A multi-component active enzyme leech freeze-dried powder and a preparation method thereof

Abstract

The present application relates to the field of freeze-dried preparations of leeches, and provides a freeze-dried powder containing multiple active enzymes and a preparation method thereof, to solve the problem that the prior art cannot simultaneously ensure the release of active ingredients and the maintenance of the stability of active ingredients, comprising the following steps: S100, cooling leeches to obtain freeze-dried leeches; S200, putting the freeze-dried leeches into a solution system, crushing, centrifuging, and taking the supernatant; the pH value of the solution system is 6.5-8.5; the solution system at least comprises water; the crushing speed is 150-250 r / min, and the time is 5-8 min; S300, after the supernatant is separated and purified, freeze-drying is performed to obtain the freeze-dried powder of leeches. The present application adopts a freeze-crushing process, and after freezing, not only can the crushing operation be simply realized, but also the loss of active ingredients caused by high temperature can be reduced by utilizing the crushing process at low temperature of raw materials.

Description

Technical Field

[0001] This invention relates to the field of lyophilized leech preparations, and more specifically, to a lyophilized leech powder containing multiple active enzymes and its preparation method. Background Technology

[0002] Leeches are a traditional medicinal animal whose saliva contains various active polypeptides, such as hirudin. Hirudin's effects include anticoagulation, promoting blood circulation, and anti-inflammation. Leech powder contains various active polypeptides, such as hirudin, hirudin, and leech extract as the main active peptides, as well as some auxiliary components, such as hirudin peptides, hyaluronic acid, and heparin-like substances. Leech powder has anticoagulant, thrombolytic, and anti-inflammatory effects. Because the active ingredients in leeches are mainly polypeptides, current technologies often employ vacuum freeze-drying to avoid inactivation and prevent the effects of high temperatures on polypeptide activity.

[0003] Existing technologies generally fall into two categories. One involves killing and cleaning the leech, then directly freeze-drying it and pulverizing it into powder. The other involves killing and cleaning the leech, pulverizing it into a homogenate, and then extracting, purifying, and freeze-drying it to obtain a freeze-dried powder. The freeze-dried powder obtained by the second method typically has a higher antithrombin activity per gram, but its corresponding process is also more complex.

[0004] Regarding the second method mentioned above, the main purpose of the homogenization process is to release active peptides from the tissue for subsequent separation and purification. However, leeches contain a large amount of mucilage, resulting in high viscosity of the homogenate. Furthermore, leeches are mollusks, making it difficult for tissue cells to rupture completely, thus affecting the release of active ingredients. Increasing the rotation speed of the grinding blades to promote the release of active ingredients can easily lead to a decrease in the activity of some active ingredients due to the increased equipment temperature. Therefore, it is difficult to simultaneously improve the release and stability of active ingredients. Summary of the Invention

[0005] The purpose of this invention is to provide a lyophilized leech powder containing multiple active enzymes and its preparation method, thereby solving the problem that existing technologies cannot simultaneously ensure the release of active ingredients and the maintenance of their stability.

[0006] The embodiments of the present invention are achieved through the following technical solutions:

[0007] A method for preparing a lyophilized leech powder containing multiple active enzymes includes the following steps:

[0008] S100 and leeches were cooled to -30~-40℃ at a rate of less than 5℃ / min and kept at that temperature for 2~4h. Then, under a vacuum of 10~40Pa, the temperature was increased to -20~0℃ at a rate of 1~3℃ / min and kept at that temperature for 3~5h to obtain freeze-dried leeches.

[0009] S200. Freeze-dried leeches are added to a solution system, pulverized, and then centrifuged and filtered to obtain the supernatant. The pH value of the solution system is 6.5~8.5. The solution system contains at least water. The pulverization speed is 150~250 r / min and the time is 5~8 min.

[0010] After separation and purification of the supernatant by S300, freeze-drying is used to obtain lyophilized leech powder.

[0011] The applicant conceived of a freeze-drying process, which simplifies the pulverization process after freezing and reduces the loss of active ingredients caused by high temperatures during the pulverization process at low temperatures. However, this presents a problem: excessively low temperatures can lead to high hardness in the leeches, hindering pulverization. Therefore, temperature control is crucial. This invention utilizes the principle of freeze-drying, freezing the leeches into a hard block and then subjecting them to appropriate rewarming. This process allows for the evaporation of moisture and other volatile substances, reducing the fishy smell and creating porosity in the leech structure, thus lowering its strength and facilitating subsequent pulverization. Furthermore, the formation of ice crystals during freezing causes cell rupture, aiding in the release of subsequent active ingredients. The difference between the freezing process in this invention and the first prior art is that this invention promotes cell rupture through ice crystal formation, facilitating the release of subsequent active ingredients. In contrast, prior art freeze-drying of leeches typically does not involve the release of subsequent active peptides; it focuses more on preventing the loss of active peptides due to cell rupture. Consequently, prior art often employs rapid freezing to reduce the volume of ice crystals.

