CN102262136A - Method for analyzing change rule of metabolites during fermentation of cephalosporium acremonium - Google Patents
Method for analyzing change rule of metabolites during fermentation of cephalosporium acremonium Download PDFInfo
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Abstract
The invention discloses a method for analyzing a change rule of metabolites during fermentation of cephalosporium acremonium. The method comprises the following steps of: sampling and centrifuging before fermentation, during fermentation and after fermentation of cephalosporium acremonium generated by cephalosporin C, mixing phosphate buffer solution into sediment uniformly and centrifuging; quenching by using liquid nitrogen and grinding; adding powdered cells into methanol aqueous solution and butanedioic acid aqueous solution which is marked by deuterium and is used as an internal standard substance, and mixing uniformly; freezing and melting, centrifugally collecting the supernate and drying; performing oximation reaction; performing silanization reaction; determining components and relative content of a sample by using a gas chromatography-flying time mass spectrometer; analyzing principal components of preprocessed data by using software so as to analyze metabolism difference of the samples; performing cluster analysis on the preprocessed data by using the software so as to analyze the metabolism difference of metabolites among the samples; determining the nature of the metabolites to classify the metabolites; and analyzing the change rule of all types of the metabolites during the fermentation so as to comprehensively disclose metabolism information of the cephalosporium acremonium according to a large amount of metabolism physiome data.
Description
Technical field
The present invention relates to the analytical approach of metabolin Changing Pattern in a kind of cephalosporium sp sweat.
Background technology
Cephalosporin analog antibiotic is a kind of of beta-lactam antibiotic, reaches the purpose of sterilization by the generation that suppresses bacteria cell wall.Since its high antibacterial infection activity and low spinoff incidence, widespread use clinically.As the synthetic raw material of cephalosporins medicine, the achievement far away exploitation of the cephalo medicine of too late a new generation obtained to the fermentation research of cephalosporin.Therefore need more research, to obtain bigger cynnematin fermentation titer about the production run of cynnematin.
Metabolism group can be come postgraduate's objects system by the variation of its metabolic product after investigation living things system irriate or the disturbance.The key of metabolism group is to test the mass data information excavating process behind that obtains.The information that traditional data processing method obtains is single relatively, and still needs a large amount of manually-operateds, and a large amount of valuable information are run off.The metabolism group data analysing method of seeking a kind of comparatively system is particularly important with regard to what show.
Summary of the invention
The analytical approach that the purpose of this invention is to provide metabolin Changing Pattern in a kind of cephalosporium sp sweat.
Technical scheme of the present invention is summarized as follows:
The analytical approach of metabolin Changing Pattern comprises the steps: in the cephalosporium sp sweat
(1) produce earlier fermentation, mid-term and the later stage of bacterial strain cephalosporium sp at cephalosporin, respectively getting 2-3 sampling spot, to get the 100-300ml fermentation liquor centrifugal, abandons supernatant;
(2) to add 100-300ml pH value be 7.2 phosphate buffer for precipitation part, centrifugal behind the mixing; Sedimentation cell in liquid nitrogen cancellation to stop the metabolic response of cell, with liquid nitrogen grinding to fine-powdered;
(3) get the fine-powdered cell 20-80mg that step (2) obtains and place centrifuge tube, the volumetric concentration that adds 0.5-1.5ml-40 ℃～-50 ℃ is that 50% methanol aqueous solution is an extract, and the deuterium-labelled succinic acid aqueous solution of the 0.14mg/ml that adds 10 μ l～20 μ l is internal standard compound, mixing; Place the liquid nitrogen multigelation 2-4 time, freeze 0.5-2.5min at every turn; Centrifugal collection supernatant and freeze drying;
(4) the 20mg/ml methoxy amine hydrochlorate pyridine solution of adding 40-60 μ l in the freeze-drying sample that step (3) obtains, oximation reaction 60-100min is carried out in 30-40 ℃ of water-bath; Add N-methyl-N-trimethyl silane trifluoroacetamide of 50-100 μ l again, Silanization reaction 50-70min is carried out in 30-40 ℃ of water-bath;
(5) with gas chromatography-flight time mass spectrum combined instrument working sample composition and relative content:
The sample of 1 μ l silanization is entered in the gas chromatography, chromatographic column is DB-5MS, the specification of described chromatographic column is 30m * 0.25mm i.d., utilization Masslynx software carries out structure prediction to the metabolin in the sample, a kind of multiple mass spectrogram that predicts the outcome of metabolin is analyzed with final definite metabolin structure, according to qualitative results and retention time the various metabolin areas in each sample are obtained, with each metabolin peak area in each sample with compare with the internal standard compound peak area in the individual sample, obtain the relative peak area of each metabolin, can represent the relative content of metabolin in the sample;
(6) utilize Matlab software that pretreated data are carried out principal component analysis (PCA), the metabolism difference of each sample room of holistic approach; Utilize Expander software that pretreated data are carried out cluster analysis, to analyze the metabolism difference of each metabolin at sample room; Qualitative by metabolin, and then metabolin classified, each metabolite Changing Pattern is during the fermentation analyzed.
