CN103808841A - Method for detecting organic acid, amino acid and sugar in fermented liquid by gas chromatography-mass spectrometry - Google Patents
Method for detecting organic acid, amino acid and sugar in fermented liquid by gas chromatography-mass spectrometry Download PDFInfo
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- CN103808841A CN103808841A CN201310651252.6A CN201310651252A CN103808841A CN 103808841 A CN103808841 A CN 103808841A CN 201310651252 A CN201310651252 A CN 201310651252A CN 103808841 A CN103808841 A CN 103808841A
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Abstract
The invention relates to a method for detecting an organic acid, an amino acid and sugar in fermented liquid by gas chromatography-mass spectrometry, belonging to the technical field of biochemical examination and determination. The detection method comprises the following steps: (1) centrifuging taken fermentation liquid at a high speed and respectively collecting cells and extracellular fluid; (2) preparing a fermentation liquid sample: adding acetonitrile in a liquid supernatant prepared in the step (1) to remove protein; carrying out vortex shaking, and centrifuging and collecting liquid supernatant III; adding an internal standard substance ribitol solution and drying at a room temperature in vacuum to obtain an extracellular fluid sample II; (3) deriving the sample adding a sugar deriving agent and an amino acid deriving agent; and (4) carrying out the gas chromatography-mass spectrometry and data acquisition. The method provided by the invention has the advantages of simplicity in sample treatment, simplicity and convenience in operation, short detection time of organic acid, amino acid and sugar, high sensitivity, high repeatability of a detection result, capability of preparing a plurality of samples and the like.
Description
Technical field
The invention belongs to biochemical test determination techniques field, particularly relate to a kind of method of organic acid, amino acid, sugar in gas chromatography-mass spectrum detection fermentation liquor.
Background technology
Utilizing gas chromatography-mass spectrum to detect amino acid, glycometabolism object space face, at present existing correlation technique report, as patent 201210046953.2, announce a kind of method of gas chromatography-mass spectrum detection urinary organic acid, comprise the steps: that (1) completes the processing of urine enzyme to urine specimen to be checked; (2) add interior mark product, adopt frozen ethanol to carry out the processing of protein precipitation, sample is dried up; (3) above-mentioned sample is carried out to methyl-monosilane derivation process; (4) adopt the step process standard organic acid of above-mentioned 1-3, obtain standard organic acid sample; (5) adopt gas chromatograph-mass spectrometer (GCMS) 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, the organic acid of urine specimen to be checked is carried out quantitatively with conventional algorithm.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 detection research of metabolin in extracellular microbial, outside born of the same parents, can disclose microorganism metabolic rule under fermentation conditions, the especially fermentation condition rule that affects for microorganism target product.The output of 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 is synthetic.
Be used for specific certain class material for the metabolite analysis detection method limitation in fermentation liquor at present, can not realize multiclass metabolin and detect simultaneously.Because each component content complexity in fermentation liquor is various, disposal route for sample is very crucial, at present also in relevant fermentation liquor, the system of extracellular metabolin is processed detection method, make to develop a kind of more sensitive, fermentating metabolism object detecting method wide spectrum, general, identify the compound structure of various spectrums 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 the blank to metabolin system detecting method in microorganism fermentation at present, overcome the shortcomings such as existing detection technique disturbing factor is a lot, complex operation, testing cost is high, sensitivity is low, sensing range is narrow.
The solution of the present invention is by such realization: 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, collects respectively thalline and supernatant I;
(2) fermentation broth sample preparation: get the supernatant I that 60～350ul step 1) obtains, in clear liquid I, add acetonitrile to remove deproteinized, supernatant I and acetonitrile volume ratio are 1:0.5～1.5, vortex vibration, centrifugal collection supernatant III, adds internal standard compound ribitol solution again, and supernatant III volume and internal standard compound ribitol part by weight are 50～300ul:20 μ g, room temperature vacuum drying, obtains extracellular fluid sample II;
(3) analyte derivative: in step 2) in the sample II that obtains, add respectively 50～150ul sugar derivating agent, constant temperature is placed 1.5～4h, add respectively more amino acid derived dose of 50～150ul, constant temperature is placed and is spent the night, derivative complete, the sample II after centrifugal deriving, collects supernatant and obtains sample II in born of the same parents to be measured;
(4) utilize sample II in the born of the same parents to be measured that gas chromatography-mass spectrum obtains step 3), analyze and data acquisition.
