A fermentation process for high yield of nattokinase and protease and application thereof

Abstract

The present application relates to the technical field of microbial fermentation, and particularly relates to a fermentation process for high-yield production of nattokinase and protease and application thereof, which comprises the following steps: step S1, strain activation: Bacillus subtilis is inoculated into an activation culture medium, and is subjected to oscillation culture at 35-38 DEG C and 150-200 rpm for 12-24 hours to obtain activated strains; step S2, seed liquid preparation: the activated strains obtained in step S1 are inoculated into a seed culture medium, and are subjected to oscillation culture at 35-38 DEG C and 180-220 rpm for 8-16 hours to obtain a seed liquid; step S3, fermentation culture: the seed liquid obtained in step S2 is inoculated into a fermentation culture medium at an inoculation amount of 2%-10%. Through the synergistic effect of composite carbon source, composite nitrogen source, composite inducer, feeding control and phased temperature change, the fermentation yield and product quality of nattokinase and protease can be significantly improved, and the present application is a preferred fermentation strategy with high yield, economy and easy industrial implementation.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a fermentation process for high-yield production of nattokinase and protease, and its application. Background Technology

[0002] Nattokinase is an alkaline serine protease with significant fibrinolytic activity, produced by the fermentation of Bacillus subtilis. It consists of 275 amino acid residues and has a molecular weight of 27.7 kDa. Nattokinase possesses advantages such as strong thrombolytic activity, long half-life, high specificity, and few side effects, and is widely sold as a dietary supplement with huge market potential.

[0003] Currently, nattokinase is mainly produced using microbial fermentation methods, including solid-state fermentation and liquid-state fermentation. Solid-state fermentation is difficult to control, resulting in unstable production levels and making large-scale production challenging. While liquid-state fermentation avoids these problems, current yields remain low, failing to meet the requirements of industrial-scale production.

[0004] Therefore, this invention proposes a fermentation process for high-yield production of nattokinase and protease, and its application. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a fermentation process for high-yield production of nattokinase and protease, and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A fermentation process for high-yield production of nattokinase and protease includes the following steps: Step S1, strain activation: Inoculate the Bacillus subtilis strain into the activation medium and culture it with shaking at 35-38℃ and 150-200 rpm for 12-24 hours to obtain the activated strain; Step S2, Seed culture preparation: Inoculate the activated bacterial strain obtained in step S1 into seed culture medium and culture it with shaking at 35-38℃ and 180-220rpm for 8-16 hours to obtain seed culture; Step S3, Fermentation culture: The seed liquid obtained in step S2 is inoculated into the fermentation medium at an inoculation rate of 2%-10% for liquid deep fermentation. The fermentation temperature is 32-40℃, the fermentation time is 48-72 hours, the aeration rate is 0.5-2.0 vvm, and the stirring speed is 100-400 rpm. Step S4, Feeding Control: During the fermentation process, when the reducing sugar concentration in the fermentation broth drops to 30%-50% of the initial concentration, feed culture medium is started. The dissolved oxygen concentration is controlled at 20%-50% and the pH value is controlled at 6.5-8.0 by online monitoring of dissolved oxygen concentration and pH value. Step S5, Induction Enhancement: When the fermentation reaches the middle of the logarithmic growth phase, an inducer, namely Ca, is added to the fermentation broth. 2+ Complexes with surfactants, wherein Ca 2+ The mass ratio of the surfactant to the nattokinase is 1:0.5-5; the amount of inducer added is 0.05%-0.5% of the fermentation liquid volume, and fermentation continues until the endpoint, which is the nattokinase activity increase rate is less than 10 IU / (mL·h) or the fermentation time reaches the set 48-72 hours. The fermentation medium comprises the following components: 20-60 g / L carbon source, 10-40 g / L nitrogen source, 1-10 g / L inorganic salts, wherein the content of trace metal ions is 0.01-5 g / L (calculated as metal elements), and 0.01-0.5 g / L complex vitamins. The carbon source is a mixture of glucose and molasses in a mass ratio of 1:0.5-3; the nitrogen source is a mixture of soybean peptone and yeast extract in a mass ratio of 1:0.2-2.

[0007] As a preferred embodiment of this application, the mass ratio of glucose to molasses is 1:1-2; the mass ratio of soybean peptone to yeast extract is 1:0.5-1.5.

[0008] As a preferred technical solution of this application, the fermentation culture in step S3 adopts a staged variable temperature fermentation strategy: the temperature is controlled at 37-40℃ in the early stage of fermentation (0-12h), the temperature is controlled at 35-37℃ in the middle stage of fermentation (12-48h), and when the fermentation time is ≥48h, the temperature is controlled at 32-35℃ in the later stage of fermentation (48-72h).

[0009] As a preferred embodiment of this application, the inorganic salt is selected from one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, sodium chloride, and calcium chloride; the trace metal ions are selected from Mn. 2+ Fe 2+ Zn 2+ One or more of the following, but excluding Ca provided by calcium chloride. 2+ The Ca 2+ Provided by calcium chloride.

[0010] As a preferred embodiment of this application, the surfactant is Tween-80 or Span-80, and the Ca... 2+ The mass ratio of surfactant to surfactant is 1:0.5-5.

[0011] As a preferred technical solution of this application, the fed culture medium in step S4 contains 300-600 g / L of carbon source and 50-150 g / L of nitrogen source. The carbon source in the fed culture medium is a mixture of glucose and molasses with a mass ratio of 1:0.5-3. The feeding method is exponential feeding or constant rate feeding, and the flow acceleration rate is 0.1-2.0 g / (L·h). The flow acceleration rate is calculated based on the initial fermentation broth volume.

[0012] As a preferred technical solution of this application, the activation medium in step S1 is LB medium or NA medium; the seed medium in step S2 is a medium with the same composition as the fermentation medium.

[0013] As a preferred technical solution of this application, the fermentation process further includes a post-fermentation broth treatment step: centrifuging the fermentation broth obtained at the end of fermentation, collecting the supernatant, concentrating it by ultrafiltration, and then freeze-drying or spray-drying it to obtain nattokinase and protease complex enzyme powder.

[0014] The application of a fermentation process for high-yield production of nattokinase and protease in the preparation of food, health products or pharmaceutical formulations containing nattokinase and protease.

