Neurospora intermedia and application thereof in production of plasmin or preparation of products for preventing and treating thrombus

Abstract

The invention provides neurospora intermedia and application of the neurospora intermedia in production of plasmin or preparation of products for preventing and treating thrombus, and belongs to the technical field of plasmin preparation. The plasmin-producing neurospora intermedia provided by the invention is neurospora intermedia L01, and the preservation number of the neurospora intermedia is CGMCC (China General Microbiological Culture Collection Center) No.41253. The neurospora intermedia L01 provided by the invention can produce plasmin at high yield, and the produced plasmin has the advantage of high activity. According to the plasmin-rich matrix provided by the invention, a neurospora intermedia strain L01 is used as a fermentation strain, corn flour and peanut cake meal are used as a culture medium, and the matrix prepared through a fermentation step is rich in mycelia and plasmin, and the plasmin activity reaches 305.13 + / -4.68 U / g. The plasmin-rich matrix raw material provided by the invention is safe and reliable, short in preparation time and easy to realize, can be used as a functional raw material in products for preventing and / or treating thrombosis 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 Neurospora intermedia and its application in producing plasmin or preparing products for preventing and treating thrombosis. Background Art

[0002] At present, with the improvement of people's living standards and the change of lifestyle, the incidence of thrombus diseases has been increasing year by year. Thrombosis is the main cause of stroke, myocardial infarction, venous thromboembolism, etc., with extremely high fatality and disability rates. Currently, the most commonly used thrombolytic drug in clinical practice is tissue-type plasminogen activator. The commercially available thrombolytic drugs on the market currently have disadvantages such as low specificity, easy bleeding, short half-life, and drug resistance. Therefore, developing thrombolytic compounds with high efficiency, low toxicity, fast drug efficacy, and high specificity will be of great significance to human health. Plasmin is an important protease and has various functions in preventing and treating thrombosis, such as dissolving thrombus, preventing thrombus expansion, and maintaining vascular patency. Plasmin is of great significance for maintaining the normal coagulation-fibrinolysis balance in the human body, preventing thrombosis formation, and dissolving the formed thrombus, and is one of the key factors to ensure the health of the cardiovascular system.

[0003] Neurospora intermedia is a filamentous fungus with orange-yellow spores. Due to its extremely high safety and rapid growth, Neurospora intermedia has gradually become an important strain for fermented foods. It can be used to ferment and prepare various metabolites, such as producing cellulase, producing carotene, hemicellulase, xylose reductase, etc. However, there is currently no report on obtaining highly active plasmin from the mycelium of Neurospora intermedia. Summary of the Invention

[0004] In view of this, one of the purposes of the present invention is to provide a Neurospora intermedia that can produce high yields of plasmin and has high activity of the produced plasmin and its application.

[0005] Another purpose of the present invention is to provide a matrix rich in plasmin and its application.

[0006] The third purpose of the present invention is to provide a method for producing a plasmin solution and its application.

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

[0008] The present invention provides a Neurospora intermedia that produces plasmin. The Neurospora intermedia is Neurospora intermedia L01, classified and named as Neurospora intermedia, deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 41253, and the deposit time is July 23, 2024.

[0009] The present invention also provides the use of the above-mentioned Neurospora intermedia in the production of fibrinolytic enzyme or in the preparation of products for preventing and treating thrombosis.

[0010] The present invention also provides a matrix rich in fibrinolytic enzyme. The preparation method of the matrix comprises the following steps: inoculating the above-mentioned Neurospora intermedia into a fermentation medium for fermentation to obtain a matrix rich in fibrinolytic enzyme.

[0011] Preferably, the fermentation medium comprises nutrient raw materials and water. The nutrient raw materials include corn flour, peanut cake meal, and a quick-acting carbon source. The quick-acting carbon source includes any one or more of glucose, fructose, D-maltose, and sucrose.

[0012] Preferably, the mass ratio of the corn flour to the peanut cake meal is (5:1) to (1:5); the mass concentration of the quick-acting carbon source is 0.5% to 2%.

[0013] Preferably, the mass-to-volume ratio of the nutrient raw materials to water is 1 g: 0.25 to 2 mL.

[0014] Preferably, the temperature of the fermentation is 26°C to 34°C, and the time of the fermentation is 2 days to 5 days.

[0015] Preferably, the inoculation amount is 1×10 5 spores to 9.9×10 6 spores per gram of the fermentation medium.

[0016] The present invention also provides a method for producing a fibrinolytic enzyme solution, which comprises the following steps: mixing and leaching the above-mentioned matrix with PBS buffer solution, and discarding the precipitate to obtain a fibrinolytic enzyme solution.

[0017] The present invention also provides the use of the above-mentioned matrix or the above-mentioned method in the preparation of products for preventing and treating thrombosis.