The present invention has following advantage:
(1) can be accurately carry out qualitative to the fermentating metabolism thing of cephalosporium sp;
(2) utilize internal standard compound that the metabolin of each sample of sweat is carried out ratio analysis, can reach the identical effect of absolute quantitation, and more convenient;
(3) utilize various software analysis, can study the metabolic characteristic of cephalosporium sp fermentation, make the analysis of metabolic rule more complete from a plurality of angles.
Description of drawings
Fig. 1 is the total ion current figure of cephalosporium sp sweat metabolin.
Fig. 2 is the mass spectrogram of 15.92 minutes metabolin for retention time.
Fig. 3 is the characteristic peak chromatogram that comprises the special characteristic fragment.
Fig. 4 is for to carry out the qualitative different qualitative results that obtain to characteristic peak.
Fig. 5 is the principal component analysis (PCA) result of cephalosporium sp different fermentations metabolic characteristics in period.
Fig. 6 is that cephalosporium sp different metabolic thing is in the different fermentations analysis of trend result in period.
Fig. 7 is the variation diagram that synthesizes closely-related precursor amino acid and related amino acid in cephalosporin pilot scale and the production run in the born of the same parents with cephalosporin.A is an alpha-Aminoadipic acid, and b is a halfcystine, and c is a valine, and d is a lysine, and e is a methionine.
Embodiment
The following examples are in order to make those skilled in the art understand the present invention better but are not limited to the present invention, for example: cephalosporium sp, except that disclosed by the invention several, other the cephalosporium sp that is used for fermentative production of cephalosporin C also can be used for the present invention.
The present invention is further described below in conjunction with specific embodiment:
Embodiment 1
The analytical approach of metabolin Changing Pattern comprises the steps: in the cephalosporium sp sweat
(1) respectively get 3 sampling spots in earlier fermentation, mid-term, the later stage of cephalosporin production bacterial strain cephalosporium sp (Cephalosporium acremonium) CICC No 40515, it is centrifugal to get the 100ml fermentation liquor, abandons supernatant.
(2) to add 100ml pH value be 7.2 phosphate buffer for precipitation part, centrifugal behind the mixing.Sedimentation cell in liquid nitrogen cancellation to stop the metabolic response of cell, with liquid nitrogen grinding to fine-powdered;
(3) fine-powdered cell 20mg is placed centrifuge tube, the volumetric concentration that adds 0.5ml-40 ℃ of preservation is that 50% methanol aqueous solution is an extract, and the deuterium-labelled succinic acid aqueous solution of 0.14mg/ml that adds 10 μ l is internal standard compound, mixing; Place the liquid nitrogen multigelation 2 times, freeze 0.5min at every turn; Centrifugal collection supernatant and freeze drying;
(4) the 20mg/ml methoxy amine hydrochlorate pyridine solution of adding 40 μ l in the sample of freeze-drying, oximation reaction 60min is carried out in 30 ℃ of water-baths; Add N-methyl-N-trimethyl silane trifluoroacetamide of 50 μ l again, Silanization reaction 50min is carried out in 30 ℃ of water-baths;
(5) with gas chromatography-flight time mass spectrum combined instrument working sample composition and relative content:
1 μ l silanization sample is entered in the gas chromatography, and chromatographic column is DB-5MS, and the specification of described chromatographic column is 30m * 0.25mm i.d..Utilization Masslynx software carries out structure prediction to the metabolin in the sample, a kind of multiple mass spectrogram that predicts the outcome of metabolin is analyzed with final definite metabolin structure, according to qualitative results and retention time the various metabolin areas in each sample are obtained, with each metabolin peak area in each sample with compare with the internal standard compound peak area in the individual sample, obtain the relative peak area of each metabolin, can represent the relative content of metabolin in the sample.
