CN115851533B - Bacillus thuringiensis and application thereof in inhibiting livestock and poultry feed mould - Google Patents
Bacillus thuringiensis and application thereof in inhibiting livestock and poultry feed mould Download PDFInfo
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Abstract
The invention relates to a bacillus thuringiensis and application in inhibiting livestock and poultry feed mould, which belongs to the technical field of microorganisms, wherein a strain of bacteria is isolated from an ecological pig farm, the strain is identified as bacillus thuringiensis (Bacillus thuringiensis) through 16S rDNA sequencing comparison, the strain is named as bacillus thuringiensis zhu-64, and the strain is delivered to China center for preservation of typical culture collection of Wuhan in China for 25 days in 2022, wherein the preservation number is CCTCC NO: m20221173. The bacillus thuringiensis can effectively inhibit the hypha growth and spore production of mould, has obvious mould inhibition effect on livestock and poultry feed, and has wide application prospect.
Description
Technical Field
The invention relates to bacillus thuringiensis and application thereof in inhibiting livestock and poultry feed mould, belonging to the technical field of microorganisms.
Background
Mycotoxins (aflatoxins, vomitoxins, etc.) are secondary metabolites produced by certain aspergillus fungi and have strong teratogenic, oncogenic and mutagenic effects. Aspergillus flavus is an important storage mould, and the aflatoxin B1 produced by the aspergillus flavus has extremely strong toxicity and can seriously damage the digestive function and the immune system of livestock and poultry organisms. The livestock and poultry feed is generally severe in storage and use environment, high in temperature and humidity, high in mildew possibility, easy to cause generation of mycotoxins such as aflatoxin and the like, the feed conversion rate is affected, the livestock and poultry production performance is reduced, and the mycotoxins can be accumulated in organs and tissues such as livers, kidneys and muscles of livestock and poultry, so that the health of the livestock and poultry and the food safety of human beings are seriously endangered.
Disclosure of Invention
The invention provides the bacillus thuringiensis and the application thereof in inhibiting the fungus of the livestock and poultry feed, which can effectively inhibit the hypha growth and spore production of the fungus and has remarkable effect on inhibiting the fungus in the livestock and poultry feed.
The technical scheme for solving the technical problems is as follows:
one of the objects of the present invention is to provide a Bacillus thuringiensis (Bacillus thuringiensis) strain designated zhu-64, which has been submitted for preservation of the strain.
[ biological preservation Specification ]
Preservation unit: china center for type culture Collection;
preservation address: chinese university of Wuhan;
preservation date: 2022, 25 th 07;
preservation number: cctccc NO: m20221173;
classification naming: bacillus thuringiensis zhu-64Bacillus thuringiensiszhu-64。
The 16S rDNA of the bacillus thuringiensis zhu-64 has a sequence table SEQ ID NO:1, and a nucleotide sequence shown in the specification.
The second purpose of the invention is to provide the application of the bacillus thuringiensis in inhibiting the livestock and poultry feed mould.
Further, the feed includes corn.
The invention separates a strain of bacteria from a pig farm, identifies the bacteria as bacillus thuringiensis (Bacillus thuringiensis), and names the bacteria as bacillus thuringiensis zhu-64.
Culturing Bacillus thuringiensis zhu-64 on NA culture medium at 25deg.C for 48 hr, wherein colony is round or elliptical, has irregular edge, and has opaque micro-bump in the form of drop wax; gram staining is purple, is gram-negative bacteria, and has oval rod shape under microscope, size of (1.2-1.8) μm x (3.0-5.0) μm, short chain or long chain arrangement, oval spore, close to middle growth, and micro-enlargement of spore capsule.
The invention discovers that bacillus thuringiensis zhu-64 has obvious inhibition effect on the hypha growth and spore colony of the mould, and can effectively inhibit the hypha growth and spore production of the mould.
Experiments show that the bacillus thuringiensis zhu-64 has obvious inhibition effect on the growth of mould in the feed, and the feed treated by the bacillus thuringiensis zhu-64 does not agglomerate.
The invention has the beneficial effects that: the bacillus thuringiensis zhu-64 separated by the invention has obvious inhibition effect on the hypha growth and spore colony of the mould, and the feed treated by the bacillus thuringiensis zhu-64 has no mould hypha and no caking basically, and has good antibacterial effect on mould control applied to livestock and poultry feed and wide application prospect.
Drawings
FIG. 1 is a photograph showing the inhibitory effect of a strain on the growth of mycelium and spore colonies of Aspergillus flavus 1-3;
FIG. 2 is a photograph showing the inhibitory effect of the strain on the growth of Aspergillus flavus in livestock and poultry feed;
FIG. 3 is a genetic evolution analysis of Bacillus thuringiensis zhu-64.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
1. Separation and purification of bacillus thuringiensis
1.1 test sample
6 parts of pig manure and an environmental sample are collected from a longevity ecological pig farm.
1.2 treatment of samples
1g of each fresh sample is taken, 10mL of sterile water is added into each sample, the mixture is sufficiently vibrated to obtain suspension, 100ul of suspension is sucked and coated on a solid NA culture medium plate by using a coater, and the suspension is cultured for 24 hours at the constant temperature of 37 ℃. And (3) selecting a typical colony (namely, the colony is round or oval, the edge is irregular, and the opaque micro-bulge is in a drop wax shape) for purification, so as to obtain a pure culture strain.
1.3 16S rDNA sequence analysis and identification
The extracted genomic DNA of the strain was used as a template, and bacterial 16S rDNA primers (27F (5)
'-AGAGTTTGATCCTCCTCAG-3') and 1492R (5'-GGATACCTT GTTACGACTT-3')), the PCR products are detected by using a 1.0% agarose gel electrophoresis method, the size of the target fragment to be detected is primarily judged according to the relative position of the Marker, and the length of the DNA fragment obtained by PCR amplification is 750-1500 bp.
The amplified PCR product was sent to the China big gene company for DNA sequence determination.
The determined base sequences were subjected to BLAST alignment in GenBank.
1.4 Aspergillus flavus separation and purification
Selecting moldy feed, washing with sterile water for 3-5 times, air drying, sterilizing with 75% alcohol, sterilizing with 0.1% mercuric solution surface, washing with sterile water, inoculating into PDA culture medium, culturing and observing in 4-6 pieces per dish in a constant temperature oven at 28deg.C. After hypha germination, a typical colony (namely, the front color of the colony changes from white to yellow and yellow-green along with the growth of the colony, and is semi-fluff) is selected and transferred for 3 times, and after 7d, a puncher with the inner diameter of 0.5cm is used for punching a bacterial cake from the edge of the colony, and transferred to a PDA flat plate for purification culture, and the standard is 1-3.
Strains with dense hypha growth, regular colony and no pollution on the PDA plate are stored in a 1.8ml centrifuge tube and stored in a refrigerator at 4 ℃.
1.5 morphological identification of Aspergillus flavus
The front color of the colony changes from white to yellow and yellow-green along with the growth of the colony, and the colony is semi-villiated. The spore turns brown after maturing, the surface of the spore is flat or has radial grooves, the back surface of the spore is colorless or brown, and the conidiophore is loose and radial under observation of a low power microscope, and then is loose and columnar. And the conidiophores are rough when observed by a film-making microscopic examination. The top bag is flask-shaped or nearly spherical. The conidium grows on the small peduncles in a chain shape, and small protrusions, spheres and roughness are arranged around the conidium, so that the conidium can be judged to be aspergillus flavus Aspergillus flavus (1-3).
1.6Zhu-64 inhibition of growth of Aspergillus flavus 1-3
The PDA plate is perforated by a cross method, four holes are perforated, and the distance from the center point to the holes is 2.5cm. The center of the plate was inoculated with 1-3 cakes (0.5 cm) of Aspergillus flavus and four wells were inoculated with zhu-64 bacteria solution for 3 replicates. A blank CK was set up and inoculated with sterile water in four wells. After 5-7d, the length of the yellow aspergillus 1-3 bacterial cake is measured by using a crisscross method, and calculation and analysis are carried out on the data.
1.7Zhu-64 inhibition of mould growth in livestock and poultry feeds
Selecting feed (comprising 63% of corn, 16% of soybean meal, 6% of northeast DDGS (distillers dried grains with corn) and soluble matters thereof, 5% of wheat middling, 4% of bran, 3% of northeast rice bran and 3% of premix compound) for treatment.
10mL zhu-64 bacteria are added into 100g feed and mixed uniformly; the addition amount is 10 8 -10 9 Individual/kg feed.
The control group was prepared without mold inhibitor and with commercial calcium propionate (2 g/1kg feed) as mold inhibitor, 10mL of water was added separately and mixed well.
