阅读说明：本技术 基于代谢组学建立阿莫西林浓度对嗜酸乳杆菌影响的方法 (Method for establishing influence of amoxicillin concentration on lactobacillus acidophilus based on metabonomics ) 是由 苏志恒 郭玥 刘西 黄慧敏 宋慧 郑华 梁永红 冯氏岁 蒙明薇 于 2020-04-17 设计创作，主要内容包括：本发明公开了一种基于代谢组学建立阿莫西林浓度对嗜酸乳杆菌影响的方法,包括：以不同浓度阿莫西林给药后的嗜酸乳杆菌胞内代谢物进行液质联用分析,得到代谢物的碎片信息；通过建立正交偏最小二乘法判别分析模型对代谢物的碎片信息进行筛选,得到潜在的生物标志物；其中,筛选条件为变量重要性投影值大于1、差异倍数绝对值大于1、p值小于0.05；对潜在的生物标志物进行代谢通路富集,得到不同浓度阿莫西林引起嗜酸乳杆菌代谢紊乱发生发展的相关重要代谢通路；将重要的代谢通路里的潜在生物标志物通过代谢通路连接起来,得到综合代谢网络。本发明建立阿莫西林通过影响嗜酸乳杆菌代谢而造成损伤的方法,得到在阿莫西林用于嗜酸乳杆菌时,嗜酸乳杆菌的代谢物以及代谢通路之间的联系,以便对阿莫西林诱导的嗜酸乳杆菌损伤的作用机理进一步的研究。(The invention discloses a method for establishing the influence of amoxicillin concentration on lactobacillus acidophilus based on metabonomics, which comprises the following steps: carrying out LC-MS analysis on intracellular metabolites of lactobacillus acidophilus after the amoxicillin with different concentrations is administrated, and obtaining fragment information of the metabolites; screening fragment information of the metabolite by establishing an orthogonal partial least square method discriminant analysis model to obtain a potential biomarker; wherein the screening conditions are that the projection value of the variable importance is greater than 1, the absolute value of the difference multiple is greater than 1, and the p value is less than 0.05; carrying out metabolic pathway enrichment on the potential biomarker to obtain relevant important metabolic pathways for development of lactobacillus acidophilus metabolic disorder caused by amoxicillin with different concentrations; potential biomarkers in important metabolic pathways are connected through the metabolic pathways to obtain the comprehensive metabolic network. The invention establishes a method for causing damage to amoxicillin by influencing the metabolism of lactobacillus acidophilus, and obtains the relation between the metabolite of lactobacillus acidophilus and the metabolic pathway when amoxicillin is used for lactobacillus acidophilus, so as to further research the action mechanism of the lactobacillus acidophilus damage induced by amoxicillin.)

1. A method for establishing the influence of amoxicillin concentration on lactobacillus acidophilus based on metabonomics is characterized by comprising the following steps:

carrying out LC-MS analysis on intracellular metabolites of lactobacillus acidophilus after the amoxicillin with different concentrations is administrated, and obtaining fragment information of the metabolites;

screening fragment information of the metabolite by establishing an orthogonal partial least square method discriminant analysis model to obtain a potential biomarker; wherein the screening conditions are that the projection value of the variable importance is greater than 1, the absolute value of the difference multiple is greater than 1, and the p value is less than 0.05;

carrying out metabolic pathway enrichment on the potential biomarker to obtain relevant important metabolic pathways for development of lactobacillus acidophilus metabolic disorder caused by amoxicillin with different concentrations;

potential biomarkers in important metabolic pathways are connected through the metabolic pathways to obtain the comprehensive metabolic network.

2. The metabolomics-based method for establishing the effect of amoxicillin concentration on lactobacillus acidophilus according to claim 1, wherein the intracellular metabolites of lactobacillus acidophilus after the administration of amoxicillin at different concentrations are obtained by:

dividing Lactobacillus acidophilus cultured to logarithmic phase into 4 groups of 8 biological repeats, respectively feeding to 1.0 × 10^-4，1.0×10^-5，1.0×10^-6Amoxicillin at M concentration, collected 24 hours after dosing.

