were measured (pH was measured after 60 s of reconstitution). 0.5 mL of the reconstituted solution was added to 4.5 mL of 50 mM neutral sodium phosphate buffer and the enzyme activity after reconstitution (%) was tested = activity after reconstitution / bottled activity × 100%. Three independent samples were set for each batch of tests and the results were averaged. The reconstitution time and enzyme activity after reconstitution were as follows: Figure 1 As shown; after reconstitution, the clarity and pH are as Figure 2 shown.

[0021] Depend on Figure 1 and Figure 2The results show that the lyoprotectant prepared in Example 3 of the present invention is superior to the control group and the comparative example in terms of resolubility performance: resolubility time, resolubility activity, clarity and pH stability are significantly ahead. Analysis of the results of Comparative Example 1 and Example 3 shows that the resolubility time and clarity are close to those of Example 1, but the resolubility activity and pH change are large. This may be due to the lack of GSSG / NAC, which causes the redox protection system to collapse. The absence of GSSG prevents the active cysteine ​​in the freeze-dried product from forming a reversible S-glutathionyl temporary seal, and irreversible oxidation occurs before resolubility; the lack of NAC results in a lack of reducing agent to restore the oxidized sites during resolubility, and the loss of free radical scavenging ability; the accumulation of oxidation byproducts lowers the pH. Analysis of the results from Comparative Example 2 and Example 3 showed a significant increase in reconstitution time, severe turbidity, and a significant decrease in activity. This may be due to the loss of the mannitol / sorbitol / glycine crystalline framework, resulting in cascading failure. Trehalose, in turn, formed a dense amorphous glass, which resulted in zero porosity and a dramatic increase in water penetration resistance, prolonging reconstitution time. The freeze-dried cake structure collapsed, producing microcracks and flakes. During reconstitution, irreversible protein aggregates formed, leading to deteriorated clarity. Water aggregation caused shear denaturation and hydrolysis of the enzyme. Analysis of the results from Comparative Example 3 and Example 3 showed a prolonged reconstitution time, decreased clarity, significant pH fluctuations, and significant activity loss. This may be due to the direct exposure of NAC to moisture and oxidation, which caused coupled damage due to water-oxidation. The hygroscopicity of NAC destroyed the microcapsule barrier, leading to migration and aggregation of the fat-soluble antioxidants. This failure of interfacial protection resulted in increased reconstitution turbidity. Premature oxidation of NAC to generate acidic byproducts led to a decrease in pH. GSSG was excessively consumed during the freeze-drying stage, lacking sulfhydryl reducing capacity during reconstitution, leading to cumulative oxidation of active sites.

[0022] Freeze-drying stability test: The freeze-dried samples of Examples 1 to 3, Comparative Examples 1 to 3, and a control group (conventional freeze-drying protective agent trehalose + mannitol + buffer + polysorbate-20) were prepared in the same manner as the freeze-drying reconstitution test above. They were then vacuum bottled and subjected to accelerated testing at 42°C / 75% humidity. The sampling points were day 0, day 7, day 14, day 21, day 42, and day 63, and freeze-dried reconstitution tests were performed respectively (the protease activity on day 0 was recorded as 100%). The change in protease activity (%) and the residual water rate (%) were calculated. Three independent samples were set for each batch of tests, and the results were averaged. The results are shown in the figure. Figure 3 and Figure 4 As shown; freeze-thaw cycle test was carried out at -20℃ to simulate accelerated freezing transportation, freeze-thaw cycle was carried out once every 14 days (-20℃ to 25℃ and then to -20℃), a total of 3 cycles, after completing all cycles, calculate the protease activity change (%) and measure the residual water rate (%), set three independent samples for each batch test, and take the average value of the results. The results are shown in Figure 5 and Figure 6 shown.

