Preparation process of freeze-dried nattokinase

Abstract

The invention particularly discloses a preparation process of freeze-dried nattokinase. The preparation process of the freeze-dried nattokinase comprises the following steps: S1, mixing a nattokinase stock solution with a composite freeze-drying protective agent to obtain a mixed solution; s2, performing pre-freezing treatment operation on the mixed solution twice to obtain a pre-frozen product; s3, carrying out sublimation treatment operation on the pre-frozen product at a sublimation rate of 0.1-0.5 g / h.cm < 2 >, a sublimation temperature of-30 DEG C to-10 DEG C and a sublimation vacuum degree of 20-25 Pa to obtain a sublimation treatment product; S4, carrying out desorption drying treatment operation on the sublimation treatment product at a drying temperature of 15-20 DEG C and a drying vacuum degree of 20-30 Pa to obtain a sublimation treatment product; freeze-drying the obtained nattokinase; wherein the composite freeze-drying protective additive comprises sugar, amino acid, an antioxidant and an excipient. The nattokinase freeze-drying agent has the advantage of improving the freeze-drying activity and storage stability of nattokinase.

Description

Technical Field

[0001] The present application relates to the technical field of natto processing, and in particular to a preparation process for freeze-drying nattokinase. Background Art

[0002] As a bioactive substance with important medicinal and health care values, nattokinase has broad application prospects in the fields of medicine, health care products, etc. However, it faces many challenges during processing, storage, and transportation. In particular, it is extremely sensitive to environmental factors (such as temperature, humidity, oxygen, etc.) and is prone to problems such as denaturation and inactivation, which greatly limits the effective utilization of nattokinase.

[0003] Freeze-drying technology is one of the effective means to solve the stability problems of bioactive substances. Currently, traditional nattokinase freeze-drying processes usually adopt fixed procedures and parameter settings, which will result in uneven freezing degrees during freeze-drying, thus affecting the quality and stability of the products. In order to improve the quality of freeze-dried products, a method of adding freeze-drying protectants is currently used to improve in terms of freeze-drying protectants. However, existing single protectants or simply combined protectants are difficult to comprehensively and effectively solve all problems of nattokinase during freeze-drying, and cannot completely solve the denaturation problem throughout the preparation process of nattokinase, which is not conducive to the improvement of the enzyme activity and storage stability of nattokinase freeze-dried products. Summary of the Invention

[0004] To solve the problems in the prior art, the present application provides a preparation process for freeze-drying nattokinase.

[0005] In a first aspect, the present application provides a preparation process for freeze-drying nattokinase, adopting the following technical solution:

[0006] A preparation process for freeze-drying nattokinase includes the following steps:

[0007] S1, mixing the nattokinase stock solution with a composite freeze-drying protectant to obtain a mixed solution;

[0008] S2, performing a pre-freezing treatment operation on the mixed solution. In the pre-freezing treatment operation, first cool down to a first freezing temperature point at a first temperature gradient and maintain the frozen state at the first freezing temperature point; then continue to cool down to a second freezing temperature point at a second temperature gradient and maintain the frozen state at the second freezing temperature point to obtain a pre-frozen product;

[0009] S3, performing a sublimation treatment operation on the pre-frozen product. The sublimation rate in the sublimation treatment operation is 0.1 - 0.5 g / h·cm 2 , the sublimation temperature is -30°C to -10°C, and the sublimation vacuum degree is 20 - 25 Pa to obtain the sublimation treatment product; as the sublimation rate increases, the sublimation temperature and the sublimation vacuum degree gradually increase;

[0010] S4. Perform an analytical drying operation on the sublimation treatment product. The drying temperature in the analytical drying operation is 15 - 20°C, and the drying vacuum degree is 20 - 30 Pa to obtain freeze-dried nattokinase. As the water content in the sublimation treatment product decreases, the drying temperature gradually increases, and the drying vacuum degree gradually decreases.

[0011] Among them, the composite freeze-drying protectant includes sugar, amino acids, antioxidants, and excipients; the sugar includes at least three of sophorolipid, sucrose, lactose, and skim milk powder; the amino acids include at least one of arginine, serine, and glycine; the antioxidants include at least three of tea polyphenols, phytic acid, rosemary extract, tert-butylhydroquinone, dibutylhydroxytoluene, and propyl gallate; the excipients include at least one of glycerol and gelatin.

[0012] In this application, by controlling the use of the composite freeze-drying protectant and combining with the processing technology of this application, first, through two pre-freezing treatments, the first pre-freezing treatment can initially freeze the water on the surface of the sample to form an "ice shell" to protect the internal structure, and the second pre-freezing treatment then freezes the internal water. Under the action of the composite protectant, the occurrence of ice crystals in the nattokinase stock solution can be significantly reduced, and a stable molecular structure can be formed in the system, thus helping to improve the stability of the enzyme preparation. Secondly, by setting the sublimation temperature and sublimation vacuum degree in the sublimation treatment operation to gradually increase as the sublimation rate increases, it can ensure that the mixed solution sublimates at a relatively stable speed in the initial stage of the sublimation treatment operation, avoiding damage to the activity of the mixed solution caused by too high sublimation temperature and too high sublimation vacuum degree. In the later stage of the sublimation treatment operation, as the sublimation rate gradually rises to the upper limit, by increasing the sublimation temperature and sublimation vacuum degree, the situation of further increasing the sublimation rate can be inhibited, thereby ensuring the stability of the mixed solution throughout the sublimation treatment operation, and further ensuring the enzyme activity of the target product. Finally, as the analytical drying process progresses, the water content in the sublimation treatment product gradually decreases. In this process, timely adjusting the drying temperature to gradually increase and the drying vacuum degree to gradually decrease can significantly improve the drying uniformity of the sublimation treatment product. Moreover, under the action of the composite protectant, it can ensure the charge stable state of the protein and prevent protein aggregation and denaturation caused by the charge change of the protein with temperature changes during the sublimation and analytical drying operations. By using the composite freeze-drying protectant and cooperating with the freeze-drying preparation process in this application, the initial activity and storage activity of the freeze-dried nattokinase can be significantly improved.

[0013] Preferably, after the nattokinase stock solution is mixed with the composite lyoprotectant, it is stirred at a speed of 100-200 rpm to obtain the mixed solution. By stirring within the above rotation speed range, excessive bubbles can be avoided, the possibility of the hydrophobic groups of proteins being exposed to the non-polar environment at the gas-liquid interface can be reduced, and the forces maintaining the stability of the protein structure can be prevented from being damaged, thereby maintaining the structural stability of the active center of the enzyme and avoiding the reduction of the activity of the nattokinase stock solution.

[0014] Preferably, the sugar includes sophorolipid, sucrose and lactose, and the mass ratio of sophorolipid, sucrose and lactose is 0.1:3:10; the antioxidant includes tea polyphenols, phytic acid and tert-butylhydroquinone, and the mass ratio of tea polyphenols, phytic acid and tert-butylhydroquinone is 5:3:1.

[0015] Preferably, in S1, the mass proportion of the composite lyoprotectant in the mixed solution is 18%-23%.

[0016] Preferably, the mass ratio of the sugar, the amino acid, the antioxidant and the excipient in the composite lyoprotectant is 10-25:1-3:1-3:1-5.

[0017] By adjusting the mass proportion of each component in the composite lyoprotectant, on the one hand, the structural stability of the nattokinase stock solution at each stage during the processing can be significantly improved to ensure the enzyme activity, and on the other hand, the synergistic cooperation of each component can be significantly improved to increase the initial enzyme activity and storage enzyme activity of the prepared nattokinase freeze-dried product.

[0018] Preferably, in S1, the first temperature gradient is 2-2.5°C / min, the first freezing temperature point is -10°C, and freezing is maintained for 1-2 h at the first freezing temperature point; the second temperature gradient is 1-1.5°C / min, the second temperature point is -40°C, and freezing is maintained for 4-6 h at the second freezing temperature point.

[0019] By selecting appropriate first temperature gradient, first freezing temperature point, second temperature gradient and second freezing temperature point, it can be ensured that the free water in the sample can be fully frozen and no ice crystals will be generated to damage the enzyme structure, which helps to improve the enzyme activity.

[0020] Preferably, when the temperature is close to the second freezing temperature point, the freezing power is adjusted to 0.8-1.2 kW.

[0021] Preferably, during the pre-freezing treatment operation, when the temperature is between -39°C and -40°C, the freezing power is adjusted to 0.6-0.9 kW.

[0022] By further fine-tuning the freezing power and freezing at the above temperature, the degree of solidification in the pre-frozen product can be further improved, and damage to the enzyme structure caused by ice crystal components in the pre-frozen product can be avoided.

[0023] Preferably, in S3, during the process where the sublimation rate rises from 0.1 g / h·cm 2 to 0.3 g / h·cm 2 , the sublimation temperature rises from -30°C to -20°C, and the sublimation vacuum degree rises from 20 Pa to 22 Pa; during the process where the sublimation rate rises from 0.3 g / h·cm 2 to 0.5 g / h·cm 2 , the sublimation temperature rises from -20°C to -10°C, and the sublimation vacuum degree rises from 22 Pa to 25 Pa.

[0024] By using a lower sublimation temperature in the initial stage, nattokinase can start to sublime at a relatively stable rate in the initial stage, avoiding damage to nattokinase activity caused by too high sublimation temperature or too fast sublimation rate and being unable to be effectively controlled; further, as the sublimation rate gradually increases, appropriately increasing the sublimation temperature and the sublimation vacuum degree can slow down the sublimation speed and make the sublimation rate return to a more ideal range; thus ensuring that the nattokinase stock solution completes the sublimation drying process at a stable sublimation rate and guaranteeing the enzyme activity of freeze-dried nattokinase.

[0025] Preferably, when the sublimation vacuum degree in S3 remains stable within 3 - 4 h, the sublimation treatment operation ends.

[0026] Preferably, in S4, during the process where the water content in the sublimation treatment product drops from 10% to 6%, the drying temperature rises from 15°C to 18°C, and the drying vacuum degree drops from 30 Pa to 25 Pa; during the process where the water content in the sublimation treatment product drops from 6% to 3%, the drying temperature rises from 18°C to 20°C, and the drying vacuum degree drops from 25 Pa to 20 Pa.

[0027] In the stage with relatively high water content, lower temperature and higher vacuum degree can avoid the inactivation of nattokinase due to too high temperature or too rapid pressure change while ensuring the drying efficiency. This is because, as the water content decreases, appropriately increasing the temperature can accelerate the sublimation and evaporation speed of water, and reducing the vacuum degree can balance the influence brought by the increase in temperature to a certain extent, making the drying process smoother; at the same time, such adjustment can improve the drying efficiency on the premise of ensuring the activity of nattokinase; as the desorption drying process progresses, since the water content in the sublimation treatment product is already relatively low and nattokinase is relatively stable, appropriately increasing the temperature and reducing the vacuum degree can further remove the residual water, making the product reach the ideal drying degree and improving the storage activity of freeze-dried nattokinase.

[0028] Preferably, when the quality of the sublimation treatment product remains stable within 0.8 - 1.2 h in S4, the analytical drying treatment operation ends.

