Substrate rich in plasmin as well as preparation method and application thereof

Abstract

The invention provides a plasmin-rich matrix as well as a preparation method and application thereof, and belongs to the technical field of plasmin preparation. The method for preparing the plasmin-rich matrix provided by the invention comprises the following steps: inoculating a fermentation medium with neurospora intermedia L01 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.41253, and fermenting to obtain the plasmin-rich matrix; the fermentation medium comprises nutritional raw materials and water, and the nutritional raw materials are composed of brewer's grains, bran and a quick-acting carbon source and / or a quick-acting nitrogen source. According to the method, the neurospora intermedia L01 serves as a fermentation strain, the brewer's grains and the bran serve as raw materials of a fermentation culture medium, the prepared matrix is rich in neurospora intermedia L01 mycelia and plasmin, and the activity of the plasmin reaches 468.53 + / -7.19 U / g. The solid fermentation substrate prepared by the method disclosed by the invention can be used as a functional raw material in products for preventing and / or treating thrombus diseases, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of plasmin preparation, and specifically relates to a matrix rich in plasmin, a preparation method thereof, and an application thereof. Background Art

[0002] A thrombus refers to a blood clot formed by blood components in the blood vessels under certain conditions, causing thrombosis. As one of the main diseases endangering human life and health, thromboembolic diseases not only have a relatively high prevalence rate, but also have a high fatality rate. Due to the continuous changes in people's lifestyles and the continuous improvement of living standards, excessive intake of high protein and high fat in people's daily diets has promoted the increase in the incidence of thromboembolic diseases.

[0003] Currently, the most commonly used thrombolytic drug in clinical practice is tissue-type plasminogen activator. Currently, commercially available thrombolytic drugs have disadvantages such as low specificity, easy bleeding, short half-life, and drug resistance. As a thrombolytic agent for the prevention and treatment of thromboembolic diseases, thrombolytic agents of microbial origin show broad prospects due to their good efficacy and low side effects. Therefore, it is of great significance to develop new and safe products with a tendency to reduce thrombosis by fermenting agricultural product processing by-products with microorganisms from safe sources to reduce the number of people with thromboembolism.

[0004] Neurospora intermedia is a filamentous fungus with orange-yellow spores and has been used to make traditional fermented foods. Currently, there is no report at home and abroad on preparing highly active plasmin using Neurospora intermedia as a fermentation strain and the by-product brewer's grains in the beer brewing process as the main raw material. Summary of the Invention

[0005] In view of this, one of the purposes of the present invention is to provide a matrix rich in plasmin, a preparation method thereof, and an application thereof.

[0006] Another purpose of the present invention is to provide a method for producing a plasmin solution and an application thereof.

[0007] In order to achieve the above-mentioned invention purposes, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a matrix rich in plasmin, comprising the following steps: inoculating Neurospora intermedia L01 into a fermentation medium for fermentation to obtain a matrix rich in plasmin;

[0009] The Neurospora intermedia L01 is taxonomically named Neurospora intermedia, deposited in the China General Microbiological Culture Collection Center, with the address being Beijing, China, and the deposit number being CGMCC No. 41253, and the deposit date being July 23, 2024;

[0010] The fermentation medium comprises nutritional raw materials and water, and the nutritional raw materials are composed of brewer's grains, wheat bran, and a readily available carbon source and / or a readily available nitrogen source.

[0011] Preferably, the mass ratio of the brewer's grains to the wheat bran is (5:1) - (1:5).

[0012] Preferably, the mass concentration of the readily available carbon source is 0.5% - 2%; the readily available carbon source includes any one or more of glucose, fructose, D-maltose, and sucrose.

[0013] Preferably, the mass concentration of the readily available nitrogen source is 0.1% - 1%; the readily available nitrogen source includes any one or more of yeast extract powder, ammonium chloride, urea, ammonium sulfate, and tryptone.

[0014] Preferably, the mass-volume ratio of the nutritional raw materials to water is 1 g:0.25 - 2 mL.

[0015] Preferably, the temperature of the fermentation is 26°C - 34°C, and the time of the fermentation is 2 days - 5 days; the inoculation amount is 1×10 5 spores - 1×10 6 spores per g of the fermentation medium.

[0016] The present invention also provides a matrix rich in plasmin, and the matrix is obtained by the above method.

[0017] The present invention also provides the application of the above method or the above matrix in the production of plasmin or in the preparation of products for preventing and treating thrombosis.

