WO2021000247A1 - 一种纳豆激酶的生产菌株及其生产方法 - Google Patents

一种纳豆激酶的生产菌株及其生产方法 Download PDF

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WO2021000247A1
WO2021000247A1 PCT/CN2019/094334 CN2019094334W WO2021000247A1 WO 2021000247 A1 WO2021000247 A1 WO 2021000247A1 CN 2019094334 W CN2019094334 W CN 2019094334W WO 2021000247 A1 WO2021000247 A1 WO 2021000247A1
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nattokinase
source material
strain
powder
natto
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PCT/CN2019/094334
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English (en)
French (fr)
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陈杰鹏
段丽丽
洪琳
纪烨瑜
陈鸿锐
蔡春丽
胡留松
许志锴
陈煜藩
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广东双骏生物科技有限公司
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Priority to US17/424,761 priority Critical patent/US20220088154A1/en
Priority to PCT/CN2019/094334 priority patent/WO2021000247A1/zh
Priority to JP2021540418A priority patent/JP7259050B2/ja
Priority to EP19936116.3A priority patent/EP3892715A4/en
Publication of WO2021000247A1 publication Critical patent/WO2021000247A1/zh

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Definitions

  • This application relates to but not limited to the field of microbial fermentation. Specifically, this application relates to but not limited to a nattokinase production strain and a nattokinase product production method.
  • Nattokinase is internationally recognized as one of the most important active molecules for the prevention, health care and treatment of human cardiovascular and cerebrovascular diseases. It is a trace active substance produced by the fermentation of Bacillus subtilis natto. Over the years, it has been widely used in food, dietary supplements and domestic health products markets. The raw materials of nattokinase dietary supplements are in huge demand in the international and domestic markets.
  • nattokinase the main producing areas of nattokinase internationally are Japan, Taiwan and the United States. Nattokinase produced in Japan is extracted from Bacillus subtilis natto (Bacillus subtilis natto) fermented with natto. The activity is 10000-22000FU/g, which is equivalent to 67000-147400IU/g.
  • Japan there are four main manufacturers producing nattokinase: Nabaien, Biotech, Yamato, and a subsidiary of Honda.
  • the earliest and largest producer of nattokinase was a biotechnology company, occupying most of the markets in Taiwan and Japan. Daiwa's nattokinase powder also occupies a certain market share in Japan.
  • the purpose of this application is to provide a new strain that can produce nattokinase.
  • Bacillus subtilis natto strain from fresh natto on the market, and used it as a starting strain to mutate a new Bacillus subtilis natto (Bacillus subtilis natto) ST1086
  • the mutagenic strain was deposited under CGMCC No. 17895 in the General Microbiology Center of China Microbial Culture Collection Management Committee, Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, preservation date: June 2019 On the 5th.
  • the ST102 strain was used as the starting strain to undergo UV mutagenesis. After 50 generations of UV mutagenesis, the morphological variant ST1086 was obtained, and the yield of nattokinase was increased by 11 times. The above morphological variation and the yield of nattokinase are relatively stable after several generations of subculture.
  • the new strain CGMCC No. 17895 provided by this application has the following microbiological characteristics: Gram-positive bacteria, spores grow in the middle, the spore size is 0.6-0.8 ⁇ m ⁇ 1.0-1.5 ⁇ m, the cell width is 1 ⁇ m, and the length is 2-3 ⁇ m.
  • the surface of the colony is full, wrinkled, white, convex, capable of drawing silk, the diameter of the colony is 0.3-0.5cm, there is no pigment, and spores appear when the bacteria are cultured for 10 hours.
  • a method for producing nattokinase products using the new strain CGMCC No. 17895 of the present application includes culturing the strain CGMCC No. 17895 of the present application in a medium to produce sodium in the medium.
  • Bean kinase. Cultivation can be carried out under conventional or known devices and conditions in the art, for example, a shake flask can be used at a conventional or known rotational speed in the art; it can also be carried out in a conventional fermenter, such as 5L fermentor, 5T fermentation tank.
  • the culture medium includes a carbon source material and a nitrogen source material, and the ratio of the carbon source material to the nitrogen source material is 10:1 to 1:2.
  • the carbon source material is selected from one or more of glucose, sucrose, maltose, fructose, and glycerin
  • the nitrogen source material is selected from one or more of yeast powder, peptone, soybean powder and chickpea powder. Many kinds.
  • the culture medium further contains organic matter, inorganic matter, or a mixture of organic matter and inorganic matter that promote the growth of microorganisms and increase the yield of nattokinase.
  • the organic substance is one or more of serine, glycine, and alanine
  • the inorganic substance is a magnesium salt or a sodium salt.
  • the inorganic substance is magnesium sulfate, magnesium chloride, sodium chloride.
  • the cultivation is performed at 35-45°C, preferably 37-40°C. In some embodiments, the culture lasts 10-48 hours, preferably 12-24 hours.
  • the method includes adding a carbon source material or a nitrogen source material, or a mixture of a carbon source material and a nitrogen source material during the fermentation process.
  • the method further includes the following steps: (1) solid-liquid separation to separate bacterial cells and supernatant; (2) separation with an ultrafiltration membrane to obtain a nattokinase concentrate, the ultrafiltration The molecular weight range of the membrane is preferably between 1,000 and 50,000 D, more preferably between 10,000 and 30,000 D; (3) Wash with 1 mmol/L NaCl solution so that the conductivity of the nattokinase concentrate is ⁇ 300 ⁇ s/cm, preferably ⁇ 200 ⁇ s/cm, more preferably ⁇ 100 ⁇ s/cm; and (4) drying, which is preferably spray drying, freeze drying, or vacuum drying.
  • solid-liquid separation is performed using solid-liquid separation methods conventional or known in the art, such as ceramic membrane separation or centrifugation.
  • a protective agent is added in the drying step, and the protective agent is preferably soy protein powder, collagen powder, dietary fiber, microcrystalline cellulose, corn starch or a combination thereof, more preferably 5-25% Soy protein powder and 2.5-12.5% dietary fiber.
  • the activity of the nattokinase product obtained by the method of the present application is 65,000-750,000 FU/g, which is equivalent to 436,000 IU/g-5025 million IU/g.
  • the present application also provides a method for preparing nattokinase products from the culture broth of the new strain CGMCC No. 17895 of the present application.
  • the method includes the following steps:
  • Solid-liquid separation which uses conventional or known solid-liquid separation methods in the art, such as ceramic membrane separation and centrifugation, to separate bacterial cells and fermentation supernatant;
  • the molecular weight of the ultrafiltration membrane is preferably between 1,000 and 50,000 D, more preferably between 10,000 and 30,000 D;
  • the drying is preferably spray drying, freeze drying, or vacuum drying.
  • a protective agent conventional or known in the art is added during the drying process, such as soy protein powder, collagen powder, dietary fiber, microcrystalline cellulose, corn starch or a combination thereof, preferably 5-25 % Soy protein powder and 2.5-12.5% dietary fiber.
  • the cycle is as short as 16-24 hours, the output is as high as 12000IU/ml (equivalent to 1791.0FU/ml), and the final nattokinase product activity is 65,000-750,000 FU/g, and more stable in the preparation process.
  • nattokinase activity is measured by the following method.
  • PBS phosphate buffer saline, phosphate buffer saline
  • phosphate buffer 0.01mol/L phosphate buffer (pH 7.5): Weigh 3.58g of disodium hydrogen phosphate (Na 2 HPO 4 ⁇ 12H 2 O), add double distilled water to dissolve and dilute to 1000mL as liquid I; take diphosphate Sodium hydrogen (NaH 2 PO 4 ⁇ 2H 2 O) 0.78g, add double distilled water to dissolve and dilute to 500mL as liquid II; take about 84mL of liquid I and about 16mL of liquid II, and mix the two until the pH is 7.5.
  • disodium hydrogen phosphate Na 2 HPO 4 ⁇ 12H 2 O
  • diphosphate Sodium hydrogen NaH 2 PO 4 ⁇ 2H 2 O
  • agarose solution take 1.5 g of agarose, add 100 mL of PBS buffer solution, heat to dissolve, and keep it in a water bath at 50°C.
  • Fibrinogen solution Take an appropriate amount of fibrinogen and add PBS buffer solution to make a solution containing 1.5 mg of condensable protein per 1 mL.
  • Thrombin solution Take thrombin, add 0.9% sodium chloride solution to make a solution containing 1 BP unit per 1 mL.
  • Urokinase standard solution 1000IU/mL: Take a bottle of urokinase, add PBS buffer solution according to the labeled titer to dissolve, that is, 1000IU/mL urokinase standard solution.
  • nattokinase activity accurately weigh the nattokinase sample in a volumetric flask, dissolve it with an appropriate amount of PBS buffer solution, and dilute to the mark after sonicating for 15 minutes so that the final spot concentration is 200-400IU/mL.
  • Accurately measure 10 ⁇ L of the nattokinase sample solution place it on the agarose fibrin plate, cover it, and react in a 37°C incubator for 18 hours. After taking it out, measure the diameter of the dissolved circle, calculate the area of the dissolved circle, and substitute the sample dissolved circle area into the regression equation to calculate the nattokinase activity of the sample solution.
  • X sample nattokinase activity, IU/g
  • V Total volume of sample dilution, mL
  • nattokinase activity in terms of FU is defined as:
  • the nattokinase activity was analyzed according to the Nattokinase Activity Analysis Method (No. 104022640) of the Japan Food Research Laboratory.
  • step (3) When the solution of step (2) is accurately reacted for the 10th minute, add 0.1mL of the test sample solution accurately, mix well, carry out the enzyme reaction in a 37 ⁇ 0.3°C water bath for 60 minutes, and shake it up at 30min and 50min. once.
  • step (2) When the solution in step (2) is accurately reacted to the 60th minute, add 2mL 0.2M trichloroacetic acid solution to stop the enzyme reaction, and react in a 37 ⁇ 0.3°C water bath for 20 minutes.
  • test sample solution 0.1 mL
  • test sample solution 0.1 mL
  • mix well 0.1 mL
  • test tube is centrifuged at 12000 rpm for 10 min.
  • nattokinase The activity of nattokinase is calculated in the following way:
  • X sample nattokinase activity, FU/g or FU/mL;
  • Ar-Ac The value must be between 0.050 and 0.080;
  • Example 1 Isolation of Nattokinase-producing Bacillus subtilis from commercially available natto
  • Dissolve natto (purchased from Japan Biotech) with sterile water, apply it to a solid LB medium plate after dilution, and incubate at 37°C for 24 hours. White colonies grow on the surface of the plate. phenomenon. Transfer the white colonies to the LB slant medium, incubate at 37°C for 24 hours, and inoculate the fermentation medium with an inoculation loop (100ml triangular flask with 20ml, medium formula: glucose 2%, sucrose 2%, soybean powder 3%, Magnesium sulfate 0.01%, sodium chloride 0.5%, serine 0.08%), 37°C, 270rpm shake for 20 hours, centrifuge to take the supernatant, use agarose fibrin plate method to determine the content of nattokinase, select the larger diameter Strains, and finally identified Bacillus subtilis nattokinase producing nattokinase.
  • inoculation loop 100ml triangular flas
  • UV mutagenesis was performed: UV wavelength 200-300nm, irradiation distance 15-30cm, irradiation time 20s, after 50 generations, the mutant strain ST-1086 was induced.
  • the mutant ST-1086 has the following microbiological characteristics: Gram-positive bacteria, spores grow in the middle, the spore size is 0.6-0.8 ⁇ m ⁇ 1.0-1.5 ⁇ m, the bacteria width is 1 ⁇ m, and the length is 2-3 ⁇ m.
  • the surface of the colony is full, wrinkled, white, and convex, capable of drawing, with a diameter of 0.3-0.5cm, no pigment, and spores appear after the bacteria are cultured for 10 hours.
  • the mutant ST-1086 is deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, preservation date: June 5, 2019, The deposit number is: CGMCC No.17895.
  • the starting strain and the CGMCC No.17895 strain obtained in Example 2 were respectively inoculated into 20ml seed culture medium (see Table 1), and cultured in an incubator at 37-40°C and 150-300rpm for 3-16 hours. Seed culture solution.
  • the seed culture solution obtained in Example 3 was inoculated into the basal medium (see Table 2) according to the inoculum amount accounting for 20% of the fermentation medium amount, and cultivated at 40°C. After 30 minutes of incubation, the feed medium (see Table 3), adding 200 ml of the feed medium every half an hour for 10.5 hours, and add 2L of the feed medium.
  • the pH value of the cultivation process is natural, and the dissolved oxygen concentration is controlled above 30%.
  • the fermentation cycle is 17 hours. Fibrin plate method was used to determine the content of nattokinase.
  • the starting strain was used this method to obtain a nattokinase output of 800IU/ml (equivalent to 119.4FU/ml), the resulting fermentation broth was separated by a ceramic membrane, and the resulting dialysate was concentrated with a 10000D molecular weight ultrafiltration membrane.
  • Nattokinase activity is 7000IU/ml (equivalent to 1044.8FU/ml).
  • Nattokinase concentrate is added with 15% soybean protein powder and 5% dietary fiber (purchased from French Roquette, wheat-derived water-soluble dietary fiber) After dissolution, spray drying is performed. The natto powder with an activity of 1500FU/g (equivalent to 10050IU/g) was obtained.
  • the CGMCC No.17895 strain of this application has a nattokinase output of 7500IU/ml (equivalent to 1119.4FU/ml).
  • the resulting fermentation broth is separated by a ceramic membrane for solid-liquid separation, and the resulting dialysate is concentrated with a 10,000D molecular weight ultrafiltration membrane ,
  • the concentration of nattokinase activity is 150000IU/ml (equivalent to 22388.1FU/ml).
  • the obtained nattokinase concentrate is spray-dried by adding 12% soy protein powder and 6% dietary fiber.
  • the natto powder with an activity of 58000FU/g (equivalent to 390000IU/g) was obtained.
  • Example 4 The difference from Example 4 is that the feed medium contains 0.16% serine.
