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Wild enterococcus faecium ZK03 and electric shock transformation method and application thereof

A technology of Enterococcus faecium and ZK03, applied in the field of microorganisms, can solve the problems of difficulty in obtaining positive transformation, low transformation efficiency, and improving transformation difficulty.

Pending Publication Date: 2022-05-13
ZHONGKAI UNIV OF AGRI & ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are not many methods specifically suitable for electroporation of Enterococcus faecium at present. Therefore, the electroporation of Enterococcus faecium is usually referred to the method of electroporation of Lactococcus lactis. Due to the relatively large heterogeneity of Enterococcus faecium, the electroporation conditions are different. Uniformity, so when the electroporation method of Lactococcus lactis is applied to Enterococcus faecium, there is a problem of low transformation efficiency, which increases the difficulty of transformation, and the existing electroporation method is cumbersome to operate, and it is even difficult to obtain positive transformants

Method used

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  • Wild enterococcus faecium ZK03 and electric shock transformation method and application thereof
  • Wild enterococcus faecium ZK03 and electric shock transformation method and application thereof
  • Wild enterococcus faecium ZK03 and electric shock transformation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Isolation and identification of wild-type Enterococcus faecium (Enterococcus faecium) ZK03

[0030] 1. Screening

[0031] (1) Collect the feces of healthy pigs, apply them in Enterococcus screening medium after appropriate dilution, purify and culture, isolate pure colonies, and store them at -80°C.

[0032] (2) Through the antibacterial test or other tests, several strains were screened out, and the strain ZK03 with the best effect was selected among them.

[0033] 2. Identification

[0034] Biochemical identification was performed using conventional biochemical identification methods. Identification methods specifically include identification of culture characteristics, identification of morphological characteristics, and molecular identification, as follows:

[0035] (1) Identification of culture characteristics:

[0036] The colony is cultured in BHI medium, with a diameter of 0.5-1 mm. The surface of the colony is smooth, the edges are neat, white or mi...

Embodiment 2

[0044] Example 2 Detection of virulence genes of wild-type Enterococcus faecium (Enterococcus faecium) ZK03 according to the existing literature (Jia Xiaojiong, investigation of risk factors of linezolid-resistant Enterococcus and vancomycin-resistant Enterococcus faecium virulence factors and transposition Substructure analysis, the virulence gene detection method in Chongqing Medical University, 2016) and the disclosed virulence gene detection primers were carried out to the wild-type Enterococcus faecium (Enterococcus faecium) ZK03 after routine PCR electrophoresis, and Enterococcus faecium ATCC29212 ( Provided by the research group of Mr. Wang Wei, Institute of Healthy Breeding Innovation, Zhongkai College of Agricultural Engineering), as a positive control.

[0045] Enterococcus faecium ATCC29212 was used as a positive control to verify the virulence gene PCR of wild-type Enterococcus faecium (Enterococcus faecium) ZK03, and the PCR results were as follows: figure 2 As s...

Embodiment 3

[0046] Example 3 Acid resistance, bile salt resistance, high temperature resistance, and normal saline test of wild-type Enterococcus faecium (Enterococcus faecium) ZK03

[0047] According to the test methods of acid resistance, bile salt resistance, high temperature resistance and physiological saline in the existing literature (Zhai Mengkai, the probiotic characteristics and safety research of a strain of Enterococcus faecium, Shandong Agricultural University, 2017), the wild type Enterococcus faecium (Enterococcus faecium ) ZK03 for detection.

[0048] Test results: the tolerance characteristics of the wild-type Enterococcus faecium (Enterococcus faecium) ZK03 are as follows: in the physiological saline with a pH of 2.0, some colonies still survive; in 5% pig bile salt culture for 12 hours, the survival rate remains the same. 3.4% or more; at a temperature of 60°C, the survival rate is above 24%; when cultured in normal saline for 12 hours, the survival rate is above 73%. ...

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Abstract

The invention discloses wild enterococcus faecium ZK03 as well as an electric shock transformation method and application thereof, and belongs to the field of microorganisms. The strain is preserved in Guangdong Microbial Culture Collection Center on January 5, 2022, and the preservation number is GDMCC (China General Microbiological Culture Collection Center) No. 62192. The strain does not contain enterococcus virulence factors such as Asa1, cylA, efaA, esp, gelE and hyl. The strain has the characteristics of good acid resistance, swine bile salt resistance, high temperature resistance and normal saline resistance, and can survive for a long time in normal saline at room temperature; the strain has a certain inhibition effect on staphylococcus aureus, salmonella and escherichia coli, can be used for preparing a bacteriostatic agent, and can also be used as a feed additive for fermentation production of feed. The electric shock transformation method of the wild enterococcus faecium ZK03 is simple to operate, high in transformation efficiency and strong in repeatability.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of wild-type Enterococcus faecium ZK03 and its electric shock transformation method and application. Background technique [0002] Enterococcus Faecium (E. faecium) is one of the normal intestinal flora of humans and animals. It belongs to Gram-positive cocci without capsule and flagella. It has good acid resistance, high temperature resistance and bile salt resistance. According to a large number of literature reports, Enterococcus faecium can produce lactic acid, bacteriocin, hydrogen peroxide and other substances, and has a certain inhibitory effect on Escherichia coli, Salmonella, Staphylococcus aureus, etc. In food, the fermentation of Enterococcus faecium contributes to the development of aroma and flavor, prolongs shelf life, and improves the microbial environment. In animal husbandry, Enterococcus faecium is often used as one of the tecan prod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N13/00C12N15/74A01N63/20A01P1/00A01P3/00A23K10/18C12R1/01
CPCC12N13/00C12N15/746A01N63/20A23K10/18
Inventor 王玮谢改杰金鑫鑫张雪黄燕华曲庆
Owner ZHONGKAI UNIV OF AGRI & ENG
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