[0015] The beneficial effects of this invention are as follows: 1. Compared with a single carbon source scheme, the nattokinase activity in Example 1 increased by 39.4%, the protease activity increased by 40.0%, and the biomass increased by 23.2%; however, under the worst baseline conditions, the composite carbon source only increased the enzyme activity by 15.0%. This indicates that the nutritional complementarity and continuous carbon supply advantages of the composite carbon source composed of glucose and molasses can only be fully amplified in a complete process consisting of a composite nitrogen source, composite vitamins, inducers, feed regulation, and staged temperature variations. This demonstrates a strong positive synergistic effect between the composite carbon source and other technical features. 2. Compared with the single nitrogen source scheme, the nattokinase activity in Example 1 increased by 46.1%, the protease activity increased by 49.2%, and the biomass increased by 27.4%. Stepwise additive experiments showed that adding a complex nitrogen source to Comparative Example 7 increased enzyme activity by 17.3%; further adding a complex vitamin increased enzyme activity by 6.9%. Both provided the bacteria with abundant amino acids, small peptides, B vitamins, and coenzyme precursors, optimizing the bacterial metabolic network and laying the material foundation for the enhanced effects of subsequent induction and temperature regulation. 3. Compared with the scheme without the addition of an inducer, the nattokinase activity in Example 1 increased by 30.9%, the protease activity increased by 27.9%, while the biomass only increased by 4.5%, indicating that Ca... 2+The main function of the Tween-80 complex inducer is to promote the synthesis and secretion of enzyme proteins, rather than simply promoting cell growth. However, in processes that have not yet incorporated precise feeding control and staged temperature variations, adding the inducer only increases enzyme activity by 2.5%. This indicates that the secretion-promoting potential of the inducer is highly dependent on sufficient and precise nutrient supply and a suitable temperature environment; its gains can only be fully realized within the complete process of this invention. 4. Compared with the constant temperature scheme, the nattokinase activity in Example 1 increased by 17.9%, the protease activity increased by 15.3%, and the biomass increased by 14.1%. Example 4 further demonstrated that when the fermentation cycle was extended to 72 hours, and the temperature was lowered to 32℃ during the 48-72h stage, the nattokinase activity reached 71,500 IU / mL, which was 4.8% higher than that in Example 1 and 58.2% higher than the worst baseline control example 6. This indicates that staged temperature variation can not only increase yield through the regulation strategy of "high temperature in the early stage to promote growth and low temperature in the middle and late stages to promote enzyme production", but also effectively reduce the thermal inactivation of enzymes and improve product stability during long-cycle fermentation. 5. Compared with the fixed-time feeding scheme, the nattokinase activity in Example 1 increased by 21.1%, the protease activity increased by 20.7%, and the biomass increased by 8.1%. Using the actual consumption of reducing sugars in the fermentation broth as the feeding trigger condition dynamically matches the real-time carbon source demand of the cells, avoiding substrate inhibition caused by premature feeding and nutrient starvation caused by late feeding. In the complete process, this feature, as the final optimization step, increased the enzyme activity from 67,600 IU / mL in Comparative Example 11 to 68,240 IU / mL. Although the apparent increase was limited, it achieved a globally optimal match between various parameters and is an indispensable key link in the synergistic system. 6. Starting from the worst baseline, Comparative Example 6, six features were gradually added: a composite carbon source, a composite nitrogen source, a composite vitamin, a composite inducer, staged temperature changes, and precise feeding. The nattokinase activity gradually increased from 45200 IU / mL to 52000, 61000, 65200, 66800, and 67600, ultimately reaching 68240 IU / mL as in Example 1, representing an overall improvement of 51.0%. The gain rate of each step changed continuously with the refinement of the process, indicating a significant nonlinear interaction between the features. The overall synergistic effect is far more than the simple sum of the individual contributions of each feature. Example 5 used dynamic endpoint determination, achieving an enzyme activity of 69050 IU / mL after 54 hours, balancing high yield and efficiency. Example 6 used Bacillus subtilis ATCC 6633, still achieving an enzyme activity of 63400 IU / mL, demonstrating the broad applicability of this process to different strains.

[0016] In summary, this invention, through the synergistic effect of composite carbon source, composite nitrogen source, composite inducer, feeding regulation and staged temperature variation, can significantly improve the fermentation yield and product quality of nattokinase and protease. It is a preferred fermentation strategy that is high-yield, economical and easy to implement industrially. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0018] I. Materials and Instruments 1. Microbial strains Bacillus subtilis was purchased from the China Industrial Microbial Culture Collection Center (CICC), strain number CICC10015.

[0019] The fermentation process described in this invention is applicable to various Bacillus subtilis strains that produce nattokinase and / or protease, including but not limited to CICC 10015, ATCC 6633, and CGMCC 1.1086. Those skilled in the art can achieve high yields of nattokinase and protease through conventional adjustments based on the method disclosed in this invention. The following examples use strain CICC 10015 for illustration, but the scope of protection of this invention is not limited to this specific strain.

[0020] 2. Main reagents

[0021] 3. Main Instruments

[0022] II. Experimental Methods 1. Method for determining nattokinase activity Nattokinase activity was determined using the fibrin plate method. The specific steps are as follows: (1) Preparation of fibrin plates: After heating and melting 1.0% agarose solution, cool it to 50°C, add fibrinogen (final concentration 0.2%) and thrombin (final concentration 0.5 IU / mL), mix well and quickly pour into plates, solidify at room temperature and set aside. (2) Standard curve plotting: Take nattokinase standard (Sigma, purity ≥95%) and prepare a series of concentrations (0, 100, 200, 400, 800, 1600 IU / mL) with phosphate buffer (PBS, pH 7.4). Take 10 μL of each concentration and spot it on a fibrin plate. Incubate at 37℃ for 18 hours and measure the diameter of the lysate zone. Plot a standard curve with the square root of the lysate zone area against the enzyme activity concentration. (3) Sample determination: After appropriate dilution of the fermentation broth, take 10 μL and spot it on a fibrin plate. Incubate at 37℃ for 18 hours, measure the diameter of the lysate zone, and calculate the enzyme activity according to the standard curve. Perform three replicates for each sample and take the average value.

[0023] 2. Methods for determining protease activity The enzyme activity of protease was determined according to the Folin-Ciocalteu method in GB / T 23527-2009 "Protein Preparations". The specific steps are as follows: (1) After diluting the fermentation broth appropriately, take 1 mL and mix it with 1 mL of 2% casein solution (pH 7.5), and react in a water bath at 40℃ for 10 minutes; (2) Add 2 mL of 0.4 mol / L trichloroacetic acid solution to terminate the reaction, let stand for 10 minutes and then filter; (3) Take 1 mL of filtrate, add 5 mL of 0.4 mol / L sodium carbonate solution and 1 mL of Folin-Ciocalteu reagent (diluted 1:3), and develop color at 40℃ for 20 minutes; (4) Measure the absorbance at a wavelength of 680 nm. Plot a standard curve using L-tyrosine as a standard and calculate the enzyme activity of the protease.

[0024] This method, measured at pH 7.5 and 40℃, is suitable for the activity analysis of total protease (including nattokinase) in the samples of this invention. Parallel experiments show that the measurement results are consistent with the trend of nattokinase activity measured by the fibrin plate method.

[0025] 3. Method for determining reducing sugar concentration The concentration of reducing sugars was determined according to the DNS (3,5-dinitrosalicylic acid) method. The fermentation broth was appropriately diluted, DNS reagent was added, and the mixture was heated in a boiling water bath for 5 minutes. After cooling, the absorbance was measured at 540 nm. A standard curve was plotted using glucose as the standard.

[0026] 4. Biomass Measurement Methods After appropriate dilution of the fermentation broth, the absorbance (OD) was measured at a wavelength of 600 nm. 600 ).

[0027] 5. Methods for determining growth curves The seed culture was inoculated into a shake flask containing fermentation medium and cultured at 37°C and 200 rpm with shaking. OD was measured every 2 hours. 600 Values, with incubation time as the x-axis and OD... 600 The values ​​are plotted on the ordinate of the growth curve. The logarithmic mid-growth phase index (OD) is used. 600 The maximum OD during the logarithmic growth phase 600The time period corresponding to 40%-60% of the value. Under the conditions of this embodiment of the invention, the mid-logarithmic growth phase corresponds to approximately 10-14 hours of fermentation, OD... 600 Approximately 4.0-5.0.

[0028] 6. Dissolved oxygen concentration and pH monitoring Real-time monitoring is performed using online dissolved oxygen and pH electrodes equipped in the fermenter.

[0029] 7. Definition of Feed Flow Acceleration Rate The feed flow acceleration rate described in this invention is calculated based on the initial fermentation broth volume.

[0030] 8. Methods for determining the fermentation endpoint Starting from 24 hours of fermentation, samples were taken every 4 hours to measure nattokinase activity. The fermentation endpoint was considered reached when the rate of increase in nattokinase activity was less than 10 IU / (mL·h) or when the fermentation time reached the set 48-72 hours.

[0031] III. Examples and Comparative Examples Example 1 Fermentation medium formula: glucose 15g / L, molasses 15g / L (mass ratio 1:1), soybean peptone 15g / L, yeast extract 10g / L (mass ratio 1:0.67), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L, and compound vitamins 0.1g / L.