  • the nattokinase fermentation yield of the CGMCC No. 17895 strain of the present application is 12000IU/ml (equivalent to 1791.0F U/ml).
  • Example 4 The difference from Example 4 is that the feed medium contains 0.16% glycine.
  • the fermentation yield of nattokinase of the CGMCC No. 17895 strain of this application is 9000 IU/ml (equivalent to 1343.3 FU/ml).
  • Example 4 The difference from Example 4 is that the feed medium contains 0.16% alanine.
  • the nattokinase fermentation yield of the CGMCC No. 17895 strain of the present application is 10000IU/ml (equivalent to 1492.5FU/ml).
  • Example 4 The difference from Example 4 is that the feed medium contains 0.16% serine and 0.16% glycine.
  • the nattokinase fermentation yield of the CGMCC No. 17895 strain of this application is 10500IU/ml (equivalent to 1567.2FU/ml).
  • Example 4 The difference from Example 4 is that the feed medium contains 0.16% serine and 0.16% alanine.
  • the nattokinase fermentation yield of the CGMCC No. 17895 strain of the present application is 11000IU/ml (equivalent to 1641.8FU/ml).
  • Example 4 The difference from Example 4 is that the feed medium contains 0.16% glycine and 0.16% alanine.
  • the nattokinase fermentation yield of the CGMCC No. 17895 strain of the present application is 9500IU/ml (equivalent to 1417.9FU/ml).
  • Example 4 The difference from Example 4 is that the feed medium contains 0.16% serine, 0.16% glycine and 0.16% alanine.
  • the nattokinase fermentation yield of the CGMCC No. 17895 strain of the present application is 10300IU/ml (equivalent to 1537.3FU/ml).
  • Example 4 Take the fermentation broth of CGMCC No. 17895 in Example 4 and separate it from the solid-liquid through a ceramic membrane with a pore size of 0.1 ⁇ m to remove the bacteria, culture medium and other solid particles to obtain a nattokinase-containing liquid; the resulting nattokinase-containing liquid
  • the liquid is filtered and concentrated with an ultrafiltration membrane with a molecular weight cut-off of 10000D to obtain a nattokinase concentrate;
  • the obtained nattokinase concentrate is purified with 30% saturated ammonium sulfate, and then 70% saturated ammonium sulfate is used to precipitate natto Kinase, remove some pigments and polysaccharides to obtain nattokinase precipitation; Desalt the nattokinase concentrate precipitation with Sephadex G25 packing, elution with phosphate buffer, loading volume 20% CV (column volume), flow rate 40cm/ h.
  • the desalted nattokinase solution is purified with SP sepharose FF packing (purchased from GE), the loading amount is 80-120mg/ml, the flow rate is 120cm/h, and the pigmented polysaccharide part is obtained Contaminated protein nattokinase solution; take the concentrated solution from which pigment polysaccharides are removed, and perform column chromatography with molecular sieve filler superdex75 to remove the remaining contaminated protein bands.
  • the loading volume is 5% CV, the flow rate is 20 cm/h, and the collection contains natto A collection solution of a single band of kinase; the collection solution of a single band of nattokinase is freeze-dried with a freeze dryer to obtain 7 million FU/g (equivalent to 46.9 million IU/g) pure nattokinase powder.
  • the purpose of this example is to purify the nattokinase of CGMCC No. 17895 strain according to the literature purification method, and investigate the changes in the activity of nattokinase during the purification process.
  • Example 4 Take the fermentation broth of CGMCC No.17895 in Example 4 and separate it from the solid-liquid through a ceramic membrane with a pore size of 0.1 ⁇ m to remove the bacteria, culture medium and other solid particles to obtain a nattokinase-containing liquid;
  • the ultrafiltration membrane with a molecular weight cut-off of 10000D was filtered and concentrated to obtain a nattokinase concentrate of 7000IU/ml.
  • the concentrated solution was precipitated with 30% ammonium sulfate and then with 60% ammonium sulfate to remove part of the pigment and polysaccharides to obtain nattokinase precipitation.
  • the precipitation was dissolved into a 5% solution with 2M ammonium sulfate solution, and then used Phenyl Sepharose hydrophobic column chromatography.
  • the loading amount was 20-60mg/ml
  • the flow rate was 60cm/h
  • the linear gradient was eluted with 2M ⁇ 0M ammonium sulfate solution.
  • 20 times the column volume, flow rate 100cm/h collect the eluate step by step, use SDS-PAGE to detect the purity of nattokinase in the eluate, obtain 95% pure nattokinase, the chromatographic effluent is directly frozen After drying, 5 million FU/g (33.5 million IU/g) pure nattokinase powder was obtained.
  • Example 12 The difference from Example 12 is that the fermentation broth of the starting strain in Example 4 is taken to obtain 6 million FU/g (equivalent to 40.2 million IU/g) pure nattokinase powder.
  • Example 12 Compared with the prior art method, the purification method of the present application can significantly increase the active concentration of pure nattokinase.
  • Example 12 It can be seen from the results of Example 12 and Example 14 that compared with the original strain, the CGMCC No. 17895 strain of the present application can obtain a pure product with a higher concentration of nattokinase activity.
  • Example 5 The difference from Example 5 is that the fermentation broth obtained by fermentation with the strain CGMCC No. 17895 of the present application is concentrated with a 10000D ultrafiltration membrane and washed with a 0.1 mmol/L NaCl solution until the conductivity is 300 ⁇ s/cm. The conductivity is measured with a conductivity meter.
  • the resulting nattokinase concentrate has an activity of 300,000 IU/ml (equivalent to 45,000 FU/ml).
  • the concentrate is spray dried by adding 5% microcrystalline cellulose and 5% dietary fiber. Spray drying conditions: inlet air temperature 200°C, outlet air temperature 45°C, fan speed 70R/min, feed speed 45L/h, drying yield 45%.
  • the obtained natto powder has an activity of 101,000 FU/g (678,000 IU/g).
  • Example 5 The difference from Example 5 is that the fermentation broth obtained by fermentation with the strain CGMCC No. 17895 of the present application is concentrated with a 10000D ultrafiltration membrane and washed with a 0.1 mmol/L NaCl solution until the conductivity is 300 ⁇ s/cm. The conductivity is measured with a conductivity meter.
  • the resulting nattokinase concentrate has an activity of 300,000 IU/ml (equivalent to 45,000 FU/ml).
  • the concentrate is spray-dried by adding 5% soybean protein powder and 5% dietary fiber, and the drying yield is 80%.
  • the obtained natto powder has an activity of 180,000 FU/g (equivalent to 1.206 million IU/g). Soy protein powder, as a protein protective agent, can improve the stability of nattokinase; microcrystalline cellulose is only an excipient to increase the solid content of the concentrated solution, which is beneficial to the spray drying process.
  • Example 5 The difference from Example 5 is that the fermentation broth obtained by fermentation with the CGMCC No. 17895 strain of the present application is concentrated with a 10000D ultrafiltration membrane and then washed with a 0.1 mmol/L NaCl solution until the conductivity is 200 ⁇ s/cm.
