Salmonella bacteriophage and application thereof
A Salmonella and phage technology, applied in virus/bacteriophage, application, food science, etc., can solve problems such as human health hazards and Salmonella pollution, and achieve high safety, small toxic and side effects, and strong cracking activity.
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Embodiment 1
[0023] Embodiment 1, the separation and preparation of bacteriophage PSA-6
[0024] The fecal liquid sewage sample among the present invention is collected from the animal experiment field of Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences;
[0025] The host bacteria was Salmonella enteritidis ATCC 13076.
[0026] Fecal sewage samples were filtered through double-layer filter paper, centrifuged at 8000 rpm for 20 min, and then the supernatant was filtered with 0.45 μm and 0.22 μm filter membranes respectively.
[0027] Take 50mL supernatant, add 500μl phage host bacteria (Salmonella) overnight culture, then add CaCl 2 After mixing the mother solution to a final concentration of 1mM, add 20mL of LB medium and place it at 37°C for 12-16h. On the next day, the above-mentioned culture was centrifuged at 12000 rpm for 10 min, and the supernatant was taken and 0.3% chloroform was added to form a phage stock solution.
[0028] Take three 1.5% ordinary nu...
Embodiment 2
[0030] Embodiment 2, amplification culture and purification of phage
[0031] On the double-layer plate forming phage plaques, use the tip of a pipette to pick up a single phage plaque with a larger diameter, inoculate it in 3-5mL LB medium, add 0.1mL of phage host bacterial solution, mix well, Incubate at room temperature for 15 minutes, incubate at 37°C for 10 to 14 hours, centrifuge at 12,000 rpm at 4°C for 10 minutes, take the supernatant, and add 0.3% chloroform.
[0032] Take 1 mL of freshly cultured host bacteria and add 0.1 mL of phage lysate (the ratio of a single phage culture to host bacteria is 1:1, 1:10 and 1:100, respectively). Incubate at 37°C for 20 minutes to make the phage particles adsorb to the host bacteria; add 100mL LB liquid medium, then add CaCl 2 The mother liquor was prepared to a final concentration of 1 mM, cultured with shaking at 37°C for 12 to 16 hours, centrifuged at 12,000 rpm at 4°C for 10 minutes, and the supernatant was collected to obtain...
Embodiment 3
[0043] Embodiment 3, the influence of temperature and pH on PSA-6 bacteriophage stability
[0044] Based on the PSA-6 phage of Example 2, it was formulated into 6×10 9 pfu / mL of purified phage, take 1.0mL respectively in 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C water bath for 1 hour, extract the sample and measure its titer after cooling in an ice bath, The result is as Figure 4 As shown, the activity of PSA-6 phages did not change significantly after being treated at 30-60°C for 1 hour; after being treated at 70°C for 1 hour, the activity decreased; after being treated at 80°C, all phages were inactivated.
[0045] Based on the PSA-6 phage of Example 2, it was formulated into 8×10 7 pfu / mL purified phage for use; prepare peptone water with pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 for use; : 9 volume ratio mixed, 37 ℃ of water bath action 2h, detect their potency respectively, the result is as follows Figure 5 As shown, when the pH of PSA-6 phage ...
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