CN102943060B

CN102943060B - Strain for nattokinase with high activity and thermal stability and fermented product thereof

Info

Publication number: CN102943060B
Application number: CN201210479382.1A
Authority: CN
Inventors: 唐选明; 李淑英; 聂莹
Original assignee: Institute of Food Science and Technology of CAAS
Current assignee: Institute of Food Science and Technology of CAAS
Priority date: 2012-11-22
Filing date: 2012-11-22
Publication date: 2014-08-20
Anticipated expiration: 2032-11-22
Also published as: CN102943060A

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

High thermostability Nattokinase bacterial strain and the fermented product thereof of living

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 the earliest from traditional Japanese soybean leavened prod natto.Compared with the thrombolytic drug of clinical application, have cellulolytic activity high, 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 optimization of fermentation conditions to improve the output of Nattokinase, the research such as purity, the vigor that improves Nattokinase by strain improvement that heterogenous expression improves Nattokinase, but the research that improves the thermally-stabilised aspect of Nattokinase is rarely had to report.

The bacterial strain that really can be used for industrialization production Nattokinase in practice is little.Particularly, because Nattokinase is to thermally labile, be to limit the bottleneck of this enzyme in protective foods, field of medicaments application always.

Therefore, obtain the Nattokinase bacterial strain demand of high reactivity and thermostability very urgent.

Summary of the invention:

The object of this invention is to provide new height work, thermostability Nattokinase and produce bacterium; Direct object 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 casein plate method through repeatedly putting the plate repeated screening of going down to posterity, obtain high work, thermostability mutant strain in conjunction with 65 DEG C of processing---Bacillus subtilis natto (Bacillus natto) BSNK-T160, this new bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's 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 LB flat board, diameter 2-4mm, and oyster white, opaque, edge is irregular, and surface drying has gauffer, and choosing bacterium colony with transfering loop has wire drawing phenomenon, and bacterium colony is not combined with substratum, and bacterium colony pros and cons color is identical.Under microscope, 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, gemma ellipse, middle life or near middle raw, is shown in Fig. 1 and Fig. 2.

Cultural characteristic:

The BSNK-T160 substratum used of growing is LB substratum, and irradiation mutagenesis screening culture medium used is casein substratum, and culture temperature is 30-37 DEG C.

The formula of described casein substratum is: casein 1%, yeast extract 0.5%, NaCl 0.9%, agar 2%, pH7.2-7.4,121 DEG C of sterilizing 20min, matching while using.

Solid fermentation of the present invention substratum used is soybean, and leavening temperature is 38-40 DEG C.

The fermented product being produced by Bacillus subtilis natto BSNK-T160 fermentation also belong to this patent protection domain.Described fermented product can be leavened prod itself, 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.

Invention effect:

Screen through casein plate method, fibrin plate method detects fibrinolytic activity and the thermostability of bacterium, the experiments such as the fermentation capacity of solid fermentation checking thalline confirm: compared with wild type strain BSNK-5, mutant strain BSNK-T160 has following beneficial effect:

1, Caseinolytic activity and thermostability have been improved

Improve 46.9%, 65 DEG C of thermostability at the Caseinolytic activity of 37 DEG C and improved approximately 8 times (Fig. 3)

2, improved fibrinolytic activity, particularly the activity under 65 DEG C of high temperature

Fibrin plate detected result show BSNK-T160 at the dissolving circle area change of 37 DEG C 20.93%, improved 29.62% with respect to the activity of urokinase; After 65 DEG C of thermal treatments dissolve circle area change 32.13%, improved 82.31%(Fig. 4 with respect to the activity of urokinase).

3, the fermented product quality being produced by Bacillus subtilis natto BSNK-T160 fermentation is good

Solid state fermentation experiment further confirms just to form one deck tunica albuginea on soybean surface after 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, good thermostability;

3, fermenting speed is fast, within 24 hours, just can produce natto by fermented soybean, be greater than market fermenting bacillus natto and produce the minimum 48h of needs of natto.

4, fermentation gained natto quality better, does not have ammonia stink, has sauce fragrance.

Therefore, BSNK-T160 bacterial strain is desirable research, the material bacterial strain that sets out of application.In the production field of Nattokinase and natto, there is the very big possibility that realizes its industrialization, and there are wide medicine, food applications and market outlook.

Brief description of the 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 treatment of different temperature;

Fig. 4 be after treatment of different temperature BSNK-T160 with respect to the activity figure of urokinase;

Fig. 5 is that 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's common micro-organisms center

The preservation time: on October 29th, 2012.

Embodiment

Following examples only, for illustrating method of the present invention, do not limit the scope of the invention.

The high thermostability mutant strain BSNK-T160 screening of living of embodiment 1

1. material

1.1 bacterial classification

BSNK-5, the Bacillus subtilis natto by 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

Casein plate method: flat board (9cm × 9cm) is placed on to level-off on water level gauge, with the agarose of 10mM pH7.2PBS solution preparation 1%, microwave-oven-heating makes it to melt, add the equal-volume casein solution of pH8.9Tris-HCl damping fluid preparation, making final concentration is 3mg/ml, pour into while hot in flat board, bubble is rushed to edge, under room temperature, solidify.With the punch tool punching of diameter 5mm, testing sample is put in hand-hole, 37 DEG C are incubated 18h, the careful flat board that takes out, coomassie brilliant blue staining, 7% glacial acetic acid decolouring, after casein is hydrolyzed, the circle that shows transparency around point sample hole, all the other positions are blue.