[0018] The present invention also provides a method for producing a plasmin solution, comprising the following steps: mixing and leaching the above matrix with PBS buffer solution, and discarding the precipitate to obtain the plasmin solution.

[0019] The present invention also provides the application of the above method in the preparation of products for preventing and treating thrombosis.

[0020] Advantages of the present invention:

[0021] Using Neurospora intermedia L01 as the fermentation strain and brewer's grains and wheat bran as the raw materials of the fermentation medium, through solid fermentation, the obtained matrix is rich in Neurospora intermedia L01 mycelia and plasmin, and the plasmin activity reaches 468.53 ± 7.19 U / g. The strain and raw materials of the method of the present invention are safe and reliable, the cultivation time is short, and it is easy to implement; the prepared solid fermentation matrix can be used as a functional raw material in products for preventing and / or treating thrombosis diseases, and has good application prospects.

[0022] Deposition description

[0023] The present invention relates to Neurospora intermedia L01, which is classified and named as Neurospora intermedia. It is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The preservation time is July 23, 2024. The preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation number is CGMCC No. 41253. Description of the Drawings

[0024] Figure 1 It shows the results of the fibrin plate lysis test with the fibrinolytic enzyme solution of Neurospora intermedia. Among them, 1 is the fibrinolytic enzyme solution of Neurospora intermedia, and 2 is normal saline.

[0025] Figure 2 It shows the effect of different ratios of spent beer grains and wheat bran in the fermentation medium on the enzyme activity of fibrinolytic enzyme of Neurospora intermedia. If any two groups are compared, different letters on the column indicate significant differences, p ≤ 0.05. If the letters on the column are the same, it indicates no significant difference, p > 0.05.

[0026] Figure 3 It shows the effect of the types of readily available carbon sources on the enzyme activity of fibrinolytic enzyme of Neurospora intermedia. If any two groups are compared, different letters on the column indicate significant differences, p ≤ 0.05. If the letters on the column are the same, it indicates no significant difference, p > 0.05.

[0027] Figure 4 It shows the effect of the addition amount of readily available carbon sources on the enzyme activity of fibrinolytic enzyme of Neurospora intermedia. If any two groups are compared, different letters on the column indicate significant differences, p ≤ 0.05. If the letters on the column are the same, it indicates no significant difference, p > 0.05.

[0028] Figure 5 It shows the effect of the types of readily available nitrogen sources on the enzyme activity of fibrinolytic enzyme of Neurospora intermedia. If any two groups are compared, different letters on the column indicate significant differences, p ≤ 0.05. If the letters on the column are the same, it indicates no significant difference, p > 0.05.

[0029] Figure 6 It shows the effect of the addition amount of readily available nitrogen sources on the enzyme activity of fibrinolytic enzyme of Neurospora intermedia. If any two groups are compared, different letters on the column indicate significant differences, p ≤ 0.05. If the letters on the column are the same, it indicates no significant difference, p > 0.05. Detailed Embodiments

[0030] The present invention provides a method for preparing a matrix rich in fibrinolytic enzyme, which includes the following steps: inoculating Neurospora intermedia L01 into a fermentation medium for fermentation to obtain a matrix rich in fibrinolytic enzyme.

[0031] The classification and naming of the Neurospora intermedia L01 is Neurospora intermedia, which is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation number of CGMCC No. 41253 and the preservation date of July 23, 2024;

[0032] The fermentation medium includes nutritional raw materials and water, and the nutritional raw materials are composed of brewery grains, wheat bran, and readily available carbon source and / or readily available nitrogen source.

[0033] The Neurospora intermedia L01 of the present invention is isolated and screened from traditional fermented soybean paste in Northeast China, and has the advantages of high yield of fibrinolytic enzyme and high activity of the produced fibrinolytic enzyme.