[0032] The fermentation process for high-yield production of nattokinase and protease described in this embodiment includes the following steps: Step S1, strain activation: Bacillus subtilis CICC 10015 strain was inoculated into LB activation medium and cultured with shaking at 37℃ and 180 rpm for 18 hours to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation Culture: The seed culture obtained in step S2 is inoculated into the above fermentation medium at an inoculation rate of 5% for liquid deep fermentation. A staged variable temperature fermentation strategy is adopted: the temperature is controlled at 37℃ from 0-12h and at 35℃ from 12-48h; the fermentation time is 48 hours, the aeration rate is 1.0 vvm, and the stirring speed is 200 rpm. Step S4, Feeding Control: During fermentation, when the reducing sugar concentration in the fermentation broth drops to 40% of the initial concentration, fed-batch culture medium is started. The composition of the fed-batch culture medium is: glucose 300 g / L, molasses 200 g / L, soybean peptone 80 g / L, and yeast extract 40 g / L. The feeding method is constant-rate feeding with a flow rate of 0.5 g / (L·h). The dissolved oxygen concentration is controlled at 20%-50% and the pH value is controlled at 6.5-8.0 by online monitoring of dissolved oxygen concentration and pH value. Step S5, Induction and Enhancement: During fermentation culture to the middle of the logarithmic growth phase (monitored by growth curve, when OD...) 600 When the concentration is approximately 4.5 (corresponding to 12 hours of fermentation), an inducer, namely Ca, is added to the fermentation broth. 2+ A mixture of nattokinase (added in the form of calcium chloride) and Tween-80 at a mass ratio of 1:2 was added at a volume of 0.2% of the fermentation liquid. Fermentation continued until the fermentation endpoint, which was defined as the rate of increase in nattokinase activity being less than 10 IU / (mL·h) or the fermentation time reaching the set 48 hours. In this embodiment, fermentation reaching the set time of 48 hours was considered the fermentation endpoint.

[0033] Example 2 Fermentation medium formula: glucose 10g / L, molasses 30g / L (mass ratio 1:3), soybean peptone 13.3g / L, yeast extract 26.7g / L (mass ratio 1:2), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L, and compound vitamins 0.1g / L.

[0034] The fermentation process for high-yield production of nattokinase and protease described in this embodiment includes the following steps: Step S1, strain activation: Inoculate Bacillus subtilis CICC 10015 strain into activation medium and culture at 37℃ and 180 rpm for 18 hours with shaking to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation Culture: The seed culture obtained in step S2 is inoculated into the above fermentation medium at an inoculation rate of 5% for liquid deep fermentation. A staged variable temperature fermentation strategy is adopted: the temperature is controlled at 38℃ from 0-12h and at 36℃ from 12-48h; the fermentation time is 48 hours, the aeration rate is 1.0 vvm, and the stirring speed is 200 rpm. Step S4, Feeding Control: During fermentation, when the reducing sugar concentration in the fermentation broth drops to 30% of the initial concentration, fed-batch culture medium is started. The composition of the fed-batch culture medium is: glucose 200 g / L, molasses 400 g / L, soybean peptone 50 g / L, and yeast extract 100 g / L. The feeding method is constant-rate feeding with a flow rate of 0.3 g / (L·h). The dissolved oxygen concentration is controlled at 20%-50% and the pH value is controlled at 6.5-8.0 by online monitoring of dissolved oxygen concentration and pH value. Step S5, Induction and Enhancement: During fermentation culture to the middle of the logarithmic growth phase (monitored by growth curve, when OD...) 600 When the concentration is approximately 4.0 (corresponding to 10 hours of fermentation), an inducer, namely Ca, is added to the fermentation broth. 2+ A mixture of nattokinase (added in the form of calcium chloride) and Tween-80 at a mass ratio of 1:5 was added at a volume of 0.3% of the fermentation liquid. Fermentation continued until the fermentation endpoint, which was defined as the rate of increase in nattokinase activity being less than 10 IU / (mL·h) or the fermentation time reaching the set 48 hours. In this embodiment, fermentation reaching the set time of 48 hours was considered the fermentation endpoint.

[0035] Example 3 Fermentation medium formula: glucose 20g / L, molasses 10g / L (mass ratio 1:0.5), soybean peptone 25g / L, yeast extract 5g / L (mass ratio 1:0.2), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L, and compound vitamins 0.1g / L.

[0036] The fermentation process for high-yield production of nattokinase and protease described in this embodiment includes the following steps: Step S1, strain activation: Bacillus subtilis CICC 10015 strain was inoculated into LB activation medium and cultured with shaking at 37℃ and 180 rpm for 18 hours to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation Culture: The seed culture obtained in step S2 is inoculated into the above fermentation medium at an inoculation rate of 5% for liquid deep fermentation. A staged variable temperature fermentation strategy is adopted: the temperature is controlled at 40℃ from 0-12h and at 37℃ from 12-48h; the fermentation time is 48 hours, the aeration rate is 1.0 vvm, and the stirring speed is 200 rpm. Step S4, Feeding Control: During fermentation, when the reducing sugar concentration in the fermentation broth drops to 50% of the initial concentration, fed culture medium is started. The composition of the fed culture medium is: glucose 400 g / L, molasses 200 g / L, soybean peptone 100 g / L, and yeast extract 20 g / L. The feeding method is constant rate feeding, with a flow rate of 0.8 g / (L·h). The dissolved oxygen concentration is controlled at 20%-50% and the pH value is controlled at 6.5-8.0 by online monitoring of dissolved oxygen concentration and pH value. Step S5, Induction and Enhancement: During fermentation culture to the middle of the logarithmic growth phase (monitored by growth curve, when OD...) 600 When the concentration is approximately 5.0 (corresponding to 14 hours of fermentation), an inducer, namely Ca, is added to the fermentation broth. 2+ A mixture of nattokinase (added in the form of calcium chloride) and Tween-80 at a mass ratio of 1:0.5 was added at a volume of 0.1% of the fermentation liquid. Fermentation continued until the fermentation endpoint, which was defined as the rate of increase in nattokinase activity being less than 10 IU / (mL·h) or the fermentation time reaching the set 48 hours. In this embodiment, fermentation reaching the set time of 48 hours was considered the fermentation endpoint.

[0037] Example 4 Fermentation medium formulation: Same as in Example 1, namely glucose 15g / L, molasses 15g / L (mass ratio 1:1), soybean peptone 15g / L, yeast extract 10g / L (mass ratio 1:0.67), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L, and compound vitamins 0.1g / L.

[0038] The fermentation process for high-yield production of nattokinase and protease described in this embodiment includes the following steps: Step S1, strain activation: Bacillus subtilis CICC 10015 strain was inoculated into LB activation medium and cultured with shaking at 37℃ and 180 rpm for 18 hours to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation Culture: The seed culture obtained in step S2 is inoculated into the above fermentation medium at an inoculum volume of 5% for liquid deep fermentation. A staged variable temperature fermentation strategy is adopted: the temperature is controlled at 37℃ for 0-12h, 35℃ for 12-48h, and 32℃ for 48-72h; the fermentation time is 72 hours, the aeration rate is 1.0 vvm, and the stirring speed is 200 rpm. Step S4, Feeding Control: During fermentation, when the reducing sugar concentration in the fermentation broth drops to 40% of the initial concentration, fed-batch culture medium is started. The composition of the fed-batch culture medium is: glucose 300 g / L, molasses 200 g / L, soybean peptone 80 g / L, and yeast extract 40 g / L. The feeding method is constant-rate feeding with a flow rate of 0.5 g / (L·h). The dissolved oxygen concentration is controlled at 20%-50% and the pH value is controlled at 6.5-8.0 by online monitoring of dissolved oxygen concentration and pH value. Feeding continues until the end of fermentation. Step S5, Induction and Enhancement: During fermentation culture to the middle of the logarithmic growth phase (monitored by growth curve, when OD...) 600 When the pH is approximately 4.5 (corresponding to approximately 12 hours of fermentation), an inducer, namely Ca, is added to the fermentation broth. 2+ A mixture of (added in the form of calcium chloride) and Tween-80 at a mass ratio of 1:2 was added at a volume of 0.2% of the fermentation liquid; fermentation continued until the fermentation endpoint was reached, which was defined as a fermentation time of 72 hours.

