CN108998437B - Nattokinase liquid state fermentation method - Google Patents
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Abstract
The invention discloses a method for producing nattokinase, which comprises the following steps: 1) adding water into bean dregs to adjust the water content to 80-90%; 2) inoculating activated Bacillus natto according to the inoculation amount of 3-10%; 3) shake culturing at 36-38 deg.C for 30-40 hr; 4) centrifuging to obtain supernatant, filtering the supernatant for sterilization to obtain crude enzyme solution; adding ammonium sulfate into the obtained crude enzyme liquid until the saturation reaches 50-90%, standing at 0-4 ℃ for 10-20 h, centrifuging at 9000-10000 rpm/min for 8-15 min, taking the precipitate, re-dissolving with a buffer solution, and dialyzing to remove salt to obtain nattokinase; mixing with trehalose maltodextrin protective agent, and spray drying to obtain nattokinase powder; the specific enzyme activity of the nattokinase prepared by the invention can reach 385.14 IU/mg, and the stability is good.
Description
Technical Field
The invention belongs to the field of food processing, and particularly relates to a production method of nattokinase.
Background
In 1987, Nattokinase (NK), an enzyme which can effectively dissolve thrombus in heart and brain, was discovered in Nattokinase by Sumi of Japan, and application and research on NK began. The nattokinase is produced by extracting from food natto or fermenting natto bacteria, is a protein with molecular weight far less than UK, SK and tPA, is serine protease produced by natto in the fermentation process, can hydrolyze fibrin into small peptide and amino acid, has good thrombolysis capability, and has certain curative effect in the aspects of lowering blood pressure, preventing cardiovascular and cerebrovascular diseases and the like. Currently, the thrombolytic drugs that are medically approved for use are: streptokinase (SK) and Urokinase (UK) have some defects, such as short drug action time and easy bleeding. Compared with the existing thrombolytic drugs, the nattokinase is derived from traditional foods, has low price, high safety, long half-life period in vivo, can be absorbed by intestinal tracts, is not easy to cause bleeding, has quick action and long duration in vivo, can activate tPA in vivo, and mildly and continuously improves the fibrinolytic activity of blood, thereby being widely concerned by biomedicine and the food industry. In recent years, the nattokinase capsules in domestic markets are various, but the solubility fineness and the activity of the nattokinase are relatively low.
At present, the nattokinase mainly comprises two methods of solid fermentation and liquid fermentation. The solid fermentation is a traditional fermentation method, the yield is low, the labor intensity is high, and the liquid fermentation can not meet the requirement although the yield of the nattokinase is improved. The main method for improving the yield of the nattokinase comprises the following steps of (1) selecting a high-yield nattokinase strain through mutagenesis and screening; (2) optimizing a fermentation culture medium and fermentation conditions; (3) the high-yield enzyme engineering bacteria are obtained by using a genetic engineering technology. The fermented natto kinase is treated through centrifuging to eliminate thallus, salting out to eliminate impurity protein, dialysis, ultrafiltering, gel column and other steps to eliminate salt and purify the enzyme liquid.
The nattokinase drying method mainly comprises vacuum freeze drying, spray drying and other methods, the activity of the nattokinase freeze dried in vacuum is high, in addition, the activity of the nattokinase can be better kept by adding a proper protective agent, and meanwhile, the higher survival rate of the bacillus natto can be ensured.
The bean dregs are main by-products in the bean product industry, at present, in the domestic soybean food industry, thousands of tons of wet bean dregs can be generated every year, and the bean dregs contain more nutrient substances such as polysaccharide, dietary fiber, isoflavone, protein and the like, so the bean dregs are a healthy and green novel health-care food raw material. Has effects in lowering blood pressure, reducing cerebrovascular diseases, and improving insulin level of diabetic patients. Although the nutrition effect is well known, the bean dregs have extremely high water content, extremely high viscosity, easy decay, easy environmental pollution and great processing difficulty. In recent years, a fermentation treatment method, a screw extrusion method, a pulverization treatment method, and the like are widely used as bean dregs processing methods. However, the chemical structure of dietary fiber in the bean dregs contains a large amount of hydrophilic groups and has high bound water content, so that a large amount of energy is consumed in the screw extrusion method and the crushing treatment process, and no economic benefit is obtained. The fermentation method is used for processing the bean dregs to produce a product such as nattokinase, so that the process can be simplified, the energy can be saved, the production cost can be reduced, and great economic and social benefits can be brought.