3. The metabolomics-based method for establishing the effect of amoxicillin concentration on lactobacillus acidophilus according to claim 1, characterized in that: the conditions of the LC-MS are as follows:

adopting 100 × 2.1.1 mm, 1.8 μm particle size and HSS T3C 18 chromatographic column as chromatographic column, wherein the column temperature of the chromatographic column is 40 deg.C;

the mobile phase A is ammonium acetate, the mobile phase B is acetonitrile, and the flow rate of the mobile phase is 0.3m L & min^-1；

The elution procedure comprises the steps of eluting for 0-1.5 minutes by using 90-70% of the solution A, then continuously eluting for 1.5-8 minutes by using 70-2% of the solution A, then eluting for 8-9 minutes by using 2-90% of the solution A, and finally eluting for 9-10 minutes by using 90% of the solution A, wherein the sample injection temperature is 4 ℃, and the sample injection amount is 5 mu L;

the mass number range of the collected mass spectrum is 50-1200 Da, and all data are collected in a positive ion mode and a negative ion mode of an electrospray ion source, wherein the capillary voltage is 3.0kV, the foramen voltage is 40kV, the extraction voltage is 4.0kV, the ion source temperature is 100 ℃, the desolvation gas temperature is 350 ℃, the taper hole gas flow is 40L & h^-1Desolventizing agent flow rate 800L ·h^-1。

4. The metabolomics-based method for establishing the effect of amoxicillin concentration on lactobacillus acidophilus according to claim 1, wherein the specific steps for obtaining the potential biomarkers are:

grouping according to the intracellular metabolites of lactobacillus acidophilus after administration at different concentrations, setting a blank control group, and respectively performing principal component analysis, partial least square method discriminant analysis and orthogonal partial least square method discriminant analysis on each group;

screening potential biomarkers among groups, and selecting variables of which the variable importance projection value of the differential metabolite is greater than 1, the absolute value of the difference multiple is greater than 1 and the p value is less than 0.05 as the potential biomarkers;

the partial least square method discriminant analysis model fitting ability indexes are respectively as follows:

in positive ion mode: r²X＝0.554，R²Y＝0.921，Q²＝0.659；

In the negative ion mode: r²X＝0.613，R²Y＝0.854，Q²＝0.536。

5. The metabolomics-based method for establishing the effect of amoxicillin concentration on lactobacillus acidophilus according to claim 1, wherein the specific steps of metabolic pathway enrichment for potential biomarkers are:

uploading the potential biomarkers to a MetabioAnalyst analysis webpage for enrichment of relevant pathways.

6. The metabolomics-based method for establishing the effect of amoxicillin concentration on lactobacillus acidophilus according to claim 1, which further comprises, before the LC-MS, a pretreatment of the intracellular metabolites of lactobacillus acidophilus:

centrifuging the acidophilic lactobacillus liquid with different concentrations at low speed and low temperature for 10 minutes under the condition of 2000 revolutions respectively to obtain precipitates;

washing the precipitate with phosphate buffer solution for 3 times, adding 3 times volume of glacial methanol to extract protein for 20min, ultrasonically crushing for 15 min, centrifuging at high speed and low temperature for 20min at 12000 r, and collecting supernatant;

the supernatant was dried with nitrogen, redissolved with an equal volume of 400 μ L acetonitrile in water before injection, and filtered through a 0.22 μm nylon filter.

7. The metabonomics-based method for establishing the effect of amoxicillin concentration on lactobacillus acidophilus according to claim 4, wherein before the principal component analysis, the fragment information of metabolites is preprocessed, the data is processed by 80% filtering principle, and the variables with blank value more than 80% are removed;

wherein the preprocessing is carried out on Marker L ynx 4.1.1 software and MetabioAnalyst website;

the parameters of the pretreatment are as follows: the retention time is 0-10 min; the mass number is 50-1200 Da; a mass number tolerance of 0.01; the mass number window is 0.02; the noise removal level was 6.