[0029] In a second aspect, the present application provides a nattokinase freeze-drying, adopting the following technical solution:

[0030] A nattokinase freeze-drying is prepared by using the process described above. Description of the Drawings

[0031] Figure 1 It is the determination of the enzyme activity of the nattokinase freeze-drying prepared in Example 1, Examples 4 - 5 and Comparative Example 1 of the present application.

[0032] Figure 2 It is the determination of the enzyme activity of the nattokinase freeze-drying prepared in Examples 1 - 2 and Comparative Example 2 of the present application.

[0033] Figure 3 It is the determination of the enzyme activity of the nattokinase freeze-drying prepared in Example 1, Example 3 and Comparative Example 3 of the present application.

[0034] Figure 4 It is the determination of the enzyme activity of the nattokinase freeze-drying prepared in Example 1 and Comparative Examples 4 - 7 of the present application.

[0035] Figure 5 It is the determination of the enzyme activity of the nattokinase freeze-drying prepared in Example 1 and Example 6 of the present application.

[0036] Figure 6 It is the determination of the enzyme activity of the nattokinase freeze-drying prepared in Example 1 and Examples 7 - 10 of the present application. Detailed Embodiments

[0037] For better understanding and implementation, the technical solutions of the present application will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all of the embodiments.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs. The terms used in the description of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0039] Unless otherwise specified, all numerical values expressing the amounts of components, reaction conditions, etc. used in the description and claims are understood to be modified by the term "about". Therefore, unless otherwise indicated, the numerical parameters set forth herein are approximate values that can vary as required to obtain the desired performance.

[0040] As used herein, "and / or" means one or all of the recited elements.

[0041] As used herein, "comprising" and "including" cover the case where only the recited elements are present and the case where there are other unrecited elements in addition to the recited elements.

[0042] All percentages in this application are by weight, unless otherwise specified.

[0043] Unless otherwise specified, the articles "a", "an", "one" and "the" as used in this specification are intended to include "at least one" or "one or more". For example, "a component" means one or more components, so more than one component may be contemplated and may be employed or used in the implementation of the described embodiments.

[0044] Example 1

[0045] A nattokinase freeze-dried product is prepared by the following process:

[0046] S1. Mix the nattokinase stock solution with a composite lyoprotectant and stir at a speed of 150 rpm to obtain a mixed solution. The mass ratio of the composite lyoprotectant in the mixed solution is 21%; the composite lyoprotectant includes sugar (the mass ratio of sophorolipid, sucrose and lactose is 0.1:3:10), amino acid (arginine), antioxidant (the mass ratio of tea polyphenols, phytic acid and tert-butylhydroquinone is 5:3:1), and excipient (gelatin) with a mass ratio of 15:1.5:1.5:3.

[0047] S2. Perform a pre-freezing operation on the above mixed solution. In the pre-freezing operation, first cool down at a cooling rate of 2 °C / min to -10 °C and maintain the frozen state at -10 °C for 1 h; then continue to cool down at a cooling rate of 1 °C / min to -40 °C, and adjust the freezing power to 0.8 kW during the process of the temperature dropping from -39 °C to -40 °C, and maintain the frozen state at -40 °C for 5 h; obtain a pre-frozen product.

[0048] S3. Perform a sublimation operation on the above pre-frozen product. During the process of the sublimation rate rising from 0.1 g / h·cm 2 to 0.3 g / h·cm 2 , the sublimation temperature rises from -30 °C to -20 °C and the sublimation vacuum degree rises from 20 Pa to 22 Pa; during the process of the sublimation rate rising from 0.3 g / h·cm 2 to 0.5 g / h·cm 2 , the sublimation temperature rises from -20 °C to -10 °C and the sublimation vacuum degree rises from 22 Pa to 25 Pa; when the sublimation vacuum degree remains stable within 4 h, the sublimation operation ends.

[0049] S4. Perform analytical drying treatment on the above sublimation treatment product. During the process of reducing the water content in the sublimation treatment product from 10% to 6%, the drying temperature rises from 15°C to 18°C, and the drying vacuum degree drops from 30 Pa to 25 Pa; during the process of reducing the water content in the sublimation treatment product from 6% to 3%, the drying temperature rises from 18°C to 20°C, and the drying vacuum degree drops from 25 Pa to 20 Pa; when the sublimation vacuum degree remains stable within 4 h and the mass of the sublimation treatment product remains stable within 1 h, the analytical drying treatment operation ends to obtain nattokinase freeze-dried. The test results of the nattokinase freeze-dried enzyme activity prepared in this example are as Figure 1 shown.

[0050] Example 2

[0051] The difference between this example and Example 1 is that the sugar in the composite freeze-drying protectant is obtained by mixing sophorolipid, sucrose, and lactose according to a mass ratio of 0.3:3:1; other steps and parameter settings are the same as those in Example 1. The test results of the nattokinase freeze-dried enzyme activity prepared in this example are as Figure 2 shown.

[0052] Example 3

[0053] The difference between this example and Example 1 is that the mass ratio of the composite freeze-drying protectant in the mixed solution is 21%; the composite freeze-drying protectant includes sugar (the mass ratio of sophorolipid, sucrose, and lactose is 0.1:3:10), amino acid (arginine), antioxidant (the mass ratio of tea polyphenols, phytic acid, and tert-butylhydroquinone is 5:3:1), and excipient (gelatin) with a mass ratio of 15:1.5:5:3; other steps and parameter settings are the same as those in Example 1. The test results of the nattokinase freeze-dried enzyme activity prepared in this example are as Figure 1 shown.

