Method for producing douchi fibrinolytic enzyme through microbial fermentation

A microbial fermentation, thrombolytic enzyme technology, applied in the direction of hydrolase, etc.

Inactive Publication Date: 2012-09-05
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The industrialization of microbial fermentation production of NK, large-scale production and application as an oral thrombolytic agent have not been reported yet

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] (1) Culture medium preparation

[0013] ① Strain activation medium: peptone 15.0g, beef extract 10.0g, NaCl 10.0g, agar 20.0g, distilled water 1.0L, pH value 7.2-7.4, sterilized by high pressure steam at 121℃ for 30min.

[0014] ② Directed acclimation medium for strain products: 3.0-7.0g peptone, 3.0-7.0g beef extract, 10.0-20.0g fibrin, 5.0-15.0g NaCl, 20g agar, 1.0L distilled water, natural pH, high-pressure steam at 121°C Sterilize for 30min.

[0015] ③Liquid seed medium: tryptone 8.0-12.0g, yeast powder 4.0-6.0g, NaCl 8.0-10.0g, tap water 1.0L, pH value natural, sterilized by high pressure steam at 121℃ for 30min.

[0016] ④ Fermentation enzyme production medium: glucose 13.0-17.0g, peptone 13.0-17.0g, yeast powder 1.2-1.7g, CaCl 2 2H 2 O 0.1~0.3g, K 2 HPO 4 2.0~3.0g, MgSO 4 ·5H 2 O 0.3~0.7g, KH 2 PO 4 0.3-0.7g, 1.0L tap water, natural pH, sterilized by high-pressure steam at 121℃ for 30min.

[0017] (2) The strains producing NK were initially activated ac...

Embodiment 2

[0024] (1) Culture medium preparation

[0025] ① Strain activation medium: peptone 15.0g, beef extract 10.0g, NaCl 10.0g, agar 20.0g, distilled water 1.0L, pH value 7.2-7.4, sterilized by high pressure steam at 121℃ for 30min.

[0026] ② Directed acclimation medium for strain products: 3.0-7.0g peptone, 3.0-7.0g beef extract, 10.0-20.0g fibrin, 5.0-15.0g NaCl, 20g agar, 1.0L distilled water, natural pH, high-pressure steam at 121°C Sterilize for 30min.

[0027] ③Liquid seed medium: tryptone 8.0-12.0g, yeast powder 4.0-6.0g, NaCl 8.0-10.0g, tap water 1.0L, pH value natural, sterilized by high pressure steam at 121℃ for 30min.

[0028] ④ Fermentation enzyme production medium: glucose 13.0-17.0g, peptone 13.0-17.0g, yeast powder 1.2-1.7g, CaCl 2 2H 2 O 0.1~0.3g, K 2 HPO 4 2.0~3.0g, MgSO 4 ·5H 2 O 0.3~0.7g, KH 2 PO 4 0.3-0.7g, 1.0L tap water, natural pH, sterilized by high-pressure steam at 121℃ for 30min.

[0029] (2) The strains producing NK were initially activated ac...

Embodiment 3

[0036] (1) Culture medium preparation

[0037] ① Strain activation medium: peptone 15.0g, beef extract 10.0g, NaCl 10.0g, agar 20.0g, distilled water 1.0L, pH value 7.2-7.4, sterilized by high pressure steam at 121℃ for 30min.

[0038] ② Directed acclimation medium for strain products: 3.0-7.0g peptone, 3.0-7.0g beef extract, 10.0-20.0g fibrin, 5.0-15.0g NaCl, 20g agar, 1.0L distilled water, natural pH, high-pressure steam at 121°C Sterilize for 30min.

[0039] ③Liquid seed medium: tryptone 8.0-12.0g, yeast powder 4.0-6.0g, NaCl 8.0-10.0g, tap water 1.0L, pH value natural, sterilized by high pressure steam at 121℃ for 30min.

[0040]④ Fermentation enzyme production medium: glucose 13.0-17.0g, peptone 13.0-17.0g, yeast powder 1.2-1.7g, CaCl 2 2H 2 O 0.1~0.3g, K 2 HPO 4 2.0~3.0g, MgSO 4 ·5H 2 O 0.3~0.7g, KH 2 PO 4 0.3-0.7g, 1.0L tap water, natural pH, sterilized by high-pressure steam at 121℃ for 30min.

[0041] (2) The strains producing NK were initially activated acc...

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Abstract

The invention discloses a method for producing douchi fibrinolytic enzyme (NK) through microbial fermentation. The method comprises the following steps of: activating the microorganism generating NK for 6-10 times, and performing directional acclimation of the product for 4-6 times so that the product grows well in the environment at 26-32 DEG C; performing gradual amplification culture of the NK after the directional acclimation of the product at 26-32 DEG C, inoculating the product into a liquid fermentation medium according to an inoculation amount of 3-9% of the fermentation broth volume, and culturing at 26-32 DEG C for 72-144 hours to end the production of NK through microbial fermentation; centrifuging the fermentation broth at 4,000-8,000rpm and collecting the liquid which is the crude enzyme liquid; and performing further concentration, separation and purification of the crude enzyme liquid according to different needs and different objects to obtain the enzymic preparations different in activity, purity and form.

Description

technical field [0001] The invention relates to the fields of microbiology, enzyme engineering, fermentation engineering, biochemistry and the like, in particular to a microbial fermentation production method of tempeh thrombolytic enzyme. The low-temperature tempeh thrombolytic enzyme produced by the method has unique advantages in dissolving thrombus and activating plasminogen in the body, and can be widely used in the production of thrombolytic drugs and thrombolytic health products. Background technique [0002] Soybean thrombolytic enzyme (Nattokinase, referred to as NK) is an alkaline serine protease, non-toxic, can be taken orally (Li Jiangwei et al., Chinese Journal of Pharmaceutical Sciences, 1999), easily absorbed by the body, can directly dissolve fibrin, and has a strong thrombolytic effect. function (Hao Zhenghong et al., Chinese Journal of Agricultural Engineering, 2008; Peng Yong et al., High-Tech Communication, 2002), and the thrombolytic action time is long,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50
Inventor 迟乃玉张庆芳王晓辉窦少华于爽王贵鹏
Owner DALIAN UNIV
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