A highly stable protein glutaminase preparation and a method for its preparation

Abstract

The present application relates to the technical field of enzyme preparation, and discloses a high-stability protein glutamine enzyme preparation and a preparation method thereof.The preparation is a freeze-dried solid preparation, and according to 100 parts of total dry matter, the preparation comprises 10-20 parts of protein glutamine enzyme concentrate solid, 10-18 parts of trehalose, 2-5 parts of betaine, 0.3-1.5 parts of L-proline, 1.5-4 parts of pullulan, 0.2-1.0 parts of citrate buffer component and the rest low-DE-value maltodextrin.The enzyme liquid is clarified, concentrated by ultrafiltration and replaced by supplement liquid, trehalose and auxiliary stabilizing liquid are sequentially added, and after the conditions of OD600 increment correction and soluble enzyme activity retention rate are reached, pullulan and low-DE-value maltodextrin are added, and then the preparation is obtained through degassing, pre-freezing and freeze-drying, the obtained preparation has a water content of 2.5-4.0%, a water activity of 0.18-0.28 at 25 DEG C, and good storage stability and reconstitution.

Description

Technical Field

[0001] This invention relates to the field of enzyme preparation technology, and in particular to a highly stable protein glutaminase preparation and its preparation method. Background Technology

[0002] Protein glutaminase catalyzes the hydrolysis of the amide group on the side chain of glutamine residues in proteins or peptides, converting glutamine residues into glutamate residues and releasing ammonia. This alters the charge distribution, hydration state, and intermolecular interactions of the protein. This enzyme can be used to modify soybean protein, pea protein, wheat protein, and milk protein. In industrial production, protein glutaminase is usually prepared into liquid or solid enzyme preparations after microbial fermentation, solid-liquid separation, ultrafiltration concentration, and stabilization for storage, transportation, measurement, and use.

[0003] Existing technologies often improve the storage stability of enzyme preparations by adding sugars, polyols, inorganic salts, amino acids, or dextrin carriers, along with pH adjustment, coating, cross-linking, freeze-drying, or spray drying. The residual enzyme activity measured by standard small molecule substrates is usually used as the basis for quality evaluation. However, enzyme preparations may repeatedly experience heating, moisture absorption, and cooling during summer transportation, diurnal temperature changes, opening and weighing, and resealing. At this time, even if the preparation can still maintain a high standard substrate enzyme activity, the enzyme protein may reduce its actual contact ability with the intact protein substrate due to local aggregation, matrix binding, or incomplete resolution, resulting in a discrepancy between the labeled enzyme activity and the actual deamidation efficiency.

[0004] In addition, the use of protein glutaminase is also affected by the reconstitution water temperature, water hardness, and the buffering capacity and aggregation state of the protein raw materials. The process water temperature differs between winter and summer, and the water quality varies between different plant areas. The same type of plant protein may also exhibit different dispersion states due to different raw material sources and processing history. When the enzyme preparation enters the high solids content protein slurry, if the release and dispersion processes are inconsistent, the protein near the feeding point may undergo deamidation first, causing changes in local charge, viscosity, and hydration state, which in turn changes the subsequent contact process between the enzyme and the substrate. As a result, even with the same dosage, there may still be problems such as fluctuations in the overall degree of deamidation, uneven local treatment, and unstable subsequent emulsification or dispersion performance. Summary of the Invention

[0005] The technical problem to be solved by this invention is that existing protein glutaminase preparations are prone to a decrease in actual deamidation ability of intact protein substrates when storage, transportation and reconstitution conditions change. To address this, we propose a highly stable protein glutaminase preparation and its preparation method.

[0006] To solve the above-mentioned technical problems, the present invention provides a highly stable protein glutaminase preparation, which is a freeze-dried solid preparation containing protein glutaminase. The protein glutaminase has the activity of catalyzing the hydrolysis of the amide group of the side chain of glutamine residue in protein or polypeptide.

[0007] Based on a total dry matter mass of 100 parts, the formulation includes 10-20 parts protein glutaminase concentrate solids, 10-18 parts trehalose, 2-5 parts betaine, 0.3-1.5 parts L-proline, 1.5-4 parts pullulan, 0.2-1.0 parts citrate buffer, and the balance low DE value maltodextrin.

[0008] The mass ratio of trehalose to betaine is 3:1 to 7:1, the mass ratio of betaine to L-proline is 2:1 to 8:1, and the mass ratio of pullulan to low DE value maltodextrin is 1:15 to 1:35, wherein the DE value of the low DE value maltodextrin is 4 to 10.

[0009] The water content of the formulation is 2.5% to 4.0%, the water activity at 25℃ is 0.18 to 0.28, and the pH of the formulation after reconstitution at a 1% mass-volume concentration is 5.4 to 6.2.

[0010] Protein glutaminase concentrate solids include protein glutaminase and soluble protein components that are retained along with protein glutaminase during the concentration process.

[0011] The citrate buffer component consists of citric acid and sodium citrate.

[0012] The formulation is a freeze-dried powder obtained by sieving, wherein the freeze-dried powder passes through a 40-mesh sieve and is retained on an 80-mesh sieve.

[0013] Preferably, based on a total dry matter mass of 100 parts, the formulation comprises 12-18 parts protein glutaminase concentrate solids, 13-16 parts trehalose, 2.5-4 parts betaine, 0.5-1.0 parts L-proline, 2-3 parts pullulan, 0.3-0.8 parts citrate buffer, and the balance low DE value maltodextrin.

[0014] The present invention also provides a method for preparing the above-mentioned highly stable protein glutaminase preparation, comprising the following steps:

[0015] Step 1: Clarify and concentrate the enzyme solution containing protein glutaminase by ultrafiltration, and replace the solution with citrate buffer to obtain an enzyme concentrate with a total solids concentration of 100-180 g / L, protein glutaminase activity of 60-120 U / mL, pH of 5.6-6.1, and conductivity of 1.2-2.5 mS / cm at 25℃.

[0016] Step 2: Add trehalose solution to enzyme concentrate at 4-10℃, mix, then add auxiliary stabilizing solution containing betaine, L-proline and citrate buffer components, and continue mixing for 20-40 minutes to obtain pre-equilibrium solution.

[0017] Step 3: When the corrected OD600 increment of the pre-equilibration solution relative to the enzyme solution before addition is not greater than 0.02, and the soluble enzyme activity retention rate of the pre-equilibration solution supernatant after centrifugation is not less than 95%, add pullulan polysaccharide solution to the pre-equilibration solution, mix, and then add low DE value maltodextrin solution in batches to obtain a dry matrix precursor solution with a total solids mass fraction of 26% to 30%.

[0018] Step 4: Degas, pre-freeze, perform primary drying and secondary drying sequentially on the dry matrix precursor solution until the water content of the resulting freeze-dried solid preparation is 2.5% to 4.0% and the water activity at 25°C is 0.18 to 0.28.

[0019] Step 5: The freeze-dried solid preparation is subjected to low-shear crushing and sieving to obtain freeze-dried powder.

[0020] In S1, the enzyme solution containing protein glutaminase is either fermentation supernatant or recombinant expression enzyme solution. The enzyme solution is maintained at 4–10°C and clarified by low-temperature centrifugation or microfiltration with a pore size of 0.2–0.45 μm, followed by concentration using an ultrafiltration membrane with a molecular weight cutoff of 5–10 kDa. The concentration of citrate buffer is 5–10 mmol / L, and the pH is 5.6–6.1.

[0021] In step S2, trehalose is prepared into a 25%–35% trehalose solution. After cooling to 4–10°C, the enzyme concentrate is added within 10–20 minutes, and the mixture is stirred for 10–20 minutes after the addition is complete. A 20%–35% auxiliary stabilizing solution is prepared from a betaine, L-proline, and citrate buffer group. After cooling to 4–10°C, the enzyme solution containing trehalose is added within 10–20 minutes.

[0022] In S3, the enzyme solution before addition and the pre-equilibration solution were taken and diluted 10 times with a buffer solution with the same pH and conductivity as the corresponding sample. The absorbance at 600 nm was measured using a 1 cm path length cuvette. Blank correction was performed using an excipient solution containing the same proportions of trehalose, betaine, L-proline and citrate buffer components but without protein glutaminase to obtain the corrected OD600 increment.

[0023] The pre-equilibrated solution was centrifuged at 4℃ and 10000×g for 10 min. The percentage of the total enzyme activity of the supernatant after volume correction to the total enzyme activity of the enzyme solution before feeding was taken as the soluble enzyme activity retention rate.

[0024] Pullulan solution has a mass fraction of 5%–10%, and is hydrated at 35–45°C and then cooled to no higher than 12°C. Low DE value maltodextrin solution has a mass fraction of 35%–45%, and is dissolved at 40–50°C and then cooled to no higher than 12°C, and added in 2–4 batches.

[0025] In S4, the dry matrix precursor liquid is intermittently degassed under vacuum at 4–10°C for 5–15 min, and then loaded into a tray with a material layer thickness of 8–12 mm. The core temperature of the material is then reduced to no higher than -40°C and maintained for at least 1 h.

[0026] The product is dried once under 30-80 Pa conditions, with the shelf temperature gradually increased from -35°C to -25°C, and the product temperature kept not higher than -20°C; then the shelf temperature is increased to 20-25°C, and a second drying is carried out under 5-30 Pa conditions for 4-8 hours.

[0027] The freeze-dried material obtained from drying is subjected to low-shear crushing and passed through a 40-80 mesh sieve. Based on the measured enzyme activity of the obtained preparation, enzyme activity standardization is performed using the low DE value maltodextrin reserved in the formula. The reserved low DE value maltodextrin is 0-5 parts and is included in the total amount of low DE value maltodextrin.

[0028] The technical effects and advantages of this invention are as follows:

[0029] In this invention, trehalose, betaine, and L-proline form a small-molecule stable system, while pullulan and low-DE maltodextrin form a drying matrix system. By limiting the amount and mass ratio of each component, the small-molecule stable component and the polymer carrier maintain a relatively stable complexation relationship in the formulation. The resulting freeze-dried solid formulation has a low water content and water activity, which can reduce moisture absorption, clumping, and enzyme activity decline during storage.

[0030] In the preparation process, the total solids, pH, and conductivity of the original enzyme solution are first standardized. Then, trehalose and an auxiliary stabilizing solution containing betaine and L-proline are added sequentially to allow the protein glutaminase to complete low-temperature pre-equilibration before the addition of the polymeric carrier. The state of the pre-equilibrated solution is judged by the corrected OD600 increment and the retention rate of soluble enzyme activity. This can identify the increase in turbidity, protein aggregation, or loss of soluble activity that occurs during the addition of excipients, and avoid continuing to add polymeric carriers when the solution state does not meet the requirements.

[0031] Pullulan was added after pre-equilibration, and low-DE maltodextrin was added in batches in solution form. This reduced local agglomeration, uneven mixing, and enzyme-protein encapsulation caused by pre-thickening of high molecular weight substances. By controlling the solid content of the drying matrix precursor solution, pre-freezing conditions, primary drying product temperature, and secondary drying endpoint, the water content and water activity of the resulting formulation were kept stable within the specified range. Attached Figure Description

[0032] The disclosure of this invention is illustrated with reference to the accompanying drawings. It should be understood that the drawings are for illustrative purposes only and are not intended to limit the scope of protection of this invention. In the drawings, the same reference numerals are used to refer to the same parts: Figure 1 This is a schematic flowchart of the method for preparing a highly stable protein glutaminase preparation according to the present invention. Figure 2 A comparison chart showing the retention rates of standard substrate enzyme activity, intact protein functional activity, and intact protein functional coefficient of each embodiment and comparative example after storage at 40°C for 30 days. Figure 3 This is a comparison chart showing the relative values ​​of the intact protein functional coefficients of each embodiment and comparative example under different water temperature and water hardness resolution conditions. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments. It should be understood that the listed embodiments are used to illustrate the formulation composition, preparation sequence, and process control of the present invention, and are not intended to limit the scope of protection of the present invention. Unless otherwise stated, all raw materials used are food-grade or enzyme-grade, and the water used is purified water. All percentages are mass percentages, and all mass parts are calculated on a dry matter basis.

[0034] The protein glutaminase described in this invention catalyzes the hydrolysis of the amide group on the side chain of glutamine residues in proteins or peptides, converting glutamine residues into glutamate residues and releasing ammonia, without targeting the peptide bonds of the protein backbone. During raw material acceptance and finished product testing, the protein glutaminase activity should be distinguished from the activity of accompanying proteases to avoid interference from protein hydrolysis on the application results of intact proteins.

