Screening and application of fish-derived enterococcus faecium R8

A technology for separation and screening of Enterococcus faecium, applied in the fields of application, bacteria, antibacterial drugs, etc., can solve the problems of inability to colonize and unstable of probiotic strains, reduce the impact of pollution and human health, improve the survival rate and increase the growth rate. The effect of reducing the incidence of bacterial drug resistance

Active Publication Date: 2018-10-16
TIANJIN AGRICULTURE COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many probiotics used in aquaculture are not screened from aquatic animals or living environments, but from some terrestrial animals, which makes the probiotic strains unable to colonize or unstable
Only the indigenous microorganisms screened from the host itself can better adapt to the environment and inhibit pathogenic bacteria more efficiently, while those probiotic strains that are not isolated from the digestive tract of fish often cannot stably colonize in the digestive tract of fish

Method used

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  • Screening and application of fish-derived enterococcus faecium R8
  • Screening and application of fish-derived enterococcus faecium R8
  • Screening and application of fish-derived enterococcus faecium R8

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Isolation and identification of probiotic strains

[0027] 1. Isolation of strains

[0028] The samples were taken from healthy crucian carp purchased in the market. After the body surface was sterilized with 75% medical alcohol, the crucian carp was aseptically dissected and the intestines were taken out, and pre-cooled sterilized saline was added at a ratio of 1:10. Homogenate, the homogenate is serially diluted by 10 times, spread on the MRS medium plate, and observe after 24-36 hours of constant temperature cultivation at 37°C. Select colonies with clear edges, scattered and about 1 mm in size for pure culture. The pure cultures were preliminarily judged by morphological observation and Gram staining, and the Gram-positive cocci in single, paired or chained form were passaged and preserved. The growth characteristic of the bacterial strain of this invention on MRS agar culture sees figure 1 , see Gram staining properties figure 2 .

[0029] The stra...

Embodiment 2

[0046]Embodiment 2: the antibacterial test of Enterococcus faecium bacterial strain R8

[0047] Aeromonas vernerii, Staphylococcus hemolyticus, Vibrio parahaemolyticus and Vibrio vulnificus (provided by Aquatic Animal Disease and Immunology Laboratory, Fisheries College, Tianjin Agricultural University) were used as indicator bacteria. Adopt spot method (Smith P, 1993) primary screening, the method of Oxford cup method (Liu Dongmei, 2006) secondary screening to carry out bacteriostasis test, concrete steps are as follows: adjust indicator bacteria liquid concentration to be 10 6 cfu / mL, draw 0.1mL and spread it on the LB agar medium, then pick a single strain to be tested with the inoculation loop and spot it on the plate. Incubate at 37°C for 24 hours and observe the inhibition zone.

[0048] The Oxford cup method was used for re-sieving. Adjust the concentration of the strain to be tested after 24h of activation to 10 7 cfu / mL, adjust the concentration of indicator bacter...

Embodiment 3

[0052] Example 3: Safety evaluation of Enterococcus faecium strains

[0053] 1. Hemolysis test

[0054] Spot the R8 bacterial solution after 24 hours of cultivation on the rabbit blood plate, incubate at 37°C for 24 hours, and judge the production of hemolysin according to the formation of transparent circles around the colonies. As a result, there was no transparent circle and no hemolytic reaction.

[0055] 2. Animal tests

[0056] The R8a strain was inoculated on LB agar medium, cultured at 37°C for 24 hours, and then eluted with 0.75% sterile saline to obtain a bacterial suspension. The bacterial suspension was serially diluted 10 times, and the bacterial suspension of different dilutions was used aseptic Inject 0.1 mL of each crucian carp intraperitoneally with a syringe, and the concentration of the bacteria solution is 1.0×10 8 cfu / mL, 12 tails were injected into each group as the test group. The same volume of sterile saline was injected as the control group. Put ...

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Abstract

The invention provides enterococcus faecium R8 that is screened and separated from a crucian intestinal tract and has safe and prebiotic characteristics. The enterococcus faecium is collected in ChinaGeneral Microbiological Culture Collection Center (CGMCC) of China Committee for Culture Collection of Microorganisms on January 17, 2018 and has a collection number of CGMCC NO. 15229. The enterococcus faecium is high in heat resistance, acid-base resistance and stress resistance and more significant in probiotic performance, has a relatively obvious inhibiting effect on common aquatic pathogenic bacteria such as aeromonas veronii, staphylococcus haemolyticus, vibrio parahaemolyticus and vibrio vulnificus and can be widely applied to aquaculture as a feed additive.

Description

technical field [0001] The invention belongs to the technical field of microbial additives for aquatic products, and relates to an enterococcus faecium (Enterococcus faecium) which has obvious inhibitory effect on common aquatic pathogens Aeromonas vietori, staphylococcus hemolyticus, vibrio parahaemolyticus and vibrio vulnificus ) isolation and identification of strains, safety tests and identification and application of stress resistance. Background technique [0002] At present, with people's gradual recognition of micro-ecological products and the government's stricter restrictions on the use of antibiotics in recent years, when people are looking for alternatives to antibiotic products, they are turning more attention to green, environmentally friendly and efficient products. Microecological products. As an important class of probiotics, lactic acid bacteria are also an important dominant flora in the intestinal tract of animals. Because of their non-toxic, non-residua...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A61P31/04A23K10/18A23K50/80C12R1/01
CPCA23K10/18A23K50/80A61P31/04C12N1/205C12R2001/46
Inventor 孙敬锋毛晴
Owner TIANJIN AGRICULTURE COLLEGE
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