Method for screening anti-bacteriophage Lactobacillus plantarum strain
A Lactobacillus plantarum, anti-phage technology, applied in the field of microorganisms, can solve problems such as complicated operation, achieve the effects of simple operation, high safety, and improve the success rate of separation
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Embodiment 1
[0026] Embodiment 1: the isolation of plant lactobacillus phage
[0027] (1) In order to obtain Lactobacillus plantarum phage, different batches of fermented milk samples were obtained from Hangzhou Wahaha Company. After fully mixing the obtained fermented milk samples, take 10 mL of fermented milk in 100 mL of MRS-Ca liquid medium, and incubate at 37° C. for 24 h. The culture solution was taken in a sterile centrifuge tube and centrifuged at 12,000 g for 10 min, the supernatant was filtered through a 0.45 μm sterile microporous membrane, and the filtrate was placed in a 4°C refrigerator for later use.
[0028] (2) Enrichment of phage: Take 15 mL of the filtrate from step (1), add 2× TSB liquid medium and 1 mL of Lactobacillus plantarum cultured to logarithmic growth phase, mix well and place in a sterile Erlenmeyer flask , cultivate overnight at 37°C, take out the culture in a 50mL sterile centrifuge tube, centrifuge at 12000g for 30min at 4°C, filter the supernatant with a ...
Embodiment 2
[0031] Embodiment 2: Screening of anti-phage Lactobacillus plantarum strains
[0032] Lactobacillus plantarum spontaneously mutated anti-phage strains were screened by secondary infection method. Lactobacillus plantarum was inoculated into MRS-Ca liquid medium and cultured at 37°C until logarithmic growth phase. After adding the phage according to the multiplicity of infection of 0.1, culture at 37°C until the culture medium is completely lysed. Continue to culture at 37°C for 48 hours. The lysate was spread on the MRS solid plate and incubated at 37°C for 24h. The single colony obtained by picking was cultured in MRS liquid medium. After the culture medium was streaked three times to obtain pure strains, the resulting strains were cultured in MRS-Ca liquid medium, and phages were added to observe the lysis. If in the presence of phage, it can grow normally for three consecutive passages, then the strain is determined as a phage-resistant strain.
[0033] The experimental...
Embodiment 3
[0034] Example 3: Molecular identification of anti-phage Lactobacillus plantarum strains
[0035] (1) Genomic DNA extraction: The single colonies of Lactobacillus plantarum spontaneously mutated anti-phage strains were cultured in 5 mL MRS liquid medium to the logarithmic growth phase, and the genomes of the phage-resistant strains were extracted with bacterial genomic DNA extraction reagents.
[0036] (2) PCR reaction system (25 μL): Ex Taq DNA polymerase 0.25 μL, 10×Ex PCR buffer 2.5 μL, dNTPs 2.0 μL, template 2 μL, upstream primer 1.5 μL, downstream primer 1.5 μL, sterilized deionized water 15.25 μL . Primers for bacterial amplification were universal primers 27F and 1492R.
[0037] (3) The PCR amplification program was set as follows: pre-denaturation at 94°C for 5 min, followed by PCR cycle; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, and 30 cycles; and final extension at 72°C for 10 min.
[0038] (4) Log in the NCBI database ...
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