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Bacillus cereus phage DLn1 and application thereof

A technology of Bacillus cereus and phage, which is applied in the direction of phage, virus/phage, application, etc., can solve the problem of limited phage, and achieve the effects of not easy inactivation, strong environmental adaptability, and good inhibitory effect

Active Publication Date: 2021-10-22
JINAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although many phage preparations have been used to prevent and control microbial contamination, the current use of phages for the prevention and control of Bacillus cereus contamination is still very limited. Therefore, it is urgent to isolate phages that can infect Bacillus cereus and can be used in food processing and production Phages and their combination preparations

Method used

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  • Bacillus cereus phage DLn1 and application thereof
  • Bacillus cereus phage DLn1 and application thereof
  • Bacillus cereus phage DLn1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1, Isolation, purification and propagation of bacteriophage DLn1

[0029] 1. Sample source and processing

[0030] The samples used to isolate phages came from sewage in Guangzhou City, Guangdong Province. After the water samples were collected, they were centrifuged at 10,000×g for 20 minutes to sink most of the impurities to the bottom. The supernatant was filtered through a 0.45 μm filter membrane to remove most of the environmental bacteria. Add 50mM magnesium sulfate to the supernatant, let it stand for 30min, and filter it with a 0.22μm filter membrane to make the phage adsorb to the filter membrane. Then soak the filter membrane with the eluent (1% beef extract, 3% Tween 80) and sonicate for 5 minutes, filter the eluent with a 0.22 μm filter membrane to remove bacteria, and save the filtrate for future use.

[0031] 2. Isolation and purification of phage

[0032] In 3mL TSB medium (containing 1mM CaCl 2 ) was added 30 μL of Bacillus cereus 2177 culture...

Embodiment 2

[0036] Embodiment 2, Morphological observation of bacteriophage DLn1

[0037] Take 10 μL of high-titer phage DLn1 and place it on the copper film of the carbon-coated mesh, let it stand naturally for 15 minutes, and absorb the excess liquid with clean filter paper. After drying in the air, stain with 2% tungsten phosphate for 10 min, and after natural drying, observe the prepared stained slice under a transmission electron microscope ( figure 2 ). The length and width of the phage head were 60.1±3.6nm and 36.5±3.6nm, respectively, and the length and width of the phage tail were 36.4±3.7nm and 4.7±1.0nm, respectively.

Embodiment 3

[0038] Embodiment 3, phage DLn1 genome analysis

[0039] Genomic DNA of phage DLn1 was extracted for analysis. The phage DLn1 obtained above was concentrated with polyethylene glycol and sodium chloride to obtain a higher titer phage. Then the phage DNA was extracted by the phenol-chloroform method, and the DNA was sequenced and analyzed by Illumina Miseq. According to analysis, the phage DLn1 has a nucleotide sequence such as SEQ ID NO:1. The similarity of phage DLn1 to reported phages was determined by NCBI BLASTn.

[0040]According to sequencing and analysis, the genome size of phage DLn1 is 25379bp, and it is linear DNA. The comparison results between phage DLn1 and NCBI are shown in Table 1, where the nucleic acid identity is the product of Query Cover and Per.ident. It can be seen from Table 1 that the similarity between the phage and the known phage is less than 95%, indicating that the phage belongs to a new type of phage. Combined with electron microscope observa...

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Abstract

The invention discloses a bacillus cereus phage DLn1 and application of the bacillus cereus phage DLn1. The preservation number of the phage DLn1 is GDMCC No: 61638-B1. The virulent phage DLn1 provided by the invention can be used for specifically cracking bacillus cereus, and the cracking amount of the phage DLn1 is relatively large. The phage with large lysis amount can proliferate in a short time and can be better used for resisting pollution of bacillus cereus. Meanwhile, the phage DLn1 has tolerance to high temperature and acid-base, has strong adaptive capacity to environment, is not easy to inactivate in the application process, and has a good inhibition effect on target bacteria in milk. In conclusion, the phage DLn1 is expected to become a bacteriostatic preparation for preventing and controlling the pollution of bacillus cereus in food.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a Bacillus cereus phage DLn1 and its application. Background technique: [0002] Bacteria are the main cause of foodborne illnesses, seriously endangering people's health, and the overuse of antibiotics has further exacerbated this situation. The main food-borne pathogenic bacteria in my country include Vibrio parahaemolyticus, Salmonella, Staphylococcus aureus, Bacillus cereus, and diarrhea-causing Escherichia coli. Among them, Bacillus cereus has attracted more and more attention. According to statistics, from 2003 to 2019, there were 504 foodborne outbreaks caused by Bacillus cereus in my country, with 6 deaths, ranking fourth among foodborne pathogens. When the human body ingests food contaminated by Bacillus cereus, it can cause vomiting or diarrhea symptoms, and in severe cases, it can lead to death. The production of spores is an important reason why Bacillus c...

Claims

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Application Information

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IPC IPC(8): C12N7/00A23L5/20A23C7/04C12R1/92
CPCC12N7/00A23L5/28A23C7/04C12N2795/00021
Inventor 丁郁李娜吴清平王涓朱振军李淳袁晓鸣张菊梅曾海燕杨美艳陈博陈谋通薛亮
Owner JINAN UNIVERSITY
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