Preparation method of fermented soya bean plasmin composition

Abstract

The invention discloses a preparation method of a fermented soya bean plasmin composition. The invention relates to a fermented soya bean plasmin composition and a preparation method thereof. The fermented soya bean plasmin composition simultaneously contains fermented soya bean plasmin and fermented soya bean plasmin crude extraction residual solid dry powder. Wherein the fermented soya bean plasmin crude extraction residual solid dry powder is a byproduct for preparing fermented soya bean plasmin, can be used as a bitter taste flavoring agent, a solubilizer or a lipid regulator, and can reduce or avoid repeated addition of corresponding auxiliary components in functional foods or special medical foods. And secondary development and utilization of byproducts for preparing the plasmin of the fermented soya beans are realized, so that the additional value of a final product is improved while the emission of fermentation wastes is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of synthetic biology, and in particular to a method for preparing fermented black bean curd fibrinolytic enzyme. Background Art

[0002] In recent years, various bacterially produced plasmins from fermented foods have been reported. These bacterial plasmins exhibit high plasminogen activators, are generally safe, have minimal side effects, and are inexpensive, offering promising market prospects and medical value. The low prevalence of thrombotic disorders in Asians is closely linked to their frequent consumption of fermented foods such as natto, fermented black beans, soybean paste, and tempeh.

[0003] Douchi (fermented black beans) is a traditional fermented soybean food in my country. According to the Chinese Pharmacopoeia, it has the benefits of stimulating appetite, promoting digestion, dispelling stagnation, dispelling wind and cold, and preventing cardiovascular disease. In recent years, domestic researchers have discovered that thrombolytic enzymes are also present in Douchi (fermented black beans). Several strains have been isolated from these enzymes. The resulting Douchi fibrinolytic enzymes exhibit high fibrinolytic activity and excellent anticoagulant and thrombolytic effects. They do not dissolve blood cells or cause internal bleeding, have a long half-life in vivo, and are directly absorbed through the digestive tract. Furthermore, the enzyme is derived from traditional foods, is safe, reliable, non-toxic, and can be mass-produced using bacterial or fungal fermentation. Therefore, it has significant medicinal value and promising application prospects. Currently, there is an urgent need for industrialized methods for preparing Douchi fibrinolytic enzymes. Summary of the Invention

[0004] One object of the present invention is to provide a method for preparing a fermented black bean fibrinolytic enzyme composition, which is suitable for industrial production and can be prepared efficiently and cost-effectively. To address the differences between old and new soybeans, the present invention provides different process combinations for the pretreatment and fermentation steps, respectively, to improve the stability of the fermented product and reduce batch-to-batch variability.

[0005] Another object of the present invention is to provide a dry powder of the residual solids from a crude extract of fermented black bean fibrinolytic enzyme, a byproduct of the fermented black bean fibrinolytic enzyme composition, and its use. This dry powder can be used as a bitter flavoring agent, solubilizer, or lipid regulator, reducing or eliminating the need for repeated addition of corresponding auxiliary ingredients in functional foods or special medical foods. This present invention redevelops and utilizes the byproduct of fermented black bean fibrinolytic enzyme production, reducing the discharge of fermentation waste while increasing the added value of the final product.

[0006] The present invention specifically provides the following embodiments:

[0007] Embodiment 1. A method for preparing a fermented black bean curd fibrinolytic enzyme composition, characterized by comprising the following steps:

[0008] S1. Soybean pretreatment: Wash and swell the soybeans, then put them into the pretreatment equipment and steam them at a temperature of 100-105°C.

[0009] S2. Culture: Place the selected Bacillus subtilis strain and culture medium in a culture container and culture at 34-38°C for 20-48 hours to expand the strain;

[0010] S3, fermentation: put the cooked soybeans and cultured bacteria into the fermentation equipment, ferment at a temperature of 45-52 ° C and high humidity for 20-48 hours, and obtain a solid fermentation product.

[0011] S4, crudely extracting the solid fermentation product to obtain a crude product of fermented black bean curd fibrinolytic enzyme;

[0012] S5. Dry the remaining solids after the crude extraction in step S4 under vacuum at 48-50° C. and grind them to obtain a by-product dry powder;

[0013] S6, placing the crude fermented black bean curd fibrinolytic enzyme into a purification and separation system for secondary purification to obtain fermented black bean curd fibrinolytic enzyme with higher purity;

[0014] S7, drying the fermented black bean curd fibrinolytic enzyme prepared in S6;

[0015] S8. Mix the fermented black bean curd fibrinolytic enzyme dry powder prepared in S7 with the by-product dry powder obtained in step S5, and package them.

