Separated coliphage and application as biological fungicide in foods and anti-infection contamination thereof
A technology of Escherichia coli and bacteriophage, applied in the field of bioengineering to prevent pollution
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Embodiment 1
[0022] Phage Screening and Purification
[0023] (1) Sample collection and processing
[0024] The samples in the present invention were collected from Zhuhai, including untreated sewage from the Fifth Affiliated Hospital of Sun Yat-Sen University, sewer sewage in Zhuhai City, seawater at the sewage discharge estuary, poultry farms in Zhuhai City, pig farms in Zhuhai City, and slaughterhouses in Zhuhai City.
[0025] The collected samples were adjusted with NaOH to make the pH about 7.0, centrifuged at 4000g / min for 15min, and filtered with a 0.45um filter membrane to obtain the supernatant. . Take 75ml of the sample after filter sterilization, and add 25ml of 4 times LB medium.
[0026] (2) Phage-specific amplification of Escherichia coli in the sample
[0027] Add pre-amplified Escherichia coli (OD about 1.0, adding ratio 1:100) to the processed sample, place it on a constant temperature shaker at 37° C., and cultivate it at 250 g / min for 6-8 hours.
[0028] (3) Determina...
Embodiment 2
[0037] Phage count method (titer)
[0038]Dilute the obtained phage sample by 10 times, take 100 ul of the sample with a certain dilution ratio, lay a double-layer plate with the method in the step (4) described in Example 1, get the appropriate proportion of the plate to calculate the number of plaques, one A plaque represents a phage monomer.
[0039] Bacteria counting method (titer)
[0040] Counting by standard plate method
[0041] Phage Stability Assay
[0042] 1. Effect of temperature on phage
[0043] On the day before the experiment, the titer of the phage was detected, and the phage samples were placed at 4°C, room temperature, 37°C, 50°C, and 70°C respectively, and the titer was measured after incubation for 1 hour. The effect of temperature on the phage sample was thus obtained.
[0044] Incubate the phage at 4°C, room temperature and 37°C, and measure the titer at 1d, 5d, 10d, 15d, and 30d
[0045] The results showed that the titer of phage BPH-EC-5 hardly c...
Embodiment 3
[0061] Toxicology experiment
[0062] Kunming mice, clean grade, weighing 22±3g, 2 months old (certificate number: SCXK (Beijing) 2004-0001), were provided by the Experimental Animal Research Center of Peking Union Medical College. The experimental animals were kept in a clean-grade feeding room with a room temperature of 18-22°C, a relative humidity of 40%-70%, and a 12-h sunshine and dark cycle. 20 experimental mice, half male and half male, after 3 days of adaptive feeding, were randomly divided into two groups (phage group, control group), 10 mice in each group (5 male and female), and the phage dose of 1011pfu / kg was given to the phage group. BPH-EC-5 and the control group were given the same amount of normal saline for 15 days, and the experimental mice were killed by neck dislocation, and the internal organs were checked.
[0063] The observation results showed that this amount of phage BPH-EC-5 had no effect on the daily behavior of mice. Anatomical examination of in...
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