Bacillus subtilis for degrading AFB1 (Aflatoxius B1)

A technology of Bacillus subtilis and microbial strains, applied in the direction of bacteria, microorganism-based methods, microorganisms, etc., can solve problems such as difficult large-scale production, unstable effects, and loss of nutrients

Active Publication Date: 2014-05-28
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Traditional aflatoxin detoxification methods include physical and chemical methods, including ammonification method, alkali method, high temperature method, oxidation method, and adsorbent method. These methods have unstable effects, large loss of nutrients, and difficulty in large-scale production. Disadvantages; therefore, the control of aflatoxin pollution urgently needs a technology with high efficiency, strong specificity and no pollution to feed and environment

Method used

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  • Bacillus subtilis for degrading AFB1 (Aflatoxius B1)
  • Bacillus subtilis for degrading AFB1 (Aflatoxius B1)
  • Bacillus subtilis for degrading AFB1 (Aflatoxius B1)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1. Isolation of Bacillus subtilis Taishan

[0018] The starch-rich Taishan soil was collected, treated in a water bath at 80°C for 1 hour, and then diluted and coated on a starch-containing plate medium (beef extract 5.0g, peptone 10.0g, sodium chloride 5.0g, soluble starch 5.0g, agar) 20g and distilled water 1000ml pH=7.2) were cultured for 1-3 days and observed after culturing. Then carry out primary screening and purification, and identify whether it is Bacillus subtilis by using a microscope to carry out Gram staining identification. Then re-screen, determine its amylase activity, and finally determine the strain.

[0019] Experimental process: pouring plate - preparation of gradient dilution - coating - culture - primary screening - re-screening - purification - preservation

[0020] (1) Preparation of soil dilution

[0021] Take soil samples near the flower beds in Tianwai Village, Mount Tai, remove 5 cm of the surface layer, and take 5-15 cm. Sampling...

Embodiment 2

[0034] Example 2. Identification of Bacillus subtilis Taishan

[0035](1) Taishan Bacillus subtilis was cultured on BPY agar plate (5g of beef extract, 10g of peptone, 5g of sodium chloride, 5g of glucose, 5g of yeast extract powder, 15g of agar and 1L of distilled water, pH=7.0) at 37°C for 48h Afterwards, its shape and structure were observed by optical microscope.

[0036] (2) Design of primers for PCR amplification of 16S rDNA sequence

[0037] Eubac27F: AGA GTT TGA TCC TGC CTC AG;

[0038] Eubac1492R: GGA TAC CTT GTT ACG ACT T

[0039] (3) PCR reaction system: 10×PCR buffer (containing 20mmol / L Mg2+) 5μl, 20umol / L dNTP 4μl, 0.1OD / ml primer 1μl, 5u / μl Taq enzyme 1μl, ddH2O33μl, total DNA template 50μl.

[0040] Reaction conditions: denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 48.2°C for 1 min, extension at 72°C for 2 min, 35 cycles; extension at 72°C for 10 min. After the reaction, 5 ul of the PCR product was electrophoresed in 1% agaros...

Embodiment 3

[0041] Example 3. Determination of Taishan Bacillus subtilis Degradation Aflatoxin Active Component

[0042] Pick Taishan Bacillus subtilis and culture it in 50ml BPY liquid medium for 8 hours, then inoculate the bacterium solution in 100ml BPY liquid medium with 6% inoculum amount, shake the flask for 24 hours at 37°C and 180r / min to obtain Bacillus subtilis Bacillus fermentation broth. Take 5ml of fermentation broth, centrifuge at 4°C for 20min (8000r / min), separate the supernatant and bacteria, and store the supernatant at 4°C for later use; wash the centrifuged bacteria with distilled water, then centrifuge, take the bacteria and add 5ml of distilled water to prepare The bacterial suspension was obtained for later use; then take 5ml of fermentation broth, centrifuge at 4°C for 20min (8000r / min), separate the supernatant and bacteria, add 5ml of distilled water to the bacteria, perform ultrasonic crushing on the cells, freeze and centrifuge the centrifuged supernatant The ...

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Abstract

The invention relates to a Taishan bacillus subtilis for degrading AFB1 (Aflatoxius B1). The bacillus subtilis is preserved in the China General Microbiological Culture Collection Center (CGMCC) since September 13, 2013, with a preservation number of CGMCC No. 8186, and the 16srDNA is shown in SEQ.NO.1. Experiments prove that after a fermentation liquor of the bacillus subtilis works with foldy feed at a temperature of 37 DEG C for 72 hours, active substances produced by the fermentation liquor can degrade AFB1 in the moldy feed; the bacillus subtilis has the advantages of high efficiency, mild acting effect, high safety, no influence on the original quality, simplicity of operations, low cost and the like, and is suitable for scale application in feed.

Description

(1) Field of Invention [0001] The present invention relates to a strain of Bacillus subtilis that degrades AFB1. (2) Background of the invention [0002] Aflatoxius (AF) is a secondary metabolite produced by several fungi such as Aspergillus flavus, Aspergillums nomius, A. nomius, and A. pseudotamarii. , it is very harmful to the health of humans and livestock. In June 1960, 100,000 turkeys died suddenly in a suburb of London, England, after peanut meal imported from Brazil was contaminated with a poisonous substance from a fungus. An autopsy revealed hemorrhage of the liver and swelling of the kidneys, which was classified as turkey x disease due to unknown etiology. After research, it was found that the cause of death of turkeys was caused by a fluorescent substance produced by Aspergillus flavus isolated from the feed, and the substance was named aflatoxin, which attracted worldwide attention. Later studies by many scholars have proved that aflatoxin can not only cause...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A23L1/015C12R1/125A23L5/20
Inventor 柴同杰孙玲玉
Owner SHANDONG AGRICULTURAL UNIVERSITY
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