[0054] Example 4

[0055] A nattokinase freeze-dried product is prepared by the following process:

[0056] S1. Mix the nattokinase stock solution with the composite freeze-drying protectant and stir at a speed of 100 rpm to obtain a mixed solution. The mass ratio of the composite freeze-drying protectant in the mixed solution is 18%; the composite freeze-drying protectant includes sugar (the mass ratio of sophorolipid, lactose, and skim milk powder is 0.1:3:10), amino acid (serine), antioxidant (the mass ratio of tea polyphenols, phytic acid, and rosemary extract is 5:3:1), and excipient (gelatin) with a mass ratio of 25:3:3:5;

[0057] S2. Perform a pre-freezing operation on the above mixed solution. In the pre-freezing operation, first cool down at a temperature reduction gradient of 2 °C / min to -10 °C, and maintain the frozen state at -10 °C for 1 h; then continue to cool down at a temperature reduction gradient of 1 °C / min to -40 °C, and adjust the freezing power to 0.6 kW during the process of the temperature dropping from -39 °C to -40 °C, and maintain the frozen state at -40 °C for 5 h to obtain a pre-frozen product;

[0058] S3. Perform a sublimation operation on the above pre-frozen product. During the process where the sublimation rate rises from 0.1 g / h·cm 2 to 0.3 g / h·cm 2 , the sublimation temperature rises from -30 °C to -20 °C, and the sublimation vacuum degree rises from 20 Pa to 22 Pa; during the process where the sublimation rate rises from 0.3 g / h·cm 2 to 0.5 g / h·cm 2 , the sublimation temperature rises from -20 °C to -10 °C, and the sublimation vacuum degree rises from 22 Pa to 25 Pa; when the sublimation vacuum degree remains stable within 4 h, the sublimation operation ends;

[0059] S4. Perform an analytical drying operation on the above sublimation-treated product. During the process where the water content in the sublimation-treated product drops from 10% to 6%, the drying temperature rises from 15 °C to 18 °C, and the drying vacuum degree drops from 30 Pa to 25 Pa; during the process where the water content in the sublimation-treated product drops from 6% to 3%, the drying temperature rises from 18 °C to 20 °C, and the drying vacuum degree drops from 25 Pa to 20 Pa; when the sublimation vacuum degree remains stable within 4 h and the mass of the sublimation-treated product remains stable within 1.2 h, the analytical drying operation ends to obtain nattokinase freeze-dried powder.

[0060] Example 5

[0061] A kind of nattokinase freeze-drying is prepared by the following process:

[0062] S1. Mix the nattokinase stock solution with a composite freeze-drying protectant and stir at a speed of 200 rpm to obtain a mixed solution. The mass proportion of the composite freeze-drying protectant in the mixed solution is 23%; the composite freeze-drying protectant includes sugar (the mass ratio of sucrose, lactose, and skim milk powder is 0.1:3:10), amino acid (glycine), antioxidant (the mass ratio of tert-butylhydroquinone, dibutylhydroxytoluene, and propyl gallate is 5:3:1), and excipient (glycerol) with a mass ratio of 10:1:1:1;

[0063] S2. Perform a pre-freezing treatment operation on the above mixed solution. In the pre-freezing treatment operation, first cool down at a cooling rate of 2 °C / min to -10 °C, and maintain the frozen state at -10 °C for 1 h; then continue to cool down at a cooling rate of 1 °C / min to -40 °C, and adjust the freezing power to 0.6 kW during the process of the temperature dropping from -39 °C to -40 °C, and maintain the frozen state at -40 °C for 5 h to obtain a pre-frozen product;

[0064] S3. Perform a sublimation treatment operation on the above pre-frozen product. During the process where the sublimation rate rises from 0.1 g / h·cm 2 to 0.3 g / h·cm 2 , the sublimation temperature rises from -30 °C to -20 °C, and the sublimation vacuum degree rises from 20 Pa to 22 Pa; during the process where the sublimation rate rises from 0.3 g / h·cm 2 to 0.5 g / h·cm 2 , the sublimation temperature rises from -20 °C to -10 °C, and the sublimation vacuum degree rises from 22 Pa to 25 Pa; when the sublimation vacuum degree remains stable within 4 h, the sublimation treatment operation ends;

[0065] S4. Perform an analytical drying treatment operation on the above sublimation treatment product. During the process where the water content in the sublimation treatment product drops from 10% to 6%, the drying temperature rises from 15 °C to 18 °C, and the drying vacuum degree drops from 30 Pa to 25 Pa; during the process where the water content in the sublimation treatment product drops from 6% to 3%, the drying temperature rises from 18 °C to 20 °C, and the drying vacuum degree drops from 25 Pa to 20 Pa; when the sublimation vacuum degree remains stable within 4 h and the mass of the sublimation treatment product remains stable within 0.8 h, the analytical drying treatment operation ends to obtain nattokinase freeze-dried powder.

[0066] Example 6

[0067] The difference between this example and Example 1 is that the mass ratio of sugar, amino acid, antioxidant, and excipient in the composite freeze-drying protectant is 30:0.5:0.5:6; other parameter and step settings are the same as those in Example 1. The test results of the freeze-dried enzyme activity of nattokinase prepared in this example are as Figure 5 shown.

[0068] Example 7

[0069] The difference between this example and Example 1 is that the pre-freezing treatment operation in S2 is as follows: in the pre-freezing treatment operation, first cool down at a cooling rate of 3 °C / min to -10 °C, and maintain the frozen state at -10 °C for 1 h; then continue to cool down at a cooling rate of 2 °C / min to -40 °C, and maintain the frozen state at -40 °C for 5 h to obtain a pre-frozen product; other step and parameter settings are the same as those in Example 1. The test results of the freeze-dried enzyme activity of nattokinase prepared in this example are as Figure 6 shown.

