A freeze-drying protective agent for bromelain and a method for preparing the same

Abstract

The present application 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 composition comprises the following components: 30-50 parts of trehalose, 8-10 parts of pullulan, 3-10 parts of malt dextrin, 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. The present application takes trehalose-pullulan polysaccharide as a main skeleton, inhibits ice crystal growth and reduces water activity; mannitol constructs a crystal phase support network, and polysorbate-20 stabilizes the interface; oxidized glutathione temporarily seals mercapto groups, and microencapsulated N-acetylcysteine realizes controllable reduction during redissolution; and a sodium phosphate buffer system maintains pH stability.

Description

TECHNICAL FIELD

[0001] The present application 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

[0002] Bromelain is a sulfur-rich cysteine protease with a neutral optimum reaction environment, and is widely used in medicine, food and biological catalysis industries due to its high hydrolysis capacity for various protein substrates. However, the conformation of bromelain depends on disulfide bonds, free sulfhydryl groups and surrounding hydration layers, and it is rapidly inactivated when exposed to high temperature, extreme pH, metal ions or oxidative stress. Therefore, mild and accurate drying and storage processes must be used in industrial production. Freeze-drying technology significantly delays the degradation reaction by first freezing at low temperature and then sublimating water under reduced pressure. However, during the freezing, primary drying and secondary drying stages, the enzyme molecules are subjected to ice crystal shearing, solute concentration, interface exposure and residual water disturbance. To alleviate these stresses, a complex additive, freeze-drying enzyme protective agent, is added before freeze-drying. The freeze-drying enzyme protective agent limits molecular motion by forming a glass or crystalline scaffold in the dry state, replaces water molecules with polyols or non-reducing sugars to maintain the hydrogen bond network, reduces the conformational destruction of the air-liquid surface and ice-liquid interface by using surfactants, and maintains the chemical environment by using antioxidants, sulfhydryl protective agents and buffer salt systems, thereby synergistically improving the stability. However, the traditional formula usually relies on a single sugar or protein filler, which is insufficient to protect against the dual stresses of ice crystal growth and freezing concentration, resulting in a loss of activity of up to 30-40% after freeze-drying. If the residual water content is high, the active sulfhydryl groups can easily trigger self-cleavage and disulfide bond rearrangement, making it difficult to achieve a shelf life of more than one year at room temperature. Reducing sugars can undergo the Maillard reaction during secondary drying, resulting in browning and further reducing enzyme activity. If the crystallinity or glass transition temperature of the support agent is not properly controlled, the freeze-dried cake is prone to collapse and powdering, the reconstitution is slow, and the activity recovery is incomplete. The lack of interfacial stabilizers can also induce adsorption inactivation and foam generation during filling and transportation. Based on the above deficiencies, it is extremely necessary to develop a high-stability freeze-drying protective agent that can provide efficient protection during the entire freeze-drying process and quickly restore the activity of bromelain during reconstitution. SUMMARY

[0003] In view of the defects of the prior art, the present application aims to provide a bromelain freeze-drying protective agent and a preparation method thereof. The traditional freeze-drying protective system mainly based on trehalose-mannitol often forms a dense amorphous glass, and the interconnected capillary pores are limited, resulting in slow wetting of the freeze-dried cake, long reconstitution time and many suspended particles. Based on this problem, the present application introduces an oxidized glutathione reversible sulfhydryl temporary sealing / N-acetylcysteine microcapsule delayed reduction double module, and further cooperates with a microenvironment regulation of low ionic strength sodium phosphate buffer to solve the risk of pH drift caused by freeze fractionation, and to comprehensively improve the clarity, activity retention and pH stability of freeze-dried bromelain.