  • the resulting nattokinase concentrate has an activity of 300,000 IU/ml (equivalent to 45,000 FU/ml).
  • the concentrate is spray-dried by adding 15% soybean protein powder and 7.5% dietary fiber, and the drying yield is 90%.
  • the obtained natto powder has an activity of 129,000 FU/g (861,000 IU/g).
  • Example 5 The difference from Example 5 is that the fermentation broth obtained by fermentation with the CGMCC No. 17895 strain of the present application is concentrated with a 10000D ultrafiltration membrane and then washed with a 0.1 mmol/L NaCl solution until the conductivity is 100 ⁇ s/cm.
  • the resulting nattokinase concentrate has an activity of 300,000 IU/ml (equivalent to 45,000 FU/ml).
  • the concentrate was spray-dried by adding 15% soybean protein powder, 7.5% dietary fiber and 5% microcrystalline cellulose, and the drying yield was 98%.
  • the obtained natto powder has an activity of 124,000 FU/g (equivalent to 832,000 IU/g).
  • Example 5 The difference from Example 5 is that the fermentation broth obtained by fermentation with the strain CGMCC No. 17895 of the present application is concentrated with a 10000D ultrafiltration membrane and washed with purified water until the conductivity is 300 ⁇ s/cm.
  • the resulting nattokinase concentrate has an activity of 250,000 IU/ml (37,000 FU/ml).
  • the concentrate is spray-dried with 5% soy protein powder and 5% dietary fiber, and the drying yield is 80%.
  • the obtained natto powder has an activity of 164,000 FU/g (1.1 million IU/g).
  • Example 5 The difference from Example 5 is that the fermentation broth obtained by fermentation with the CGMCC No. 17895 strain of the present application is concentrated with a 10000D ultrafiltration membrane and washed with purified water until the conductivity is 200 ⁇ s/cm.
  • the obtained nattokinase concentrate has an activity of 240,000 IU/ml (36,000 FU/ml).
  • the concentrate is spray-dried by adding 15% soybean protein powder and 7.5% dietary fiber, and the drying yield is 90%.
  • the obtained natto powder has an activity of 108,000 FU/g (724,000 IU/g).
  • Example 5 The difference from Example 5 is that the fermentation broth obtained by fermentation with the strain CGMCC No. 17895 of the present application is concentrated with a 10000D ultrafiltration membrane and washed with purified water until the conductivity is 100 ⁇ s/cm.
  • the obtained nattokinase concentrate has an activity of 230,000 IU/ml (34,000 FU/ml).
  • the concentrate was spray-dried by adding 15% soybean protein powder, 7.5% dietary fiber and 5% microcrystalline cellulose, and the drying yield was 98%.
  • the obtained natto powder has an activity of 97,000 FU/g (647,000 IU/g).
  • Example 5 The difference from Example 5 is that the fermentation broth obtained by fermentation with the strain CGMCC No. 17895 of the present application is concentrated with a 10000D ultrafiltration membrane and washed with purified water until the conductivity is 100 ⁇ s/cm.
  • the obtained nattokinase concentrate has an activity of 230,000 IU/ml (34,000 FU/ml).
  • the concentrate was spray-dried, and the drying yield was 50%.
  • the obtained natto powder has an activity of 210,000 FU/g (1.42 million IU/g).