[0070] Example 8

[0071] The difference between this example and Example 1 lies in that the pre-freezing treatment operation in S2 is as follows: in the pre-freezing treatment operation, first cool down to -20°C at a cooling rate of 2°C / min, maintain the frozen state at -20°C for 0.5 h; then continue to cool down to -40°C at a cooling rate of 1°C / min, and maintain the frozen state at -40°C for 5 h to obtain the pre-frozen product; other steps and parameter settings are the same as those in Example 1. The test results of the lyophilized enzyme activity of nattokinase prepared in this example are as Figure 6 shown.

[0072] Example 9

[0073] The difference between this example and Example 1 lies in that the sublimation treatment operation in S3 is as follows:

[0074] During the process of the sublimation rate rising from 0.1 g / h·cm 2 to 0.3 g / h·cm 2 , the sublimation temperature rises from -30°C to -15°C and the sublimation vacuum degree rises from 20 Pa to 21 Pa; during the process of the sublimation rate rising from 0.3 g / h·cm 2 to 0.5 g / h·cm 2 , the sublimation temperature rises from -15°C to -10°C and the sublimation vacuum degree rises from 21 Pa to 25 Pa; when the sublimation vacuum degree remains stable within 4 h, the sublimation treatment operation ends; other steps and parameter settings are the same as those in Example 1. The test results of the lyophilized enzyme activity of nattokinase prepared in this example are as Figure 6 shown.

[0075] Example 10

[0076] The difference between this example and Example 1 lies in that the analytical drying treatment operation in S4 is as follows: during the process of the water content in the sublimation treatment product decreasing from 10% to 6%, the drying temperature rises from 15°C to 16°C and the drying vacuum degree drops from 30 Pa to 28 Pa; during the process of the water content in the sublimation treatment product decreasing from 6% to 3%, the drying temperature rises from 16°C to 20°C and the drying vacuum degree drops from 28 Pa to 20 Pa; when the sublimation vacuum degree remains stable within 4 h and the mass of the sublimation treatment product remains stable within 1 h, the analytical drying treatment operation ends to obtain nattokinase freeze-dried; other steps and parameter settings are the same as those in Example 1. The test results of the lyophilized enzyme activity of nattokinase prepared in this example are as Figure 6 shown.

[0077] Comparative Example 1

[0078] The difference between this comparative example and Example 1 lies in that the process during the preparation of freeze-dried nattokinase is the traditional freeze-drying process. The specific steps are as follows: After mixing the nattokinase stock solution with the cryoprotectant, it is placed in a -40°C refrigerator for pre-freezing for 12 hours to obtain a pre-frozen product; the pre-frozen product is subjected to sublimation drying at -25°C and a vacuum degree of 20 Pa for 14 hours to obtain a sublimation-treated product; the sublimation-treated product is placed at 30°C for desorption drying for 2 hours to obtain freeze-dried nattokinase;

[0079] All other steps and parameter settings are the same as those in Example 1. The test results of the enzyme activity of the freeze-dried nattokinase prepared in this comparative example are as Figure 1 shown.

[0080] Comparative Example 2

[0081] The difference between this comparative example and Example 1 lies in that the sugar in the composite cryoprotectant is only sucrose; all other steps and parameter settings are the same as those in Example 1. The test results of the enzyme activity of the freeze-dried nattokinase prepared in this comparative example are as Figure 2 shown.

[0082] Comparative Example 3

[0083] The difference between this comparative example and Example 1 lies in that the antioxidant in the composite cryoprotectant is only tea polyphenols; all other steps and parameter settings are the same as those in Example 1. The test results of the enzyme activity of the freeze-dried nattokinase prepared in this comparative example are as Figure 3 shown.

[0084] Comparative Example 4

[0085] The difference between this comparative example and Example 1 lies in that the composite cryoprotectant does not contain amino acids, antioxidants, and excipients, and only sugar (the mass ratio of sophorolipid, sucrose, and lactose is 0.1:3:10) is used as the cryoprotectant; all other steps and parameter settings are the same as those in Example 1. The test results of the enzyme activity of the freeze-dried nattokinase prepared in this comparative example are as Figure 4 shown.

[0086] Comparative Example 5

[0087] The difference between this comparative example and Example 1 lies in that the composite cryoprotectant does not contain sugar, antioxidants, and excipients, and the amino acid is lysine; all other steps and parameter settings are the same as those in Example 1. The test results of the enzyme activity of the freeze-dried nattokinase prepared in this comparative example are as Figure 4 shown.

[0088] Comparative Example 6

[0089] The difference between this comparative example and Example 1 is that the composite lyophilization protectant does not contain sugars, amino acids, and excipients, and only uses antioxidants (the mass ratio of tea polyphenols, phytic acid, and tert-butylhydroquinone is 5:3:1) as the lyophilization protectant; other steps and parameter settings are the same as those in Example 1. The test results of the lyophilized enzyme activity of nattokinase prepared in this comparative example are as Figure 4 shown.

[0090] Comparative Example 7

[0091] The difference between this comparative example and Example 1 is that the composite lyophilization protectant does not contain sugars, amino acid antioxidants, and only uses an excipient (gelatin) as the lyophilization protectant; other steps and parameter settings are the same as those in Example 1. The test results of the lyophilized enzyme activity of nattokinase prepared in this comparative example are as Figure 4 shown.