[0004] The technical effect of the present application is realized by 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 malt dextrin, 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 malt dextrin is 5-10, preferably 6-8;

[0006] Preferably, the PEG is PEG-4000;

[0007] Preferably, the buffer is any one of a sodium citrate buffer system and a sodium phosphate buffer system, preferably the sodium phosphate buffer system;

[0008] Preferably, the antioxidant is any one of alpha-tocopherol acetate and ascorbic acid palmitate;

[0009] Preferably, another aspect of the present application provides a preparation method of the bromelain freeze-drying protective agent, comprising the following steps:

[0010] S1: cool pure water to 5-10°C, sequentially dissolve trehalose, pullulan and malt dextrin, add a buffer to adjust the pH to 4.8-5.2, add oxidized glutathione, 0.22μm filter, vacuum thin film evaporation to concentrate to a solid content of 45-55%, spray drying to obtain a primary powder;

[0011] S2: first dissolve 99% of the mannitol and glycine, sorbitol in pure water in proportion, adjust the solid content to 25-30% by adding water, heat to 60°C in a water bath, stir until completely clear, filter, then slowly cool to the point where crystals begin to precipitate, then start adding the remaining weight of mannitol, continue stirring, cool to room temperature and age for 30-60min, centrifuge at 1000-2000g for 5-10min, remove the supernatant, take the precipitate and spray freeze with nitrogen, vacuum freeze-dry, sieve to obtain 50-200μm free-flowing crystal grains;

[0012] S3: nitrogen protection, heating PEG to 60℃ to obtain a clear melt, adding antioxidant, cooling to 35-40℃, adding N-acetyl cysteine 2000rpm for 30-60s, forming a semi-solid suspension; adding the semi-solid suspension into 0.5% polysorbate-20 buffer cooled to 5-10℃, 10000rpm for 60-120s, emulsifying into fine emulsion, low-temperature spray drying, fluidized bed film coating with trehalose / pullulan aqueous solution, screening through a 40-mesh sieve, obtaining microcapsules;

[0013] S4: in a clean environment, controlling humidity at 15%, under nitrogen atmosphere, adding the primary powder of step S1 into a mixer, pre-mixing for 2-3min, then slowly adding the free-flowing crystal grains of step S2, low-speed mixing for 5-15min, continuously spraying the microcapsules of step S3 within 15-30min, low-speed mixing for 5-8min, screening through a 20-mesh sieve, packaging and storing, obtaining a freeze-drying protective agent;

[0014] Preferably, in step S1, the spray drying parameters are: inlet 100-120℃, outlet 60-70℃, atomization pressure 0.6-0.8MPa;

[0015] Preferably, in step S2, the vacuum freeze-drying parameters are: sublimation at -20℃, then secondary drying at 25℃ until the water content is less than 2%;

[0016] Preferably, in step S3, the 0.5% polysorbate-20 buffer is prepared by adding polysorbate-20 into a buffer;

[0017] Preferably, in step S3, the low-temperature spray drying parameters are: nozzle 0.7mm, inlet 70-90℃, outlet 40-50℃, atomization pressure 0.6-0.8MPa;

[0018] Preferably, in step S3, the fluidized bed film coating is specifically operated as follows: using 5-8% trehalose / pullulan aqueous solution, inlet air temperature 32-35℃, bed temperature 28-32℃, spraying speed 0.5-1g / min•kg, atomization pressure 1-1.5bar;

[0019] Preferably, in step S4, the continuous spraying parameters are: carrier gas pressure 0.1-0.2MPa, spray gun distance from material surface 10-20cm.

[0020] The beneficial effects of the present application are as follows:

[0021] The freeze-drying protective system used in the present application mainly uses trehalose with non-reducing and high glass transition temperature as the main framework, and is combined with high molecular weight pullulan to form a dense hydrogen bond network and a viscous unfrozen phase, which effectively limits the migration of free water and buffers the initial growth of ice crystals in the early freezing stage, and forms a glass matrix with limited molecular motion in the dry state, to improve the solubility and dry powder forming. A small proportion of low-reducing maltodextrin is added to adjust the rheological properties. Due to the dominance of trehalose / pullulan and the significant reduction of water activity in the dry state, the potential Maillard reaction rate of maltodextrin can be effectively inhibited, thereby balancing the process adaptability and color / activity stability. Mannitol as the main crystal filler can spontaneously crystallize under conventional freeze-drying conditions to form discrete support domains, thereby improving the mechanical integrity of the freeze-dried cake and providing a lower density flow path for water immersion during reconstitution. By controlling the amount of sorbitol added, only the viscosity of the pre-freezing solution and the flowability of the frozen concentrate are adjusted to avoid affecting the overall dry glass transition temperature. Glycine mainly plays a role in assisting molding and reducing the risk of collapse in this system. The interface inhibition module uses polysorbate-20, which reduces the interfacial tension of the newly formed gas-liquid / ice-liquid and competes for adsorption to reduce the interface-induced enzyme conformation unfolding and aggregation.