Disclosure of Invention
The invention aims to provide a method for producing nattokinase.
1. A method for producing nattokinase, which comprises the following steps: 1) Adding water into the bean dregs to adjust the water content to 80-90%, and sterilizing;
2) inoculating activated bacillus natto according to the inoculation amount of 3-10%;
3) shake culturing at 36-38 deg.C for 30-40 hr; 4) Centrifuging to obtain supernatant, filtering the supernatant for sterilization to obtain crude enzyme solution;
4) drying;
the inoculation amount in the step 2) is 8 percent;
the activation in the step 2) is as follows: dissolving Bacillus natto powder in sterile water, and diluting to 10%-3~10-6Gradient, streaking a plate on an LB culture medium containing 2% agar for culturing for 18h, then selecting a single colony to be inoculated into an LB liquid culture medium, activating three generations, and then inoculating the colony in the LB liquid culture medium according to the inoculum size of 3% at 120rpm and culturing for 12h at 37 ℃; centrifuging the bacterial liquid in a high-speed refrigerated centrifuge at 4 ℃ at 9500r/min for 10 min; after being centrifugedThe bacteria were washed twice with sterile water and resuspended to adjust the concentration of the bacterial suspension to 107CFU/mL；
The sterilization in the step 1) is performed for 20min at the temperature of 121 ℃;
culturing the mixture in the step 3) for 36 hours at the temperature of 37 ℃ by using a shaking table;
filtering by using a microporous filter membrane of which the filtering is 0.45-0.22 mu m in the step 4);
the purification in the step 4) is to add ammonium sulfate until the saturation reaches 50-90%, to stand for 10-20 h at 0-4 ℃, to centrifuge for 8-15 min at 9000-10000 rpm/min, to take the precipitate, to redissolve with a buffer solution, and to dialyze for desalting;
adding trehalose and maltodextrin mixed protective agent into the dialyzed nattokinase solution according to the volume ratio of 1: 2-4, and performing spray drying to obtain nattokinase powder;
the trehalose maltodextrin mixed protective agent is as follows: 6g of maltodextrin and 12g of trehalose, and 100mL of distilled water is added;
the volume ratio is 1: 3.
The invention aims to provide a method for producing nattokinase, which comprises the following steps: 1) adding water into bean dregs to adjust the water content to 80-90%; 2) inoculating activated Bacillus natto according to the inoculation amount of 3-10%; 3) shake culturing at 36-38 deg.C for 30-40 hr; 4) centrifuging to obtain supernatant, filtering the supernatant for sterilization to obtain crude enzyme solution; adding ammonium sulfate into the obtained crude enzyme liquid until the saturation reaches 50-90%, standing at 0-4 ℃ for 10-20 h, centrifuging at 9000-10000 rpm/min for 8-15 min, taking the precipitate, re-dissolving with a buffer solution, and dialyzing to remove salt to obtain nattokinase; mixing with trehalose maltodextrin protective agent, and spray drying to obtain nattokinase powder; the specific enzyme activity of the nattokinase prepared by the invention can reach 385.14 IU/mg, and the stability is good.
Drawings
FIG. 1A microscopic view of Bacillus natto;
FIG. 2 results of a fermentation single factor test;
FIG. 3 results of viable count of different media;
FIG. 4 shows the results of nattokinase activity produced by different culture media;
FIG. 5 is a graph showing salting-out results;
FIG. 6 comparison of nattokinase stability after freeze-drying under different protective agents; (a) a trehalose protective agent, (b) a maltodextrin protective agent, (c) a whey protein powder protective agent, (d) a mixed protective agent I, (e) a mixed protective agent II;
FIG. 7 comparison of nattokinase stability after spray drying under different protective agents; (a) trehalose protective agent, (b) maltodextrin protective agent, (c) whey protein powder protective agent, (d) mixed protective agent I, and (e) mixed protective agent II.
Detailed Description
EXAMPLE 1 preparation of crude enzyme solution of Nattokinase
1. Preparation of leavening agent
Purchasing Bacillus natto powder, dissolving in sterile water, and diluting to 10%-3To 10-6Streaking different gradient plates on LB culture medium containing 2% agar for 18h, performing microscopic examination as shown in FIG. 1, then selecting single colony, inoculating into LB liquid culture medium, activating for three generations, inoculating into LB liquid culture medium at 120rpm according to 3% inoculum size, and culturing at 37 deg.C for 12 h; centrifuging the bacterial liquid in a high-speed refrigerated centrifuge at 4 ℃ at 9500r/min for 10 min; washing the centrifuged thallus twice with sterile water, and resuspending to adjust the concentration of the suspension to 107CFU/mL as starter.
