A method for producing nattokinase by solid-state fermentation of hemp seed meal

Abstract

The invention discloses a method for producing nattokinase by solid-state fermentation of hemp seed meal. Hemp seed meal is used as the only matrix, nattokinase producing bacteria are inoculated on the solid culture medium, the temperature is controlled at 30-40°C, the humidity is controlled at 60-80°C, and the fermentation is carried out for 16-24 hours, and the enzyme production of nattokinase reaches 9592.8IU / g. The invention uses hemp seed meal, which is a waste of oil extraction, as the only fermentation matrix, greatly reduces the production cost of nattokinase, simplifies the fermentation process, and realizes the high-value utilization of hemp seed meal. The fresh fragrance of hemp seeds well neutralizes the ammonia smell generated during the fermentation process of nattokinase, making nattokinase more acceptable. The method is the first to use hemp seed meal solid-state fermentation to produce nattokinase, provides a new method for the production of nattokinase, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and in particular relates to a method for producing nattokinase by solid-state fermentation of hemp seed meal. Technical Background

[0002] Hemp seeds are often pressed to make hemp oil because they are rich in oil and unsaturated fatty acids. The hemp seed meal produced by making hemp oil is often directly discarded as processing waste or sold as animal feed at a very low price. This is a huge waste of limited hemp resources. Hemp seed meal contains about 60% crude protein, 20% dietary fiber, 7% to 8% lipids, 10% carbohydrates, and a variety of vitamins and minerals. It is an excellent carbon source. Previously, hemp seed meal was rarely used, and it has not been used as a solid fermentation matrix for microbial cultivation. Using hemp seed meal as the only fermentation matrix to produce nattokinase achieves efficient utilization of waste, reduces the production cost of nattokinase, and turns waste into treasure.

[0003] Nattokinase (NK) is a serine protease. When added to food, NK can reduce the viscosity of blood and plasma in the human body, thereby preventing and treating thrombotic diseases. Compared with existing fibrinolytic enzymes, NK has attracted much attention due to its high fibrinolytic activity, no side effects, and easier absorption by the human body. However, the large demand, low yield, and high cost of NK hinder its widespread application. Improving the yield and activity of NK has always been the focus of promoting the development of NK.

[0004] Patent 201110034654.2 "A method for industrial production of nattokinase using chickpeas" records that after soaking chickpeas in a nutrient solution containing lactose and glucose, solid-state fermentation is carried out with it, and the activity of crude nattokinase is 3270IU / g. Patent 200910040896.5 "A solid fermentation method for nattokinase" records that soybeans are used as a solid fermentation matrix, and the nattokinase enzyme activity produced is 700IU / g. Patent 200810231137.2 "A solid fermentation method for nattokinase" records that bean dregs are used as a solid fermentation matrix, and the unit fermentation enzyme activity is as high as 5311.72IU / g.

[0005] Patent 202210267411.1 "A high F value oligopeptide from hemp seed meal, its preparation method and application in increasing the production of nattokinase" uses hemp seed meal as raw material, first separates and prepares high F value hemp seed meal oligopeptide, and adds it to the fermentation broth of Bacillus subtilis, which can significantly increase its nattokinase production, but the hemp seed meal needs to be processed first, and the process is complicated. Summary of the invention

[0006] The object of the present invention is to provide a method for producing nattokinase by solid-state fermentation of hemp seed meal, the method process is simple, and the activity of the produced nattokinase is high, and the hemp seed meal of oil extraction waste can be effectively utilized, the production cost is reduced and the efficient utilization of the hemp seed meal is realized. In addition, the solid-state fermentation time of the hemp seed meal is short, and the nattokinase activity just reaches the highest in about 20h, which reduces the fermentation time. Compared with other solid-state fermentation substrates, the hemp seed meal has a fresh smell, can neutralize the ammonia smell produced in the fermentation process, and makes nattokinase more acceptable.