[0022] The polysorbate-20 used in the present application does not drive the active orientation migration of hydrophobic antioxidants, but can help the pre-dispersed fat-soluble antioxidants to be quickly wetted and re-dispersed during reconstitution; these fat-soluble antioxidants spontaneously distribute to hydrophobic microdomains or interfaces according to their hydrophobic affinity, thereby more effectively intercepting free radicals and synergistically protecting the active cysteine residues of bromelain. PEG-4000 is added in a low amount, mainly for adjusting the rheological properties of the pre-freezing solution and helping to disperse the fat-soluble antioxidant microparticles; its interface protection effect is limited, and because its polyether peroxide impurities can damage sulfhydryl groups, the amount is controlled and synergized with polysorbate-20 / fat-soluble antioxidant to reduce the overall oxidative load. The buffer system uses sodium phosphate, by adjusting the initial pH to the interval of 5-6 and limiting the ionic strength, both the enzyme activity can be preserved and the severe pH drift caused by freeze-induced fractionation can be reduced. The buffer system is synergized with trehalose / pullulan glass matrix to maintain the reconstitution pH close to the initial filling value after freezing, concentration and drying, avoiding the exposure of the enzyme to unfavorable extreme pH. In view of the inherent weakness of the active cysteine site being easily oxidized, a low equivalent amount of oxidized glutathione (GSSG) is added during the liquid phase preparation of the system, forming a partially reversible S-glutathione temporary seal in a slightly acidic environment to reduce the risk of sulfhydryl oxidation and self-cleavage during storage; at the same time, N-acetylcysteine (NAC) is physically isolated from the main glass phase in the form of dry microcapsules, so that it is gradually released after water absorption during reconstitution, and by establishing a mild reducing potential, NAC and GSSG undergo controlled sulfur-disulfide exchange, removing sulfhydryl shielding, restoring catalytic activity and capturing residual oxidants, avoiding premature reaction between the two in the liquid preparation and freeze-drying stage to weaken the protection efficiency. After the above multi-module pre-encoding of restraint, support, interface stabilization, anti-oxidation-reduction and pH microenvironment adjustment at the formulation level, a freeze-dried bromelain preparation with complete structure, stable color, controllable residual water and rapid reconstitution can be obtained under conventional freeze-drying process. Its dry activity retention and reconstituted activity recovery are both significantly better than traditional systems that rely on single sugar alcohols or simple excipients. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only a part of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0024] Figure 1 is a graph of the freeze-drying reconstitution time and reconstitution activity change results of the freeze-drying protectants of examples 1-3 and comparative examples 1-3 and the control group of the present application;

[0025] Figure 2is a freeze-dried re-dissolution clarity and pH change result chart of the freeze-drying protectants of the inventive examples 1-3 and comparative examples 1-3 and the control group;

[0026] Figure 3 is an accelerated test protease activity change result chart of the freeze-drying protectants of the inventive examples 1-3 and comparative examples 1-3 and the control group;

[0027] Figure 4 is an accelerated test residual water rate change result chart of the freeze-drying protectants of the inventive examples 1-3 and comparative examples 1-3 and the control group;

[0028] Figure 5 is a freeze-thaw cycle test protease activity change result chart of the freeze-drying protectants of the inventive examples 1-3 and comparative examples 1-3 and the control group;

[0029] Figure 6 is a freeze-thaw cycle test residual water rate change result chart of the freeze-drying protectants of the inventive examples 1-3 and comparative examples 1-3 and the control group. DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. It should be noted that, unless otherwise specified, the raw materials involved in the present application are purchased through conventional commercial channels.

