A production process for highly active nattokinase

Abstract

This invention discloses a production process for highly active nattokinase. The process includes the preparation of seed liquid, raw material processing and preparation, liquid fermentation, and spray drying. In this process, the soybean milk is treated with 0.05%–4.5% neutral protease for enzymatic hydrolysis, which effectively improves the enzyme activity of nattokinase. The spray drying process, with the addition of 20%–35% maltodextrin and inulin as protective agents, offers advantages such as minimal enzyme activity loss at high temperatures, low energy consumption, and high spray drying efficiency. The nattokinase spray-dried powder produced by this process has high activity and high viable cell count, with an enzyme activity reaching 62900 IU / g and a colony count of 7.4*10⁻⁶. 11 CFU / g or higher. Compared with other fermentation methods, this method has simpler raw material processing, lower cost, lower energy consumption, less pollution, higher production efficiency, and simpler process. Therefore, this invention has broad market prospects and industrial production application value.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a production process for highly active nattokinase. Background Technology

[0002] Currently, reports indicate that thrombotic diseases account for one-third of all deaths worldwide. As people's living standards improve, their diets frequently include foods high in cholesterol, fat, and sugar. Long-term consumption of such foods can lead to thrombosis, negatively impacting health. Thrombosis is caused by the adhesion of four types of proteins in the blood (red blood cells, fibrin, platelets, and white blood cells), resulting in blood clots, blockages, and impaired organ function. Therefore, these patients may die or suffer disability or paralysis. Treatment for thrombosis both domestically and internationally typically involves three methods: conservative therapy, thrombolysis, and surgery. Thrombolysis is the most commonly used. Currently, commercially available thrombolytic drugs are mainly traditional medications such as urokinase and lumbrokinase. These have significant side effects and short half-lives; their high price limits their consumer base. However, nattokinase has a clear advantage in terms of market appeal and efficacy, particularly in its fibrinolytic effect. Its function can be manifested through direct and indirect effects. The direct effect refers to nattokinase's direct response to plasminogen activating, thereby increasing plasmin levels and achieving a greater thrombolytic effect. Compared to traditional thrombolytic agents, this enzyme can be obtained through microbial fermentation, offering advantages such as higher safety, lower cost, and rapid dissolution. It also exhibits excellent stability in the gastrointestinal tract, making it a promising candidate for oral thrombolytic therapy. This type of enzyme is quite safe in production and application, and is considered by both domestic and international researchers to be one of the most valuable dietary supplements.

[0003] Nattokinase, discovered by Japanese expert Hiroyuki Sumi in 1980, possesses a strong thrombolytic effect, even stronger than that of urokinase. It also boasts a long duration of action, safety, and no side effects. Furthermore, it can be directly fermented from Bacillus subtilis in natto, resulting in low production costs. Therefore, nattokinase is a promising new thrombolytic drug. The fermentation process for nattokinase generally falls into two categories: solid-state fermentation and liquid-state fermentation. Compared to solid-state fermentation, which suffers from problems such as susceptibility to bacterial contamination, difficulty in heat dissipation, low recovery rate, unpleasant odor, and difficulty meeting stringent production requirements for pharmaceuticals, especially biological agents, liquid fermentation offers advantages such as low cost, high purity, and minimal environmental pollution. Currently, the production process of nattokinase involves separating and concentrating the fermentation broth to increase enzyme activity, which increases production costs and is time-consuming and labor-intensive. However, this technology utilizes yellow liquid containing enzymatically hydrolyzed soybean milk for fermentation, not only improving enzyme activity through over-fermentation but also solving the environmental pollution problem.