As further restriction of the present invention, its sampling amount of described thalline reaches 0.2～1.0g/ml for dissolve artifact amount dry weight with cold methanol; Described cold methanol is for being chilled in advance-40 ℃ through cryostat groove; Described low-temperature extraction extracts 2～7h at-40 ℃ to-50 ℃.
As a further improvement on the present invention, described sugared derivating agent is that 0.1～0.3mg methoxamine hydrochloric acid is dissolved in 10ml pyridine solution preparation and obtains; 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 analysis condition of described gas chromatography-mass spectrum is: gas chromatography: the HP-5MS that chromatographic column is 30m × 0.25mm or DB-5MS capillary column, 300 ℃ of injector temperatures, 250 ℃ of detector temperatures, column temperature rise program equilibration time 3min → 80 ℃ maintain 1min → 2 ℃/min and are warming up to 100 ℃ → 15 ℃/min and are warming up to 220 ℃ → 30 ℃/min and are warming up to 300 ℃ → 300 ℃ and maintain 3min, sampling volume 1 μ L; Mass spectrum: ion gun EI, detecting device is level Four bar, ionization energy 70eV, 280 ℃ of ion gun surface temperatures.
As a further improvement on the present invention, the method is applied to organic acid, amino acid, the sugar in the fermentation liquor in the biological fermentation process that detects saccharomycete, lactic acid bacteria, clostridium, mould.
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: when (1) the method can realize organic acid in fermentation liquor, amino acids material, detect, do not need fermentation liquor to carry out repeatedly complex process, save time and simplify the operation step, obtain in time microbial metabolism rule in sweat, 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 good, obtains more metabolites kinds in fermentation liquor, is conducive to the analysis of metabolic rule.(3) the chromatographic peak analytical effect that sample analysis obtains is good, can the organic acids such as succinic acid, malic acid, citric acid, pyruvic acid, fumaric acid be realized and being detected; Can the sugar such as glucose, fructose, wood sugar be realized and being detected; Can the amino acid such as glutamic acid, glycocoll, halfcystine, serine, threonine, leucine, isoleucine, phenylalanine, valine, lysine, alanine, histidine, methionine, arginine, glutamine, asparatate, asparagine, tyrosine, proline be realized and being detected.
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
To further describe the present invention by embodiment below, these descriptions are not that content of the present invention is further limited.
In following examples, centrifugal condition is: the centrifugal 10min of 10000rpm at-4 ℃.
In following examples, its instrumental analysis condition of gas chromatography-mass spectrum is: gas chromatography: the HP-5MS that chromatographic column is 30m × 0.25mm or DB-5MS capillary column, 300 ℃ of injector temperatures, 250 ℃ of detector temperatures, column temperature rise program equilibration time 3min → 80 ℃ maintain 1min → 2 ℃/min and are warming up to 100 ℃ → 15 ℃/min and are warming up to 220 ℃ → 30 ℃/min and are warming up to 300 ℃ → 300 ℃ and maintain 3min, sampling volume 1 μ L; Mass spectrum: ion gun EI, detecting device is level Four bar, ionization energy 70eV, 280 ℃ of ion gun surface temperatures.
Following examples all can realize and detecting organic acids such as succinic acid, malic acid, citric acid, pyruvic acid, fumaric acid; Can the sugar such as glucose, fructose, wood sugar be realized and being detected; Can the amino acid such as glutamic acid, glycocoll, halfcystine, serine, threonine, leucine, isoleucine, phenylalanine, valine, lysine, alanine, histidine, methionine, arginine, glutamine, asparatate, asparagine, tyrosine, proline be realized and being detected.
Get clostridium fermentation liquor.
