CN1273274A - Process for preparing natto kinase and its application - Google Patents
Process for preparing natto kinase and its application Download PDFInfo
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Abstract
A natto kinase is prepared from (Bacillus natto) X-501 (preserved in CGMCC no.0449) through culturing in soybean culture medium, and implementing the process of solid or liquid fermentation, extraction and purification according to a defined specification. After processed, said natto kinase can be used to make capsules, health-care food or its additive, and medical injection.
Description
The employing bacillus natto that the present invention proposes produces production method of Nattokinase and uses thereof and belongs to microbial technology field, specially refers to the production method of superior strain fermentation and enzyme.
Natto is a kind of nutritious high-quality leavened food, and it is to utilize the soybean of Bacillus natto inoculation boiling to obtain at the proper temperature bottom fermentation, and as traditional food, and natto is used to prevention and treats diseases such as cardiovascular in that Japan is among the people in Japan.Japan must find to exist and the similar enzyme of thrombus dissolving enzyme in the natto by Jian Shi in 1987, and called after Nattokinase (Nattokinase), he has confirmed Nattokinase through edible also very stable to the long like this time of blood later on, and from then on Nattokinase is by extensive concern.
Describe among the Japanese Patent JP6261744A (1994), adopt China production soybean to carry out solid fermentation and produce Nattokinase, its fermentation condition is at first 50 ℃, 18 hours, 5 ℃ then, 24 hours.
Record adopts liquid fermentation process to produce Nattokinase among the Japanese Patent JP6153977A (1994), and its fermention medium consists of: 2% fructose, 1.5% soy peptone, 0.2% yeast extract, 0.1%KH
2PO
4, 0.3%K
2HPO
4, 0.05%MgSO
45H
2O, 0.02%CaCl
22H
2O, leavening temperature 30 ℃, 40 hours.Described natto kinase activity unit is 0.42-1.48u/ml.Butyl-agarose gel 4B (Butyl Sepharose 4B), sephadex G-25 (Sephadex G-25) and propylene dextrane gel S-200HR (Sephacryl S-200HR) are adopted in large-scale separation and purification.
Wild natto bacterial strain and to obtain the general production method of Nattokinase thus not ideal enough mainly is that productive rate is high and be unsuitable for industrial production.
Purpose of the present invention overcomes the deficiencies in the prior art exactly, proposes a kind of high-yield method that adopts bacillus natto X-501 superior strain to carry out solid and liquid fermenting generation Nattokinase.
Purpose of the present invention reaches by the following technical programs: 1. bacillus natto, it is characterized in that this bacterial strain is defined as bacillus natto (Bacillusnatto) X-501, and this bacterial strain is preserved in the CGMCC of Pekinese, and preserving number is 0449.2. cultivate the production method that produces Nattokinase by the described bacillus natto X-501 of claim 1 for one kind, it is characterized in that: 1) bacillus natto X-501 is to carry out solid or liquid fermenting on the main medium soybean or soya products; 2) solid fermentation or liquid fermenting
Solid fermentation:
Adopt water jaundice beans broken or not broken, directly inoculate after the sterilization and ferment, soybean cake powder, bean dregs, defatted soyflour also can be used as fermentation raw material; Temperature is controlled to be 22-40 ℃ during fermentation, and optimum temps is 36-38 ℃, and relative humidity is controlled to be 80-95%, and fermentation period is 18-60 hour;
Liquid fermenting:
Adopt soya-bean milk to do fermented substrate, can dilute or not dilute and ferment; Utilize soyflour, soybean cake powder, bean dregs usage quantity to be 1-8%, optimum amount is 2-5%; Add extractum carnis or corn steep liquor and can promote the secretion of bacillus natto X-501 to Nattokinase, the extractum carnis usage quantity is 0.2-2%, and best for 1-1.5% corn steep liquor usage quantity is 0.5-1.5%, the best is 0.5-1.0%; Temperature is controlled to be 22-40 ℃ during fermentation, and optimum temps is 36-38 ℃, and fermentation period 18-48 hour, the pH value was controlled to be 6.8-7.0;
Shake-flask culture condition during test:
The 500ml triangle shakes bottle, substratum loading amount 60-100ml, and when eccentricity is the rotary shaker of 2.54cm, revolution 250-350rpm, when adopting eccentricity to be the rotary shaker of 5.0cm, revolution is 180-240rpm;
