CN103808841B - Gas chromatography-mass spectrum detects the method for organic acid in fermentation liquor, amino acid, sugar - Google Patents
Gas chromatography-mass spectrum detects the method for organic acid in fermentation liquor, amino acid, sugar Download PDFInfo
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Abstract
The present invention relates to a kind of method that gas chromatography-mass spectrum detects organic acid in fermentation liquor, amino acid, sugar, belong to technical field of biochemistry test measurement.Detection method step comprises: (1) gets fermentation liquor, high speed centrifugation, respectively collecting cell and extracellular fluid; (2) fermentation broth sample preparation: get the supernatant that step 1) obtains and add acetonitrile except deproteinized, vortex oscillation, then collected by centrifugation supernatant III, add internal standard compound ribitol solution, and room temperature in vacuo is dried, and obtains extracellular fluid sample II; (3) analyte derivative, add sugared derivating agent, amino acid derived dose; (4) gas chromatography-mass spectrometry analysis and data acquisition.It is simple that the present invention has sample preparation, easy and simple to handle, and detect organic acid, amino acid, sugar time short, highly sensitive, testing result repeatability is high, can realize the advantages such as multiple sample preparations.
Description
Technical field
The invention belongs to technical field of biochemistry test measurement, particularly relate to a kind of method that gas chromatography-mass spectrum detects organic acid in fermentation liquor, amino acid, sugar.
Background technology
Amino acid, glycometabolism object space face is detected utilizing gas chromatography-mass spectrum, existing correlation technique report at present, as patent 201210046953.2, disclose a kind of method that gas chromatography-mass spectrum detects urinary organic acid, comprise the steps: that (1) completes urine ferment treatment to urine specimen to be checked; (2) add interior mark product, adopt frozen ethanol to carry out the process of protein precipitation, sample is dried up; (3) silylation derivation process is carried out to above-mentioned sample; (4) adopt the step process standard organic acid of above-mentioned 1-3, obtain standard organic acid sample; (5) gas chromatograph-mass spectrometer (GCMS) is adopted to detect standard organic acid sample and urine specimen to be checked; (6) using the testing result of standard organic acid sample as reference curve, with conventional algorithm, the organic acid of urine specimen to be checked is carried out quantitatively.This patented invention main application fields is urine examination, and this detection method is only confined to the detection of organic acid metabolite, can not detect amino acid, sugared two metabolites simultaneously.
For the detect delay of metabolin in extracellular microbial, outside born of the same parents, can disclose microorganism metabolic rule under fermentation conditions, especially fermentation condition is for the affecting laws of microorganism target product.The output grasping the promotion target product that the metabolic rule of microorganism can be not only favourable promotes, and can determine that endobacillary metabolic flux distributes, and then utilizing genetic engineering means to realize the optimization and reconstruction of bacterium passway of metabolism, realize target product high-performance bio synthesizes.
At present certain class material specific is used for for the metabolite analysis detection method limitation in fermentation liquor, multiclass metabolin can not be realized and detect simultaneously.Because component content complexity each in fermentation liquor is various, disposal route for sample is very crucial, at present also in relevant fermentation liquor, the system process detection method of extracellular metabolin, make to develop a kind of more sensitive, wide spectrum, general fermentating metabolism object detecting method, identify the compound structure of various spectrum peak mapping, and with the integration of other dummy model, become the focus of microbial metabolism group research.
Summary of the invention
The object of this invention is to provide a kind of method that gas chromatography-mass spectrum detects organic acid in fermentation liquor, amino acid, sugar, make up at present in fermentable to the blank of metabolin system detecting method, overcome the shortcomings such as existing detection technique disturbing factor is a lot, complex operation, testing cost are high, sensitivity is low, sensing range is narrow.