[0012] During pulverization, the temperature of the system should not exceed 20℃.

[0013] The cooling rate in the S100 can be selected as 2-4℃ / min.

[0014] By controlling the final temperature of the leech during the rewarming process, the impact of the operating temperature of the subsequent pulverizing device on the active ingredients can be reduced or avoided. The combination of freezing and rewarming can also increase the applicable pulverizing speed and improve the pulverizing effect.

[0015] During the pulverization process, the present invention promotes the release of effective substances into the solution system. At this time, the mucin is in a frozen state, which will not affect the pulverization process. After the system temperature recovers, the mucin is re-dissolved in water. In the present invention, the mucin mainly refers to hyaluronic acid.

[0016] Since the mucin is not directly removed in this invention, the applicant devised a scheme to remove fishy odor using mucin. In the process of removing fishy odor molecules with mucin, it is necessary to reduce or avoid the simultaneous removal of active peptides such as hirudin by mucin. Therefore, this invention adjusts the pH value of the solution system so that the mucin is negatively charged and the active peptides such as hirudin are negatively charged or electrically neutral. This allows the mucin to mainly adsorb fishy odor molecules, while the active peptides such as hirudin are excluded from the mucin network structure. Finally, the heavier mucin and other tissue precipitates are removed by centrifugation and filtration, leaving the supernatant. At this time, the fishy odor molecules in the supernatant are greatly reduced.

[0017] Hyaluronic acid typically begins to release hydrogen ions when the system pH is above 5, gradually increasing its negative charge. Hirudin typically exhibits a negative charge when the system pH is above 4.6. Therefore, when hirudin is neutrally charged, the system is acidic. Although hirudin has good acid resistance, other active peptides may denature. This invention utilizes mucin and hirudin, both of which carry negative charges, to remove fishy odor molecules. The negative charge of both also makes it easier to exclude hirudin from the hyaluronic acid network structure. Based on the aforementioned concept, the applicant has experimentally determined the optimal pH range, ensuring that each gram of product contains at least 200.0 U of antithrombin activity while minimizing the fishy odor. The highest antithrombin activity is observed at pH 8.

[0018] Preferably, the centrifugal filtration speed is 10000~12000 rpm and the time is 8~12 min.

[0019] Preferably, step S200 includes: after pulverizing, adding 0.5% to 2% salt to the solution system, and then centrifuging and filtering to obtain the supernatant; the centrifugation and filtration speed is 5000 to 7000 rpm and the time is 10 to 12 min.

[0020] The purpose of adding salt is at least to: cause the mucus to aggregate, improve the stability of capturing multiple fishy odor molecules, and facilitate subsequent centrifugation. Once the mucus is easily separated, the centrifugation speed can be reduced, and the reduction in centrifugation speed can reduce the amount of fishy odor molecules released from the mucus.

[0021] 0.5% to 2% is the percentage between the weight of the salt and the volume of the solution system.

[0022] Preferably, the salt comprises sodium chloride or sodium citrate.

[0023] Sodium citrate has antioxidant properties, which can prevent oxidation, especially the oxidation of fatty acids, and reduce the fishy smell of products.

[0024] Preferably, step S200 includes: after pulverization, adding a salt solution in two stages to a solution system at 2~10℃, first adding 50vt%~60vt% salt solution for 1~3 min, and second adding the remaining salt solution for 5~8 min; the mass of salt in the salt solution accounts for 0.5%~2% of the volume of the solution system, and the salt solution concentration is 0.1~0.2 mol / L.

[0025] Because excessively high salt concentrations can affect proteins, the amount of salt used should not be too high, especially the instantaneous amount. Hyaluronic acid has a high charge density, so salt ions will preferentially penetrate the hydration layer of hyaluronic acid, while proteins will be affected. In addition, regulating the temperature of the system can also reduce or avoid the effects of salt on proteins.

[0026] Because there is a relatively large amount of unneutralized hyaluronic acid in the early stages, the addition rate can be relatively fast. However, as the competitiveness of hyaluronic acid weakens in the later stages, the salt addition rate needs to be reduced to minimize its impact on the protein. Using a salt solution makes it easier to control the addition rate compared to directly adding solid salt. Both stages of addition were performed at a constant rate.

[0027] Preferably, when adding the salt solution, the system is in a two-stage stirring state. The stirring speed in the first stage is 50~80 r / min and the stirring time is 1~3 min. The stirring speed in the second stage is 120~150 r / min and the stirring time is 7~10 min.

[0028] In the initial stage of adding salt solution, the hyaluronic acid structure is not very compact, resulting in insufficient capture of odor molecules. Therefore, the stirring speed should not be too high. At this time, there are also many unbound negative charges on the hyaluronic acid, so even a low stirring speed will not easily lead to excessively high local salt concentrations in the solution system. As the hyaluronic acid aggregates, its stability in capturing odor molecules increases, and the stirring speed can be appropriately increased. In addition, increasing the stirring speed at this time can also reduce the increased impact of salt on proteins due to the decrease in the negative charge of hyaluronic acid.

