Bacillus pumilus JY2 and applications thereof
A Bacillus pumilus, JY2 technology, applied in applications, bacteria, enzymes, etc., can solve the problems of polluting the air environment, affecting metabolism, waste of resources, etc., and achieve the effects of rapid growth, reduced crude fiber content, and short fermentation cycle
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Embodiment 1
[0032] Embodiment one: bacillus pumilus ( Bacillus pumilus ) Isolation and screening of JY2 CGMCC No.9150
[0033] 1. Isolation: Bacillus pumilus JY2 of the present invention was isolated from a winter wheat field (N 72 ° 50' 70.01 "E 40 ° 26' 51.73 ") in Osh Racecourse, Kyrgyz Republic, randomly selected multiple depths in the farmland A soil sample of 3cm-20cm was mixed, put into a sterile container, transported to the laboratory in a car refrigerator, and a strain of Bacillus pumilus was isolated by the plate dilution coating method, numbered JY2.
[0034] Separation steps: According to the gradient dilution method, weigh 10g of soil samples in 90mL sterile physiological saline, and perform gradient dilution after activation at 30°C for 30min. Select 10 -2 、10 -3 、10 -4 The diluted solution was spread on the LB agar medium plate, and each treatment was replicated three times, and cultured at 37°C. After the colonies grow out, pick colonies of different shapes, sizes, ...
Embodiment 2
[0041] Embodiment two: bacillus pumilus ( Bacillus pumilus ) Sequence determination and phylogenetic analysis of JY2 CGMCC No. 9150
[0042] 1. DNA extraction: JY2 genomic DNA was extracted by CTAB / NaCl method. The strain was inoculated in LB medium (peptone 10g, yeast extract / powder 5g, NaCl 10g, agar 17g, distilled water 1000mL natural pH, sterilized at 121°C for 20 minutes.), 37°C shaking culture overnight. Take 1.5ml culture and centrifuge at 12000rpm for 2min. Add 567 ul of TE buffer to the precipitate, pipette repeatedly to resuspend it, add 30 ul of 10% SDS and 15 ul of proteinase K, mix well, and incubate at 37°C for 1 h.
[0043] Add 100ul 5mol / L NaCl, mix well, then add 80ul CTAB / NaCl solution, mix well, and then incubate at 65°C for 10min. Add an equal volume of phenol / chloroform / isoamyl alcohol and mix well, centrifuge for 4-5min, transfer the supernatant to a new tube, add 0.6-0.8 times the volume of isopropanol, mix gently until the DNA precipitates, Can be...
Embodiment 3
[0051] Embodiment three: bacillus pumilus ( Bacillus pumilus ) Antibacterial performance test of JY2 CGMCC No. 9150
[0052]Escherichia coli 1565 and Staphylococcus aureus were used as indicator bacteria to test the antibacterial performance in vitro. Take 0.5 mL of Escherichia coli, Salmonella and Bacillus pumilus JY2 bacterial fluid, and inoculate it in 5 mL common broth medium at the same time, and inoculate 0.5 mL of Escherichia coli and Salmonella bacterial fluid in the same common broth culture medium respectively. cultured as a control group. After shaking culture at 37°C for 18 hours, the CFU method was counted by serial dilution. The results showed that after JY2 was co-cultivated with Escherichia coli, the number of viable bacteria was 16.00% lower than that of single-inoculation Escherichia coli; after JY2 was co-cultured with Salmonella, the number of viable bacteria was 11.91% lower than that of single-inoculation Salmonella. Therefore, Bacillus pumilus (Baci...
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