The solution of the present invention is by realizing like this: a kind of gas chromatography-mass spectrum detects the method for organic acid in fermentation liquor, amino acid, sugar, and detection method step comprises:
(1) get fermentation liquor, high speed centrifugation, collect thalline and supernatant I respectively;
(2) fermentation broth sample preparation: get the supernatant I that 60 ~ 350ul step 1) obtains, acetonitrile is added except deproteinized in clear liquid I, supernatant I and acetonitrile volume ratio are 1:0.5 ~ 1.5, vortex oscillation, collected by centrifugation supernatant III again, adds internal standard compound ribitol solution, and supernatant III volume and internal standard compound ribitol part by weight are 50 ~ 300ul:20 μ g, room temperature in vacuo is dried, and obtains extracellular fluid sample II;
(3) analyte derivative: in step 2) in the sample II that obtains, add 50 ~ 150ul sugar derivating agent respectively, constant temperature places 1.5 ~ 4h, add amino acid derived dose of 50 ~ 150ul more respectively, constant temperature is placed and is spent the night, derivative complete, centrifugal derivative after sample II, collect supernatant and obtain sample II in born of the same parents to be measured;
(4) sample II in the born of the same parents to be measured utilizing gas chromatography-mass spectrum to obtain step 3), carries out analyzing and data acquisition.
As further restriction of the present invention, its sampling amount of described thalline reaches 0.2 ~ 1.0g/ml for dissolving artifact amount dry weight with cold methanol; Described cold methanol is for be chilled to-40 DEG C in advance through cryostat groove; Described low-temperature extraction is extract 2 ~ 7h at-40 DEG C to-50 DEG C.
As a further improvement on the present invention, described sugared derivating agent is that 0.1 ~ 0.3mg methoxamine hydrochloric acid to be dissolved in 10ml pyridine solution preparation and to obtain; Described amino acid derived dose is that 50 ~ 200ul trimethyl chlorosilane is dissolved in the trifluoroacetamide of 10ml.
As a further improvement on the present invention, its instrumental conditions of described gas chromatography-mass spectrum is: gas chromatography: chromatographic column is HP-5MS or the DB-5MS capillary column of 30m × 0.25mm, injector temperature 300 DEG C, detector temperature 250 DEG C, column temperature rise program equilibration time 3min → 80 DEG C maintain 1min → 2 DEG C/min and are warming up to 100 DEG C → 15 DEG C/min and are warming up to 220 DEG C → 30 DEG C/min and are warming up to 300 DEG C → 300 DEG C and maintain 3min, sampling volume 1 μ L; Mass spectrum: ion gun EI, detecting device is level Four bar, ionization energy 70eV, ion gun surface temperature 280 DEG C.
As a further improvement on the present invention, the method be applied to detect saccharomycete, lactic acid bacteria, clostridium, mould biological fermentation process in fermentation liquor in organic acid, amino acid, sugar.
As a further improvement on the present invention, it is characterized in that, described organic acid is succinic acid, malic acid, citric acid, pyruvic acid, fumaric acid; Described amino acid is glutamic acid, glycocoll, halfcystine, serine, threonine, leucine, isoleucine, phenylalanine, valine, lysine, alanine, histidine, methionine, arginine, glutamine, asparatate, asparagine, tyrosine, proline; Described sugar is glucose, fructose, wood sugar.
Substantive distinguishing features of the present invention and marked improvement are: detect the while that (1) the method can realizing organic acid in fermentation liquor, amino acids material, do not need to carry out repeatedly complex process to fermentation liquor, save time and simplify the operation step, obtain microbial metabolism rule in sweat in time, analyze born of the same parents' intracellular metabolite flow.(2) the method adopts metabolin method in cold methanol extractive fermentation liquid, and its effect of extracting is excellent, obtains more metabolites kinds in fermentation liquor, is conducive to the analysis of metabolic rule.(3) the chromatographic peak analytical effect that obtains of sample analysis is good, can realize detecting to organic acids such as succinic acid, malic acid, citric acid, pyruvic acid, fumaric acid; Can realize detecting to sugar such as glucose, fructose, wood sugars; Can realize detecting to amino acid such as glutamic acid, glycocoll, halfcystine, serine, threonine, leucine, isoleucine, phenylalanine, valine, lysine, alanine, histidine, methionine, arginine, glutamine, asparatate, asparagine, tyrosine, proline.