[0007] To achieve the above purpose, the technical method adopted by the present invention is as follows:

[0008] A method for producing nattokinase by solid-state fermentation of hemp seed meal comprises the following steps:

[0009] 1) crushing the hemp seed meal and adding a certain amount of distilled water to soak it;

[0010] 2) sterilizing the soaked hemp seed meal;

[0011] 3) inoculating the nattokinase-producing bacteria on the cooled hemp seed meal, controlling the fermentation temperature and humidity, and performing solid-state fermentation;

[0012] 4) Add a certain amount of distilled water to the fermented product for extraction and centrifugation to obtain nattokinase.

[0013] In step 1), the hemp seed meal is crushed, and distilled water with a ratio of 1: (1-5) (w / v, g / mL) is added and soaked for 2-5 hours.

[0014] In step 2), the crushed hemp seed meal is sterilized by high-pressure steam, the temperature is controlled at 100-120° C., the sterilization time is 20-30 minutes, and the mixture is cooled to 30-40° C. for inoculation.

[0015] The nattokinase producing bacteria include at least one of Bacillus subtilis CGMCC No. 13932, Bacillus natto BNCC194961 and Bacillus licheniformis BNCC132622.

[0016] In step 3), the seed solution of nattokinase producing bacteria is inoculated into the fermentation medium at a seed solution inoculation rate of 5-15%, and stirred. The culture temperature is 30-40° C., the humidity of the incubator is 60-80%, and the fermentation is carried out for 16-24 hours.

[0017] Step 4) Add 1: (5-10) (w / v, g / mL) distilled water for 2-5 hours, centrifuge at 10000-12000 r / min for 10-20 minutes. Obtain nattokinase in the supernatant.

[0018] Preferably, in step 2), the crushed hemp seed meal is sterilized by high-pressure steam, the temperature is controlled at 100-120° C., the sterilization time is 20-30 min, after cooling, protease is added, and the pH and temperature are adjusted for enzymatic hydrolysis;

[0019] The protease can be any one of neutral protease, alkaline protease and papain, the enzymolysis temperature is 40-55° C., and the amount of protease added is 5-10% (mass percentage).

[0020] Preferably, the inoculation order of the nattokinase producing bacteria is that Bacillus subtilis CGMCC No.13932 is first inoculated into the culture medium, the culture temperature is 30-40°C, the humidity of the incubator is 60-80%, and after fermentation for 16-24 hours, Bacillus natto BNCC194961 and / or Bacillus licheniformis BNCC132622 is inoculated to continue fermentation for 16-24 hours.

[0021] Alternatively, Bacillus subtilis CGMCC No. 13932 and Bacillus licheniformis BNCC132622 are added at the same time, the culture temperature is 30-40° C., the humidity of the incubator is 60-80%, and after fermentation for 16-24 hours, Bacillus natto BNCC194961 is added to continue fermentation for 16-24 hours.

[0022] The order of adding strains has a great influence on the activity of nattokinase. When Bacillus subtilis CGMCC No.13932 and Bacillus licheniformis BNCC132622 are added first, they can secrete a variety of proteases and cellulases, turning hemp seed protein into more usable polypeptides, which is beneficial to the growth of subsequent strains and the production of nattokinase. Bacillus natto BNCC194961 has strong acid resistance and can still maintain high activity when added in the late fermentation. The effect of mixed bacteria using solid-state fermentation of hemp seed meal to produce nattokinase is better than single bacteria fermentation.

[0023] The present invention uses hemp seed meal as the only fermentation substrate for the first time to carry out solid-state fermentation of nattokinase, thereby increasing the yield of nattokinase and realizing the high-value utilization of hemp seed meal, a byproduct of oil extraction. The production cost of nattokinase is reduced, the production process is simplified, and the technology has broad application prospects.

[0024] Beneficial effects:

[0025] (1) After solid fermentation of hemp seed meal, the yield of nattokinase is high.

[0026] (2) Hemp seed meal is used as the only fermentation matrix, and no other nutrients need to be added, which simplifies the fermentation process.

[0027] (3) Using hemp seed meal as the only fermentation substrate reduces the production cost of nattokinase.

[0028] (4) The high-value utilization of hemp seed meal, a waste product from oil extraction, has been achieved.