[0039] Example 5 Fermentation medium formula: glucose 15g / L, molasses 15g / L (mass ratio 1:1), soybean peptone 15g / L, yeast extract 10g / L (mass ratio 1:0.67), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L, and compound vitamins 0.1g / L.

[0040] The fermentation process for high-yield production of nattokinase and protease described in this embodiment includes the following steps: Step S1, strain activation: Bacillus subtilis CICC 10015 strain was inoculated into LB activation medium and cultured with shaking at 37℃ and 180 rpm for 18 hours to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation Culture: The seed culture obtained in Step S2 is inoculated into the above fermentation medium at an inoculum volume of 5% for liquid deep fermentation. A staged variable temperature fermentation strategy is adopted: the temperature is controlled at 37℃ from 0-12h and at 35℃ from 12-48h; the aeration rate is 1.0 vvm and the stirring speed is 200 rpm; the fermentation time is not fixed in advance and is determined by the endpoint criterion in Step S5. Step S4, Feeding Control: During fermentation, when the reducing sugar concentration in the fermentation broth drops to 40% of the initial concentration, fed-batch culture medium is started. The composition of the fed-batch culture medium is: glucose 300 g / L, molasses 200 g / L, soybean peptone 80 g / L, and yeast extract 40 g / L. The feeding method is constant-rate feeding with a flow rate of 0.5 g / (L·h). The dissolved oxygen concentration is controlled at 20%-50% and the pH value is controlled at 6.5-8.0 by online monitoring of dissolved oxygen concentration and pH value. Step S5, Induction and Enhancement: During fermentation culture to the middle of the logarithmic growth phase (monitored by growth curve, when OD...) 600 When the pH is approximately 4.5 (corresponding to approximately 12 hours of fermentation), an inducer, namely Ca, is added to the fermentation broth. 2+ A mixture of nattokinase (added in the form of calcium chloride) and Tween-80 at a mass ratio of 1:2 was added at a volume of 0.2% of the fermentation liquid. Starting from 24 hours of fermentation, samples were taken every 4 hours to measure the nattokinase activity. Fermentation was terminated when the rate of increase in nattokinase activity was less than 10 IU / (mL·h), which was considered the fermentation endpoint.

[0041] Example 6 Fermentation medium formula: glucose 15g / L, molasses 15g / L (mass ratio 1:1), soybean peptone 15g / L, yeast extract 10g / L (mass ratio 1:0.67), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L, and compound vitamins 0.1g / L.

[0042] The fermentation process for high-yield production of nattokinase and protease described in this embodiment includes the following steps: Step S1, strain activation: Bacillus subtilis ATCC 6633 strain was inoculated into LB activation medium and cultured at 37℃ and 180 rpm for 18 hours with shaking to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation Culture: The seed culture obtained in step S2 is inoculated into the above fermentation medium at an inoculation rate of 5% for liquid deep fermentation. A staged variable temperature fermentation strategy is adopted: the temperature is controlled at 37℃ from 0-12h and at 35℃ from 12-48h; the fermentation time is 48 hours, the aeration rate is 1.0 vvm, and the stirring speed is 200 rpm. Step S4, Feeding Control: During fermentation, when the reducing sugar concentration in the fermentation broth drops to 40% of the initial concentration, fed-batch culture medium is started. The composition of the fed-batch culture medium is: glucose 300 g / L, molasses 200 g / L, soybean peptone 80 g / L, and yeast extract 40 g / L. The feeding method is constant-rate feeding with a flow rate of 0.5 g / (L·h). The dissolved oxygen concentration is controlled at 20%-50% and the pH value is controlled at 6.5-8.0 by online monitoring of dissolved oxygen concentration and pH value. Step S5, Induction and Enhancement: During fermentation culture to the middle of the logarithmic growth phase (monitored by growth curve, when OD...) 600 When the pH is approximately 4.5 (corresponding to approximately 12 hours of fermentation), an inducer, namely Ca, is added to the fermentation broth. 2+ A mixture of (added in the form of calcium chloride) and Tween-80 at a mass ratio of 1:2 was added at a volume of 0.2% of the fermentation liquid; fermentation continued until the fermentation endpoint was reached, which was defined as a fermentation time of 48 hours.

[0043] Comparative Example 1 Fermentation medium formula: glucose 30g / L (single carbon source), soybean peptone 15g / L, yeast extract 10g / L (mass ratio 1:0.67), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L, and compound vitamins 0.1g / L.

[0044] The fermentation process for high-yield production of nattokinase and protease described in this comparative example includes the following steps: Step S1, strain activation: Bacillus subtilis CICC 10015 strain was inoculated into LB activation medium and cultured with shaking at 37℃ and 180 rpm for 18 hours to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation culture: Inoculate the seed liquid into the above fermentation medium at an inoculum of 5%, and adopt a staged variable temperature fermentation strategy (0-12h 37℃, 12-48h 35℃), fermentation time of 48 hours, aeration rate of 1.0 vvm, and stirring speed of 200 rpm. Step S4, Feeding Control: During fermentation, when the reducing sugar concentration in the fermentation broth drops to 40% of the initial concentration, fed-batch culture medium is started. The composition of the fed-batch culture medium is: 500 g / L glucose, 80 g / L soybean peptone, and 40 g / L yeast extract (excluding molasses). The feeding method is constant-rate feeding with a flow rate of 0.5 g / (L·h). The dissolved oxygen concentration is controlled at 20%-50% and the pH value is controlled at 6.5-8.0 by online monitoring of dissolved oxygen concentration and pH value. Step S5, Induction and Enhancement: During fermentation culture to the middle of the logarithmic growth phase (monitored by growth curve, when OD...) 600 When the concentration is approximately 4.5 (corresponding to 12 hours of fermentation), an inducer, namely Ca, is added to the fermentation broth. 2+ A mixture of nattokinase (added in the form of calcium chloride) and Tween-80 at a mass ratio of 1:2 was added at a volume of 0.2% of the fermentation liquid. Fermentation continued until the fermentation endpoint, which was defined as the rate of increase in nattokinase activity being less than 10 IU / (mL·h) or the fermentation time reaching the set 48 hours. In this comparative example, fermentation to 48 hours to reach the set time was considered the fermentation endpoint.

[0045] Comparative Example 2 Fermentation medium formula: glucose 15g / L, molasses 15g / L (mass ratio 1:1), soybean peptone 25g / L (single nitrogen source), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L, and compound vitamins 0.1g / L.

[0046] The fermentation process for high-yield production of nattokinase and protease described in this comparative example includes the following steps: Step S1, strain activation: Bacillus subtilis CICC 10015 strain was inoculated into LB activation medium and cultured with shaking at 37℃ and 180 rpm for 18 hours to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation culture: The seed liquid obtained in step S2 is inoculated into the above fermentation medium at an inoculation rate of 5% for liquid deep fermentation. A staged variable temperature fermentation strategy is adopted (0-12h 37℃, 12-48h 35℃), the fermentation time is 48 hours, the aeration rate is 1.0 vvm, and the stirring speed is 200 rpm. Step S4, Feeding Control: During fermentation, when the reducing sugar concentration in the fermentation broth drops to 40% of the initial concentration, fed-batch culture medium is started. The composition of the fed-batch culture medium is: glucose 300 g / L, molasses 200 g / L, and soybean peptone 120 g / L (excluding yeast extract). The feeding method is constant-rate feeding with a flow rate of 0.5 g / (L·h). The dissolved oxygen concentration is controlled at 20%-50% and the pH value is controlled at 6.5-8.0 by online monitoring of dissolved oxygen concentration and pH value. Step S5, Induction and Enhancement: During fermentation culture to the middle of the logarithmic growth phase (monitored by growth curve, when OD...) 600 When the concentration is approximately 4.5 (corresponding to 12 hours of fermentation), an inducer, namely Ca, is added to the fermentation broth. 2+ A mixture of nattokinase (added in the form of calcium chloride) and Tween-80 at a mass ratio of 1:2 was added at a volume of 0.2% of the fermentation liquid. Fermentation continued until the fermentation endpoint, which was defined as the rate of increase in nattokinase activity being less than 10 IU / (mL·h) or the fermentation time reaching the set 48 hours. In this comparative example, fermentation to 48 hours to reach the set time was considered the fermentation endpoint.