Accompanying drawing explanation
Fig. 1 .GC-MS detects collection of illustrative plates.In figure, 1 is malonic acid; 2 is glycocoll; 3 is tryptophane; 4 is alanine; 5 is glycerine; 6 is propylene glycol; 7 is butylene glycol; 8 is xylitol; 9 is ribitol; 10 is proline; 11 is 4-Aminobutanoicacid; 12 is gummy saccharic acid; 13 is ketoglutaric acid; 14 is succinic acid; 15 is lactic acid.
Embodiment
Further will be described the present invention by embodiment below, these descriptions are not be further limited content of the present invention.
In following examples, centrifugal condition is: the centrifugal 10min of 10000rpm at-4 DEG C.
In following examples, its instrumental conditions of gas chromatography-mass spectrum is: gas chromatography: chromatographic column is HP-5MS or the DB-5MS capillary column of 30m × 0.25mm, injector temperature 300 DEG C, detector temperature 250 DEG C, column temperature rise program equilibration time 3min → 80 DEG C maintain 1min → 2 DEG C/min and are warming up to 100 DEG C → 15 DEG C/min and are warming up to 220 DEG C → 30 DEG C/min and are warming up to 300 DEG C → 300 DEG C and maintain 3min, sampling volume 1 μ L; Mass spectrum: ion gun EI, detecting device is level Four bar, ionization energy 70eV, ion gun surface temperature 280 DEG C.
Following examples all can realize detecting to organic acids such as succinic acid, malic acid, citric acid, pyruvic acid, fumaric acid; Can realize detecting to sugar such as glucose, fructose, wood sugars; Can realize detecting to amino acid such as glutamic acid, glycocoll, halfcystine, serine, threonine, leucine, isoleucine, phenylalanine, valine, lysine, alanine, histidine, methionine, arginine, glutamine, asparatate, asparagine, tyrosine, proline.
Embodiment 1
Get clostridial fermentation liquid.
(1) endocellular metabolism matter sample preparation: get 5ml fermentation liquor, centrifugally obtain thalline, thalline is cleaned 2 times with physiological saline,-40 DEG C of cold methanols dissolve artifact amount dry weight and reach 0.8g/ml, ultrasonication is to lysis, obtain lysate, low-temperature extraction lysate, cryogenic conditions is: extract 2h at-40 DEG C to-50 DEG C, cracking supernatant I after collected by centrifugation 50ul extracts, add internal standard compound ribitol solution, cracking supernatant I volume and internal standard compound ribitol part by weight are 50ul:20 μ g, room temperature in vacuo is dried, and obtains endocellular metabolism matter sample I.
(2) extracellular fluid sample preparation: get fermentation liquor, centrifugally obtain supernatant II, get 200ul supernatant II, in clear liquid II, add acetonitrile except deproteinized, supernatant II and acetonitrile volume ratio are 1:1.0, vortex oscillation, collected by centrifugation obtains supernatant III again, adds internal standard compound ribitol solution, and supernatant III volume and internal standard compound ribitol part by weight are 150ul:20 μ g, room temperature in vacuo is dried, and obtains extracellular fluid sample II.
(3) analyte derivative: in step 1) and step 2) in the sample I that obtains and sample II, add 120ul sugar derivating agent (sugared derivating agent be 0.2mg methoxamine hydrochloric acid to be dissolved in 10ml pyridine solution preparation obtain) respectively, constant temperature places 3.0h, add amino acid derived dose of 80ul (amino acid derived dose for 50ul trimethyl chlorosilane to be dissolved in the trifluoroacetamide of 10ml preparation and obtain) more respectively, constant temperature is placed and is spent the night, derivative complete, respectively centrifugal derivative after sample I and sample II, correspondingly collect respective supernatant and obtain sample I and the outer sample II of born of the same parents to be measured in born of the same parents to be measured.