[0035] Existing solid protein glutaminase preparations typically include sugars, inorganic salts, or dextrin carriers and are prepared by freeze-drying or spray-drying. Stability is usually evaluated by the residual enzyme activity of small molecule standard substrates. This embodiment retains the standard enzyme activity detection while adding evaluation of the functional activity of intact protein substrates. Furthermore, by unifying the original enzyme solution state, pre-equilibrating the small molecule stable components, and adding the polymer matrix later, the influence of drying, storage, and reconstitution processes on the deamidation ability of intact proteins is reduced.

[0036] Protein glutaminase can be derived from fermentation supernatant, recombinant expression enzyme solution, or commercial enzyme preparations. When using commercial enzyme preparations that already contain dextrin or other carriers, the total solids and original carriers should be measured, and this part should be included in the final total carrier amount. The enzyme concentrate used for formulation preferably meets the following requirements: total solids concentration of 100-180 g / L, enzyme activity of 60-120 U / mL, pH of 5.6-6.1, conductivity of 1.2-2.5 mS / cm at 25℃, and no visible flocculent matter.

[0037] Based on a final dry matter total mass of 100 parts, the formulation comprises 10-20 parts protein glutaminase concentrate solids, 10-18 parts trehalose, 2-5 parts betaine, 0.3-1.5 parts L-proline, 1.5-4 parts pullulan, 0.2-1.0 parts citrate buffer, and up to 100 parts low DE value maltodextrin.

[0038] The preferred composition includes 12-18 parts enzyme concentrate solids, 13-16 parts trehalose, 2.5-4 parts betaine, 0.5-1.0 parts L-proline, 2-3 parts pullulan, 0.3-0.8 parts citrate buffer, and the balance low DE value maltodextrin.

[0039] Trehalose, betaine, and L-proline constitute a small molecule stable system; pullulan and low DE value maltodextrin constitute a dry matrix precursor system.

[0040] The mass ratio of trehalose to betaine is 3:1 to 7:1, the mass ratio of betaine to L-proline is 2:1 to 8:1, the mass ratio of pullulan to low DE value maltodextrin is 1:15 to 1:35, and the DE value of low DE value maltodextrin is 4 to 10.

[0041] Pullulan and L-proline are used in lower amounts with low DE value maltodextrin as the main carriers to coordinate the continuity of the drying matrix, the viscosity of the precursor solution, and the reconstitution and dispersibility of the formulation.

[0042] Trehalose is mainly used to mitigate the rapid changes in the hydration layer around enzyme proteins during freezing and dehydration; betaine and L-proline are used to supplement the small molecule stabilizing environment; pullulan improves the continuity and reconstitution dispersibility of the matrix after drying at a lower ratio; and low DE value maltodextrin serves as the main carrier, regulates solids, and standardizes enzyme activity.

[0043] All excipients can be used in enzyme preparations. Pullulan and L-proline are used in a low ratio to control the viscosity of the precursor solution and improve the reconstitution performance of the preparation. The amounts of pullulan and L-proline in the central formulation are controlled at 2.5 parts and 0.8 parts, respectively, to avoid excessive pullulan dosage leading to increased viscosity of the precursor solution and to control the effect of L-proline on the solid content ratio of the preparation.

[0044] When trehalose content is below 10 parts, its protection during the freeze-drying process may be insufficient; when it is above 18 parts, the hygroscopicity of the formulation may increase, and the increase in stabilizing effect slows down. When pullulan content is below 1.5 parts, its improvement on reconstitution and dispersion is not readily apparent; when it is above 4 parts, it increases the viscosity of the solution and the reconstitution time. Therefore, this embodiment does not rely on the large-dose use of a single excipient, but rather utilizes the coordination of different components in the feeding sequence and drying stage.

[0045] The resulting formulation had a water activity of 0.18–0.28 at 25°C and a water content of 2.5%–4.0%. After reconstitution at a 1% mass-volume concentration, the pH of the reconstituted solution was 5.4–6.2, and a uniform dispersion was formed within 5 minutes without any visible flocculent precipitate.

[0046] like Figure 1 As shown, the preparation method includes the following steps:

[0047] Step S1: The fermentation supernatant or recombinant expression enzyme solution containing protein glutaminase is kept at 4–10°C and insoluble matter is removed by low-temperature centrifugation or 0.2–0.45 μm microfiltration. Then, it is concentrated using an ultrafiltration membrane with a molecular weight cutoff of 5–10 kDa. During the concentration process, 5–10 mmol / L citrate buffer solution with pH 5.6–6.1 is used to replenish and replace the solution so that the total solids, enzyme activity, pH and conductivity of the enzyme concentrate are within the aforementioned range. This step is used to reduce the differences in fermentation salts, free ammonia and the original buffer system, and high-purity chromatographic purification is not required.

[0048] If the conductivity remains above 2.5 mS / cm after replenishment, continue adding buffer and ultrafiltration; if the total solids or enzyme activity concentration is below the required level, continue low-temperature concentration. If free ammonia or ammonium salts are present in the concentrate, set a sample background blank and subtract it from the standard enzyme activity and intact protein functional activity assays to avoid misidentifying residual ammonia in the original solution as an enzyme reaction product.

[0049] Step S2: Prepare a 25%–35% aqueous solution of trehalose, cool it to 4–10°C, and add the enzyme concentrate over 10–20 minutes while stirring at low speed. After the addition is complete, continue mixing for another 10–20 minutes. Adding trehalose first ensures that the enzyme protein is in a sugar-hydrated environment before the addition of other stable components and polymer carriers.

[0050] Step S3: Prepare a 20%–35% auxiliary stabilizing solution by dispensing betaine, L-proline, and citrate buffer groups. After cooling to 4–10°C, add the enzyme solution containing trehalose within 10–20 min and continue mixing at low temperature for 20–40 min. After pre-equilibration, test the corrected OD600 and soluble enzyme activity: After diluting the sample with buffer of the same pH and conductivity and subtracting the blank without enzyme excipients, the increase in OD600 relative to the value before adding the additives should not be greater than 0.02; After centrifuging the sample at 4°C and 10000×g for 10 min, the soluble enzyme activity retention rate of the supernatant should not be less than 95%. If the requirements are not met, stop adding the carrier and check the state of the original enzyme solution, the addition rate, and the temperature of the solution.

[0051] Step S4: Prepare a 5%–10% solution of pullulan in water at 35–45°C, and cool it to no higher than 12°C after hydration. Prepare a 35%–45% solution of low DE value maltodextrin in water at 40–50°C and cool it. After the enzyme solution passes the pre-equilibrium check, first add the pullulan solution and mix for 15–30 minutes. Then add the maltodextrin solution used before drying in 2–4 batches, preferably in three batches. The amount added in the first batch is greater than the amount added in each subsequent batch. When adding in three batches, the amount added in each batch is 40%, 30%, and 30% of the total mass of the maltodextrin solution, respectively. Mix for 10–20 minutes after each batch is added.

