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Preparation and application of bifidobacterium longum with ampicillin resistance and its preparation

A bifidobacterium longum and ampicillin technology, applied in the field of biomedicine, can solve the problem of lack of systemic resistance genes of lactic acid bacteria

Inactive Publication Date: 2019-02-12
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, human research on antibiotic resistance is mostly focused on pathogenic bacteria, and there is a lack of systematic research on the antibiotic resistance of lactic acid bacteria and whether the resistance gene can be transferred horizontally. With the popularization and wide application of probiotic lactic The research on bacteria is getting more and more in-depth, and the antibiotic resistance of lactic acid bacteria is getting more and more attention

Method used

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  • Preparation and application of bifidobacterium longum with ampicillin resistance and its preparation
  • Preparation and application of bifidobacterium longum with ampicillin resistance and its preparation
  • Preparation and application of bifidobacterium longum with ampicillin resistance and its preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Preliminary Screening of Probiotics

[0050] The specific operation steps of strain isolation (including culture temperature, time, etc.) feature description)

[0051] 1. Culture medium:

[0052] Lactobacillus MRS separation solid medium: Accurately weigh 10.0g of beef extract peptone, 10.0g of tryptone, 5.0g of yeast extract, 5.0g of anhydrous sodium acetate (NaAc), 2.0g of triammonium citrate, K 2 HPO 4 2.0g, MgSO 4 ·7H 2 O0.58g, MnSO 4 ·H 2 O 0.28g, glucose 15.0g, 1.0mL Tween-80 into the beaker, distilled water and stir evenly, add 15.0g agar and stir, after the drug is completely dissolved, add distilled water to 1000mL, monitor and adjust the pH of the medium to 6.2 with a pH meter ~6.4, 121°C damp heat sterilization for 15 minutes, set aside.

[0053] Bifidobacterium improved MRS separation solid medium: Accurately weigh 10.0g beef extract peptone, 10.0g tryptone, 5.0g yeast extract, 15.0g glucose, 5.0g sodium acetate, 2.0g triammonium citrate, ...

Embodiment 2

[0089] The biological activity evaluation of embodiment 2 probiotics

[0090] The bacteria to be tested were Bifidobacterium longum HB25 and Lactobacillus acidophilus HL9 obtained in Example 1, and the control strain was Lactobacillus rhamnosus GG (LGG for short, ATCC 53103). Melt in a water bath at 37°C, and take 100 μL of each bacteria in 100 mL of lactic acid bacteria MRS enrichment broth in a biological safety cabinet for activation and recovery. Shake the bacteria at 200rpm on an air-bath shaker at 37°C for 12h, centrifuge the bacteria solution at 4000rpm for 15min, remove the supernatant after centrifugation, wash the bacterial pellet with sterile saline for 3 times, and then transfer the bacterial pellet to the standard turbidimetric solution In the same test tube as the centrifuge tube, use sterile physiological saline to dilute to the concentration that needs to be higher than the standard turbidity solution (bacterial turbidity and 10 wheat turbidity standard tube eq...

Embodiment 3

[0127] 1. Analysis of strain plasmids

[0128] According to Wan Qian's method (Wan Qian, Cao Jian, Wu Xingquan, etc. Optimization of the extraction method of natural plasmids of lactic acid bacteria [J]. China Biotechnology Journal, 2010,30(2):94-99.) Carry out plasmid extraction, take Centrifuge 5-10 mL of the strain to be tested overnight, discard the supernatant, resuspend with 250 μL of solution P1 (add lysozyme, to a final concentration of 20 mg / mL), and bathe in water at 37°C for 30 minutes; add 250 μL of solution P2 and mix quickly, then add 350 μL Solution P3, mix well, and centrifuge at 12000rpm for 10min; take the supernatant and add it to the spin column, centrifuge at 9000rpm for 1min, discard the liquid in the centrifuge tube; wash the spin column twice with 500μL Wash Buffer, put the spin column into a new EP tube and centrifuge at 12000rpm 2min, then add 50μL Elution Buffer (preheated at 60°C), let stand for 2min, centrifuge, and set aside. Detected by 1% agaro...

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Abstract

The present invention discloses lactic acid bacteria having ampicillin resistance, and preparation and applications of a preparation of the lactic acid bacteria having ampicillin resistance. According to the present invention, human manure is separated under antibiotic pressure to obtain two strains of lactic acid bacteria have drug resistance, an antibiotic sensitivity test is performed, and whether plasmid DNA exists is detected; PCR amplification is performed to analyze the drug resistance gene localization conditions of the strains and explore the molecular mechanisms of the antibiotic resistance of the lactic acid bacteria, and safety evaluation is performed on the screened lactic acid bacteria having the resistance gene; whether the screened resistance strain provides an adjustment effect on intestinal tract imbalance is verified, and an antibiotic type intestinal tract flora imbalance model is used so as to provide potential treatment methods for diarrhea, flora imbalance and other diseases caused by the use of antibiotics in the clinic; and according to the obtained strain of the present invention, the intestinal tract probiotics can survive under the antibiotic pressure when the antibiotic is clinically used again while the ordinary probiotics can not survive, such that the treatment method can be provided for the diarrhea caused by the antibiotics in the clinic.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to the preparation and application of ampicillin-resistant Bifidobacterium longum and its preparation. Background technique [0002] There are a large number of microorganisms living in the human body, the main ones are bacteria, as we know, they can settle in the human intestinal tract and form complex groups or micro-ecosystems. According to statistics, there are 1014 bacteria living in the intestinal tract of healthy adults, which is 10 to 20 times the total number of human cells, and there are as many as nearly 500 species of bacteria. Intestinal bacteria are the largest and most important part of the human body. The bacteria count in the stool reaches 10 12 . The body's host cells, gut microbes, and other microbes constitute a complex system that interacts to form a variety of biological effects. [0003] Intestinal probiotics (Probiotics) are a type of intestinal bacteria that ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A61K35/747A61K35/745A61P1/00C12R1/01C12R1/23
Inventor 孙晗笑田鹏杨妍利时雨丁清韦丕金刘照兵黄俊丽
Owner JINAN UNIVERSITY