(4) according to its instrumental conditions of gas chromatography-mass spectrum, sample I in born of the same parents to be measured and the outer sample II of born of the same parents to be measured are analyzed and data acquisition.
Testing result is as Fig. 1.Due to derivatization, in figure, 8 derive the xylitol of rear formation for wood sugar.
Embodiment 2
Extracting lactic acid fermented liquid.
(1) endocellular metabolism matter sample preparation: get 5ml fermentation liquor, centrifugally obtain thalline, thalline is cleaned 3 times with physiological saline,-40 DEG C of cold methanols dissolve artifact amount dry weight and reach 1.0g/ml, ultrasonication is to lysis, obtain lysate, low-temperature extraction lysate, cryogenic conditions is: extract 5h at-40 DEG C to-50 DEG C, cracking supernatant I after collected by centrifugation 100ul extracts, add internal standard compound ribitol solution, cracking supernatant I volume and internal standard compound ribitol part by weight are 100ul:20 μ g, room temperature in vacuo is dried, and obtains endocellular metabolism matter sample I.
(2) extracellular fluid sample preparation: get fermentation liquor, centrifugally obtain supernatant II, get 350ul supernatant II, in clear liquid II, add acetonitrile except deproteinized, supernatant II and acetonitrile volume ratio are 1:0.7, vortex oscillation, collected by centrifugation obtains supernatant III again, adds internal standard compound ribitol solution, and supernatant III volume and internal standard compound ribitol part by weight are 200ul:20 μ g, room temperature in vacuo is dried, and obtains extracellular fluid sample II.
(3) analyte derivative: in step 1) and step 2) in the sample I that obtains and sample II, add 50ul sugar derivating agent (sugared derivating agent be 0.3mg methoxamine hydrochloric acid to be dissolved in 10ml pyridine solution preparation obtain) respectively, constant temperature places 2.0h, add amino acid derived dose of 150ul (amino acid derived dose for 150ul trimethyl chlorosilane to be dissolved in the trifluoroacetamide of 10ml preparation and obtain) more respectively, constant temperature is placed and is spent the night, derivative complete, distinguish the sample I after centrifugal deriving and sample II, the respective supernatant of corresponding collection obtains sample I and the outer sample II of born of the same parents to be measured in born of the same parents to be measured.
(4) according to its instrumental conditions of gas chromatography-mass spectrum, sample I in born of the same parents to be measured and the outer sample II of born of the same parents to be measured are analyzed and data acquisition.
Embodiment 3
Get mold fermentation liquid.
(1) endocellular metabolism matter sample preparation: get 5ml fermentation liquor, centrifugally obtain thalline, thalline is cleaned 3 times with physiological saline,-40 DEG C of cold methanols dissolve artifact amount dry weight and reach 0.6g/ml, ultrasonication is to lysis, obtain lysate, low-temperature extraction lysate, cryogenic conditions is: extract 3h at-40 DEG C to-50 DEG C, cracking supernatant I after collected by centrifugation 200ul extracts, add internal standard compound ribitol solution, cracking supernatant I volume and internal standard compound ribitol part by weight are 200ul:20 μ g, room temperature in vacuo is dried, and obtains endocellular metabolism matter sample I.
(2) extracellular fluid sample preparation: get fermentation liquor, centrifugally obtain supernatant II, get 60ul supernatant II, in clear liquid II, add acetonitrile except deproteinized, supernatant II and acetonitrile volume ratio are 1:1.5, vortex oscillation, collected by centrifugation obtains supernatant III again, adds internal standard compound ribitol solution, and supernatant III volume and internal standard compound ribitol part by weight are 50ul:20 μ g, room temperature in vacuo is dried, and obtains extracellular fluid sample II.