[0004] Therefore, our laboratory still uses the traditional liquid fermentation method. However, during the fermentation process, adding different proportions of soy milk to the yellow liquid of soy product waste and enzymatically decomposing it into small peptides can promote enzyme production, greatly save costs, and improve the production efficiency and enzyme activity of nattokinase. Summary of the Invention

[0005] The purpose of this invention is to provide a production process for highly active nattokinase. The nattokinase spray-dried powder prepared by this process has high activity and contains a large number of probiotics. It utilizes enzymatically hydrolyzed soybean milk and employs fermentation and spray-drying processes to prepare the nattokinase spray-dried powder. This method is low-cost, safe, environmentally friendly, produces minimal pollution, has high production efficiency, and is simple to implement.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A production process for highly active nattokinase, the production process including the preparation of seed liquid, processing and preparation of raw materials, liquid fermentation production, and spray drying process.

[0008] The preparation method of the initial seed culture is as follows: Under aseptic conditions, Bacillus subtilis is used as the bacterial strain. The strain is inoculated into the aseptic seed culture at a rate of one loop. The culture temperature is 37℃, the incubation speed is 120 rpm to 250 rpm, and the culture time is 8 h to 18 h. The colony count of the seed culture is 10. 7 cfu / mL or higher.

[0009] The raw material processing and preparation process is as follows: 2% to 15% soybean milk is added to the yellow slurry, and 0.05% to 4.5% neutral protease is added for hydrolysis. The hydrolysis temperature is 50 to 60°C, the time is 600 to 800 minutes, and the pH is 6.5 to 7.5.

[0010] The liquid fermentation production process is as follows: the volume of the fermentation medium is 60%–65%, the inoculation amount of the fermentation medium is 2%–10%, and the main components of the fermentation medium are: 10g / L–30g / L glucose, 0.1g / L–1g / L calcium chloride, 0.1g / L–0.2g / L magnesium sulfate, and 2g / L–10g / L phosphate. The medium is dissolved in soybean milk yellow liquid with 2%–15% added for enzymatic hydrolysis, sterilized, cooled to room temperature for inoculation, fermented at 37℃, with an aeration rate of 0.5vvm–1vvm, a fermentation time of 8–16 hours, and a stirring speed of 200rpm–300rpm.

[0011] The spray drying process includes the following steps:

[0012] 1) After fermentation, add a mixture of maltodextrin and inulin as a protective agent and stir evenly;

[0013] 2) Set the inlet air temperature of the spray dryer to 140-200℃ and the atomization frequency to 250Hz-300Hz to obtain nattokinase spray-dried powder.

[0014] The mass ratio of maltodextrin to inulin in the protective agent is 1-6:1-3.

[0015] The preferred Bacillus subtilis used is CGMCC No. 13932.

[0016] The enzymatically hydrolyzed raw material solution must be used within 3 days.

[0017] In the raw material processing step, it is preferable to add 4.5%–12% soybean milk to the yellow fermented liquid and 2%–4.5% neutral protease for hydrolysis; more preferably, 8%–12% soybean milk is added to the yellow fermented liquid, and even more preferably, 10% soybean milk is added. Adding a certain amount of enzymatically hydrolyzed soybean milk to the yellow fermented liquid makes the enzymatically hydrolyzed peptides and other substances more conducive to fermentation, resulting in better absorption and growth of the bacterial strain and a significant increase in enzyme activity.

[0018] The yellow liquid was mixed with 10% soy milk and 4.5% neutral protease for hydrolysis before being added to the fermentation medium. After fermentation, the nattokinase activity reached 62900 IU / g, and the colony count reached 7.4*10⁻⁶. 11 CFU / g or higher.

[0019] In this process, the raw materials are treated by adding protease for enzymatic hydrolysis, which can effectively improve the enzyme activity of nattokinase. In the spray drying process, maltodextrin and inulin are added in a ratio of 1-6:1-3 as a protective agent, which can prevent the loss of enzyme activity of nattokinase under high temperature environment, and has the advantages of low energy consumption and high spray drying efficiency.

[0020] The mass ratio of maltodextrin to inulin in the protective agent is 1-6:1-3, preferably 2-3:1.

[0021] The amount of the protective agent added is 20-40%. More preferably, the amount of the protective agent added is 30%.

