CN107475233B - Method for producing nattokinase by using bean curd yellow serofluid - Google Patents

Method for producing nattokinase by using bean curd yellow serofluid Download PDF

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CN107475233B
CN107475233B CN201710914669.5A CN201710914669A CN107475233B CN 107475233 B CN107475233 B CN 107475233B CN 201710914669 A CN201710914669 A CN 201710914669A CN 107475233 B CN107475233 B CN 107475233B
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郝宁
郭格格
刘兆星
李艳
陈可泉
欧阳平凯
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Nanjing Tech University
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Abstract

The invention discloses a method for producing nattokinase by using bean curd yellow serofluid, which comprises the steps of taking the bean curd yellow serofluid as a unique fermentation substrate, and adjusting the pH value to 6-8; inoculating the natto kinase producing strain into bean curd yellow slurry water, fermenting at the temperature of 25-40 ℃ for 20-72 h, wherein the enzyme production amount of the natto kinase reaches 47031.6 Fu/g. The nattokinase-producing strain comprises at least one of bacillus licheniformis, bacillus subtilis, bacillus natto and bacillus thuringiensis. The method can fully recycle the waste bean curd yellow serofluid, so that the waste bean curd yellow serofluid can be comprehensively utilized, the environmental pollution is reduced, the waste is changed into valuable, and the economic benefit is improved; in the aspect of fermentation raw materials, the raw materials of the bean curd yellow serofluid are utilized to reduce the cost, and the method has the advantages of low energy consumption, little pollution, high production efficiency, simple process and convenient operation. Therefore, the invention has wide market prospect and industrial production application value.

Description

Method for producing nattokinase by using bean curd yellow serofluid
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to a method for producing nattokinase by using bean curd yellow serofluid as a fermentation raw material.
Background
The yellow serofluid of the bean curd is mainly derived from waste water drained in the bean curd processing production process, and the yellow serofluid of the bean curd contains abundant organic matters. At present, most food processing enterprises discharge yellow soybean milk water generated in the soybean milk processing process directly through a sewer, directly increase COD and BOD values of water quality, pollute the environment and waste resources. Through detection, the basic chemical components of the bean curd yellow serofluid comprise protein, crude fat, metal ions such as K, Na, Mg, Ca, Fe and the like, carbonate, phosphate radical and the like, and the protein, soybean oligosaccharide, soybean isoflavone and soybean saponin in the components belong to nutritional components, wherein the soybean oligosaccharide has the effects of promoting the proliferation of bifidobacteria, promoting the growth of beneficial bacteria in intestines and stomach, preventing and improving constipation, promoting the absorption of trace elements such as calcium, magnesium and the like, and simultaneously has lower calorie, so the bean curd yellow serofluid is an ideal weight-reducing food. In addition, soy isoflavones have proven beneficial in preventing certain malignancies, preventing cardiovascular disease, lowering cholesterol, preventing osteoporosis, and alleviating symptoms of climacteric syndrome in women. Therefore, the method has practical significance for using safe strains to industrially breed pure fermented yellow serofluid.
Meanwhile, due to the huge number of bean product factories in various places, the processed bean curd yellow serofluid has rich nutrition, the COD and BOD values seriously exceed the standard, and if the bean curd yellow serofluid is directly discharged, the COD and BOD values of the water body are obviously increased, and the environment is seriously polluted. If the yellow serofluid of the bean curd in a bean product factory is recycled and fully utilized, not only can waste be changed into valuable, but also the cost is reduced, the environmental pollution is reduced, and the comprehensive benefit is improved.