  • natto powder obtained in Example 22, Example 15, Example 16, Example 17, and Example 18 were used to compress tablets respectively to investigate the stability of different excipients to the formulation process.
  • Tableting process 30% fish collagen, 10% maltodextrin, 10% lactose, 30% microcrystalline cellulose are added to natto powder, the raw materials and auxiliary materials are respectively passed through 80 mesh sieve-mixing-granulating-tableting to determine the sodium The activity of bean kinase before and after compression. The results are as follows:

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Abstract

一种纳豆激酶的生产菌株及其生产方法。具体地，涉及一种可生产纳豆激酶的新菌种，纳豆枯草芽孢杆菌(Bacillus subtils natto)ST-1086，其以CGMCC No.17895保藏于中国微生物菌种保藏管理委员会普通微生物中心。还涉及应用本申请的新菌株CGMCC No.17895生产纳豆激酶产品的方法，所得纳豆激酶产品可以用作溶解血栓的药品。还涉及本申请的纳豆激酶产品用于制备用于溶解血栓的组合物的用途以及用于治疗血栓的方法。

Description

一种纳豆激酶的生产菌株及其生产方法 技术领域
本申请涉及但不限于微生物发酵领域。具体地，本申请涉及但不限于一种纳豆激酶的生产菌株及纳豆激酶产品的生产方法。
背景技术
纳豆激酶为国际上公认的用于人类心脑血管疾病的预防、保健和治疗的最重要的活性分子之一，为纳豆枯草芽孢杆菌发酵所产生的微量活性物质。多年来，已广泛应用于食品、膳食补充剂和国内的保健品市场。纳豆激酶膳食补充剂原料国际国内市场需求量巨大。
目前国际范围内纳豆激酶主要产地有日本、台湾和美国。日本产的纳豆激酶由纳豆枯草芽孢杆菌(Bacillus subtilis natto)发酵纳豆中提取，活性为10000-22000FU/g，相当于67000-147400IU/g。在日本，主要有四个厂家生产纳豆激酶：纳百恩、生物科技、大和及本田下属一个子公司。生产纳豆激酶最早、产量最大的是生物科技公司，占据了台湾和日本的大部分市场。大和公司生产的纳豆激酶粉末，在日本也占据一定的市场份额。这两家的纳豆激酶原料自用不对外销售。生物科技公司以及大和公司使用的技术老、产品功效单一、缺少粘物质的保护、容易失活、稳定性差，但成本低廉。本田下属子公司基本同生物科技，拥有纳豆激酶专利。日本纳百恩公司是生产纳豆激酶后起之秀，纳豆激酶产品的活性达到22000FU/g，相当于147400IU/g，但价格昂贵。台湾产纳豆激酶的技术来源于日本。美国产纳豆激酶由曲霉菌发酵物提取，产品中不含纳豆菌和维生素K2等粘物质，可称为“溶栓酶”。
文献报道，利用纳豆枯草芽孢杆菌进行液态发酵，纳豆激酶的产量为3232IU/ml(相当于482.4FU/ml)(参见熊强等人，纳豆激酶液态发酵条件 的研究，生物加工过程，2012年，第10卷第4期，第26-29页)。
在中国，纳豆产业正处于快速发展阶段，新鲜纳豆以及以纳豆提取粉为主要原料的复合饮料、糖果、饼干及保健食品等纳豆系列食品成为一大消费热点。但是，由于纳豆激酶不稳定，纯化过程易变性，目前获得纳豆激酶纯品非常困难，目前中国及外国尚未有纳豆激酶纯品或其制剂销售，也未有纳豆激酶的药品。因此，本申请旨在提供一种生产、纯化纳豆激酶的方法。
发明内容
以下是对本文详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。
为了克服现有技术的不足和满足市场需求，本申请的目的在于提供一种可以产生纳豆激酶的新菌种。
本申请人经过多年大量深入细致地研究，从市售新鲜纳豆中分离纳豆枯草芽孢杆菌菌株，以其作为出发菌株，诱变出一种新的纳豆枯草芽孢杆菌(Bacillus subtilis natto)ST1086，该诱变菌株以CGMCC No.17895保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏日期：2019年6月5日。本申请人从市售新鲜纳豆中分离菌株，得到产纳豆激酶的菌种ST102。以ST102菌株作为出发菌株，进行紫外诱变，紫外诱变50代后得到形态变异株ST1086，纳豆激酶产量提高11倍。以上形态变异以及纳豆激酶的产量经数代传代培养较稳定。
本申请提供的新菌株CGMCC No.17895具有以下微生物学特性：革兰氏阳性菌，芽孢中间生，芽孢大小0.6～0.8μm×1.0～1.5μm，菌体宽1μm，长2-3μm。在LB琼脂培养基上，菌落表面饱满、褶皱、白，凸起，能拉丝，菌落直径0.3-0.5cm，无色素，菌体培养10小时出现芽孢。
在本申请的一个方面，提供了应用本申请的新菌株CGMCC No.17895生产纳豆激酶产品的方法，包括在培养基中培养本申请的菌株CGMCC No. 17895以在所述培养基中产生纳豆激酶。培养可以在本领域常规或已知的装置和条件下进行，例如可以使用摇瓶在本领域常规或已知的转速下进行；也可以在常规的发酵罐中进行，例如5L发酵罐、5T发酵罐。
在一些实施方案中，培养基包含碳源物质和氮源物质，且所述碳源物质与所述氮源物质的比为10:1～1:2。在一些实施方案中，碳源物质选自葡萄糖、蔗糖、麦芽糖、果糖和甘油中的一种或多种，氮源物质选自酵母粉、蛋白胨、黄豆粉和鹰嘴豆粉中的一种或多种。
在又一些实施方案中，培养基还包含促进微生物的生长和提高纳豆激酶的产率的有机物、无机物、或有机物和无机物的混合物。在一些实施方案中，有机物为丝氨酸、甘氨酸、和丙氨酸中的一种或多种，无机物为镁盐或钠盐。优选地，无机物为硫酸镁、氯化镁、氯化钠。
在另一些实施方案中，培养在35-45℃，优选37-40℃进行。在一些实施方案中，培养持续10-48小时，优选12-24小时。
根据一些实施方案，所述方法包括在发酵过程中补加碳源物质或氮源物质，或碳源物质和氮源物质的混合物。在一些实施方案中，所述方法还包括以下步骤：(1)固液分离，以分离菌体和上清液；(2)用超滤膜分离，得到纳豆激酶浓缩液，所述超滤膜的分子量范围优选在1000～50000D之间，更优选在10000～30000D之间；(3)用1mmol/L NaCl溶液洗涤，使所述纳豆激酶浓缩液的电导率≤300μs/cm，优选≤200μs/cm，更优选≤100μs/cm；以及(4)干燥，所述干燥优选为喷雾干燥、冷冻干燥、真空干燥。在一些实施方案中，固液分离使用本领域常规或已知的固液分离方法进行，诸如陶瓷膜分离或离心。在一些实施方案中，在干燥步骤中加入保护剂，所述保护剂优选为大豆蛋白粉、胶原蛋白粉、膳食纤维、微晶纤维素、玉米淀粉或者其组合物，更优选为5-25％的大豆蛋白粉和2.5-12.5％膳食纤维。