Technical Field

The invention belongs to the technical field of analysis, and particularly relates to a method for screening an amoxicillin concentration by using a lactobacillus acidophilus metabolite.

Background

Antibiotics have some adverse reactions in clinical use, wherein the gastrointestinal reaction is one of the more common reactions. Such as antibiotic-associated diarrhea. The reason why antibiotics cause diarrhea is that the antibiotics destroy the steady balance of intestinal flora while having an antibacterial effect, and harm probiotics while killing harmful bacteria. It has been shown that lactobacilli are beneficial probiotics for health and cause damage to antibiotics after administration.

The amoxicillin is one of the most commonly used semi-synthetic penicillins, namely broad-spectrum β -lactam antibiotics, is stable under acidic conditions, has the gastrointestinal absorption rate of 90%, has strong sterilization effect and strong cell membrane penetrating capability, is one of the oral semi-synthetic penicillins widely applied at present, and has preparations such as capsules, tablets, granules, dispersible tablets and the like.

Lactobacillus acidophilus is the most representative strain of lactic acid bacteria, is one of few beneficial bacteria in human intestinal tracts, can improve the balance of intestinal flora, enhance immunity, reduce cholesterol level, relieve lactose intolerance and inhibit tumor formation, is considered as a food additive safe to human bodies, and is widely applied to various foods. However, in recent years, in the course of treating diseases, the use of large amounts of antibiotics causes a disturbance in intestinal microflora, a decrease in beneficial bacteria, an increase in pathogenic bacteria, a decrease in the diversity of microorganisms, a change in the metabolic function of the microflora, and in severe cases, intestinal diseases and the like.

Therefore, a method for establishing the influence of amoxicillin concentration on lactobacillus acidophilus based on metabonomics is needed.

Disclosure of Invention

It is an object of the present invention to address at least the above-mentioned deficiencies and to provide at least the advantages which will be described hereinafter.

The invention also aims to provide a method for establishing the influence of the concentration of amoxicillin on lactobacillus acidophilus based on metabonomics, which can clarify the action mechanism of amoxicillin-induced lactobacillus acidophilus damage.

To achieve these objects and other advantages in accordance with the present invention, there is provided a method for establishing the effect of amoxicillin concentration on lactobacillus acidophilus based on metabolomics, comprising:

carrying out LC-MS analysis on the intracellular metabolites of lactobacillus acidophilus after the amoxicillin with different concentrations is administrated, and obtaining fragment information of the metabolites.

Screening fragment information of the metabolite by establishing an orthogonal partial least square method discriminant analysis model to obtain a potential biomarker; wherein, the screening conditions are that the projection value of the variable importance is more than 1, the absolute value of the difference multiple is more than 1, and the p value is less than 0.05.

And (3) carrying out metabolic pathway enrichment on the potential biomarker to obtain relevant important metabolic pathways for development of lactobacillus acidophilus metabolic disorder caused by amoxicillin with different concentrations.

Potential biomarkers in important metabolic pathways are connected through the metabolic pathways to obtain the comprehensive metabolic network.

In the scheme, firstly, the intracellular metabolites of the lactobacillus acidophilus damaged by amoxicillin with different concentrations are analyzed by an ultra-high performance liquid chromatography-tandem mass spectrometry technology to obtain information containing retention time and mass-to-charge ratio; establishing an orthogonal partial least square method discriminant analysis model to screen data according to the conditions that the variable importance projection value is greater than 1, the difference multiple absolute value is greater than 1 and the p value is less than 0.05 to obtain a potential biomarker; analyzing chemical structural formulas of retention time, accurate molecular mass and mass spectrum fragment information of the metabolites, and verifying the chemical structural formulas through an online database to obtain metabolites with statistical differences; carrying out metabolic pathway enrichment according to the different metabolites to obtain relevant important metabolic pathways for development of lactobacillus acidophilus metabolic disorder caused by amoxicillin with different concentrations; important metabolic pathways are connected to obtain a comprehensive metabolic network to prompt that clinical medication dosage is emphasized, and a foundation is laid for research, development and use of gastrointestinal tract protection drugs.