[0092] Test method

[0093] I. Method for determining the enzyme activity of nattokinase lyophilized preparation

[0094] The enzyme activity of the nattokinase lyophilized preparation was determined using ultraviolet spectrophotometry, and the specific method is as follows:

[0095] Add a certain proportion of Tris-HCl buffer solution and fibrinogen solution to a test tube, and incubate in a water bath at 37°C for 5 min; add 0.1 mL of thrombin, and incubate in a water bath at 37°C for 10 min to form an artificial thrombus; add 100 μL of the sample to be tested, and incubate at 37°C for 60 min; add trichloroacetic acid and let stand for 20 min to terminate the reaction. After centrifuging at 10,000 r / min for 10 min, take the supernatant and measure the absorbance at a wavelength of 275 nm. Definition of enzyme activity (FU / g): The amount of enzyme required to increase the absorbance by 0.01 per minute at 275 nm is defined as 1 unit of fibrinolytic enzyme activity.

[0096] Table 1

[0097]

[0098] Combined with Example 1, 4-5, Comparative Example 1, Table 1 and Figure 1, it can be seen that the initial enzyme activity of nattokinase fermentation broth is 20,947 FU / g. The initial enzyme activity of nattokinase lyophilized product prepared by the lyophilization process of this application reaches 18,300.92 FU / g, while the initial enzyme activity of nattokinase lyophilized product prepared by the traditional lyophilization process (Comparative Example 1) is only 12,006.24 FU / g. The decline in the initial enzyme activity of nattokinase lyophilized product prepared by the traditional lyophilization process is relatively large compared to that of the nattokinase lyophilized product prepared by the lyophilization process of this application, indicating that the lyophilization process of this application is beneficial to improving the stability of nattokinase lyophilized preparation, can more effectively maintain the storage enzyme activity of nattokinase lyophilized product, has significant advantages in the production and processing of nattokinase lyophilized product and product storage, and can ensure the quality and efficacy of the product.

[0099] Combined with Examples 1-2, Comparative Example 2, Table 1 and Figure 2 , it can be seen that the effects of sugars with different concentrations and different combinations on enzyme activity are different. In Example 1, when the mass ratio of sophorolipid, sucrose and lactose is 0.1:3:10, the effect of maintaining enzyme activity is the best. After storing the freeze-dried powder for 30 days, the enzyme activity of nattokinase lyophilized product remains at 16,225.68 FU / g, still maintaining a relatively high enzyme activity. In Example 2, the dosage of sophorolipid increases, and the initial enzyme activity and storage enzyme activity of nattokinase lyophilized product decrease to some extent. However, the enzyme activity of nattokinase lyophilized product in Example 2 is still higher than that of nattokinase lyophilized product in Comparative Example 2 using single sucrose, indicating that the addition of sophorolipid can reduce the surface tension of the freeze-drying protective solution, make components such as sugar in the freeze-drying protective agent better dispersed in the solution, avoid the situation of too high or too low local sugar concentration, and at the same time play a synergistic role with sugar to form a glassy state during the freeze-drying process to protect bioactive substances, form a more stable protective film on its surface, and reduce the damage of factors such as oxygen and moisture to active substances during the processing of nattokinase lyophilization process, so as to better maintain the initial activity and storage stability of nattokinase lyophilized product.

[0100] Although sophorolipid in sugar can be used as a surfactant to improve the wettability of the composite freeze-drying protectant and ensure that nattokinase lyophilized product can quickly restore its active ingredients, it can be seen from the comparison of Examples 1-2 that with the increase of the dosage of sophorolipid, the initial enzyme activity and storage enzyme activity of nattokinase lyophilized product both show a downward trend. Therefore, the dosage of sophorolipid should be kept within a more appropriate range, which can not only avoid the excessive interaction between the hydrophobic groups of the added excessive sophorolipid and the hydrophobic region of the enzyme protein, thus changing the conformation of the enzyme protein, resulting in the change of the structure of the enzyme active center and the inability of the substrate to bind normally, leading to the decrease of enzyme activity, but also avoid the formation of micelle-like structures by excessive sophorolipid in the solution, thereby changing the microenvironment around the enzyme protein and causing the decrease of enzyme activity due to the deviation of the pH and ionic strength around lysozyme from its optimal conditions.

[0101] Combined with Example 1, Example 3, Comparative Example 3, Table 1 and Figure 3 , it can be seen that the effects of antioxidants with different concentrations and different compositions on enzyme activity during the freeze-drying process are different. By adding antioxidants to the composite freeze-drying protectant, the antioxidants themselves are oxidized to prevent the oxidation of enzyme proteins, thereby maintaining the integrity of the enzyme protein structure and improving the stability of the enzyme preparation. The antioxidant composed of tea polyphenols, phytic acid and tert-butylhydroquinone can also play a synergistic protective role with sugars and amino acids. This synergistic effect can more effectively protect the enzyme preparation and improve its stability during the freeze-drying preparation and storage of nattokinase. Comparing Example 1 and Comparative Example 3, the use of a single type of antioxidant (only tea polyphenols) will significantly reduce the effect of the antioxidant in increasing enzyme activity. The stored enzyme activity of freeze-dried nattokinase in Example 1 is 16225.68 FU / g, which is much higher than the stored enzyme activity of freeze-dried nattokinase in Comparative Example 3, which is 13945.4 FU / g.