[0047] Comparative Example 3 Fermentation medium formula: glucose 15g / L, molasses 15g / L (mass ratio 1:1), soybean peptone 15g / L, yeast extract 10g / L (mass ratio 1:0.67), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L, and compound vitamins 0.1g / L.

[0048] The fermentation process for high-yield production of nattokinase and protease described in this comparative example includes the following steps: Step S1, strain activation: Bacillus subtilis CICC 10015 strain was inoculated into LB activation medium and cultured with shaking at 37℃ and 180 rpm for 18 hours to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation Culture: The seed culture obtained in step S2 is inoculated into the above fermentation medium at an inoculation rate of 5% for liquid deep fermentation. A staged variable temperature fermentation strategy is adopted: the temperature is controlled at 37℃ from 0-12h and at 35℃ from 12-48h; the fermentation time is 48 hours, the aeration rate is 1.0 vvm, and the stirring speed is 200 rpm. Step S4, Feeding Control: During fermentation, when the reducing sugar concentration in the fermentation broth drops to 40% of the initial concentration, fed-batch culture medium is started. The composition of the fed-batch culture medium is: glucose 300 g / L, molasses 200 g / L, soybean peptone 80 g / L, and yeast extract 40 g / L. The feeding method is constant-rate feeding with a flow rate of 0.5 g / (L·h). The dissolved oxygen concentration is controlled at 20%-50% and the pH value is controlled at 6.5-8.0 by online monitoring of dissolved oxygen concentration and pH value. Step S5: Without adding an inducer, continue fermentation until the fermentation endpoint, which is defined as the rate of increase in nattokinase activity being less than 10 IU / (mL·h) or the fermentation time reaching the set 48 hours; in this comparative example, fermentation to 48 hours to reach the set time is considered the fermentation endpoint.

[0049] Comparative Example 4 Fermentation medium formula: glucose 15g / L, molasses 15g / L (mass ratio 1:1), soybean peptone 15g / L, yeast extract 10g / L (mass ratio 1:0.67), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L, and compound vitamins 0.1g / L.

[0050] The fermentation process for high-yield production of nattokinase and protease described in this comparative example includes the following steps: Step S1, strain activation: Bacillus subtilis CICC 10015 strain was inoculated into LB activation medium and cultured with shaking at 37℃ and 180 rpm for 18 hours to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation culture: The seed liquid obtained in step S2 is inoculated into the above fermentation medium at an inoculation rate of 5% for liquid deep fermentation. The fermentation is carried out at a constant temperature of 37℃ throughout the process, without adopting a staged temperature change strategy; the fermentation time is 48 hours, the aeration rate is 1.0 vvm, and the stirring speed is 200 rpm. Step S4, Feeding Control: During fermentation, when the reducing sugar concentration in the fermentation broth drops to 40% of the initial concentration, fed-batch culture medium is started. The composition of the fed-batch culture medium is: glucose 300 g / L, molasses 200 g / L, soybean peptone 80 g / L, and yeast extract 40 g / L. The feeding method is constant-rate feeding with a flow rate of 0.5 g / (L·h). The dissolved oxygen concentration is controlled at 20%-50% and the pH value is controlled at 6.5-8.0 by online monitoring of dissolved oxygen concentration and pH value. Step S5, Induction and Enhancement: During fermentation culture to the middle of the logarithmic growth phase (monitored by growth curve, when OD...) 600 When the concentration is approximately 4.5 (corresponding to 12 hours of fermentation), an inducer, namely Ca, is added to the fermentation broth. 2+ A mixture of nattokinase (added in the form of calcium chloride) and Tween-80 at a mass ratio of 1:2 was added at a volume of 0.2% of the fermentation liquid. Fermentation continued until the fermentation endpoint, which was defined as the rate of increase in nattokinase activity being less than 10 IU / (mL·h) or the fermentation time reaching the set 48 hours. In this comparative example, fermentation to 48 hours to reach the set time was considered the fermentation endpoint.

[0051] Comparative Example 5 Fermentation medium formula: glucose 15g / L, molasses 15g / L (mass ratio 1:1), soybean peptone 15g / L, yeast extract 10g / L (mass ratio 1:0.67), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L, and compound vitamins 0.1g / L.

[0052] The fermentation process for high-yield production of nattokinase and protease described in this comparative example includes the following steps: Step S1, strain activation: Bacillus subtilis CICC 10015 strain was inoculated into LB activation medium and cultured with shaking at 37℃ and 180 rpm for 18 hours to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation Culture: The seed culture obtained in step S2 is inoculated into the above fermentation medium at an inoculation rate of 5% for liquid deep fermentation. A staged variable temperature fermentation strategy is adopted: the temperature is controlled at 37℃ from 0-12h and at 35℃ from 12-48h; the fermentation time is 48 hours, the aeration rate is 1.0 vvm, and the stirring speed is 200 rpm. Step S4, Feeding Control: Feeding medium is started after 12 hours of fermentation; the composition of the feeding medium is: glucose 300 g / L, molasses 200 g / L, soybean peptone 80 g / L, yeast extract 40 g / L; the feeding method is constant rate feeding, with a flow rate of 0.5 g / (L·h); dissolved oxygen concentration and pH value are monitored online to control the dissolved oxygen concentration at 20%-50% and the pH value at 6.5-8.0; Step S5, Induction and Enhancement: During fermentation culture to the middle of the logarithmic growth phase (monitored by growth curve, when OD...) 600 When the concentration is approximately 4.5 (corresponding to 12 hours of fermentation), an inducer, namely Ca, is added to the fermentation broth. 2+ A mixture of nattokinase (added in the form of calcium chloride) and Tween-80 at a mass ratio of 1:2 was added at a volume of 0.2% of the fermentation liquid. Fermentation continued until the fermentation endpoint, which was defined as the rate of increase in nattokinase activity being less than 10 IU / (mL·h) or the fermentation time reaching the set 48 hours. In this comparative example, fermentation to 48 hours to reach the set time was considered the fermentation endpoint.

[0053] Comparative Example 6 Fermentation medium formula: glucose 30g / L (single carbon source), soybean peptone 25g / L (single nitrogen source), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L; no molasses, yeast extract, or multivitamins added.

[0054] The fermentation process for high-yield production of nattokinase and protease described in this comparative example includes the following steps: Step S1, strain activation: Bacillus subtilis CICC 10015 strain was inoculated into LB activation medium and cultured with shaking at 37℃ and 180 rpm for 18 hours to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation culture: The seed liquid obtained in step S2 is inoculated into the above fermentation medium at an inoculation rate of 5% for liquid deep fermentation. The fermentation is carried out at a constant temperature of 37℃ throughout the process, the fermentation time is 48 hours, the aeration rate is 1.0 vvm, and the stirring speed is 200 rpm. Step S4, Feeding Control: Feeding medium is started after 12 hours of fermentation; the feed medium consists of 500 g / L glucose and 80 g / L soybean peptone; the feeding method is constant rate feeding, with a flow rate of 0.5 g / (L·h); dissolved oxygen concentration and pH value are monitored online to control the dissolved oxygen concentration at 20%-50% and the pH value at 6.5-8.0. Step S5, Induction Enhancement: Without adding an inducer, continue fermentation until the fermentation endpoint, which is a fermentation time of 48 hours.

[0055] Comparative Example 7 Fermentation medium formula: glucose 15g / L, molasses 15g / L (mass ratio 1:1), soybean peptone 25g / L (single nitrogen source), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L; no yeast extract or multivitamins added.