[0023] Depend on Figure 3 and Figure 4 According to the analysis of the results, the lyophilized protective agent prepared in the embodiment of the present invention showed excellent protection effect in the accelerated test, and its long-term stability was significantly improved compared with the control group: the residual water rate was always maintained at a low level, the protease activity showed a gentle downward trend, and entered a quasi-steady state in the later period. According to the analysis of the results of Comparative Example 1 and Example 3, the activity of Comparative Example 1 dropped rapidly before 14 days, and it continued to be inactivated in the later period. This may be due to the lack of oxidized glutathione (GSSG) and N-acetylcysteine ​​(NAC), which led to the failure of double protection: the lack of GSSG made it impossible for the active cysteine ​​site to form a reversible S-glutathionylation temporary seal, and the sulfhydryl group was directly exposed to the oxidative environment under freeze-thaw / high temperature; the lack of NAC caused the system to lose the reduction activation ability after redissolution, and the residual free radicals continued to attack the active center of the enzyme. Analysis of the results from Comparative Example 2 and Example 3 shows a sharp drop in residual water content and a precipitous drop in activity in Comparative Example 2. This is likely due to the loss of the mannitol / sorbitol / glycine crystalline framework, which is then replaced by trehalose to form a highly hygroscopic amorphous phase, accelerating water intrusion and causing structural collapse in the freeze-dried cake. Repeated dissolution and recrystallization of the amorphous region during freeze-thaw cycles triggers microcrack propagation and water accumulation. Uncontrolled water exposure exposes the enzyme molecules to water-mediated shear denaturation and oxidation, leading to premature depletion of NAC / GSSG due to localized high water activity. Analysis of the results from Comparative Example 3 and Example 3 shows a rapid decline in activity early in the reaction, accelerated inactivation later in the reaction, and a significant increase in residual water content in Comparative Example 3. This is likely due to the direct exposure of NAC to moisture, which releases its hygroscopic properties during the initial moisture absorption phase, disrupting the microcapsule barrier. Water intrusion triggers migration and aggregation of fat-soluble antioxidants. Premature NAC depletion leads to a depletion of reducing capacity in the later stages of freeze-thaw cycles, resulting in cumulative oxidative damage to active sites.

[0024] Depend on Figure 5 and Figure 6The results show that the lyophilized protective agent of Example 3 still maintains extremely high protease activity after three freeze-thaw cycles, which is significantly better than the comparative examples and the control group. According to the results of Comparative Example 1 and Example 3, the activity of Comparative Example 1 decreased significantly. This may be due to the fact that the absence of GSSG results in the inability to form a temporary seal of sulfhydryl groups, and the active oxygen (such as •OH) at the ice crystal interface during freeze-thaw directly attacks the cysteine ​​site; NAC is also missing, and there is a lack of reducing agent to restore the oxidized sulfhydryl groups during redissolution, resulting in irreversible inactivation. According to the results of Comparative Example 2 and Example 3, the activity of Comparative Example 2 decreased dramatically. This may be due to the lack of mannitol / sorbitol support, and the trehalose filling to form a hygroscopic amorphous phase. The amount of water intrusion during freeze-thaw suddenly increased, and the freeze-dried cake structure collapsed; repeated phase changes lead to the expansion of microcracks, and the enzyme molecules are exposed to ice crystal shear stress; water accumulation accelerates enzyme aggregation denaturation and free radical hydrolysis reactions. Analysis of the results of Comparative Example 3 and Example 3 showed that the activity of Comparative Example 3 continued to decline, while the residual water rate increased significantly. This may be due to the fact that NAC exposure triggered moisture-oxidation coupled damage: the hygroscopicity of unencapsulated NAC caused early destruction of the microcapsule barrier; water penetration caused the migration and failure of the fat-soluble antioxidants, resulting in loss of interfacial protection ability; NAC was consumed prematurely during freeze-thaw, and later oxidative free radicals accumulated and attacked the enzyme active center.

