CN102943060A - Strain for nattokinase with high activity and thermal stability and fermented product thereof - Google Patents
Strain for nattokinase with high activity and thermal stability and fermented product thereof Download PDFInfo
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- CN102943060A CN102943060A CN2012104793821A CN201210479382A CN102943060A CN 102943060 A CN102943060 A CN 102943060A CN 2012104793821 A CN2012104793821 A CN 2012104793821A CN 201210479382 A CN201210479382 A CN 201210479382A CN 102943060 A CN102943060 A CN 102943060A
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Abstract
A novel natto bacillus subtilis BSNK-T160 is obtained through irradiation mutagenesis. Compared with the wild fungus BSNK-5, the casein hydrolytic activity of the BSNK-T160 is improved by 46.9% and the thermal stability at a temperature of 65 DEG C is improved by eight times; the fibrin activity is improved by 29.62% and the thermal stability is improved by 82.31% compared with a urokinase. The strain BSNK-T160 can be used for producing the nattokinase with the high activity and thermal stability. The nattoes with superior quality can be produced through fermentation of soybeans for 24h by using the stain.
Description
Technical field:
The invention belongs to microorganism field, particularly relate to a kind of new height work, thermostability Nattokinase production bacterial strain and the application aspect fermentative production natto and Nattokinase thereof, the invention still further relates to the fermented product that this bacterial strain produces.
Background technology:
Nattokinase (nattokinase, NK) is the high-efficiency fiber protein resolvase by Bacillus subtilis natto (Bacillus natto) secreting, expressing, finds from traditional Japanese soybean leavened prod natto the earliest.Compare with the thrombolytic drug of clinical application, have the cellulolytic activity height, safe and reliable, have no side effect, long half time, in gi tract good stability, the advantages such as low production cost.
Although Chinese scholars Isolation and screening from different fermented soybean products has gone out some high-yield nattokinase bacterial strains, and carried out the researchs such as vigor that optimization of fermentation conditions improves Nattokinase with the output that improves Nattokinase, purity that heterogenous expression improves Nattokinase, by strain improvement, but the research that improves the thermally-stabilised aspect of Nattokinase rarely had report.
Really can be used for the bacterial strain of industrialization production Nattokinase in the practice seldom.Particularly, because Nattokinase to thermally labile, is to limit the bottleneck that this enzyme is used at protective foods, field of medicaments always.
Therefore, it is very urgent to obtain the Nattokinase bacterial strain demand of high reactivity and thermostability.
Summary of the invention:
The purpose of this invention is to provide new height work, thermostability Nattokinase and produce bacterium; Direct purpose of the present invention is to filter out a kind of new Bacillus subtilis natto, makes it can be used for relatively large production natto and high alive, thermostability Nattokinase.
The inventor passes through 800Gy
60Co-gamma-ray irradiation mutagenesis Bacillus subtilis natto BSNK-5, adopt the casein plate method through repeatedly putting the plate repeated screening of going down to posterity, obtained high work, thermostability mutant strain in conjunction with 65 ℃ of processing---Bacillus subtilis natto (Bacillus natto) BSNK-T160, this new bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 29th, 2012, and deposit number is CGMCC No.6714.
Bacillus subtilis natto BSNK-T160 of the present invention is characterized as:
Morphological specificity:
The bacterium colony of bacterial strain is rounded on the LB flat board, diameter 2-4mm, and oyster white, opaque, the edge is irregular, and surface drying has gauffer, and choosing bacterium colony with transfering loop has the wire drawing phenomenon, bacterium colony and substratum debond, bacterium colony pros and cons color is identical.The microscopically individual morphology is observed, and bacterial strain is shaft-like, long 3-4 μ m, and wide 0.8-1 μ m, the blunt circle in two ends, gramstaining is positive, and produces gemma, and gemma is oval, and middle life or nearly middle giving birth to are seen Fig. 1 and Fig. 2.