[0016] Embodiment 2. The method for preparing the fermented black bean fibrinolytic enzyme composition according to embodiment 1 is characterized in that the cooking temperature in step S1 is 100-105°C.

[0017] Embodiment 3. The method for preparing the fermented black bean fibrinolytic enzyme composition according to embodiment 1 or 2 is characterized in that the pretreatment in step S1 includes washing, packaging, swelling and steaming.

[0018] Embodiment 4. The method for preparing the fermented black bean fibrinolytic enzyme composition described in any one of Embodiments 1 to 3, when using aged soybeans harvested in the current season for more than 6 months, further adding a glucose or maltose solution with a concentration of 0.5% to 2% w / w to the surface of the soybeans steamed in S1, and then performing step S3.

[0019] Embodiment 5. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of embodiments 1 to 4,

[0020] When soybeans harvested 1-6 months in season are used, the liquid culture medium in step S2 comprises the following components: 2 kg of soybeans are washed, soaked, and ground into soy milk, water is added to make up to 100 L, and NaCl, CaCl2, and MgCl2 are added to form a solution of 0.1% w / w NaCl, 0.2 mg / kg CaCl2, and 0.3 mg / kg MgCl2; or

[0021] When aged soybeans harvested in the current season and stored for more than 6 months are used, the composition of the liquid culture medium in step S2 is as follows: 2 kg of soybeans are washed, soaked, and then ground into soy milk, water is added to 100 L, and glucose or sucrose, NaCl, CaCl2, and MgCl2 are added to form 0.5% to 2% w / w of glucose, 0.1% w / w of NaCl, 0.2 mg / kg of CaCl2, and 0.3 mg / kg of MgCl2, or 0.5% to 2% w / w of sucrose, 0.1% w / w of NaCl, 0.2 mg / kg of CaCl2, and 0.3 mg / kg of MgCl2.

[0022] Embodiment 6. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of Embodiments 1 to 5, characterized in that the culture temperature in step S2 is 34-38°C.

[0023] Embodiment 7. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of Embodiments 1 to 6, characterized in that the culture temperature in step S2 is 34-38°C and the number of days is 20-48 hours.

[0024] Embodiment 8. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of Embodiments 1 to 7 is characterized in that: the crude extraction described in step S4 is to crush the solid fermentation product and then extract it with physiological saline, and collect the supernatant by centrifugation, and add ammonium sulfate to the supernatant to a saturation of 60%, stir it thoroughly and place it at a low temperature overnight, and centrifuge it again to collect the precipitate, dialyze the precipitate for desalination, and then centrifuge it at 1000-3000r / min to obtain the supernatant.

[0025] Embodiment 9. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of Embodiments 1 to 8 is characterized in that: the secondary purification step in step S5 is to elute the centrifuged supernatant after dialysis in step S4 using a dextran gel chromatography column, the eluent is 7.4 phosphate buffer, and the components with absorbance at 280 nm are collected.

[0026] Embodiment 10. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of Embodiments 1 to 9, characterized in that the drying in step S7 is spray drying or freeze drying.

[0027] Embodiment 11. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of Embodiments 1 to 10, characterized in that: alginate and gelatin are further added in step S8.

[0028] Embodiment 12. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of Embodiments 1 to 11, characterized in that the packaging in step S8 is sterilized packaging.

[0029] Embodiment 13. Dry powder of solids remaining after crude extraction of fermented black bean curd fibrinolytic enzyme, characterized in that the dry powder of solids remaining after crude extraction of fermented black bean curd fibrinolytic enzyme is prepared by step S5 of any one of claims 1-8.

[0030] Embodiment 14. Use of dry powder of solids remaining after crude extraction of fermented black bean curd fibrinolytic enzyme in a flavoring agent, wherein the dry powder of solids remaining after crude extraction of fermented black bean curd fibrinolytic enzyme is prepared by step S5 of any one of embodiments 1-8.

[0031] Embodiment 15. Use of the dry powder of the residual solids from the crude extraction of fermented black bean curd fibrinolytic enzyme as a dissolution promoter, wherein the dry powder of the residual solids from the crude extraction of fermented black bean curd fibrinolytic enzyme is prepared by step S5 described in any one of Embodiments 1-8.