2 methods

2.1 collecting cells

From the dull and stereotyped order bacterium colony of BSNK-5 activating to 20ml LB liquid nutrient medium, cultivate about 8-10h, be 0.8-1.0 to OD600 for 30 DEG C, take out centrifugal collection thalline (6000rpm, 5min), physiological saline cleans 2 times, uses equal-volume physiological saline solution, 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 to doubling dilution, get 100 μ l and be coated with casein plate, each gradient is coated with 10 casein plates, 30 DEG C of incubated overnight.

2.3 high live strain screenings

High live strain primary dcreening operation: choose at random irradiate after growth bacterial strain point casein plate, taking without irradiation bacterial strain as contrast, 30 DEG C of incubated overnight.Take out next day, chooses positive mutating strain (being that casein plate dissolves loop diameter and colony diameter odds ratio control strain dissolves loop diameter and colony diameter ratio increases 10% bacterial strain), repeats a casein plate repeatedly, dissolves circle and observe.

High live strain sieves again: will finally confirm that blast resistance cultivates 5h through 3ml LB liquid nutrient medium, 30 DEG C, 220rpm, get fermented liquid 10 μ l point casein plates, cultivate 18h for 30 DEG C, calculate dissolving circle area, the bacterial strain of choosing mutagenic strain dissolving circle Area Ratio contrast bacterium dissolving circle area increase 10% carries out thermal stability analysis.

2.4 thermostability bacterial strain screenings

By bacterium fermented liquid, through 37,55,60 and 65 DEG C of processing 15min, casein plate detection of active, using the bacterial strain that after thermal treatment, dissolving circle Area Ratio control strain is large as gain mutant bacterial strain.

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).It is 123.44mm2 that wild type strain BSNK-5 dissolves circle area at the casein of 37 DEG C, and mutant strain BSNK-T160 is 181.34mm2, and activity has improved 46.9%; Process after 15min for 65 DEG C, the dissolving circle area of mutant strain BSNK-T160 casein plate is still 37.09mm2, 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 DEG C of water bath with thermostatic control 45min, add the equal-volume of water-bath 10min at zymoplasm (final active 0.05IU/ml) and same temperature to contain 0.1% fibrinogenic pH7.2PBS solution, mix.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 DEG C of constant temperature culture 18h, measure the diameter that dissolves circle, and the area of circle is dissolved in calculating, calculates the unit of activity of enzyme according to urokinase typical curve.

Urokinase standard curve making method

The each l0ul point sample of urokinase sample (100,200,300,400,500U/m1), on the fibrin plate of new preparation, is placed 10min, moves into 37 DEG C of incubators, after insulation 18h, takes out, and measures and dissolves the perpendicular diameter of enclosing, and calculates each dissolving circle area.To dissolve circle area as X-coordinate, the mapping taking enzyme activity as ordinate zou, according to typical curve calculation sample vigor.

Fermented liquid preparation method

By BSNK-5 and BSNK-T160 after flat board activation, access 3ml LB liquid nutrient medium, 30 DEG C of incubated overnight.Take out next day, and with 2% inoculum size access 20ml LB liquid nutrient medium, 30 DEG C, 220rpm are cultivated 5h, centrifuging and taking fermented liquid 10 μ l point fibrin plates, cultivate 18h for 37 DEG C, use vernier caliper measurement perpendicular diameter, calculate and dissolve circle area and the unit of activity with respect to urokinase.

Heat treating method is with 2.4 of embodiment 1.

2, result

Fibrin plate detected result shows that BSNK-5 is 194.09mm at the dissolving circle area of 37 DEG C ², be 289.53IU/ml with respect to the activity of urokinase; It is 234.72mm that BSNK-T160 dissolves circle area ², be 375.28IU/ml with respect to the activity of urokinase; Compared 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 DEG C of thermal treatments, the dissolving circle area of BSNK-5 is 93.42mm2, is 77IU/ml with respect to the activity of urokinase; It is 123.44mm2 that BSNK-T160 dissolves circle area, is 140.38IU/ml with respect to the activity of urokinase; Compared with BSNK-5, BSNK-T160 dissolve 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

Soy material one is cleaned-is soaked a boiling one and inoculates fermentation one after-ripening one natto; Get 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 3ml liquid nutrient medium, cultivate 8-9h for 37 DEG C, 2% the bacterium amount that connects access 20mlLB liquid nutrient medium, cultivates 4-6h for 37 DEG C, centrifugal collection thalline, physiological saline or sterile water wash three times, resuspended thalline, for solid fermentation.

2.2 Soybean Pretreatment

Get 50g washing soybean, remove residue and inferior beans, add immersions of spending the night of 100ml ultrapure water, removal excessive moisture, 121 DEG C of autoclaving 20min.

2.3 fermentation

The soybean of the bacterium of having gone out is cooled and puts 38-42 DEG C, and the bacteria suspension that access prepares, cultivates 24h for 38 DEG C, and there is one deck tunica albuginea on soybean surface; Stop fermentation, put into 4 DEG C of refrigerator after-ripening 12h.

2.4 natto organoleptic analysises

BSNK-T160 bacterial strain makes natto through above technique fermented soybean 24h, finished product golden yellow color, and taste lubrication, has sauce fragrance, has very long wire drawing sample mucosubstance with chopsticks while provoking, with commercially available natto local flavor quite similar (Fig. 5).

Claims

Bacillus subtilis natto ( bacillus natto) BSNK-T160, CGMCC No.6714.
2. Bacillus subtilis natto claimed in claim 1 is produced the purposes of natto.
3. the natto obtaining with Bacillus subtilis natto claimed in claim 1.
4. produce a method for natto, taking soybean as raw material, obtain through immersion, boiling, inoculation fermentation bacterium, fermentation, it is characterized by: zymophyte used be Bacillus subtilis natto ( bacillus natto) BSNK-T160, CGMCC No.6714.