(6) utilize Matlab software that pretreated data are carried out principal component analysis (PCA), the metabolism difference of each sample room of holistic approach.Utilize Expander that pretreated data are carried out cluster analysis, to analyze the metabolism difference of each metabolin at sample room.Qualitative by metabolin, and then metabolin classified, each metabolite Changing Pattern is during the fermentation analyzed.
With retention time is that the ion fragment analysis of locating in 15.92 minutes is an example, selects 204.152 to be its characteristic fragment according to its mass spectrogram (Fig. 2), and then obtains containing all characteristic peak chromatograms (Fig. 3) of this characteristic fragment.Carry out qualitative according to this chromatogram to metabolin.Obtain two kinds of different qualitative results (Fig. 4), according to its result's characteristic fragment with the matching degree of the mass spectrogram of target metabolin, find that the metabolin of the gained of Fig. 4-1 meets the ion fragment of target metabolin more.Its structure is studied, determined that this metabolin is a serine.
Fig. 5-1 and 5-2 are the principal component analysis (PCA) result who utilizes cephalosporium sp metabolic characteristic in industry that Matlab obtains and the pilot scale process.Its objective is the whole metabolic characteristics of each sample is analyzed.Major component result shows, utilizes metabolic characteristics each sweat can be divided into three periods, and institute's branch meets early stage, mid-term, the later stage that the fermentation experience obtains period and distributes.
Fig. 6 is the Changing Pattern cluster analysis result of part metabolin in each sample that utilizes Expander software to obtain.Find that from figure the metabolite content in the industrial fermentation process is higher than the content of metabolin in the pilot scale process, and all be that the metabolite content of ferment middle is higher in two processes.Cluster result has embodied the rule that each metabolin changes in the cephalosporium sp sweat, shows the metabolic activity that ferment middle is higher.
Fig. 7 is the comparison to the metabolin relative content.Three seed amino acid precursors and relevant amino acid thereof are analyzed: find that three seed amino acid precursors all are that the pilot scale process is higher.Alpha-Aminoadipic acid can be used for synthetic cephalosporin in cephalosporium sp, but also synthetic lysine.The mensuration of lysine is found that it increased since 80 hours, and beginning sharply increases after 100 hours, and this phenomenon is especially obvious in industrial process.Show the metabolism stream that begins more to flow to synthetic lysine since 80 hours alpha-Aminoadipic acids, can select the corresponding time point metabolism stream that lysine is synthetic to suppress, make alpha-Aminoadipic acid flow to the synthetic metabolism stream of a C according to this result; Halfcystine can be synthetic by the methionine transsulfuration.The analysis of methionine and halfcystine has been shown the content of two these two seed amino acids of process, can further adjust its feed supplement amount and time, to obtain better precursor conversion effect.
The analytical approach of metabolin Changing Pattern comprises the steps: in the cephalosporium sp sweat
(1) respectively get 3 sampling spots in earlier fermentation, mid-term, the later stage of cephalosporin production bacterial strain cephalosporium sp (Cephalosporium acremonium) CICC No 40514, it is centrifugal to get the 200ml fermentation liquor, abandons supernatant.
(2) to add 200ml pH value be 7.2 phosphate buffer for precipitation part, centrifugal behind the mixing.Sedimentation cell in liquid nitrogen cancellation to stop the metabolic response of cell, with liquid nitrogen grinding to fine-powdered.
(3) fine-powdered cell 50mg is placed centrifuge tube, the volumetric concentration that adds 1ml-45 ℃ of preservation is that 50% methanol aqueous solution is an extract, and the deuterium-labelled succinic acid aqueous solution of 0.14mg/ml that adds 15 μ l is internal standard compound, mixing; Place the liquid nitrogen multigelation 3 times, freeze 1.5min at every turn; Centrifugal collection supernatant and freeze drying.
(4) the 20mg/ml methoxy amine hydrochlorate pyridine solution of adding 50 μ l in the sample of freeze-drying, oximation reaction 80min is carried out in 35 ℃ of water-baths; Add N-methyl-N-trimethyl silane trifluoroacetamide of 75 μ l again, Silanization reaction 60min is carried out in 35 ℃ of water-baths.