The nattokinase has strong function of dissolving thrombus, and compared with the thrombolytic drugs such as urokinase, streptokinase and the like clinically used at present, the nattokinase has the advantages of good safety, easy absorption by human bodies, direct and rapid action, long duration of drug effect, low manufacturing cost and the like, and can be directly produced by fermenting the natto bacillus subtilis directly. The liquid fermentation has the advantages of low cost, high purity, less environmental pollution, etc. Currently used strains for producing nattokinase are Lactobacillus bulgaricus (L.) (Lactobacillus bulgaricus) Bacillus subtilis preparation (B)Bacillus subtilis) Streptomyces (I), (II)Streptomyces) Lactic acid bacteria (A)lactic acid bacteria) Escherichia coli (E.coli)Escherichia coli) Pichia pastoris (A), (B), (C), (Pichia pastoris) In the production of nattokinase by liquid fermentation, the by-product contains a part of purine. Patent 201110099838.7 describes that the enzyme activity of the obtained nattokinase reaches as high as 5670FU/g in the method for producing nattokinase by fermenting bacillus subtilis for producing nattokinase and the strain. In general, the time for the liquid fermentation of nattokinase is about 72 h. At present, the production mode of nattokinase mainly adopts liquid fermentation, has the defects of large energy consumption, low enzyme production amount and the like, and the defects can be compensated by utilizing bean curd yellow slurry for fermentation.
In patent 201210588335.0, a method for producing sour milk of bean curd by fermenting starch-dissolving lactic acid bacteria, it is described that the fermentation of yellow milk of bean curd with water is mainly used for the mass growth of lactic acid bacteria, and Liugui duckweed, etc. discloses research on the production of yeast single-cell protein by fermenting yellow milk of bean curd, Shenyang chemical university report, 2015.06, and it is described that the fermentation of yellow milk of bean curd with water is mainly used for the growth and culture of yeast to produce single-cell protein. At present, bean curd yellow serofluid is mainly used as a fermentation culture medium for lactic acid bacteria and yeast. The effect is to perform fermentation growth as a fermentation medium.
Disclosure of Invention
The invention aims to provide a method for producing nattokinase by using bean curd yellow serofluid, the nattokinase produced by the method has high activity, can effectively utilize wastes such as the bean curd yellow serofluid, reduces the production cost, is safe and environment-friendly, and simultaneously solves the problem of environmental pollution caused by the bean curd yellow serofluid.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for producing nattokinase by using bean curd yellow serofluid comprises the following steps:
1) taking bean curd yellow serofluid, and adjusting the pH value to 6-8;
2) inoculating the nattokinase production strain into bean curd yellow slurry water, and fermenting at the fermentation temperature of 25-40 ℃ for 20-72 h to obtain the nattokinase fermentation liquor. Directly carrying out vacuum freeze-drying or spray drying on the nattokinase fermentation liquor to obtain a crude enzyme liquid.
Before liquid fermentation, the yellow serofluid of the bean curd in the step 1) can be fermented without sterilization or with sterilization, and the sterilization is preferably performed under the following conditions: sterilizing at 121 deg.C for 20 min.
The nattokinase-producing strain comprises Bacillus licheniformis (Bacillus licheniformis)Bacillus licheniformis) Bacillus subtilis preparation (B)Bacillus subtilis) Bacillus natto (Bacillus natto) (II)Bacillus natto) Bacillus thuringiensis (B.thuringiensis)Bacillus thuringiensis) At least one of (1).
The bean curd yellow serofluid is wastewater generated after any bean product is produced in the market, and comprises but is not limited to yellow serofluid for producing gypsum bean curd, lactone bean curd or brine bean curd. Bean curd yellow serofluid is used as the only fermentation substrate.
Preferably introducing air in the fermentation process in the step 2), wherein the ventilation quantity is 1.0-2.5 v/vm, and the rotating speed is 200-500 rpm.
The bean curd yellow serofluid can be used as fermentation culture medium, and can also be usedFurther adding various substances which assist the growth of the strain or produce enzyme, including but not limited to amino acid, metal ion and the like, wherein the amino acid can be aspartic acid, serine and the like; the metal ions include but are not limited to Co2+, Fe2+, Mn2+, Ca2+, Fe3+, Al3+, Cu2+， Ni+And the like.