[0029] Preferably, in step S200, the mass-volume percentage of the freeze-dried leech and solution system is 40% to 60%.

[0030] The amount of freeze-dried leeches used affects the content of mucin and active peptides in the system. Reasonable control of its amount and its coordination with the salt addition process can improve the antithrombin activity of the product and facilitate the removal of fishy odor molecules.

[0031] Preferably, the separation and purification operation includes: adjusting the supernatant to neutral, passing it through an 8-10 kDa ultrafiltration membrane, and collecting the permeate; passing the permeate through a 1-2 kDa ultrafiltration membrane, and collecting the retentate; and freeze-drying the retentate to obtain lyophilized leech powder.

[0032] Preferably, after adding 2wt%~5wt% trehalose to the retentate, it is pre-cooled to -40~-50℃ and kept at that temperature for 2~3 hours. Then, under a vacuum of 10~30Pa, it is heated to -20℃~-10℃ and kept at that temperature for 12~15 hours. Finally, under a vacuum of 1~5Pa, it is heated to 10~20℃ and kept at that temperature for 16~20 hours to obtain lyophilized leech powder.

[0033] Trehalose can protect bioactive peptides.

[0034] A lyophilized leech powder prepared by the aforementioned method.

[0035] The present invention has at least the following beneficial effects:

[0036] This invention employs a freeze-pulverization process. Freezing not only simplifies the pulverization operation but also reduces the loss of active ingredients caused by high temperatures during the pulverization process at low temperatures. Utilizing the principle of freeze-drying, the leech is frozen into a hard block and then appropriately reheated. This allows for the evaporation of moisture and other volatile substances, reducing the fishy smell and creating porosity in the leech structure, thus lowering its strength and facilitating subsequent pulverization. Furthermore, the formation of ice crystals during freezing causes cell rupture, aiding in the release of subsequent active ingredients. By controlling the final temperature of the leech during the reheating process, the influence of the operating temperature of the pulverizing device on the active ingredients can be reduced or avoided. By combining freezing and thawing, the applicable pulverization speed can be increased, thus improving the pulverization effect. In this invention, the mucus is not directly removed, but rather the odor is removed by utilizing the mucus in reverse. During the process of removing odor molecules with the mucus, it is necessary to reduce or avoid the simultaneous removal of active peptides such as hirudin by the mucus. Therefore, this invention adjusts the pH value of the solution system so that the mucus is negatively charged and the active peptides such as hirudin are negatively charged or electrically neutral. This allows the mucus to mainly adsorb odor molecules, while the active peptides such as hirudin are excluded from the mucus network structure. Finally, the heavier mucus and other tissue precipitates are removed by centrifugation and filtration, leaving the supernatant. At this point, the odor molecules in the supernatant are significantly reduced. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 A diagram illustrating the product prepared according to the method provided in Example 10;

[0039] Figure 2 This is a test report for the product prepared according to the preparation method provided in Example 10. Detailed Implementation

[0040] To make the objectives, methods, and advantages of the embodiments of the present invention clearer, the methods in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0041] Example 1: A method for preparing a lyophilized leech powder containing multiple active enzymes, comprising the following steps:

[0042] After killing and cleaning the S100 leeches, the temperature was lowered to -30℃ at a rate of 4℃ / min and kept at that temperature for 2 hours. Then, under a vacuum of 10Pa, the temperature was raised to -20℃ at a rate of 1℃ / min and kept at that temperature for 3 hours to obtain freeze-dried leeches.

[0043] S200: Freeze-dried leeches are added to the solution system, pulverized, and then centrifuged and filtered to obtain the supernatant; the pH value of the solution system is 6.5; the solvent of the solution system is pure water; the centrifugation and filtration speed is 10000 rpm and the time is 8 min; the mass-volume percentage of freeze-dried leeches to the solution system is 40%; the pulverization speed is 250 r / min and the time is 5 min;

[0044] After separation and purification of the supernatant by S300, freeze-drying is used to obtain lyophilized leech powder.

[0045] The separation and purification process includes: adjusting the supernatant to neutral, passing it through an 8kDa ultrafiltration membrane, and collecting the permeate; passing the permeate through a 1kDa ultrafiltration membrane and collecting the retentate.

[0046] After adding 2wt% trehalose to the retentate, it was pre-cooled to -40℃ and kept at that temperature for 2 hours. Then, under a vacuum of 10 Pa, it was heated to -20℃ and kept at that temperature for 12 hours. Finally, under a vacuum of 5 Pa, it was heated to 20℃ and kept at that temperature for 20 hours to obtain lyophilized leech powder.

[0047] Example 2: A method for preparing a lyophilized leech powder containing multiple active enzymes, comprising the following steps:

[0048] After killing and cleaning the S100 leeches, the temperature was lowered to -40℃ at a rate of 2℃ / min and kept at that temperature for 4 hours. Then, under a vacuum of 40Pa, the temperature was raised to 0℃ at a rate of 3℃ / min and kept at that temperature for 5 hours to obtain freeze-dried leeches.