2. Preparation of fermentation Medium
Soaking at room temperature for 13h at a mass ratio of soybean to water of 1:5, draining off water, grinding the soaked soybean and water of 100 deg.C in a processor for 3min, filtering to obtain fresh bean dregs, and adjusting water content of the bean dregs to about 85%. And preparing the pretreated fresh bean dregs and water into a liquid fermentation culture medium according to different proportions.
3. Preparation of nattokinase crude enzyme liquid
Placing the fermentation medium into a semi-automatic high-pressure steam sterilization pot, and sterilizing at 121 deg.C for 20 min; after cooling, the mixture was inoculated with an inoculum size of 8% (v/w) 107CFU/mL Bacillus natto starter, then putting into a shaking table with set rotating speed (160 rpm) and temperature (37 ℃) for culturing for required time, taking out fermentation liquor, centrifuging at 9500rpm for 10min, and using 0.45 mu m and 0.And (3) sterilizing by using a microporous filter membrane of 22 mu m, collecting the crude nattokinase obtained by removing the supernatant of the thallus, and storing in a refrigerator at 4 ℃.
Example 2 Nattokinase liquid fermentation Process optimization
Optimization of fermentation conditions of nattokinase
Investigating the influence of five factors of inoculation quantity, fermentation temperature, fermentation time, table rotating speed and solid-liquid ratio on the activity of the nattokinase, obtaining the parameter range of the optimum enzyme activity of the nattokinase as shown in figure 2, and performing response surface test design on the three main factors of the fermentation time, the table rotating speed and the inoculation quantity according to the Box-Behnken principle on the basis of a single-factor test to obtain the test factor level shown in table 1 and the test result shown in table 2;
the enzyme activity reaches the highest at 37 ℃ through a single-factor test, which is related to the influence of temperature on the metabolism of thalli, and the thalli metabolism is slow and the enzyme production is less when the temperature is low; as the temperature rises, the consumption of nutrient substances is accelerated along with the acceleration of metabolism, and meanwhile, enzyme protein is easily inactivated by high temperature; the solid-liquid ratio reaches the highest enzyme activity when being 1:3, but the solid matter accounts for too large proportion and is not beneficial to industrial production, so the solid-liquid ratio is reasonable when being 1: 5; therefore, response surface optimization is carried out by selecting three factors with larger influence on fermentation time, table rotating speed and inoculation amount. The optimal time 36h for producing the single factor of the nattokinase is taken as the central point of a response surface of the fermentation time, and the slightly lower rotating speed 160rpm is taken as the central point of the response surface of the rotating speed of the shaking table in consideration of the actual selection of industrial production. The central point of the response surface of the inoculation amount is selected to be 8% of the relatively stable enzyme production.
TABLE 1 Box-Behnken test factor level Table
TABLE 2 Box-Behnken test results
And (3) carrying out regression analysis on the data of the table 2 by Design-Expert 8.05.B software to obtain an analysis of variance table of a secondary regression model, wherein a quadratic polynomial regression equation of the activity of the Y nattokinase to the fermentation time A, the rotating speed of a shaking table B and the inoculation amount C is as follows: y = +273.15-20.65 a + 108.7B + 28.26C-5.43 a B +40.22 a C + 16.30B C-88.36 a2-47.05*B2-34.01*C2(ii) a The p value of the model is obvious, the p value of the failure term does not obviously indicate that the modeling is established, and R2A value of 0.9288 indicates a better model fit; the significance analysis of the regression model shows that the influence of the rotating speed of the shaking table is very significant relative to the activity of the nattokinase, and then the inoculation amount and then the fermentation time are obtained; the final optimization result is that the highest enzyme activity of the natto kinase produced by 9 percent of inoculation amount at 36h, 170rpm reaches 318IU/mL, and the difference between the predicted value and the actual value is not obvious after three parallel tests.
Second, method for measuring natto kinase
1. Urokinase standard curve
Diluting 50000 IU/g urokinase standard substance into different enzyme activity gradients, establishing the correlation between the urokinase activity unit and the size of a fibrin plate lysis ring, and finally presenting a good linear relation at 50-350IU/mL to obtain a regression equation y =0.0023x +0.8214, R2=0.997。
2. Nattokinase Activity measurement
The fibrinolysis activity of the enzyme is measured by adopting a fibrin plate method, and the activity of the nattokinase is expressed by the diameter of a fibrinolysis ring; accurately sucking 10 mu L of crude enzyme solution by a pipette, injecting the crude enzyme solution into a fibrin plate, putting the fibrin plate into a constant-temperature incubator at 37 ℃ for 18h, taking out the fibrin plate to measure the diameter of a dissolving ring, and comparing the diameter with the diameter of a urokinase standard curve to determine the fibrinolytic activity of the nattokinase.
Fibrin plate components: 20mL of 1% agarose solution (dissolved by 0.9% NaCl solution), 2mL of bovine serum fibrinogen (7.5 mg/mL, dissolved by 0.1mol/L pH7.4 phosphate buffer solution: 0.9% NaCl solution =1:17 mixed working solution), 200. mu.L thrombin (250U/mL).
Example 3 comparison of enzyme production and viable count of different fermentation media
The method is used for investigating the influence of the type of the culture medium on the activity of the nattokinase, and comprises the following specific operation steps:
will reach 107Inoculating a bacillus natto starter with the same viable count of cfu/mL into a bean dreg culture medium, a corn bean pulp culture medium and an LB culture medium respectively with the same inoculation amount, culturing in a shaking table with the same temperature and the same rotating speed, centrifuging three liquid culture media respectively every 12 hours, taking supernate to measure the nattokinase activity, simultaneously taking partial supernate to dilute in a 10-fold gradient series, selecting 2-3 proper gradients to evenly coat a flat plate, performing three parallel tests on each gradient, and taking the average value to count viable colonies; the nattokinase activity results are shown in FIG. 3, and the viable count results are shown in FIG. 4.
As can be seen from the figure, the trends of the viable count of the three culture media are approximately consistent and reach the maximum viable count within 60 hours, in the aspect of enzyme activity, the LB culture medium has no enzyme activity all the time, and although the enzyme activity of the corn bean pulp culture medium is higher than that of the bean dreg culture medium in the general trend, the cost of the corn bean dreg culture medium is higher than that of the bean dreg culture medium.
The corn soybean meal culture medium comprises 80g of corn flour, 40g of soybean meal, 8g of disodium hydrogen phosphate, 4g of ammonium sulfate, 2g of calcium chloride, 1.5g of ammonium chloride and 1000mL of deionized water, and the pH value of the culture medium is 7.0-7.2; the LB culture medium comprises 3g of beef extract, 10g of peptone, 5g of sodium chloride, 10g of glucose and 1000mL of deionized water, and the pH value of the culture medium is 7.2-7.4.
Example 4 preliminary purification of Nattokinase
Primarily purifying nattokinase by an ammonium sulfate separation and precipitation method, taking 30mL of crude enzyme liquid, adding ammonium sulfate to ensure that the saturation degrees of the crude enzyme liquid reach 50%, 60%, 70%, 80% and 90% respectively, standing the crude enzyme liquid in a refrigerator at 4 ℃ for 12h, centrifuging the crude enzyme liquid at 9500rpm/min for 10min, redissolving the precipitate by using a buffer solution, respectively measuring the enzyme activity and the protein content of the nattokinase in supernatant and the precipitate, and determining the saturation point of ammonium sulfate fractional precipitation by using relative enzyme activity; and dialyzing the crude enzyme solution after the ammonium sulfate is primarily purified to remove salt. The result is shown in figure 5, the relative enzyme activity of the nattokinase with the saturation degree of the ammonium sulfate of 50-60% is obviously improved, and the relative enzyme activity starts to be reduced when the saturation degree of the ammonium sulfate is 70%, so that 60% is selected as the salting-out saturation degree; the enzyme activity and protein concentration of the fermentation supernatant, 60% of salted-out nattokinase and dialyzed nattokinase are respectively measured, and the results are shown in table 3, which shows that the enzyme activity is improved by 2.23 times after salting-out dialysis.