Cultural characteristic:
The used substratum of BSNK-T160 growth is the LB substratum, and the used screening culture medium of irradiation mutagenesis is the casein substratum, and culture temperature is 30-37 ℃.
The prescription of described casein substratum is: casein 1%, yeast extract 0.5%, NaCl 0.9%, agar 2%, pH7.2-7.4,121 ℃ of sterilization 20min, matching while using.
The used substratum of solid fermentation of the present invention is soybean, and leavening temperature is 38-40 ℃.
The fermented product that is produced by Bacillus subtilis natto BSNK-T160 fermentation also belong to this patent protection domain.Described fermented product can be leavened prod itself, the leavened prod through diluting or purified leavened prod; Described fermented product can adopt the fluid profiles such as the solid profiles such as particle, powder, tablet or liquid, pasty state, glue.
The invention effect:
Screen through the casein plate method, the fibrin plate method detects fibrinolytic activity and the thermostability of bacterium, the experiment confirms such as fermentation capacity of solid fermentation checking thalline: compare with wild type strain BSNK-5, mutant strain BSNK-T160 has following beneficial effect:
1, Caseinolytic activity and thermostability have been improved
Improved 46.9%, 65 ℃ of thermostability at 37 ℃ Caseinolytic activities and improved about 8 times (Fig. 3)
2, improved fibrinolytic activity, particularly the activity under 65 ℃ of high temperature
The fibrin plate detected result show BSNK-T160 at 37 ℃ dissolving circle area change 20.93%, improved 29.62% with respect to the activity of urokinase; After 65 ℃ of thermal treatments dissolving circle area change 32.13%, improved 82.31%(Fig. 4 with respect to the activity of urokinase).
3, the fermented product quality that is produced by Bacillus subtilis natto BSNK-T160 fermentation is good
The solid state fermentation experiment further confirms just to form one deck tunica albuginea on the soybean surface behind the inoculation BSNK-T160 bacterial strain 24h, is viscosity, and wire drawing is longer, and the jaundice of gained natto color has metalluster, has sauce fragrance (Fig. 5).
The advantage of BSNK-T160 of the present invention is as follows:
1, higher fibrinolytic activity;
2, thermostability preferably;
3, fermenting speed is fast, but 24 hours just fermented soybean produce natto, produce the minimum 48h that needs of natto greater than the market fermenting bacillus natto.
4, fermentation gained natto quality better does not have the ammonia stink, has sauce fragrance.
Therefore, the BSNK-T160 bacterial strain is desirable research, the material bacterial strain that sets out of application.In the production field of Nattokinase and natto, have the very big possibility that realizes its industrialization, and have wide medicine, food applications and market outlook.
Description of drawings:
Fig. 1 is the colonial morphology figure of Bacillus subtilis natto BSNK-T160;
Fig. 2 is the thalli morphology figure of Bacillus subtilis natto BSNK-T160;
Fig. 3 is the Caseinolytic activity figure of BSNK-T160 after the treatment of different temperature;
Fig. 4 be after the treatment of different temperature BSNK-T160 with respect to the activity figure of urokinase;
Fig. 5 is that the BSNK-T160 solid fermentation produces natto figure.
Biomaterial preservation information:
Title: Bacillus subtilis natto (Bacillus sutilis natto) BSNK-T160
Deposit number: CGMCC No.6714
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center
The preservation time: on October 29th, 2012.
Embodiment
Following examples only are used for illustrating method of the present invention, do not limit the scope of the invention.
The embodiment 1 high thermostability mutant strain BSNK-T160 screening of living
1. material
1.1 bacterial classification
BSNK-5 is by the Bacillus subtilis natto of contriver laboratory from China's black soya bean fermented soya bean separation, screening and identification.
1.2 substratum
Seed and liquid fermentation medium: LB substratum;
Screening culture medium: casein plate substratum.