The feed which is not treated by the mould inhibitor, treated by the mould inhibitor (calcium propionate) and treated by zhu-64 is firstly exposed to air for 24 hours, and then the feed is put into a plastic bottle for sealing and is cultured at the temperature of 40 ℃. 1g of feed was weighed every 1d, diluted 10000 times with sterile water, and counted by a blood cell counting plate.
2 results and analysis
2.1 16S rDNA sequence analysis and identification
A strain of bacteria was isolated from pig farm and the 16S rDNA sequence was sequenced after PCR reaction. Sequencing results BLAST alignment was performed in GenBank.
After alignment, other bacillus gene sequences (Bacillus thuringiensis: MG709181.1, KU898281.1; bacillus toyonensis: ON933850.1, ON 933868.1) closest to zhu-64 homology were selected to build a evolutionary tree, and analyzed. Referring to FIG. 3, zhu-64 has a homology of >99% with respect to MG709181.1 of Bacillus thuringiensis. It can be identified as Bacillus thuringiensis (Bacillus thuringiensis), designated as Bacillus thuringiensis zhu-64, and delivered to China center for type culture collection, with a collection number of CCTCCNO: m20221173.
2.2 inhibition of growth of Aspergillus flavus 1-3 by Bacillus thuringiensis zhu-64
TABLE 1zhu-64 inhibition of growth of Aspergillus flavus 1-3
As can be seen from the results of Table 1 and FIG. 1, the colony diameter of the inoculated cake of the facing cultured Aspergillus flavus 1-3 was significantly smaller than that of the control group. The antibacterial rate of the bacillus thuringiensis zhu-64 on the aspergillus flavus 1-3 reaches 60%, and the antibacterial effect is obvious. Referring to FIG. 1, the opposing cultured Aspergillus flavus 1-3 are substantially free of scattered colonies. The inhibition rate of zhu-64 to the scattered colony of aspergillus flavus 1-3 reaches 100%, so that bacillus thuringiensis zhu-64 can effectively inhibit the hypha growth and spore production of aspergillus flavus.
2.3 inhibition of mould growth in livestock and poultry feeds by Bacillus thuringiensis zhu-64
(1) Influence of Bacillus thuringiensis zhu-64 on the number of mould spores in livestock and poultry feed
The number of mold spores in 1.7 above was recorded by hemocytometer.
Referring to Table 2, it was found through experiments that after treatment with Bacillus thuringiensis zhu-64, the number of mould spores of the feed was significantly reduced, and the spore inhibition rate was between 29% and 58%. Compared with the calcium propionate mould inhibitor, the inhibition rate is higher than 16%, and the bacillus thuringiensis zhu-64 has better inhibition effect on mould spore growth, and can be applied to mould control of livestock and poultry feed.
TABLE 2Zhu-64 inhibition of mold spore growth in feeds
(2) Effect of Bacillus thuringiensis zhu-64 on mold growth in livestock and poultry feeds
Referring to FIG. 2, the results of the test in the blank, commercially available mold inhibitor treated and Bacillus thuringiensis zhu-64 treated groups are shown in the order from left to right. After 30d of experimental treatment, the growth of mould in the feed was observed, and it can be seen that mould in the left blank control treated feed (no bacteriostatic agent added) and the left commercial mould inhibitor (calcium propionate) treated feed grew rapidly, covered the feed surface, and the feed had caking. Whereas the right Bacillus thuringiensis zhu-64 treated feed was substantially free of mold hyphae and did not cake.
Therefore, the bacillus thuringiensis zhu-64 provided by the invention can inhibit the generation of mycotoxin mainly by inhibiting the growth of hyphae and spores of mould, can effectively inhibit the growth of the hyphae and spores of mould, has a better lasting inhibition effect, and has a better inhibition effect than the conventional mould inhibitor calcium propionate on the market, and the bacillus thuringiensis zhu-64 has little influence on feed, does not cause the phenomena of feed caking and the like, and has a good application prospect when being used as a mould inhibitor for preventing and controlling the mould of livestock and poultry feed.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (3)
1. Bacillus thuringiensis @Bacillus thuringiensis) zhu-64, deposited with the China center for type culture Collection, having a accession number of university of Wuhan, china, and having a accession number of: cctccc NO: m20221173.
2. Use of bacillus thuringiensis according to claim 1 for inhibiting mould in livestock and poultry feed.
3. The use according to claim 2, wherein the feed comprises corn.
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