TABLE 3 Nattokinase purification results
EXAMPLE 5 preparation of Nattokinase Dry protectant
Respectively preparing a trehalose protective agent, a mixed protective agent I, a mixed protective agent II, a maltodextrin protective agent and a whey protein powder protective agent for subsequent experiments, wherein the preparation method specifically comprises the following steps:
1. trehalose protective agent: weighing 18g of trehalose, adding into 100mL of distilled water, and uniformly stirring until no particles exist;
2. mixed protective agent I: respectively weighing 6g of maltodextrin and 12g of trehalose, adding into 100mL of distilled water, and uniformly stirring until no particles exist;
3. and (3) mixing a protective agent II: respectively weighing 6g of maltodextrin, 2g of beta-cyclodextrin and 10g of whey protein powder, adding into 100mL of distilled water, and uniformly stirring until no particles exist;
4. maltodextrin protectant: weighing 18g of maltodextrin, adding into 100mL of distilled water, and uniformly stirring until no particles exist;
5. whey protein powder protective agent: weighing 18g of whey protein powder, adding into 100mL of distilled water, and stirring uniformly until no particles exist.
Example 6 Nattokinase crude enzyme liquid drying technique experiment
Optimization of spray drying condition of nattokinase crude enzyme liquid
Mixing the crude enzyme solution of nattokinase and various protective agents prepared in the embodiment 5 according to the volume ratio of 1:3 (V/V), homogenizing for 2 minutes at 10000r/min, then carrying out spray drying, setting the inlet temperature of a spray dryer to be 140, 150 and 160 ℃, the outlet temperature to be 75 ℃, the rotating speed of a peristaltic pump to be 240, 340 and 440mL/h, the fan speed to be 70Hz, the firing pin time to be 1s and the firing pin interval to be 1s, collecting enzyme powder after drying, carrying out activity determination, and inspecting the influence of the type of the protective agents, the inlet temperature of spray drying and the rotating speed of the spray drying peristaltic pump on the nattokinase activity.
Determining spray drying conditions suitable for each single factor by taking the nattokinase activity as an assessment index, providing a reference range for condition optimization of spray drying nattokinase, and setting 3 parallels for each factor of experimental design; the research on the activity of the nattokinase under different spray drying conditions shows that under the conditions that the protective agent is the same (mixed protective agent II) and the rotating speed of a peristaltic pump is 340mL/h, the nattokinase is partially inactivated and the enzyme activity is gradually reduced along with the increase of the inlet temperature, and when the inlet temperature is lower than 140 ℃, a small amount of pasty object is generated at the collection bottle mouth to influence the spray drying result. Paste-like objects may be incompletely dried due to lower spray drying temperature or faster peristaltic pump speed; the result shows that under the conditions of the same inlet temperature (150 ℃) and the same protective agent (trehalose protective agent), the optimum peristaltic pump rotating speed is 340mL/h, and the activity of the nattokinase is the maximum at the moment; when the rotation speed of the peristaltic pump is 240mL/h, the nattokinase is gradually inactivated under the high-temperature condition possibly due to the over-slow rotation speed of the peristaltic pump; when the rotating speed of the peristaltic pump is 440mL/h or 540mL/h, the enzyme activity is reduced probably because the rotating speed of the peristaltic pump is too high, the crude enzyme liquid is not completely dried, and the yield is reduced; the result shows that the activity of the nattokinase is maximum under the conditions of the same inlet temperature (150 ℃) and the rotating speed of a peristaltic pump (340 mL/h); the test is repeated for three times, and the average value of the enzyme activity of the nattokinase is (210200 +/-19126) FU/g.
The fibrinolysis activity of the nattokinase is measured by adopting a fibrin plate method, and the nattokinase activity is represented by the diameter of a dissolving ring generated by dissolving the fibrin plate by the nattokinase; the activity of the nattokinase is expressed by activity units relative to urokinase, and a linear regression equation of the activity of the urokinase is established as follows: y =0.0023x +0.8207, R2=0.998；
Enzyme activity (FU/g) of nattokinaseA×4In the formula:A-calculating the corresponding nattokinase activity unit (FU/g) by using a urokinase standard curve measured by the sample;4natto kinase to be testedDilution factor of enzyme sample.