[0031] Example 1: A bromelain freeze-drying protectant, which comprises the following components by weight parts: 30 parts of trehalose, 8 parts of pullulan, 3 parts of malt dextrin, 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.

[0032] The DE value of the malt dextrin is 5 to 10;

[0033] The preparation of the bromelain freeze-drying protectant comprises the following steps:

[0034] S1: cool pure water to 10℃, dissolve trehalose, pullulan and malt dextrin in sequence, add sodium phosphate buffer to adjust pH to 4.8, add oxidized glutathione, 0.22μm filtration, vacuum thin film evaporation to concentrate to solid content 55%, inlet 120℃, outlet 60℃, spray drying at atomization pressure 0.8MPa to obtain primary powder;

[0035] S2: First, dissolve 99% of the weight of mannitol and glycine, sorbitol in pure water in proportion, adjust the solid content to 30% by adding water, heat to 60℃ in water bath, stir until completely clear, filter, then slowly cool to the temperature at which the crystals begin to precipitate, then start adding the remaining weight of mannitol, continue stirring, cool to room temperature and age for 60 min, centrifuge at 2000g for 5 min, remove the supernatant, take the precipitate and spray freeze, sublimate at -20℃, then warm up to 25℃ for secondary drying until the water content is less than 2%, sieve to obtain 50 to 200μm free-flowing crystal grains;

[0036] S3: Under nitrogen protection, heat PEG-4000 to 60℃ to obtain a clear melt, add ascorbyl palmitate, cool to 40℃, add N-acetylcysteine at 2000rpm for 60s to form a semi-solid suspension; add the semi-solid suspension to a sodium phosphate buffer containing 0.5% polysorbate-20 cooled to 10℃, treat at 10000rpm for 120s, emulsify into fine emulsion, nozzle 0.7mm, inlet 90℃, outlet 40℃, low-temperature spray drying at atomizing pressure 0.8MPa, use 8% trehalose / pruaine aqueous solution, inlet temperature 35℃, bed temperature 32℃, spray rate 1g / min•kg, atomizing pressure 1.5bar, fluidized bed film coating, sieve through 40 mesh, obtain microcapsules;

[0037] S4: In a clean environment, control humidity at 15%, nitrogen atmosphere, add the primary powder of step S1 to the mixer, premix for 3min, then slowly add the free-flowing crystal grains of step S2, mix at low speed for 15min; load gas pressure 0.2MPa, spray gun distance from material surface 20cm, continuously add the microcapsules of step S3 within 15min, mix at low speed for 8min, sieve through 20 mesh, package and store, obtain lyophilization protectant.

[0038] Example 2: A bromelain lyophilization protectant, which consists of the following components by weight: 50 parts of trehalose, 10 parts of pullulan, 10 parts of malt dextrin, 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.

[0039] The DE value of the malt dextrin is 5 to 10;

[0040] The preparation of the bromelain lyophilization protectant includes the following steps:

[0041] S1: cool pure water to 5℃, dissolve trehalose, pullulan and maltodextrin in sequence, add sodium citrate buffer to adjust pH to 4.8, add oxidized glutathione, 0.22 μm filtration, vacuum thin film evaporation to concentrate to solid content 45%, inlet 100℃, outlet 70℃, spray dry at atomization pressure 0.6 MPa, to obtain primary powder;

[0042] S2: first dissolve 99% of mannitol, glycine and sorbitol in pure water in proportion, adjust solid content to 25% by adding water, heat to 60℃ in water bath, stir until completely clear, filter, then slowly cool to start crystallization, then start adding the remaining weight of mannitol, continue stirring, cool to room temperature and age for 30 min, centrifuge 1000 g for 10 min, remove supernatant, take the precipitate and nitrogen spray freeze, sublimate at -20℃, then warm to 25℃ for secondary drying to water content less than 2%, sieve to obtain 50 to 200 μm free-flowing crystal grains;

