Sample treatment method for metabonomics research of enterococcus raffinosus

A technology of enterococcus saccharides and metabolomics, which can be applied in scientific instruments, measuring devices, instruments, etc., and can solve problems such as research gaps in the metabolomics of Enterococcus raffinoses

Pending Publication Date: 2021-05-18
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the methods of studying various microorganisms through metabolomics are often reported, but the metabolomics research of Enterococcus raffinose is still blank

Method used

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  • Sample treatment method for metabonomics research of enterococcus raffinosus
  • Sample treatment method for metabonomics research of enterococcus raffinosus
  • Sample treatment method for metabonomics research of enterococcus raffinosus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Pretreatment method for metabolome analysis of Enterococcus raffinose

[0040] (1) Activation culture of Enterococcus raffinose CGMCC No.5584

[0041] Inoculate the preservation solution of Enterococcus raffinose CGMCC No.5584 glycerol tube on the slant medium, cultivate at 30°C for 12-20 hours, pick a single colony from a sterile loop and inoculate it into the seed culture medium, and place it at 30°C for static culture 16 to 24 hours to obtain a sample of Enterococcus raffinose bacteria liquid;

[0042] main medium

[0043] Incline medium (g / L): peptone 15-20, beef extract 10-12, yeast extract powder 5-7, glucose 10-15, sodium acetate 5-7, ammonium citrate 2-2.5, manganese sulfate 0.05, sulfuric acid Magnesium 0.2, agar 15-18.

[0044] Seed medium (g / L): peptone 10~20, yeast extract powder 5~7, glucose 10~15, sodium acetate 0.1, ammonium citrate 2~2.5, manganese sulfate 0.05, magnesium sulfate 0.2, Tween-801~ 2mL.

[0045] (2) Bacterial liquid quenching

[0046]...

Embodiment 2

[0066] Effect of cleaning times on the extract of Enterococcus raffinose CGMCC No.5584

[0067] With reference to Example 1, the number of times when the cells were washed with 0.6-0.85% NaCl was changed to 2, 3, and 4 times respectively, and other conditions were unchanged. The sample obtained after the treatment was detected by GC-MS, and the metabolite chromatogram Such as figure 2 As shown, the times of cleaning from top to bottom are 2 times, 3 times, and 4 times in turn, and the overlay of metabolite chromatograms is shown in image 3 The number of metabolites is shown in Table 2.

[0068] Table 2 The number of metabolites in samples with different washing times

[0069]

[0070] The detection of intracellular metabolites by GC-MS found that when the number of washings was 2 times, 238 substances were detected; when the number of washings was 3 times, 253 substances were detected; when the number of washings was 4 times, 197 substances were detected. When the numb...

Embodiment 3

[0072] Effect of freeze-thaw time on the extract of Enterococcus raffinose CGMCC No.5584

[0073] With reference to Example 1, the freeze-thaw time was changed to 2min, 3min, and 4min respectively, and other conditions were unchanged. After the treatment, the sample obtained was detected by GC-MS, and the metabolite chromatogram was as follows: Figure 4 As shown, the freeze-thaw time from top to bottom is washing 2min, 3min, 4min in turn, and the metabolite chromatogram overlay is as follows Figure 5 The number of metabolites is shown in Table 3.

[0074] Table 3 The number of metabolites in samples with different freeze-thaw time

[0075]

[0076] When the freeze-thaw time was controlled at 2 min, 229 metabolites were detected; when the freeze-thaw time was controlled at 3 min, 253 metabolites were detected; when the freeze-thaw time was controlled at 4 min, 247 metabolites were detected. After comparing the experimental results, it is found that the amount of metaboli...

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Abstract

The invention discloses a sample treatment method for the metabonomics research of enterococcus raffinosus, and belongs to the field of chemical analysis and detection. The method comprises the steps of culture of the enterococcus raffinosus, quenching of bacterial liquid, cleaning of thalli, extraction and derivatization of intracellular metabolites, GC-MS detection and metabolite analysis. The method comprises the following steps: quenching a bacterial liquid sample by adopting a precooled methanol-ultrapure water quenching liquid; adopting a NaCl solution with the mass fraction being 0.6%-0.85% for cleaning the thalli; crushing the thalli by using a liquid nitrogen grinding method; repeatedly freezing and thawing intracellular small molecule metabolites by adopting a mixed solution of chloroform, ultrapure water and ethanol to extract; carrying out derivatization treatment on a sample through a methoxyammonium hydrochloride / pyridine solution and N-methyl-N-(trimethylsilane) trifluoroacetamide to obtain a pretreatment sample, and obtaining the quantity maximization of intracellular metabolites through GC-MS computer detection. The method can be used for researching the metabolic mechanism of the enterococcus raffinosus by a metabonomics method, and provides technical method support and scientific experimental basis for the metabonomics research of the enterococcus raffinosus in the future.

Description

technical field [0001] This patent relates to the detection of intracellular small molecule metabolites of Enterococcus raffinose by using GC-MS, which belongs to the field of chemical detection and analysis. Background technique [0002] Enterococcus raffinosus is a bacterium of the genus Enterococcus, which belongs to the facultative anaerobic Gram-positive bacteria. It is widely distributed in various environments in nature. It is also widespread and is part of the intestinal flora, which can lead to infection when parts of the body are dysfunctional. Although Enterococcus raffinose has certain pathogenicity, clinical infection cases are rare, and it is less pathogenic than strains of the same genera such as Enterococcus faecium and Enterococcus faecalis. Therefore, it is safer to use Enterococcus raffinose to carry out experiments related to Enterococcus. Therefore, this strain will become an important strain for the study of Enterococcus and other related strains. [...

Claims

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Application Information

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IPC IPC(8): G01N30/06
CPCG01N30/06
Inventor高强杨新达牛肖凡尤田朱芳芳刘之毅高幸朱燕
OwnerTIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY