Screening and application of enterococcus faecium G12

A technology of Enterococcus faecium and G12, applied in the field of microbial additives for aquatic products, can solve the problems of increased overall drug resistance and unfavorable treatment of animal diseases, and achieve the effect of sustainable development, significant probiotics, and high resistance to stress.

Inactive Publication Date: 2019-11-05
TIANJIN AGRICULTURE COLLEGE
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Problems solved by technology

On the other hand, the resistance of bacteria to antibiotics has a horizontal transmission effect, and it is likely to transmit the level of drug resistance to...
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Abstract

The invention provides enterococcus faecium G12. The enterococcus faecium G12 is isolated from hepato-pancreas of shellfish, is safe and has probiotic characteristics. A strain enterococcus faecium isnamed the enterococcus faecium G12, and is deposited in the China General Microbiological Culture Collection Center, the deposit number is CGMCC NO.15230, and the date of deposit is January 17, 2018.The enterococcus faecium G12 has high heat resistance, high resistance to stress and remarkable probiotic properties, has an obvious inhibitory effect on a common aquatic pathogenic bacterium aeromonas veronii, is sensitive to most broad-spectrum antibiotics, can tolerate a small intestine high-bile-salt environment and is colonized in the intestine, and is suitable for being used as a feed additive to be widely applied to aquaculture, thereby being capable of greatly reducing the pollution to the environmental and the adverse effects on human health caused by the addition of traditional antibiotics in the aquaculture, and achieving sustainable development of the aquaculture.

Application Domain

Technology Topic

Laboratory cultureAeromonas veronii +20

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  • Screening and application of enterococcus faecium G12
  • Screening and application of enterococcus faecium G12
  • Screening and application of enterococcus faecium G12

Examples

  • Experimental program(6)

Example Embodiment

[0027] Example 1: Screening, isolation and identification of strains
[0028] Screening and isolation: A strain of Enterococcus faecium was selected from the hepatopancreas of shellfish, especially the hepatopancreas of Philippine clam. The specific operation of the screening is as follows: the body surface of the clam clams is sterilized with 75% medical alcohol and then dissected, the liver and pancreas are taken, and then 1 mL of pre-chilled sterile normal saline is added, homogenized thoroughly, centrifuged, and the supernatant is taken 10 times series dilution. Take stock solution, 10 -1 , 10 -2 The samples of the three dilutions were each 100uL, and they were coated on the MRS plate. Each dilution gradient was set to 3 parallel, and the samples were incubated at 37°C for 24h. Pick a single colony and purify the bacteria by streaking multiple times. The purified strain was made into a bacterial suspension with 40% glycerol and MRS liquid medium 1:1, and stored at -80°C for later use. The growth status of this strain on MRS agar culture is as follows figure 1 As shown, the Gram staining positive reaction is as attached figure 2 Shown. It can be seen that the mycological characteristics of the bacteria are as follows: the strain belongs to Gram-positive cocci; as attached figure 1 As shown, the colony shape of the bacteria on the MRS solid agar medium is round, arranged in pairs or short chains, and is milky white, with a smooth surface and neat edges. Observed under the oil microscope, the strain cells were spherical, solitary or in pairs, with few pairs and chains. No spores, no flagella.
[0029] Preliminary identification: The obtained strain was named Enterococcus faecium G12, and it was identified through a bacterial microbiochemical reaction tube (purchased from Hangzhou Binhe Microbial Reagent Co., Ltd.), including: 65°C growth test, peroxidase test, glucose, xylose, Sucrose, lactose, sorbitol, arabinose, raffinose, mannitol, arginine dihydrolase, bile esculin hydrolysis test. The bile esculin hydrolysis test of this strain was positive, it can hydrolyze arabinose, ferment sorbitol, etc. The identification results are shown in Table 1. Compared with the "Manual for Identification of Common Bacterial Systems" (edited by Dong Xiuzhu, Cai Miaoying, etc., Science Press, 2001), it was found that the description of this strain was basically consistent with the description of Enterococcus faecium species, and the strain was preliminarily determined as Enterococcus faecium.
[0030] Table 1 Biochemical identification results of Enterococcus faecium G12
[0031]
[0032] Gene determination: The 16S rDNA gene sequence of the bacteria was detected, and the phylogenetic tree analysis was constructed to further confirm the bacterial species. First, use the boiling method (the application of the boiling method to extract bacterial DNA in the detection of brucellosis pathogens, Chen Guangli, Liu Hailin, Ma Jian, "Journal of Animal Husbandry and Veterinary Science and Technology Information", 2015 Issue 1) to extract the total DNA of the isolate; Design of primers for amplifying 16SrDNA gene: Amplification primers adopt bacterial universal primers, forward primer F: 5'-AGAGTTTGATCATGGCTCAG-3', reverse primer R: 5'-GGTTACCTTGTTACGACTT-3'; again, PCR amplification of 16S rDNA gene: The 16S rDNA genome of the strain Enterococcus faecium genome was amplified with the above primers. The reaction system is shown in Table 2. The PCR reaction procedure is: 95℃ 10min, 94℃ 5min, 55℃ 40s, 72℃ 40s, 30 cycles, 72℃ 10min, End the reaction at 4°C; take 4μL of PCR product on a 1.5% agarose gel for electrophoresis detection, gel electrophoresis image analysis system to observe the results, and submit the product to Shanghai Bioengineering Technology Company for PCR product purification and cloning sequencing; finally, based on 16S rDNA sequence to construct phylogenetic tree: Through gel electrophoresis test, a target band of 1522bp was detected (see attached image 3 Shown); Blast alignment shows that the sequence homology with Enterococcus faecium (AM157434.1) is 99%. Submit the sequencing result to GenBank (https://www.ncbi.nlm.nih.gov) to obtain the accession number (MF784082) ), screened 7 sequences with relatively close homology and 1 sequence of an outer group, established an alignment model, and constructed a phylogenetic tree with the Neighbor-Joining method using Mega 5 software, and the analysis showed that the strains naturally clustered with Enterococcus faecium (As attached Figure 4 Shown), and further judged as Enterococcus faecium.
[0033] Table 2 PCR reaction system of 16S rDNA of Enterococcus faecium
[0034]