[0043] S3: under nitrogen protection, heat PEG-4000 to 60℃ to obtain a clear melt, add α-tocopherol acetate, cool to 35℃, then add N-acetylcysteine at 2000 rpm for 30 s to form a semi-solid suspension; add the semi-solid suspension to sodium citrate buffer containing 0.5% polysorbate-20 cooled to 5℃, emulsify at 10000 rpm for 60 s to form fine emulsion, low-temperature spray dry at nozzle 0.7 mm, inlet 70℃, outlet 50℃, atomization pressure 0.6 MPa, use 5% trehalose / pullulan aqueous solution, inlet air temperature 32℃, bed temperature 28℃, spray rate 0.5 g / min•kg, atomization pressure 1 bar, fluidized bed film coating, sieve through 40 mesh to obtain microcapsules;

[0044] S4: in a clean environment, control humidity 15%, nitrogen atmosphere, add primary powder of step S1 to the mixer, premix for 2 min, then slowly add free-flowing crystal grains of step S2, mix at low speed for 5 min; load gas pressure 0.1 MPa, spray gun distance from material surface 10 cm, continuously add microcapsules of step S3 in 30 min, mix at low speed for 5 min, sieve through 20 mesh, control moisture less than 3%, package and store to obtain lyophilization protectant.

[0045] Example 3: A bromelain lyophilization protectant, which consists of the following components by weight: 45 parts trehalose, 9 parts pullulan, 6 parts maltodextrin, 20 parts mannitol, 2 parts sorbitol, 8 parts glycine, 0.12 parts polysorbate-20, 3 parts buffer, 0.6 parts N-acetylcysteine, 0.15 parts oxidized glutathione, 1.5 parts PEG and 0.18 parts antioxidant.

[0046] The maltodextrin has a DE value of 6 to 8.

[0047] The preparation of the bromelain freeze-drying protectant comprises the following steps:

[0048] S1: cool pure water to 8℃, dissolve trehalose, pullulan and maltodextrin in sequence, add sodium phosphate buffer to adjust pH to 5, add oxidized glutathione, 0.22μm filtration, vacuum thin film evaporation to concentrate to solid content 50%, inlet 110℃, outlet 65℃, spray drying at atomization pressure 0.7MPa, to obtain primary powder;

[0049] S2: first dissolve 99% of mannitol and glycine, sorbitol in pure water according to proportion, adjust solid content to 28% by adding water, heat to 60℃ in water bath, stir until completely clear, filter, slowly cool to start crystallization, then start adding the remaining weight of mannitol, continue stirring, cool to room temperature and age for 45min, centrifuge at 1500g for 8min, remove supernatant, take the precipitate liquid to spray freeze, sublimate at -20℃, then warm to 25℃ for secondary drying to water content less than 2%, sieve to obtain 50-200μm free-flowing crystal grains;

[0050] S3: under nitrogen protection, heat PEG-4000 to 60℃ to obtain a clear melt, add ascorbic acid palmitate, cool to 38℃, add N-acetylcysteine at 2000rpm for 50s to form a semi-solid suspension; add the semi-solid suspension into sodium phosphate buffer containing 0.5% polysorbate-20 cooled to 8℃, treat at 10000rpm for 100s, emulsify into fine emulsion, low-temperature spray dry at nozzle 0.7mm, inlet 80℃, outlet 45℃, atomization pressure 0.7MPa, using 6% trehalose / pullulan aqueous solution, inlet air temperature 34℃, bed temperature 30℃, spray rate 0.8g / min•kg, atomization pressure 1.2bar, fluidized bed film coating, sieve through 40 mesh, to obtain microcapsules;

[0051] S4: in a clean environment, control humidity 15%, nitrogen atmosphere, add primary powder of step S1 to the mixer, premix for 2.5min, then slowly add free-flowing crystal grains of step S2, mix at low speed for 10min; load gas pressure 0.15MPa, spray gun distance from material surface 15cm, continuously add microcapsules of step S3 within 25min, mix at low speed for 6min, sieve through 20 mesh, package and store, to obtain freeze-drying protectant.

[0052] Comparative Example 1: the operation process parameters of Comparative Example 1 and Example 3 are basically the same, the main difference is that N-acetylcysteine and oxidized glutathione are removed in Comparative Example 1, and trehalose is used to make up for the missing proportion, and the related operation process is also removed.