[0032] Embodiment 16. Use of the dry powder of the residual solids of the crude extract of fermented black bean fibrinolytic enzyme as a flavoring agent and dissolution promoter in an oral combination, wherein the dry powder of the residual solids of the crude extract of fermented black bean fibrinolytic enzyme is prepared by step S5 of any one of Embodiments 1-8.

[0033] Embodiment 17. A method for preparing dry powder of the remaining solids from the crude extraction of fermented black bean curd fibrinolytic enzyme. The dry powder of the remaining solids from the crude extraction of fermented black bean curd fibrinolytic enzyme is obtained by the step S5 described in any one of Embodiments 1-8.

[0034] Compared with the prior art, the preparation method of the fermented black bean curd fibrinolytic enzyme composition provided by the present invention has the following beneficial effects:

[0035] 1. Suitable for industrial production, capable of preparing fermented black bean fibrinolytic enzyme composition efficiently and at low cost

[0036] 2. To address the differences between old and new beans, different process combinations are provided in the pretreatment and fermentation steps, improving the stability of the fermented product and reducing batch-to-batch variability.

[0037] 3. A new application has been developed for the dry powder of the residual solids from the crude fermented ... BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A test chart showing the solubilization effect of the residual solids from the crude extract of fermented black bean curd fibrinolytic enzyme on curcumin. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0040] Example 1

[0041] The fermented black bean curd fibrinolytic enzyme composition was prepared using the following steps:

[0042] S1. Soybean pretreatment: Soybeans harvested 1-6 months into the season are cleaned, then washed, packaged, soaked, and steamed in containers at a temperature of 100°C.

[0043] S2. Seed Culture: Wash and soak 2 kg of soybeans, grind them into soy milk, add water to 100 L, and add NaCl, CaCl2, and MgCl2 to prepare a culture solution with 0.1% w / w NaCl, 0.2 mg / kg CaCl2, and 0.3 mg / kg MgCl2 as the seed culture. Place the Bacillus subtilis strain and culture medium in a culture container and incubate at 35°C for 36 hours to expand the production of the strain.

[0044] S3, fermentation: put the soybeans pretreated in step S1 and the bacterial liquid obtained in step S2 into a fermentation device, and ferment them at a temperature of 45-50°C for 48 hours to obtain a solid fermentation product.

[0045] S4, crushing the solid fermentation product and extracting it with physiological saline, and centrifuging and collecting the supernatant, and adding ammonium sulfate to the supernatant to a saturation of 60%, stirring thoroughly and then placing it at a low temperature overnight, and centrifuging again to collect the precipitate, dialyzing the precipitate for desalination, and centrifuging again at 2000 r / min to obtain the supernatant to obtain a crude product of fermented black bean curd fibrinolytic enzyme;

[0046] S5. Dry the remaining solids after extraction with physiological saline in step S4 under vacuum at 48-50° C. and grind them to obtain a by-product dry powder;

[0047] S6, eluting the centrifugal supernatant after dialysis in step S4 using a dextran gel chromatography column, using a 7.4% phosphate buffer as the eluent, collecting the fraction with an absorbance of 280 nm to obtain purified fermented black bean curd fibrinolytic enzyme;

[0048] S7, spray-drying the fermented black bean curd fibrinolytic enzyme prepared in S6 at 35-40 degrees Celsius to obtain fermented black bean curd fibrinolytic enzyme dry powder;

[0049] The fermented black bean curd fibrinolytic enzyme dry powder prepared in S8 and S7 is mixed with the by-product dry powder obtained in step S5 and packaged.

[0050] Example 2

[0051] The fermented black bean curd fibrinolytic enzyme composition was prepared using the following steps:

[0052] S1. Soybean pretreatment: 18-month-old soybeans were cleaned, packaged, soaked, and steamed in containers at 100°C. After steaming, 2% w / w glucose was sprayed on the surface of the soybeans.

[0053] S2. Seed Culture: Wash and soak 2 kg of soybeans, then grind them into soy milk. Add water to 100 L. Add glucose, NaCl, CaCl₂, and MgCl₂ to create a culture solution containing 1.5% w / w glucose, 0.1% w / w NaCl, 0.2 mg / kg CaCl₂, and 0.3 mg / kg MgCl₂ as the seed culture. Place the Bacillus subtilis strain and culture medium in a culture container and incubate at 35°C for one day to expand the production of the strain.