1.3 vigour-testing method
The casein plate method: (9cm * 9cm) is placed on level-off on the water level gauge with flat board, agarose with 10mM pH7.2PBS solution preparation 1%, microwave-oven-heating makes it to melt, the equal-volume casein solution that adds the preparation of pH8.9Tris-HCl damping fluid, making final concentration is 3mg/ml, pour into while hot in the flat board, bubble is rushed to the edge, solidify under the room temperature.With the punch tool punching of diameter 5mm, testing sample is put in the hand-hole, 37 ℃ of insulation 18h, the careful flat board that takes out, coomassie brilliant blue staining, the decolouring of 7% glacial acetic acid, after casein is hydrolyzed, the circle that shows transparency around the point sample hole, all the other positions are blue.
2 methods
2.1 collecting cells
From the dull and stereotyped order bacterium colony of the BSNK-5 that activates to 20ml LB liquid nutrient medium, cultivate about 8-10h, be 0.8-1.0 to OD600 for 30 ℃, take out centrifugal collection thalline (6000rpm, 5min), physiological saline cleans 2 times, uses the equal-volume physiological saline solution, and is for subsequent use.
2.2
60The mutagenesis of Co-gamma-ray irradiation
Get the above-mentioned bacterium liquid of 1ml, add 9ml physiological saline, 800Gy carries out irradiation mutagenesis.Irradiation mutagenesis bacterium liquid is carried out doubling dilution, get 100 μ l and be coated with casein plate, each gradient is coated with 10 casein plates, 30 ℃ of incubated overnight.
2.3 high live strain screening
High live strain primary dcreening operation: choose at random growth bacterial strain point casein plate after the irradiation, take without shining bacterial strain as contrast, 30 ℃ of incubated overnight.Take out next day, chooses positive mutating strain (being that casein plate dissolving loop diameter and colony diameter odds ratio control strain dissolving loop diameter and colony diameter ratio increase 10% bacterial strain), repeats a casein plate repeatedly, dissolves circle and observe.
High live strain sieves again: will confirm finally that blast resistance cultivates 5h through 3ml LB liquid nutrient medium, 30 ℃, 220rpm, get fermented liquid 10 μ l point casein plates, cultivate 18h for 30 ℃, calculate dissolving circle area, choose mutagenic strain dissolving circle Area Ratio contrast bacterium dissolving circle area and increase 10% bacterial strain and carry out thermal stability analysis.
2.4 thermostability bacterial strain screening
Through 37,55,60 and 65 ℃ of processing 15min, the casein plate detection of active will dissolving be enclosed the large bacterial strain of Area Ratio control strain as the gain mutant bacterial strain after the thermal treatment with the bacterium fermented liquid.
3 results
Utilize 800Gy
60Co-γ irradiation BSNK-5 bacterial strain, in conjunction with thermal treatment, casein plate method sieve bacterium obtains activity and thermally-stabilised raising bacterial strain BSNK-T160(bacterium colony and thalli morphology and sees Fig. 1 and 2).Wild type strain BSNK-5 is 123.44mm2 at 37 ℃ casein dissolving circle area, and mutant strain BSNK-T160 is 181.34mm2, and activity has improved 46.9%; Behind 65 ℃ of processing 15min, the dissolving circle area of mutant strain BSNK-T160 casein plate is 37.09mm2 still, and the dissolving circle area of BSNK-5 is 4.12mm2, and thermostability has improved 8 times (Fig. 3).
Embodiment 2BSNK-T160 fibrinolytic activity is measured
1, vigour-testing method
The preparation of agarose-fibrin plate
The pH7.2PBS solution that contains 1% agarose, 50 ℃ of water bath with thermostatic control 45min, the equal-volume that adds water-bath 10min under zymoplasm (final active 0.05IU/ml) and the same temperature contains 0.1% fibrinogenic pH7.2PBS solution, mixing.The culture dish of 9cm * 9cm adds this solution of 35ml, solidifies the rear diameter 5mm punch tool punching of using, loading 10ul, and 37 ℃ of constant temperature culture 18h measure the diameter that dissolves circle, and the area of calculating dissolving circle is according to urokinase typical curve calculating enzyme activity unit.