[0029] (5) The fresh aroma of hemp seeds effectively neutralizes the ammonia smell produced during the fermentation process, making the product better in color and taste. DETAILED DESCRIPTION

[0030] In order to better understand the present invention, the present invention is further described below in conjunction with specific embodiments. The embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention.

[0031] The nattokinase producing bacteria adopted in the following examples include Bacillus subtilis CGMCC No.13932 (from China General Microbiological Culture Collection Center), Bacillus natto BNCC194961 (purchased from Beina Biological), and Bacillus licheniformis BNCC132622 (purchased from Beina Biological).

[0032] Example 1 Production of Nattokinase by Solid-State Fermentation of Hemp Seed Meal Using Different Strains

[0033] Plate culture medium: peptone 10g / L, yeast extract 5g / L, Nacl 10g / L, agar 20g / L, add distilled water to 1000ml, pH natural, sterilize at 121℃ for 20min.

[0034] Seed culture medium: glucose 12g / L, peptone 15g / L, K2HPO4 1.2g / L, KH2PO4 1g / L, MgSO40.40g / L, CaCl2 0.25g / L, MnSO4 0.001g / L, add distilled water to 1000ml, pH is 7.4, sterilize at 121℃ for 20min.

[0035] Bacillus subtilis CGMCC No.13932, Bacillus natto BNCC194961, and Bacillus licheniformis BNCC132622 were activated on plate culture media, respectively. After culturing at 37°C for 24 hours, two loops of well-growing bacteria were taken and cultured in seed solution at 37°C and 200 r / min for 12 hours.

[0036] 10 kg of hemp seed meal was crushed, added into 20 L of distilled water and soaked for 3 h. After soaking, it was sterilized by high pressure steam at 110 °C for 20 min and cooled to 40 °C.

[0037] The three seed solutions were inoculated into the solid culture medium at a seed solution inoculation rate of 10%, and stirred. The culture temperature was 37° C., the humidity of the incubator was 75%, and the fermentation was carried out for 20 hours.

[0038] Add 1:5 (w / v, g / mL) distilled water for 3 hours, centrifuge at 10000 r / min for 10 minutes, and use the centrifuged supernatant to test the activity of nattokinase.

[0039] The nattokinase produced by fermentation of Bacillus subtilis CGMCC No.13932 is 6732.1 IU / g, the nattokinase produced by fermentation of Bacillus natto BNCC194961 is 4548.4 IU / g, and the nattokinase produced by fermentation of Bacillus licheniformis BNCC132622 is 2960.2 IU / g.

[0040] Example 2 Production of Nattokinase by Solid-State Fermentation of Bacillus subtilis CGMCC No.13932 Using Different Substrates and Sensory Evaluation

[0041] Plate culture medium: peptone 10g / L, yeast extract 5g / L, Nacl 10g / L, agar 20g / L, add distilled water to 1000ml, pH natural, sterilize at 121℃ for 20min.

[0042] Seed culture medium: glucose 12g / L, peptone 15g / L, K2HPO4 1.2g / L, KH2PO4 1g / L, MgSO40.40g / L, CaCl2 0.25g / L, MnSO4 0.001g / L, add distilled water to 1000ml, pH is 7.4, sterilize at 121℃ for 20min.

[0043] Bacillus subtilis CGMCC No. 13932 was activated on a plate culture medium, cultured at 37°C for 24 hours, and two loops of well-grown cells were taken and cultured in seed solution, and cultured at 37°C and 200 r / min for 12 hours.

[0044] 10 kg of hemp seed meal and soybean meal were crushed separately, added to 20 L of distilled water and soaked for 3 hours. After soaking, they were sterilized by high pressure steam at 110 ° C for 20 minutes and cooled to 40 ° C. 10 kg of soybeans were washed, soaked until fully absorbed, drained, sterilized by high pressure steam at 110 ° C for 20 minutes, and cooled to 40 ° C.

