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Culture medium for separating and screening lactobacillus fermentum in human intestinal canals and application thereof

A medium and yeast powder technology, applied in the field of microorganisms, can solve the problems of low abundance, inability to accurately reach the level of strains, and no separation and screening, etc., to achieve the effect of improving efficiency

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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lactobacillus, including Lactobacillus fermentum, is an important probiotic in the intestinal tract. There are no less than 20 species of Lactobacillus commonly found in the human intestinal tract, but their abundance is often low and it is not easy to screen
At present, the most effective medium for screening Lactobacillus from intestinal contents is LAMVAB (Lactobacillus Fermentum MRS with Arabinose and Three Antibiotics) medium, which inhibits the growth of other bacteria other than lactic acid bacteria by reducing the pH and adding vancomycin. But it is limited to the screening of lactic acid bacteria and cannot be accurate to the strain level
Therefore, there is currently no specific medium for the isolation and screening of Lactobacillus fermentum in the human intestinal tract

Method used

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  • Culture medium for separating and screening lactobacillus fermentum in human intestinal canals and application thereof
  • Culture medium for separating and screening lactobacillus fermentum in human intestinal canals and application thereof
  • Culture medium for separating and screening lactobacillus fermentum in human intestinal canals and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Exploring the composition of Lactobacillus in human intestinal flora based on Lactobacillus metagenomics

[0030] Human intestinal flora Lactobacillus metagenomic sequencing technology process:

[0031] (1) Take out samples of intestinal contents from 10 people in Nantong area from -80°C freezer, and after thawing, take out 0.2g samples of intestinal contents with a pipette tip for DNA extraction;

[0032] (2) Using the Feces DNA Kit (FastDNA Spin Kit For Feces, MP) to isolate and extract genomic DNA from the 10 intestinal contents described in (1);

[0033] (3) Lactobacillus groel gene amplification:

[0034] (I) Take 1uL of genomic DNA described in step (2) as a PCR template.

[0035] (II) The PCR primers used were Lactobacillus groel gene-specific amplification primers: 308F-TGAAGAAYGTNRYNGCYGG, 806R-AANGTNCCVCGVATCTTGT, and the length of the amplified fragment was 499bp.

[0036] (Ⅲ) PCR reaction system: 25uL Premix Taq (TaKaRa Taq Version 2.0plus dye,...

Embodiment 2

[0056] Example 2: Application of bacterial species screening of intestinal content samples on Lactobacillus basal medium LAMVAB

[0057] The formula of LAMVAB medium includes the following components (calculated in 1 L): peptone 10g, beef extract 10g, yeast powder 5g, dipotassium hydrogen phosphate 2.6g, sodium acetate 2g, diammonium citrate 2g, magnesium sulfate heptahydrate 0.5, a Manganese sulfate water 0.25, arabinose 20g, vancomycin 20×10 -3 g, L-cysteine ​​0.5g, bromocresol green 0.05g, Tween 80 1g, water 1000mL.

[0058] The preparation steps of the above-mentioned culture medium are as follows:

[0059] (1) Weigh 10g of peptone, 10g of beef extract, 5g of yeast powder, 2.6g of dipotassium hydrogen phosphate, 2g of sodium acetate, 2g of diammonium hydrogen citrate, 0.5g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, and 20g of glucose , put 0.5g of L-cysteine ​​and 20g of agar into a container, add distilled water to make up to 969mL, adjus...

Embodiment 3

[0074] Embodiment 3: the carbon source selection and optimization of culture medium

[0075] As described in Examples 1-2, there are about 25 types of lactobacilli commonly found in the human intestinal tract and lactic acid bacteria that can grow on the existing lactobacillus-specific medium LAMVAB. For these 25 species of bacteria, we first explored their utilization of different carbon sources. The experimental results are as follows:

[0076] Table 3 Utilization of different sugars by 25 common lactic acid bacteria in the human intestinal tract

[0077]

[0078]

[0079] Note: "+" means available; "-" means not available; " / " means unknown.

[0080] According to the utilization of different sugars by various bacteria in the human intestinal tract, we chose arabinose as the carbon source of the selection medium. In addition to Lactobacillus fermentum that can utilize arabinose, there are 11 strains that can also utilize arabinose, namely Lactobacillus fermentum, La...

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Abstract

The invention discloses a culture medium for separating and screening lactobacillus fermentum in human intestinal canals and application thereof, and belongs to the technical field of microorganism. The culture medium inhibits the growth of common lactobacillus and other lactic acid bacteria in the human intestinal canals and ensures growth of lactobacillus fermentum in the culture medium on the basis of a traditional lactobacillus screened culture medium LAMVAB culture medium by replacing a carbon source and adding streptomycin with the concentration being 0.256*10-3mg / mL and gentamicin withthe concentration being 6.4*10-6mg / mL. The culture medium solves the problem that the lactobacillus fermentum in the human intestinal canals is difficult to screen, and improves the screening efficiency of the lactobacillus fermentum in the human intestinal canals.

Description

technical field [0001] The invention relates to a culture medium for separating and screening lactobacillus fermentum in the human intestinal tract and an application thereof, belonging to the technical field of microbes. Background technique [0002] Lactobacillus fermentum (Lactobacillus fermentum) belongs to the Lactobacillus genus in the Lactobacillus family, is a heterofermentative Lactobacillus, Gram staining is positive, the metabolic type is facultative anaerobic, and the optimum growth temperature is 30-37°C. Lactobacillus fermentum is one of the resident flora in the human intestinal tract, which mainly adheres to the epithelial cells of the small intestine. In 2011, Lactobacillus fermentum was included in my country's "List of Strains Applicable to Food", and it also passed the GRAS (Generally Recognized as Safe Substance) certification of the US Food and Drug Administration. Among them, Lactobacillus fermentum CECT5716 was included in the "List of Edible Strains...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12N1/02C12R1/225
CPCC12N1/02C12Q1/045G01N2333/335
Inventor 翟齐啸陈卫赵岩陆文伟唐晓姝赵建新张灏
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