Bacillus subtilis AP139 and fermented microbial inoculum thereof, and application method of bacillus subtilis AP139 and fermented microbial inoculum
A technology of Bacillus subtilis and AP139, applied in botany equipment and methods, microbial-based methods, biochemical equipment and methods, etc., can solve problems such as double infection, endogenous infection, and secondary pollution, and achieve reduction Incidence and Harm Effects
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Embodiment 1
[0034] Screening of a Bacillus subtilis AP139
[0035]More than 200 strains of bacillus were isolated from the intestinal tract of animals, and these more than 200 strains of bacillus were tested for Escherichia coli, Staphylococcus aureus, Salmonella paratyphi, Pasteurella 439 (low-toxic Pasteurella), and Pasteurella 2010 ( Toxic Pasteurella), Pasteurella 2011 (virulent Pasteurella) bacteriostasis test, the results showed that the Bacillus 139 strain had the best inhibitory effect on Pasteurella, reaching 18-25mm, and the Bacillus 139 strain was tested by biochemistry and molecular biology. It was identified as Bacillus subtilis and named as Bacillus subtilis AP139. It was preserved in China Center for Type Culture Collection as CCTCC M2014053, and its 16S rDNA sequence is shown as SEQ ID NO:1.
[0036] Table 1. The inhibitory effects of some Bacillus strains on Pasteurella and other pathogenic bacteria
[0037]
Embodiment 2
[0039] A preparation method of Bacillus subtilis (Bacillus subtilis) AP139 fermentation agent:
[0040] A. Bacillus subtilis AP139 (preservation number: CCTCC M2014053) was streak cultured on LB solid medium, the culture temperature was 30-37°C, and the culture time was 18-36h;
[0041] B, the preparation of bacillus subtilis (Bacillus subtilis) AP139 fermented liquid: the bacillus subtilis (Bacillus subtilis) AP139 that step A is cultivated is picked single clone, is placed in 10ml EP tube (4ml LB substratum is housed), The culture temperature is 37°C, the rotation speed is 200r / min, and the culture time is 24h. Then use LB liquid medium to adjust the bacterial concentration to 10 10 CFU / ml, take 4ml and inoculate in 100ml medium for shake flask fermentation culture, the fermentation temperature is 37°C, the rotation speed is 200r / min, and the fermentation time is 24h. The shake flask fermentation medium is LB medium, and its composition is: tryptone 10g, yeast extract 5g, ...
Embodiment 3
[0044] Bacillus subtilis (Bacillus subtilis) AP139 antibacterial activity verification:
[0045] A. Take 3 LB nutrient agar plates, take 20 μL of activated Pasteurella bacteria liquid, and evenly spread it on one LB solid plate, take 20 μL of activated Salmonella bacteria liquid, evenly spread it on another LB solid plate, take 20 μL of activated Escherichia coli liquid was evenly spread on the third LB solid plate, and placed in an incubator at 37°C for 10 minutes;
[0046] B. Utilize a puncher to punch holes in the culture dish, and the hole diameter is 5 mm;
[0047] C. After centrifuging the Bacillus subtilis AP139 fermentation broth at 12,000 r / m for 1 min, take 20 μL of the supernatant and add it to the well of the culture dish, and incubate in an incubator at 37° C. for 24 h;
[0048] D, the results show that the diameters of the inhibition zone of Bacillus subtilis (Bacillus subtilis) AP139 to Pasteurella, Staphylococcus aureus, Escherichia coli and Salmonella are 22m...
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