Fermentor cultivation condition during production:
Ventilation is controlled to be 1: 0.4-1: 1v/v/min, stir 200-300rpm, warm 36-38 ℃ in jar, tank pressure 0.03-0.05MPa;
3) extract
For solid fermentation, earlier the sodium chloride solution of the 0.1-0.15M that doubly measures with 5-10 stirs extracting earlier thick pre-flock after 1-2 hour down at 4-10 ℃, removes the macrobead fermented substrate, press filtration or centrifugal removal thalline; Add 0.02-0.05%CaCl in filtrate or the supernatant liquor
2, and carry out ultrafiltration and concentration 20-50 at low temperatures more doubly, add cold alcohol to ethanol content and reach 75-80%, or add solid ammonium sulfate and make saturation ratio reach 80% gradually, so that Nattokinase precipitates;
For liquid fermenting, then directly carry out press filtration or the centrifugal thalline of removing; Add 0.02-0.05%CaCl in filtrate or the supernatant liquor
2, and carry out ultrafiltration and concentration 20-50 at low temperatures more doubly, add cold alcohol to ethanol content and reach 75-80%, or add solid ammonium sulfate and make saturation ratio reach 80% gradually, so that Nattokinase precipitates;
4) purifying
The throw out that alcohol precipitation through 75% or solid ammonium sulfate are precipitated out adds solid ammonium sulfate and reaches 80% saturation ratio gradually, high speed centrifugation, collecting precipitation thing with the phosphoric acid buffer dissolving of 0.05M PH7.6.Throw out is with the dissolving of 0.05M PH6.5 phosphoric acid buffer and dialyse chromatography on the CM32 ion exchange column, collection active ingredient.This component is carried out ultrafiltration and concentration, obtains the enzyme liquid of high density, carries out the SephacrylS-100HR gel filtration chromatography again, collects active ingredient, and this component obtains the purified product of Nattokinase through the ultrafiltration and concentration dialysis.
3. the purposes of a Nattokinase of cultivating as method as described in the claim 2, it is characterized in that, after this Nattokinase throw out dissolving desalination, can be directly and auxiliary material bean powder, milk powder, soymilk powder, dextrin mixing preparation to the enzyme of the necessary requirement unit that lives, after lyophilize, make Nattokinase capsule or other forms of protective foods; Also can directly carry out lyophilize after the Nattokinase throw out dissolving desalination, make food supplement with a small amount of glucose or lactose allotment to desired enzyme unit alive again.
4. the purposes of a Nattokinase of cultivating as method as described in the claim 2 is characterized in that the purified product of above-mentioned Nattokinase is made the medical injection injection through lyophilize.
More detailed description below technical scheme of the present invention:
Bacillus natto (Bacillus natto) X-501 that the present invention proposes belongs to the natto bacterial strain, and this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 11st, 2000, abbreviates CGMCC as, and preserving number is 0449.
This fungus characteristic is as follows:
1. morphological specificity
In 30 ℃ cultivate 2-4 days after, the X-501 thalline is a rod-short, chaining not, straight or straight shape: 0.6～0.8 * 1.1～1.5 μ m, can move, the flagellum adnation has gemma, life is not expanded in the gemma, in a sporocyst cell, no more than one of spore; Gram-positive; The acid-fast stain feminine gender.
2. cultural characteristic
Strain X-501 on four kinds of substratum 30 ℃ the results are shown in Table 1 after cultivating 3～5 days.
The cultural characteristic of table 1 strain X-501
| Substratum | Colonial morphology |
| Analysis for soybean powder agar Sang Tasi agar beef broth agar yeast extract paste malt extract agar | The little yellow of bacterium colony, the thick film shape, edge smooth colony almond white, the edge is smooth, surface irregularity, and the strong bacterium colony creamy edge of ridge is smooth, surface irregularity, the strong bacterium colony canescence edge of ridge is smooth, surface irregularity, and ridge is strong |
3. chemical classification:
The result who carries out the analysis of full cell hydrolyzed solution by Lechevalier thin plate chromatography shows: strain X-501 contains meso-DAP (diaminopimelic acid, Diaminopinelic acid), glycine; Atypism sugar (sugared type C).