(5) with gas chromatography-flight time mass spectrum combined instrument working sample composition and relative content:
The sample of 1 μ l silanization is entered in the gas chromatography, and chromatographic column is DB-5MS, and the specification of described chromatographic column is 30m * 0.25mm i.d..Utilization Masslynx software carries out structure prediction to the metabolin in the sample, a kind of multiple mass spectrogram that predicts the outcome of metabolin is analyzed with final definite metabolin structure, according to qualitative results and retention time the various metabolin areas in each sample are obtained, with each metabolin peak area in each sample with compare with the internal standard compound peak area in the individual sample, obtain the relative peak area of each metabolin, can represent the relative content of metabolin in the sample.
(6) utilize Matlab software that pretreated data are carried out principal component analysis (PCA), the metabolism difference of each sample room of holistic approach.Utilize Expander software that pretreated data are carried out cluster analysis, to analyze the metabolism difference of each metabolin at sample room.Qualitative by metabolin, and then metabolin classified, each metabolite Changing Pattern is during the fermentation analyzed.Proof by experiment, the method for present embodiment also can obtain the result similar to embodiment 1.
Embodiment 3
The analytical approach of metabolin Changing Pattern comprises the steps: in the cephalosporium sp sweat
(1) respectively get 3 sampling spots in earlier fermentation, mid-term, the later stage of cephalosporin production bacterial strain cephalosporium sp (Cephalosporium acremonium) CICC No 2444, it is centrifugal to get the 300ml fermentation liquor, abandons supernatant.
(2) to add the 300mlpH value be 7.2 phosphate buffer for precipitation part, centrifugal behind the mixing.Sedimentation cell in liquid nitrogen cancellation to stop the metabolic response of cell, with liquid nitrogen grinding to fine-powdered.
(3) fine-powdered cell 80mg is placed centrifuge tube, the volumetric concentration that adds 1.5ml-50 ℃ of preservation is that 50% methanol aqueous solution is an extract, and the deuterium-labelled succinic acid aqueous solution of 0.14mg/ml that adds 20 μ l is internal standard compound, mixing; Place the liquid nitrogen multigelation 4 times, freeze 2.5min at every turn; Centrifugal collection supernatant and freeze drying.
(4) the 20mg/ml methoxy amine hydrochlorate pyridine solution of adding 60 μ l in the sample of freeze-drying, oximation reaction 100min is carried out in 40 ℃ of water-baths; Add N-methyl-N-trimethyl silane trifluoroacetamide of 100 μ l again, Silanization reaction 70min is carried out in 40 ℃ of water-baths.
(5) with gas chromatography-flight time mass spectrum combined instrument working sample composition and relative content:
The sample of 1 μ l silanization is entered in the gas chromatography, and chromatographic column is DB-5MS, and the specification of described chromatographic column is 30m * 0.25mm i.d..Utilization Masslynx software carries out structure prediction to the metabolin in the sample, a kind of multiple mass spectrogram that predicts the outcome of metabolin is analyzed with final definite metabolin structure, according to qualitative results and retention time the various metabolin areas in each sample are obtained, with each metabolin peak area in each sample with compare with the internal standard compound peak area in the individual sample, obtain the relative peak area of each metabolin, can represent the relative content of metabolin in the sample.
(6) utilize Matlab software that pretreated data are carried out principal component analysis (PCA), the metabolism difference of each sample room of holistic approach.Utilize Expander software that pretreated data are carried out cluster analysis, to analyze the metabolism difference of each metabolin at sample room.Qualitative by metabolin, and then metabolin classified, each metabolite Changing Pattern is during the fermentation analyzed.Proof by experiment, the method for present embodiment also can obtain the result similar to embodiment 1.
