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A strain of Bacillus subtilis that degrades AFB1

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, loss of nutrients, and unstable effects

Active Publication Date: 2015-10-14
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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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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  • A strain of Bacillus subtilis that degrades AFB1
  • A strain of Bacillus subtilis that degrades AFB1
  • A strain of Bacillus subtilis that degrades AFB1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1. Isolation of Taishan Bacillus subtilis

[0018] Collect the starch-rich Taishan soil, treat it in a water bath at 80°C for 1 hour, and use the dilution coating method on the starch-containing plate medium (5.0g of beef extract, 10.0g of peptone, 5.0g of sodium chloride, 5.0g of soluble starch, agar 20g and 1000ml of distilled water (pH=7.2) were cultivated for 1-3 days and then observed. Then carry out preliminary screening and purification, and use microscope to carry out Gram staining identification to determine whether it is Bacillus subtilis. Re-screen again, measure its amylase activity, and finally determine the bacterial strain.

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

[0020] (1) Preparation of soil dilution

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

Embodiment 2

[0034] Embodiment 2. Identification of Taishan Bacillus subtilis

[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: 5μl of 10×PCR buffer (containing 20mmol / L Mg2+), 4μl of 20umol / L dNTP, 1μl of each primer at 0.1OD / ml, 1μl of 5u / μl Taq enzyme, 3μl of ddH2O3, and 50μl of total DNA template.

[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...

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] Aflatoxins (Aflatoxius, AF) are secondary metabolites produced by several fungi such as Aspergillus flavus, Aspergillums nomius, A.nomius, and A.pseudotamarii , which is very harmful to the health of humans and livestock. In June 1960, 100,000 turkeys died suddenly in the suburbs of London, England. The reason was that the peanut meal imported from Brazil was contaminated by a poisonous substance from a fungus. After the autopsy, it was found that the liver was bleeding and the kidneys were swollen. Because the cause was unknown, it was determined to be Turkey X disease. After research, it was found that the cause of death of turkeys was caused by the fluorescent substance produced by Aspergillus flavus isolated from the feed, and this substance was named aflatoxin, which attracted worldwide attention. Later, research by...

Claims

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Application Information

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