[0045] According to the seed liquid inoculation amount of 10%, Bacillus subtilis CGMCC No.13932 was inoculated into hemp seed meal, soybean meal and soybean solid culture medium respectively, and stirred. The culture temperature was 37° C., the humidity of the incubator was 80%, and the fermentation was carried out for 20 hours.

[0046] The three solid culture media were added with 1:5 (w / v, g / mL) distilled water for 3 h, and centrifuged at 10000 r / min for 10 min. The supernatant was used for nattokinase activity detection.

[0047] Bacillus subtilis CGMCC No.13932 fermented hemp seed meal to produce 6905.4IU / g of nattokinase, and Bacillus subtilis CGMCC No.13932 fermented soybean meal to produce 2279.3IU / g of nattokinase. Bacillus subtilis CGMCC No.13932 fermented soybean to produce 583.2IU / g of nattokinase. The results show that compared with other solid matrices, hemp seed meal contains more complete nutrients and is more conducive to the solid-state fermentation of nattokinase.

[0048] The sensory evaluation of different fermentation matrices is shown in Table 1, and the evaluation results are shown in Table 2.

[0049] Table 1 Evaluation criteria for solid fermentation matrix of nattokinase

[0050]

[0051] Table 2 Evaluation results of nattokinase solid fermentation matrix

[0052]

[0053]

[0054] As shown in Table 2, hemp seed meal had the highest sensory evaluation score, higher than soybean meal and soybeans.

[0055] Example 3 Effect of different protease enzymatic hydrolysis of hemp seed meal on the production of nattokinase by solid-state fermentation of Bacillus subtilis CGMCC No. 13932

[0056] Plate culture medium: peptone 10g / L, yeast extract 5g / L, Nacl 10g / L, agar 20g / L, add distilled water to 1000ml, pH natural, sterilize at 121℃ for 20min.

[0057] Seed culture medium: glucose 12g / L, peptone 15g / L, K2HPO4 1.2g / L, KH2PO4 1g / L, MgSO40.40g / L, CaCl2 0.25g / L, MnSO4 0.001g / L, add distilled water to 1000ml, pH is 7.4, sterilize at 121℃ for 20min.

[0058] Bacillus subtilis CGMCC No. 13932 was activated on a plate culture medium, cultured at 37°C for 24 hours, and two loops of well-grown cells were taken and cultured in seed solution, and cultured at 37°C and 200 r / min for 12 hours.

[0059] 10 kg of hemp seed meal was crushed, added into 20 L of distilled water, and soaked for 3 h. After soaking, it was sterilized by high pressure steam at 110 °C for 20 min. After cooling, the pH and temperature were adjusted according to different proteases according to Table 3.

[0060] Table 3 Optimal pH of different proteases

[0061]

[0062] Before inoculation, 5% (w / w, g / g) of different proteases were added and enzymolysis was carried out at their respective optimal temperatures for 3 h.

[0063] Bacillus subtilis CGMCC No. 13932 was inoculated into solid culture media treated with different enzymes at a seed liquid inoculation rate of 10%, and stirred. The culture temperature was 37° C., the humidity of the culture box was 80%, and the fermentation was carried out for 20 hours.

[0064] The three solid culture media were added with 1:5 (w / v, g / mL) distilled water for 3 h, and centrifuged at 10000 r / min for 10 min. The supernatant was used for nattokinase activity detection.

[0065] After treatment with neutral protease, the yield of nattokinase was 7217.3 IU / g, after treatment with alkaline protease, the yield of nattokinase was 7782.2 IU / g, after treatment with papain, the yield of nattokinase was 7092.8 IU / g, and after treatment with pepsin, the yield of nattokinase was 5738.4 IU / g. The results showed that hemp seed meal treated with neutral protease, alkaline protease and papain could increase the yield of nattokinase, but pepsin acted under acidic conditions, which was not conducive to the production of nattokinase by the strain.

[0066] Example 4 Effect of the order of adding nattokinase-producing strains on the production of nattokinase by solid-state fermentation of hemp seed meal

[0067] Plate culture medium: peptone 10g / L, yeast extract 5g / L, Nacl 10g / L, agar 20g / L, add distilled water to 1000ml, pH natural, sterilize at 121℃ for 20min.