Claims (1)
1. the analytical approach of metabolin Changing Pattern in the cephalosporium sp sweat, its feature comprises the steps:
(1) produce earlier fermentation, mid-term and the later stage of bacterial strain cephalosporium sp at cephalosporin, respectively getting 2-3 sampling spot, to get the 100-300ml fermentation liquor centrifugal, abandons supernatant;
(2) to add 100-300ml pH value be 7.2 phosphate buffer for precipitation part, centrifugal behind the mixing; Sedimentation cell in liquid nitrogen cancellation to stop the metabolic response of cell, with liquid nitrogen grinding to fine-powdered;
(3) get the fine-powdered cell 20-80mg that step (2) obtains and place centrifuge tube, the volumetric concentration that adds 0.5-1.5ml-40 ℃～-50 ℃ is that 50% methanol aqueous solution is an extract, and the deuterium-labelled succinic acid aqueous solution of the 0.14mg/ml that adds 10 μ l～20 μ l is internal standard compound, mixing; Place the liquid nitrogen multigelation 2-4 time, freeze 0.5-2.5min at every turn; Centrifugal collection supernatant and freeze drying;
(4) the 20mg/ml methoxy amine hydrochlorate pyridine solution of adding 40-60 μ l in the freeze-drying sample that step (3) obtains, oximation reaction 60-100min is carried out in 30-40 ℃ of water-bath; Add N-methyl-N-trimethyl silane trifluoroacetamide of 50-100 μ l again, Silanization reaction 50-70min is carried out in 30-40 ℃ of water-bath;
(5) with gas chromatography-flight time mass spectrum combined instrument working sample composition and relative content:
The sample of 1 μ l silanization is entered in the gas chromatography, chromatographic column is DB-5MS, the specification of described chromatographic column is 30m * 0.25mm i.d., utilization Masslynx software carries out structure prediction to the metabolin in the sample, a kind of multiple mass spectrogram that predicts the outcome of metabolin is analyzed with final definite metabolin structure, according to qualitative results and retention time the various metabolin areas in each sample are obtained, with each metabolin peak area in each sample with compare with the internal standard compound peak area in the individual sample, obtain the relative peak area of each metabolin, can represent the relative content of metabolin in the sample;
(6) utilize Matlab software that pretreated data are carried out principal component analysis (PCA), the metabolism difference of each sample room of holistic approach; Utilize Expander software that pretreated data are carried out cluster analysis, to analyze the metabolism difference of each metabolin at sample room; Qualitative by metabolin, and then metabolin classified, each metabolite Changing Pattern is during the fermentation analyzed.
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| CN104450834A (en) * | 2014-12-04 | 2015-03-25 | 天津大学 | Method of increasing yield of spinosad by improving fermentation condition of saccharopolyspora spinosa based on metabonomics |
| CN106442812A (en) * | 2016-09-18 | 2017-02-22 | 天津北洋百川生物技术有限公司 | Sample treatment method for GC-MS metabonomics research of aureobasidium pullulans |
| CN107860857A (en) * | 2017-10-27 | 2018-03-30 | 中国科学院青岛生物能源与过程研究所 | A kind of extraction and analytical method of yellow silk frustule intracellular metabolite thing |
| CN108776186A (en) * | 2018-07-30 | 2018-11-09 | 天津科技大学 | A kind of analysis method of Corynebacterium glutamicum metabolism group |
| CN112630363A (en) * | 2020-12-31 | 2021-04-09 | 四川大学华西医院 | MXene biological response characteristic metabonomics analysis method |
| CN112980911A (en) * | 2019-12-18 | 2021-06-18 | 万华化学集团股份有限公司 | Method for producing rhamnolipid by low-residue oil fermentation |
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| CN104450834A (en) * | 2014-12-04 | 2015-03-25 | 天津大学 | Method of increasing yield of spinosad by improving fermentation condition of saccharopolyspora spinosa based on metabonomics |
| CN106442812A (en) * | 2016-09-18 | 2017-02-22 | 天津北洋百川生物技术有限公司 | Sample treatment method for GC-MS metabonomics research of aureobasidium pullulans |
| CN107860857A (en) * | 2017-10-27 | 2018-03-30 | 中国科学院青岛生物能源与过程研究所 | A kind of extraction and analytical method of yellow silk frustule intracellular metabolite thing |
| CN108776186A (en) * | 2018-07-30 | 2018-11-09 | 天津科技大学 | A kind of analysis method of Corynebacterium glutamicum metabolism group |
| CN112980911A (en) * | 2019-12-18 | 2021-06-18 | 万华化学集团股份有限公司 | Method for producing rhamnolipid by low-residue oil fermentation |
| CN112630363A (en) * | 2020-12-31 | 2021-04-09 | 四川大学华西医院 | MXene biological response characteristic metabonomics analysis method |
| CN113109280A (en) * | 2021-04-08 | 2021-07-13 | 新疆师范大学 | Method for analyzing antioxidant activity of peganum harmala plants in different producing areas |
| CN113109280B (en) * | 2021-04-08 | 2023-09-15 | 新疆师范大学 | Analysis method for antioxidant activity of peganum harmala plants in different producing areas |
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