Example Embodiment

[0035] Example 2: Bacteriostatic test
[0036] Aeromonas victorini, Pseudomonas aeruginosa and Vibrio vulnificus (the above-mentioned strains are provided by the Aquatic Animal Disease and Immunology Laboratory of the Fisheries College of Tianjin Agricultural College) as indicator bacteria. Methods Refer to the literature (Evidence for the competitive exclusion of Aeromonas salmonicida from fish with a tressinducible furunculosis by a fluorescent pseudomonad. Smith P and Dabey S, "Journal of Fish Diseases", 1993 Issue 16), and the activated indicator bacteria and the test Strains were cultured in LB liquid medium for 24h, diluted with saline to 1×10 7 cfu/mL, take 0.2mL of indicator strain and spread it on fresh LB solid medium, and then take 2μL of test strain to spot on the above plate, and observe whether there is obvious inhibition zone around the spot seeding area for 24h.
[0037] The Oxford cup method (using the Oxford cup method to determine the antibacterial activity of probiotics, Liu Dongmei, Li Li, Yang Xiaoquan, "Food Research and Development", Issue 27, 2006) was used for re-screening. Adjust the concentration of the strain to be tested 24h after activation to 1×10 7 cfu/mL, adjust the concentration of the indicator bacteria solution to 1×10 6 cfu/mL, pipette 0.1mL on LB agar medium. Use sterile tweezers to take the sterilized Oxford cup and place it on a plate coated with indicator bacteria. Press lightly to make the bottom of the cup close to the culture medium. Add 200 μL of the pre-screened probiotic bacteria liquid to each Oxford cup, and then put it in a 37°C incubator for 24 hours to measure the size of the inhibition zone. The experimental results showed that Enterococcus faecium G12 had an antibacterial effect on Aeromonas velvetii (the results are shown in Table 3).
[0038] Table 3 In vitro antibacterial test of Enterococcus faecium G12
[0039]

Example Embodiment

[0040] Example 3: Safety test
[0041] Hemolysis test: Streak the tested strain on LB solid medium for 24h, scrape a single colony with an inoculation loop and spot it on the blood plate, incubate at 37℃ for 24h, observe whether a transparent circle is formed around the colony, and judge the production of hemolysin . The experimental results showed that: no hemolysis was observed on the blood plate, indicating that the strain did not produce hemolysin and had no potential pathogenicity.
[0042] Animal test: refer to the literature, select the immersion test of Penaeus monodon juveniles, select 1×10 7 , 1×10 5 The concentration of cfu/mL bacterial solution was soaked, the positive control was selected as Vibrio vulnificus, and the negative control group was selected as normal saline. After being soaked in the bacterial suspension for four hours, the juvenile shrimp were transferred to violent water. Each treatment group made three parallel observations for 7 days, and recorded the death of juvenile shrimp every day. The results showed that the concentration of juvenile shrimp was 1×10 7 cfu/mL and 1×10 5 There was no death after soaking in cfu/mL Enterococcus faecium liquid (Table 4). It shows that Enterococcus faecium G12 has no toxic effect on Penaeus monodon juveniles.
[0043] Table 4 Cumulative mortality of each Penaeus monodon in the animal test of Enterococcus faecium G12 (%)
[0044]
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