根据一些实施方案，通过本申请的方法得到的纳豆激酶产品的活性为6.5万-75万FU/g，相当于43.6万IU/g-502.5万IU/g。
在另一个方面，本申请还提供了由本申请的新菌株CGMCC No.17895 发酵培养的培养液制备纳豆激酶产品的方法。在一些实施方案中，所述方法包括以下步骤：
(1)固液分离，所述固液分离使用本领域常规或已知的固液分离方法进行，诸如陶瓷膜分离、离心，以分离菌体和发酵上清液；
(2)用超滤膜分离，得到纳豆激酶浓缩液，所述超滤膜的分子量范围优选在1000～50000D之间，更优选在10000～30000D之间；
(3)用等渗盐溶液洗涤，使所述纳豆激酶浓缩液的电导率≤300μs/cm，优选≤200μs/cm，更优选≤100μs/cm；以及
(4)干燥，所述干燥优选为喷雾干燥、冷冻干燥、真空干燥。
在一些实施方案中，在干燥过程中加入本领域常规的或者已知的保护剂，例如大豆蛋白粉、胶原蛋白粉、膳食纤维、微晶纤维素、玉米淀粉或者其组合物，优选5-25％的大豆蛋白粉和2.5-12.5％膳食纤维。
采用本申请的菌种CGMCC No.17895及本申请的发酵方法，周期短16-24小时，产量高达12000IU/ml(相当于1791.0FU/ml)，最终纳豆激酶产品活性在6.5万～75万FU/g，且在制剂工艺过程更加稳定。
在阅读并理解了具体实施方式后，可以明白其他方面。
具体实施方式
下面将通过实施例对本申请做进一步描述，这些描述并不是对本申请内容做进一步限定。本领域的普通技术人员应当理解，可以对本申请的技术方案进行修改或者等同替换，而不脱离本申请技术方案的精神和范围，均应涵盖在本申请的权利要求范围当中。
在本申请中，纳豆激酶活性通过以下方法测定。
纳豆激酶活性的测定方法(一)
试液
1.PBS(phosphate buffer saline，磷酸缓冲盐溶液)缓冲液：
0.01mol/L磷酸盐缓冲液(pH7.5)：称取磷酸氢二钠(Na 2HPO 4·12H 2O)3.58g，加双蒸水使溶解并稀释至1000mL为I液；取磷酸二氢钠(NaH 2PO 4·2H 2O)0.78g，加双蒸水使溶解并稀释至500mL为II液；取I液约84mL，II液约16mL，将二者混合至pH值为7.5。
将0.01mol/L磷酸盐缓冲液(pH7.5)与0.9％氯化钠溶液(1:17)混合，得到PBS缓冲液。
2.1.5％琼脂糖溶液：取琼脂糖1.5g，加PBS缓冲溶液100mL，加热溶解，50℃水浴保温。
3.纤维蛋白原溶液：取纤维蛋白原适量，加PBS缓冲溶液制成每1mL中含1.5mg的可凝蛋白溶液。
4.凝血酶溶液：取凝血酶，加0.9％氯化钠溶液制成每1mL中含1BP单位的溶液。
5.尿激酶标准品溶液的制备：
5.1尿激酶标准溶液(1000IU/mL)：取尿激酶一瓶，按标示效价加入PBS缓冲溶液溶解，即为1000IU/mL尿激酶标准溶液。
5.2尿激酶工作标准溶液的配制，如下：
表1：
Figure PCTCN2019094334-appb-000001
制备平板
取于50℃水浴5min的纤维蛋白原溶液39mL，置烧杯中，边搅拌边加入50℃琼脂糖溶液39mL、凝血酶溶液3.0mL，立即混匀，快速全部倒入14cm的培养皿中，室温水平放置1小时，用直径为3mm的不锈钢小管(打孔器)，在纤维蛋白平板上打若干个孔。
测定
精密量取不同浓度的尿激酶标准品溶液各10μL，分别点在同一琼脂糖纤维蛋白平板中，加盖，置37℃恒温箱中反应18小时。取出测量溶圈直径。计算溶圈面积，以溶圈面积对数为横坐标，浓度对数为纵坐标作回归曲线，得出相应的回归方程。
根据预先估算的纳豆激酶活性，精密称取纳豆激酶样品于容量瓶中，用适量PBS缓冲溶液溶解，超声15分钟后定容至刻度，使最终点样浓度在200～400IU/mL。精密量取纳豆激酶样品溶液10μL，点在琼脂糖纤维蛋白平板中，加盖，于37℃恒温箱中反应18小时。取出后测量溶圈直径，计算溶圈面积，将样品溶圈面积代入回归方程，计算样品溶液的纳豆激酶活性。
纳豆激酶活性的计算：
X＝C×V/M
其中：X：样品纳豆激酶活性，IU/g；
C：通过回归方程计算的上样样品液中纳豆激酶活性，IU/mL；
V：样品稀释总体积，mL；
M：样品质量，g。
纳豆激酶活性的测定方法(二)
以FU计的纳豆激酶活性定义为：
Figure PCTCN2019094334-appb-000002
依据日本食品研究实验室之纳豆激酶活性分析方法(第104022640号)分析纳豆激酶活性。
酶反应组
(1)取试管加入1.4mL PBS缓冲液及0.4mL 0.72％纤维蛋白原溶液，混合均匀，于37±0.3℃水浴中反应5min。
(2)在上述试管中再加入0.1mL 20U/mL凝血酶溶液，并充分混合均匀，于37±0.3℃水浴中反应10min。
(3)在步骤(2)的溶液准确反应第10分钟时，准确加入0.1mL测试样品溶液，混合均匀，于37±0.3℃水浴中进行酶反应60分钟，并于30min和50min时各摇匀一次。
(4)在步骤(2)的溶液准确反应至第60min时加入2mL 0.2M三氯乙酸溶液终止酶反应，于37±0.3℃水浴中反应20min。
阴性对照管
(1)与酶反应组的步骤(1)和(2)相同，至准确反应第10分钟时，首先加入2mL 0.2M三氯乙酸溶液。
(2)接着加入0.1mL测试样品溶液，并充分混匀，于37±0.3℃水浴中反应20min。
(3)反应终止后，将试管以12000rpm离心10min。
(4)将上清液转移到干净的试管中，为阴性对照管。以阴性对照管为空白，于275nm处测定酶反应组的吸光值(OD，Optical Density)，并记录。
纳豆激酶的活性以下列方式计算:
Figure PCTCN2019094334-appb-000003
其中：X：样品纳豆激酶活性，FU/g或FU/mL；
Ar：酶反应组OD值；
Ac：阴性对照组OD值；
Ar-Ac：数值必须介于0.050～0.080之间；
60：表示反应时间(min)；
0.1：表示样品体积(mL)。
实施例1：从市售纳豆中分离产纳豆激酶的纳豆枯草芽孢杆菌
将纳豆(购自日本生物科技公司)用无菌水溶解、稀释后涂抹在固体LB培养基平板上，于37℃恒温培养24小时，平板表面长出白色菌落，用接种针挑取有拉丝现象。把白色菌落转移到LB斜面培养基上，于37℃培养24小时，用接种环接种于发酵培养基(100ml三角瓶装量20ml，培养基配方：葡萄糖2％，蔗糖2％，黄豆粉3％，硫酸镁0.01％，氯化钠0.5％，丝氨酸0.08％)，37℃，270rpm振摇20小时，离心取上清，用琼脂糖纤维蛋白平板法测定纳豆激酶含量，挑选溶圈直径较大的菌株，最终鉴定出产纳豆激酶的纳豆枯草芽孢杆菌。
实施例2：产纳豆激酶的菌种诱变
以实施例1所得的纳豆枯草芽孢杆菌为出发菌株，进行紫外诱变：紫外波长200-300nm，照射距离15-30cm，照射时间20s，经过50代诱变得变异株ST-1086。该变异株ST-1086具有以下微生物学特性：革兰氏阳性菌，芽孢中间生，芽孢大小0.6～0.8μm×1.0～1.5μm，菌宽1μm，长2-3μm。在LB琼脂培养基上，菌落表面饱满、褶皱、白，凸起，能拉丝，直径0.3-0.5cm，无色素，菌体培养10小时出现芽孢。该变异株ST-1086保藏在中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏日期：2019年6月5日，保藏号为：CGMCC No.17895。
实施例3：种子制备