4. the physiological and biochemical property of strain X-501 sees Table 2:
The physiological and biochemical property of table 2 strain X-501
5 Detection of Stability:
| Feature result | Utilize sugar to produce sour result |
| Gelatin liquefaction+Starch Hydrolysis-indole reaction-nitrate reduction-catalase+cellulose growth-VP test-lecithin+tyrosine decomposition-casein hydrolysis+ | Melibiose-galactolipin+ribose+maltose+winged euonymus sugar-inositol-sucrose+wood sugar-arabinose-glucose+ |
5.1X-501 bacterial strain covers after shining 72 hours under the UV-light across plate, thalli growth is good, and it is transferred second, third generation, and its vigor is good, and the cell wall chemical composition is identical with reference strain.
5.2 the growth temperature test: strain X-501 sees Table 3 at the growing state of differing temps.
Table 3 strain X-501 is at the growing state of differing temps
5.3 ethanol tolerance test: the results are shown in Table 4.
| Growth temperature (℃) growing state | Growth temperature (℃) growing state |
| ????-20????????????- ????-10????????????- ????-5?????????????+ ????10?????????????+ ????15?????????????+ ????20?????????????+ ????30?????????????+ | ????35???????????+ ????40???????????+ ????45???????????+ ????50???????????- ????60???????????- ????65???????????- |
The ethanol tolerance of table 4 X-501 bacterial strain
5.4 the acid-basicity test: the growing state of X-501 bacterial strain under different pH sees Table 5.
| Soak time (h) | ????10 | ????24 | ????36 | ????48 | ????64 | ????72 | ????84 |
| Growing state | ????+ | ????+ | ????+ | ????+ | ????+ | ????- | ????- |
The growing state of table 5 X-501 bacterial strain under the pH difference.
5.5 the salt tolerant experiment: the growing state of X-501 bacterial strain under different N aCl concentration sees Table 6.
| ??pH | ??3 | ??4 | ??5 | ??6 | ??7 | ??8 | ??9 | ??10 | ??11 | ??12 |
| Growing state | ?- | ??- | ??+ | ??+ | ??+ | ??+ | ??+ | ??- | ??- | ??- |
The growing state of table 6 X-501 bacterial strain under different salt concn
| Salt concn (%) | ????2 | ????4 | ????6 | ????8 | ????10 |
| Growing state | ????+ | ????+ | ????+ | ????- | ????- |
The production method of bacillus natto X-501 of the present invention is as follows:
Strain X-501 is carried out solid or liquid fermenting soybean or soya products (comprising soyflour, soybean cake powder, defatted soyflour, bean dregs, soya-bean milk etc.) on the substratum of main matrix.
Fermentation
Through broken or not broken, after sterilization, direct inoculation ferments for solid fermentation used water jaundice beans.Soybean cake powder, soyflour, bean dregs, defatted soyflour also can be used as fermentation raw material, and temperature is controlled to be 22-40 ℃ during fermentation, and optimum temperuture 36-38 ℃ of relative humidity is controlled to be 80-95%, and fermentation period is 18-60 hour.
Can ferment through diluting or not diluting as fermented substrate for the also available soya-bean milk of liquid fermenting.Utilize usage quantitys such as soyflour, soybean cake powder, bean dregs to be 1-8%, optimum amount is 2-5%.Interpolation extractum carnis or corn steep liquor can promote the secretion of Bacillus natto to Nattokinase, corn steep liquor usage quantity 0.5-1.5%, optimum amount 0.5-1.0% during liquid fermenting; 1.0%; Extractum carnis 0.5-2%, optimum dose 1-1.5%.Controllable Temperature is built in 22-40 ℃ during liquid fermenting, and optimum temperuture 36-38 ℃, fermentation period 18-48 hour.PH value can be controlled in 6.8-7.0.