[0022] The seed culture must reach the logarithmic growth phase before transfer, at which point the colony count is 2.3 x 10⁻⁶. 7 cfu / mL or higher;

[0023] The culture medium was sterilized at a temperature of 121℃ for 20 to 30 minutes.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0025] 1. Compared with other fermentation processes, the fermentation raw material processing method of the present invention, which adds a certain amount of soybean milk treated with neutral protease as a key component of the fermentation culture medium, significantly improves the fermentation production of nattokinase.

[0026] 2. Compared with the traditional liquid fermentation method, the raw materials for cultivation are replaced with soybean peptone and yellow soy milk containing a certain amount of enzymatically hydrolyzed soybean milk. From the perspective of fermentation process, the small peptides and other substances after enzymatic hydrolysis are more conducive to fermentation production, the strains absorb and grow better, and the enzyme activity is significantly increased.

[0027] 3. Spray drying can maintain the activity of nattokinase for a long time while avoiding the problem of contamination by other bacteria during solid-state fermentation. Detailed Implementation

[0028] The invention can be better understood through the following embodiments. It will then be readily understood by those skilled in the art that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0029] The Bacillus subtilis used in the following examples is CGMCC No. 13932.

[0030] Example 1

[0031] Add 4.5% soy milk and 1.2% neutral proteolytic enzyme (mainly purchased from Beijing Aoboxing Biotechnology Co., Ltd.) to the soy milk for hydrolysis. The hydrolysis temperature is 55℃, the time is 720 min, and the pH is 6.5-7. The raw material is used as a nitrogen source and water substitute for fermentation. The fermentation medium has a liquid volume of 60% and an inoculum size of 2.5%. The main components of the fermentation medium are 20 g / L glucose, 0.1 g / L calcium chloride, 0.15 g / L magnesium sulfate, and 2 g / L phosphate. The enzymatically hydrolyzed raw material solution was dissolved and sterilized at 121℃ for 20 minutes. After cooling to room temperature, it was inoculated. The fermentation temperature was 37℃, the aeration rate was 0.8 vvm, the stirring speed was 200 rpm, and the fermentation time was 10 hours. After fermentation, 28% maltodextrin and inulin (ratio 1.5:1.2) were added as a protectant to the fermentation broth and stirred evenly. The inlet air temperature for spray drying was set at 140℃ and the atomization frequency at 250 Hz. Spray drying was then performed to obtain nattokinase spray-dried powder. The spray-dried powder was tested, and the nattokinase activity reached over 35000 IU / g, with a colony count of 3.5*10⁻⁶. 11 CFU / g or higher.

[0032] Example 2

[0033] Add 8% soy milk and 2% neutral protease to the soy milk whey for hydrolysis at 55℃ for 600 minutes, with a pH of 6.5–7. This raw material is used as the nitrogen source and water substitute for fermentation. The fermentation medium has a volume of 62% and an inoculum size of 5%. The main components of the fermentation medium are 25 g / L glucose, 0.5 g / L calcium chloride, 0.15 g / L magnesium sulfate, and 4 g / L phosphate. The raw material is dissolved in the enzymatically hydrolyzed liquid for inactivation. The bacteria were sterilized at 121℃ for 30 minutes, cooled to room temperature, and then inoculated. Fermentation was carried out at 37℃ with an aeration rate of 0.8 vvm, a stirring speed of 250 rpm, and a fermentation time of 10 hours. After fermentation, 25% maltodextrin and inulin (in a ratio of 2.2:1) were added as a preservative to the fermentation broth and stirred evenly. The inlet air temperature for spray drying was set at 180℃, and the atomization frequency at 300 Hz. Spray drying was then performed to obtain nattokinase spray-dried powder. The spray-dried powder was tested, and the nattokinase activity reached 49400 IU / g, with a colony count of 5.8*10⁻⁶. 11 cfu / g.