(3) analyte derivative: in step 1) and step 2) in the sample I that obtains and sample II, add 150ul sugar derivating agent (sugared derivating agent be 0.15mg methoxamine hydrochloric acid to be dissolved in 10ml pyridine solution preparation obtain) respectively, constant temperature places 2.5h, add amino acid derived dose of 50ul (amino acid derived dose for 200ul trimethyl chlorosilane to be dissolved in the trifluoroacetamide of 10ml preparation and obtain) more respectively, constant temperature is placed and is spent the night, derivative complete, distinguish the sample I after centrifugal deriving and sample II, the respective supernatant of corresponding collection obtains sample I and the outer sample II of born of the same parents to be measured in born of the same parents to be measured.
(4) according to its instrumental conditions of gas chromatography-mass spectrum, sample I in born of the same parents to be measured and the outer sample II of born of the same parents to be measured are analyzed and data acquisition.
Embodiment 4
Get saccharomycetes to make fermentation liquid.
(1) endocellular metabolism matter sample preparation: get 5ml fermentation liquor, centrifugally obtain thalline, thalline is cleaned 2 times with physiological saline,-40 DEG C of cold methanols dissolve artifact amount dry weight and reach 0.2g/ml, ultrasonication is to lysis, obtain lysate, low-temperature extraction lysate, cryogenic conditions is: extract 4h at-40 DEG C to-50 DEG C, cracking supernatant I after collected by centrifugation 150ul extracts, add internal standard compound ribitol solution, cracking supernatant I volume and internal standard compound ribitol part by weight are 150ul:20 μ g, room temperature in vacuo is dried, and obtains endocellular metabolism matter sample I.
(2) extracellular fluid sample preparation: get fermentation liquor, centrifugally obtain supernatant II, get 300ul supernatant II, in clear liquid II, add acetonitrile except deproteinized, supernatant II and acetonitrile volume ratio are 1:1.2, vortex oscillation, collected by centrifugation obtains supernatant III again, adds internal standard compound ribitol solution, and supernatant III volume and internal standard compound ribitol part by weight are 100ul:20 μ g, room temperature in vacuo is dried, and obtains extracellular fluid sample II.
(3) analyte derivative: in step 1) and step 2) in the sample I that obtains and sample II, add 100ul sugar derivating agent (sugared derivating agent be 0.1mg methoxamine hydrochloric acid to be dissolved in 10ml pyridine solution preparation obtain) respectively, constant temperature places 1.5h, add amino acid derived dose of 100ul (amino acid derived dose for 100ul trimethyl chlorosilane to be dissolved in the trifluoroacetamide of 10ml preparation and obtain) more respectively, constant temperature is placed and is spent the night, derivative complete, distinguish the sample I after centrifugal deriving and sample II, the respective supernatant of corresponding collection obtains sample I and the outer sample II of born of the same parents to be measured in born of the same parents to be measured.
(4) according to its instrumental conditions of gas chromatography-mass spectrum, sample I in born of the same parents to be measured and the outer sample II of born of the same parents to be measured are analyzed and data acquisition.
Embodiment 5
Get saccharomycetes to make fermentation liquid.
(1) endocellular metabolism matter sample preparation: get 5ml fermentation liquor, centrifugally obtain thalline, thalline is cleaned 3 times with physiological saline,-40 DEG C of cold methanols dissolve artifact amount dry weight and reach 0.5g/ml, ultrasonication is to lysis, obtain lysate, low-temperature extraction lysate, cryogenic conditions is: extract 3h at-40 DEG C to-50 DEG C, cracking supernatant I after collected by centrifugation 200ul extracts, add internal standard compound ribitol solution, cracking supernatant I volume and internal standard compound ribitol part by weight are 150ul:20 μ g, room temperature in vacuo is dried, and obtains endocellular metabolism matter sample I.
(2) extracellular fluid sample preparation: get fermentation liquor, centrifugally obtain supernatant II, get 300ul supernatant II, in clear liquid II, add acetonitrile except deproteinized, supernatant II and acetonitrile volume ratio are 1:0.5, vortex oscillation, collected by centrifugation obtains supernatant III again, adds internal standard compound ribitol solution, and supernatant III volume and internal standard compound ribitol part by weight are 300ul:20 μ g, room temperature in vacuo is dried, and obtains extracellular fluid sample II.
