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Method for fermenting vegetable soybeans and other foreign products by bacillus natto to produce nattokinase

A technology for producing nattokinase and vegetable soybeans, which is applied in the field of microbial fermentation, can solve problems such as uncertain production conditions, and achieve the effects of simple fermentation conditions, good thrombolytic effect in vitro, and high enzyme production activity

Active Publication Date: 2020-07-28
HEILONGJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the research on natto products is mainly based on soybean as a fermentation raw material to produce a series of products including nattokinase. There are also pigeon peas, red beans, chickpeas, etc. used as production raw materials to study the production of nattokinase, but vegetable There is no report on the production of nattokinase using soybeans and other off-products as fermentation raw materials. Therefore, the production conditions of nattokinase and various products including isoflavones are uncertain using off-products such as vegetable soybeans as substrates.

Method used

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  • Method for fermenting vegetable soybeans and other foreign products by bacillus natto to produce nattokinase
  • Method for fermenting vegetable soybeans and other foreign products by bacillus natto to produce nattokinase
  • Method for fermenting vegetable soybeans and other foreign products by bacillus natto to produce nattokinase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] A method for producing nattokinase by bacillus natto fermenting vegetable soybeans and other foreign products, comprising:

[0038] (1) Raw material processing: After selection, washing and weighing of vegetable soybeans and other foreign products, put them into a sterilizing pot for sterilizing by high-pressure steam at 121°C for 20 minutes and steam them until cooked;

[0039] (2) Inoculation: Bacillus subtilis natto bacterium liquid is mixed with natto bacterium powder with warm water, inoculated by 2% of the weight of foreign products such as vegetable soybeans, and the bacterium liquid is poured into when the temperature of beans is maintained at 50°C;

[0040] (3) Fermentation: Put the foreign products such as vegetable soybeans poured into the bacterial liquid into a constant temperature incubator at 37°C to ferment for 36 hours,

[0041] (4) post-ripening: after the fermentation is completed, place it in a refrigerator at 4°C for post-ripening for 18 hours to ob...

Embodiment 2

[0046] Determination of plasmin activity.

[0047] 1. Agarose-fibrin plate method

[0048] 1. Preparation of fibrin plate: refer to the method of Astrup et al. and improve it. Dissolve 0.3% fibrinogen in 10 mL pH 7.4 phosphate buffer solution and keep warm at 50°C for 5-10 min. Dissolve 20 U of thrombin in 1 mL of pH 7.4 phosphate buffer solution to prepare thrombin buffer solution. Prepare 1.5% agarose solution, keep it warm at 50°C for 10 minutes, mix 1mL 20U / mL thrombin buffer solution, 10mL fibrinogen solution and 10mL agarose solution quickly, pour the plate immediately, and punch holes after solidification. Dilute the sample on a fibrin plate, and measure the diameter of the fibrinolytic circle with a vernier caliper after incubating in a 37°C incubator for 18 hours.

[0049] 2. Drawing of the standard curve: Dilute the urokinase standard product accordingly to a concentration gradient of 100, 200, 400, 800, 1600 IU / mL, spot the sample on the prepared fibrin plate, an...

Embodiment 3

[0054] Single-factor experiment on the production of nattokinase from vegetable soybean fermented by Bacillus natto.

[0055] Bacillus natto was inoculated in steamed vegetable soybeans for fermentation, and the effects of four factors including fermentation time, inoculum size, fermentation temperature and post-ripening time on the activity of nattokinase were investigated. After being screened by each single factor experiment, it provides the preliminary experimental basis for the next orthogonal optimization experiment.

[0056] 1. Effect of fermentation time on nattokinase activity

[0057] Inoculate 2% Bacillus natto bacteria liquid into sterilized and steamed vegetable soybeans, put them into a constant temperature incubator with a preset temperature of 37°C, and cultivate them for 12h, 24h, 36h and 48h respectively, and put them into After ripening in a refrigerator at 4°C for 12 hours, three parallel experiments were carried out for each group, and then the finished p...

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Abstract

The invention relates to the technical field of microbial fermentation, in particular to a method for fermenting vegetable soybeans and other foreign products by bacillus natto to produce nattokinase.The method comprises the steps of inoculating 2% of prepared bacillus natto solution into selected, cleaned, steamed and sterilized vegetable soybeans and other foreign products; carrying out fermentation in a constant-temperature incubator of 37 DEG C for 36 hours; carrying out after-ripening for 18 hours; carrying out salting-in and centrifuging to obtain an original extracting solution; carrying out ammonium sulfate precipitation and preliminary purification to obtain a crude enzyme solution; and then carrying out further purification by using DEAE ion-exchange column chromatography. The invention aims to provide a new idea for producing the nattokinase through fermentation and to provide a scheme for solving the problem of processing the foreign products of a company. The activity ofthe nattokinase produced under an optimum fermentation process can reach 2326.60 IU / g; an SDS-PAGE electrophoresis test verifies that the molecular weight of the extracted nattokinase is 25-35 kDa, and the recovery rate and the purification multiple are 4.9% and 11.2 separately; and a theoretical basis is provided for production of the nattokinase and follow-up in vivo tests.

Description

technical field [0001] The invention relates to the technical field of microbial fermentation, in particular to a method for producing nattokinase by bacillus natto by fermenting vegetable soybeans and other external products. Background technique [0002] As a traditional food with a history of 2,000 years, natto has many health care functions such as preventing osteoporosis and promoting blood coagulation, anti-pathogenic bacteria, delaying aging, preventing and fighting cancer, and adjusting intestinal function. The main active ingredient, nattokinase, has attracted extensive attention because of its good thrombolytic properties and safety. Nattokinase has good thrombolytic properties, a wide range of sources, is safe and non-toxic, and has a small molecular weight and is easily absorbed by the human body. As a microbial metabolite, it has the possibility of large-scale production and has broad development and application prospects. At present, the research on natto prod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/56C12R1/125
CPCC12N9/54C12Y304/21062
Inventor 李秀凉朱玉孙庆申靳文凤
Owner HEILONGJIANG UNIV
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