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Method for rapidly screening high-titer tylosin strain by taking lipase activity as index and method for rapidly screening tylosin medium by taking lipase activity as index

A technology of tylosin and high titer, which is applied in the field of rapid screening of high titer strains and culture media of tylosin, can solve the problems of difficult optimization of breeding media, long fermentation period and other problems, and achieve the improvement of fermentation titer , reduce workload and improve work efficiency

Inactive Publication Date: 2014-03-19
宁夏泰瑞制药股份有限公司
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This screening method often leads to many excellent strains being ignored in the screening process; in addition, in the screening process, due to the long fermentation period, it brings difficulties to the selection of strains and the optimization of the medium

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: strain screening

[0019] 1. The spore suspension of Streptomyces freundii is obtained as follows: Add 9 mL of sterile water to the fresh slant of the cultured Streptomyces freundii strain, scrape off the spores indicated by the culture with an inoculation loop, first gently, then gradually force Scrape to resuspend the spores. Pour the crude spore suspension into a centrifuge tube, shake vigorously on the shaker for about 1 min, filter the suspension with a test tube equipped with absorbent cotton, pour the filtrate into a centrifuge tube, and centrifuge at 3000r / min for 10min To make the spores precipitate, after stopping the centrifugation, pour off the supernatant immediately, shake the centrifuge tube on the shaker for a few seconds, disperse the spore sediment in the remaining sterile water, and then wash it twice with sterile water, Put it into a triangular flask with glass beads and shake it for 10-15min to break up the spore mass, filter it with ...

Embodiment 2

[0025] Example 2: Medium Screening

[0026] Take 3-6 cm 2 Streptomyces freundii strains on fresh slant were inserted into seed medium (seed bottle medium: bean cake powder 4 g / L; yeast extract 4 g / L; corn steep liquor 5 g / L; calcium carbonate 3 g / L. 120°C Sterilize for 30 min), cultivate for 44-50 h under the conditions of temperature 27-29 ℃, humidity 30-50%, shaker speed 230 r / min.

[0027] Inoculate the cultivated seed liquid with 5% inoculum amount into the shake flask medium based on different nitrogen sources, shake at 220r / min, 29°C, and ferment for 144-160h. The lipase activity is measured every 24 hours after fermentation and cultivation. The determination method refers to the determination method of lipase in the industry standard "General Test Method for Industrial Enzyme Preparations" approved and issued by the Ministry of Industry, and the enzyme activity on the first day is used as an index to screen the new formula. , combined with the shake-flask titer after ...

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Abstract

The invention relates to a method for rapidly screening high-titer tylosin strain by taking lipase activity as an index and a method for rapidly screening a tylosin medium by taking lipase activity as an index. The method for rapidly screening high-titer tylosin strain by taking lipase activity as the index comprises: adding rhodamine B into a separation plate medium with plant oil as a main carbon source, coating with streptomyces fradiae spore suspension for plate culture, after culture is finished, choosing a single colony with a large ratio of bacterium colony hydrolysis circle diameter to bacterium colony diameter; inoculating a portion of the single colony to an inclined surface, inoculating the other portion of the single colony to a flask-shaking medium, and investigating the tilter. By adding rhodamine B into the medium the hydrolysis circle shows pink color; and according to the ratio of the bacterium colony hydrolysis circle diameter to the bacterium colony diameter, an excellent bacterial strain accord with requirements is chosen, and thus the workload is reduced, the screening efficiency is improved and the probability that a high-yield bacterial strain is chosen is increased. In a similar way, by utilizing lipase activity under different nitrogen-source conditions of streptomyces fradiae as the index, a medium formula suitable for tylosin growth is chosen, and thus the fermentation tilter is improved.

Description

technical field [0001] The invention belongs to the technical field of biological fermentation, and in particular relates to a method for rapidly screening strains with high titer of tylosin and a culture medium by using lipase activity as an index. Background technique [0002] Tylosin (Tylosin) is a special macrolide antibiotic for poultry and livestock, which was discovered by Mocuire in 1955 in Streptomyces freundii ( Streptomyces fradiae ) found in the culture medium. Tylosin is effective against Gram-positive bacteria and certain negative bacteria (such as Staphylococcus aureus, Streptococcus pyogenes, Diplococcus pneumoniae, Corynebacterium pyogenes, etc.), and has special effects on spirochetes and mycoplasma. It not only has a unique curative effect on chicken septicemia, porcine epidemic pneumonia, red disease and pneumonia caused by Mycoplasma bovis, but also has a significant role in promoting the growth of livestock and poultry, so it is widely used in livestoc...

Claims

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Application Information

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IPC IPC(8): C12Q1/44C12Q1/04C12R1/54
Inventor 王义李康乐
Owner 宁夏泰瑞制药股份有限公司
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