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Bacillus subtilis high in nattokinase secretion and application thereof

A Bacillus subtilis, nattokinase technology, applied in the directions of enzymes, peptidases, bacteria, etc., can solve the problems of difficult to meet strict drug production requirements, unacceptable odor, low recovery rate, etc., and achieve high nattokinase activity, The effect of reducing waste discharge and cost saving

Active Publication Date: 2017-08-29
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with solid-state fermentation, it is prone to bacterial contamination, difficult to dissipate heat, low recovery rate, unacceptable odor, and difficult to meet the strict production requirements of pharmaceuticals, especially biological agents, etc.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Bacillus subtilis ( Bacillus subtilis ) mutagenesis screening of gs-11061

[0024] Five strains of Bacillus subtilis were isolated from natto products bought in the market, and they were cultured in liquid LB medium. After culturing at 37°C for 1 day, the biomass and the secretion of nattokinase were measured simultaneously, and one strain was obtained. The most vigorous strain, named best-1, was used as the dominant strain.

[0025] Best-1 was cultured in liquid LB seed medium. Dilute the cultured Bacillus subtilis to a cell number of 10 by the 10-fold dilution method 9 / mL, take 0.1 mL of the bacterial solution and evenly spread it on a sterile empty plate, and carry out N + ion beam implantation, N + The ion beam implant dose is (95, 140, 185, 230, 275)×2.6×10 13 N + / cm 2 , N+ ion beam implantation energy is 15keV. After the irradiation, the cells were washed with 1 mL of sterile water, diluted according to the 10-fold dilution method, and then ap...

Embodiment 2

[0026] Embodiment 2 Bacillus subtilis ( Bacillus subtilis ) culture and physiological characteristics of gs-11061

[0027] The strains are cultivated in an aerobic way. The carbon source used to cultivate the bacterial strain can be glucose, sucrose, fructose, lactose, soluble starch and other materials; the nitrogen source used to cultivate the bacterial strain can be yeast extract, beef extract, tryptone, soybean peptone, whole soybean powder, okara and other materials. The optimal temperature range for the growth of the strain is 25-50° C., the pH range is 5-9, and the optimal pH range is 6-8. Dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, magnesium phosphate and other inorganic salts can also be added during the strain cultivation process.

[0028] The Bacillus subtilis ( Bacillus subtilis ) The physiological and morphological characteristics of gs-11061 are:

[0029]Bacillus subtilis grows vigorously on the LB plate, and it is ob...

Embodiment 3

[0030] Example 3 Bacillus subtilis ( Bacillus subtilis ) gs-11061 fermented to produce nattokinase

[0031] Plate medium: peptone 15 g / L, yeast extract 7.5 g / L, NacL 15 g / L, agar 20 g / L, pH natural.

[0032] Aerobic fermentation medium: xylose mother liquor 15g / L, peptone 20g / L, K 2 HPO 4 3H 2 O 1.2g / L, KH 2 PO41.6g / L, MgSO 4 7H 2 O 0.45g / L, Cacl 2 0.21g / L, MnSO 4 0.001g / L, FeCl 2 0.002g / L, the rest is water, the initial pH is 7.4. The liquid volume in a 500mL Erlenmeyer bottle is 80mL, and it is sterilized at 121°C for 20 minutes. Xylose mother liquor was purchased from Weike Food Factory in Julu County.

[0033] The initial strain and CGMCC No. 13932 bacteria were firstly activated on the plate at 35°C, and after 24 hours, two rings of well-grown bacteria were put into 80mL fermentation medium, cultivated at 35°C, 180rpm for 11h, and then according to 2.5% (v / v ) The inoculum amount is transferred to a 80ml fermentation broth / 500ml Erlenmeyer flask, cultured at ...

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Abstract

The invention discloses Bacillus subtilis high in nattokinase secretion and application thereof. The strain is named as Bacillus subtilis gs-11061 and is preserved in China General Microbiological Culture Collection Center, and the collection number of the Bacillus subtilis gs-11061 is CGMCC No. 13932. The invention further discloses a method using the Bacillus subtilis high in nattokinase yield to produce nattokinase. Compared with other fermentation methods, the method has the advantages that the Bacillus subtilis can be fermented under high temperature, and the fermentation cycle is shortened evidently; in terms of a culture medium, a xylose mother liquor coarse raw material is used to lower cost, and low energy consumption and low pollution are achieved; compared with original bacteria, the secretion of the nattokinase is increased evidently, fermentation products can be processed easily, and few byproducts are produced.

Description

technical field [0001] The invention belongs to the technical field of biological fermentation, and in particular relates to a bacillus subtilis highly secreting nattokinase. Background technique [0002] Bacillus natto (Bacillus natto) is a strain isolated from Japanese traditional food, and its original strain is the same as Bacillus subtilis, which is a subspecies of Bacillus subtilis. Bacillus natto not only has the ability to decompose macromolecular substances such as proteins, carbohydrates, and fats, but also makes the fermented products rich in amino acids, organic acids, oligosaccharides, and other components that are easily absorbed by the human body. The discovery of some physiologically active substances makes natto have a variety of health functions, such as anti-tumor, lowering blood pressure, antibacterial, etc. It can also prevent osteoporosis, improve protein digestibility, and resist oxidation. The most important thing is that Bacillus natto can produce n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/54C12R1/125
CPCC12N9/54C12Y304/21062C12R2001/125C12N1/205
Inventor 郝宁郭格格许琳欧阳平凯
Owner NANJING UNIV OF TECH
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