[0018] Advantages of the present invention:

[0019] The present invention provides a Neurospora intermedia L01, which can produce a high yield of fibrinolytic enzyme, and the produced fibrinolytic enzyme has the advantage of high activity. The matrix rich in fibrinolytic enzyme provided by the present invention uses the Neurospora intermedia strain L01 as a fermentation strain, uses corn flour and peanut cake meal as a culture medium, and through a fermentation step, the obtained matrix is rich in Neurospora intermedia L01 mycelia and fibrinolytic enzyme, and the fibrinolytic enzyme activity reaches 305.13±4.68 U / g. The matrix rich in fibrinolytic enzyme provided by the present invention has safe and reliable raw materials, short preparation time, and is easy to implement, and can be used as a functional raw material in products for preventing and / or treating thrombus diseases, and has good application prospects.

[0020] Depositing description

[0021] The Neurospora intermedia strain L01 of the present invention, classified and named as Neurospora intermedia, was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on July 23, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 41253. Description of the Drawings

[0022] Figure 1 It is the result 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 physiological saline.

[0023] Figure 2 It is the effect of different ratios of corn flour and peanut cake meal 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, and the same letters on the column indicate no significant differences, p>0.05.

[0024] Figure 3 It is the effect of the type and addition amount of rapid-acting carbon source 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, and the same letters on the column indicate no significant differences, p>0.05.

[0025] Figure 4 It is the effect of fermentation temperature 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, and the same letters on the column indicate no significant differences, p>0.05.

[0026] Figure 5 It is the effect of fermentation time 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, and the same letters on the column indicate no significant differences, p>0.05.

[0027] Figure 6 It is the effect of different ratios of nutrient raw materials and water 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, and the same letters on the column indicate no significant differences, p>0.05.

[0028] Figure 7 It is the effect of inoculum size 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, and the same letters on the column indicate no significant differences, p>0.05. Detailed implementation mode

[0029] The present invention provides a Neurospora intermedia strain capable of producing plasmin. The Neurospora intermedia strain is 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, with the preservation number of CGMCC No. 41253 and the preservation date of July 23, 2024.

[0030] 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 plasmin and high activity of the produced plasmin.

[0031] The present invention also provides the application of the above-mentioned Neurospora intermedia strain 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.

[0032] The present invention provides a matrix rich in plasmin. The preparation method of the matrix includes the following steps: inoculating the above-mentioned Neurospora intermedia strain into a fermentation medium for fermentation to obtain a matrix rich in plasmin.

[0033] In the present invention, the Neurospora intermedia strain 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 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 be kept 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 be kept natural. In the present invention, the temperature of the seed culture is preferably 26-34 °C, more preferably 28-30 °C, and the time of the seed culture is preferably 2-5 days, more preferably 3-4 days.

[0034] In the present invention, the fermentation medium preferably comprises a nutrient raw material and water. The nutrient raw material includes corn flour, peanut cake meal, and a quick-acting carbon source. The quick-acting carbon source includes any one or more of glucose, fructose, D-maltose, and sucrose. In the present invention, the mass ratio of the corn flour to the peanut cake meal is preferably (5:1) to (1:5), more preferably (4:1) to (1:4); the mass concentration of the quick-acting carbon source is preferably 0.5% to 2%, more preferably 1% to 1.4%. The mass concentration of the quick-acting carbon source refers to the mass concentration of the quick-acting carbon source in the nutrient raw material. In the present invention, the mass-volume ratio of the nutrient raw material to water is preferably 1 g: 0.25 to 2 mL, more preferably 1 g: 0.5 to 1.5 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 9.9×10 6 spores per g of the fermentation medium, and more preferably 3×10 5 spores to 8×10 6 spores per g of the fermentation medium.

[0035] The present invention also provides a method for producing a fibrinolytic enzyme solution, comprising the following steps: mixing and extracting the above-mentioned substrate with PBS buffer solution, and discarding the precipitate to obtain the fibrinolytic enzyme solution.

[0036] In the present invention, the mass-volume ratio of the substrate 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 10,000 r / min.

[0037] The present invention also provides the application of the above-mentioned substrate or the above-mentioned method in the preparation of a product for preventing and treating thrombosis. In the present invention, the type of the product preferably includes drugs.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] In the following embodiments, unless otherwise specified, all are conventional methods.

[0040] The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0041] Example 1

[0042] Screening and Isolation of Neurospora intermedia L01 of the Present Invention

[0043] Take an appropriate amount of traditional fermented soybean paste from Northeast China and dilute it serially in sterile water, then coat it on PDA medium to obtain a series of strains.