[0052] Pullulan and maltodextrin should not be added directly to the low-temperature enzyme solution in powder form. Hydration or dissolution should be completed at an appropriate temperature first, followed by cooling before contact with the enzyme solution. This reduces agglomeration, localized thickening, and the high-permeability environment near the feeding point. During scale-up production, the stirring speed should not be directly copied from the small-scale test values. The mixing should be confirmed by ensuring that the solids or conductivity deviation from the top, middle, and bottom of the tank does not exceed 5%.

[0053] Step S5: Degas the dry matrix precursor liquid intermittently under vacuum at 4-10℃ for 5-15 minutes, load it into a freeze-drying tray with a material layer thickness of 8-12 mm, lower the core temperature of the material to no higher than -40℃ and maintain it for no less than 1 hour to complete the pre-freezing.

[0054] Step S6: Perform a first drying at 30–80 Pa, gradually increasing the shelf temperature from -35°C to -25°C, while controlling the product temperature to not exceed -20°C. The first drying time is 10–18 hours. Subsequently, raise the shelf temperature to 20–25°C and perform a second drying at 5–30 Pa for 4–8 hours. The endpoint of the first drying is determined based on the product temperature and pressure rise test. After the second drying, the endpoint is defined as the simultaneous attainment of the specified range in both moisture content and water activity.

[0055] During freeze-drying scale-up, maintain similar material layer thickness, tray density, and heat transfer path, and reconfirm the product temperature and the endpoint of the first drying cycle. Do not simply extend the drying time linearly according to the loading amount. If the product temperature is close to the collapse temperature of the formula, the shelf heating rate or shelf temperature should be reduced to avoid material layer collapse and slower remelting.

[0056] Step S7: Discharge the freeze-dried block in an environment with a relative humidity not exceeding 30%. Perform low-shear crushing on the freeze-dried block, then sieve it sequentially using a 40-mesh sieve and an 80-mesh sieve. Collect the freeze-dried powder that passes through the 40-mesh sieve but is retained on the 80-mesh sieve. After determining the enzyme activity per unit mass, standardize the enzyme activity using 0-5 parts of low DE-value maltodextrin reserved in the formula, which is included in the total maltodextrin amount. After mixing, store in moisture-proof sealed packaging.

[0057] The standard substrate enzyme activity (As) was determined using the Z-Gln-Gly ammonia release method. 1.0 mL of 0.2 mol / L phosphate buffer (pH 6.5) containing 30 mmol / L Z-Gln-Gly was added, along with 0.1 mL of appropriately diluted test sample. The reaction was incubated at 37°C for 10 min, followed by the addition of 1.0 mL of 0.4 mol / L trichloroacetic acid to terminate the reaction. A sample blank was prepared by first adding the trichloroacetic acid stop solution, then adding the test sample, to subtract the ammonia present in the test sample itself. The amount of ammonia generated in the reaction system was determined using an ammonia detection kit. One unit of protein glutaminase activity (U) was defined as the amount of enzyme required to catalyze the release of 1 μmol NH3 per minute under the above reaction conditions. The standard substrate enzyme activity (As) is expressed as protein glutaminase activity per unit mass of the formulation, in U / g of formulation.

[0058] The functional activity Ap of intact protein was determined using a fixed batch of soy protein isolate as the substrate. Soy protein isolate was prepared as a 5% (w / v) dispersion, pH adjusted to 7.0, and pre-equilibrated at 44℃. The test formulation was added at a concentration of 40 U As / g protein. Samples were taken at 0 min, 10 min, and 20 min, and the reaction was terminated by adding an equal volume of 0.4 mol / L trichloroacetic acid. Enzyme-free substrate blanks, heat-inactivated enzyme blanks, and ammonia background blanks were set up. After subtracting ammonia from each blank, a linear fit was performed on the reaction time using ammonia production. The coefficient of determination R0 was calculated. 2 The ammonia generation rate obtained from the fitting is not less than 0.98. The ammonia generation rate obtained is divided by the mass of the formulation added to the reaction system to obtain the complete protein functional activity Ap, in U / g formulation, where 1U represents the activity of catalyzing the release of 1μmol NH3 per minute.

[0059] The functional coefficient Kp of a complete protein is calculated according to the following formula:

[0060] Kp = Ap ÷ As.

[0061] Where Ap represents the functional activity of the intact protein per unit mass of the formulation as measured against the intact protein substrate, As represents the enzyme activity of the standard substrate per unit mass of the formulation as measured against the Z-Gln-Gly standard substrate, and Kp is used to evaluate the correspondence between the actual deamidation ability of the same formulation against the intact protein substrate and the enzyme activity of the standard substrate.

[0062] Example 1

[0063] Based on a final dry matter of 10.00 kg, this embodiment includes 1.50 kg of protein glutaminase concentrate solids, 1.50 kg of trehalose, 0.30 kg of betaine, 0.08 kg of L-proline, 0.25 kg of pullulan, 0.05 kg of citrate buffer, and 6.32 kg of low DE value maltodextrin. Of the low DE value maltodextrin, 5.82 kg is added before drying, and 0.50 kg is reserved as a standardization allowance after drying.

[0064] Take 10.0 L of the clarified and replenished protein glutaminase concentrate, which has a total solids concentration of 150 g / L, an enzyme activity concentration of 100 U / mL, a pH of 5.8, and a conductivity of 1.8 mS / cm. This feed provides 1.50 kg of concentrate solids and approximately 1.0 × 10^6 U of protein glutaminase activity.

[0065] Add 1.50 kg of trehalose to 3.50 kg of purified water and stir at 25 °C until completely dissolved. Add water to make the solution weigh 5.00 kg. After filtration, cool to 8 °C. Add the trehalose solution to the enzyme concentrate within 15 min, stirring at a low speed during the addition process. After the addition is complete, continue mixing for 15 min.

[0066] Add 0.30 kg betaine, 0.08 kg L-proline and 0.05 kg citrate buffer to 1.00 kg purified water, stir to dissolve and cool to 8°C. Add enzyme solution containing trehalose within 10 min, continue mixing at low temperature for 30 min, take samples for testing, the corrected OD600 increment is 0.009, the change is 0.003 over 15 min, the soluble enzyme activity retention rate is 97.2%, meeting the carrier feeding conditions.