将出发菌株和实施例2所得的CGMCC No.17895菌株分别接种至装有20ml种子培养基(见表1)，于37-40℃，150-300rpm振摇培养箱培养3-16小时，即得种子培养液。
表2：种子培养基：
成分 含量％
葡萄糖 1.0
胰蛋白胨 1.0
酵母提取物 0.5
NaCl 1.0
纯水 补足至体积100ml
pH 7.0
实施例4：5L罐发酵
将实施例3所得种子培养液按照接种量占发酵培养基量的20％接种至基础培养基(见表2)中，40℃培养，培养30分钟后开始流加补入补料培养基(见表3)，每半小时加入补料培养基200ml，持续到10.5小时，共补入2L补料培养基。培养过程pH值自然，溶解氧浓度控制在30％以上。发酵周期17小时。纤维蛋白平板法测定纳豆激酶含量。
表3：基础培养基
成分 含量％
黄豆粉 0.5
蔗糖 0.9
葡萄糖 0.1
硫酸镁 0.05
氯化钠 0.5
丝氨酸 0.01
氢氧化钠 0.003
纯水 补足至体积100ml
表4：补料培养基
成分 含量％
黄豆粉 10
葡萄糖 8
蔗糖 8
氢氧化钠 0.04
纯水 补足至体积100ml
出发菌株用本方法得纳豆激酶产量为800IU/ml(相当于119.4FU/ml)，将所得发酵液用陶瓷膜进行固液分离，所得透析液用10000D分子量的超滤膜进行浓缩，浓缩液纳豆激酶活性为7000IU/ml(相当于1044.8FU/ml)，纳豆激酶浓缩液加入15％大豆蛋白粉和5％膳食纤维(购自法国罗盖特公司，小麦来源的水溶性膳食纤维)溶解后进行喷雾干燥。得到活性为1500FU/g(相当于10050IU/g)的纳豆粉。
本申请的CGMCC No.17895菌株的纳豆激酶产量为7500IU/ml(相当于1119.4FU/ml)，将所得发酵液用陶瓷膜进行固液分离，所得透析液用10000D分子量的超滤膜进行浓缩，浓缩液纳豆激酶活性为150000IU/ml(相当于22388.1FU/ml)。所得纳豆激酶浓缩液加入12％的大豆蛋白粉和6％膳食纤维进行喷雾干燥。得活性为58000FU/g(相当于390000IU/g)的纳豆粉。
实施例5
与实施例4不同之处在于，补料培养基中含有0.16％丝氨酸。本申请的CGMCC No.17895菌株的纳豆激酶发酵产量12000IU/ml(相当于1791.0F U/ml)。
实施例6
与实施例4不同之处在于，补料培养基中含有0.16％甘氨酸。本申请的CGMCC No.17895菌株的纳豆激酶发酵产量9000IU/ml(相当于1343.3FU/ml)。
实施例7
与实施例4不同之处在于，补料培养基中含有0.16％丙氨酸。本申请的CGMCC No.17895菌株的纳豆激酶发酵产量10000IU/ml(相当于1492.5FU/ml)。
实施例8
与实施例4不同之处在于，补料培养基中含有0.16％丝氨酸和0.16％甘氨酸。本申请的CGMCC No.17895菌株的纳豆激酶发酵产量10500IU/ml(相当于1567.2FU/ml)。
实施例9
与实施例4不同之处在于，补料培养基中含有0.16％丝氨酸和0.16％丙氨酸。本申请的CGMCC No.17895菌株的纳豆激酶发酵产量11000IU/ml(相当于1641.8FU/ml)。
实施例10
与实施例4不同之处在于，补料培养基中含有0.16％甘氨酸和0.16％丙氨酸。本申请的CGMCC No.17895菌株的纳豆激酶发酵产量9500IU/ml(相当于1417.9FU/ml)。
实施例11
与实施例4不同之处在于，补料培养基中含有0.16％丝氨酸、0.16％甘氨酸和0.16％丙氨酸。本申请的CGMCC No.17895菌株的纳豆激酶发酵产量10300IU/ml(相当于1537.3FU/ml)。
实施例12
取实施例4中CGMCC No.17895的发酵液，经孔径为0.1μm的陶瓷膜进行固液分离，除去菌体，培养基等固体颗粒，得到含纳豆激酶的液体；所得的含纳豆激酶的液体用截留分子量为10000D的超滤膜过滤、浓缩得到纳豆激酶浓缩液；所得纳豆激酶浓缩液用30％饱和度的硫酸铵除杂，再用70％饱和度的硫酸铵沉淀纳豆激酶，去除部分色素及多糖，得到纳豆激酶沉淀；将纳豆激酶浓缩液沉淀用sephadex G25填料进行脱盐，用磷酸缓冲液进行洗脱，上样量20％CV(柱体积)，流速40cm/h，收集脱盐的纳豆激酶液；脱盐后的纳豆激酶液，用SP sepharose FF填料(购自GE)进行纯化，上样量80～120mg/ml，流速120cm/h，得到去除色素多糖部分杂蛋白的纳豆激酶液；取去除色素多糖的浓缩液，用分子筛填料superdex75进行柱层析，去除剩余的杂蛋白条带，上样量5％CV，流速20cm/h,收集得到含纳豆激酶单一条带的收集液；将单一条带的纳豆激酶的收集液用冻干机进行冷冻干燥，得到700万FU/g(相当于4690万IU/g)的纳豆激酶纯品粉末。
表5：
  活性浓度IU/ml(g) 回收率％
发酵液 7500 100％
浓缩液 1.5×10 5 95％
30％硫酸铵盐析 1.4×10 5 93％
70％硫酸铵盐析 2.0×10 5 90％
Sephadex G25柱层析 1.4×10 5 81％
SP sepharose FF 3.1×10 5 50％
Superdex 75 5.2×10 5 42％
冻干粉 4.7×10 7 38％
实施例13
本实施例目的是CGMCC No.17895菌株的纳豆激酶按文献纯化方法进行纯化，考察纯化过程中纳豆激酶活性的变化情况。
取实施例4中CGMCC No.17895的发酵液，经孔径为0.1μm的陶瓷膜进行固液分离，除去菌体，培养基等固体颗粒，得到含纳豆激酶的液体；所得纳豆激酶液体用截留分子量为10000D的超滤膜过滤、浓缩得到纳豆激酶浓缩液7000IU/ml，浓缩液经过30％硫酸铵沉淀，再经过60％硫酸铵沉淀，去除部分色素及多糖，得到纳豆激酶沉淀。沉淀用2M硫酸铵溶液溶解成5％的溶液，用Phenyl Sepharose疏水柱层析，上样量20～60mg/ml，流速60cm/h，用2M～0M的硫酸铵溶液线性梯度洗脱，洗脱20倍柱体积，流速100cm/h，分步收集洗脱液，用SDS-PAGE检测洗脱液中纳豆激酶纯度，得到了95％纯度的纳豆激酶纯品，层析流出液直接进行冷冻干燥，得到500万FU/g(3350万IU/g)的纳豆激酶纯品粉末。
表6：
Figure PCTCN2019094334-appb-000004
实施例14
与实施例12不同之处在于，取实施例4中出发菌株的发酵液，得到600万FU/g(相当于4020万IU/g)的纳豆激酶纯品粉末。
表7：
  活性浓度IU/ml(g) 回收率％
发酵液 8.0×10 2 100％
浓缩液 1.1×10 4 95％
30％硫酸铵盐析 1.4×10 4 93％
70％硫酸铵盐析 2.0×10 4 90％
Sephadex G25柱层析 1.4×10 4 83％
SP sepharose FF 2.9×10 4 49％
Superdex 75 4.6×10 4 35％
冻干粉 4.0×10 7 32％
从实施例12、实施例13的结果可以看出，与现有技术方法相比，本申请的纯化方法能显著提高纳豆激酶纯品的活性浓度。
从实施例12、实施例14的结果可以看出，与出发菌株相比，本申请的CGMCC No.17895菌株能够得到纳豆激酶活性浓度更高的纯品。
实施例15
与实施例5不同之处在于，将用本申请的CGMCC No.17895菌株发酵所得的发酵液用10000D超滤膜浓缩后用0.1mmol/L NaCl溶液进行洗涤，直到电导率为300μs/cm。电导率用电导计进行测量。所得纳豆激酶浓缩液活性为30万IU/ml(相当于4.5万FU/ml)。浓缩液加入5％微晶纤维素和5％的膳食纤维进行喷雾干燥。喷雾干燥条件：进风温度200℃，出风温度45℃，风机速度70R/min，进料速度45L/h，干燥收率为45％。所得纳豆粉活性为10.1万FU/g(67.8万IU/g)。