(3) analyte derivative: in step 1) and step 2) in the sample I that obtains and sample II, add 100ul sugar derivating agent (sugared derivating agent be 0.2mg methoxamine hydrochloric acid to be dissolved in 10ml pyridine solution preparation obtain) respectively, constant temperature places 4.0h, add amino acid derived dose of 120ul (amino acid derived dose for 120ul trimethyl chlorosilane to be dissolved in the trifluoroacetamide of 10ml preparation and obtain) more respectively, constant temperature is placed and is spent the night, derivative complete, distinguish the sample I after centrifugal deriving and sample II, the respective supernatant of corresponding collection obtains sample I and the outer sample II of born of the same parents to be measured in born of the same parents to be measured.
(4) according to its instrumental conditions of gas chromatography-mass spectrum, sample I in born of the same parents to be measured and the outer sample II of born of the same parents to be measured are analyzed and data acquisition.
Claims (2)
1. gas chromatography-mass spectrum detects a method for organic acid in fermentation liquor, amino acid, sugar, and it is characterized in that, detection method step comprises:
1) get fermentation liquor, high speed centrifugation, collect thalline and supernatant I respectively;
2) fermentation broth sample preparation: get the supernatant I that 60 ~ 350 μ L step 1) obtain, acetonitrile is added except deproteinized in supernatant I, supernatant I and acetonitrile volume ratio are 1:0.5 ~ 1.5, vortex oscillation, collected by centrifugation supernatant III again, adds internal standard compound ribitol solution, and supernatant III volume and internal standard compound ribitol part by weight are 50 ~ 300 μ L:20 μ g, room temperature in vacuo is dried, and obtains extracellular fluid sample II;
3) analyte derivative: in step 2) in the sample II that obtains, add 50 ~ 150 μ L sugar derivating agents respectively, constant temperature places 1.5 ~ 4h, add amino acid derived dose of 50 ~ 150 μ L more respectively, constant temperature is placed and is spent the night, derivative complete, centrifugal derivative after sample II, collect supernatant and obtain the outer sample II of born of the same parents to be measured;
4) the outer sample II of born of the same parents to be measured utilizing gas chromatography-mass spectrum to obtain step 3), carries out analyzing and data acquisition;
Described sugared derivating agent is that 0.1 ~ 0.3mg methoxamine hydrochloric acid to be dissolved in 10ml pyridine solution preparation and to obtain; Described amino acid derived dose is that 50 ~ 200 μ L trimethyl chlorosilanes are dissolved in the trifluoroacetamide of 10ml;
Its instrumental conditions of described gas chromatography-mass spectrum is: gas chromatography: chromatographic column is HP-5MS or the DB-5MS capillary column of 30m × 0.25mm, injector temperature 300 DEG C, detector temperature 250 DEG C, column temperature rise program equilibration time 3min → 80 DEG C maintain 1min → 2 DEG C/min and are warming up to 100 DEG C → 15 DEG C/min and are warming up to 220 DEG C → 30 DEG C/min and are warming up to 300 DEG C → 300 DEG C and maintain 3min, sampling volume 1 μ L; Mass spectrum: ion gun EI, detecting device is level Four bar, ionization energy 70eV, ion gun surface temperature 280 DEG C;
Described organic acid is succinic acid, malic acid, citric acid, pyruvic acid, fumaric acid; Described amino acid is glutamic acid, glycocoll, halfcystine, serine, threonine, leucine, isoleucine, phenylalanine, valine, lysine, alanine, histidine, methionine, arginine, glutamine, asparatate, asparagine, tyrosine, proline; Described sugar is glucose, fructose, wood sugar.
2. a kind of gas chromatography-mass spectrum detects the method for organic acid in fermentation liquor, amino acid, sugar according to claim 1, it is characterized in that, the method is applied to organic acid, amino acid, sugar in the fermentation liquor in the biological fermentation process detecting saccharomycete, lactic acid bacteria, clostridium, mould.
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