[0049] S200: Freeze-dried leeches are added to the solution system, pulverized, and then centrifuged and filtered to obtain the supernatant; the pH value of the solution system is 8.5; the solvent of the solution system is pure water; the centrifugation and filtration speed is 12000 rpm and the time is 12 min; the mass-volume percentage of freeze-dried leeches to the solution system is 60%; the pulverization speed is 150 r / min and the time is 8 min;

[0050] After separation and purification of the supernatant by S300, freeze-drying is used to obtain lyophilized leech powder.

[0051] The separation and purification process includes: adjusting the supernatant to neutral, passing it through a 10kDa ultrafiltration membrane, and collecting the permeate; passing the permeate through a 2kDa ultrafiltration membrane and collecting the retentate.

[0052] After adding 5 wt% trehalose to the retentate, it was pre-cooled to -50℃ and kept at that temperature for 3 hours. Then, under a vacuum of 30 Pa, it was heated to -10℃ and kept at that temperature for 15 hours. Finally, under a vacuum of 1 Pa, it was heated to 10℃ and kept at that temperature for 16 hours to obtain lyophilized leech powder.

[0053] Example 3: A method for preparing a lyophilized leech powder containing multiple active enzymes, comprising the following steps:

[0054] After killing and cleaning the S100 leeches, the temperature was lowered to -35℃ at a rate of 2.5℃ / min and kept at that temperature for 3 hours. Then, under a vacuum of 25Pa, the temperature was raised to -10℃ at a rate of 2℃ / min and kept at that temperature for 4 hours to obtain freeze-dried leeches.

[0055] S200: The freeze-dried leech is added to the solution system, pulverized, and then centrifuged and filtered to obtain the supernatant; the pH value of the solution system is 8; the solvent of the solution system is pure water; the centrifugation and filtration speed is 11000 rpm and the time is 10 min; the mass-volume percentage of the freeze-dried leech to the solution system is 50%; the pulverization speed is 180 r / min and the time is 6.5 min;

[0056] After separation and purification of the supernatant by S300, freeze-drying is used to obtain lyophilized leech powder.

[0057] The separation and purification process includes: adjusting the supernatant to neutral, passing it through a 9kDa ultrafiltration membrane, and collecting the permeate; passing the permeate through a 1kDa ultrafiltration membrane and collecting the retentate.

[0058] After adding 3wt% trehalose to the retentate, it was pre-cooled to -45℃ and kept at that temperature for 2.5h. Then, under a vacuum of 29Pa, it was heated to -15℃ and kept at that temperature for 14h. Finally, under a vacuum of 3Pa, it was heated to 15℃ and kept at that temperature for 18h to obtain lyophilized leech powder.

[0059] Comparative Example 1: A method for preparing a lyophilized leech powder containing multiple active enzymes, comprising the following steps:

[0060] S100, after killing and cleaning the leeches, is added to the solution system, crushed, and the supernatant is collected by centrifugation and filtration; the pH value of the solution system is 8; the solvent of the solution system is pure water; the centrifugation and filtration speed is 11000 rpm and the time is 10 min; the mass-volume percentage of leeches to the solution system is 50%; the crushing speed is 180 r / min and the time is 6.5 min;

[0061] After separation and purification of the supernatant by S200, freeze-drying is used to obtain lyophilized leech powder.

[0062] The separation and purification process includes: adjusting the supernatant to neutral, passing it through a 9kDa ultrafiltration membrane, and collecting the permeate; passing the permeate through a 1kDa ultrafiltration membrane and collecting the retentate.

[0063] After adding 3wt% trehalose to the retentate, it was pre-cooled to -45℃ and kept at that temperature for 2.5h. Then, under a vacuum of 29Pa, it was heated to -15℃ and kept at that temperature for 14h. Finally, under a vacuum of 3Pa, it was heated to 15℃ and kept at that temperature for 18h to obtain lyophilized leech powder.

[0064] Comparative Example 2: The difference from Example 3 is that the cooling rate in S100 is 8°C / min.

[0065] Comparative Example 3: The pH value of the solution system is 4.

[0066] Experiment 1: Lyophilized leech powder was prepared according to the preparation methods provided in Examples 1-3 and Comparative Examples 1-2. The content of antithrombin activity in each g of lyophilized leech powder was determined. The test results are shown in Table 1.

[0067] Table 1

[0068]

[0069] As can be seen from the experimental results of Examples 1-3, the lyophilized leech powder prepared by the preparation method provided by the present invention has a good anticoagulant effect.

[0070] A comparison of the results from Comparative Example 1 and Example 3 shows that the anticoagulant effect of the product was significantly reduced when the leech was not freeze-dried. The applicant hypothesizes that this is because, under the same pulverization speed and time, the active peptides are difficult to release effectively.

