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Method for purifying bacillus spores

A bacillus and purification method technology, applied in the field of microbial spore preparation, can solve the problems of 100% purity, process amplification attenuation, poor reproducibility, etc., and achieve the effects of long-term preservation, easy process, and small attenuation

Active Publication Date: 2017-02-22
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the operability of these three methods is poor, the purity cannot reach 100%, the process amplification attenuation is serious, and the reproducibility is poor

Method used

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Effect test

Embodiment 1

[0033] A method for purifying bacillus spores, comprising the following steps:

[0034] 1) Sterile water washing: the crude bacillus spores (injection amount 1g) were centrifuged and washed with sterile water at 5000rpm and 4°C; and the concentration of the bacillus spores was adjusted to 10 8 / mL, to obtain the spore mixture;

[0035] 2) Heavy salt washing: add 15 times the weight of heavy salt washing solution to the spore mixture, mix well, centrifuge at 5000 rpm and 4°C, remove the supernatant, and obtain a precipitate;

[0036] 3) Enzyme solution incubation: add enzyme digestion solution to the precipitation one, suspend the precipitation, adjust the concentration of spores to 10 6 / mL, after heating in a water bath at 37°C for 1 hour, centrifuge at 5000 rpm at 4°C, remove the supernatant, and obtain Precipitate II;

[0037] 4) Washing with surfactant solution: add surfactant solution to precipitation two to make the concentration of spores reach 10 6 / mL, after mixing...

Embodiment 2

[0046] A method for purifying bacillus spores, comprising the following steps:

[0047] 1) Sterile water washing: the crude bacillus spores (injection amount 1g) were centrifuged and washed with sterile water at 5000rpm and 4°C; and the concentration of the bacillus spores was adjusted to 10 5 / mL, to obtain the spore mixture;

[0048] 2) Heavy salt washing: add 5 times the weight of heavy salt washing solution to the spore mixture, mix well, centrifuge at 5000 rpm and 4°C, remove the supernatant, and obtain a precipitate;

[0049] 3) Enzyme solution incubation: add enzyme digestion solution to the precipitation one, suspend the precipitation, adjust the concentration of spores to 10 4 / mL, after heating in a water bath at 37°C for 1 hour, centrifuge at 5000 rpm at 4°C, remove the supernatant, and obtain Precipitate II;

[0050] 4) Washing with surfactant solution: add surfactant solution to precipitation two to make the concentration of spores reach 10 4 / mL, after mixing, c...

Embodiment 3

[0059] The dosage of embodiment 1 is enlarged 100 times, all the other are identical with embodiment 1.

[0060] Result analysis: before purification, the spore purity of the bacillus spore crude product was 89%; after purification, the spore purity was 96%, and the number of spores remained at 10 8 / mL, no loss occurred.

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Abstract

The invention relates to a method for purifying bacillus spores. The method includes the steps that the bacillus spores are washed with bacterium-free water and concentrated; the product is washed with double salt washing liquid; the product is incubated with an enzyme solution; the product is washed with a surfactant solution; the product is washed with an enzyme inhibitor solution; the product is washed again with bacterium-free water; the product is put into a water bath at the temperature of 80 DEG C and sterilized; the finally-obtained bacillus spores are suspended with the bacterium-free water and stored. According to method, the purity of the spores is greatly increased while the quantity of the prepared spores is guaranteed; by means of the purifying method, active substances such as thallus nutritive cells, medium residual substances and protease released by fracture nutritive cells can be effectively removed, and induced transformation of the substances for the spores is effectively avoided; long-stem storage of the spore suspension liquid can be better achieved; the technology is easy to operate, easy to magnify, small in attenuation, good in repeatability and easy to industrially produce.

Description

technical field [0001] The invention relates to the field of preparation of microbial spores, in particular to a method for purifying bacillus spores. Background technique [0002] Bacillus is a highly stress-resistant dormant body formed by highly concentrated and dehydrated bacterial cytoplasm under certain conditions. It has strong stress resistance and can withstand high temperature, low temperature, dryness, chemical drugs, and nutritional deficiencies. and other harsh environments; according to its characteristics, the spores of Bacillus are widely used in various fields. [0003] Because the spores of Bacillus spores have high stability and long storage time during the processing of granules and powders and in acidic environments, some probiotic Bacillus spores are used as microbial additives and are used in feed additives, oral probiotic preparations, etc. such as Bacillus subtilis. At the same time, spores are currently the most stress-resistant living organisms i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N3/00C12R1/07
CPCC12N3/00
Inventor 尹焕才田晶晶
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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