[0054] S3, fermentation: the soybeans pre-treated in step S1 and the bacterial liquid obtained in step S2 are placed in a fermentation device and fermented at 50°C for 48 hours to obtain a solid fermentation product.

[0055] S4, crushing the solid fermentation product and extracting it with physiological saline, and centrifuging and collecting the supernatant, and adding ammonium sulfate to the supernatant to a saturation of 60%, stirring thoroughly and then placing it at a low temperature overnight, and centrifuging again to collect the precipitate, dialyzing the precipitate for desalination, and centrifuging again at 3000 r / min to obtain the supernatant to obtain a crude product of fermented black bean curd fibrinolytic enzyme;

[0056] S5. Dry the remaining solids after extraction with physiological saline in step S4 under vacuum at 48-50° C. and grind them to obtain a by-product dry powder;

[0057] S6, eluting the centrifugal supernatant after dialysis in step S4 using a dextran gel chromatography column, using a 7.4% phosphate buffer as the eluent, collecting the fraction with an absorbance of 280 nm to obtain a high-purity fermented black bean fibrinolytic enzyme;

[0058] S7, spray-drying the fermented black bean curd fibrinolytic enzyme prepared in S6 at 35-40 degrees Celsius to obtain fermented black bean curd fibrinolytic enzyme dry powder;

[0059] The fermented black bean curd fibrinolytic enzyme dry powder prepared in S8 and S7 is mixed with the by-product dry powder obtained in step S5 and packaged.

[0060] Example 3

[0061] The fermented black bean curd fibrinolytic enzyme composition was prepared using the following steps:

[0062] S1. Soybean pretreatment: 18-month-old soybeans were cleaned, then washed, packaged, soaked, and steamed at 100° C. 2% w / w maltose was sprayed on the surface of the steamed soybeans.

[0063] S2. Culture: Wash and soak 2 kg of soybeans, then grind them into soy milk. Add water to 100 L. Add sucrose, NaCl, CaCl₂, and MgCl₂ to create a culture solution containing 1.5% w / w sucrose, 0.1% w / w NaCl, 0.2 mg / kg CaCl₂, and 0.3 mg / kg MgCl₂ as the seed culture. Place the Bacillus subtilis strain and culture medium in a culture container and incubate at 35°C for one day to expand the production of the strain.

[0064] S3, fermentation: the soybeans pre-treated in step S1 and the bacterial liquid obtained in step S2 are placed in a fermentation device and fermented at 50°C for 48 hours to obtain a solid fermentation product.

[0065] S4, crushing the solid fermentation product and extracting it with physiological saline, and centrifuging and collecting the supernatant, and adding ammonium sulfate to the supernatant to a saturation of 60%, stirring thoroughly and then placing it at a low temperature overnight, and centrifuging again to collect the precipitate, dialyzing the precipitate for desalination, and centrifuging again at 2000 r / min to obtain the supernatant to obtain a crude product of fermented black bean curd fibrinolytic enzyme;

[0066] S5. Dry the remaining solids after extraction with physiological saline in step S4 under vacuum at 48-50° C. and grind them to obtain a by-product dry powder;

[0067] S6, eluting the centrifugal supernatant after dialysis in step S4 using a dextran gel chromatography column, using a 7.4% phosphate buffer as the eluent, collecting the fraction with an absorbance of 280 nm to obtain a high-purity fermented black bean fibrinolytic enzyme;

[0068] S7, spray-drying the fermented black bean curd fibrinolytic enzyme prepared in S6 at 35-40 degrees Celsius to obtain fermented black bean curd fibrinolytic enzyme dry powder;

[0069] The fermented black bean curd fibrinolytic enzyme dry powder prepared in S8 and S7 is mixed with the by-product dry powder obtained in step S5 and packaged.

[0070] Example 4

[0071] Optimization of soybean usage in step S2

[0072] Experimental steps: According to step S2 of Example 1, 0.5 kg, 2 kg, 5 kg and 20 kg of soybeans were weighed respectively, ground into soy milk and poured into a liquid fermentation tank. Under the same conditions, liquid fermentation was carried out to expand the strain and solid fermentation was carried out to produce fermented black bean curd fibrinolytic enzyme according to the steps of Example 1. The amount of mycelium was observed after 20 hours of solid fermentation.