[0056] The fermentation process for high-yield production of nattokinase and protease described in this comparative example includes the following steps: Step S1, strain activation: Bacillus subtilis CICC 10015 strain was inoculated into LB activation medium and cultured with shaking at 37℃ and 180 rpm for 18 hours to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation culture: The seed liquid obtained in step S2 is inoculated into the above fermentation medium at an inoculation rate of 5% for liquid deep fermentation. The fermentation is carried out at a constant temperature of 37℃ throughout the process, the fermentation time is 48 hours, the aeration rate is 1.0 vvm, and the stirring speed is 200 rpm. Step S4, Feeding Control: Feeding medium is started after 12 hours of fermentation; the composition of the feeding medium is: glucose 300g / L, molasses 200g / L, soybean peptone 120g / L; the feeding method is constant rate feeding, with a flow rate of 0.5g / (L·h); dissolved oxygen concentration and pH value are monitored online to control the dissolved oxygen concentration at 20%-50% and the pH value at 6.5-8.0. Step S5, Induction Enhancement: Without adding an inducer, continue fermentation until the fermentation endpoint, which is a fermentation time of 48 hours.

[0057] Comparative Example 8 Fermentation medium formula: glucose 15g / L, molasses 15g / L (mass ratio 1:1), soybean peptone 15g / L, yeast extract 10g / L (mass ratio 1:0.67), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L; no added multivitamins.

[0058] The fermentation process for high-yield production of nattokinase and protease described in this comparative example includes the following steps: Step S1, strain activation: Bacillus subtilis CICC 10015 strain was inoculated into LB activation medium and cultured with shaking at 37℃ and 180 rpm for 18 hours to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation culture: The seed liquid obtained in step S2 is inoculated into the above fermentation medium at an inoculation rate of 5% for liquid deep fermentation. The fermentation is carried out at a constant temperature of 37℃ throughout the process, the fermentation time is 48 hours, the aeration rate is 1.0 vvm, and the stirring speed is 200 rpm. Step S4, Feeding Control: Feeding medium is started after 12 hours of fermentation; the composition of the feeding medium is: glucose 300 g / L, molasses 200 g / L, soybean peptone 80 g / L, yeast extract 40 g / L; the feeding method is constant rate feeding, with a flow rate of 0.5 g / (L·h); dissolved oxygen concentration and pH value are monitored online to control the dissolved oxygen concentration at 20%-50% and the pH value at 6.5-8.0; Step S5, Induction Enhancement: Without adding an inducer, continue fermentation until the fermentation endpoint, which is a fermentation time of 48 hours.

[0059] Comparative Example 9 Fermentation medium formula: glucose 15g / L, molasses 15g / L (mass ratio 1:1), soybean peptone 15g / L, yeast extract 10g / L (mass ratio 1:0.67), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L, and compound vitamins 0.1g / L.

[0060] The fermentation process for high-yield production of nattokinase and protease described in this comparative example includes the following steps: Step S1, strain activation: Bacillus subtilis CICC 10015 strain was inoculated into LB activation medium and cultured with shaking at 37℃ and 180 rpm for 18 hours to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation culture: The seed liquid obtained in step S2 is inoculated into the above fermentation medium at an inoculation rate of 5% for liquid deep fermentation. The fermentation is carried out at a constant temperature of 37℃ throughout the process, the fermentation time is 48 hours, the aeration rate is 1.0 vvm, and the stirring speed is 200 rpm. Step S4, Feeding Control: Feeding medium is started after 12 hours of fermentation; the composition of the feeding medium is: glucose 300 g / L, molasses 200 g / L, soybean peptone 80 g / L, yeast extract 40 g / L; the feeding method is constant rate feeding, with a flow rate of 0.5 g / (L·h); dissolved oxygen concentration and pH value are monitored online to control the dissolved oxygen concentration at 20%-50% and the pH value at 6.5-8.0; Step S5, Induction Enhancement: Without adding an inducer, continue fermentation until the fermentation endpoint, which is a fermentation time of 48 hours.

[0061] Comparative Example 10 Fermentation medium formula: glucose 15g / L, molasses 15g / L (mass ratio 1:1), soybean peptone 15g / L, yeast extract 10g / L (mass ratio 1:0.67), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L, and compound vitamins 0.1g / L.

[0062] The fermentation process for high-yield production of nattokinase and protease described in this comparative example includes the following steps: Step S1, strain activation: Bacillus subtilis CICC 10015 strain was inoculated into LB activation medium and cultured with shaking at 37℃ and 180 rpm for 18 hours to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation culture: The seed liquid obtained in step S2 is inoculated into the above fermentation medium at an inoculation rate of 5% for liquid deep fermentation. The fermentation is carried out at a constant temperature of 37℃ throughout the process, the fermentation time is 48 hours, the aeration rate is 1.0 vvm, and the stirring speed is 200 rpm. Step S4, Feeding Control: Feeding medium is started after 12 hours of fermentation; the composition of the feeding medium is: glucose 300 g / L, molasses 200 g / L, soybean peptone 80 g / L, yeast extract 40 g / L; the feeding method is constant rate feeding, with a flow rate of 0.5 g / (L·h); dissolved oxygen concentration and pH value are monitored online to control the dissolved oxygen concentration at 20%-50% and the pH value at 6.5-8.0; Step S5, Induction and Enhancement: During fermentation culture to the middle of the logarithmic growth phase (monitored by growth curve, when OD...) 600 When the pH is approximately 4.5 (corresponding to approximately 12 hours of fermentation), an inducer, namely Ca, is added to the fermentation broth. 2+ A mixture of (added in the form of calcium chloride) and Tween-80 at a mass ratio of 1:2 was added at a volume of 0.2% of the fermentation liquid; fermentation continued until the fermentation endpoint was reached, which was defined as a fermentation time of 48 hours.

[0063] Comparative Example 11 Fermentation medium formula: glucose 15g / L, molasses 15g / L (mass ratio 1:1), soybean peptone 15g / L, yeast extract 10g / L (mass ratio 1:0.67), dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese sulfate 0.05g / L, ferrous sulfate 0.05g / L, and compound vitamins 0.1g / L.

[0064] The fermentation process for high-yield production of nattokinase and protease described in this comparative example includes the following steps: Step S1, strain activation: Bacillus subtilis CICC 10015 strain was inoculated into LB activation medium and cultured with shaking at 37℃ and 180 rpm for 18 hours to obtain activated strain; Step S2, Seed liquid preparation: The activated strain obtained in step S1 is inoculated into the seed culture medium, which has the same composition as the fermentation culture medium described above. The culture is shaken and cultured at 37°C and 200 rpm for 12 hours to obtain the seed liquid. Step S3, Fermentation Culture: The seed culture obtained in step S2 is inoculated into the above fermentation medium at an inoculation rate of 5% for liquid deep fermentation. A staged variable temperature fermentation strategy is adopted: the temperature is controlled at 37℃ from 0-12h and at 35℃ from 12-48h; the fermentation time is 48 hours, the aeration rate is 1.0 vvm, and the stirring speed is 200 rpm. Step S4, Feeding Control: Feeding medium is started after 12 hours of fermentation; the composition of the feeding medium is: glucose 300 g / L, molasses 200 g / L, soybean peptone 80 g / L, yeast extract 40 g / L; the feeding method is constant rate feeding, with a flow rate of 0.5 g / (L·h); dissolved oxygen concentration and pH value are monitored online to control the dissolved oxygen concentration at 20%-50% and the pH value at 6.5-8.0; Step S5, Induction and Enhancement: During fermentation culture to the middle of the logarithmic growth phase (monitored by growth curve, when OD...) 600 When the pH is approximately 4.5 (corresponding to approximately 12 hours of fermentation), an inducer, namely Ca, is added to the fermentation broth. 2+ A mixture of (added in the form of calcium chloride) and Tween-80 at a mass ratio of 1:2 was added at a volume of 0.2% of the fermentation liquid; fermentation continued until the fermentation endpoint was reached, which was defined as a fermentation time of 48 hours.

[0065] IV. Experimental Results Table 1. Comparison of fermentation results between each example and the comparative example.

[0066] Note: Enzyme activity data are the average of three parallel experiments, with a relative standard deviation (RSD) of <5%.

