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Screening method of leuconostoc mesenteroides and application of leuconostoc mesenteroides in preparation of aquatic product bacteriostatic agent

A technology of Leuconostoc enterococcus and screening method, which is applied in the field of aquatic microorganism application, can solve the problems of the small amount and limited number of antibacterial substances of lactic acid bacteria, achieve high application prospects and practical value, wide pH tolerance range, and high acid production capacity Effect

Pending Publication Date: 2022-04-01
HENAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the number of lactic acid bacteria antibacterial substances found so far is not large and the reports on the application in the prevention and control of aquatic bacterial diseases are quite limited.

Method used

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  • Screening method of leuconostoc mesenteroides and application of leuconostoc mesenteroides in preparation of aquatic product bacteriostatic agent
  • Screening method of leuconostoc mesenteroides and application of leuconostoc mesenteroides in preparation of aquatic product bacteriostatic agent
  • Screening method of leuconostoc mesenteroides and application of leuconostoc mesenteroides in preparation of aquatic product bacteriostatic agent

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Isolation, Screening and Identification of Leuconostoc Enteritidis

[0026] (1) Make kimchi juice with sterile water for 10 -1 -10 -5 Gradient dilution, apply different gradient dilutions to the MRS solid plate with calcium carbonate, incubate at 37°C for 48 hours, pick a single colony with a calcium-dissolving circle, streak and purify it on the MRS solid plate, repeat the operation 2-3 The second time, purebred strains were obtained, and the purebred strains were stored in glycerol at -80°C for subsequent experiments.

[0027] (2) The genomic DNA of the screened strains was extracted as a template, and the 16S rRNA gene was amplified by PCR. The PCR amplification conditions were: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 45 s, 30 cycles, and extension at 72°C for 2 min. The PCR products were purified and sent to the company for sequencing. The sequencing results were compared with BLAST on N...

Embodiment 2

[0030] Study on the Growth and Acid Production Ability of Leuconostoc Enteritidis at Different pH

[0031] (1) After activation in MRS, Leuconostoc enterococcus was inserted into MRS liquid medium with pH = 2, pH = 4, pH = 6, and pH = 8, mixed evenly, and cultured at 37°C with constant temperature and shaking. Samples were taken after a period of time, and their absorbance values ​​at 600nm were measured ( figure 2 Middle A). The results showed that in the environment of pH=8, the strain entered the logarithmic growth phase after 12 hours of growth, and entered the stable phase after 20 hours; in the growth environment of pH=4, the lag period of the strain was shorter, and the strain entered the logarithmic growth phase faster, and After 10 hours, it enters the stable period; the growth condition at pH=6 is roughly similar to that at pH=4. In contrast, the environment of pH=6 is more suitable for the growth of lactic acid bacteria, and the environment of pH=2 hardly grows la...

Embodiment 3

[0034] Bacteriostatic Ability of Leuconostoc Enteritidis

[0035] The pathogenic bacteria Escherichia coli, Pseudomonas aeruginosa, Salmonella, Aeromonas salmonicida Z2, Shewanella Z3, and Aeromonas victoria Z12 were activated in LB liquid, and Leuconostoc enterica was cultured in MRS liquid Activated in medium, cultivated overnight at 37°C, took the fermentation broth of Leuconostoc enterococci, centrifuged at 12000rpm for 3min, and took the supernatant. Draw 100 μL of pathogenic bacteria fermentation broth and spread it on different LB solid media, and spread the bacteria solution evenly with a coating stick. A piece of sterile filter paper was placed on each plate, 10 μL of lactic acid sterile supernatant was added dropwise as a treatment group, and 50 μL of MRS was added dropwise as a blank control. Three parallels were set up for each pathogen treatment and control group. Place the plate in a 37°C incubator and cultivate overnight, and observe the size of the inhibition ...

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Abstract

The invention discloses a screening method of leuconostoc mesenteroides and application of the leuconostoc mesenteroides in preparation of a bacteriostatic agent for preventing and treating aquatic bacterial diseases, which comprises the following steps: gradient dilution is performed on pickle juice with sterile water, the diluent is coated on an MRS solid plate added with calcium carbonate, and culture is performed at 37 DEG C; selecting a single colony with a calcium dissolving ring, and performing streak purification on an MRS solid plate to obtain a pure strain; extracting genome DNA of the screened strain as a template, and carrying out PCR amplification on the 16S rRNA gene of the screened strain; and inoculating the screened strains after sequencing to an MRS solid plate, and culturing at a constant temperature of 37 DEG C until bacterial colonies are formed. The invention also discloses application of the leuconostoc mesenteroides in preparation of a bacteriostatic agent for preventing and treating aquatic bacterial diseases. The screened leuconostoc mesenteroides has an inhibition effect on various aquatic pathogenic bacteria by generating extracellular antibacterial substances, the survival rate of fishes under the stress of aeromonas can be increased, and the survival rate of bacteria is increased by preparing a viable bacterium agent.

Description

technical field [0001] The invention belongs to the technical field of application of aquatic microorganisms, and in particular relates to a screening method for Leuconostoc enterica and its application in the preparation of a bacteriostatic agent for preventing and controlling aquatic bacterial diseases. Background technique [0002] Freshwater aquaculture is an important part of the inland aquaculture industry. It provides humans with a large amount of high-quality animal protein. It is one of the main sources of high-quality animal protein for human beings in the future, and it is of great significance to improve the food structure of the people. Freshwater aquaculture products are deeply loved by consumers because of their low fat content, fresh and tender meat, and rich in vitamins. The output and scale of the freshwater aquaculture industry are increasing. The area and output of freshwater aquaculture in my country rank first in the world. Henan is located in the middl...

Claims

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Application Information

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IPC IPC(8): C12N1/02C12N1/20C12N1/04C12Q1/689A61K35/744A61P31/04C12R1/01
Inventor 李祎王海磊
Owner HENAN NORMAL UNIV
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