Preferably, 0.1-0.5 g/L of aspartic acid and 0.1-0.5 g/L of serine are added into the bean curd yellow slurry water to serve as mixed fermentation liquor.
Preferably Cacl in bean curd yellow serofluid2 0.20~0.45 g/L,MnSO40.001-0.055 g/L, and adjusting pH = 6.5-7.5 to obtain the mixed fermentation liquor.
The natto kinase producing strain is bacillus subtilis (Bacillus subtilis)Bacillus subtilis) gs-11061, preparation of Bacillus subtilis (B.) (Bacillus subtilis) The gs-11061 is inoculated into the bean curd yellow serofluid according to the inoculation amount of 2-10% (V/V), air is introduced for 1.0-2.5V/vm, the rotating speed is 200-500 rpm, the fermentation temperature is 35 ℃, aerobic fermentation is carried out for 20-72 h, and the produced natto kinase amount is 47031.6 Fu/g.
The invention takes the bean curd yellow serofluid as the only fermentation culture medium to ferment and produce the nattokinase, the final fermentation liquor directly obtains the crude enzyme liquid in a vacuum freeze-drying or spray-drying mode, and simultaneously solves the hidden trouble of environmental pollution caused by the direct discharge of the fermentation liquor. Compared with other fermentation methods, the method can fully recycle the waste bean curd yellow serofluid, reduce the potential safety hazards of pathogenic bacteria and the like in the natural fermentation of the yellow serofluid in the bean product processing, simultaneously comprehensively utilize the yellow serofluid, reduce the environmental pollution, simultaneously change waste into valuable and improve the economic benefit; in the aspect of fermentation raw materials, the raw materials of the bean curd yellow serofluid are utilized to reduce the cost, and the method has the advantages of low energy consumption, little pollution, high production efficiency, simple process and convenient operation. Therefore, the invention has wide market prospect and industrial production application value.
Has the advantages that:
(1) after the strain is fermented in bean curd yellow serofluid, the produced nattokinase has high activity, and is safe and environment-friendly.
(2) Compared with the traditional liquid fermentation method, the strain can utilize bean curd yellow serofluid for fermentation, and the cost is greatly reduced.
(3) A large amount of yellow serofluid generated in the bean product processing is comprehensively utilized, the wastewater discharge is reduced, the environmental pollution is reduced, the waste is changed into the valuable, and the comprehensive benefits of enterprises and production are improved;
(4) the fermentation process is simple and easy to operate.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results described in the examples are merely illustrative of the invention and should not be construed to limit the invention as described in the claims.
Example 1 Bacillus subtilis ((B))Bacillus subtilis) gs-11061 fermentation of bean curd yellow slurry to produce natto kinase
Plate culture medium: 15g/L of peptone, 7.5 g/L of yeast extract, 15g/L of NaCl, 20g/L of agar and natural pH;
seed culture medium: glucose 12 g/L, K2HPO4•3H2O1.1 g/L, peptone 15g/L, KH2PO4 1.2 g/L，MgSO4•7H2O 0.40 g/L,Cacl2 0.25 g/L,MnSO40.001 g/L, balance water, pH = 7.4.
Fermentation medium: taking fresh bean curd yellow serofluid (the initial pH of the wastewater after the production of common bean products in the market is 2-4), filtering the fresh bean curd yellow serofluid by using 6 layers of gauze, adjusting the pH to be 6.5-7.5, and subpackaging the bean curd yellow serofluid in 500mL conical bottles, wherein the liquid filling amount is 80 mL, the temperature is 121 ℃, and the sterilization is carried out for 20 min.