[0067] 0.25 kg pullulan was dispersed in 2.875 kg of purified water and hydrated at 40 °C for 45 min to obtain an 8% pullulan solution, which was then cooled to 10 °C. 5.82 kg of low DE value maltodextrin was added to 8.73 kg of purified water and dissolved at 45 °C to obtain a 40% maltodextrin solution, which was then cooled to 10 °C.

[0068] First, add pullulan polysaccharide solution within 15 minutes and continue mixing for 20 minutes; then add maltodextrin solution in three portions of 40%, 30% and 30%, mixing for 15 minutes after each addition. The total solids of the resulting dry matrix precursor solution is about 28%, the pH is 5.9, the temperature is 9℃, the solution can be pumped smoothly and no flocculation is observed.

[0069] The liquid material was intermittently degassed for 10 minutes under an absolute pressure of 20 kPa. It was then loaded into trays with a material layer thickness of 10 mm. The shelf temperature was lowered to -48°C. After the product center temperature dropped to -42°C, it was maintained for 1.5 hours. During the first drying stage, the system pressure was controlled at 50 Pa, and the shelf temperature was raised from -35°C to -25°C and maintained for 13 hours. After the pressure increase test confirmed that the main ice crystal sublimation was completed, the shelf temperature was raised to 23°C and a second drying was carried out for 6 hours.

[0070] The dried powder had a moisture content of 3.1% and a water activity of 0.22 at 25°C. The freeze-dried blocks were subjected to low-shear crushing and sieving. Based on the measured enzyme activity per unit mass, the required amount of low DE value maltodextrin for standardization was determined to be 0.50 kg. All of the 0.50 kg of low DE value maltodextrin was added and mixed evenly to obtain the formulation of Example 1 with a dry matter mass of 10.00 kg.

[0071] Example 2

[0072] Based on 100 parts of final dry matter, it includes 12.00 parts of protein glutaminase concentrate solids, 13.00 parts of trehalose, 2.50 parts of betaine, 0.50 parts of L-proline, 2.00 parts of pullulan, 0.50 parts of citrate buffer, and 69.50 parts of low DE value maltodextrin. 4.00 parts of the low DE value maltodextrin are reserved as a standardization allowance after drying.

[0073] Take a protein glutaminase concentrate with a total solids concentration of 120 g / L, an enzyme activity concentration of 85 U / mL, a pH of 5.7, and a conductivity of 1.4 mS / cm, and calculate the actual feed volume based on 12.00 parts of the concentrate solids.

[0074] Trehalose was prepared as a 28% solution and added over 18 minutes at 8°C. A 25% auxiliary stabilizing solution was prepared using a buffer of betaine, L-proline, and citrate and added over 12 minutes. The mixture was then allowed to equilibrate at low temperature for another 35 minutes. The corrected OD600 increment was 0.012, and the soluble enzyme activity retention rate was 96.1%.

[0075] Pullulan was prepared into a 6% solution, and low DE-value maltodextrin was prepared into a 38% solution. After being added in the prescribed order, the total solids of the drying matrix precursor solution were controlled to 26%. The mixture was then packed into trays with a 9mm layer thickness and pre-frozen at -45°C. After the core temperature of the material dropped to no higher than -40°C, it was held for 1 hour. The primary drying pressure was 45 Pa, and the primary drying shelf temperature program, product temperature control, and endpoint determination method were the same as in Example 1. The secondary drying shelf temperature was 22°C, and the secondary drying pressure and drying time were the same as in Example 1. The resulting formulation had a water content of 3.4% and a water activity of 0.24 at 25°C.

[0076] Example 3

[0077] Based on 100 parts of final dry matter, it includes 18.00 parts protein glutaminase concentrate solids, 17.00 parts trehalose, 4.00 parts betaine, 1.20 parts L-proline, 3.50 parts pullulan, 0.80 parts citrate buffer, and 55.50 parts low DE value maltodextrin.

[0078] Take a protein glutaminase concentrate with a total solids concentration of 175 g / L, an enzyme activity concentration of 115 U / mL, a pH of 6.0, and a conductivity of 2.3 mS / cm, and add it to the concentrate at a concentration of 18.00 parts solids.

[0079] Trehalose was prepared into a 32% solution and added over 12 minutes at 10°C. A 30% auxiliary stabilizing solution was prepared by dispersing betaine, L-proline and citrate buffer and added. The solution was equilibrated at low temperature for 30 minutes. The corrected OD600 increment was 0.008, and the soluble enzyme activity retention rate was 97.7%.

[0080] Pullulan solution and maltodextrin solution were cooled to 10°C and added sequentially to achieve a total solids content of 30% in the dried matrix precursor solution. The solution was packed into trays with an 11mm layer thickness. The pre-freezing temperature and holding conditions were the same as in Example 1. The first drying time was 14 hours, and the first drying pressure, shelf temperature program, and product temperature control were the same as in Example 1. The second drying time was 6 hours, and the second drying pressure and shelf temperature were the same as in Example 1. The resulting formulation had a water content of 2.9% and a water activity of 0.21 at 25°C.

[0081] Example 4

[0082] Example 1 was scaled up to a final dry matter of 50 kg. A mixing tank with a cooling jacket was used to maintain the temperature of the original enzyme solution and the feed solution after addition at 4-10°C. The temperature of the feed solution during the addition of pullulan and low DE value maltodextrin was maintained at no higher than 12°C. The dry matter ratios of protein glutaminase concentrate solids, trehalose, betaine, L-proline, pullulan, citrate buffer component and low DE value maltodextrin were the same as in Example 1.

[0083] Each excipient solution is prepared in an independent dissolving tank. Pullulan and maltodextrin are hydrated and dissolved in warm water, and then cooled to no higher than 12°C by a plate heat exchanger or jacket. Trehalose solution is added within 20 minutes, and auxiliary stabilizing solution is added within 20 minutes.

[0084] The stirring speed was not directly copied from the small-scale test values. Instead, the mixing verification was carried out based on the tank diameter, impeller type, and liquid volume. The criterion for complete mixing was that the relative deviation of the conductivity and soluble solids of the samples at the top, middle, and bottom of the tank was no more than 5%.

[0085] During freeze-drying scale-up, a material layer thickness of 8–12 mm was maintained, and product temperature probes were set at different locations. The endpoint of the first drying cycle was determined by a combination of product temperature and pressure rise tests and vacuum gauge readings. The formulation obtained from the scale-up batch had a water content of 3.2% and a water activity of 0.23.

