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Fibrinolytic enzyme-producing Bacillus subtilis and fermentation method and application thereof

A technology of Bacillus subtilis and plasmin, applied in the field of microorganisms, can solve problems such as low activity, and achieve the effects of high enzyme activity, short fermentation period and simple formula

Active Publication Date: 2016-01-13
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the activity of domestic plasmin products is generally low

Method used

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  • Fibrinolytic enzyme-producing Bacillus subtilis and fermentation method and application thereof
  • Fibrinolytic enzyme-producing Bacillus subtilis and fermentation method and application thereof
  • Fibrinolytic enzyme-producing Bacillus subtilis and fermentation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] A kind of acquisition of Bacillus subtilis DC27:

[0040] 1) Primary screening

[0041]Take 1g of Douchi from each of the 5 Douchi samples sold in Chongqing, put them into a 100mL sterile saline flask filled with 4 to 6 glass beads, vibrate on a shaker at 150r / min at 37°C for 20min, then After 10 minutes in a water bath at 80°C, centrifuge at 4000 r / min for 5 minutes. Collect the supernatant, take 1mL of the treatment solution and add 9mL of sterile distilled water to dilute 5 gradients (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 ), respectively absorb 0.1mL on the casein primary screening medium plate, take it out after inverting at 37°C for 18 hours, observe the plate, pick the strain with a larger ratio of the diameter of the transparent circle to the diameter of the colony on the primary screening plate, and transfer it to Streak pure culture on LB plates, inoculate the obtained pure culture strains on LB slant medium for culture, culture at 37°C for 24h, and store...

Embodiment 2

[0052] The determination of plasmin activity adopts the fibrin plate method, and the steps are as follows:

[0053] 1) Make a fibrin plate

[0054] According to the Astrup method (Astrup, 1952) slightly modified. Weigh 0.150g of agarose and dissolve it in 10mL of 0.06mol / L PBS phosphate buffer solution, dissolve it in a boiling water bath, shake it well during the dissolution process, so that the agarose can be completely dissolved, and the solution is transparent. Dissolve 0.015g of fibrinogen in 10mL of 0.06mol / L PBS phosphate buffer, preheat the dissolved agarose, fibrinogen and thrombin in a water bath at 50°C for 5min, then add 1mL of thrombin Add it into the agarose and shake it gently by hand, mix it completely and pour it into the fibrinogen solution quickly. After mixing evenly, slowly pour the liquid into a clean plate with a diameter of 9cm. After the plate is solidified at natural temperature for 5 minutes, put it into a 37°C incubator to solidify for 30 minutes,...

Embodiment 3

[0060] Preservation of strains

[0061] Glycerin cryopreservation method. First, use a flame-sterilized inoculation loop to take 1 ring of inclined-plane strains, streak on a plate to separate a single colony, place the plate upside down in a 37°C constant temperature incubator, and incubate for 24-48 hours until the size of a single colony is about 3mm. Pick one A single colony was inoculated in a Erlenmeyer flask filled with 50mL of LB medium, cultured with shaking at 37°C for 10-15h, until the bacterial density was OD 600 1.0 to 1.5. Then use a flame-sterilized inoculation loop to take a small amount of seed liquid, smear it, do Gram staining, and observe the shape of the bacteria under a microscope, and whether there are any miscellaneous bacteria. Finally, add 30% glycerol (sterilized) to the bacterial solution at a ratio of 1:1 (v / v), mix and distribute into sterilized strain preservation tubes, 1-2mL / tube, store at -80°C or stored in liquid nitrogen.

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Abstract

The invention discloses a fibrinolytic enzyme-producing Bacillus subtilis and a fermentation method and application thereof. The Bacillus subtilis is isolated and sieved from a traditional Chongqing fermented food, i.e., fermented soya beans. The Bacillus subtilis DC27 is collected in China Center for Type Culture Collection under CCTCC NO: M 2015573. The invention further provides a liquid fermentation method of the Bacillus subtilis, and the fibrinolytic enzyme in obtained fermentation broth has an activity of up to 4506 IU / ml. The Bacillus subtilis is from foods and can secrete the high-activity fibrinolytic enzyme; the Bacillus subtilis is safe and nontoxic, and is applicable to the development of functional foods or drugs for preventing or relieving thrombotic diseases; and the Bacillus subtilis has a promising application prospect.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a plasminase-producing Bacillus subtilis and its fermentation method and application. The Bacillus subtilis in the invention is isolated and screened into fermented soya bean, has the ability of high-yield fibrinolytic enzyme in fermented soya bean, and can be used for the prevention or treatment of In the development of functional food or medicine for alleviating thrombotic diseases. Background technique [0002] Cardiovascular diseases (cardiovascular disease, CVDs), such as acute myocardial infarction, ischemic heart disease and hypertension are one of the leading causes of human death in the world. According to the World Health Organization report in 2001, about 17 million people die from CVDs every year, and by 2020, the annual death toll due to CVDs will increase to 25 million. Among different types of CVDs, thrombotic disease has become one of the most import...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/68A61K38/48A61P7/02C12R1/125
Inventor 胡咏梅王昭婷毛娉梁运祥葛向阳赵述淼王绩梅余霞陈正军彭楠
Owner HUAZHONG AGRI UNIV
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