Bacillus subtilis (A), (B) and (C)Bacillus subtilis) gs-11061 (accession number: CGMCC No. 13932) firstly activates strains on a plate at 35 ℃, strains with good growth of two rings are inoculated into 50 mL of seed culture medium after 24 h, the strains are cultured at 35 ℃ and 180 rpm for 12 h, then the strains are inoculated into a triangular flask with the liquid loading capacity of 80 mL of fermentation medium/500 mL according to the inoculation amount of 3.5% (v/v), the strains are cultured at 35 ℃ and the rotation speed of 180 rpm, and after fermentation for 36 h, the nattokinase in the fermentation liquid is 27961.2 Fu/g. Most preferablyThe final fermentation liquid is directly freeze-dried or spray-dried in vacuum to obtain crude enzyme liquid, and simultaneously, the hidden trouble of environmental pollution caused by direct discharge of the fermentation liquid is also solved.
Example 2 Bacillus natto (II)Bacillus natto) BNCC194961 method for producing nattokinase by fermenting bean curd yellow slurry
Plate culture medium: 15g/L of peptone, 7.5 g/L of yeast extract, 15g/L of NacL and 20g/L of agar, and the pH value is natural;
seed culture medium: glucose 12 g/L, K2HPO4•3H2O1.1 g/L, peptone 15g/L, KH2PO4 1.2 g/L，MgSO4•7H2O 0.40 g/L,Cacl2 0.25 g/L,MnSO40.001 g/L, balance water, pH = 7.4.
Fermentation medium: taking fresh bean curd yellow serofluid (the initial pH of the wastewater after the production of common bean products in the market is 2-4), filtering the fresh bean curd yellow serofluid by using 6 layers of gauze, adjusting the pH to be 6.5-7.5, and subpackaging the bean curd yellow serofluid in 500mL conical bottles, wherein the liquid filling amount is 80 mL, the temperature is 121 ℃, and the sterilization is carried out for 20 min.
Mixing Bacillus natto (Bacillus natto) ((Bacillus natto) BNCC194961, purchased from Beina organisms, firstly plate activated strains at 35 ℃, inoculating two rings of well-grown strains into 50 mL of seed culture medium after 24 h, culturing at 35 ℃ and 180 rpm for 12 h, then inoculating into a triangular flask with the liquid loading amount of 80 mL of fermentation medium/500 mL according to the inoculation amount of 3.5% (v/v), culturing at 35 ℃ and the rotating speed of 180 rpm, and after fermenting for 36 h, the nattokinase in the fermentation liquid is 26389.2 Fu/g. The final fermentation liquor is directly freeze-dried or spray-dried in vacuum to obtain crude enzyme liquid, and simultaneously, the hidden trouble of environmental pollution caused by direct discharge of the fermentation liquor is also solved.
Example 3 Bacillus licheniformis: (Bacillus licheniformis) BNCC132622 utilizes bean curd yellow slurry to ferment and produce nattokinase
Plate culture medium: 15g/L of peptone, 7.5 g/L of yeast extract, 15g/L of NacL and 20g/L of agar, and the pH value is natural;
seed culture medium: glucose 12 g/L, K2HPO4•3H2O1.1 g/L, peptone 15g/L, KH2PO4 1.2 g/L，MgSO4•7H2O 0.40 g/L,Cacl2 0.25 g/L,MnSO40.001 g/L, balance water, pH = 7.4.
Fermentation medium: taking fresh bean curd yellow serofluid (the initial pH of the wastewater after the production of common bean products in the market is 2-4), filtering the fresh bean curd yellow serofluid by using 6 layers of gauze, adjusting the pH to be 6.5-7.5, and subpackaging the bean curd yellow serofluid in 500mL conical bottles, wherein the liquid filling amount is 80 mL, the temperature is 121 ℃, and the sterilization is carried out for 20 min.