(1) endocellular metabolism matter sample preparation: get 5ml fermentation liquor, the centrifugal thalline that obtains, clean thalline 2 times with physiological saline,-40 ℃ 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: at-40 ℃ to-50 ℃, extract 2h, cracking supernatant I after centrifugal collection 50ul extraction, adds internal standard compound ribitol solution, and cracking supernatant I volume and internal standard compound ribitol part by weight are 50ul:20 μ g, room temperature vacuum drying, obtains endocellular metabolism matter sample I.
(2) extracellular fluid sample preparation: get fermentation liquor, the centrifugal supernatant II that obtains, get 200ul supernatant II, in clear liquid II, add acetonitrile to remove deproteinized, supernatant II and acetonitrile volume ratio are 1:1.0, vortex vibration, the centrifugal supernatant III of collecting to obtain, adds internal standard compound ribitol solution again, and supernatant III volume and internal standard compound ribitol part by weight are 150ul:20 μ g, room temperature vacuum drying, obtains extracellular fluid sample II.
(3) analyte derivative: in step 1) and step 2) in the sample I and sample II that obtain, add respectively 120ul sugar derivating agent (sugared derivating agent be 0.2mg methoxamine hydrochloric acid be dissolved in preparation in 10ml pyridine solution obtain), constant temperature is placed 3.0h, add respectively more amino acid derived dose of 80ul (prepare and obtain for 50ul trimethyl chlorosilane is dissolved in the trifluoroacetamide of 10ml for amino acid derived dose), constant temperature is placed and is spent the night, derivative complete, sample I and sample II after centrifugal deriving respectively, correspondence is collected supernatant separately and is obtained 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 analysis condition 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 is the derivative rear xylitol forming of wood sugar.
Extracting lactic acid fermented liquid.
(1) endocellular metabolism matter sample preparation: get 5ml fermentation liquor, the centrifugal thalline that obtains, clean thalline 3 times with physiological saline,-40 ℃ 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: at-40 ℃ to-50 ℃, extract 5h, cracking supernatant I after centrifugal collection 100ul extraction, adds internal standard compound ribitol solution, and cracking supernatant I volume and internal standard compound ribitol part by weight are 100ul:20 μ g, room temperature vacuum drying, obtains endocellular metabolism matter sample I.
(2) extracellular fluid sample preparation: get fermentation liquor, the centrifugal supernatant II that obtains, get 350ul supernatant II, in clear liquid II, add acetonitrile to remove deproteinized, supernatant II and acetonitrile volume ratio are 1:0.7, vortex vibration, the centrifugal supernatant III of collecting to obtain, adds internal standard compound ribitol solution again, and supernatant III volume and internal standard compound ribitol part by weight are 200ul:20 μ g, room temperature vacuum drying, obtains extracellular fluid sample II.
(3) analyte derivative: in step 1) and step 2) in the sample I and sample II that obtain, add respectively 50ul sugar derivating agent (sugared derivating agent be 0.3mg methoxamine hydrochloric acid be dissolved in preparation in 10ml pyridine solution obtain), constant temperature is placed 2.0h, add respectively more amino acid derived dose of 150ul (prepare and obtain for 150ul trimethyl chlorosilane is dissolved in the trifluoroacetamide of 10ml for amino acid derived dose), constant temperature is placed and is spent the night, derivative complete, sample I and sample II after centrifugal deriving respectively, correspondence is collected supernatant separately and is obtained 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 analysis condition 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.
Get mold fermentation liquid.
(1) endocellular metabolism matter sample preparation: get 5ml fermentation liquor, the centrifugal thalline that obtains, clean thalline 3 times with physiological saline,-40 ℃ 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: at-40 ℃ to-50 ℃, extract 3h, cracking supernatant I after centrifugal collection 200ul extraction, adds internal standard compound ribitol solution, and cracking supernatant I volume and internal standard compound ribitol part by weight are 200ul:20 μ g, room temperature vacuum drying, obtains endocellular metabolism matter sample I.