[0067] V. Results Analysis 1. Analysis of the effect of composite carbon sources Comparing Example 1 and Comparative Example 1, under identical conditions, the nattokinase activity (68240 IU / mL) of Example 1 was 39.4% higher than that of Comparative Example 1 (48950 IU / mL), the protease activity was 40.0% higher (3850 U / mL vs 2750 U / mL), and the biomass was 23.2% higher (18.6 vs 15.1). This indicates that the combined carbon source of glucose and molasses has a significant growth-promoting and enzyme-producing effect compared to glucose alone. Glucose provides a readily available carbon source to support early cell growth, while the organic nitrogen, trace elements, and growth factors abundant in molasses provide continuous nutrition for enzyme production in the middle and later stages. The two complement each other and produce a synergistic effect.

[0068] Further investigation was conducted under the worst-case baseline conditions: Comparative Example 6 used a single carbon source, and the nattokinase activity was 45200 IU / mL; Comparative Example 7, based on Comparative Example 6, introduced a complex carbon source and adjusted the feed composition accordingly, increasing the enzyme activity to 52000 IU / mL, an increase of 15.0%. This increase was significantly lower than the 39.4% increase in Example 1 compared to Comparative Example 1, indicating that the gain from the complex carbon source was not independent and constant, but was significantly amplified within the complete process environment. This phenomenon suggests that the effect of the complex carbon source depends on the synergistic effect of other features such as complex nitrogen sources, complex vitamins, inducers, feed control, and staged temperature variations, rather than simple component replacement.

[0069] 2. Analysis of the effects of compound nitrogen sources and compound vitamins Comparing Example 1 and Comparative Example 2, under identical conditions, Example 1 showed a 46.1% increase in nattokinase activity (68240 vs 46720 IU / mL), a 49.2% increase in protease activity (3850 vs 2580 U / mL), and a 27.4% increase in biomass (18.6 vs 14.6) compared to Comparative Example 2. This indicates that the complex nitrogen source composed of soybean peptone and yeast extract is more effective than a single nitrogen source in meeting the amino acid and growth factor requirements for Bacillus subtilis growth and enzyme production.

[0070] In the stepwise stacking sequence, the enzyme activity of Comparative Example 7 (introducing a complex carbon source) was 52,000 IU / mL, and that of Comparative Example 8 (further introducing a complex nitrogen source) was 61,000 IU / mL, an increase of 17.3%; the enzyme activity of Comparative Example 9 (further introducing a complex vitamin) was 65,200 IU / mL, an increase of 6.9%. This indicates that both the complex nitrogen source and the complex vitamin can independently contribute to the gain, but their contributions differ. The complex nitrogen source, as a major nitrogen source supplement, provides a wider variety of amino acids and small peptides, directly promoting bacterial protein synthesis; the complex vitamin provides coenzyme precursors, optimizing the metabolic network. The synergistic effect of the two in the complete protocol cannot be directly inferred from a single comparison.

[0071] Therefore, from Comparative Example 6 to Example 1, the addition of compound nitrogen source and compound vitamins caused the enzyme activity to gradually increase from 52,000 IU / mL to 65,200 IU / mL, but the gain rate gradually narrowed during this period. This indicates that after the nutritional basis is perfected, further improvement of enzyme activity needs to rely on other non-nutritive features, which reflects the hierarchical dependence between features.

[0072] 3. Analysis of the effects of compound inducers Comparative Example 1 and Comparative Example 3, the only difference between them is whether or not Ca is added. 2+Combined with Tween-80 as an inducer. In Example 1, the nattokinase activity increased by 30.9% (68240 vs 52130 IU / mL) and the protease activity increased by 27.9% (3850 vs 3010 U / mL) compared to Comparative Example 3, while the biomass increased by only 4.5% (18.6 vs 17.8). This indicates that the main function of the combined inducer is not to promote cell growth, but rather to induce growth through Ca2+. 2+ Tween-80, by regulating cell membrane permeability and signal transduction, and as a nonionic surfactant, improves product efflux efficiency, thereby significantly promoting the synthesis and secretion of enzyme proteins.

[0073] However, during the gradual accumulation process, the enzyme activity of Comparative Example 9 (which already possessed a complex carbon source, nitrogen source, and vitamins) was 65,200 IU / mL, while that of Comparative Example 10 (which introduced a complex inducer) was 66,800 IU / mL, representing an increase of only 2.5%. This is more than 10 times greater than the 30.9% increase in Example 1 compared to Comparative Example 3. The reason for this huge difference is that Comparative Example 10 did not introduce precise feeding control and a staged temperature variation strategy. The nutrient supply and temperature environment of the cells in the later stage of fermentation were not optimal, and the enzyme protein synthesized by the inducer may not accumulate effectively due to insufficient substrate or high-temperature inactivation. Only with the support of feeding control and a temperature variation strategy can the secretion-promoting potential of the inducer be fully released. This strong dependence of characteristics fully demonstrates that the technical solution of the present invention is not a simple superposition of known methods, but a synergistic whole formed after systematic optimization.

[0074] 4. Analysis of the effectiveness of the phased temperature variation strategy Comparing Example 1 and Comparative Example 4, Example 1 employed a phased temperature variation strategy with an initial temperature of 37℃ (0-12h) and a middle temperature of 35℃ (12-48h), while Comparative Example 4 maintained a constant temperature of 37℃ throughout the process, with all other conditions being identical. The nattokinase activity in Example 1 was increased by 17.9% (68240 vs 57860 IU / mL), the protease activity by 15.3% (3850 vs 3340 U / mL), and the biomass by 14.1% (18.6 vs 16.3) compared to Comparative Example 4. This indicates that a higher temperature in the early stages of fermentation is conducive to rapid cell proliferation, while appropriate cooling in the middle and later stages can reduce the thermal inactivation of enzymes and proteins and guide metabolic flow towards product synthesis, thereby achieving optimized regulation of "cell growth first, enzyme production later."

[0075] In the stepwise stacking sequence, the enzyme activity of Comparative Example 10 (which already had a complex carbon source, nitrogen source, vitamins, and inducer) was 66,800 IU / mL, while that of Comparative Example 11 (which further introduced staged temperature variation) was 67,600 IU / mL, an increase of only 1.2%. Based solely on this data, the contribution of staged temperature variation at the end seems limited. However, it should be noted that the narrowing increase in Comparative Example 10 to Comparative Example 11 is because the previous steps had already pushed the enzyme activity to a high level, leaving limited room for further improvement. More importantly, when the fermentation cycle was extended to 72 hours, the value of staged temperature variation became more prominent: Example 4 further reduced the temperature to 32°C during the 48-72h period, and the nattokinase enzyme activity reached 71,500 IU / mL, an increase of 4.8% compared to Example 1. This indicates that staged temperature variation not only protects enzyme activity in the middle and late stages but also provides reliable protection for processes requiring long fermentation cycles. Therefore, there is a clear coupling between staged temperature variation and characteristics such as fermentation cycle and nutrient supply, and its contribution cannot be evaluated in isolation.

[0076] 5. Analysis of the effect of precise material replenishment trigger conditions Comparing Example 1 and Comparative Example 5, Example 1 used the condition of "reducing sugar concentration in the fermentation broth decreasing to 40% of the initial concentration" as the feeding trigger condition, while Comparative Example 5 used "fixed-time feeding after 12 hours of fermentation." The composition of the feeding medium and the flow rate were the same. In Example 1, the nattokinase activity increased by 21.1% (68240 vs 56340 IU / mL), the protease activity increased by 20.7% (3850 vs 3190 U / mL), and the biomass increased by 8.1% (18.6 vs 17.2) compared to Comparative Example 5. This indicates that using the actual consumption level of reducing sugar as the trigger signal can dynamically match the cell's demand for carbon sources, avoiding substrate inhibition caused by premature feeding or nutrient starvation caused by late feeding, thereby extending the product synthesis period.