[0053] 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 trehalose is used to make up the proportion of the absence, that is, the free-flowing crystal grains prepared in step S2 are removed.

[0054] 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.

[0055] Performance test:

[0056] Freeze-dried reconstitution test: Take 6g of freeze-dried protectant samples prepared in Examples 1-3 and Comparative Examples 1-3 and the control group (conventional freeze-dried protectant trehalose + mannitol + buffer + polysorbate-20) and add 100mL of pure water, stir to dissolve uniformly, adjust the pH of the buffer to 5, and remove the particles with a 0.45µm PES filter to obtain a protectant solution. Take 8mL of 20mg / mL bromelain solution and add it to 92mL of protectant solution, mix evenly by light shaking, then stand at 5°C for 30min, pre-freeze at -40°C for 60min, place the product in -25°C, and warm up to -15°C at a speed of 1°C / h under a vacuum of 10Pa for 2h; then warm up to 25°C at a speed of 0.2°C / min for 6h to obtain a freeze-dried sample; add 10mL of pure water to each sample, shake once for 5s, start the stopwatch to record the reconstitution time, and measure the clarity (NTU) and pH fluctuation (pH is measured 60s after reconstitution) after reconstitution; take 0.5mL of the reconstituted solution and add it to 4.5mL of 50mM neutral sodium phosphate buffer, and test the enzyme activity (%) after reconstitution = reconstitution activity / bottling activity x 100%; each batch test sets three independent samples, and the results are averaged, the reconstitution time and enzyme activity after reconstitution are shown in Figure 1 , and the clarity and pH after reconstitution are shown in Figure 2 .

[0057] From Figure 1 and Figure 2The results show that the freeze-drying protectant prepared in Example 3 is superior to the control group and the comparative examples in the reconstitution performance: the reconstitution time, reconstitution activity, clarity and pH stability are significantly ahead. From the result analysis of Comparative Example 1 and Example 3, the reconstitution time and clarity are close to Example 1, but the reconstitution activity and pH change are large, which may be due to the collapse of the redox protection system caused by the absence of GSSG / NAC. The absence of GSSG makes the active cysteine in freeze-drying unable to form reversible S-glutathione temporary sealing, and irreversible oxidation occurs before reconstitution. The absence of NAC leads to the lack of reducing agent to restore the oxidized site during reconstitution, and the loss of free radical scavenging ability. The accumulation of oxidation byproducts reduces the pH. From the result analysis of Comparative Example 2 and Example 3, the reconstitution time is significantly improved, the clarity is severely turbid, and the activity is significantly decreased, which may be due to the failure of the chain caused by the absence of mannitol / sorbitol / glycine crystalline skeleton, and the dense amorphous glass body formed by trehalose filling, the porosity tends to zero, causing the water permeation resistance to increase dramatically, the reconstitution time is prolonged; the structure of the freeze-dried cake collapses to produce micro-cracks and peeling pieces, which form irreversible protein aggregates during reconstitution, leading to the deterioration of clarity; water aggregation causes enzyme shear denaturation and hydrolysis. From the result analysis of Comparative Example 3 and Example 3, the reconstitution time of Comparative Example 3 is prolonged, the clarity is decreased, the pH fluctuation is large, and the activity loss is obvious; this may be due to the water-oxidation coupling damage caused by the direct exposure of NAC: the hygroscopicity of NAC destroys the microcapsule barrier, leading to the migration and aggregation of lipid-soluble antioxidants, the interface protection failure leads to the increase of reconstitution turbidity; NAC is oxidized in advance to generate acidic byproducts, leading to the decrease of pH; GSSG is excessively consumed during freeze-drying, and the thiol reducing ability is lacking during reconstitution, and the active site accumulates oxidative.