(2) extracellular fluid sample preparation: get fermentation liquor, the centrifugal supernatant II that obtains, get 60ul supernatant II, in clear liquid II, add acetonitrile to remove deproteinized, supernatant II and acetonitrile volume ratio are 1:1.5, vortex vibration, the centrifugal supernatant III of collecting to obtain, adds internal standard compound ribitol solution again, and supernatant III volume and internal standard compound ribitol part by weight are 50ul:20 μ g, room temperature vacuum drying, obtains extracellular fluid sample II.
(3) analyte derivative: in step 1) and step 2) in the sample I and sample II that obtain, add respectively 150ul sugar derivating agent (sugared derivating agent be 0.15mg methoxamine hydrochloric acid be dissolved in preparation in 10ml pyridine solution obtain), constant temperature is placed 2.5h, add respectively more amino acid derived dose of 50ul (prepare and obtain for 200ul trimethyl chlorosilane is dissolved in the trifluoroacetamide of 10ml for amino acid derived dose), constant temperature is placed and is spent the night, derivative complete, sample I and sample II after centrifugal deriving respectively, correspondence is collected supernatant separately and is obtained 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 analysis condition 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, the centrifugal thalline that obtains, clean thalline 2 times with physiological saline,-40 ℃ 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: at-40 ℃ to-50 ℃, extract 4h, cracking supernatant I after centrifugal collection 150ul extraction, adds internal standard compound ribitol solution, and cracking supernatant I volume and internal standard compound ribitol part by weight are 150ul:20 μ g, room temperature vacuum drying, obtains endocellular metabolism matter sample I.
(2) extracellular fluid sample preparation: get fermentation liquor, the centrifugal supernatant II that obtains, get 300ul supernatant II, in clear liquid II, add acetonitrile to remove deproteinized, supernatant II and acetonitrile volume ratio are 1:1.2, vortex vibration, the centrifugal supernatant III of collecting to obtain, adds internal standard compound ribitol solution again, and supernatant III volume and internal standard compound ribitol part by weight are 100ul:20 μ g, room temperature vacuum drying, obtains extracellular fluid sample II.
(3) analyte derivative: in step 1) and step 2) in the sample I and sample II that obtain, add respectively 100ul sugar derivating agent (sugared derivating agent be 0.1mg methoxamine hydrochloric acid be dissolved in preparation in 10ml pyridine solution obtain), constant temperature is placed 1.5h, add respectively more amino acid derived dose of 100ul (prepare and obtain for 100ul trimethyl chlorosilane is dissolved in the trifluoroacetamide of 10ml for amino acid derived dose), constant temperature is placed and is spent the night, derivative complete, sample I and sample II after centrifugal deriving respectively, correspondence is collected supernatant separately and is obtained 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 analysis condition 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.
Get saccharomycetes to make fermentation liquid.
(1) endocellular metabolism matter sample preparation: get 5ml fermentation liquor, the centrifugal thalline that obtains, clean thalline 3 times with physiological saline,-40 ℃ 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: at-40 ℃ to-50 ℃, extract 3h, cracking supernatant I after centrifugal collection 200ul extraction, adds internal standard compound ribitol solution, and cracking supernatant I volume and internal standard compound ribitol part by weight are 150ul:20 μ g, room temperature vacuum drying, obtains endocellular metabolism matter sample I.
(2) extracellular fluid sample preparation: get fermentation liquor, the centrifugal supernatant II that obtains, get 300ul supernatant II, in clear liquid II, add acetonitrile to remove deproteinized, supernatant II and acetonitrile volume ratio are 1:0.5, vortex vibration, the centrifugal supernatant III of collecting to obtain, adds internal standard compound ribitol solution again, and supernatant III volume and internal standard compound ribitol part by weight are 300ul:20 μ g, room temperature vacuum drying, obtains extracellular fluid sample II.