[0068] Seed culture medium: glucose 12g / L, peptone 15g / L, K2HPO4 1.2g / L, KH2PO4 1g / L, MgSO40.40g / L, CaCl2 0.25g / L, MnSO4 0.001g / L, add distilled water to 1000ml, pH is 7.4, sterilize at 121℃ for 20min.

[0069] Bacillus subtilis CGMCC No.13932, Bacillus natto BNCC194961, and Bacillus licheniformis BNCC132622 were activated on plate culture media, respectively. After culturing at 37°C for 24 hours, two loops of well-growing bacteria were taken and cultured in seed solution at 37°C and 200 r / min for 12 hours.

[0070] 10 kg of hemp seed meal and soybean meal were crushed separately, added to 20 L of distilled water and soaked for 3 hours. After soaking, they were sterilized by high pressure steam at 110 ° C for 20 minutes and cooled to 40 ° C. 10 kg of soybeans were washed, soaked until fully absorbed, drained, sterilized by high pressure steam at 110 ° C for 20 minutes, and cooled to 40 ° C.

[0071] Add according to the different addition order in Table 4

[0072] Table 4 Order of adding bacteria

[0073]

[0074]

[0075] Note: In Table 4, A refers to Bacillus subtilis CGMCC No.13932, B refers to Bacillus natto BNCC194961, and C refers to Bacillus licheniformis BNCC132622. Simultaneous addition means that several strains are inoculated into the solid culture medium at a seed liquid inoculation rate of 5% at the same time, and stirred, cultured at 37°C, incubator humidity of 80%, and fermented for 20 hours. The remaining numbers are added in sequence, the inoculation rate is 5%, and the fermentation conditions are the same. After the first strain is fermented, the remaining strains are inoculated in turn and fermented for 20 hours.

[0076] The above different solid culture media were added with 1:5 (w / v, g / mL) distilled water for 3 h, and centrifuged at 10000 r / min for 10 min. The centrifuged supernatant was tested for nattokinase activity. The results are shown in Table 5

[0077] Table 5 Effect of the order of adding nattokinase-producing strains on the production of nattokinase by solid-state fermentation of hemp seed meal

[0078] serial number Nattokinase activity (IU / g) 1 8382.1 2 8621.9 3 9214.3 4 8031.2 5 7829.5 6 8148.4 7 7918.3 8 9056.6 9 9592.8

[0079] According to the results in Table 5, the order of adding strains has a great influence on the activity of nattokinase. When Bacillus subtilis CGMCC No.13932 and Bacillus licheniformis BNCC132622 are added first, they can secrete a variety of proteases and cellulases, turning hemp seed protein into more usable polypeptides, which is beneficial to the growth of subsequent strains and the production of nattokinase. Bacillus natto BNCC194961 has strong acid resistance and can still maintain high activity when added in the late fermentation. The effect of mixed bacteria on the production of nattokinase using solid-state fermentation of hemp seed meal is better than that of single bacteria fermentation.

[0080] Example 5 Production of Nattokinase by Solid-State Fermentation of Hemp Seed Meal Using Different Inoculation Rates of Bacillus subtilis CGMCC No.13932

[0081] Plate culture medium: peptone 10g / L, yeast extract 5g / L, Nacl 10g / L, agar 20g / L, add distilled water to 1000ml, pH natural, sterilize at 121℃ for 20min.

[0082] Seed culture medium: glucose 12g / L, peptone 15g / L, K2HPO4 1.2g / L, KH2PO4 1g / L, MgSO40.40g / L, CaCl2 0.25g / L, MnSO4 0.001g / L, add distilled water to 1000ml, pH is 7.4, sterilize at 121℃ for 20min.

[0083] Bacillus subtilis CGMCC No. 13932 was activated on a plate culture medium, cultured at 37°C for 24 hours, and two loops of well-grown cells were taken and cultured in seed solution, and cultured at 37°C and 200 r / min for 12 hours.