Bacillus licheniformis (A), (B), (C)Bacillus licheniformis) BNCC132622, purchased from North Nami biology, firstly plate activating strains at 35 ℃, inoculating two rings of well-grown thalli into 50 mL of seed culture medium after 24 h, culturing at 35 ℃ and 180 rpm for 12 h, then inoculating into a triangular flask with the liquid loading amount of 80 mL of fermentation medium/500 mL according to the inoculation amount of 3.5% (v/v), culturing at 35 ℃ and the rotation speed of 180 rpm, and fermenting for 36 h, wherein the nattokinase in the fermentation liquid is 18123.2 Fu/g. The final fermentation liquor is directly freeze-dried or spray-dried in vacuum to obtain crude enzyme liquid, and simultaneously, the hidden trouble of environmental pollution caused by direct discharge of the fermentation liquor is also solved.
Example 4 Bacillus subtilis ((B))Bacillus subtilis) gs-11061 utilizes bean curd yellow slurry water with different concentrations to ferment and produce nattokinase
Plate culture medium: 15g/L of peptone, 7.5 g/L of yeast extract, 15g/L of NacL and 20g/L of agar, and the pH value is natural.
Seed culture medium: glucose 12 g/L, K2HPO4•3H2O1.1 g/L, peptone 15g/L, KH2PO4 1.2 g/L，MgSO4•7H2O 0.40 g/L,Cacl2 0.25 g/L,MnSO40.001 g/L, balance water, pH = 7.4.
Aerobic fermentation culture medium: 25% (v/v) of bean curd yellow serofluid, 50% (v/v) of bean curd yellow serofluid, 75% (v/v) of bean curd yellow serofluid and 100% (v/v) of bean curd yellow serofluid, respectively taking fresh bean curd yellow serofluid with different concentrations, adjusting the pH to be 6.5-7.5, subpackaging in 500mL conical flasks, filling 80 mL, sterilizing at 121 ℃ for 20 min.
Bacillus subtilis (A), (B) and (C)Bacillus subtilis) gs-11061 (Bao)Tibetan code: CGMCC No. 13932) is firstly activated by a flat plate at 35 ℃, after 24 hours, two rings of well-grown thalli are inoculated into 50 mL of seed culture medium, the culture is carried out for 12 hours at 35 ℃ and 180 rpm, then the thalli are inoculated into a triangular flask with the liquid loading amount of 80 mL of fermentation liquid/500 mL according to the inoculation amount of 3.5% (v/v), and the culture is carried out at 35 ℃ and the rotating speed of 180 rpm. The bean curd yellow serofluid with the volume concentration of 25%, 50%, 75% and 100% is respectively used as a fermentation main body, and after fermentation for 36 h, the nattokinase in the fermentation liquor is respectively 6678.1 Fu/g, 12309.4 Fu/g, 18732.5 Fu/g and 28409.8 Fu/g. In conclusion, the fermentation is carried out by using bean curd yellow serofluid with different concentrations, and the higher the concentration is, the better the fermentation is.
Example 5 Bacillus subtilis ((B))Bacillus subtilis) gs-11061 fermentation of bean curd yellow slurry to produce natto kinase
Plate culture medium: 15g/L of peptone, 7.5 g/L of yeast extract, 15g/L of NacL and 20g/L of agar, and the pH value is natural.
Seed culture medium: glucose 12 g/L, K2HPO4•3H2O1.1 g/L, peptone 15g/L, KH2PO4 1.2 g/L，MgSO4•7H2O 0.40 g/L,Cacl2 0.25 g/L,MnSO40.001 g/L, balance water, pH = 7.4.
Aerobic fermentation culture medium: taking fresh bean curd yellow serofluid, adjusting the pH to be 6.5-7.5, putting the fresh bean curd yellow serofluid into a fermentation tank, filling the fermentation tank with a liquid volume of 3L, sterilizing at 121 ℃ for 20 min.
Firstly, plate activating a strain by an initial strain CGMCC No. 13932 at 35 ℃, inoculating two rings of well-grown thalli after 24 hours into 50 mL of seed culture medium, inoculating 6 bottles together, culturing at 35 ℃ and 180 rpm for 12 hours, then inoculating 3L of fermentation culture medium according to 10% (v/v) inoculum concentration, introducing air for 1.1v/vm, culturing at the rotation speed of 300 rpm and at 35 ℃, and fermenting for 22 hours to obtain the CGMCC No. 13932 with the fermentation enzyme production of 47031.6 Fu/g.
