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Screening and identification of microorganism for degradation of deoxynivalenol (DON) in feeds

A technology for microbial screening and vomitoxin, which is applied in the determination/testing of microorganisms, microorganisms, and separation of microorganisms, etc. It can solve the problems of low DON degradation efficiency, long degradation time, and difficult to popularize applications, etc.

Inactive Publication Date: 2019-04-12
JIANGSU VOCATION & TECHNICAL COLLEGE OF FINANCE & ECONOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, researchers at home and abroad have screened and isolated different strains to degrade DON and made some research progress. However, the degradation efficiency of DON is not high, the degradation time is long, and there are application defects that are not easy to promote.

Method used

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  • Screening and identification of microorganism for degradation of deoxynivalenol (DON) in feeds
  • Screening and identification of microorganism for degradation of deoxynivalenol (DON) in feeds
  • Screening and identification of microorganism for degradation of deoxynivalenol (DON) in feeds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The screening of degrading DON microorganisms, the specific implementation steps are as follows:

[0034] 1. DON standard curve and its standard high performance liquid chromatography

[0035] The HPLC detection conditions are: DAD detector, column temperature 35°C, injection volume 50 μL, mobile phase: 20% methanol; flow rate: 0.8mL / min; detection wavelength: 218nm. attached figure 1 As shown, the standard curve obtained by HPLC-DAD measuring different concentrations of DON standard products, the relationship between the peak area and the DON concentration obtained from this curve is: y=20.116x+0.7005, the linear relationship coefficient R2=0.9999, indicating that DON standard products The concentration was highly linear with the peak area detected by DAD.

[0036] attached figure 2 Shown is the chromatogram of DON standard substance with a concentration of 20 μg / mL. The peak time of DON standard product is 5.975 minutes. Therefore, when detecting the content of DON ...

Embodiment 2

[0045] Select the strains with significant degradation effect on DON to carry out the degradation effect test, respectively take the bacteria suspension with the concentration of 1×107 CFU / mL that has the degradation effect after re-screening, and inoculate them with a final concentration of 20 μg / mL according to the 10% inoculum amount. Shake flask culture at 120r / min at 30°C in salt liquid medium, take samples at 6h, 12h, 24h, 36h, and 48h to measure the DON content, calculate the degradation rate, and compare the degradation effects of the target strains for re-screening. The strain with the best DON degradation effect was the final target strain.

[0046] The DON content was determined by HPLC, and the DON degradation rate was calculated, and the degradation rate was calculated according to formula 1. The chromatographic column is Agilent ZorbaxSB-C18 column, the specification is 4.6mm×150mm, 5μm; HPLC detection conditions: DAD detector, column temperature 35℃, injection v...

Embodiment 3

[0058] The identification of bacterial strain, specific implementation steps are as follows:

[0059] 1. Morphological identification: mark the target strain on the LB plate, culture at 30°C until a single colony grows, and observe its colony morphology on the surface of the LB medium.

[0060] attached Figure 8 The colony form of the DON-degrading strain JC-5 in LB medium is characterized by: the colony is milky white, with neat edges, round, opaque, round and smooth surface, uniform texture, and easy to pick.

[0061] attached Figure 9 It is the thalline morphology of the bacterium under the microscope after Gram staining. The bacterium is a Gram-negative bacterium with a short rod shape and no spores.

[0062] 2. 16SrDNA identification: Use the Omega Bacteria Genomic DNA Extraction Kit to extract the total DNA of the strain and use bacterial 16sr DNA amplification primers 27F: 5-AGAGTTTGATCCTGGCTCAG-3 and 1492R: 5-GGTTACCTTGTTACGACTT-3 to amplify the 16s r DNA fragment,...

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Abstract

The present invention discloses a screening and identification of a microorganism for degradation of deoxynivalenol (DON) in feeds. The screening and identification is characterized by comprising thefollowing steps: (1) a strain of Jiangdu comamonas is screened and separated from chicken intestinal tracts; (2) the Jiangdu comamonas is subjected to an ICR mouse acute toxicity test to determine that the Jiangdu comamonas is highly safe and harmless to human body; and (3) after the Jiangdu comamonas samples are diluted properly with sterile physiological saline, the diluted Jiangdu comamonas samples inoculate into a culture medium with a certain concentration of DON with an inoculation amount of 10%, and shake culture is conducted at 120 r / min and 30 DEG C. Advantages are as follows: the microorganism has a DON degradation rate of 56.56% within 12 h, has the DON degradation rate of 91.12% within 24 h and has the DON degradation rate as high as 96.76% within 36 h. The successful screeningof the microorganism provides a feasible reference research method for isolation and screening of other toxin-producing molds, and also lays a foundation for an application of DON biodegradation.

Description

technical field [0001] The invention relates to the technical field of biodegradation, in particular to screening and identification of feed vomitoxin degrading microorganisms. Background technique [0002] Deoxynivalenol (DON for short), also known as vomitoxin, scientific name is 3,7,15-trihydroxy-12,13-epoxytrichothecene-9-en-8-one, It is a secondary metabolite of filamentous fungi such as Fusarium graminearum and Fusarium yellow that contaminate wheat, corn and other cereal molds. DON is highly harmful to humans and animals, and can cause abdominal pain, diarrhea, vomiting, gastroenteritis and other diseases in humans and animals. Its toxicity is related to the epoxy groups on C12 / C13 and C3-OH on the DON molecule. Studies have shown that when the DON content in feed or grain exceeds 1000 μg / kg, it will cause harm to human and animal health. The Ministry of Agriculture of my country made clear regulations on the limit standard of DON in animal feed in 2011, and its valu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/02C12N1/20C12Q1/04C12R1/00
CPCC12N1/02C12N1/20C12Q1/04
Inventor 窦勇胡佩红李逸鹤顾鹏程董静姚妙爱
Owner JIANGSU VOCATION & TECHNICAL COLLEGE OF FINANCE & ECONOMICS
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