[0034] The present invention has no special limitation on the specific sources of each raw material in the fermentation medium. In the present invention, the mass ratio of the brewery grains to the wheat bran is preferably (5:1) to (1:5), more preferably (2:1) to (1:3). In the present invention, the mass concentration of the readily available carbon source is preferably 0.5% to 2%, and the mass concentration of the readily available carbon source refers to the mass concentration in the nutritional raw materials. In specific embodiments, it can be 0.5%, 0.8%, 1.2%, 1.5%, 1.8% or 2%; the readily available carbon source preferably includes any one or more of glucose, fructose, D-maltose and sucrose. In the present invention, the mass concentration of the readily available nitrogen source is preferably 0.1% to 1%, more preferably 0.5% to 0.8%, and the mass concentration of the readily available nitrogen source refers to the mass concentration in the nutritional raw materials; the readily available nitrogen source preferably includes any one or more of yeast extract powder, ammonium chloride, urea, ammonium sulfate and tryptone. In the present invention, the mass-to-volume ratio of the nutritional raw materials to water is preferably 1 g:0.25 to 2 mL, more preferably 1 g:1 mL. In the present invention, the fermentation temperature is preferably 26°C to 34°C, more preferably 28°C to 32°C; the fermentation time is preferably 2 days to 5 days, more preferably 3 days to 4 days; the fermentation method is preferably static light-avoiding solid fermentation. In the present invention, the inoculation amount is preferably 1×10 5 spores to 1×10 6 spores per g of the fermentation medium, and more preferably 2.5×10 5 spores to 8×10 5 spores per g of the fermentation medium.

[0035] In the present invention, the Neurospora intermedia L01 preferably needs to be activated and subjected to seed culture first to obtain a seed solution, and then inoculated into a fermentation medium for fermentation. In the present invention, the activation medium for activation preferably includes: 20% potato solution and glucose, and the volume-mass ratio of the 20% potato solution and glucose is preferably 100 mL: 2 g; the present invention has no special requirements for the pH of the activation medium, and it can remain natural; the activation temperature is preferably 28-32 °C, more preferably 29-31 °C, the activation rotation speed is preferably 170-190 r / min, more preferably 175-180 r / min, and the activation time is preferably 3-5 days, more preferably 4 days. The seed medium for seed culture preferably includes 20% potato solution, glucose and agar, and the volume-mass ratio of the 20% potato solution, glucose and agar is preferably 100 mL: 1 g: 1.5 g. The present invention has no special requirements for the pH of the seed medium, and it can remain natural. In the present invention, the temperature for seed culture is preferably 26-34 °C, more preferably 28-30 °C, and the time for seed culture is preferably 2-5 days, more preferably 3-4 days.

[0036] The present invention also provides a matrix rich in plasmin, which is prepared by the above method.

[0037] The present invention also provides the application of the above method or the above matrix in the production of plasmin or in the preparation of products for preventing and treating thrombosis. In the present invention, the type of the product preferably includes drugs.

[0038] The present invention also provides a method for producing a plasmin solution, which includes the following steps: mixing and extracting the above matrix with PBS buffer solution, and discarding the precipitate to obtain a plasmin solution.

[0039] In the present invention, the mass-volume ratio of the matrix to the PBS buffer solution is preferably 1 g: 5 mL; the extraction time is preferably 2 h to 4 h, more preferably 2.5 h to 3.5 h; the extraction temperature is preferably 20 °C to 25 °C, more preferably 22 °C to 24 °C. In the present invention, after the extraction is completed, it is preferably centrifuged to discard the precipitate. The centrifugation temperature is preferably 4 °C, and the centrifugation speed is preferably 10000 r / min.

[0040] The present invention also provides the application of the above method in the preparation of products for preventing and treating thrombosis. In the present invention, the type of the product preferably includes drugs.

[0041] The following examples are used to illustrate in detail the technical solutions provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0042] In the following examples, unless otherwise specified, they are all conventional methods.

[0043] Materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0044] Example 1

[0045] A matrix rich in plasmin, which is prepared by the following method:

[0046] (1) Activation of Neurospora intermedia L01: Transfer 500 μL of activation medium (the composition of 100 mL of activation medium is: 100 mL of 20% potato solution and 2 g of glucose, natural pH) into the freeze-dried tube containing Neurospora intermedia L01 obtained in Example 1 to completely dissolve the freeze-dried powder. Transfer the dissolved bacterial suspension to a 250 mL conical flask containing 150 mL of activation medium, and culture it with shaking at 30 °C and 150 r / min for 3 days to obtain the primary bacterial liquid. Take 100 μL of the primary bacterial liquid and place it in a 250 mL conical flask containing 150 mL of activation medium, culture it with shaking at 30 °C and 150 r / min for 3 days, and transfer it to the 4th generation according to the above method to obtain the activated Neurospora intermedia L01.

[0047] (2) Inoculate the activated Neurospora intermedia L01 in step (1) into the seed medium (the composition of the seed medium is: 100 mL of 20% potato solution, 1.5 g of agar, 1 g of glucose, natural pH), and culture it at 28 °C for 3 days. After rinsing the inclined plane with sterile water, obtain the Neurospora intermedia L01 seed liquid.