[0084] 10 kg of hemp seed meal and soybean meal were crushed separately, added into 20 L of distilled water and soaked for 3 h. After soaking, they were sterilized by high pressure steam at 110 °C for 20 min and cooled to 40 °C.

[0085] Bacillus subtilis CGMCC No. 13932 was inoculated into the solid culture medium of hemp seed meal at a seed liquid inoculation rate of 10%, 15% and 20% respectively, and stirred. The culture temperature was 37° C., the humidity of the culture box was 70%, and the fermentation was carried out for 20 hours.

[0086] The three fermented products were added to distilled water at a ratio of 1:5 (w / v, g / mL) and extracted for 3 hours, and centrifuged at 10000 r / min for 10 minutes. The supernatant was centrifuged for nattokinase activity detection.

[0087] When the inoculation amount was 10%, the nattokinase produced by hemp seed meal fermented by Bacillus subtilis CGMCC No.13932 was 6163.8 IU / g; when the inoculation amount was 15%, the nattokinase produced by hemp seed meal fermented by Bacillus subtilis CGMCC No.13932 was 8069.4 IU / g; when the inoculation amount was 20%, the nattokinase produced by hemp seed meal fermented by Bacillus subtilis CGMCC No.13932 was 7853.8 IU / g.

[0088] Example 6 Production of Nattokinase by Solid-State Fermentation of Hemp Seed Meal Using Bacillus subtilis CGMCC No.13932 at Different Fermentation Times

[0089] Plate culture medium: peptone 10g / L, yeast extract 5g / L, Nacl 10g / L, agar 20g / L, add distilled water to 1000ml, pH natural, sterilize at 121℃ for 20min.

[0090] Seed culture medium: glucose 12g / L, peptone 15g / L, K2HPO4 1.2g / L, KH2PO4 1g / L, MgSO40.40g / L, CaCl2 0.25g / L, MnSO4 0.001g / L, add distilled water to 1000ml, pH is 7.4, sterilize at 121℃ for 20min.

[0091] Bacillus subtilis CGMCC No. 13932 was activated on a plate culture medium, cultured at 37°C for 24 hours, and two loops of well-grown cells were taken and cultured in seed solution, and cultured at 37°C and 200 r / min for 12 hours.

[0092] 10 kg of hemp seed meal was crushed, added into 20 L of distilled water and soaked for 3 h. After soaking, it was sterilized by high pressure steam at 110 °C for 20 min and cooled to 40 °C.

[0093] The seed liquid was inoculated into the solid culture medium at a seed liquid inoculation rate of 15% and stirred. The culture temperature was 37°C and the humidity of the incubator was 80% for fermentation for 16, 20, and 24 hours.

[0094] Add 1:5 (w / v, g / mL) distilled water for 3 hours, centrifuge at 10000r / min for 10 minutes. The supernatant was used for nattokinase activity detection at different fermentation times.

[0095] When the fermentation time was 16h, the nattokinase produced by hemp seed meal fermented by Bacillus subtilis CGMCC No.13932 was 2037.3IU / g; when the fermentation time was 20h, the nattokinase produced by hemp seed meal fermented by Bacillus subtilis CGMCC No.13932 was 8421.8IU / g; when the fermentation time was 24h, the nattokinase produced by hemp seed meal fermented by Bacillus subtilis CGMCC No.13932 was 8219.5IU / g.

[0096] Enzyme activity detection steps:

[0097] Preparation of reagents and solutions: phosphate buffer (pH = 7.8) disodium hydrogen phosphate 0.895g is dissolved in water and diluted to 250mL as solution A; weigh 0.789g sodium dihydrogen phosphate and dissolve it in water to 125mL as solution B, mix A and B to make its pH = 7.8; preparation of 0.9% sodium chloride solution: weigh 3.6g sodium chloride and dissolve it in water to 400mL; preparation of working solution: mix A+B mixed solution with 0.9% sodium chloride solution at a ratio of 1:17; 1.5% agarose solution: weigh 1.5g agarose and dissolve it in 100mL working solution, sterilize it at high temperature and place it in a 60℃ oven; preparation of fibrinogen solution: take 103mg fibrinogen and add 68.67mL working solution to dissolve it to make a 1.5mg / mL coagulable protein solution; preparation of thrombin solution: 160bp Dissolve thrombin in 20 mL of 0.9% sodium chloride solution to prepare a thrombin solution with a concentration of 8 bp / mL, divide it into 20 vials, and store it in a -20°C refrigerator. Take out one vial and dilute it to 8 mL each time to make a thrombin solution with a concentration of 1 bp / mL. Take out 5.28 mL and set aside.