Preferably, the intracellular metabolites of lactobacillus acidophilus after administration at different concentrations are obtained by:

dividing Lactobacillus acidophilus cultured to logarithmic phase into 3 groups, and respectively feeding to 1.0 × 10^-4，1.0×10^-5，1.0×10^-6Amoxicillin at M concentration, collected 24 hours after dosing.

In the scheme, the lactobacillus acidophilus is taken as a research object, and is administrated in the logarithmic growth phase, so that the damage effect of the lactobacillus acidophilus on administration due to the influence of self factors in the analysis process is avoided.

Preferably, the conditions of the LC-MS are as follows:

adopting 100 × 2.1.1 mm, 1.8 μm particle size and HSS T3C 18 chromatographic column as chromatographic column, wherein the column temperature of the chromatographic column is 40 deg.C;

the mobile phase A is ammonium acetate, the mobile phase B is acetonitrile, and the flow rate of the mobile phase is 0.3m L & min^-1；

The elution procedure comprises the steps of eluting for 0-1.5 minutes by using 90-70% of the solution A, then continuously eluting for 1.5-8 minutes by using 70-2% of the solution A, then eluting for 8-9 minutes by using 2-90% of the solution A, and finally eluting for 9-10 minutes by using 90% of the solution A, wherein the sample injection temperature is 4 ℃, and the sample injection amount is 5 mu L;

the mass number range of the collected mass spectrum is 50-1200 Da, and all data are collected in a positive ion mode and a negative ion mode of an electrospray ion source, wherein the capillary voltage is 3.0kV, the foramen voltage is 40kV, the extraction voltage is 4.0kV, the ion source temperature is 100 ℃, the desolvation gas temperature is 350 ℃, the taper hole gas flow is 40L & h^-1Desolventizing rate 800L. multidot.h^-1。

Preferably, the specific steps for obtaining the differential metabolite are:

grouping lactobacillus acidophilus intracellular metabolites after amoxicillin administration with different concentrations, setting a blank control group, and respectively performing principal component analysis, partial least square method discriminant analysis and orthogonal partial least square method discriminant analysis on each group;

screening differential metabolites among groups, and selecting variables of which the variable importance projection value is greater than 1, the absolute value of the difference multiple is greater than 1 and the p value is less than 0.05 as the differential metabolites;

the partial least square method discriminant analysis model fitting ability indexes are respectively as follows:

in positive ion mode: r²X＝0.554，R²Y＝0.921，Q²＝0.659；

In the negative ion mode: r²X＝0.613，R²Y＝0.854，Q²＝0.536。

Preferably, the specific steps for enriching the metabolic pathways of the differential metabolites are as follows: uploading the different metabolites to a metabolic component online analysis webpage, recording the name of each different metabolite, and enriching related pathways.

Preferably, the method further comprises the step of pretreating the intracellular metabolites of lactobacillus acidophilus:

and (3) centrifuging the intracellular metabolites of the lactobacillus acidophilus after the amoxicillin with different concentrations is administered for 10 minutes at low speed and low temperature under the condition of 2000 revolutions respectively to obtain precipitates.

Washing the precipitate with phosphate buffer solution for 3 times, adding 3 times volume of glacial methanol to extract protein for 20min, performing ultrasonication for 15 min, centrifuging at high speed and low temperature at 12000 r for 20min, and collecting supernatant.

The supernatant was dried with nitrogen, redissolved with an equal volume of 400 μ L acetonitrile in water before injection, and filtered through a 0.22 μm nylon filter.