[0071] A comparison of the results from Comparative Example 2 and Example 3 shows that the cooling rate affects the final product's effectiveness. The applicant hypothesizes that this is because an excessively rapid cooling rate results in finer ice crystals, making it difficult to effectively break down cell walls and hindering the release of active peptides.

[0072] A comparison of the results from Comparative Example 3 and Example 3 shows that the pH value of the solution system slightly affects the product's efficacy. The applicant hypothesizes that this is because pH affects the solubility of the active peptides.

[0073] Experiment 2: Freeze-dried leech powder was prepared according to the preparation methods provided in Examples 1-3 and Comparative Examples 1-2. The fishy smell of the freeze-dried leech powder was evaluated by sensory evaluation test. The test results are shown in Table 2.

[0074] Table 2

[0075]

[0076] Explanation: Using distilled water as the standard for no fishy smell, the more "+" signs there are, the stronger the fishy smell. The product with the strongest fishy smell is "+++++"; the product with the least fishy smell is " / ". It should be noted that " / " does not mean completely no fishy smell, but rather a slight fishy smell.

[0077] The experimental results of Examples 1-3 show that the optimal pH value of the solution system is 8.

[0078] A comparison of the results of Comparative Example 3 and Example 3 shows that when the pH of the solution system is too low, the fishy smell of the product is difficult to remove effectively.

[0079] Example 4: A method for preparing a lyophilized leech powder containing multiple active enzymes, comprising the following steps:

[0080] After killing and cleaning the S100 leeches, the temperature was lowered to -35℃ at a rate of 2.5℃ / min and kept at that temperature for 3 hours. Then, under a vacuum of 25Pa, the temperature was raised to -10℃ at a rate of 2℃ / min and kept at that temperature for 4 hours to obtain freeze-dried leeches.

[0081] S200: Freeze-dried leeches are added to a solution system and pulverized. Then, 0.5% sodium chloride is added to the solution system over 8 minutes. The solution is then centrifuged and filtered to obtain the supernatant. The centrifugation speed is 5000 rpm for 10 minutes. The pH of the solution system is 8. The solvent in the solution system is pure water. The mass-volume percentage of freeze-dried leeches to the solution system is 50%. The pulverization speed is 180 rpm for 6.5 minutes.

[0082] After separation and purification of the supernatant by S300, freeze-drying is used to obtain lyophilized leech powder.

[0083] The separation and purification process includes: adjusting the supernatant to neutral, passing it through a 9kDa ultrafiltration membrane, and collecting the permeate; passing the permeate through a 1kDa ultrafiltration membrane and collecting the retentate.

[0084] After adding 3wt% trehalose to the retentate, it was pre-cooled to -45℃ and kept at that temperature for 2.5h. Then, under a vacuum of 29Pa, it was heated to -15℃ and kept at that temperature for 14h. Finally, under a vacuum of 3Pa, it was heated to 15℃ and kept at that temperature for 18h to obtain lyophilized leech powder.

[0085] Example 5: A method for preparing a lyophilized leech powder containing multiple active enzymes, comprising the following steps:

[0086] After killing and cleaning the S100 leeches, the temperature was lowered to -35℃ at a rate of 2.5℃ / min and kept at that temperature for 3 hours. Then, under a vacuum of 25Pa, the temperature was raised to -10℃ at a rate of 2℃ / min and kept at that temperature for 4 hours to obtain freeze-dried leeches.

[0087] S200: Freeze-dried leeches are added to the solution system and pulverized. Then, 2% sodium chloride is added to the solution system over 8 minutes. The solution is then centrifuged and filtered to obtain the supernatant. The centrifugation speed is 7000 rpm for 12 minutes. The pH of the solution system is 8. The solvent in the solution system is pure water. The mass-volume percentage of freeze-dried leeches to the solution system is 50%. The pulverization speed is 180 rpm for 6.5 minutes.

[0088] After separation and purification of the supernatant by S300, freeze-drying is used to obtain lyophilized leech powder.

[0089] The separation and purification process includes: adjusting the supernatant to neutral, passing it through a 9kDa ultrafiltration membrane, and collecting the permeate; passing the permeate through a 1kDa ultrafiltration membrane and collecting the retentate.

[0090] After adding 3wt% trehalose to the retentate, it was pre-cooled to -45℃ and kept at that temperature for 2.5h. Then, under a vacuum of 29Pa, it was heated to -15℃ and kept at that temperature for 14h. Finally, under a vacuum of 3Pa, it was heated to 15℃ and kept at that temperature for 18h to obtain lyophilized leech powder.

[0091] Example 6: A method for preparing a lyophilized leech powder containing multiple active enzymes, comprising the following steps:

[0092] After killing and cleaning the S100 leeches, the temperature was lowered to -35℃ at a rate of 2.5℃ / min and kept at that temperature for 3 hours. Then, under a vacuum of 25Pa, the temperature was raised to -10℃ at a rate of 2℃ / min and kept at that temperature for 4 hours to obtain freeze-dried leeches.