[0098] Preparation of agarose-fibrinogen plate: Preheat the prepared fibrinogen solution and thrombin solution in a water bath, quickly take the agarose solution, add the preheated fibrinogen solution and thrombin solution, mix quickly, and place at room temperature for 1 hour until solidified before punching.

[0099] Sample testing: Use a pipette to accurately measure 15 μL of the standard and sample, respectively, and place them in the same plate. Cover the plate and place it in a constant temperature incubator at 37°C for 14 to 16 hours.

Claims

1. A method for producing nattokinase by solid-state fermentation of hemp seed meal, characterized in that: The steps include: 1) Crush the hemp seed meal and add a certain amount of distilled water to soak it; 2) sterilizing the soaked hemp seed meal; 3) Inoculate the nattokinase-producing bacteria on the cooled hemp seed meal, control the fermentation temperature and humidity, and perform solid-state fermentation; 4) Add a certain amount of distilled water to the fermented product for extraction and centrifugation to obtain nattokinase; The nattokinase producing bacteria in step 3) is Bacillus subtilis ( Bacillus subtilis )CGMCCNo.13932, Bacillus natto( Bacillus natto ) BNCC194961 and Bacillus licheniformis ( Bacillus licheniformis )BNCC132622; The inoculation order of the nattokinase production bacteria is as follows: Bacillus subtilis CGMCC No. 13932 is first inoculated into the culture medium, the culture temperature is 30-40° C., the humidity of the incubator is 60-80%, and after fermentation for 16-24 hours, Bacillus natto BNCC194961 and Bacillus licheniformis BNCC132622 are inoculated and fermentation is continued for 16-24 hours; Alternatively, Bacillus subtilis CGMCC No.13932 and Bacillus licheniformis BNCC132622 are added at the same time, the culture temperature is 30-40°C, the humidity of the incubator is 60-80%, and after fermentation for 16-24 hours, Bacillus natto BNCC194961 is added to continue fermentation for 16-24 hours.

2. A method for producing nattokinase by solid-state fermentation of hemp seed meal according to claim 1, characterized in that: Step 1) Crush the hemp seed meal and add 1: (1-5) (w / v, g / mL) distilled water to soak for 2-5 h.

3. A method for producing nattokinase by solid-state fermentation of hemp seed meal according to claim 1, characterized in that: Step 2) sterilize the hemp seed meal with high pressure steam, control the temperature at 100-120° C., and sterilize for 20-30 minutes.

4. A method for producing nattokinase by solid-state fermentation of hemp seed meal according to claim 1, characterized in that: Step 3) Inoculate the nattokinase producing bacteria seed liquid into the fermentation medium at a seed liquid inoculation rate of 5-15%.

5. A method for producing nattokinase by solid-state fermentation of hemp seed meal according to claim 1, characterized in that: Step 4) Add 1: (5-10) (w / v, g / mL) distilled water and extract for 2-5 h, centrifuge at 10000-12000 r / min for 10-20 min, and use the centrifuged supernatant to detect nattokinase activity.

6. A method for producing nattokinase by solid-state fermentation of hemp seed meal according to claim 3, characterized in that: In step 2), protease is added to the sterilized hemp seed meal for enzymolysis.

7. A method for producing nattokinase by solid-state fermentation of hemp seed meal according to claim 6, characterized in that: The protease is any one of neutral protease, alkaline protease and papain, the enzymolysis temperature is 40-55° C., and the amount of protease added is 5-10%.

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