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Bacillus velezensis bred by ultrasonic-assisted adaptive evolution and application of bacillus velezensis

A technology of adaptive evolution and bacillus, applied in the field of microbial engineering, can solve the problems of high requirements for experimental equipment and operation level, unfavorable industrial promotion and use, low mutation efficiency, etc., to shorten the fermentation cycle, no mutagen and radioactivity Residue, initiation and termination of rapid effects

Pending Publication Date: 2022-02-08
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The essence of ALE is internal gene mutation and external environmental screening. The speed of evolution depends on the gene mutation rate and screening conditions, but the natural mutation rate of general genes is 10 -6 ~10 -10 , there are disadvantages of low mutation efficiency and high time cost
Therefore, it is necessary to combine ALE with cell sorting technology, genome modeling technology and other technologies to quickly obtain beneficial mutant phenotypes, but these technologies require high levels of experimental equipment and operation, which is not conducive to industrial promotion and use

Method used

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  • Bacillus velezensis bred by ultrasonic-assisted adaptive evolution and application of bacillus velezensis
  • Bacillus velezensis bred by ultrasonic-assisted adaptive evolution and application of bacillus velezensis
  • Bacillus velezensis bred by ultrasonic-assisted adaptive evolution and application of bacillus velezensis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Ultrasound-assisted adaptive evolution selection of Bacillus velei

[0044] Take the strain B. velezensis CICC 23571 (purchased from the China Industrial Microbiology Culture Collection Management Center) as the starting strain (and record it as the original strain BV0), and make three parallel samples at the same time, according to the inoculation of 2% (v / v) Quantitatively transfer the seed solution cultivated for 24 hours to CM50 medium and cultivate it on a shaker at 33°C and 150r / min until the mid-logarithmic phase (6h), then perform low-intensity ultrasound (10-60W / mL) + short-term cold shock treatment (0.5-5min) to implement ultrasonic-assisted adaptive evolution selection, and optimize the experimental parameters to obtain the optimal ultrasonic + cold shock treatment conditions.

[0045] The conditions of the optimal ultrasonic+cold shock treatment include condition 1 and condition 2:

[0046] Condition 1: Sonicate at 20W / mL, 40kHz for 5min, immedia...

Embodiment 2

[0053] Example 2. Morphological observation and identification of 16s rDNA bacterial species of Bacillus Velez US-ALE-BV3

[0054] (1) Morphological observation of Bacillus veleisi US-ALE-BV3

[0055] In the present embodiment, the morphological observation of Bacillus velei US-ALE-BV3 is carried out, figure 1 It is the colony morphology diagram of Bacillus Velez US-ALE-BV3 on nutrient agar. It can be seen from the figure that the colony of US-ALE-BV3 is milky white, translucent, with a small amount of bulges and wrinkles; compared with the original strain For example, the colonies of US-ALE-BV3 became thicker, the colony diameter became smaller, and the jagged edges became less obvious.

[0056] (2) Identification of 16s rDNA strains of Bacillus veleisi US-ALE-BV3

[0057] In this example, the 16s rDNA strain identification of Bacillus Velez US-ALE-BV3 was also carried out, and the specific steps were as follows: extract the whole strain of Bacillus Velez US-ALE-BV3 through...

Embodiment 3

[0059] Embodiment 3. The comparison of the growth situation of Bacillus Velez US-ALE-BV3 and original bacterial strain BV0

[0060] In the present embodiment, the growth conditions of Bacillus velezensis US-ALE-BV3 and the original bacterial strain BV0 (B. velezensis CICC 23571) were respectively investigated and compared. The specific steps are:

[0061] (1) The original strain BV0 of Bacillus Velez and the Bacillus Velez US-ALE-BV3 obtained in Example 1 were respectively inoculated in LB liquid medium for activation, and cultured in a shaker at 33°C at 150r / m overnight.

[0062] (2) Transfer the seed solution obtained in the above (1) to 50 mL casein and starch mixed medium (3:1, v / v) at an inoculation amount of 1% (v / v) (250 mL triangular bottle), cultured on a shaker at 33°C and 150r / min.

[0063] (3) During the cultivation process in step (2), samples were taken every 3 hours, the OD value at a wavelength of 600nm was measured, and the growth curve was drawn.

[0064] ...

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Abstract

The invention provides bacillus velezensis bred through ultrasonic-assisted adaptive evolution and application of the bacillus velezensis, and belongs to the technical field of microbial engineering. In the invention, a bacillus velezensis strain (B.velezensis CICC 23571) is taken as a starting strain, an ultrasonic-assisted adaptive evolution method is utilized, the proportion of organic nitrogen in a culture medium is continuously reduced, and the proportion of inorganic nitrogen to a sugar source is increased, so that a bacillus velezensis mutant strain with high yield of polypeptide is obtained, and the mutant strain strengthens different metabolic pathways, the thalli can efficiently convert inorganic nitrogen into organic nitrogen or reduce the deamination effect on the organic nitrogen, and finally, the yield of the polypeptide is greatly increased.

Description

technical field [0001] The invention belongs to the technical field of microbial engineering, and in particular relates to Bacillus Velez selected and bred by ultrasonic-assisted adaptive evolution and its application. Background technique [0002] Polypeptide is a compound formed by connecting amino acids through peptide bonds, and the size is between amino acids and proteins. In recent years, bioactive peptides have been widely used in the fields of medicine and health such as food, health products and medicines due to their safety, functionality, and easy absorption. [0003] At present, the main preparation methods of polypeptides include synthesis method and hydrolysis method. The microbial fermentation method in the hydrolysis method directly uses the protease system produced in the microbial fermentation process to degrade the protein, and the steps of enzyme production, enzymatic hydrolysis and debittering are carried out simultaneously. It greatly simplifies the pr...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N13/00A23K10/18A23L29/00C12R1/07
CPCC12N13/00C12N1/20A23K10/18A23L29/065
Inventor 阮思煜马海乐李云亮纪耀勇王晓静周安奇谢鹏飞刘晓霜
Owner JIANGSU UNIV
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