Example 6 Bacillus subtilis ((B))Bacillus subtilis) gs-11061 utilizes bean curd yellow slurry from different sources to ferment and produce nattokinase
Plate culture medium: 15g/L of peptone, 7.5 g/L of yeast extract, 15g/L of NacL and 20g/L of agar, and the pH value is natural.
Seed cultureAnd (3) nutrient medium: glucose 12 g/L, K2HPO4•3H2O1.1 g/L, peptone 15g/L, KH2PO4 1.2 g/L，MgSO4•7H2O 0.40 g/L,Cacl2 0.25 g/L,MnSO40.001 g/L, balance water, pH = 7.4.
Aerobic fermentation culture medium: three different bean curd yellow serofluid (gypsum bean curd, lactone bean curd and brine bean curd) after bean curd production are respectively taken and three different (gypsum bean curd, lactone bean curd and brine bean curd) fresh bean curd yellow serofluid (pH = 6.5-7.5) are respectively packaged in 500mL conical flasks, the liquid filling amount is 80 mL, and the sterilization is carried out at 121 ℃ for 20 min.
Firstly activating strain by a plate at 35 ℃ for CGMCC No. 13932, inoculating two rings of well-grown strains into 50 mL of seed culture medium after 24 h, culturing at 35 ℃ and 180 rpm for 12 h, then inoculating into a triangular flask with the liquid loading amount of 80 mL of fermentation liquid/500 mL according to the inoculation amount of 3.5% (v/v), and culturing at 35 ℃ and the rotating speed of 180 rpm. Bean curd yellow serofluid from different sources is used as a fermentation main body, and after fermentation for 36 h, fermentation liquor of CGMCC No. 13932 is used for producing natto kinase which is 26881.1 Fu/g, 27509.5 Fu/g and 26093.4 Fu/g respectively. In conclusion, the enzyme production of the bean curd yellow slurry from different sources by fermentation is not greatly different.
Example 7 Bacillus subtilis ((B))Bacillus subtilis) gs-11061 utilizes different fermentation media to produce natto kinase
Plate culture medium: 15g/L of peptone, 7.5 g/L of yeast extract, 15g/L of NacL and 20g/L of agar, and the pH value is natural.
Seed culture medium: glucose 12 g/L, K2HPO4•3H2O1.1 g/L, peptone 15g/L, KH2PO4 1.2 g/L，MgSO4•7H2O 0.40 g/L,Cacl2 0.25 g/L,MnSO40.001 g/L, balance water, pH = 7.4.
Aerobic fermentation culture medium: (1) glucose 15g/L, soybean meal 20g/L, K2HPO4•3H2O 1.2g/L，KH2PO41.6g/L，MgSO4•7H2O 0.45g/L,CaCl2 0.21g/L, the balance being water, pH =7.4,
（2）Glucose 15g/L, whole soybean powder 20g/L, K2HPO4•3H2O 1.2g/L，KH2PO41.6g/L，MgSO4•7H2O 0.45g/L,CaCl2 0.21g/L, balance water, pH =7.4
(3) Glucose 15g/L, soybean peptone 20g/L, K2HPO4•3H2O 1.2g/L，KH2PO41.6g/L，MgSO4•7H2O 0.45g/L,CaCl2 0.21g/L, balance water, pH =7.4
(4) Bean curd yellow serofluid.
And respectively taking four different fermentation media (pH = 6.5-7.5) and respectively filling the four different fermentation media into 500mL conical flasks, wherein the liquid filling amount is 80 mL, and sterilizing the flasks for 20min at 121 ℃.