[0086] Comparative Example 1

[0087] The final components and amounts of this comparative example are the same as those in Example 1. During preparation, trehalose, betaine, L-proline, pullulan, and low DE value maltodextrin used before drying are mixed together to form a solution, which is then added in one go within 30 minutes to a protein glutaminase concentrate that has been uniformly prepared.

[0088] After adding all components, mix for 60 minutes without performing pre-equilibration of the small molecule stable system or intermediate release checks. Then, degassing, pre-freezing, freeze-drying, granulation, and standardization are carried out according to the conditions of Example 1.

[0089] Comparative Example 2

[0090] The final components and amounts in this comparative example are the same as in Example 1. The difference is that after the original enzyme solution is concentrated, it is not replaced with citrate buffer, but is directly stabilized using a concentrated protein glutaminase solution with a pH of 6.1 and a conductivity of 6.2 mS / cm. The remaining order of addition and drying conditions are the same as in Example 1.

[0091] Comparative Example 3

[0092] The final components and amounts of this comparative example are the same as those in Example 1. The difference is that pullulan polysaccharide solution and low DE value maltodextrin solution are added first, and trehalose, betaine and L-proline are added after mixing. The remaining degassing, pre-freezing and freeze-drying conditions are the same as those in Example 1.

[0093] Comparative Example 4

[0094] The final components and amounts in this comparative example are the same as in Example 1. The difference is that after the auxiliary stabilizing solution is added, it is mixed for only 5 minutes, and the OD600 correction and soluble enzyme activity tests are not performed. Pullulan and low DE value maltodextrin are added immediately.

[0095] Comparative Example 5

[0096] This comparative example does not include betaine and is made up to 100 parts by equal mass of low DE value maltodextrin. The remaining components, order of addition, and drying conditions are the same as in Example 1.

[0097] Comparative Example 6

[0098] This comparative example does not include L-proline and is made up to 100 parts with an equal mass of low DE value maltodextrin. All other conditions are the same as in Example 1.

[0099] Comparative Example 7

[0100] This comparative example did not include pullulan, and was made up to 100 parts by weight of an equal mass of low DE value maltodextrin. The addition and pre-equilibration process of the small molecule stabilizing system was the same as in Example 1.

[0101] Comparative Example 8

[0102] Based on 100 parts of final dry matter, this comparative example includes 15.00 parts of protein glutaminase concentrate solids, 2.50 parts of magnesium chloride, and 82.50 parts of low DE value maltodextrin.

[0103] Magnesium chloride was prepared into an aqueous solution and then added to a concentrated protein glutaminase solution. The mixture was stirred for 20 minutes, followed by the addition of maltodextrin solution to bring the total solids before drying close to that of Example 1. A control formulation was prepared following the same pre-freezing, single drying, and double drying endpoints as in Example 1.

[0104] To evaluate the stability of the formulations obtained in each example and comparative example during formulation preparation, storage, reconstitution, and application of intact proteins, the following methods were used for testing.

[0105] Test Example 1

[0106] After the small molecule stable system was pre-equilibrium, the corrected OD600 and the soluble enzyme activity retention rate were measured. After freeze-drying, the water content, water activity, and the recovery rate of dried enzyme activity were measured. The water content was determined by the Karl Fischer method, and the water activity was measured at 25°C.

[0107] The recovery rate of desiccant activity is calculated using the following formula:

[0108] Dry enzyme activity recovery rate = (Total enzyme activity of the sample after drying and reconstitution ÷ Total enzyme activity of the solution before drying) × 100%

[0109] The calculation was corrected for sampling loss, amount of standardized carrier added after drying, and reconstitution volume. The test results are shown in Table 1.

[0110] Table 1 Formulation and Drying Properties

[0111] As shown in Table 1, the corrected OD600 increment of Examples 1 to 4 was 0.008 to 0.012, the soluble enzyme activity retention rate was 96.1% to 97.7%, and the drying enzyme activity recovery rate was 81.9% to 86.3%. Even after reducing the amount of pullulan and L-proline, the examples still maintained a good level of drying recovery.

[0112] Comparative Example 1 used the same final components and amounts as Example 1, but added all components at once. Its corrected OD600 increment increased to 0.027, and the drying enzyme activity recovery rate decreased to 70.2%. In Comparative Example 3, the polymer carrier was added first, and the corrected OD600 increment and drying activity loss were more obvious. This shows that the stabilization effect is not only related to the final formulation, but also to the contact sequence of the small molecule stabilizing components and the polymer carrier.

[0113] Test Example 2

[0114] like Figure 2 As shown, each group of samples was sealed in the same moisture-proof packaging and stored at 40°C for 30 days. Accelerated storage at 40°C was used for relative comparison of different formulations and is not directly equivalent to determining the product shelf life.

[0115] After storage, the standard substrate enzyme activity (As), intact protein functional activity (Ap), intact protein functional coefficient (Kp), reconstitution time, and reconstitution appearance were measured. The retention rates of standard enzyme activity, intact protein functional activity, and Kp were calculated as the ratios of the values ​​after storage to those before storage. The test results are shown in Table 2.

[0116] Table 2. Stability after storage at 40℃ for 30 days

[0117] After storage at 40°C for 30 days, the As retention rate in Examples 1-4 was 81.5%-85.7%, the Ap retention rate was 76.9%-82.2%, and the Kp retention rate was 94.4%-95.9%. The decrease in the functional activity of the intact protein was slightly greater than that of the standard substrate enzyme activity, but the two still maintained a relatively stable correspondence.

[0118] Comparative Example 8, using a magnesium chloride and maltodextrin stabilization route, achieved an As retention rate of 81.2%, similar to Example 2, but its Ap and Kp retention rates were lower than those of Example 2. This result indicates that relying solely on the enzyme activity of small molecule standard substrates may be insufficient to reflect the actual ability of the formulation to act on intact protein substrates after storage.

[0119] Test Example 3

[0120] like Figure 3 As shown, soft water and hard water are prepared using calcium chloride and magnesium sulfate. 2+ With Mg 2+ The molar ratio was fixed at 4:1, and the hardness was set at 50 mg / L and 300 mg / L, respectively, both calculated as CaCO3. The water temperature was adjusted to 10℃ and 25℃, respectively.

[0121] Each formulation was prepared into a 1% (w / v) reconstituted solution. The solutions were stirred at the same speed for 5 minutes. Then, a fixed batch of soy protein isolate substrate was added with the same As. Ap and Kp were measured. The Kp of Example 1 under soft water conditions at 25°C was taken as 100%. The relative Kp under each condition was calculated. The results are shown in Table 3.