In the above scheme, the culture solution and the like are removed by pretreating the intracellular metabolite of lactobacillus acidophilus to ensure the stability of the intracellular metabolite of lactobacillus acidophilus for LC-MS analysis.

Preferably, before the principal component analysis, the fragment information of the metabolite is processed, the data is processed by an 80% filtering principle, the variable with blank value more than 80% is removed, and preprocessing such as normalization, peak alignment and the like is performed; wherein the preprocessing is performed at the MetabioAnalyst website;

the parameters of the pretreatment are as follows: the retention time is 0-10 min; the mass number is 50-1200 Da; a mass number tolerance of 0.01; the mass number window is 0.02; the noise removal level was 6.

In the scheme, the variable with blank value more than 80% is removed to reduce the missing value caused by the peak which does not exist in the chromatogram so as to influence the judgment of the result.

The invention at least comprises the following beneficial effects:

firstly, analyzing intracellular metabolites of lactobacillus acidophilus damaged by amoxicillin with different concentrations by using an ultra-high performance liquid chromatography-tandem mass spectrometry technology to obtain fragment information containing retention time and a mass-to-charge ratio, establishing a model, and screening data according to the conditions that a variable importance projection value is greater than 1, a difference multiple absolute value is greater than 1 and a p value is less than 0.05 to obtain a potential biomarker; analyzing chemical structural formulas of retention time, accurate molecular mass and mass spectrum fragment information of the metabolites, and verifying the chemical structural formulas through an online database to obtain metabolites with statistical differences; carrying out metabolic pathway enrichment according to the different metabolites to obtain relevant important metabolic pathways for development of lactobacillus acidophilus metabolic disorder caused by amoxicillin with different concentrations; important metabolic pathways are connected to obtain a comprehensive metabolic network to prompt that clinical medication dosage is emphasized, and a foundation is laid for research, development and use of gastrointestinal tract protection drugs.

Secondly, taking lactobacillus acidophilus as a research object, and administering the lactobacillus acidophilus in the logarithmic growth phase to avoid the damage effect of the lactobacillus acidophilus on administration due to self factors in the analysis process.

Furthermore, the culture fluid and the like are removed by pretreatment of the intracellular metabolites of lactobacillus acidophilus to ensure the stability of the intracellular metabolites of lactobacillus acidophilus for the LC-MS analysis.

Finally, the variable with blank value more than 80% is removed to reduce the missing value caused by the peak which does not exist in the chromatogram so as to influence the judgment of the result.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a graph of the MTT assay for Lactobacillus acidophilus following administration of different concentrations in an example of the present invention;

FIG. 2 is a scanning map of the plasma reactivity of Lactobacillus acidophilus after administration of different concentrations in an embodiment of the present invention;

FIG. 3 is a flow chart of each set of total ions collected in a positive ion mode and a negative ion mode based on ultra performance liquid chromatography-tandem mass spectrometry in an embodiment of the present invention;

FIG. 4 is a three-dimensional PCA plot of Lactobacillus acidophilus metabolites after various concentrations of amoxicillin administration in accordance with an embodiment of the present invention;

FIG. 5 is a plot of P L S-DA of Lactobacillus acidophilus metabolites after various concentrations of amoxicillin administration in accordance with embodiments of the present invention;

FIG. 6 is a graph of 200 displacement assays of Lactobacillus acidophilus metabolites after various concentrations of amoxicillin administration in accordance with an embodiment of the present invention;

FIG. 7 is a graph of the OP L S-DA profile of Lactobacillus acidophilus metabolites with respect to a control group, respectively, after administration of varying concentrations of amoxicillin in accordance with embodiments of the present invention;

FIG. 8 is an enrichment map of metabolic pathways constructed on-line on the metaboanalyst platform according to an embodiment of the present invention;

FIG. 9 is a diagram of the comprehensive metabolic network established by the embodiment of the present invention.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.