Shake-flask culture condition during test:
The 500ml triangle shakes bottle, substratum loading amount 60-100ml, and when adopting eccentricity to be the rotary shaker of 2.54cm, revolution 250-350rpm, when adopting eccentricity to be the rotary shaker of 5.0cm, revolution is 180-240rpm;
Fermentor cultivation condition during production:
Ventilation is controlled to be 1: 0.4-1: 1v/v/min, stir 200-300rpm, 38 ℃ of jar warm 36-, tank pressure 0.03-0.05MPa; The extraction of Nattokinase:
The 5-10 of the sodium-chlor liquid 0.1-0.15M that can be earlier doubly measures with to(for) solid fermentation stirs extracting earlier thick pre-flock after 1-2 hour down at 4-10 ℃, removes the macrobead fermented substrate, press filtration or centrifugal removal thalline.Then directly carry out press filtration or the centrifugal thalline of removing for liquid fermenting.Add 0.02-0.05%CaCl in filtrate or the supernatant liquor
2, and carry out ultrafiltration and concentration 20-50 at low temperatures more doubly, add cold alcohol to ethanol content and reach 75-80%, or add solid ammonium sulfate and make saturation ratio reach 80% gradually, so that Nattokinase precipitates.
For liquid fermenting, then directly carry out press filtration or the centrifugal thalline of removing, add 0.02-0.05%CaCl in filtrate or the supernatant liquor
2, and carry out ultrafiltration and concentration 20-50 at low temperatures more doubly, add cold alcohol to ethanol content and reach 75-80%, or add solid ammonium sulfate and make saturation ratio reach 80% gradually, so that Nattokinase precipitates.The purifying of Nattokinase:
The throw out that is precipitated out through 75% alcohol precipitation or solid ammonium sulfate adds solid ammonium sulfate and reaches 80% saturation ratio gradually with the dissolving of 0.05M PH7.6 phosphoric acid buffer.High speed centrifugation, the collecting precipitation thing.Throw out is with the dissolving of 0.05M PH6.5 phosphoric acid buffer and dialyse chromatography on the CM32 ion exchange column, collection active ingredient.This component is carried out ultrafiltration and concentration, obtains the enzyme liquid of high density, carries out Sephacryl S-100HR gel filtration chromatography again, collects active ingredient, and this component obtains the purified product of Nattokinase through the ultrafiltration and concentration dialysis.The purposes of Nattokinase:
Can be directly after the Nattokinase throw out dissolving desalination and the extremely desired enzyme work of other auxiliary materials (bean powder, milk powder, soymilk powder and dextrin etc.) mixing preparation unit, after lyophilize, make the protective foods of Nattokinase capsule or other form, also can will directly carry out lyophilize after the desalination of Nattokinase throw out, make protective foods with a small amount of glucose or lactose allotment to desired enzyme unit alive again.
The purified product of above-mentioned Nattokinase utilizes this finished product to can be made into the medical injection injection through freezing dry highly active Nattokinase finished product.
Find that after deliberation Nattokinase is actually the effect that there is no Profibrinolysin in the activation blood plasma, Nattokinase is fibrin degradation and Fibrinogen directly.Oral Nattokinase not only can enter blood circulation through intestinal absorption and directly act on thrombus, also can induce liver or blood vessel endothelium to produce TPA (histiotype plasminogen incitant) and make the activation of organism self plasmin system, thus the gentle fibrinolytic that improves blood constantly.
Activity unit's definition---the cellulolytic activity of Nattokinase can adopt the bead descent method to measure.In (see appendix I) under this mensuration system our regulation: the enzyme amount when bead drops to grumeleuse bottom and needs 300 seconds (5 minutes) from the grumeleuse top is 1 fibrinolytic unit.In this mensuration system, if the enzyme amount that adds when 1-2 molten fine unit, the time that bead descends is a straight line with the activity unit of enzyme and concerns.Therefore can come enzyme activity in the working sample by a known unit standard enzyme.