[0093] S200: Freeze-dried leeches are added to the solution system, pulverized, and then 1% sodium chloride is added to the solution system over 8 minutes. The solution is then centrifuged and filtered to obtain the supernatant. The centrifugation speed is 6000 rpm for 10 minutes. The pH of the solution system is 8. The solvent in the solution system is pure water. The mass-volume percentage of freeze-dried leeches to the solution system is 50%. The pulverization speed is 180 rpm for 6.5 minutes.

[0094] After separation and purification of the supernatant by S300, freeze-drying is used to obtain lyophilized leech powder.

[0095] The separation and purification process includes: adjusting the supernatant to neutral, passing it through a 9kDa ultrafiltration membrane, and collecting the permeate; passing the permeate through a 1kDa ultrafiltration membrane and collecting the retentate.

[0096] After adding 3wt% trehalose to the retentate, it was pre-cooled to -45℃ and kept at that temperature for 2.5h. Then, under a vacuum of 29Pa, it was heated to -15℃ and kept at that temperature for 14h. Finally, under a vacuum of 3Pa, it was heated to 15℃ and kept at that temperature for 18h to obtain lyophilized leech powder.

[0097] Example 7: A method for preparing a lyophilized leech powder containing multiple active enzymes, comprising the following steps:

[0098] After killing and cleaning the S100 leeches, the temperature was lowered to -35℃ at a rate of 2.5℃ / min and kept at that temperature for 3 hours. Then, under a vacuum of 25Pa, the temperature was raised to -10℃ at a rate of 2℃ / min and kept at that temperature for 4 hours to obtain freeze-dried leeches.

[0099] S200: Freeze-dried leeches are added to the solution system and pulverized. Then, 1% sodium citrate is added to the solution system over 8 minutes. The solution is then centrifuged and filtered to obtain the supernatant. The centrifugation speed is 6000 rpm for 10 minutes. The pH of the solution system is 8. The solvent in the solution system is pure water. The mass-volume percentage of freeze-dried leeches to the solution system is 50%. The pulverization speed is 180 rpm for 6.5 minutes.

[0100] After separation and purification of the supernatant by S300, freeze-drying is used to obtain lyophilized leech powder.

[0101] The separation and purification process includes: adjusting the supernatant to neutral, passing it through a 9kDa ultrafiltration membrane, and collecting the permeate; passing the permeate through a 1kDa ultrafiltration membrane and collecting the retentate.

[0102] After adding 3wt% trehalose to the retentate, it was pre-cooled to -45℃ and kept at that temperature for 2.5h. Then, under a vacuum of 29Pa, it was heated to -15℃ and kept at that temperature for 14h. Finally, under a vacuum of 3Pa, it was heated to 15℃ and kept at that temperature for 18h to obtain lyophilized leech powder.

[0103] Experiment 3: The freeze-dried powder of Hirudo medicinalis was prepared according to the preparation method provided in Examples 4-7. Then, the content of antithrombin activity in each g of freeze-dried Hirudo medicinalis powder was determined, and the degree of fishy smell was evaluated by sensory evaluation test. The test results are shown in Table 3.

[0104] Table 3

[0105]

[0106] As can be seen from the test results of Examples 4-7, adding salt can reduce the fishy smell of the product and improve the anticoagulant effect of the product to a certain extent.

[0107] Example 8: A method for preparing a lyophilized leech powder containing multiple active enzymes, comprising the following steps:

[0108] After killing and cleaning the S100 leeches, the temperature was lowered to -35℃ at a rate of 2.5℃ / min and kept at that temperature for 3 hours. Then, under a vacuum of 25Pa, the temperature was raised to -10℃ at a rate of 2℃ / min and kept at that temperature for 4 hours to obtain freeze-dried leeches.

[0109] S200: Freeze-dried leeches are added to the solution system, pulverized, and then 1% sodium citrate is added to the solution system. The mixture is then centrifuged and filtered to obtain the supernatant. The centrifugation speed is 6000 rpm for 10 min. The pH value of the solution system is 8. The solvent of the solution system is pure water. The mass-volume percentage of freeze-dried leeches to the solution system is 50%. The pulverization speed is 180 r / min for 6.5 min.

[0110] The S200 process includes: after pulverization, adding salt solution in two stages to a solution system at 2°C; in the first stage, adding 50% salt solution for 1 minute; and in the second stage, adding the remaining salt solution for 5 minutes; the mass of salt in the salt solution accounts for 0.5% of the volume of the solution system, and the salt solution concentration is 0.1 mol / L.

[0111] When adding the salt solution, the system was in a two-stage stirring state. The stirring speed in the first stage was 50 r / min and the stirring time was 1 min. The stirring speed in the second stage was 120 r / min and the stirring time was 7 min.

[0112] After separation and purification of the supernatant by S300, freeze-drying is used to obtain lyophilized leech powder.

[0113] The separation and purification process includes: adjusting the supernatant to neutral, passing it through a 9kDa ultrafiltration membrane, and collecting the permeate; passing the permeate through a 1kDa ultrafiltration membrane and collecting the retentate.