[0034] Example 3

[0035] Add 10% soy milk and 4.5% neutral protease to the soy milk whey for hydrolysis. The hydrolysis temperature is 60℃, pH 6.5-7, and the time is 660 min. This raw material is used as the nitrogen source and water substitute for fermentation. The fermentation medium has a liquid volume of 65% and an inoculum size of 10%. The main components of the fermentation medium are 30 g / L glucose, 0.5 g / L calcium chloride, 0.15 g / L magnesium sulfate, and 8 g / L phosphate. The enzymatically hydrolyzed raw material solution is then used for fermentation. Dissolve and sterilize at 121℃ for 30 minutes. Cool to room temperature for inoculation. Fermentation temperature is 37℃, aeration rate is 1 vvm, stirring speed is 280 rpm, and fermentation time is 9 hours. After fermentation, 28% maltodextrin and inulin (2:1 ratio) are added as a protectant to the fermentation broth and stirred evenly. The inlet air temperature for spray drying is set to 200℃ and the atomization frequency to 280 Hz. Spray drying is then performed to obtain nattokinase spray-dried powder. The spray-dried powder was tested, and the nattokinase activity reached 62900 IU / g, with a colony count of 7.4*10⁻⁶. 11 cfu / g.

[0036] Example 4: Comparison of enzyme production and growth under different basal culture media.

[0037] ① The fermentation process of a culture medium containing 10% neutral protease hydrolyzed soybean milk: 10% soybean milk and 4% neutral protease are added to the soybean milk for hydrolysis. The hydrolysis temperature is 60℃, pH 6.5-7, and the time is 720 min. The raw material is used as the nitrogen source and water substitute for fermentation. The liquid volume of the fermentation medium is 60%, and the inoculum size is 10%. The main components of the fermentation medium are 30 g / L glucose, 0.5 g / L calcium chloride, 0.15 g / L magnesium sulfate, and 8 g / L... Phosphate was dissolved in the enzymatically hydrolyzed raw material solution and sterilized at 121℃ for 30 minutes. After cooling to room temperature, inoculation was performed. The fermentation temperature was 37℃, the aeration rate was 1 vvm, the stirring speed was 250 rpm, and the fermentation time was 8 hours. After fermentation, 30% maltodextrin and inulin (ratio 2.5:1) were added as a protectant to the fermentation broth and stirred evenly. The inlet air temperature for spray drying was set to 200℃ and the atomization frequency to 290 Hz. Spray drying was then performed to obtain nattokinase spray-dried powder. The spray-dried powder was tested, and the nattokinase activity reached 60300 IU / g, with a colony count of 6.2*10⁻⁶. 11 cfu / g.

[0038] ② Fermentation process of basic culture medium with only yellow whey (without soybean milk): Yellow whey is used to replace water. The liquid volume of the fermentation culture medium is 60%, and the inoculation amount is 10%. The main components of the fermentation culture medium are 30 g / L glucose, 0.5 g / L calcium chloride, 0.15 g / L magnesium sulfate, and 8 g / L phosphate. The raw material liquid after enzymatic hydrolysis is dissolved and sterilized at 121℃ for 30 min. After cooling to room temperature, inoculation is performed. The fermentation temperature is 37℃, the aeration rate is 1 vvm, the stirring speed is 250 rpm, and the fermentation time is 8 h. After fermentation, 30% maltodextrin and inulin (ratio 2.5:1) are added as a protectant to the fermentation liquid and stirred evenly. The inlet air temperature of the spray dryer is set to 200℃ and the atomization frequency is 290 Hz. Spray drying is performed to obtain nattokinase spray-dried powder. The spray-dried powder was tested, and the nattokinase activity reached 21900 IU / g, with a colony count of 1.1*10⁻⁶. 11 cfu / g.