[0044] Take appropriate amounts of the strains and inoculate them in fibrin plates respectively, culture at 37°C for 24 hours, and observe whether there is a dissolution zone. If there is a dissolution zone, it indicates that the strain can secrete fibrinolytic enzyme. According to the results of the size of the dissolution zone, select the strain with the largest dissolution zone and send it to Shanghai Sangon Biological Engineering Co., Ltd. for sequencing and identification of the strain. After identification, it is Neurospora intermedia, preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the preservation number of CGMCC No. 41253 and the preservation time of July 23, 2024.

[0045] Example 2

[0046] A matrix rich in fibrinolytic enzyme, which is obtained by the following method:

[0047] (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, pH natural) into the freeze-dried tube containing the Neurospora intermedia strain 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 at 30°C and 150 r / min on a shaker 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 at 30°C and 150 r / min on a shaker for 3 days, and transfer it according to the above method to the 4th generation to obtain the activated Neurospora intermedia L01.

[0048] (2) Inoculate the activated Neurospora intermedia L01 obtained 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, pH natural), culture at 30°C for 3 days, and rinse the inclined plane with sterile water to obtain the Neurospora intermedia L01 seed liquid.

[0049] (3) Preparation of fermentation medium: Mix the nutrient raw materials and water in a ratio of 1 g:0.25 mL. The nutrient raw materials consist of corn flour, peanut cake meal and sucrose. The mass ratio of corn flour to peanut cake meal is 4:1, and the mass concentration of sucrose is 0.5% (here 0.5% refers to the mass concentration of sucrose in the nutrient raw materials), and the pH is the natural pH.

[0050] (4) Inoculate the Neurospora intermedia L01 seed liquid obtained in step (2) into the fermentation medium at an inoculation amount of 2.5×10 per g of fermentation medium5 Inoculate the fermentation medium obtained in step (3) with the amount of spores of 5 , and carry out static light-shielded fermentation culture at 30 °C for 3 days to obtain a matrix rich in fibrinolytic enzyme, and the matrix is also rich in Neurospora intermedia mycelium.

[0051] Example 3

[0052] A matrix rich in fibrinolytic enzyme, which is obtained by the following method:

[0053] Steps (1) and (2) are the same as steps (1) and (2) of Example 2.

[0054] (3) Preparation of fermentation medium: Mix the nutrient raw materials and water in an amount of 1 g: 0.5 mL. The nutrient raw materials consist of corn flour, peanut cake meal and sucrose. The mass ratio of corn flour to peanut cake meal is 5:1, and the mass concentration of sucrose is 0.5% (here 0.5% refers to the mass concentration of sucrose in the nutrient raw materials), and the pH is the natural pH.

[0055] (4) Inoculate the Neurospora intermedia L01 seed liquid obtained in step (2) into the fermentation medium obtained in step (3) at an amount of 5×10 5 spores per gram of fermentation medium, and carry out static light-shielded 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 mycelium.

[0056] Example 4

[0057] A matrix rich in fibrinolytic enzyme, which is obtained by the following method:

[0058] Steps (1) and (2) are the same as steps (1) and (2) of Example 2.

[0059] (3) Preparation of fermentation medium: Mix the nutrient raw materials and water in an amount of 1 g: 1.5 mL. The nutrient raw materials consist of corn flour, peanut cake meal and sucrose. The mass ratio of corn flour to peanut cake meal is 1:5, and the mass concentration of sucrose is 0.5% (here 0.5% refers to the mass concentration of sucrose in the nutrient raw materials), and the pH is the natural pH.

[0060] (4) Inoculate the Neurospora intermedia L01 seed liquid obtained in step (2) into the fermentation medium obtained in step (3) at an amount of 7.5×10 5 spores per gram of fermentation medium, and carry out static light-shielded 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 mycelium.

[0061] Example 5

[0062] A method for producing fibrinolytic enzyme solution, which consists of the following steps: Mix the matrix obtained in Example 2 with PBS buffer at a mass-to-volume ratio of 1 g:5 mL, extract at 25 °C for 2 h, then centrifuge at 4 °C and 10,000 r / min for 10 min, and discard the precipitate to obtain the fibrinolytic enzyme solution.

[0063] Determination of fibrinolytic enzyme activity in the fibrinolytic enzyme solution:

[0064] (1) Preparation of fibrin plate:

[0065] ① Fibrinogen solution (100 mL)

[0066] Prepare 100 mL of 0.04 mol / L sodium phosphate buffer (mix a solution of Na 2 HPO 4 ·12H 2 O solution and 0.04 mol / L NaH 2 PO 4 ·2H 2 O solution to make them fully mixed); adjust the pH of the volumetric sodium phosphate buffer solution 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;

[0067] ② Thrombin solution

[0068] 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;

[0069] 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.