[0114] After adding 3wt% trehalose to the retentate, it was pre-cooled to -45℃ and kept at that temperature for 2.5h. Then, under a vacuum of 29Pa, it was heated to -15℃ and kept at that temperature for 14h. Finally, under a vacuum of 3Pa, it was heated to 15℃ and kept at that temperature for 18h to obtain lyophilized leech powder.

[0115] Example 9: A method for preparing a lyophilized leech powder containing multiple active enzymes, comprising the following steps:

[0116] After killing and cleaning the S100 leeches, the temperature was lowered to -35℃ at a rate of 2.5℃ / min and kept at that temperature for 3 hours. Then, under a vacuum of 25Pa, the temperature was raised to -10℃ at a rate of 2℃ / min and kept at that temperature for 4 hours to obtain freeze-dried leeches.

[0117] S200: Freeze-dried leeches are added to the solution system, pulverized, and then 1% sodium citrate is added to the solution system. The mixture is then centrifuged and filtered to obtain the supernatant. The centrifugation speed is 6000 rpm for 10 min. The pH value of the solution system is 8. The solvent of the solution system is pure water. The mass-volume percentage of freeze-dried leeches to the solution system is 50%. The pulverization speed is 180 r / min for 6.5 min.

[0118] The S200 process includes: after pulverization, adding salt solution in two stages to a solution system at 10°C; in the first stage, adding 60% salt solution for 3 minutes, and in the second stage, adding the remaining salt solution for 8 minutes; the mass of salt in the salt solution accounts for 2% of the volume of the solution system, and the salt solution concentration is 0.2 mol / L.

[0119] When adding salt solution, the system is in a two-stage stirring state. The stirring speed in the first stage is 80 r / min and the stirring time is 3 min. The stirring speed in the second stage is 150 r / min and the stirring time is 10 min.

[0120] After separation and purification of the supernatant by S300, freeze-drying is used to obtain lyophilized leech powder.

[0121] The separation and purification process includes: adjusting the supernatant to neutral, passing it through a 9kDa ultrafiltration membrane, and collecting the permeate; passing the permeate through a 1kDa ultrafiltration membrane and collecting the retentate.

[0122] After adding 3wt% trehalose to the retentate, it was pre-cooled to -45℃ and kept at that temperature for 2.5h. Then, under a vacuum of 29Pa, it was heated to -15℃ and kept at that temperature for 14h. Finally, under a vacuum of 3Pa, it was heated to 15℃ and kept at that temperature for 18h to obtain lyophilized leech powder.

[0123] Example 10: A method for preparing a lyophilized leech powder containing multiple active enzymes, comprising the following steps:

[0124] After killing and cleaning the S100 leeches, the temperature was lowered to -35℃ at a rate of 2.5℃ / min and kept at that temperature for 3 hours. Then, under a vacuum of 25Pa, the temperature was raised to -10℃ at a rate of 2℃ / min and kept at that temperature for 4 hours to obtain freeze-dried leeches.

[0125] S200: Freeze-dried leeches are added to the solution system, pulverized, and then 1% sodium citrate is added to the solution system. The mixture is then centrifuged and filtered to obtain the supernatant. The centrifugation speed is 6000 rpm for 10 min. The pH value of the solution system is 8. The solvent of the solution system is pure water. The mass-volume percentage of freeze-dried leeches to the solution system is 50%. The pulverization speed is 180 r / min for 6.5 min.

[0126] The S200 process includes: after pulverization, adding salt solution in two stages to a solution system at 5°C; in the first stage, adding 55% salt solution for 2 minutes; and in the second stage, adding the remaining salt solution for 6 minutes; the mass of salt in the salt solution accounts for 1% of the volume of the solution system, and the salt solution concentration is 0.15 mol / L.

[0127] When adding the salt solution, the system was in a two-stage stirring state. The stirring speed in the first stage was 65 r / min and the stirring time was 2 min. The stirring speed in the second stage was 140 r / min and the stirring time was 8 min.

[0128] After separation and purification of the supernatant by S300, freeze-drying is used to obtain lyophilized leech powder.

[0129] The separation and purification process includes: adjusting the supernatant to neutral, passing it through a 9kDa ultrafiltration membrane, and collecting the permeate; passing the permeate through a 1kDa ultrafiltration membrane and collecting the retentate.

[0130] After adding 3wt% trehalose to the retentate, it was pre-cooled to -45℃ and kept at that temperature for 2.5h. Then, under a vacuum of 29Pa, it was heated to -15℃ and kept at that temperature for 14h. Finally, under a vacuum of 3Pa, it was heated to 15℃ and kept at that temperature for 18h to obtain lyophilized leech powder.

[0131] Comparative Example 4: The difference from Example 10 is that the mass of salt in the salt solution accounts for 4% of the volume of the solution system.

[0132] Comparative Example 5: The difference from Example 10 is that a 40 wt% salt solution was added.

[0133] Comparative Example 6: The difference from Example 10 is that when the salt solution was added, the system was only in a stirring state for a period of time, with a stirring speed of 65 r / min and a stirring time of 10 min.

