Bacteriophage composition, bacteriostatic agent and application thereof
A phage and composition technology, applied in the field of bioengineering, can solve problems such as ineffective prevention and control of animal intestinal diseases
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Embodiment 1
[0036] Isolation, preparation and purification of phage
[0037] 1. Phage isolation
[0038]Add peptone solution to the feces samples collected from poultry farms, shake at room temperature for 2-4 hours, and take the supernatant at 5000×g min -1 Centrifuge for 30 min, and filter the supernatant with a 0.22 μm filter membrane.
[0039] Take 1ml of the supernatant and add it to 50ml of TY medium, then add 1ml of the overnight culture of ATCC 13124 Clostridium perfringens strain (TY overnight culture), wherein the overnight culture of ATCC 13124 Clostridium perfringens strain produces perfringens The concentration of Clostridium was 1 x 10 8 cfu / ml~1×10 10 cfu / ml.
[0040] Get 1ml supernatant and add in 50ml LB culture medium respectively, then add the overnight culture of 1ml Salmonella ATCC 13076, avian Escherichia coli E-0503 (isolated from a chicken farm in Zhengzhou, Henan) and Shigella flexneri CMCC 51572 respectively, Among them, the concentration of corresponding ba...
Embodiment 2
[0048] Phage lysis in vitro
[0049] Mix Clostridium perfringens, Salmonella, Escherichia coli and Shigella cultured overnight at a concentration ratio of 1:1:1:1, and the final concentration of the mixed bacteria is about 10 9 cfu / ml, centrifuged to remove medium, adjusted to OD with 0.85% saline 600 About 0.5. After mixing the 4 kinds of phages according to the titer ratio of 1:1:1:1, the final concentration of the mixed phages is about 10 10 pfu / ml, added to the above mixed bacterial solution (MOI is 0.1 and 1 respectively), cultivated at 37°C, and set up a bacterial control group at the same time, that is, no phage composition was added to the bacterial solution; After 10 hours, take 0.2ml to detect OD 600 value, compare the OD after adding the phage composition 600 Variety.
[0050] The result is as figure 2 As shown, starting OD in MOI 0.1 and 1 groups 600 The values are 0.51 and 0.52, respectively. After acting for 2 hours, compared with the control group, th...
Embodiment 3
[0052] Bacteriostasis Effect of Phage Composition in Poultry Breeding
[0053] Forty 1-day-old chicks were selected and randomly divided into 4 groups, 10 in each group. Among them, group A was fed normally as the control group, and group B was fed normally with phage composition at the same time, about 1×10 8 pfu / 0.1ml / cat, fed continuously for 7 days, group C was fed normally and added ampicillin drinking water (0.1% W / V), drank water continuously for 7 days, group D was the blank control group. On the 14th to 21st day, groups B and C were fed with phage composition and ampicillin respectively; groups A, B, and C were respectively fed with Clostridium perfringens, Salmonella, Escherichia coli and Shigella flexneri on day 22 Bacteria composition (approximately 10 8 ~10 9 cfu / 0.1ml / bird, the ratio is 10:1:1:1) to infect, the chickens were weighed on the 25th day, and the intestines were taken to analyze the occurrence of necrotic enteritis and scored (0 was normal small int...
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