[0048] (3) Preparation of fermentation medium: Mix the nutrient raw materials and water in a ratio of 1 g:1 mL. The nutrient raw materials consist of brewer's grains, wheat bran, glucose, and yeast extract powder. The mass ratio of brewer's grains to wheat bran is 4:1, the mass concentration of glucose is 0.5% (here 0.5% refers to the mass concentration of glucose in the nutrient raw materials), the mass concentration of yeast extract powder is 0.7% (here 0.7% refers to the mass concentration of yeast extract powder in the nutrient raw materials), and the initial pH is the natural pH.

[0049] (4) Inoculate the Neurospora intermedia L01 seed liquid obtained in step (2) into the fermentation medium obtained in step (3) at a rate of 2.5×10 5 spores per gram of fermentation medium, and culture it statically and protected from light at 28 °C for 3 days to obtain a matrix rich in plasmin, and the matrix is also rich in Neurospora intermedia mycelia.

[0050] Example 2

[0051] A matrix rich in plasmin, which is prepared by the following method:

[0052] Steps (1) and (2) are the same as steps (1) and (2) of Example 1.

[0053] (3) Preparation of fermentation medium: Mix the nutrient raw materials and water in a ratio of 1 g: 0.5 mL. The nutrient raw materials consist of brewer's grains, wheat bran, glucose, and yeast extract powder. The mass ratio of brewer's grains to wheat bran is 2:1, the mass concentration of glucose is 1.5%, the mass concentration of yeast extract powder is 0.5%, and the pH is the natural pH.

[0054] (4) Inoculate the Neurospora intermedia L01 seed liquid obtained in step (2) into the fermentation medium obtained in step (3) at an inoculation amount of 5×10 5 spores per gram of the fermentation medium, and perform static light - avoiding fermentation culture at 26 °C for 3 days to obtain a matrix rich in fibrinolytic enzyme, and the matrix is also rich in Neurospora intermedia mycelia.

[0055] Example 3

[0056] A matrix rich in fibrinolytic enzyme, which is obtained by the following method:

[0057] Steps (1) and (2) are the same as steps (1) and (2) of Example 1.

[0058] (3) Preparation of fermentation medium: Mix the nutrient raw materials and water in a ratio of 1 g: 1.5 mL. The nutrient raw materials consist of brewer's grains, wheat bran, glucose, and yeast extract powder. The mass ratio of brewer's grains to wheat bran is 1:3, the mass concentration of glucose is 1%, the mass concentration of yeast extract powder is 0.3%, and the pH is the natural pH.

[0059] (4) Inoculate the Neurospora intermedia L01 seed liquid obtained in step (2) into the fermentation medium obtained in step (3) at an inoculation amount of 7.5×10 5 spores per gram of the fermentation medium, and perform static light - avoiding fermentation culture at 34 °C for 2 days to obtain a matrix rich in fibrinolytic enzyme, and the matrix is also rich in Neurospora intermedia mycelia.

[0060] Example 4

[0061] A method for producing fibrinolytic enzyme solution, which consists of the following steps: Mix the matrix obtained in Example 1 and PBS buffer in a mass - to - volume ratio of 1 g: 5 mL, extract at 25 °C for 2 h, then centrifuge at 4 °C and 10000 r / min for 10 min, and discard the precipitate to obtain the fibrinolytic enzyme solution.

[0062] Determination of fibrinolytic enzyme activity in the fibrinolytic enzyme solution:

[0063] (1) Preparation of fibrin plate:

[0064] ① Fibrinogen solution (100 mL)

[0065] Prepare 100 mL of 0.04 mol / L sodium phosphate buffer (a solution of Na 2 HPO 4 ·12H 2 O and 0.04 mol / L NaH 2 PO 4 ·2H 2 O solutions, and mix them well); adjust the pH of the volumetric sodium phosphate buffer to 7.8, then add 0.4 g of bovine fibrinogen to the solution and dissolve it, and centrifuge at 10,000 r / min for 10 min; place it in a 45°C water bath for 5 min;

[0066] ② Thrombin solution

[0067] Dissolve 0.5 g of agarose in 100 mL of physiological saline, place it in a 45°C water bath for 25 min, and then add 200 U of thrombin;

[0068] ③ Take 5 mL each of the prepared ① and ② and quickly pour them into a plate and mix. After cooling for 30 min, place the solidified plate in the refrigerator for storage and standby.