[0134] Comparative Example 7: The difference from Example 10 is that when the salt solution was added, the system was only in a two-stage stirring state, with a stirring speed of 140 r / min and a stirring time of 10 min.

[0135] Experiment 4: Freeze-dried leech powder was prepared according to the preparation methods provided in Examples 8-10 and Comparative Examples 4-7. The content of antithrombin activity in each g of freeze-dried leech powder was then determined, and the degree of fishy smell was evaluated by sensory evaluation test. The test results are shown in Table 4.

[0136] Table 4

[0137]

[0138] The experimental results from Examples 8-10 show that both the salt addition rate and the stirring speed during addition affect the removal effect of fishy odor molecules. By regulating these two factors, the loss of active peptides during salt addition can also be reduced.

[0139] A comparison of the results of Comparative Example 4 and Example 10 shows that increasing the amount of salt affects the anticoagulant effect of the product. The applicant hypothesizes that the reason is that excessively high salt concentration in the system may lead to the precipitation of active peptides, which are then filtered out during centrifugation.

[0140] A comparison of the results from Comparative Example 5 and Example 10 shows that the fishy smell of the product increased when the amount of salt solution added was relatively small. The applicant hypothesizes that this is because, when the hyaluronic acid structure is not yet sufficiently compact, increasing the stirring speed may cause the release of fishy smell molecules.

[0141] A comparison of the results from Comparative Example 6 and Example 10 shows that maintaining low-speed stirring during the addition of the salt solution had almost no effect on the fishy smell of the product, but slightly affected the content of active peptides. The applicant hypothesizes that this is because after a large number of hyaluronic acid adsorption sites are occupied, the competitiveness of salt capture decreases. In this case, it is necessary to increase the stirring speed to reduce or avoid the impact of excessively high local salt concentrations on the active peptides.

[0142] A comparison of the results from Comparative Example 7 and Example 10 shows that maintaining high-speed stirring throughout the salt solution addition process significantly impacts the fishy odor of the product. The applicant hypothesizes that this is because, when the structure of hyaluronic acid is not yet sufficiently compact, excessively fast stirring can affect its stability in capturing fishy odor molecules.

[0143] like Figure 1 As shown, the product prepared according to the preparation method provided by the present invention has good particle size uniformity.

[0144] like Figure 2 As shown, the product prepared according to the preparation method provided by the present invention conforms to the standard requirements.

[0145] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a lyophilized leech powder containing multiple active enzymes, characterized in that, Includes the following steps: S100 and leeches were cooled to -30~-40℃ at a rate of less than 5℃ / min and kept at that temperature for 2~4h. Then, under a vacuum of 10~40Pa, the temperature was increased to -20~0℃ at a rate of 1~3℃ / min and kept at that temperature for 3~5h to obtain freeze-dried leeches. S200. Freeze-dried leeches are added to a solution system and pulverized. A salt solution is then added in two stages at 2-10°C. In the first stage, 50%-60% of the salt solution is added over 1-3 minutes. In the second stage, the remaining salt solution is added over 5-8 minutes. The salt content in the solution is 0.5%-2% of the solution volume, and the salt concentration is 0.1-0.2 mol / L. The solution is then centrifuged and filtered to obtain the supernatant. The centrifugation speed is 5000-7000 rpm, and the centrifugation time is 10-12 minutes. The pH of the solution system is 6.5-8.

5. The solvent in the solution system is water. The grinding speed is 150~250 r / min, and the grinding time is 5~8 min; When adding salt solution, the system is in a two-stage stirring state. The stirring speed in the first stage is 50~80 r / min and the stirring time is 1~3 min. The stirring speed in the second stage is 120~150 r / min and the stirring time is 7~10 min. After separation and purification of the supernatant by S300, freeze-drying is used to obtain lyophilized leech powder.

2. The preparation method according to claim 1, characterized in that, The salt includes sodium chloride or sodium citrate.

3. The preparation method according to claim 1, characterized in that, In S200, the mass-volume percentage of the freeze-dried leech and solution system is 40%~60%.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The separation and purification process includes: adjusting the supernatant to neutral, passing it through an 8-10 kDa ultrafiltration membrane, and collecting the permeate; passing the permeate through a 1-2 kDa ultrafiltration membrane, and collecting the retentate; and freeze-drying the retentate to obtain lyophilized leech powder.

5. The preparation method according to claim 4, characterized in that, After adding 2wt%~5wt% trehalose to the retentate, it is pre-cooled to -40~-50℃ and kept at that temperature for 2~3 hours. Then, under a vacuum of 10~30Pa, it is heated to -20℃~-10℃ and kept at that temperature for 12~15 hours. Finally, under a vacuum of 1~5Pa, it is heated to 10~20℃ and kept at that temperature for 16~20 hours to obtain lyophilized leech powder.

6. A lyophilized leech powder prepared by the preparation method according to any one of claims 1 to 5.

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