[0102] At the same time, since the antioxidant capacity of the combined antioxidant is greater than that of the single antioxidant, adding a small amount of antioxidant to the composite freeze-drying protectant will produce a good antioxidant effect. However, with the increase in the amount of antioxidant used, it may also lead to a decrease in enzyme activity. As shown in Example 1 and Example 3, after 30 days of storage, the enzyme activity of freeze-dried nattokinase in Example 1 is 16225.68 FU / g, while the enzyme activity of freeze-dried nattokinase in Example 3 drops to 15861.64 FU / g. This may be because an increase in the amount of antioxidant used will cause a change in the structure of the enzyme protein. This structural change is similar to destroying the support structure of a building, changing the conformation of the active center of the enzyme, thereby reducing the activity of the enzyme. In addition, a higher amount of antioxidant used may also directly react with the enzyme protein, interfering with the normal catalytic mechanism of the enzyme, and thus reducing the enzyme activity; however, it should be noted that although the amount of antioxidant used in Example 3 is higher than that in Comparative Example 3, the enzyme activity of freeze-dried nattokinase in Example 3 is still higher than that of freeze-dried nattokinase in Comparative Example 3, indicating that the combined antioxidant can better maintain the enzyme activity of freeze-dried nattokinase compared to the single antioxidant.

[0103] Combined with Example 1, Comparative Examples 4-7, Table 1 and Figure 4, it can be seen that there are significant differences in the effects of the composite lyophilization protectant of the present application on enzyme activity compared with the lyophilization protectant of a single component, that is, the initial enzyme activity and the stored enzyme activity corresponding to the lyophilization protectant of a single component are significantly lower than the initial enzyme activity and the stored enzyme activity corresponding to the composite lyophilization protectant. Taking the enzyme activity changes of Comparative Example 4 and Comparative Example 7 as an example: In Comparative Example 4, sucrose was used alone as the lyophilization protectant, and the enzyme activity of nattokinase lyophilization decreased by 3552.76 FU / g within 30 days of storage, while the enzyme activity of nattokinase lyophilization using the composite lyophilization protectant in Example 1 decreased by only 2075.24 FU / g within 30 days of storage; this shows that using only sugar as the lyophilization protectant is difficult to continuously and stably maintain the activity of the enzyme preparation during long-term storage, while using the composite lyophilization protectant can, through the synergistic cooperation of different components, resist the influence of various external factors on the stability of the enzyme preparation and extend the storage life of the enzyme preparation. When gelatin was used alone as the lyophilization protectant in Comparative Example 7, the initial enzyme activity of nattokinase decreased to 13870.04 FU / g, while the initial enzyme activity of the kinase lyophilization using the composite lyophilization protectant in Example 1 reached 18300.92 FU / g, which was significantly higher than the enzyme activity of Comparative Example 7; this is because although gelatin can to a certain extent prevent the enzyme protein from being damaged due to ice crystal formation during the freezing process, it cannot effectively solve problems such as dehydration and denaturation of the enzyme protein during the lyophilization preparation process. However, after using the composite lyophilization protectant, sugar can replace water molecules to form hydrogen bonds with the enzyme protein during the drying process to maintain the structural stability of the protein; therefore, by utilizing the synergistic effect between the components in the composite lyophilization protectant, the initial enzyme activity and the stored enzyme activity of nattokinase lyophilization can be improved.

[0104] Combined with Example 1, Example 6, Table 1 and Figure 5, it can be seen that the proportion of each component of the composite lyophilization protectant of the present application has a certain degree of influence on the enzyme activity. Compared with the composite lyophilization protectant used in Example 1, the proportion of the components of sugar and excipient in the composite lyophilization protectant used in Example 6 increases, while the proportion of the components of antioxidant and amino acid decreases. At this time, the initial activity of the nattokinase lyophilized product prepared in Example 6 is 2317.52 FU / g lower than that of the nattokinase lyophilized product prepared in Example 1, and the storage activity decreases by 2683.88 FU / g. This is because as the sugar concentration in the composite lyophilization protectant increases, the osmotic pressure of the nattokinase solution will change, resulting in the dehydration of nattokinase and affecting the flexibility and activity of enzyme molecules; at the same time, as the concentration of the excipient in the composite lyophilization protectant increases, the viscosity of the solution increases, and this high-viscosity environment will limit the diffusion movement of nattokinase and substrate molecules, reducing the effective collision frequency between the substrate and the enzyme, making it difficult for the enzyme-catalyzed reaction to proceed, and thus leading to a decrease in enzyme activity; while amino acids have a protective effect on the amino acid residues of nattokinase. When the amino acid concentration in the composite lyophilization protectant decreases, the amino acid residues of nattokinase are more vulnerable to external factors, changing the structure of the enzyme active center, resulting in a decrease in the binding ability and catalytic ability of the enzyme to the substrate and a decrease in enzyme activity; at the same time, when the antioxidant concentration in the composite lyophilization protectant decreases, nattokinase is more vulnerable to oxidative damage, which may inactivate the enzyme protein containing sulfhydryl groups or cause reactions such as cross-linking of protein molecules, thereby changing the structure and function of the enzyme and leading to a decrease in enzyme activity.

[0105] Therefore, by selecting the components of the composite lyophilization protectant with appropriate matching effects, the damage to enzyme activity caused by the external environment and the composite lyophilization protectant itself can be reduced, thereby maintaining the initial enzyme activity and storage enzyme activity of nattokinase.

[0106] Combined with Example 1, Examples 7-10, Table 1 and Figure 6, can be seen that the freeze-dried preparation technology of nattokinase has a certain degree of influence on enzyme activity.With embodiment 7 and 8 as example, in the pre-freeze stage, when the temperature-falling gradient rate is too fast, sugar may not be able to form effective protective structure around nattokinase in time, cause the partial enzyme molecule to be directly exposed to under the mechanical damage of ice crystal in ice crystal formation process, affect the integrity of its active center, and then reduce initial enzyme activity and storage enzyme activity.With embodiment 9 as example, in the sublimation drying stage, temperature raising can accelerate the sublimation of water molecule, but nattokinase is a protein, and its structure is more sensitive to temperature, therefore, water molecule can also increase the risk of nattokinase thermal denaturation when being accelerated distillation, cause the active center structure of enzyme to be destroyed, and the activity of enzyme reduces. Taking Example 10 as an example, during the desorption and drying stage, a decrease in temperature will prolong the desorption and drying process, the antioxidants may be exhausted, and the amino acids may also be detached from the enzyme molecules, causing the nattokinase molecules to lose protection and the enzyme activity to decrease; at the same time, a higher vacuum degree may make the structure of the gelatin loose because the gas inside it is extracted, resulting in a decrease in its ability to wrap the nattokinase molecules and a decrease in enzyme activity.

