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Lactobacillus reuteri antagonizing Campylobacter jejuni and inhibiting flaA gene expression of Campylobacter jejuni

A technology of Lactobacillus reuteri and Campylobacter jejuni, applied in the field of microorganisms, can solve the problems of reducing Campylobacter jejuni antigens, not involving Campylobacter jejuni, not many, etc., achieving growth inhibition, good adhesion properties, and wide application prospects. Effect

Active Publication Date: 2016-01-27
JIANGNAN UNIV
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, some published patent documents relate to the antagonism of Campylobacter jejuni and its use. For example, CN104870472A discloses an antibody and its fragments specific to Campylobacter jejuni; CN102497875A discloses an antigen for reducing the colonization of Campylobacter jejuni; CN103381220A discloses A kind of medicine that can treat Campylobacter jejuni of chicken; However, there are not many patent application documents related to lactic acid bacteria antagonizing Campylobacter jejuni and inhibiting the expression of its virulence gene. CN102533618A discloses a plant with the ability to inhibit Campylobacter jejuni in vitro Lactobacillus, but the patent does not involve the expression of the flaA gene of Campylobacter jejuni and its efficacy in cell models and in vivo antagonism against Campylobacter jejuni

Method used

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  • Lactobacillus reuteri antagonizing Campylobacter jejuni and inhibiting flaA gene expression of Campylobacter jejuni
  • Lactobacillus reuteri antagonizing Campylobacter jejuni and inhibiting flaA gene expression of Campylobacter jejuni
  • Lactobacillus reuteri antagonizing Campylobacter jejuni and inhibiting flaA gene expression of Campylobacter jejuni

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1 screens lactic acid bacteria from chicken manure

[0036] The samples were taken from fresh farm chicken manure in Gonghu Avenue, Binhu District, Wuxi City, Jiangsu Province, and preserved in 30% glycerol. Draw 1ml of chicken manure suspension and add it to a test tube containing 9ml of sterilized normal saline, shake evenly, apply the plate method after gradient dilution, and spread these diluted bacterial solutions on the MRS with acid-base indicator olfactory cresyl violet On the culture medium (Qingdao Haibo Biotechnology Co., Ltd.) plate, carry out ordinary culture and anaerobic culture at a temperature of 37 ° C for 48 hours, observe the growth of the colony, and pick the typical ones that can make the olfactory cresyl violet in the culture medium turn yellow. Single colony, and perform Gram staining on the single colony, pick the Gram-positive bacteria as the primary target strain, and then transfer to the MRS plate to separate and purify until the p...

Embodiment 2

[0037] Embodiment 2 uses the Oxford cup method to screen the lactic acid bacteria that can inhibit the growth of Campylobacter jejuni

[0038] Preparation of lactic acid bacteria fermentation supernatant: Inoculate the separated and purified lactic acid bacteria into MRS liquid medium at 2% by volume based on the volume of MRS liquid medium, culture at 37°C for 18 hours, and continuously culture for three generations in the same way, and then inoculate the lactic acid bacteria The bacterial suspension was centrifuged at 8000r / min and 4°C for 8min, the supernatant was absorbed, and sterilized by filtration with a 0.22μm water filter membrane to obtain the lactic acid bacteria fermentation supernatant.

[0039] Campylobacter jejuni (Campylobacter jejuni) NCTCl11168 strain (purchased from the American Type Culture Collection ATCC) on two media (i.e. Brooke's agar (product of Qingdao Haibo Biotechnology Co., Ltd.) and brain heart infusion broth medium ( Oxoid medium)) at a tempera...

Embodiment 3

[0044] Example 3 Tolerance experiment of Lactobacillus reuteri CGMCCNo.11447 to artificial intestinal juice and artificial gastric juice

[0045] Artificial gastric juice: Dissolve 3 g of pepsin (1:10000, Sigma, USA) in 1 L of normal saline (0.5%, w / v), adjust the pH to 2.0, pass the solution through a 0.45 / 0.22 μm sterile double-layer Pore ​​filter membrane, sterile storage for future use.

[0046] Artificial intestinal juice: Weigh 1g of trypsin (P-1500, Sigma, USA) and 0.3g of ox bile salt, dissolve in 1L of normal saline (0.5%, w / v), adjust the pH to 8.0, pass the solution through 0.45 / 0.22μm sterile double-layer microporous membrane, aseptically stored for future use.

[0047] PBS solution (phosphate buffered saline): Potassium dihydrogen phosphate 0.27g, potassium chloride 0.2g, disodium hydrogen phosphate dodecahydrate 2.85g, NaCl 8.5g, distilled water 1L, pH adjusted to 7.2, pass 0.45 / 0.22μm without Bacterial double-layer microporous membrane, sterilized at 121°C fo...

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Abstract

The invention discloses Lactobacillus reuteri, which can antagonize Campylobacter jejuni and inhibit its flaA gene expression, has better acid and bile salt tolerance, can inhibit in vitro the growth of Campylobacter jejuni, and has better ability to adhere intestinal epithelial cells. Ferment supernate of Lactobacillus reuteri has the expression of flaA gene of Campylobacter jejuni down-regulated in vitro by 11.1 times when compared to a control, and the synthesis of flagella of Campylobacter jejuni can be effectively inhibited; the ferment supernate of Lactobacillus reuteri has the expression of the flaA gene of Campylobacter jejuni down-regulated in a cell model by 1.4 times when compared to a control. The Lactobacillus reuteri is applicable to the preparation and drugs for preventing and treating diseases caused by Campylobacter jejuni, for the preparation of fermented milk and the like, and has a promising application prospect.

Description

technical field [0001] The invention relates to a strain of Lactobacillus reuteri that antagonizes Campylobacter jejuni and inhibits the expression of its flaA gene, and belongs to the technical field of microorganisms. Background technique [0002] Campylobacter jejuni is a Gram-negative bacterium that is widely distributed in nature and can be transmitted through animals, food, water, milk, etc. Infection in humans can result from ingestion of undercooked or contaminated chicken, beef, partially pasteurized milk, and contaminated water. In recent years, the infection rate of Campylobacter jejuni has been on the rise all over the world. In some developed countries, the number of cases of diarrhea caused by Campylobacter jejuni infection even exceeds that of Salmonella and Shigella, becoming the most common pathogen of diarrhea; in developing countries, Campylobacter jejuni is the most common pathogen of infectious diarrhea in infants and young children . When people are ...

Claims

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Application Information

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IPC IPC(8): C12N1/20A61K35/747A61P31/04A23C9/123A23L33/00A23L19/20C12R1/225
CPCY02A50/30
Inventor 陈卫翟齐啸田丰伟张婷婷赵建新王刚张秋香刘小鸣张白曦张灏
Owner JIANGNAN UNIV
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