[0070] (2) Determination of urokinase standard curve

[0071] 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 urokinase standard product at different concentrations and spot them on the fibrin plate, with three parallels for each, place them in an incubator at 37 °C for 6 h; use an electronic vernier caliper to measure the diameter of the lysis circle, 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);

[0072] (3) Determination of the plasmin activity of the sample to be tested

[0073] Take 10 μL of the plasmin solution and spot it on the prepared fibrin plate. After placing it in an incubator at 37 °C for 6 h, read the diameter. Make three parallels and substitute the values into the urokinase standard curve. After calculation, the plasmin enzyme activity reaches 305.13 ± 4.68 U / g. At the same time, use normal saline as the control group to conduct the same experiment, and the measurement results are as Figure 1 shown.

[0074] 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.

[0075] Example 6

[0076] The difference from Example 2 is that in step (3), the mass ratio of corn flour to peanut cake meal in the fermentation medium is 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5. The nutrient raw material does not contain sucrose, and the mass-volume ratio of the nutrient raw material to water is 1 g:1 mL. The rest are the same as in Example 2 to obtain a matrix rich in plasmin.

[0077] After obtaining the plasmin solution according to the method of Example 5, measure the plasmin activity in the plasmin solution. The measurement method is the same as in Example 5. Make three parallels for each and take the average value. The results obtained are as Figure 2 shown.

[0078] Example 7

[0079] The difference from Example 6 is that the mass ratio of corn flour to peanut cake meal in the fermentation medium is 4:1, and a quick-acting carbon source is added to the nutrient raw material. The concentrations of the quick-acting carbon source are 0.5%, 1.0%, 1.5%, and 2.0% respectively; the quick-acting carbon source is glucose, fructose, D-maltose or sucrose. The rest are the same as in Example 6, and the results obtained are as Figure 3 shown.

[0080] Example 8

[0081] The difference from Example 7 is that the quick-acting carbon source is 0.5% sucrose, and the fermentation culture temperature is adjusted to 26 °C, 28 °C, 30 °C, 32 °C, and 34 °C respectively. The rest are the same as in Example 7, and the results obtained are as Figure 4 shown.

[0082] Example 9

[0083] The difference from Example 8 is that the fermentation culture temperature is 30 °C, and the fermentation culture time is 2 d, 3 d, 4 d, and 5 d respectively. The rest are the same as in Example 8, and the results obtained are as Figure 5 shown.

[0084] Example 10

[0085] The difference from Example 9 is that the fermentation culture time is 3 d, and the ratios of nutrient raw materials to water in the fermentation medium are 1:0.25, 1:0.5, 1:1, 1:1.5, and 1:2 respectively. The rest are the same as in Example 9, and the obtained results are as Figure 6 shown.

[0086] Example 11

[0087] The difference from Example 10 is that the ratio of nutrient raw materials to water in the fermentation medium is 1:0.25, and the inoculation amounts are 1.25×10 5 spores / g, 2.5×10 5 spores / g, 5×10 5 spores / g, 7.5×10 5 spores / g, 1×10 6 spores / g respectively. The rest are the same as in Example 10, and the obtained results are as Figure 7 shown.

[0088] 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 refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A fibrinolytic enzyme-producing Neurocystis sp., characterized in that: The intermedia Neurospora is Intermedia Neurospora L01, classified and named Neurospora intermedia, and deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.41253 and the deposit time July 23, 2024.

2. Use of the Neurocystis sp. according to claim 1 in producing plasmin or in preparing products for preventing and treating thrombosis.

3. A plasmin-rich matrix, characterized in that The preparation method of the matrix comprises the following steps: inoculating the Intercellular Sporangium described in claim 1 into a fermentation medium for fermentation to obtain a matrix rich in plasmin.

4. The matrix according to claim 3, characterized in that The fermentation medium comprises nutrient raw materials and water, wherein the nutrient raw materials comprise corn flour, peanut cake and a quick-acting carbon source, and the quick-acting carbon source comprises any one or more of glucose, fructose, D-maltose and sucrose.

5. The matrix according to claim 4, characterized in that The mass ratio of the corn flour to the peanut cake is (5:1) to (1:5); the mass concentration of the quick-acting carbon source is 0.5% to 2%.

6. The matrix according to claim 4, characterized in that The mass volume ratio of the nutrient raw material and water is 1g:0.25-2mL.

7. The matrix according to claim 3, characterized in that The fermentation temperature is 26° C. to 34° C., and the fermentation time is 2 to 5 days.

8. The matrix according to claim 3, characterized in that The inoculation amount is 1×10 5 spores~9.9×10 6 Spores.

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 any one of claims 3 to 8 with PBS buffer, and discarding the precipitate to obtain a fibrinolytic enzyme solution.

10. Use of the matrix according to any one of claims 3 to 8 or the method according to claim 9 in the preparation of a product for preventing and treating thrombosis.

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