[0025] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A bromelain freeze-drying protective agent, characterized in that The composition includes the following components in parts by weight: 30-50 parts of trehalose, 8-10 parts of pullulan, 3-10 parts of maltodextrin, 12-22 parts of mannitol, 1-4 parts of sorbitol, 4-12 parts of glycine, 0.05-0.2 parts of polysorbate-20, 1.5-4.5 parts of buffer, 0.2-0.8 parts of N-acetylcysteine, 0.05-0.3 parts of oxidized glutathione, 0.8-2.5 parts of PEG and 0.05-0.3 parts of antioxidant.

2. A bromelain freeze-drying protective agent according to claim 1, characterized in that, The DE value of the maltodextrin is 5-10.

3. A bromelain freeze-drying protective agent according to claim 2, characterized in that, The buffer is any one of a sodium citrate buffer system and a sodium phosphate buffer system.

4. A method for preparing a bromelain freeze-drying protective agent according to any one of claims 1 to 3, characterized in that: The steps include: S1: Cool pure water, dissolve trehalose, pullulan and maltodextrin in sequence, add buffer to adjust pH, add oxidized glutathione, filter, concentrate by vacuum thin film evaporation, and spray dry to obtain primary powder; S2: First, dissolve a portion of mannitol, glycine, and sorbitol in pure water in proportion, add water to adjust the solid content, heat in a water bath, stir until completely clear, filter, and slowly cool until crystals begin to precipitate. Then, add the remaining weight portion of mannitol, continue stirring, cool to room temperature, and age. Centrifuge, remove the supernatant, and take the precipitate for quick freezing by liquid nitrogen spray, freeze-dry under vacuum, and sieve to obtain free-flowing crystals. S3: Under nitrogen protection, PEG is heated to obtain a clear melt, an antioxidant is added, and after cooling, N-acetylcysteine ​​is added and stirred at high speed to form a semisolid suspension; the semisolid suspension is added to the cooled 0.5% polysorbate-20 buffer, emulsified at high speed to form a fine emulsion, spray-dried at low temperature, and film-coated with a trehalose / pullulan aqueous solution in a fluidized bed, and screened to obtain microcapsules; S4: In a clean environment, with controlled humidity and under a nitrogen atmosphere, the primary powder of step S1 is added to the mixer, premixed, and then the free-flowing crystals of step S2 are slowly added, mixed at a low speed, and the microcapsules of step S3 are added by continuous spraying, mixed at a low speed, screened, packaged, and stored to obtain a lyoprotectant.

5. the preparation method of bromelain freeze-drying protective agent according to claim 4, is characterized in that, In step S1, the spray drying parameters are: inlet temperature 100-120°C, outlet temperature 60-70°C, and atomization pressure 0.6-0.8 MPa.

6. the preparation method of bromelain freeze-drying protective agent according to claim 5, is characterized in that, In step S2, the vacuum freeze-drying parameters are: sublimation treatment at -20°C, followed by heating to 25°C and secondary drying until the water content is less than 2%.

7. the preparation method of bromelain freeze-drying protective agent according to claim 6 is characterized in that, In step S3, the low-temperature spray drying parameters are: nozzle 0.7 mm, inlet 70-90° C., outlet 40-50° C., and atomization pressure 0.6-0.8 MPa.

8. the preparation method of bromelain freeze-drying protective agent according to claim 7, is characterized in that, In step S3, the specific operation of the fluidized bed film coating is: using 5-8% trehalose / pullulan aqueous solution, an inlet air temperature of 32-35°C, a bed temperature of 28-32°C, a spray rate of 0.5-1 g / min•kg, and an atomization pressure of 1-1.5 bar.

9. the preparation method of bromelain freeze-drying protective agent according to claim 8, is characterized in that, In step S4, the parameters of the continuous spraying are: carrier gas pressure 0.1-0.2 MPa, and the spray gun is 10-20 cm away from the material surface.

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