A kind of Enterobacter sakazakii bacteriophage espyzu05 and use thereof
A technology of Enterobacter sakazakii and espyzu05, which is applied in the field of bioengineering, can solve problems such as pollution, dairy product production and processing hazards, and enhanced resistance, and achieve the effect of inhibiting reproduction
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Embodiment 1
[0029] Embodiment 1, phage isolation and purification preparation
[0030] Phage isolation
[0031] The sample of the present invention is collected from the sewage of Yangda Kangyuan Dairy Co., Ltd., Jiangsu Province. Take 30mL sample into a 50mL centrifuge tube, centrifuge at 5000×g for 10min, take 5mL supernatant and add it to 5mL nutrient broth liquid medium, and add 100μL Enterobacter sakazakii CICC 21569 in logarithmic growth phase at the same time, place at 37℃ Cultivate overnight on a shaker, and the next day, transfer the cultures in each test tube to a sterile centrifuge tube, centrifuge at 5000×g for 10 minutes at 4°C, take the supernatant and pass it through a 0.22 μm microporous membrane to sterilize, and obtain the phage stock solution. It can be stored in a refrigerator at 4°C.
[0032] Dilute the phage stock solution with sterile SM buffer, take 100 μL of appropriate dilution of phage liquid and 100 μL of Enterobacter sakazakii CICC 21569 in logarithmic growt...
Embodiment 2
[0043] Embodiment 2, the inhibitory effect of phage EspYZU05 on Enterobacter sakazakii bacterial biofilm
[0044] Enterobacter sakazakii in the logarithmic growth phase was diluted with nutrient broth medium, and the final concentration was 1×10 7 CFU / mL, experimental group: add diluted bacterial solution (10 7 CFU / mL) and phage EspYZU05 suspension (10 5 PFU / mL, 10 8 PFU / mL) each 150μL; control group: add diluted bacterial solution (10 7 CFU / mL) and 150 μL of nutrient broth medium; blank group: add 300 μL nutrient broth medium to each well; incubate at 37°C for 24 hours; take out the 96-well plate, suck out the suspended bacteria, and wash the 96-well plate with sterile PBS 3 Once, blow dry to fully remove planktonic cells; add 200 μL of crystal violet staining solution with a concentration of 0.2% to each well, and stain for 30 minutes; suck out the staining solution, and thoroughly wash the 96-well plate with sterile PBS until the eluate is colorless, blow Dry; add 200 μ...
Embodiment 3
[0046] Embodiment 3, the bacteriostasis of bacteriophage EspYZU05 in culture medium
[0047] The prepared nutrient broth medium was divided into 20mL stoppered test tubes and sterilized at 121°C for 15min. Enterobacter sakazakii (10 5 CFU / mL) to inoculate 100 μL into 10mL medium, add 100 μL phage EspYZU05 suspension (10 7 PFU / mL), 100 μL of SM buffer was added to the control group, and the inoculum was placed in a 37°C incubator and a 25°C incubator for constant temperature cultivation. The culture was sampled 1mL every 6h, and the wavelength at 600nm was measured with a spectrophotometer. For the absorbance value, 3 parallels were set for each group, and the average value was used for analysis.
[0048] The result is as Figure 6 As shown, as the culture time prolongs, the OD of the control group 600nm Rapid growth, reaching 0.5, 0.25 and 0.1 at 24h (37°C) and 24h (25°C) respectively, while the OD of the experimental group added with EspYZU05 suspension 600nm Always kept...
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