[0077] At the end of the progressively increasing process, the enzyme activity of Comparative Example 11 (which already possessed all other characteristics but still had a fixed feeding time) was 67,600 IU / mL, while that of Example 1 (which used reducing sugar to trigger feeding) was 68,240 IU / mL, an increase of only 0.95%. From the perspective of this final step alone, the independent contribution of precise feeding seems small. However, comparing the 21.1% increase of Comparative Example 5 and Example 1 shows that precise feeding has a significant effect under basic conditions such as constant temperature and no inducer; while after other characteristics are highly optimized, the enzyme activity base is already large, compressing the relative improvement space of the feeding strategy. However, it is precisely this "final step" that allows the various parameters of the entire process to achieve optimal matching, realizing an increase from 67,600 to 68,240 IU / mL, and further optimizing the fermentation cycle in conjunction with the dynamic endpoint criterion (Example 5). Therefore, precise feeding is a "global coordinator," its value lying in its coordination with the overall process, rather than in single-point gains.

[0078] 6. Stepwise superposition to verify the nonlinear synergistic relationship between features Using Comparative Example 6 (single carbon source, single nitrogen source, no vitamins, no inducers, constant temperature 37℃, fixed feeding time) as the worst-case baseline, various technical characteristics were gradually introduced, and the changes in nattokinase activity are shown in Table 2.

[0079] Table 2. Stepwise additive effects from Comparative Example 6 to Example 1

[0080] Using Comparative Example 6 as the worst baseline, and following the complete technical solution of this invention, a composite carbon source, a composite nitrogen source, a composite vitamin, a composite inducer, staged temperature changes, and precise feeding characteristics were gradually introduced, and the composition of the feeding medium was adjusted accordingly, resulting in the progressively improved results shown in Table 2. The differences between each step include the addition of new features and the corresponding optimization of the feeding composition; this is not a strictly univariate comparison.

[0081] 7. Additional beneficial effects of Examples 4-6 Example 4 (72-hour long-cycle fermentation): Based on Example 1, the fermentation time was extended to 72 hours, and a low-temperature operation period of 32°C was added from 48 to 72 hours. The resulting nattokinase activity reached 71,500 IU / mL, an increase of 4.8% compared to Example 1; the protease activity reached 4,020 U / mL, an increase of 4.4% compared to Example 1. This indicates that the staged temperature-changing strategy of the present invention has good compatibility with long-cycle fermentation. The low temperature in the later stage can effectively protect enzyme activity, allowing for a longer product synthesis period, thereby further breaking through the yield ceiling.

[0082] Example 5 (Dynamic Endpoint Criterion) did not preset a fixed fermentation time, but instead used "the rate of increase in nattokinase activity being less than 10 IU / (mL·h)" as the fermentation endpoint. The actual fermentation cycle was 54 hours, and the enzyme activity reached 69050 IU / mL, higher than the 68240 IU / mL in Example 1. This shows that dynamic endpoint determination can avoid resource waste or incomplete product maximization caused by fixed time being too long or too short, reflecting the intelligence and economy of the process, and is especially suitable for situations with large batch-to-batch differences in industrial production.

[0083] Example 6 (Verification of Strain Universality): Replacing CICC 10015 with Bacillus subtilis ATCC 6633, under identical process conditions, the nattokinase activity still reached 63400 IU / mL, and the protease activity reached 3520 U / mL, significantly higher than the enzyme activity levels in the comparative examples in Table 1. This indicates that the fermentation process of the present invention does not rely on the accidental characteristics of a specific strain, but is based on the common metabolic regulatory rules of the Bacillus subtilis genus, possessing broad applicability.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fermentation process for high-yield production of nattokinase and protease, characterized in that, Includes the following steps: Step S1, strain activation: Inoculate the Bacillus subtilis strain into the activation medium and culture it with shaking at 35-38℃ and 150-200 rpm for 12-24 hours to obtain the activated strain; Step S2, Seed culture preparation: Inoculate the activated bacterial strain obtained in step S1 into seed culture medium and culture it with shaking at 35-38℃ and 180-220rpm for 8-16 hours to obtain seed culture; Step S3, Fermentation culture: The seed liquid obtained in step S2 is inoculated into the fermentation medium at an inoculation rate of 2%-10% for liquid deep fermentation. The fermentation temperature is 32-40℃, the fermentation time is 48-72 hours, the aeration rate is 0.5-2.0 vvm, and the stirring speed is 100-400 rpm. Step S4, Feeding Control: During the fermentation process, when the reducing sugar concentration in the fermentation broth drops to 30%-50% of the initial concentration, feed culture medium is started. The dissolved oxygen concentration is controlled at 20%-50% and the pH value is controlled at 6.5-8.0 by online monitoring of dissolved oxygen concentration and pH value. Step S5, Induction Enhancement: When the fermentation reaches the middle of the logarithmic growth phase, an inducer, namely Ca, is added to the fermentation broth. 2+ Complexes with surfactants, wherein Ca 2+ The mass ratio of the surfactant to the nattokinase is 1:0.5-5; the amount of inducer added is 0.05%-0.5% of the fermentation liquid volume, and fermentation continues until the endpoint, which is the nattokinase activity increase rate is less than 10 IU / (mL·h) or the fermentation time reaches the set 48-72 hours. The fermentation medium comprises the following components: 20-60 g / L carbon source, 10-40 g / L nitrogen source, 1-10 g / L inorganic salts, wherein the content of trace metal ions is 0.01-5 g / L (calculated as metal elements), and 0.01-0.5 g / L complex vitamins. The carbon source is a mixture of glucose and molasses in a mass ratio of 1:0.5-3; the nitrogen source is a mixture of soybean peptone and yeast extract in a mass ratio of 1:0.2-2.

2. The fermentation process for high-yield production of nattokinase and protease according to claim 1, characterized in that, The mass ratio of glucose to molasses is 1:1-2; the mass ratio of soybean peptone to yeast extract is 1:0.5-1.

5.

3. The fermentation process for high-yield production of nattokinase and protease according to claim 1, characterized in that, The fermentation culture described in step S3 adopts a staged variable temperature fermentation strategy: the temperature is controlled at 37-40℃ during the early stage of fermentation (0-12h), 35-37℃ during the middle stage of fermentation (12-48h), and 32-35℃ during the later stage of fermentation (48-72h) when the fermentation time is ≥48h.

4. The fermentation process for high-yield production of nattokinase and protease according to claim 1, characterized in that, The inorganic salt is selected from one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, sodium chloride, and calcium chloride; the trace metal ions are selected from Mn. 2+ Fe 2+ Zn 2+ One or more of the following, but excluding Ca provided by calcium chloride. 2+ The Ca 2+ Provided by calcium chloride.

5. The fermentation process for high-yield production of nattokinase and protease according to claim 1, characterized in that, The surfactant is Tween-80 or Span-80, and the Ca... 2+ The mass ratio of surfactant to surfactant is 1:0.5-5.

6. The fermentation process for high-yield production of nattokinase and protease according to claim 1, characterized in that, The fed-batch culture medium in step S4 contains 300-600 g / L of carbon source and 50-150 g / L of nitrogen source. The carbon source in the fed-batch culture medium is a mixture of glucose and molasses with a mass ratio of 1:0.5-3. The feeding method is exponential feeding or constant-rate feeding, with a flow acceleration rate of 0.1-2.0 g / (L·h), which is calculated based on the initial fermentation broth volume.

7. The fermentation process for high-yield production of nattokinase and protease according to claim 1, characterized in that, The activation medium in step S1 is LB medium or NA medium; the seed medium in step S2 is a medium with the same composition as the fermentation medium.

8. A fermentation process for high-yield production of nattokinase and protease according to any one of claims 1-7, characterized in that, The fermentation process also includes a post-fermentation broth treatment step: the fermentation broth obtained at the end of fermentation is centrifuged, the supernatant is collected, concentrated by ultrafiltration, and then freeze-dried or spray-dried to obtain nattokinase and protease complex enzyme powder.

9. The use of the fermentation process according to any one of claims 1 to 8 in the preparation of food, health products or pharmaceutical preparations containing nattokinase and protease.

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