[0039] ③ Process flow of adding 10% yellow slurry water of untreated soybean milk to the basic culture medium: Add 10% yellow slurry water of untreated soybean milk to replace water. The liquid volume of the fermentation medium is 60%, and the inoculation amount of the fermentation medium is 10%. The main components of the fermentation medium are 30g / L glucose, 0.5g / L calcium chloride, 0.15g / L magnesium sulfate and 8g / L phosphate. Add untreated soybean milk and yellow slurry water to dissolve and sterilize. The sterilization temperature is 121℃ and the sterilization time is 30min. Cool to room temperature for inoculation. The fermentation temperature is 37℃, the fermentation aeration rate is 1vvm, the stirring speed is 250rpm, and the fermentation time is 8h. After fermentation, add 30% maltodextrin and inulin (ratio 2.5:1) as a protectant to the fermentation liquid and stir evenly. Set the inlet air temperature of spray drying to 200℃ and the atomization frequency to 290Hz, and spray dry it to obtain nattokinase spray-dried powder. The spray-dried powder was tested, and the nattokinase activity reached 31600 IU / g, with a colony count of 3.2*10⁻⁶. 11 cfu / g.

[0040] ④ Process flow of the basic culture medium without the addition of yellow slurry: Without adding yellow slurry, water is used to replace yellow slurry to prepare the fermentation culture medium. The liquid volume of the fermentation culture medium is 60%, and the inoculation amount of the fermentation culture medium is 10%. The main components of the fermentation culture medium are 30 g / L glucose, 0.5 g / L calcium chloride, 0.15 g / L magnesium sulfate, and 8 g / L phosphate. Untreated soybean milk and yellow slurry are added to dissolve and sterilize. The sterilization temperature is 121℃ and the sterilization time is 30 min. After cooling to room temperature, inoculation is carried out. The fermentation temperature is 37℃, the fermentation aeration rate is 1 vvm, the stirring speed is 250 rpm, and the fermentation time is 8 h. After fermentation, 30% maltodextrin and inulin (ratio 2.5:1) are added to the fermentation liquid as a protectant and stirred evenly. The inlet air temperature of the spray dryer is set to 200℃ and the atomization frequency is 290 Hz. Spray drying is performed to obtain nattokinase spray-dried powder. The spray-dried powder was tested, and the nattokinase activity reached 5100 IU / g, with a colony count of 4.5*10⁻⁶. 7 cfu / g.

[0041] ⑤ Processing flow for adding enzymatically hydrolyzed whole soybean milk: Add 10% neutral protease to hydrolyze the soybean milk to replace water. Hydrolysis is performed using 4% neutral protease at 60℃, pH 6.5-7, and for 720 minutes. The raw materials are prepared and used as the nitrogen source and water replacement for fermentation. The fermentation medium is 60% full, and the inoculum size is 10%. The main components of the fermentation medium are 30g / L glucose, 0.5g / L calcium chloride, 0.15g / L magnesium sulfate, and 8g / L phosphate. The enzymatically hydrolyzed soybean milk is then used... The raw material solution was dissolved and sterilized at 121℃ for 30 minutes. After cooling to room temperature, it was inoculated. The fermentation temperature was 37℃, the aeration rate was 1 vvm, the stirring speed was 250 rpm, and the fermentation time was 8 hours. After fermentation, 30% maltodextrin and inulin (ratio 2.5:1) were added as a protectant to the fermentation broth and stirred evenly. The inlet air temperature for spray drying was set to 200℃ and the atomization frequency to 290 Hz. Spray drying was then performed to obtain nattokinase spray-dried powder. The spray-dried powder was tested, and the nattokinase activity reached 21000 IU / g, with a colony count of 1.0*10⁻⁶. 11 cfu / g.

[0042] Table 1 Comparison of enzyme activity and colony count of nattokinase produced by different processes

[0043]

[0044] Example 5: The production of nattokinase and the number of bacterial colonies in fermented soybean milk after adding different proportions of enzymatic hydrolysis.