[0069] (2) Determination of urokinase standard curve

[0070] Take 250 U / mL urokinase and dilute it with physiological saline. The diluted concentrations are 7.5 U / mL, 15 U / mL, 30 U / mL, 60 U / mL, 120 U / mL, and 240 U / mL respectively; take 10 μL of the urokinase standard product at each different concentration and spot it on the fibrin plate, with three parallels for each, and place it in an incubator at a temperature of 37°C for 6 h; measure the diameter of the lysis circle with an electronic vernier caliper, take the average of the three parallels, and the obtained curve equation is y = 0.0506 × e (5.4285×X) ×100÷20, R 2 = 0.9781; where X represents the diameter of the lysis circle and y represents the enzyme activity (U / g);

[0071] (3) Determination of fibrinolytic enzyme activity of the sample to be tested

[0072] Take 10 μL of the fibrinolytic enzyme solution and spot it on the prepared fibrin plate. After placing it in a 37°C incubator for 6 h, read the diameter, make three parallels, take the average value, substitute the average value into the urokinase standard curve, and after calculation, the fibrinolytic enzyme activity is found to reach 468.53 U / g. At the same time, use physiological saline as a control group to conduct the same experiment, and the results are as Figure 1 shown.

[0073] It can be seen that the plasmin solution obtained in the present invention can dissolve the fibrin plate to produce a clear zone, while normal saline cannot dissolve fibrin, indicating that the plasmin solution of the present invention can achieve the thrombolytic effect.

[0074] Example 5

[0075] Influencing factors for preparing matrix rich in plasmin

[0076] Specific steps of fermentation culture:

[0077] Put the weighed brewery grains and wheat bran into a 250 mL Erlenmeyer flask, add a certain amount of water, put it into an autoclave, at a temperature of 121 °C, sterilize for 30 min to obtain a fermentation medium, and cool it in a sterile room.

[0078] Take the Neurospora intermedia L01 seed solution obtained according to steps (1) and (2) of Example 1, inoculate it into the cooled fermentation medium, and then put the fermentation medium into an incubator at 28 °C for static light-proof culture.

[0079] 1. Determination of mass ratio of brewery grains to wheat bran

[0080] According to the above specific steps of fermentation culture, add brewery grains and wheat bran to a 250 mL Erlenmeyer flask at mass ratios of 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, and 1:5 respectively. When the ratio of nutritional raw materials to water in the fermentation medium is 1 g:1 mL and the inoculation amount is 2.5×10 spores per g of fermentation medium 5 , culture for 3 days under the condition of a culture temperature of 28 °C to obtain a matrix rich in plasmin.

[0081] After obtaining the plasmin solution according to the method of Example 4, measure the plasmin activity in the plasmin solution. The measurement method is the same as that in Example 4. Do three parallels for each and take the average value to obtain the results as Figure 2 shown.

[0082] 2. Determination method for suitable types of rapid-acting carbon sources

[0083] According to the above specific steps of fermentation culture, add brewery grains and wheat bran to a 250 mL Erlenmeyer flask at a mass ratio of 2:1. The concentration of the rapid-acting carbon source is 1%; the rapid-acting carbon sources include glucose, fructose, D-maltose, and sucrose. When the ratio of nutritional raw materials to water in the fermentation medium is 1 g:1 mL and the inoculation amount is 2.5×10 spores per g of fermentation medium 5 , culture for 3 days under the condition of a culture temperature of 28 °C to obtain a matrix rich in plasmin. After obtaining the plasmin solution according to the method of Example 4, measure the plasmin activity in the plasmin solution. The measurement method is the same as that in Example 4. Do three parallels for each and take the average value. Use the medium without adding the rapid-acting carbon source as the blank group to obtain the results asFigure 3 。

[0084] 3. Determination method for appropriate addition amount of readily available carbon source

[0085] According to the above specific steps of fermentation culture, add brewer's grains and wheat bran into a 250 mL Erlenmeyer flask at a mass ratio of 2:1. The concentrations of the readily available carbon source are 0.5%, 1%, 1.5%, and 2%; the readily available carbon source is glucose. When the ratio of the nutrient raw materials to water in the fermentation medium is 1 g:1 mL and the inoculation amount is 2.5×10 5 spores per gram of the fermentation medium, culture for 3 days at a culture temperature of 28°C to obtain a matrix rich in fibrinolytic enzyme. After obtaining the fibrinolytic enzyme solution according to the method of Example 4, measure the fibrinolytic enzyme activity in the fibrinolytic enzyme solution. The measurement method is the same as that in Example 4. Conduct three parallel tests for each and take the average value. Use the medium without the addition of the readily available carbon source as the blank group, and the results are as follows Figure 4 。