Firstly activating strain by a plate at 35 ℃ for CGMCC No. 13932, inoculating two rings of well-grown strains into 50 mL of seed culture medium after 24 h, culturing at 35 ℃ and 180 rpm for 12 h, then inoculating into a triangular flask with the liquid loading amount of 80 mL of fermentation liquid/500 mL according to the inoculation amount of 3.5% (v/v), and culturing at 35 ℃ and the rotating speed of 180 rpm. Different fermentation media are used as fermentation main bodies, and after fermentation for 36 h, the fermentation liquor of CGMCC No. 13932 produces natto kinase which is 6021.4 Fu/g, 8901.2 Fu/g, 10709.5 Fu/g and 28789.5 Fu/g respectively. Compared with the common fermentation process for producing the nattokinase, the yield of the nattokinase adopting the bean curd yellow serofluid as the fermentation culture medium is respectively improved by 4.78 times, 3.23 times and 2.69 times. In conclusion, compared with the common fermentation process for producing the enzyme, the enzyme production amount of the bean curd yellow serofluid is the highest. Compared with the culture medium disclosed in the prior literature, the enzyme production effect of the strain is obviously improved.
Example 8 Bacillus subtilis ((B))Bacillus subtilis) gs-11061 utilizes bean curd yellow slurry water added with amino acid to ferment and produce nattokinase
Plate culture medium: 15g/L of peptone, 7.5 g/L of yeast extract, 15g/L of NaCl, 20g/L of agar and natural pH;
seed culture medium: glucose 12 g/L, K2HPO4•3H2O1.1 g/L, peptone 15g/L, KH2PO4 1.2 g/L，MgSO4•7H2O 0.40 g/L,Cacl2 0.25 g/L,MnSO40.001 g/L, balance water, pH = 7.4.
Fermentation medium: bean curd yellow serofluid, 0.2g/L aspartic acid, 0.3g/L serine, adjusting pH = 6.5-7.5, and subpackaging in 500mL conical bottles with liquid filling amount of 80 mL, 121 ℃ and sterilizing for 20 min.
Bacillus subtilis (A), (B) and (C)Bacillus subtilis) gs-11061 (accession number: CGMCC No. 13932) firstly activates strains on a plate at 35 ℃, strains with good growth of two rings are inoculated into 50 mL of seed culture medium after 24 h, the strains are cultured at 35 ℃ and 180 rpm for 12 h, then the strains are inoculated into a triangular flask with the liquid loading capacity of 80 mL of fermentation medium/500 mL according to the inoculation amount of 3.5% (v/v), the strains are cultured at 35 ℃ and the rotation speed of 180 rpm, and after fermentation for 36 h, the nattokinase in the fermentation liquid is 34234.1 Fu/g. The final fermentation liquor is directly freeze-dried or spray-dried in vacuum to obtain crude enzyme liquid, and simultaneously, the hidden trouble of environmental pollution caused by direct discharge of the fermentation liquor is also solved.
Example 9 Bacillus subtilis ((B))Bacillus subtilis) gs-11061 utilizes bean curd yellow slurry water added with metal ions to ferment and produce nattokinase
Plate culture medium: 15g/L of peptone, 7.5 g/L of yeast extract, 15g/L of NaCl, 20g/L of agar and natural pH;
seed culture medium: glucose 12 g/L, K2HPO4•3H2O1.1 g/L, peptone 15g/L, KH2PO4 1.2 g/L，MgSO4•7H2O 0.40 g/L,Cacl2 0.25 g/L,MnSO40.001 g/L, balance water, pH = 7.4.
Fermentation medium: yellow serofluid of bean curd, CaCl2 0.25g/L,MnSO40.002 g/L, adjusting pH = 6.5-7.5, and subpackaging in 500mL conical flasks, wherein the liquid filling amount is 80 mL, the temperature is 121 ℃, and the sterilization is carried out for 20 min.