[0058] Freeze-drying stability test: the freeze-dried samples of Example 1-3 and Comparative Example 1-3 and the control group (conventional freeze-drying protectant trehalose + mannitol + buffer + polysorbate-20) were prepared by the above freeze-drying reconstitution test, then vacuum bottled, and accelerated test was carried out at 42℃ / 75% humidity. The sampling points are 0 days, 7 days, 14 days, 21 days, 42 days and 63 days, respectively, and the freeze-drying reconstitution test is carried out (the protease activity of the 0th day is recorded as 100%), the protease activity change (%) and the residual water rate (%) are calculated, three independent samples are set for each batch test, the average value is taken, and the results are shown in Figure 3 and Figure 4 The freeze-thaw cycle test was carried out at-20℃ to simulate accelerated freezing transportation, and the freeze-thaw cycle was carried out once every 14 days (-20℃ to 25℃ and then back to-20℃), a total of 3 times. After all the cycles are completed, the protease activity change (%) and the residual water rate (%) are calculated, three independent samples are set for each batch test, the average value is taken, and the results are shown in Figure 5 and Figure 6 .

[0059] From Figure 3 andFigure 4 The results analysis of the accelerated test showed that the freeze-drying protective agent prepared by the embodiment of the present application exhibited excellent protection effect, and compared with the control group, the long-term stability was significantly improved: the residual water rate was always maintained at a low level, the protease activity showed a gentle downward trend, and in the later period, it entered a quasi-steady state. From the results analysis of Comparative Example 1 and Example 3, the activity of Comparative Example 1 rapidly decreased before 14 days, and continued to inactivate in the later period, which may be due to the loss of oxidized glutathione (GSSG) and N-acetylcysteine (NAC) leading to double protection failure: the loss of GSSG makes the active cysteine site unable to form reversible S-glutathionylation temporary seal, and the thiol group is directly exposed to the oxidation environment under freeze-thaw / high temperature; the loss of NAC leads to the loss of the ability of the system to activate reduction after reconstitution, and the residual free radicals continuously attack the active center of the enzyme. From the results analysis of Comparative Example 2 and Example 3, the residual water rate of Comparative Example 2 soared, and the activity dropped sharply, which may be due to the loss of mannitol / sorbitol / glycine crystalline skeleton, and then trehalose fills in to form a high-hygroscopic amorphous phase, which accelerates the water intrusion, and the freeze-dried cake structure collapses; the amorphous area repeatedly dissolves-recrystallizes in the freeze-thaw cycle, causing micro-crack expansion and water aggregation; uncontrolled water leads to exposure of enzyme molecules to water-mediated shear denaturation and oxidation, and NAC / GSSG is depleted in advance due to high local water activity. From the results analysis of Comparative Example 3 and Example 3, the activity of Comparative Example 3 decreased rapidly in the early stage, and the inactivation accelerated in the later period, and the residual water rate increased significantly, which may be due to the direct exposure of NAC not embedded, which releases the hygroscopicity at the initial stage of moisture absorption, and destroys the microcapsule barrier; water intrusion leads to the migration and aggregation of lipid-soluble antioxidants; the early consumption of NAC leads to the depletion of reduction ability in the later freeze-thaw period, and the active site is subjected to cumulative oxidative damage.

[0060] From the results analysis of Comparative Example 3 and Example 3, the activity of Comparative Example 3 decreased rapidly in the early stage, and the inactivation accelerated in the later period, and the residual water rate increased significantly, which may be due to the direct exposure of NAC not embedded, which releases the hygroscopicity at the initial stage of moisture absorption, and destroys the microcapsule barrier; water intrusion leads to the migration and aggregation of lipid-soluble antioxidants; the early consumption of NAC leads to the depletion of reduction ability in the later freeze-thaw period, and the active site is subjected to cumulative oxidative damage. Figure 5 and Figure 6The results show that the freeze-drying protectant of Example 3 still maintains very high protease activity after three freeze-thaw cycles, which is significantly better than each of the comparative examples and the control group. From the analysis of the results of Comparative Example 1 and Example 3, the activity of Comparative Example 1 decreases significantly, which may be due to the failure to form a temporary thiol seal due to the absence of GSSG, and the direct attack of ice crystal interface active oxygen (such as •OH) on cysteine sites during freeze-thaw; NAC is also absent, and there is a lack of reducing agent to restore the oxidized thiol during reconstitution, resulting in irreversible inactivation. From the analysis of the results of Comparative Example 2 and Example 3, the activity of Comparative Example 2 collapses, which may be due to the absence of mannitol / sorbitol support, and the sudden increase in water intrusion during freeze-thaw due to the formation of hygroscopic amorphous phase filled with trehalose, resulting in the collapse of the freeze-dried cake structure; repeated phase changes cause microcracks to expand, and enzyme molecules are exposed to ice crystal shear stress; water aggregation accelerates enzyme aggregation denaturation and free radical hydrolysis reaction. From the analysis of the results of Comparative Example 3 and Example 3, the activity of Comparative Example 3 continues to decrease, and the residual water rate increases significantly, which may be due to the water-oxidation coupling damage triggered by the exposure of NAC: the hygroscopicity of unembedded NAC triggers the early destruction of the microcapsule barrier; water penetration leads to the migration failure of lipid-soluble antioxidants, and the loss of interfacial protection ability; NAC is consumed in advance during freeze-thaw, and the accumulation of oxidative free radicals attacks the enzyme active center in the later stage.