Adopt bacillus natto X-501 can obtain the enzyme high tens times than wild strain in the fermentative production that with soybean/soyflour is main raw material and live, the highest enzyme work of liquid fermenting reaches 9.1u/ml.In solid fermentation, can reach the 22.9u/g soybean.The raw materials for production that adopt among the present invention are simple, low price, and processing parameter is reasonable.Therefore, can in the shortest production time, obtain higher yield of enzyme.Embodiment 1:
The 500ml triangular flask adds the 3.2g soyflour, the 1.2g extractum carnis, and the 80ml tap water is transferred 6.9,121 ℃ of sterilizations of pH 15min, inserts 10
5Bacillus natto X-501 spore liquid 0.8ml..In eccentricity 2.54cm rotary shaker, speed of rotation 300rpm, 38 ℃ of constant temperature culture recorded enzyme 7.6u/ml alive in 28 hours.Embodiment 2:
The 500ml triangular flask adds 4.0g raw soybean cake powder, the 1.5g extractum carnis, and the 100ml tap water is transferred pH6.9, and 121 ℃ of sterilization 15min insert 10
5Bacillus natto X-501 spore liquid 1.0ml, in eccentricity 2.54cm rotary shaker, speed of rotation 300rpm, 38 ℃ of constant temperature culture recorded enzyme 2.5u/ml alive in 30 hours.Embodiment 3:
The soybean that 100g spends the night through water logging bubble, (note is done: A), other gets the same soybean of handling of 100g, puts into another φ 16cm plate (note is done: B), 121 ℃ of sterilization 20min respectively insert 10 after the cooling through pulverizing in the φ 16cm plate of packing into
5Bacillus natto X-501 spore suspension 2.0ml.Stir rearmounted 37 ℃ of thermostatic chambers, controlling moisture 90% was cultivated 24 hours.Respectively get 1.0g from A, B two plates, be dissolved in respectively in the 5ml physiological saline, lixiviate is spent the night, and its molten fine vigor is respectively every gram culture A:22.9u and B:12.1u.Embodiment 4:
Liquid fermentation process
A bacillus natto X-501 activated inclined plane bacterial classification is inserted in the 300L fermentor tank (the substratum loading amount is 250L) after with 10ml sterilized water wash-out.The fermention medium component is: soyflour 4%, extractum carnis 1.5%, tap water is supplied volume to 250L, sterilized 30 minutes for 121 ℃, sterilization back PH is 6.7-7.0, and leavening temperature is 38 ℃, stirring velocity 250rpm, air flow is 1: 0.7v/v/m, fermentation time are 30 hours, and fermentation unit finally is 8.5u/ml.Embodiment 5:
Extraction process, the Nattokinase capsule is produced
250L fermented liquid → centrifugal remove thalline → on reset and add CaCl
20.02%～0.05% → centrifugal supernatant liquor → supernatant ultrafiltration is 75%, → 4 ℃ to 10L (concentrating 25 times) → add ethanol to ethanol content and deposits 8 hours → centrifugal must the precipitation → throw out and carry out suction filtration to do → adding a small amount of physiological saline solution interpolation auxiliary material (soymilk powder) → lyophilize powdering → encapsulated.Enzyme total recovery 43% alive.Embodiment 6:
Purifying process, the producing of Nattokinase medical injection injection
Will by a collection of 250L fermented liquid (fermentation unit 9.1u/ml) that obtains on the 300L fermentor tank by embodiment 5 extraction processes extract after the ethanol sedimentation thing dissolving, again through ammonium sulfate precipitation, the collecting precipitation thing carries out dialysis desalting again.Enzyme liquid after the dialysis is earlier through carboxymethyl cellulose CM32 cation exchange column chromatography, and the collecting high-activity component is concentrated into small volume through ultrafiltrated.Through the SephacrylS-100HR gel filtration chromatography, the collecting high-activity component is carried out lyophilize behind ultrafiltration and concentration, make the medical injection injection again.
Claims (4)
1. a bacillus natto is characterized in that this bacterial strain is defined as bacillus natto (Bacillus natto) X-501, and this bacterial strain is preserved in the CGMCC of Pekinese, and protecting number is 0449.