实施例16
与实施例5不同之处在于，将用本申请的CGMCC No.17895菌株发酵所得的发酵液用10000D超滤膜浓缩后用0.1mmol/L NaCl溶液进行洗涤，直到电导率为300μs/cm。电导率用电导计进行测量。所得纳豆激酶浓缩液活性为30万IU/ml(相当于4.5万FU/ml)。浓缩液加入5％大豆蛋白粉和5％的膳食纤维进行喷雾干燥，干燥收率为80％。所得纳豆粉活性为18万FU/g(相当于120.6万IU/g)。大豆蛋白粉作为蛋白保护剂，能提高纳豆激酶的稳定性；微晶纤维素只是赋形剂，提高浓缩液的固含量，有利喷雾干燥工艺。
实施例17
与实施例5不同之处在于，将用本申请的CGMCC No.17895菌株发酵所得的发酵液用10000D超滤膜浓缩后用0.1mmol/L NaCl溶液进行洗涤，直到电导率为200μs/cm。所得纳豆激酶浓缩液活性为30万IU/ml(相当于4.5万FU/ml)。浓缩液加入15％大豆蛋白粉和7.5％的膳食纤维进行喷雾干燥，干燥收率为90％。所得纳豆粉活性为12.9万FU/g(86.1万IU/g)。
实施例18
与实施例5不同之处在于，将用本申请的CGMCC No.17895菌株发酵所得的发酵液用10000D超滤膜浓缩后用0.1mmol/L NaCl溶液进行洗涤，直到电导率为100μs/cm。所得纳豆激酶浓缩液活性为30万IU/ml(相当于4.5万FU/ml)。浓缩液加入15％大豆蛋白粉和7.5％的膳食纤维和5％微晶纤维素进行喷雾干燥，干燥收率为98％。所得纳豆粉活性为12.4万FU/g(相当于83.2万IU/g)。
实施例19
与实施例5不同之处在于，将用本申请的CGMCC No.17895菌株发酵所得的发酵液用10000D超滤膜浓缩后用纯化水进行洗涤，直到电导率为300μs/cm。所得纳豆激酶浓缩液活性为25万IU/ml(3.7万FU/ml)。浓缩液5％大豆蛋白粉和5％的膳食纤维进行喷雾干燥，干燥收率为80％。所得纳豆粉活性为16.4万FU/g(110万IU/g)。
实施例20
与实施例5不同之处在于，将用本申请的CGMCC No.17895菌株发酵所得的发酵液用10000D超滤膜浓缩后用纯化水进行洗涤，直到电导率为200μs/cm。所得纳豆激酶浓缩液活性为24万IU/ml(3.6万FU/ml)。浓缩液加入15％大豆蛋白粉和7.5％的膳食纤维进行喷雾干燥，干燥收率为90％。所得纳豆粉活性为10.8万FU/g(72.4万IU/g)。
实施例21
与实施例5不同之处在于，将用本申请的CGMCC No.17895菌株发酵所得的发酵液用10000D超滤膜浓缩后用纯化水进行洗涤，直到电导率为100μs/cm。所得纳豆激酶浓缩液活性为23万IU/ml(3.4万FU/ml)。浓缩液加入15％大豆蛋白粉和7.5％的膳食纤维和5％微晶纤维素进行喷雾干燥，干燥收率为98％。所得纳豆粉活性为9.7万FU/g(64.7万IU/g)。
实施例22
与实施例5不同之处在于，将用本申请的CGMCC No.17895菌株发酵所得的发酵液用10000D超滤膜浓缩后用纯化水进行洗涤，直到电导率为100μs/cm。所得纳豆激酶浓缩液活性为23万IU/ml(3.4万FU/ml)。浓缩液进行喷雾干燥，干燥收率为50％。所得纳豆粉活性为21万FU/g(142万IU/g)。
实施例23
采用实施例22、实施例15、实施例16、实施例17、实施例18所得的纳豆粉，分别进行压片，考察不同的辅料对制剂工艺过程的稳定性。压片工艺：纳豆粉加入30％鱼胶原蛋白、10％麦芽糊精、10％乳糖、30％微晶纤维素，原、辅料分别过80目筛—混合—制粒—压片，测定纳豆激酶压片前后的活性。结果如下表：
表8：
Figure PCTCN2019094334-appb-000005

Claims (17)

  1. 一种纳豆枯草芽孢杆菌(Bacillus subtils natto)菌株，所述纳豆枯草芽孢杆菌菌株以CGMCC No.17895保藏于中国微生物菌种保藏管理委员会普通微生物中心。
  2. 一种生产纳豆激酶产品的方法，所述方法包括在培养基中培养根据权利要求1所述的纳豆枯草芽孢杆菌菌株以在所述培养基中产生纳豆激酶。
  3. 根据权利要求2所述的方法，其中所述培养基包含碳源物质和氮源物质，且所述碳源物质与所述氮源物质的比为10:1～1:2。
  4. 根据权利要求3所述的方法，其中所述碳源物质选自葡萄糖、蔗糖、麦芽糖、果糖和甘油中的一种或多种。
  5. 根据权利要求3和4中任一项所述的方法，其中所述氮源物质选自酵母粉、蛋白胨、黄豆粉和鹰嘴豆粉中的一种或多种。
  6. 根据权利要求2-5中任一项所述的方法，其中所述培养基还包含促进微生物的生长和提高纳豆激酶的产率的有机物、无机物、或有机物和无机物的混合物。
  7. 根据权利要求6所述的方法，其中所述有机物为丝氨酸、甘氨酸、和丙氨酸中的一种或多种。
  8. 根据权利要求6或7所述的方法，其中所述无机物为镁盐或钠盐，所述无机物优选为硫酸镁、氯化镁、氯化钠。
  9. 根据权利要求2-8中任一项所述的方法，其中培养在35-45℃，优选37-40℃进行。
  10. 根据权利要求2-9中任一项所述的方法，其中培养持续10-48小时，优选12-24小时。
  11. 根据权利要求2-10种任一项所述的方法，所述方法包括在发酵过程中补加碳源物质或氮源物质，或碳源物质和氮源物质的混合物。
  12. 根据权利要求2-11中任一项所述的方法，所述方法还包括以下步骤：
    (1)固液分离，以分离菌体和上清液；
    (2)用超滤膜分离，得到纳豆激酶浓缩液，所述超滤膜的分子量范围优选在1000～50000D之间，更优选在10000～30000D之间；
    (3)用1mmol/L的等渗NaCl溶液洗涤，使所述纳豆激酶浓缩液的电导率≤300μs/cm，优选≤200μs/cm，更优选≤100μs/cm；
    (4)干燥，所述干燥优选为喷雾干燥、冷冻干燥、真空干燥。
  13. 根据权利要求12所述的方法，其中固液分离使用陶瓷膜分离或离心进行。
  14. 根据权利要求12或13所述的方法，其中所述干燥步骤中加入保护剂，所述保护剂优选为大豆蛋白粉、胶原蛋白粉、膳食纤维、微晶纤维素、玉米淀粉或者其组合物，更优选为5-25％的大豆蛋白粉和2.5-12.5％膳食纤维。
  15. 一种纳豆激酶产品，所述纳豆激酶产品通过根据权利要求2-14中任一项所述的方法获得。
  16. 根据权利要求15所述的纳豆激酶产品，其中所述纳豆激酶产品的活性为6.5万-75万FU/g，相当于43.6万IU/g-502.5万IU/g。
  17. 如权利要求15或16所述的纳豆激酶产品在制备用于溶解血栓的药物中的用途。
PCT/CN2019/094334 2019-07-02 2019-07-02 一种纳豆激酶的生产菌株及其生产方法 WO2021000247A1 (zh)

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CN115404227A (zh) * 2022-09-22 2022-11-29 南京工业大学 一种利用火麻籽粕固态发酵生产纳豆激酶的方法
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