[0061] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A bromelain freeze-drying protectant, characterized in that, Its composition includes the following components by weight: 30-50 parts trehalose, 8-10 parts pullulan, 3-10 parts maltodextrin, 12-22 parts mannitol, 1-4 parts sorbitol, 4-12 parts glycine, 0.05-0.2 parts polysorbate-20, 1.5-4.5 parts buffer, 0.2-0.8 parts N-acetylcysteine, 0.05-0.3 parts oxidized glutathione, 0.8-2.5 parts PEG, and 0.05-0.3 parts antioxidant; The DE value of the maltodextrin is 5 to 10; The buffer is either a sodium citrate buffer system or a sodium phosphate buffer system; The preparation of the bromelain freeze protectant includes the following steps: S1: Cool pure water, dissolve trehalose, pullulan and maltodextrin in sequence, add buffer to adjust pH, add oxidized glutathione, filter, concentrate by vacuum membrane evaporation, spray dry to obtain primary powder; S2: First, take a portion of mannitol, glycine, and sorbitol and dissolve them in pure water in proportion. Add water to adjust the solid content. Heat in a water bath and stir until completely clear. After filtration, slowly cool until crystals begin to precipitate. Then, add the remaining mannitol by weight, continue stirring, cool to room temperature and age. Centrifuge, remove the supernatant, take the precipitate, spray with nitrogen to quick freeze, freeze dry under vacuum, and sieve to obtain free-flowing crystals. S3: Under nitrogen protection, PEG was heated to obtain a clear melt, an antioxidant was added, and after cooling, N-acetylcysteine ​​was added and stirred at high speed to form a semi-solid suspension. The semi-solid suspension was added to 0.5% polysorbate-20 buffer solution after cooling, and emulsified by high-speed shearing to form a fine emulsion. The emulsion was then spray-dried at low temperature, coated with a fluidized bed film of trehalose / pullulan aqueous solution, and screened to obtain microcapsules. S4: In a clean environment, with humidity controlled and under a nitrogen atmosphere, add the primary powder from step S1 to the mixer, premix, then slowly add the free-flowing crystals from step S2, mix at low speed, add the microcapsules from step S3 by continuous spraying, mix at low speed, screen, package and store to obtain the freeze-dried protectant. 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; In step S2, the vacuum freeze-drying parameters are: sublimation treatment at -20℃, followed by secondary drying at 25℃ until the moisture content is less than 2%; In step S3, the low-temperature spray drying parameters are: nozzle 0.7mm, inlet 70-90℃, outlet 40-50℃, and atomization pressure 0.6-0.8MPa; In step S3, the specific operation of the fluidized bed film coating is as follows: using a 5-8% trehalose / pullulan aqueous solution, an inlet air temperature of 32-35℃, a bed temperature of 28-32℃, a spray rate of 0.5-1g / min•kg, and an atomization pressure of 1-1.5bar; In step S4, the parameters for continuous spraying are: carrier gas pressure 0.1-0.2 MPa, and spray gun distance from the material surface 10-20 cm.

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