Bacillus subtilis (A), (B) and (C)Bacillus subtilis) gs-11061 (accession number: CGMCC No. 13932) firstly activates strains on a plate at 35 ℃, strains with good growth of two rings are inoculated into 50 mL of seed culture medium after 24 h, the strains are cultured at 35 ℃ and 180 rpm for 12 h, then the strains are inoculated into a triangular flask with the liquid loading capacity of 80 mL of fermentation culture medium/500 mL according to the inoculation amount of 3.5 percent (v/v), the strains are cultured at 35 ℃ and the rotation speed of 180 rpm, and the fermentation is carried outAfter 36 h, the nattokinase in the fermentation liquor is 30101.5 Fu/g. The final fermentation liquor is directly freeze-dried or spray-dried in vacuum to obtain crude enzyme liquid, and simultaneously, the hidden trouble of environmental pollution caused by direct discharge of the fermentation liquor is also solved.
The detection method of the nattokinase comprises the following steps: take 0.7 mL of 50 mmol. L-1Tris-HCl (pH 8.0) buffer with 0.2 mL of 0.72% (w.v)-1) The fibrinogen solution was mixed well and left at 37 ℃ for 5 min. To the above solution was added 0.1 mL of thrombin solution (20U. mL)-1) Mixing, and standing at 37 deg.C for 10 min. And adding 0.05 mL of diluted enzyme solution into the reaction system, fully mixing, keeping the temperature in a water bath at 37 ℃ for reaction, and respectively mixing for 10 s at 20min and 40 min after the reaction starts. After accurately timing for 60 min, 1mL of 0.2 mol.L is added-1The reaction was stopped with trichloroacetic acid and incubated in a 37 ℃ water bath for a further 20 min. The reaction solution is put at 15000 r.min-1The supernatant was centrifuged for 10 min and the absorbance of the centrifuged supernatant at 275nm was measured. One enzyme activity unit (FU) is defined as the amount of enzyme required to change absorbance at 275nm per minute by 0.01 at 37 ℃ and pH 8.0.
Specific enzyme activity definition: the number of enzyme activity units per mg of protein, i.e. specific activity = active FU · mg-1A protein.

Claims (6)

1. A method for producing nattokinase by using bean curd yellow serofluid is characterized by comprising the following steps:
1) taking bean curd yellow serofluid, and adjusting the pH value to 6-8;
2) inoculating the nattokinase production strain into bean curd yellow slurry water, and fermenting at the fermentation temperature of 20-40 ℃ for 20-72 h to obtain nattokinase fermentation liquor;
wherein: adding amino acid or metal ions into the bean curd yellow slurry water;
the amino acid is aspartic acid or serine, and the metal ion is Mn2+；
The natto kinase producing strain is bacillus subtilis (Bacillus subtilis)Bacillus subtilis) gs-11061；
And 2) introducing air at 1.0-2.5 v/vm in the fermentation process, and rotating at 200-500 rpm.
2. The method for producing nattokinase according to claim 1, wherein the tofu yellow serofluid in step 1) is sterilized at 121 ℃ for 20min before liquid fermentation.
3. The method for producing nattokinase according to claim 1, wherein the soybean milk is waste water after any commercially available soybean product is produced.
4. The method for producing nattokinase according to claim 1, wherein Bacillus subtilis (B.subtilis) is addedBacillus subtilis) The gs-11061 is inoculated into the yellow soybean milk water of the bean curd according to the inoculation amount of 2-10% (V/V), air is introduced for 1.1V/vm, the rotating speed is 300 rpm, the fermentation temperature is 35 ℃, and aerobic fermentation is carried out for 22 hours.
5. The method for producing nattokinase according to claim 1, wherein the amino acids include aspartic acid 0.1-0.5 g/L and serine 0.1-0.5 g/L.
6. The method for producing nattokinase according to claim 1, wherein the nattokinase fermentation liquor is directly subjected to vacuum freeze-drying or spray-drying to obtain crude enzyme liquid.
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