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Collagenase producing strain

A collagen and collagen tripeptide technology, applied in the direction of bacteria, microorganisms, and methods based on microorganisms, can solve the problems of uneven molecular weight and low collagen tripeptide content, and meet simple nutritional requirements, high collagenase activity, and tolerance The effect of strong temperature capability

Active Publication Date: 2013-08-07
茂名希普生物科技有限公司
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a collagenase producing bacterium, namely a new strain of Bacillus subtilis capable of efficiently expressing collagenase, and the application of the strain in collagen hydrolysis, that is, the application of the collagenase produced by the strain Collagen powder rich in collagen tripeptides is obtained by hydrolyzing collagen, which solves the shortcomings of products obtained in the prior art such as uneven molecular weight and low collagen tripeptide content

Method used

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  • Collagenase producing strain

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Effect test

preparation example Construction

[0028] 1. Preparation of collagenase

[0029] Select LB medium (the components of LB medium are as follows: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L), adjust the pH to 7.5, and expand the culture of bacteria at 37°C and 200rpm for 24 After 1 hour, the cultivation was stopped, and a seed solution was obtained.

[0030] Take 5ml seed liquid and inoculate it into 100mL enzyme production medium (the composition of enzyme production medium is as follows: glucose 20g / L, yeast extract 1.5 g / L, tryptone 10g / L, calcium chloride 0.05 g / L, magnesium chloride 0.165 g / L, dipotassium hydrogen phosphate 0.25 g / L, sodium dihydrogen phosphate 0.5 g / L, pH 7.5.), cultivated at 30°C, 200rpm for 36 hours, after the cultivation, centrifuged at 4°C, 10000rpm for 10min, collected Fermentation supernatant.

[0031] The supernatant was first precipitated with ammonium sulfate, and the precipitate was collected by centrifugation; the precipitate was redissolved in Tris-Hcl buffer sol...

Embodiment 1

[0039] Embodiment 1: Utilize Bacillus subtilis Col04 fermented liquid to the degradation of fish skin collagen

[0040]1) Preparation of seed solution:

[0041] The LB medium was selected, the pH was adjusted to 7.5, and the bacteria were expanded and cultured at 37° C. and 200 rpm for 24 hours, and the culture was stopped to obtain a seed solution.

[0042] 2) Enzyme production culture

[0043] Inoculate 10ml of seed liquid into 200mL of enzyme-producing medium, and incubate at 30°C, 200rpm for 36 hours. After the cultivation, centrifuge at 10,000rpm for 10min at 4°C, and collect the supernatant (save at 4°C for later use).

[0044] 3) Degradation of collagen

[0045] Take 100g of cod skin, descale and wash it, cut it into a size of 0.5mm×0.5mm, then add 100mL of the above-mentioned fermentation broth, adjust the pH to 7.5, place it at 37°C, and perform enzymatic hydrolysis at 100rpm for 8 hours. After the enzymolysis was completed, the enzyme was extinguished at 95°C for ...

Embodiment 2

[0046] Embodiment 2: Utilize Bacillus subtilis Col04 fermented liquid to the degradation of fish skin collagen

[0047] Compared with Example 1, the temperature was increased to 37°C during the enzyme-producing culture of the Bacillus subtilis Col04 strain, and the culture time was extended to 48 hours, while other conditions remained unchanged. The conditions for collagen degradation were unchanged. After 8 hours of degradation, the enzymatic solution was centrifuged, and the supernatant was freeze-dried to obtain 13.6 g of off-white powder with a protein content of 92.1% and a yield of 77.71%. Because after prolonging the time of enzyme-producing culture, the activity of collagenase in the fermentation broth increases, resulting in a better effect in degrading collagen.

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Abstract

The invention relates to a bacillus subtilis Col04 bacterial strain with a preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.4500. The bacillus subtilis Col04 bacterial strain has the advantages of simple nutrition requirements, quick growth, and high collagenase producing activity. According to the researches, the collagenase produced by the bacillus subtilis Col04 bacterial strain is insensitive to most metal ions, extensive in pH range, high in collagenase activity and stronger in tolerable temperature capability in comparison with the conventional collagenase from other bacterial strains. Moreover, the bacterial strain is safe and non-toxic, and therefore, the bacterial strain has extensive economic prospect in degrading collagenase or gelatin and being used in food and cosmetics industries.

Description

technical field [0001] The invention belongs to the technical field of industrial microorganism screening application, and more specifically relates to a collagenase-producing bacterium. Background technique [0002] The chemical name of collagenase is collagenase (Collagenase), which can specifically hydrolyze the three-dimensional helical structure of natural collagen under physiological pH and temperature conditions without damaging other proteins and tissues. [0003] Currently, collagenase is mainly used in medical and scientific research. Clinically, collagenase is mainly used in the treatment of burns and wounds, as well as the treatment of hypertrophic scars and lumbar disc herniation. In terms of scientific research, collagenase is mainly used to digest organ tissues of organisms to obtain primary cells. [0004] Studies have shown that in addition to being used in clinical medicine and scientific research, collagenase also participates in the metabolism of the st...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P21/06C12R1/125
Inventor 郑振琴
Owner 茂名希普生物科技有限公司
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