2. cultivate the production method that produces Nattokinase by the described bacillus natto X-501 of claim 1 for one kind, it is characterized in that:
1) bacillus natto X-501 is to carry out solid or liquid fermenting on the main medium soybean or soya products;
2) solid fermentation or liquid fermenting
Solid fermentation:
Adopt water jaundice beans broken or not broken, sterilization back direct inoculation ferments, and soybean cake powder, bean dregs, defatted soyflour also can be used as fermentation raw material; Temperature is controlled to be 22-40 ℃ during fermentation, and optimum temps is 36-38 ℃, and relative humidity is controlled to be 80-95%, and fermentation period is 18-60 hour;
Liquid fermenting:
Adopt soya-bean milk to do fermented substrate, can dilute or not dilute and ferment; Utilize soyflour, soybean cake powder, bean dregs usage quantity to be 1-8%, optimum amount is 2-5%; Add extractum carnis or corn steep liquor and can promote the secretion of bacillus natto X-501 Nattokinase, the extractum carnis usage quantity is 0.2-2%, the best is 1-1.5%, the corn steep liquor usage quantity is 0.5-1.5%, temperature was controlled to be 22-40 ℃ when the best was fermented for 0.5-1.0%, optimum temps is 36-38 ℃, and fermentation period 18-48 hour, the pH value was controlled to be 6.8-7.0;
Shake-flask culture condition during test:
The 500ml triangle shakes bottle, substratum loading amount 60-100ml, and when eccentricity is the rotary shaker of 2.54cm, revolution 250-350rpm, when adopting eccentricity to be the rotary shaker of 5.0cm, revolution is 180-240rpm;
Fermentor cultivation condition during production:
Ventilation is controlled to be 1: 0.4-1: 1v/v/min, stir 200-300rpm, warm 36-38 ℃ in jar, tank pressure 0.03-0.05MPa;
3) extract
For solid fermentation, earlier the sodium chloride solution of the 0.1-0.15M that doubly measures with 5-10 stirs extracting earlier thick pre-flock after 1-2 hour down at 4-10 ℃, removes the macrobead fermented substrate, press filtration or centrifugal removal thalline; Add 0.02-0.05%CaCl in filtrate or the supernatant liquor
2, and carry out ultrafiltration and concentration 20-50 at low temperatures more doubly, add cold alcohol to ethanol content and reach 75-80%, or add solid ammonium sulfate and make saturation ratio reach 80% gradually, so that Nattokinase precipitates;
For liquid fermenting, then directly carry out press filtration or the centrifugal thalline of removing; Add 0.02-0.05%CaCl in filtrate or the supernatant liquor
2, and carry out ultrafiltration and concentration 20-50 at low temperatures more doubly, add cold alcohol to ethanol content and reach 75-80%, or add solid ammonium sulfate and make saturation ratio reach 80% gradually, so that Nattokinase precipitates;
4) purifying
The throw out that alcohol precipitation through 75% or solid ammonium sulfate are precipitated out adds solid ammonium sulfate and reaches 80% saturation ratio gradually, high speed centrifugation, collecting precipitation thing with the phosphoric acid buffer dissolving of 0.05M PH7.6.Throw out is with the dissolving of 0.05M PH6.5 phosphoric acid buffer and dialyse chromatography on the CM32 ion exchange column, collection active ingredient.This component is carried out ultrafiltration and concentration, obtains the enzyme liquid of high density, carries out Sephacryl S-100HR gel filtration chromatography again, collects active ingredient, and this component obtains the purified product of Nattokinase through the ultrafiltration and concentration dialysis.
3. the purposes of a Nattokinase of cultivating as method as described in the claim 2, it is characterized in that, after this Nattokinase throw out dissolving desalination, can be directly and auxiliary material bean powder, milk powder, soymilk powder, dextrin mixing preparation to the enzyme of the necessary requirement unit that lives, after lyophilize, make Nattokinase capsule or other forms of protective foods; Also can directly carry out lyophilize after the Nattokinase throw out dissolving desalination, make food supplement with a small amount of glucose or lactose allotment to desired enzyme unit alive again.
4. the purposes of a Nattokinase of cultivating as method as described in the claim 2 is characterized in that the purified product of above-mentioned Nattokinase is made the medical injection injection through lyophilize.
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| JPH06251744A (en) * | 1993-02-22 | 1994-09-09 | Hitachi Ltd | Evaporation type getter pump |
| JPH08208512A (en) * | 1995-02-03 | 1996-08-13 | Nippon Chem Res Kk | Medicine for inhibiting thrombogenesis |
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2000
- 2000-05-22 CN CN00107589A patent/CN1100142C/en not_active Expired - Lifetime
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