[0045] The fermentation process for the yellow liquid culture medium containing enzymatically hydrolyzed soy milk (neutral protease hydrolyzed soy milk) with added concentrations of 2%, 4%, 6%, 8%, 10%, 12%, 15%, 20%, and 30% respectively is as follows: 2-30% soy milk and 4% neutral protease are added to the yellow liquid culture medium for hydrolysis. The hydrolysis temperature is 60℃, pH 6.5-7, and the time is 120 minutes. The raw material is used as the nitrogen source and water substitute for fermentation. The liquid volume of the fermentation medium is 60%, and the inoculum size is 10%. The main components of the fermentation medium are 30 g / L glucose and 0.5 g / L... L of calcium chloride, 0.15 g / L of magnesium sulfate, and 8 g / L of phosphate were dissolved in the enzymatically hydrolyzed raw material solution and sterilized at 121℃ for 30 min. After cooling to room temperature, inoculation was performed. The fermentation temperature was 37℃, the aeration rate was 1 vvm, the stirring speed was 250 rpm, and the fermentation time was 8 h. After fermentation, 30% maltodextrin and inulin (2:1 ratio) were added as a protectant to the fermentation broth and stirred evenly. The inlet air temperature for spray drying was set to 200℃ and the atomization frequency to 290 Hz. Spray drying was then performed to obtain nattokinase spray-dried powder. The activity and colony count of the spray-dried powder were tested, as shown in Table 2 below.

[0046] Table 2. Effects of different amounts of enzyme added to the enzymatically hydrolyzed soy milk on enzyme activity and bacterial count.

[0047]

[0048]

[0049] Example 6: Effect of adding a protectant on nattokinase activity and colony count

[0050] The fermentation process of a culture medium containing 10% neutral protease-hydrolyzed soy milk is as follows: 10% soy milk and 4% neutral protease are added to the soy milk for hydrolysis. The hydrolysis temperature is 60℃, pH 6.5–7, and the time is 720 min. The raw material is used as the nitrogen source and water substitute for fermentation. The volume of the fermentation medium is 60%, and the inoculum size is 10%. The main components of the fermentation medium are 30 g / L glucose, 0.5 g / L calcium chloride, and 0.15 g / L sulfur. Magnesium sulfate and 8 g / L phosphate were dissolved in the enzymatically hydrolyzed raw material solution and sterilized at 121℃ for 30 min. After cooling to room temperature, inoculation was performed. The fermentation temperature was 37℃, the aeration rate was 1 vvm, the stirring speed was 250 rpm, and the fermentation time was 8 h. After fermentation, different amounts of protective agents (as shown in Table 4) were added to the fermentation broth and stirred evenly. The inlet air temperature for spray drying was set to 200℃ and the atomization frequency to 290 Hz. Spray drying was then performed to obtain nattokinase spray-dried powder. The nattokinase content and colony count of the spray-dried powder were tested, and are shown in Tables 3 and 4.

[0051] Table 3. Effects of the protectant on nattokinase and colony count.

[0052]

[0053] Table 4. Effects of protectant content on nattokinase and bacterial colony count.

[0054]

[0055] Steps related to nattokinase enzyme activity detection:

[0056] Reagents and solution preparation: Phosphate buffer (pH = 7.8): Dissolve 0.895g of disodium hydrogen phosphate in water and dilute to 250mL as solution A; dissolve 0.789g of sodium dihydrogen phosphate in water to 125mL as solution B. Mix solutions A and B together to achieve pH = 7.8; Preparation of 0.9% sodium chloride solution: Dissolve 3.6g of sodium chloride in water to 400mL; Preparation of working solution: Mix the A+B mixture with the 0.9% sodium chloride solution at a ratio of 1:17; 1.5% agarose solution: Weigh 1.5g of agarose... Dissolve g in 100mL of working solution, sterilize at high temperature, and store in a 60℃ oven; Preparation of fibrinogen solution: Dissolve 103mg of fibrinogen in 68.67mL of working solution to prepare a 1.5mg / mL coagulable protein solution; Preparation of thrombin solution: Dissolve 160bp thrombin in 20mL of 0.9% sodium chloride solution to prepare a thrombin solution with a concentration of 8bp / mL, dispense into 20 vials, and store in a -20℃ refrigerator. Each time you use it, take out one vial, dilute it to 8mL to a concentration of 1bp / mL thrombin solution, and take out 5.28mL for later use.