[0122] Table 3. Relative values ​​of Kp under different reconstitution conditions

[0123] As shown in Table 3, both low temperature and high water hardness reduce the functional coefficient of the intact protein after reconstitution. However, the relative Kp values ​​of Examples 1-4 in hard water at 10°C are still 84.7%-91.3%. Comparative Example 3 decreased to 57.0% under the same conditions, indicating that the formulation obtained by adding the polymer carrier first is more sensitive to unfavorable reconstitution conditions.

[0124] Test Example 4

[0125] 5% (w / v) dispersions of soy protein isolate and pea protein isolate from the same batch were prepared, pH adjusted to 7.0, and pre-equilibrated at 44℃. Each formulation was added at a protein concentration of 40 U As / g, and the initial ammonia release rate was measured within 0–20 min. After 60 min of reaction, the samples were centrifuged at 10000 × g for 10 min, and the soluble protein content in the supernatant was determined using the BCA method. A standard curve was prepared using bovine serum albumin, and background correction was performed using the corresponding formulation reaction solution without intact protein substrate. The soluble protein ratio in the supernatant was calculated as the percentage of protein in the supernatant after reaction to the initial protein content of the reaction system.

[0126] Soy protein and pea protein were used to set up blanks for enzyme-free substrate, heat-inactivated enzyme, and formulation background ammonia. After subtracting the corresponding blanks, the reaction time was linearly fitted with the ammonia generation rate. The ammonia generation rate obtained by the fitted was divided by the protein mass in the reaction system to obtain the initial ammonia release rate R per unit mass of intact protein, in μmol NH3 / (min·g protein). The index R is used to evaluate the application effect of each formulation on intact protein under the same protein feeding conditions, and is not used as a parameter for calculating the functional activity Ap or functional coefficient Kp of intact protein. The soluble protein ratio in the supernatant is the percentage of protein in the supernatant after the reaction relative to the initial protein mass in the reaction system. The test results are shown in Table 4.

[0127] Table 4 Results of application of intact protein

[0128] Examples 1-4 show initial ammonia release rates of 0.820-0.883 μmol NH3 / (min·g protein) for soybean protein and 0.701-0.751 μmol NH3 / (min·g protein) for pea protein. The examples show similar trends across both intact protein substrates, indicating that the stabilization process is not limited to a single protein substrate.

[0129] Although the standard substrate enzyme activity could still be measured in Comparative Examples 1 and 3, the initial ammonia release rate of the intact protein and the content of soluble protein after treatment were significantly lower than those in the Examples, further illustrating that the preservation of the function of the intact protein by the formulation is related to the final formulation, the order of addition, and the matrix formation process.

[0130] As shown in Tables 1-4, Examples 1-4 standardized the solids, pH, and conductivity of the original enzyme solution. Trehalose was added first, followed by betaine and L-proline to achieve low-temperature pre-equilibration. Pullulan and low-DE maltodextrin were then added to form the dry matrix precursor solution. The resulting formulations maintained good stability during freeze-drying, accelerated storage, low-temperature hard water reconstitution, and application with intact proteins.

[0131] Example 1 used 15 parts trehalose, 3 parts betaine, 0.8 parts L-proline and 2.5 parts pullulan. Under the condition of reducing the ratio of pullulan and L-proline, the recovery rate of drying enzyme activity was 84.7%, and the Kp retention rate after storage at 40°C for 30 days was 95.8%. The results of Example 4 after scale-up were similar to those of Example 1, indicating that the feeding and freeze-drying process has the basis for scale-up implementation.

[0132] Comparative Example 1 used the same final components and amounts as Example 1, but added the small molecule stabilizing component and the polymer carrier at one time. Its dry enzyme activity recovery rate, Ap after storage and Kp under low temperature hard water conditions were significantly reduced. Therefore, the performance of the example cannot be attributed solely to the conventional parallel combination of trehalose, betaine, L-proline, pullulan and maltodextrin.

[0133] Comparative Example 2 did not undergo replenishment or replacement, and the high conductivity of the original enzyme solution caused a decline in the pre-protection process and the function after drying. In Comparative Example 3, after the polymer carrier was added first, the solution had already thickened before the addition of the small molecule stable component, resulting in insufficient pre-equilibration. In Comparative Example 4, the pre-equilibration and release check were cancelled, and the drying recovery rate and Kp also decreased. It can be seen that there is a continuous coordination relationship between the uniform state of the original enzyme solution, the order of adding stable components, and the intermediate state check.

[0134] Comparative Examples 5-7 were deficient in betaine, L-proline, or pullulan, respectively. The results were between those of the Examples and the main process comparative examples. Among them, the decrease caused by the lack of L-proline was less than that caused by the lack of betaine or pullulan, indicating that L-proline is a low-amount auxiliary component in this formulation.

[0135] Comparative Example 8 used a magnesium chloride and maltodextrin stabilization route, and its standard substrate activity retention rate was still relatively high, but the intact protein functional activity and Kp retention rate were lower than those of the Example. This result shows that in the evaluation of protein glutaminase solid dosage forms, simultaneous detection of standard substrate activity and intact protein functional activity can identify functional declines that are difficult to detect by relying solely on conventional standard enzyme activity.

[0136] This embodiment uses low DE-value maltodextrin as the main carrier, combined with a small amount of pullulan and small molecule stabilizing components, to form a stable formulation without using a high proportion of pullulan or a high amount of amino acids. Its key technical aspects include consistent batch-to-batch state of the original enzyme solution, pre-equilibration of the small molecule stabilizing system, subsequent addition of the polymer drying matrix, determination of the freeze-drying endpoint based on the product state, and simultaneous evaluation of the finished product's standard enzyme activity and intact protein functional activity.

[0137] The above are merely preferred embodiments of the present invention. Those skilled in the art can make appropriate adjustments to the excipient solution concentration, mixing time, freeze-drying shelf procedure, and standardized carrier dosage based on the source of protein glutaminase, equipment scale, and target enzyme activity without departing from the technical concept of the present invention. The above embodiments are used to illustrate the technical solution of the present invention and do not constitute a limitation on the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A highly stable protein glutaminase preparation, characterized in that, The formulation is a freeze-dried solid formulation containing protein glutaminase, wherein the protein glutaminase has the activity of catalyzing the hydrolysis of the amide group of the side chain of glutamine residue in a protein or polypeptide; Based on a total dry matter mass of 100 parts, the formulation comprises 10-20 parts protein glutaminase concentrate solids, 10-18 parts trehalose, 2-5 parts betaine, 0.3-1.5 parts L-proline, 1.5-4 parts pullulan, 0.2-1.0 parts citrate buffer, and the balance low DE value maltodextrin. The mass ratio of trehalose to betaine is 3:1 to 7:1, the mass ratio of betaine to L-proline is 2:1 to 8:1, the mass ratio of pullulan to low DE value maltodextrin is 1:15 to 1:35, and the DE value of the low DE value maltodextrin is 4 to 10. The water content of the preparation is 2.5% to 4.0%, and the water activity at 25°C is 0.18 to 0.

