High-throughput screening method of streptomyces fradiae strains capable of producing neomycin

Abstract

The invention provides a high-throughput screening method of streptomyces fradiae strains capable of producing neomycin. The method includes the steps: (1) inoculating single colony spores to perforated plates of a slant culture medium and a fermentation culture medium, taking supernate by sulfuric acid acidification and centrifugation after fermentation culture, and diluting the supernate with water; (2) performing reaction on fermentation diluents and 0.25% phosphotungstic acid to generate precipitates; (3) measuring an OD (optical density) value by a microplate reader and performing secondary screening; (4) inoculating spores of the strains to be secondarily screened to a boiling tube slant or eggplant bottle slant and a shake flask for fermentation culture, and acidifying the spores bysulfuric acid; (5) filtering and diluting acidizing fluids, performing reaction on the acidizing fluids and 0.25% phosphotungstic acid in an elisa plate and measuring the OD value; (6) precisely screening selected strains after shake-flask fermentation, and measuring biological values to obtain high-producing strains. The high-throughput screening method is convenient, rapid and accurate in operation, the screening efficiency of neomycin mutation breeding is improved to a great extent, and the probability of acquiring the neomycin high-producing strains is greatly increased.

Description

technical field [0001] The invention belongs to the technical field of microorganism culture, and in particular relates to a high-throughput screening method for neomycin-producing Streptomyces freundii strains. Background technique [0002] Neomycin sulfate is an aminoglycoside antibiotic produced by fermentation and extraction of Streptomyces freundii, which can inhibit the biosynthesis of protein, thereby inhibiting and killing bacteria. It has strong bactericidal effect on Gram-negative bacteria and some Gram-positive bacteria; it has inhibitory effect on actinomycetes, Leptospira, and amoeba. It is commonly used in the treatment and control of enteritis, diarrhea, infectious rhinitis, respiratory tract infection and other diseases caused by neomycin sulfate-sensitive bacteria infection. In the past, most of the Streptomyces freundii strains used to produce neomycin were obtained by traditional screening methods, such as shaking flask culture, liquid phase or biological...

AI Technical Summary

Problems solved by technology

In the past, most of the Streptomyces freundii strains used to produce neomycin were obtained by traditional screening methods, such as shaking flask culture, liquid phase or biological method for titer detection, which had high investment, long screening period, small screening amount, and single screening method. Low screening efficiency and other shortcomings, so the production capacity of neomycin climbed slowly

In recent years, there have been articles and patents published on the application of ultraviolet spectrophotometry in the method of rapid detection of gentamicin concentration in fermentation broth and the application of enzyme-labeled method in high-throughput screening of tylosin. There has been a breakthrough in the screening of tylosin and tylosin-producing bacteria. Chinese patent CN106244458 discloses a high-throughput screening method for tylosin-producing strains, but the application range is narrow and the screening method is rough. There are no reports on rapid and simple detection methods and high-throughput screening in strain breeding

Images