Urokinase standard curve making method
Each l0ul point sample of urokinase sample (100,200,300,400,500U/m1) is placed 10min on the fibrin plate of new preparation, moves into 37 ℃ of incubators, takes out behind the insulation 18h, measures the perpendicular diameter of dissolving circle, calculates each dissolving and encloses area.Take dissolving circle area as X-coordinate, the mapping take enzyme activity as ordinate zou is according to typical curve calculation sample vigor.
The fermented liquid preparation method
With BSNK-5 and BSNK-T160 after the flat board activation, access 3ml LB liquid nutrient medium, 30 ℃ of incubated overnight.Take out next day, the inoculum size access 20ml LB liquid nutrient medium with 2%, 30 ℃, 220rpm are cultivated 5h, centrifuging and taking fermented liquid 10 μ l point fibrin plates, cultivate 18h for 37 ℃, use the vernier caliper measurement perpendicular diameter, calculating dissolving circle area reaches the unit of activity with respect to urokinase.
Heat treating method is with 2.4 of embodiment 1.
2, result
The fibrin plate detected result shows that BSNK-5 is 194.09mm at 37 ℃ dissolving circle area
2, be 289.53IU/ml with respect to the activity of urokinase; BSNK-T160 dissolving circle area is 234.72mm
2, be 375.28IU/ml with respect to the activity of urokinase; Compare with BSNK-5, BSNK-T160 fibrinolysis circle area change 20.93%, improved 29.62%(Fig. 4 with respect to the activity of urokinase).
After 65 ℃ of thermal treatments, the dissolving circle area of BSNK-5 is 93.42mm2, is 77IU/ml with respect to the activity of urokinase; BSNK-T160 dissolving circle area is 123.44mm2, is 140.38IU/ml with respect to the activity of urokinase; Compare with BSNK-5, BSNK-T160 dissolving circle area change 32.13%, improved 82.31%(Fig. 4 with respect to the activity of urokinase).Embodiment 3BSNK-T160 fermentative production natto
1, technical process
A boiling one inoculation one fermentation one after-ripening one natto is cleaned-soaked to soy material one; Get the BSNK-T160 bacterial strain, according to above process trial natto.
2, method
2.1 collecting cells
From LB board joint BSNK-T160 bacterial strain to the 3ml liquid nutrient medium, cultivate 8-9h for 37 ℃, 2% the bacterium amount that connects access 20mlLB liquid nutrient medium is cultivated 4-6h for 37 ℃, centrifugal collection thalline, physiological saline or sterile water wash three times, resuspended thalline is used for solid fermentation.
2.2 Soybean Pretreatment
Get the 50g washing soybean, remove residue and inferior beans, add immersions of spending the night of 100ml ultrapure water, removal excessive moisture, 121 ℃ of autoclaving 20min.
2.3 fermentation
The soybean of bacterium of will having gone out cools puts 38-42 ℃, and the bacteria suspension that access prepares is cultivated 24h for 38 ℃, and there is one deck tunica albuginea on the soybean surface; Stop fermentation, put into 4 ℃ of refrigerator after-ripening 12h.
2.4 natto organoleptic analysis
The BSNK-T160 bacterial strain makes natto through above technique fermented soybean 24h, the finished product golden yellow color, and taste lubrication has sauce fragrance, and very long wire drawing sample mucosubstance is arranged when provoking with chopsticks, with commercially available natto local flavor quite similar (Fig. 5).
Claims (6)
1. Bacillus subtilis natto (Bacillus natto) BSNK-T160, CGMCC No.6714.
2. Bacillus subtilis natto claimed in claim 1 is in the purposes of producing aspect the fermented product.
3. purposes claimed in claim 2, described fermented product is natto or Nattokinase.
4. the fermentation culture goods that obtain with Bacillus subtilis natto claimed in claim 1.
5. goods claimed in claim 4 are natto or Nattokinase.