28. The preparation is prepared into a 1% (w / v) concentration reconstituted solution using purified water at 25°C. After the pH reading stabilizes, the pH of the reconstituted solution is 5.4 to 6.

2. The protein glutaminase concentrate solids include protein glutaminase and soluble protein components. The protein glutaminase concentrate solids are formed by removing water from an enzyme concentrate with a total solids concentration of 100-180 g / L and a protein glutaminase activity of 60-120 U / mL. The citrate buffer component is composed of citric acid and sodium citrate; In the preparation of the formulation, citrate buffer is used for replenishment and replacement to unify the total solids, pH and conductivity of the original enzyme solution. Trehalose and auxiliary stabilizing solutions containing betaine and L-proline are then added sequentially to allow the protein glutaminase to complete low-temperature pre-equilibration before the addition of the polymer carrier. The state of the pre-equilibration solution is judged by the correction OD600 increment and the soluble enzyme activity retention rate. Pullulan is added after the pre-equilibration is completed, and low DE value maltodextrin is added in batches in solution form.

2. The highly stable protein glutaminase preparation according to claim 1, characterized in that, The formulation is a freeze-dried powder obtained by sieving, wherein the freeze-dried powder passes through a 40-mesh sieve and is retained on an 80-mesh sieve.

3. The highly stable protein glutaminase preparation according to claim 1, characterized in that, Based on a total dry matter mass of 100 parts, the formulation comprises 12-18 parts protein glutaminase concentrate solids, 13-16 parts trehalose, 2.5-4 parts betaine, 0.5-1.0 parts L-proline, 2-3 parts pullulan, 0.3-0.8 parts citrate buffer, and the balance low DE value maltodextrin.

4. A method for preparing a highly stable protein glutaminase preparation according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1: The enzyme solution containing protein glutaminase was clarified and concentrated by ultrafiltration, and then replenished with citrate buffer to obtain an enzyme concentrate with a total solids concentration of 100-180 g / L, a protein glutaminase activity of 60-120 U / mL, a pH of 5.6-6.1, and a conductivity of 1.2-2.5 mS / cm at 25°C. S2: Add trehalose solution to enzyme concentrate at 4-10℃, mix, then add auxiliary stabilizing solution containing betaine, L-proline and citrate buffer components, and continue mixing for 20-40 min to obtain pre-equilibrium solution; S3: When the corrected OD600 increment of the pre-equilibration solution relative to the enzyme solution before feeding is not greater than 0.02, and the soluble enzyme activity retention rate of the pre-equilibration solution supernatant after centrifugation is not less than 95%, add pullulan polysaccharide solution to the pre-equilibration solution, mix, and then add low DE value maltodextrin solution in batches to obtain a dry matrix precursor solution with a total solids mass fraction of 26% to 30%. S4: The dry matrix precursor solution is degassed, pre-frozen, dried once and dried twice in sequence until the water content of the resulting freeze-dried solid preparation is 2.5% to 4.0% and the water activity at 25°C is 0.18 to 0.

28. S5: The freeze-dried solid preparation is subjected to low-shear crushing and sieving to obtain freeze-dried powder.

5. The method for preparing the highly stable protein glutaminase preparation according to claim 4, characterized in that, In S1, the fermentation supernatant containing protein glutaminase or the recombinant expression enzyme solution is kept at 4-10℃ and clarified by low-temperature centrifugation or microfiltration membrane with a pore size of 0.2-0.45μm, and then concentrated by ultrafiltration membrane with a molecular weight cutoff of 5-10kDa. The concentration of the citrate buffer is 5–10 mmol / L, and the pH is 5.6–6.

1.

6. The method for preparing the highly stable protein glutaminase preparation according to claim 4, characterized in that, In S2, trehalose is prepared into a trehalose solution with a mass fraction of 25% to 35%, cooled to 4 to 10°C, and then the enzyme concentrate is added within 10 to 20 minutes. After the addition is completed, the mixture is mixed for 10 to 20 minutes. A buffer solution containing betaine, L-proline, and citrate was prepared by dispersing the buffer group to form an auxiliary stabilizing solution with a mass fraction of 20%–35%. After cooling to 4–10°C, an enzyme solution containing trehalose was added within 10–20 minutes.

7. The method for preparing the highly stable protein glutaminase preparation according to claim 4, characterized in that, In S3, the enzyme solution before addition and the pre-equilibration solution were taken and diluted 10 times with a buffer solution having the same pH and conductivity as the corresponding sample. The absorbance at 600 nm was measured using a 1 cm path length cuvette. Blank correction was performed using an excipient solution containing the same proportions of trehalose, betaine, L-proline and citrate buffer components but without protein glutaminase to obtain the corrected OD600 increment. The pre-equilibrated solution was centrifuged at 4°C and 10,000×g for 10 min. The percentage of the total enzyme activity of the supernatant after volume correction to the total enzyme activity of the enzyme solution before feeding was taken as the soluble enzyme activity retention rate. The pullulan polysaccharide solution has a mass fraction of 5% to 10%, and is hydrated at 35 to 45°C and then cooled to no higher than 12°C. The low DE value maltodextrin solution has a mass fraction of 35% to 45%, is dissolved at 40 to 50°C, cooled to no higher than 12°C, and added in 2 to 4 batches.

8. The method for preparing the highly stable protein glutaminase preparation according to claim 4, characterized in that, In S4, the dry matrix precursor liquid is intermittently degassed under vacuum at 4-10℃ for 5-15 minutes, and then loaded into a tray with a material layer thickness of 8-12 mm. The core temperature of the material is reduced to no higher than -40℃ and maintained for no less than 1 hour. The product is dried once under 30-80 Pa conditions, with the shelf temperature gradually increased from -35℃ to -25℃, and the product temperature kept not higher than -20℃. The shelf temperature was then raised to 20–25°C, and a second drying process was carried out at 5–30 Pa for 4–8 hours.

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