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Bacillus subtilis strain for producing nattokinase and application thereof

A technology of Bacillus subtilis producing nattokinase, which is applied in the field of microorganisms, can solve the problems of low activity, reduce the incidence of thrombotic diseases, unfavorable nattokinase content, etc., and achieve the effects of increasing fermentation activity, improving food taste and good taste

Active Publication Date: 2014-04-02
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the activity of nattokinase products in China is generally low at present, and it is of great significance to screen strains with high activity nattokinase production capacity
[0003] Although natto food has good nutritional and health functions, the unique flavor of natto is not easily accepted by the people of our country, which is not conducive to people increasing the content of nattokinase in the body through diet, and is not conducive to reducing the incidence of thrombotic diseases
Traditional fermented food is rich in microbial species, has good food safety, and has formed a sensory flavor that people adapt to. It is an important source of nattokinase production strains. Chinese soybean paste is rich in Bacillus, such as B. subtilis, B. amyloliquefaciens and Baci llus licheniformis, but there is no report on the isolation of nattokinase-producing bacteria from Chinese soybean paste

Method used

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  • Bacillus subtilis strain for producing nattokinase and application thereof
  • Bacillus subtilis strain for producing nattokinase and application thereof
  • Bacillus subtilis strain for producing nattokinase and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0038] A kind of bacillus subtilis BN-05, its screening process is as follows:

[0039] 1) Primary screening

[0040] 11 samples from Huangpi fermented bean curd in Hubei Province, Jingzhou bean paste, soybean paste in Yingcheng, Laba beans in Yichang, bean paste in Shanxiang, Gongguan hot sauce, soybean paste in Wuhan, broad bean paste and tempeh. Take 2 g and place it in a 100 mL sterilized Erlenmeyer flask with a stopper, add sterile saline to dilute 10 times, shake for 30 seconds, and incubate at 37°C and 180 r / min for 24 hours. Heat the bacterial suspension in a water bath at 80-85°C for 10 minutes, make gradient dilutions of the cooled bacterial suspensions, and spread 0.1 mL each on a skim milk plate, incubate at 37°C for 24 hours, and pick out the hydrolyzed circle Gram staining and microscopic examination are performed on the colonies with larger colony diameter ratio (C / H value), and primary microscopic examination is carried out according to the description of the ...

Embodiment 2

[0056] Determination of fibrinolytic activity (nattokinase activity): using the agarose-fibrinogen plate method, the production steps of the agarose plate are as follows:

[0057] First, dissolve 1% agarose in 0.05mol / L Tris-HCl (pH7.8) buffer solution, heat until completely dissolved, take out 10mL and place it in a large test tube, wait for it to cool to about 50°C, and pour it quickly Add 10mL of 1.5mg / L bovine fibrinogen solution, oscillate continuously to make it completely mixed evenly, pour it into a 9cm diameter petri dish, then quickly add 300μL of 20IU / mL thrombin solution, shake the dish constantly to mix the three evenly, To prevent the generation of air bubbles, after standing for 1 hour, use a 2mm diameter sterile plastic pipette to punch holes on the plate.

[0058] Preparation of urokinase standard curve: Prepare different urokinase standard products (200, 400, 800, 1000, 1200, 1400, 1600, 1800, 2000 and 2400IU / ml), and spot 10 μL each on the newly prepared fib...

Embodiment 3

[0062] A kind of application of bacillus subtilis BN-05 in preparing natto, its step is as follows:

[0063] Transfer the Bacillus subtilis BN-05 strain to LB slant medium for activation at 37°C for 24 hours, use an inoculation loop to select 2-3 rings of bacterial lawn and inoculate it into the liquid seed medium (filling volume 50mL / 250mL), and inoculate at 35 Cultivate at 180r / min for 12h to the logarithmic growth phase, then inoculate 4% (v / w) inoculum on sterilized soybean culture, culture at 37°C for 36h, and stir once every 12h. The color of the fermented natto is khaki, and the bean grains are soft. When the soybean is touched, it has a long-drawn phenomenon, with a faint aroma. The activity of nattokinase kinase is 7980IU / g (wet soybean).

[0064] The liquid seed medium: 10g.L-1 glucose, 5g.L-1 yeast extract, 10g.L-1 beef extract, 5g.L-1NaCl, pH7.4-7.6.

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Abstract

The invention discloses a bacillus subtilis strain for producing nattokinase and an application thereof. The method comprises the following steps: separating and purifying from soybean paste in Hanjiang River basin, rapidly screening to obtain a nattokinase production strain at high efficiency by adopting a method for combining preliminary skim milk screening, soybean solid fermenting and re-screening, integrated nattokinase gene PCR (Polymerase Chain Reaction) with fiber activity, and finally, performing molecular identification to determine the bacillus subtilis through 16SrRNA, wherein the bacillus subtilis is BN-05 CCTCC NO:M2013530, and the strain is subjected to solid soybean fermentation and culture for 24 hours at the temperature of 35 DEG C. The natto drawing is long, the mouthfeel is good, the nattokinase production content is high, and the strain can serve as a basis of food fermentation research. The strain contributes to improving the in-vivo nattokinase content through diet and preventing and relieving the incidence rate of thromboembolic diseases.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a nattokinase producing strain, and also relates to the application of a nattokinase producing strain in preparing natto. The Bacillus subtilis described in the present invention has a high-yield natto kinase The ability can be used for the production of natto. Background technique [0002] Thrombotic disease can lead to myocardial infarction, cerebral thrombosis, stroke, acute pulmonary heart disease and other related diseases. Because of its high incidence and relapse rate, and it is difficult to cure, it has become a major disease that threatens human health. Therefore, in recent years, how to effectively cure thrombotic diseases has attracted great attention from the medical and scientific circles. Nattokinase (Nattokinase, NK) has been proven to have a highly efficient thrombolytic effect, it can not only directly reduce fibrinogen, but also promote the c...

Claims

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Application Information

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IPC IPC(8): C12N1/20A23L1/20C12N15/57C12R1/125A23L11/00
Inventor 高冰高泽鑫王常苏
Owner HUBEI UNIV OF TECH
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