[0073] Observation Results: As shown in the table above, the relationship between fermentation performance and the amount of soy culture medium used was tested. Fermentation performance was determined by the simple measurement of mycelium production. The experiment revealed that a larger amount of culture medium was not necessarily better. When the culture medium dosage was 0.5 kg, mycelium was relatively small. However, even at 20 kg, mycelium was still relatively small. When the culture medium dosage was between 2 and 5 kg, mycelium was more abundant.

[0074] Table 1

[0075]

[0076]

[0077] Example 5

[0078] Flavor correction effect of the residual solid powder of the crude extract of fermented black bean curd fibrinolytic enzyme prepared in step S5

[0079] Experimental Procedure: Weigh 5 portions of 1g curcumin and add 0.5g, 1g, 2g, 4g, and 8g of the remaining solid powder of fermented black bean curd fibrinolytic enzyme, respectively, and mix thoroughly. The tester then pours 0.3g of the mixed sample directly into their mouth and records the taste. After recording, they spit out the sample, rinse their mouth three times with room temperature drinking water, and wait 5 minutes before testing the next sample.

[0080] Observation Results: As shown in the table above, taste tests were conducted on mixtures of curcumin and dry powder of the residual solids from the crude extract of fermented black bean fibrinolytic enzyme at varying ratios. Plant extracts often have a bitter taste, and this can often be masked by adding excipients such as black bean powder, making the product more palatable. This experiment masked the bitterness of curcumin by adding dry powder of the residual solids from the crude extract of fermented black bean fibrinolytic enzyme, while maximizing the use of all available materials. The test showed that as the amount of dry powder increased, the bitterness of the curcumin decreased and the bean flavor became more pronounced. The test results are summarized in Table 2.

[0081] Table 2

[0082]

[0083] Example 6

[0084] Verification of the effect of the residual solids of the crude extract of fermented black bean curd fibrinolytic enzyme prepared in step S5 as a solubilizing agent

[0085] The residual solids of the crude extract of fermented black bean curd fibrinolytic enzyme contain soybean lecithin. Soybean lecithin was used to simulate the residual solids of the crude extract of fermented black bean curd fibrinolytic enzyme, and the solubilization effect of the residual solids of the crude extract of fermented black bean curd fibrinolytic enzyme on the insoluble substance curcumin was indirectly verified.

[0086] Experimental procedures: Weigh 1g of curcumin and add it to 10mL of water, denoted as A; weigh 1g of curcumin and 1g of soy lecithin and add them to 10mL of water, denoted as B; weigh 1g of curcumin and 2g of soy lecithin and add them to 10mL of water, denoted as C. A, B, and C were all thoroughly shaken on a vortex shaker and then allowed to stand.

[0087] Observation results: Figure 1 As shown (from left to right: A, B, C), after A was allowed to stand and then inverted, it was observed that more insoluble curcumin was deposited at the bottom of the test tube, and the liquid was light yellow and relatively transparent; after B was allowed to stand and then inverted, it was observed that less insoluble curcumin was deposited at the bottom of the test tube, and the liquid was dark yellow and opaque; after C was allowed to stand and then inverted, it was observed that there was almost no curcumin deposited at the bottom of the test tube, and the liquid was dark yellow (darker than B) and opaque.

[0088] Throughout this specification, references to terms such as "one embodiment," "example," and "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0089] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to specific embodiments. Obviously, many modifications and variations are possible based on the contents of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for preparing a fermented black bean curd fibrinolytic enzyme composition, characterized in that: The following steps are involved: S1. Soybean pretreatment: Wash and swell the soybeans, then put them into the pretreatment equipment and steam them at a temperature of 100-105°C. S2. Culture: Place the selected Bacillus subtilis strain and culture medium in a culture container and culture at 34-38°C for 20-48 hours to expand the strain; S3, fermentation: put the cooked soybeans and cultured bacteria into the fermentation equipment, ferment at a temperature of 45-52 ° C and high humidity for 20-48 hours, and obtain a solid fermentation product. S4, crudely extracting the solid fermentation product to obtain a crude product of fermented black bean curd fibrinolytic enzyme; S5. Dry the remaining solids after the crude extraction in step S4 under vacuum at 48-50° C. and grind them to obtain a by-product dry powder; S6, placing the crude fermented black bean curd fibrinolytic enzyme into a purification and separation system for secondary purification to obtain fermented black bean curd fibrinolytic enzyme with higher purity; S7, drying the fermented black bean curd fibrinolytic enzyme prepared in S6; S8. Mix the fermented black bean curd fibrinolytic enzyme dry powder prepared in S7 with the by-product dry powder obtained in step S5, and package them.

2. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to claim 1, wherein: The cooking temperature in step S1 is 100-105°C.

3. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to claim 1 or 2, characterized in that: The pretreatment in step S1 includes cleaning, packaging, swelling and steaming.

4. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of claims 1 to 3, wherein when aged soybeans harvested in the current season and stored for more than 6 months are used, a glucose or maltose solution with a concentration of 0.5%-2% w / w is further added to the surface of the soybeans cooked in S1, and then step S3 is performed.

5. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of claims 1 to 4, When soybeans harvested 1-6 months in season are used, the liquid culture medium in step S2 comprises the following components: 2 kg of soybeans are washed, soaked, and ground into soy milk, water is added to make up to 100 L, and NaCl, CaCl2, and MgCl2 are added to form a solution of 0.1% w / w NaCl, 0.2 mg / kg CaCl2, and 0.3 mg / kg MgCl2; or When aged soybeans harvested in the current season and stored for more than 6 months are used, the composition of the liquid culture medium in step S2 is as follows: 2 kg of soybeans are washed, soaked, and then ground into soy milk, water is added to 100 L, and glucose or sucrose, NaCl, CaCl2, and MgCl2 are added to form 0.5% to 2% w / w of glucose, 0.1% w / w of NaCl, 0.2 mg / kg of CaCl2, and 0.3 mg / kg of MgCl2, or 0.5% to 2% w / w of sucrose, 0.1% w / w of NaCl, 0.2 mg / kg of CaCl2, and 0.3 mg / kg of MgCl2.

6. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of claims 1 to 5, characterized in that: The culture temperature in step S2 is 34-38°C.

7. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of claims 1 to 6, characterized in that: The culture temperature in step S2 is 34-38° C., and the culture duration is 20-48 hours.

8. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of claims 1 to 7, characterized in that: The crude extraction described in step S4 is to crush the solid fermentation product and extract it with physiological saline, and then centrifuge to collect the supernatant, and add ammonium sulfate to the supernatant to a saturation of 60%, stir it thoroughly and place it at low temperature overnight, and centrifuge it again to collect the precipitate, dialyze the precipitate for desalination, and then centrifuge it at 1000-3000r / min to obtain the supernatant.

9. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of claims 1 to 8, characterized in that: The secondary purification step in step S5 is to elute the centrifugal supernatant after dialysis in step S4 using a dextran gel chromatography column, the eluent is 7.4 phosphate buffer, and the component with absorbance at 280 nm is collected.

10. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of claims 1 to 9, characterized in that: The drying in step S7 is spray drying or freeze drying.

11. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of claims 1 to 10, characterized in that: In the step S8, alginate and gelatin are further added.

12. The method for preparing the fermented black bean curd fibrinolytic enzyme composition according to any one of claims 1 to 11, characterized in that: The packaging in step S8 is sterilized packaging.

13. Dry powder of residual solids from crude extraction of fermented black bean curd fibrinolytic enzyme, characterized in that: The dry powder of the remaining solids from the crude extraction of fermented black bean curd fibrinolytic enzyme is prepared by step S5 according to any one of claims 1 to 8.

14. Use of dry powder of residual solids from crude extraction of fermented black bean curd fibrinolytic enzyme in a flavoring agent, wherein the dry powder of residual solids from crude extraction of fermented black bean curd fibrinolytic enzyme is prepared by step S5 according to any one of claims 1 to 8.

15. Use of dry powder of residual solids from crude extraction of fermented black bean curd fibrinolytic enzyme as a dissolution promoter, wherein the dry powder of residual solids from crude extraction of fermented black bean curd fibrinolytic enzyme is prepared by step S5 according to any one of claims 1 to 8.

16. Use of a dry powder of residual solids from a crude extract of fermented black beans fibrinolytic enzyme as a flavoring agent and dissolution promoter in an oral combination, wherein the dry powder of residual solids from a crude extract of fermented black beans fibrinolytic enzyme is prepared by step S5 according to any one of claims 1 to 8.

17. A method for preparing dry powder of residual solids from crude extracts of fermented black bean curd fibrinolytic enzyme, wherein the dry powder of residual solids from crude extracts of fermented black bean curd fibrinolytic enzyme is obtained by the step S5 described in any one of embodiments 1-8.

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