[0107] The freeze-dried preparation technology of nattokinase changes, and can cause enzyme activity to have a certain degree of reduction.Wherein, the nattokinase activity in embodiment 9 and embodiment 10 is larger than the reduction degree of nattokinase activity in embodiment 1, and initial enzyme activity is respectively reduced by 2262.8FU / g, 2343.48FU / g compared to embodiment 1, and storage enzyme activity is respectively reduced by 2228.04FU / g, 2246.16FU / g compared to embodiment 1, and this is similar to the influence degree of lyophilization protectant component to enzyme activity.Therefore, suitable preparation process conditions, are more conducive to the maintenance of nattokinase enzyme activity.

[0108] The above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solutions of the present application can be modified or replaced by equivalents, but these modifications or replacements are all within the scope of protection of the present application.

Claims

1. A preparation process for freeze-drying nattokinase, characterized in that: It includes the following steps: S1. Mix nattokinase stock solution with a composite freeze-drying protectant to obtain a mixed solution; S2. Perform a pre-freezing treatment operation on the mixed solution. First, cool it to a first freezing temperature point at a first temperature gradient and maintain the frozen state at the first freezing temperature point; then continue to cool it to a second freezing temperature point at a second temperature gradient and maintain the frozen state at the second freezing temperature point to obtain a pre-frozen product; S3. Perform a sublimation treatment operation on the pre-frozen product. The sublimation rate in the sublimation treatment operation is 0.1 - 0.5 g / h·cm 2 , the sublimation temperature is -30°C to -10°C, and the sublimation vacuum degree is 20 - 25 Pa to obtain the sublimation treatment product; As the sublimation rate increases, the sublimation temperature and the sublimation vacuum degree gradually increase; S4. Perform an analytical drying treatment operation on the sublimation treatment product. The drying temperature in the analytical drying treatment operation is 15 - 20 °C, and the drying vacuum degree is 20 - 30 Pa to obtain freeze-dried nattokinase; As the water content in the sublimation treatment product decreases, the drying temperature gradually increases and the drying vacuum degree gradually decreases; Among them, the composite freeze-drying protectant includes sugar, amino acids, antioxidants, and excipients; the sugar includes at least three of sophorolipid, sucrose, lactose, and skim milk powder, the amino acids include at least one of arginine, serine, and glycine, the antioxidants include at least three of tea polyphenols, phytic acid, rosemary extract, tert-butylhydroquinone, dibutylhydroxytoluene, and propyl gallate, and the excipients include at least one of glycerol and gelatin.

2. The preparation process of freeze-dried nattokinase according to claim 1, characterized in that: In S1, the mass ratio of the composite freeze-drying protectant in the mixed solution is 18% - 23%.

3. The preparation process of freeze-dried nattokinase according to claim 1, characterized in that: The mass ratio of the sugar, the amino acids, the antioxidants, and the excipients in the composite freeze-drying protectant is 10 - 25:1 - 3:1 - 3:1 - 5.

4. The preparation process of freeze-dried nattokinase according to claim 1, characterized in that: In S1, the first temperature gradient is 2 - 2.5 °C / min, the first freezing temperature point is -10 °C, and the frozen state is maintained at the first freezing temperature point for 1 - 2 h; the second temperature gradient is 1 - 1.5 °C / min, the second temperature point is -40 °C, and the frozen state is maintained at the second freezing temperature point for 4 - 6 h.

5. The preparation process of freeze-dried nattokinase according to claim 1, characterized in that: When the temperature is close to the second freezing temperature point, adjust the freezing power to 0.8 - 1.2 kW.

6. The preparation process of freeze-dried nattokinase according to claim 1, characterized in that: In S3, the sublimation rate increases from 0.1 g / h·cm 2 to 0.3 g / h·cm 2 During this process, the sublimation temperature increases from -30°C to -20°C, and the sublimation vacuum degree increases from 20 Pa to 22 Pa; when the sublimation rate increases from 0.3 g / h·cm 2 to 0.5 g / h·cm 2 During this process, the sublimation temperature increases from -20°C to -10°C, and the sublimation vacuum degree increases from 22 Pa to 25 Pa.

7. The preparation process of freeze-dried nattokinase according to claim 1, characterized in that: When the sublimation vacuum degree in S3 remains stable within 3 - 4 h, the sublimation treatment operation ends.

8. The preparation process of freeze-dried nattokinase according to claim 1, characterized in that: In S4, during the process where the water content in the sublimation treatment product decreases from 10% to 6%, the drying temperature rises from 15 °C to 18 °C and the drying vacuum degree drops from 30 Pa to 25 Pa; during the process where the water content in the sublimation treatment product decreases from 6% to 3%, the drying temperature rises from 18 °C to 20 °C and the drying vacuum degree drops from 25 Pa to 20 Pa.

9. The preparation process of freeze-dried nattokinase according to claim 1, characterized in that: When the mass of the sublimation treatment product in S4 remains stable within 0.8 - 1.2 h, the analytical drying treatment operation ends.

10. A nattokinase freeze-drying, characterized in that: Prepared by using the process according to any one of claims 1 - 9.

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