[0057] Preparation of agarose-fibrinogen plates: Preheat a 50mL graduated cylinder and Erlenmeyer flask in an oven, and simultaneously preheat a 50℃ water bath. Place seven plastic plates around the oven and open the plastic lids. Preheat the prepared fibrinogen and thrombin solutions in the water bath for 8–10 minutes. Quickly take 68.67mL of agarose solution and add it to the preheated fibrinogen and thrombin solutions, mix rapidly, and quickly pour 20mL into the plastic plates. Repeat this process for the remaining six plates. Allow to solidify at room temperature for 1 hour before punching holes.

[0058] Sample detection: Accurately measure 15 μL of standard and sample using a pipette, spot them separately into the same petri dish, cover, and incubate at 37℃ for 14–16 h.

[0059] Bacillus subtilis colony count:

[0060] Preparation of LB solid medium: Weigh 2g tryptone, 2g sodium chloride, and 1g yeast powder into a beaker, bring the volume to 200mL, and add 4g agar. After high-temperature sterilization, prepare plates and allow them to solidify before use.

[0061] Procedure: Aseptically weigh 1 mL of sample and add 9 mL of 0.85% sterile physiological saline to prepare a 1:10 initial suspension. Take 1 mL of the 1:10 initial suspension and add 9 mL of 0.85% sterile physiological saline, mixing thoroughly to prepare a 1:100 dilution. Perform further serial dilutions of 10-fold based on the bacterial count of the sample. Using a sterile pipette, pipette 0.1 mL of the inoculum onto two nutrient agar plates. Carefully and quickly spread the inoculum onto the agar surface using a spreader, ensuring the spreader does not touch the edge of the plate. Cover the plate after spreading. Incubate upside down in a 35°C incubator for 24 hours and 48 hours, observing and counting colonies.

Claims

1. A production method of high-activity nattokinase, the production method comprising preparation of a pre-stage seed liquid, treatment and preparation of raw materials, liquid fermentation production, and a spray drying process; characterized in that, The processing and preparation process of the raw material is as follows: 10% of soybean milk is added to the soybean curd yellow slurry water, and 4.5% of neutral protease is added for hydrolysis, the hydrolysis temperature is 60℃, the time is 660min, the pH is 6.5~7, and the raw material liquid is prepared; The liquid fermentation production process is as follows: the liquid volume of the fermentation medium is 65%, the inoculation amount of the fermentation medium is 10%, the main components of the fermentation medium are 30g / L glucose, 0.5g / L calcium chloride, 0.15g / L magnesium sulfate and 8g / L phosphate, the raw material liquid after enzymolysis is dissolved, sterilized, cooled to room temperature for inoculation, the fermentation temperature is 37℃, the ventilation amount of the fermentation is 1vvm, the fermentation time is 9h, the stirring speed is 280rpm, and the fermentation is formed; The spray drying process includes: after the fermentation is finished, a malt dextrin and inulin mixture is added as a protective agent, uniform stirring is carried out, and the protective agent is added in an amount of 28%; the mass ratio of malt dextrin to inulin in the protective agent is 2:

1. The strain used is Bacillus subtilis CGMCC No. 13932.

2. The method for producing high-activity nattokinase according to claim 1, characterized by, The preparation method of the pre-stage seed liquid is as follows: the strain is inoculated into a sterile seed liquid, the inoculation amount is one ring, the culture temperature is 37 DEG C, the culture speed is 120 rpm~250 rpm, the culture time is 8 h~18 h, and the colony number of the seed liquid is 10 7 cfu / mL or above.

3. The method for producing high-activity nattokinase according to claim 1, characterized by, In the spray drying process, the inlet air temperature of the spray drying is 140~200℃, and the atomization frequency is 250Hz~300Hz.

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