Secondly, detecting the stability of nattokinase
Respectively drying nattokinase by freeze drying and spray drying, and measuring enzyme activity; simultaneously investigating the influence of different types of protective agents on the enzymatic activity of the nattokinase; the specific operation is as follows:
under the conditions of same inlet temperature (160 ℃) and same peristaltic pump rotating speed (340 mL/h), the crude enzyme solution is protected by different protective agents prepared in the example 5, and spray drying is carried out; taking 10mL of the solution prepared by mixing and homogenizing each protective agent prepared in the embodiment 2 and the crude enzyme solution, pre-freezing the solution in a refrigerator at the temperature of-20 ℃ for 2h, then transferring the solution to a refrigerator at the temperature of-80 ℃ for freezing for 12h, and transferring the frozen crude enzyme solution to a vacuum freeze dryer for freeze-drying; the two methods obtain nattokinase powder, respectively putting 200mg into a 1.5mL centrifuge tube, putting the centrifuge tube and a drying agent into a bag for vacuum packaging, respectively preserving at 0 ℃, 4 ℃, normal temperature (about 18 ℃) and 37 ℃ for 40 days, and measuring the enzyme activity of the nattokinase once every ten days.
In order to further discuss the stability of the nattokinase activity generated after different protective agents are dried, the experiment researches the influence degree of the preservation temperature and the preservation time on the nattokinase activity stability; as can be seen from FIG. 6, the nattokinase activity of freeze-dried nattokinase is gradually reduced along with the increase of time under the condition of the same preservation temperature; under the condition of the same preservation time and temperature, trehalose is used as a protective agent to carry out freeze drying, so that the activity of the natto kinase is the highest.
As can be seen from FIG. 7, the nattokinase activity after spray drying is gradually reduced along with the increase of time under the condition of the same preservation temperature; under the conditions of the same preservation time and the same preservation temperature, the activity of the nattokinase generated by spray drying by using the mixed protective agent I is the highest; under the conditions of the same preservation time and different preservation temperatures, the activity of the nattokinase generated by spray drying with the same protective agent has no obvious change; in conclusion, the preservation temperature has no great influence on the activity of the nattokinase generated by spray drying of different protective agents, which is probably related to the relative stability of the enzyme activity of the nattokinase at the temperature below 45 ℃ and within the range of pH = 7-9; but with the increase of preservation time, the nattokinase activity generated by spray drying of different protective agents is gradually reduced and has the same trend with the nattokinase activity of freeze drying; therefore, the nattokinase spray-dried by taking the mixed protective agent I as the protective agent has long storage time and is relatively stable, and the utilization value of the nattokinase is greatly improved.
Claims (4)
1. A method for producing nattokinase, which comprises the following steps:
1) adding water into the bean dregs to adjust the water content to 80-90%, and sterilizing;
2) inoculating activated bacillus natto according to the inoculation amount of 3-10%;
3) shake culturing at 36-38 deg.C for 30-40 hr; 4) Centrifuging to obtain supernatant, filtering the supernatant to remove bacteria to obtain crude enzyme solution, and purifying;
4) spray drying;
adding trehalose and maltodextrin mixed protective agent into the purified enzyme solution according to the volume ratio of 1:3, and then drying;
the trehalose maltodextrin mixed protective agent is as follows: 6g of maltodextrin and 12g of trehalose, and 100mL of distilled water is added;
5) storing the dried nattokinase at 37 ℃;
the activation in the step 2) is as follows: dissolving Bacillus natto powder in sterile water, and diluting to 10%-3~10-6Gradient, streaking a plate on an LB culture medium containing 2% agar for culturing for 18h, then selecting a single colony to be inoculated into an LB liquid culture medium, activating three generations, and then inoculating the colony in the LB liquid culture medium according to the inoculum size of 3% at 120rpm and culturing for 12h at 37 ℃; centrifuging the bacterial liquid in a high-speed refrigerated centrifuge at 4 ℃ at 9500r/min for 10 min; washing the centrifuged thallus twice with sterile water, and resuspending to adjust the concentration of the suspension to 107CFU/mL；
And 4) adding ammonium sulfate into the mixture until the saturation degree reaches 50-90%, standing the mixture for 10-20 h at 0-4 ℃, centrifuging the mixture for 8-15 min at 9000-10000 rpm/min, taking the precipitate, redissolving the precipitate by using a buffer solution, and dialyzing the precipitate to remove salt.
2. The method for producing nattokinase according to claim 1, wherein: the inoculation amount in the step 2) is 8 percent.
3. The method for producing nattokinase according to claim 2, wherein: the sterilization in the step 1) is performed for 20min at the temperature of 121 ℃.
4. The method for producing nattokinase according to claim 3, wherein: filtering by using a microporous filter membrane of 0.45-0.22 mu m in the step 4).
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| CN111280418A (en) * | 2020-03-25 | 2020-06-16 | 中国农业科学院农产品加工研究所 | Vegetable and fruit jelly containing nattokinase and preparation method thereof |
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