Fibrinolytic-enzyme-producing Bacillus amyloliquefaciens strain separated from traditional fermented black beans

A technology of amylolytic spores and fibrinolytic enzymes, applied in the field of microorganisms and bioengineering, can solve the problems of easy bleeding, high price, high production cost, etc., and achieve the effects of good genetic stability, easy cultivation, and high production safety

Inactive Publication Date: 2015-09-02
JIANGXI NORMAL UNIV
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Problems solved by technology

The commonly used thrombolytic drugs (such as urokinase, streptokinase and plasminogen activator) in medical clinical treatment of cardiovascular and cerebrovascular embolism diseases have short half-life, easy bleeding, high price or high production cost, so research and development are natural. The source of plasmin has great development potential and application prospect

Method used

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  • Fibrinolytic-enzyme-producing Bacillus amyloliquefaciens strain separated from traditional fermented black beans
  • Fibrinolytic-enzyme-producing Bacillus amyloliquefaciens strain separated from traditional fermented black beans
  • Fibrinolytic-enzyme-producing Bacillus amyloliquefaciens strain separated from traditional fermented black beans

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Experimental program
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Embodiment 1

[0026] Embodiment 1: Preparation or screening of Bacillus amyloliquefaciens strain of the present invention

[0027] Step 1. Sample collection: collect fermented soybeans during the koji making process in the fermented soybean production factory, then seal them in sample bags and store them at low temperature.

[0028] Step 2. Enrichment culture: Take 1g of the sample in a sterilized test tube, add 9mL of sterile normal saline, seal it, shake it with a vortex oscillator for 5min, draw 1mL of the bacterial liquid and place it in a 30mL broth medium (according to the content of 100mL of water). Beef extract 0.5g, peptone 1g, sodium chloride 0.5g, agar 1.5g, pH 7.2-7.4) in a 250mL Erlenmeyer flask, and then placed in a shaker at 36-37°C, 200r / min for 72h.

[0029] Step 3. Primary screening of bacterial strains: using the plate dilution method, take 200 μL of the enriched and cultured bacterial solution and spread it on the sterilized skim milk powder screening medium (according t...

Embodiment 2

[0033] Embodiment 2: Microscopic morphology and molecular biological identification of Bacillus amyloliquefaciens strain of the present invention

[0034] Get the obtained bacterial strain Jxnuwx-1 and carry out microscopic morphology and molecular biology method identification, and specific process is as follows:

[0035] (1) The solid plate culture method is adopted: the solid culture is a broth medium, cultivated at 37±1°C for 24 hours, and the colony diameter is 3-6mm. White opaque colony, smooth surface, the same color on the opposite side, the edge of the colony is uplifted and regular serrated, with folds in the middle, and no pigment is produced on various media.

[0036] The microscopic morphological characteristics of Bacillus amyloliquefaciens are: the cells are short rods, the spores are elliptical, and the cells are mostly in pairs or chains. For the colony morphology of Bacillus amyloliquefaciens, see the attached figure 1 After staining with safranin and malac...

Embodiment 3

[0039] Embodiment 3: The implementation process of the 16S rDNA sequence analysis of the Bacillus amyloliquefaciens strain used for producing plasmin according to the present invention is as follows:

[0040] Primers were designed according to the most conserved sequence in bacterial 16S rDNA, wherein the forward primer was F: 5'-GAGAGTTTGATCCTGGCTCAG-3', and the reverse primer was R: 5'-AAGGAGGTGATCCAGCCGCA-3'. PCR reaction system 20μL. Thermal cycle parameters Pre-denaturation at 94°C for 5min, denaturation at 94°C for 30s, annealing at 55°C for 40s, extension at 72°C for 1.5min, 35 cycles, and extension at 72°C for 7min. The PCR product was detected by agarose gel electrophoresis, and the amplified product was a specific band with a size of about 1.4kb. The amplified sequence was sequenced, and the 16S rDNA nucleotide sequence of strain Jxnuwx-1 was 1451bp in size. The sequence was compared with the 16S rDNA nucleotide sequences of 10 strains in the GenBank database, and t...

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Abstract

The invention discloses a fibrinolytic-enzyme-producing Bacillus amyloliquefaciens strain separated from traditional fermented black beans, belonging to the technical field of bioengineering. The strain is separated and purified from traditional fermented black beans, is named Bacillus amyloliquefaciens by taxonomic identification, and is collected by China Center for Type Culture Collection on December 10th, 2014; and the collection number is CCTCC M2014638. The fibrinolytic-enzyme-producing Bacillus amyloliquefaciens strain is easy to culture, and has the advantages of high production safety and favorable hereditary stability. The enzyme activity of the strain liquid after fermentation and culture is up to 324.6-410.2 IU/mL. The metabolite fibrinolytic enzyme can be easily separated and purified. The separation of the strain lays foundation for implementing scientific axenic culture in traditional fermented black bean industry.

Description

technical field [0001] The present invention relates to the technical field of microbes and bioengineering, in particular to a strain of Bacillus amyloliquefaciens isolated and purified from traditional fermented soya bean: Jxnuwx-1, which is of great significance for the scientific purebred cultivation of the traditional fermented soya bean industry . Background technique [0002] Cardiovascular and cerebroembolic diseases have become one of the main causes of harm to human health. According to statistics, there are more than 15 million patients with thrombotic diseases worldwide, and about 3 million patients die every year. The etiology is caused by the accumulation of fibrin on the arterial wall (see reference 1 for details: Luo Mingdian. New Advances in Microbial Pharmaceutical Research [J]. Microbiology Bulletin, 1998,01:61-62.), the treatment of vascular embolism diseases Currently the most effective method is thrombolytic therapy. Now the commonly used thrombolytic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/68C12R1/07
CPCC12N1/20C12N9/6435C12Y304/21007C12N1/205C12R2001/07
Inventor 王筱兰涂宗财张志斌杨林肖前程
Owner JIANGXI NORMAL UNIV
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