[0086] 4. Determination method for appropriate type of readily available nitrogen source

[0087] According to the above specific steps of fermentation culture, add brewer's grains and wheat bran into a 250 mL Erlenmeyer flask at a mass ratio of 2:1. The readily available carbon source is 0.5% glucose, and the concentration of the readily available nitrogen source is 0.3%; the readily available nitrogen sources include yeast extract powder, ammonium chloride, urea, ammonium sulfate, and tryptone. When the ratio of the nutrient raw materials to water in the fermentation medium is 1 g:1 mL and the inoculation amount is 2.5×10 5 spores per gram of the fermentation medium, culture for 3 days at a culture temperature of 28°C to obtain a matrix rich in fibrinolytic enzyme. After obtaining the fibrinolytic enzyme solution according to the method of Example 4, measure the fibrinolytic enzyme activity in the fibrinolytic enzyme solution. The measurement method is the same as that in Example 4. Conduct three parallel tests for each and take the average value. Use the medium without the addition of the readily available nitrogen source as the blank group, and the results are as follows Figure 5 。

[0088] 5. Determination method for appropriate addition amount of readily available nitrogen source

[0089] According to the above specific steps of fermentation culture, add brewer's grains and wheat bran into a 250 mL Erlenmeyer flask at a mass ratio of 2:1. The readily available carbon source is 0.5% glucose, and the concentrations of the readily available nitrogen source are 0.1%, 0.3%, 0.5%, 0.7%, and 1%; the readily available nitrogen source is yeast extract powder. When the ratio of the nutrient raw materials to water in the fermentation medium is 1 g:1 mL and the inoculation amount is 2.5×10 5 spores per gram of the fermentation medium, culture for 3 days at a culture temperature of 28°C to obtain a matrix rich in fibrinolytic enzyme. After obtaining the fibrinolytic enzyme solution according to the method of Example 4, measure the fibrinolytic enzyme activity in the fibrinolytic enzyme solution. The measurement method is the same as that in Example 4. Conduct three parallel tests for each and take the average value. Use the medium without the addition of the readily available nitrogen source as the blank group, and the results are as follows Figure 6。

[0090] It can be seen that the fibrinolytic enzyme activity in the matrix prepared by the method of the present invention reaches 468.53 ± 7.19 U / g, and it can be used as a functional raw material for the preparation of products for preventing and / or treating thromboembolic diseases, having good application prospects.

[0091] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a plasmin-rich matrix, characterized in that: The method comprises the following steps: inoculating Neurocystis sp. L01 into a fermentation medium for fermentation to obtain a matrix rich in fibrinolytic enzyme; The classification name of the Neurospora intermedia L01 is Neurospora intermedia, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC No.41253 and a deposit date of July 23, 2024; The fermentation medium comprises nutrient raw materials and water, wherein the nutrient raw materials are composed of brewer's grains, bran and a quick-acting carbon source and / or a quick-acting nitrogen source.

2. The method according to claim 1, characterized in that The mass ratio of the brewer's grains to the bran is (5:1) to (1:5).

3. The method according to claim 1, characterized in that The mass concentration of the fast-acting carbon source is 0.5% to 2%; the fast-acting carbon source includes any one or more of glucose, fructose, D-maltose and sucrose.

4. The method according to claim 1, characterized in that: The mass concentration of the quick-acting nitrogen source is 0.1% to 1%; the quick-acting nitrogen source comprises any one or more of yeast extract powder, ammonium chloride, urea, ammonium sulfate and tryptone.

5. The method according to claim 1, characterized in that: The mass volume ratio of the nutrient raw material and water is 1g:0.25-2mL.

6. The method according to claim 1, characterized in that The fermentation temperature is 26°C to 34°C, the fermentation time is 2 days to 5 days; the inoculation amount is 1×10 5 Spores ~ 1×10 6 Spores.

7. A plasmin-rich matrix, characterized in that The matrix is ​​prepared by the method according to any one of claims 1 to 6.

8. Use of the method according to any one of claims 1 to 6 or the matrix according to claim 7 in producing plasmin or in preparing products for preventing and treating thrombosis.

9. A method for producing a fibrinolytic enzyme solution, characterized in that: The method comprises the following steps: mixing and leaching the matrix described in claim 7 with PBS buffer, and discarding the precipitate to obtain the fibrinolytic enzyme solution.

10. Use of the method according to claim 9 in preparing products for preventing and treating thrombosis.

Images