(3) analyte derivative: in step 1) and step 2) in the sample I and sample II that obtain, add respectively 100ul sugar derivating agent (sugared derivating agent be 0.2mg methoxamine hydrochloric acid be dissolved in preparation in 10ml pyridine solution obtain), constant temperature is placed 4.0h, add respectively more amino acid derived dose of 120ul (prepare and obtain for 120ul trimethyl chlorosilane is dissolved in the trifluoroacetamide of 10ml for amino acid derived dose), constant temperature is placed and is spent the night, derivative complete, sample I and sample II after centrifugal deriving respectively, correspondence is collected supernatant separately and is obtained 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 analysis condition 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 (7)
1. gas chromatography-mass spectrum detects a method for organic acid in fermentation liquor, amino acid, sugar, it is characterized in that, detection method step comprises:
(1) get fermentation liquor, high speed centrifugation, collects respectively thalline and supernatant I;
(2) fermentation broth sample preparation: get the supernatant I that 60 ~ 350ul step 1) obtains, in clear liquid I, add acetonitrile to remove deproteinized, supernatant I and acetonitrile volume ratio are 1:0.5 ~ 1.5, vortex vibration, centrifugal collection supernatant III, adds internal standard compound ribitol solution again, and supernatant III volume and internal standard compound ribitol part by weight are 50 ~ 300ul:20 μ g, room temperature vacuum drying, obtains extracellular fluid sample II;
(3) analyte derivative: in step 2) in the sample II that obtains, add respectively 50 ~ 150ul sugar derivating agent, constant temperature is placed 1.5 ~ 4h, add respectively more amino acid derived dose of 50 ~ 150ul, constant temperature is placed and is spent the night, derivative complete, sample II after centrifugal deriving, collects supernatant and obtains sample II in born of the same parents to be measured;
(4) utilize sample II in the born of the same parents to be measured that gas chromatography-mass spectrum obtains step 3), analyze and data acquisition.
2. a kind of gas chromatography-mass spectrum according to claim 1 detects the method for organic acid in fermentation liquor, amino acid, sugar, it is characterized in that, its sampling amount of described thalline reaches 0.2 ~ 1.0g/ml for dissolve artifact amount dry weight with cold methanol; Described cold methanol is for being chilled in advance-40 ℃ through cryostat groove; Described low-temperature extraction extracts 2 ~ 7h at-40 ℃ to-50 ℃.
3. a kind of gas chromatography-mass spectrum according to claim 1 and 2 detects the method for organic acid in fermentation liquor, amino acid, sugar, it is characterized in that, described sugared derivating agent is that 0.1 ~ 0.3mg methoxamine hydrochloric acid is dissolved in preparation in 10ml pyridine solution and obtains; Described amino acid derived dose is that 50 ~ 200ul trimethyl chlorosilane is dissolved in the trifluoroacetamide of 10ml.
4. a kind of gas chromatography-mass spectrum according to claim 3 detects organic acid in fermentation liquor, amino acid, the method of sugar, it is characterized in that, its instrumental analysis condition of described gas chromatography-mass spectrum is: gas chromatography: chromatographic column is HP-5MS or the DB-5MS capillary column of 30 m × 0.25 mm, 300 ℃ of injector temperatures, 250 ℃ of detector temperatures, column temperature rise program equilibration time 3 min → 80 ℃ maintain 1min → 2 ℃/min and are warming up to 100 ℃ → 15 ℃/min and are warming up to 220 ℃ → 30 ℃/min and are warming up to 300 ℃ → 300 ℃ and maintain 3 min, sampling volume 1 μ L, mass spectrum: ion gun EI, detecting device is level Four bar, ionization energy 70 eV, 280 ℃ of ion gun surface temperatures.
5. a kind of gas chromatography-mass spectrum detects the method for organic acid in fermentation liquor, amino acid, sugar according to claim 4, it is characterized in that, the method is applied to organic acid, amino acid, the sugar in the fermentation liquor in the biological fermentation process that detects saccharomycete, lactic acid bacteria, clostridium, mould.
6. the method that detects organic acid in fermentation liquor, amino acid, sugar according to a kind of gas chromatography-mass spectrum described in claim 1 ~ 2 or 4 ~ 5 any one, 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.
7. a kind of gas chromatography-mass spectrum according to claim 3 detects the method for organic acid in fermentation liquor, amino acid, sugar, 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.
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