6. a method of producing natto take soybean as raw material, obtains through immersion, boiling, inoculation fermentation bacterium, fermentation, and it is characterized by: used zymophyte is Bacillus subtilis natto (Bacillus natto) BSNK-T160, CGMCC No.6714.
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Cited By (8)
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| CN103255156A (en) * | 2013-05-30 | 2013-08-21 | 中国农业科学院农产品加工研究所 | Site mutant nattokinase gene high in activity and thermal stability, and application thereof |
| CN105087447A (en) * | 2015-09-16 | 2015-11-25 | 河南省科学院生物研究所有限责任公司 | Microwave-resisting bacillus subtilis and application thereof in preparing nattokinase |
| CN105349451A (en) * | 2015-10-22 | 2016-02-24 | 张淑莲 | Bacillus subtilis natto and breeding method and application thereof |
| CN107048335A (en) * | 2017-02-15 | 2017-08-18 | 蚌埠学院 | A kind of preparation method of wheat embryo sauce |
| CN107937372A (en) * | 2017-12-19 | 2018-04-20 | 江南大学 | The Nattokinase that a kind of acid resistance improves |
| CN107937374A (en) * | 2017-12-19 | 2018-04-20 | 江南大学 | The Nattokinase that a kind of heat endurance improves |
| CN108192834A (en) * | 2017-12-11 | 2018-06-22 | 青岛寿纳豆食品有限公司 | One plant of bacterial strain for being used to prepare odorless natto |
| CN116616414A (en) * | 2023-06-05 | 2023-08-22 | 吉林省雁鸣湖大豆生物科技有限责任公司 | Natto and preparation method of natto active peptide and product |
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Cited By (13)
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| CN103255156B (en) * | 2013-05-30 | 2014-12-17 | 中国农业科学院农产品加工研究所 | Site mutant nattokinase gene high in activity and thermal stability, and application thereof |
| CN103255156A (en) * | 2013-05-30 | 2013-08-21 | 中国农业科学院农产品加工研究所 | Site mutant nattokinase gene high in activity and thermal stability, and application thereof |
| CN105087447A (en) * | 2015-09-16 | 2015-11-25 | 河南省科学院生物研究所有限责任公司 | Microwave-resisting bacillus subtilis and application thereof in preparing nattokinase |
| CN105087447B (en) * | 2015-09-16 | 2018-08-21 | 河南省科学院生物研究所有限责任公司 | One plant of resistance to microwave bacillus subtilis and its application in Nattokinase preparation |
| CN105349451A (en) * | 2015-10-22 | 2016-02-24 | 张淑莲 | Bacillus subtilis natto and breeding method and application thereof |
| CN107048335A (en) * | 2017-02-15 | 2017-08-18 | 蚌埠学院 | A kind of preparation method of wheat embryo sauce |
| CN108192834A (en) * | 2017-12-11 | 2018-06-22 | 青岛寿纳豆食品有限公司 | One plant of bacterial strain for being used to prepare odorless natto |
| CN107937374A (en) * | 2017-12-19 | 2018-04-20 | 江南大学 | The Nattokinase that a kind of heat endurance improves |
| CN107937372A (en) * | 2017-12-19 | 2018-04-20 | 江南大学 | The Nattokinase that a kind of acid resistance improves |
| CN107937374B (en) * | 2017-12-19 | 2020-03-06 | 江南大学 | Nattokinase with improved thermal stability |
| CN107937372B (en) * | 2017-12-19 | 2020-03-24 | 江南大学 | Nattokinase with improved acid resistance |
| CN116616414A (en) * | 2023-06-05 | 2023-08-22 | 吉林省雁鸣湖大豆生物科技有限责任公司 | Natto and preparation method of natto active peptide and product |
| CN116616414B (en) * | 2023-06-05 | 2024-02-09 | 吉林省雁鸣湖大豆生物科技有限责任公司 | Natto and preparation method of natto active peptide and product |
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