CN103954726B - Method and the detection method of muramic acid is extracted from corynebacterium glutamicum bacterium slag - Google Patents
Method and the detection method of muramic acid is extracted from corynebacterium glutamicum bacterium slag Download PDFInfo
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Abstract
The invention discloses a kind of thin-layer chromatography scanning detection method of muramic acid, have the advantages such as simple, quick, accurate, reliable, stable, step is: 1) get detected sample liquid, and point sample is on High Performance Thin silica gel plate, meanwhile, by titer point sample in same High Performance Thin silica gel plate; Developping agent adds in expansion cylinder and carries out pre-equilibration, after High Performance Thin silica gel plate put into expansion cylinder carry out second outspread, taking-up is dried; 2) baking dyeing; 3) utilize thin-layer chromatogram scanner to carry out thin-layer chromatography scanning, obtain muramic acid Rf (Rf value) and integrating peak areas value; Meanwhile, calculate the regression equation of the relation of titer quality and integrating peak areas value, then the integrating peak areas value of liquid calculates the mass concentration of muramic acid in sample liquid per sample.Present invention also offers a kind of method extracting muramic acid from corynebacterium glutamicum bacterium slag, by the various process to bacterium slag such as washing, degreasing, acidolysis, extraction, obtain the muramic acid of purifying.
Description
Technical field
The present invention relates to a kind of method extracting muramic acid from corynebacterium glutamicum bacterium slag, and the thin-layer chromatography detection method of muramic acid.
Background technology
Muramic acid is the constituent of peptide glycan in bacteria cell wall.
Corynebacterium glutamicum (Corynebacteriumglutamicum) is aerobic bacteria, bar-shaped, sometimes microbend, the blunt circle in two ends, not branch, single or one-tenth Eight characters arrangement, thalline 0.7 ~ 0.9 micron × 1.0 ~ 2.5 microns, Gram-positive, without gemma, can be used for microbial fermentation engineering and produces glutamic acid and and then produce sodium glutamate (monosodium glutamate).The glutamic acid fermentation wine with dregs bacterium slag that gives up is the principal by product of monosodium glutamate industry, and annual output can reach ton up to a million, is mainly used as fertilizer and feed manufacturing, cheap.Give up wine with dregs bacterium slag for raw material with glutamic acid fermentation, detects and extraction purification muramic acid, significantly can promote comprehensive utilization value, improve the economic and social benefits.Have not yet to see and to give up the report that wine with dregs bacterium slag is raw material extraction purification muramic acid with glutamic acid fermentation.Summary of the invention
The invention provides a kind of method of extraction purification muramic acid from corynebacterium glutamicum bacterium slag, present invention also offers a kind of thin-layer chromatography detection method of muramic acid.
The present invention is achieved by the following technical solutions:
From corynebacterium glutamicum bacterium slag, extract a method for muramic acid, step is as follows:
1) (glutamic acid fermentation gives up wine with dregs bacterium slag to get fresh corynebacterium glutamicum bacterium slag, i.e. raw material bacterium slag), put in a reservoir, be first that the physiological saline of 0.9% is at room temperature repeatedly (2 ~ 5 times) washing bacterium slag by mass concentration, to remove foreign odor taste and lyotrope matter, filter, obtain filter residue;
Further, after brine, if filter residue also has foreign odor taste, then in filter residue, add distilled water, also under stirring, wash bacterium slag (realizing by constant temperature blender with magnetic force) temperature 55 DEG C, wash 2 ~ 5 times, filter, obtain filter residue;
2) by step 1) gained filter residue puts into closed container, the mass concentration adding raw material bacterium slag amount 3 ~ 5 times is the solution of trichloroacetic acid of 10%, at temperature 45 ~ 60 DEG C, stir 15min ~ 1h (to realize by constant temperature blender with magnetic force, the preferred middling speed of stirring rate), with cracking thalline, remove LTA, filter, obtain filter residue;
3) by step 2) the distilled water washing 1 ~ 2 time of gained filter residue, to remove trichloroacetic acid, then the methyl alcohol of raw material bacterium slag amount 2 ~ 10 times, methanol-chloroform mixed liquor (methyl alcohol, chloroform by volume 1:1 mix), chloroform degreasing is used successively, each 2 times; Obtain degreasing filter residue;
4) to step 3) add the hydrochloric acid that concentration is 5 ~ 8mol/L in gained degreasing filter residue, add 4 ~ 10 milliliters of hydrochloric acid by every gram of raw material bacterium slag, be placed in water-bath, acidolysis 4 ~ 8h at temperature 90 ~ 100 DEG C; After acidolysis, obtain the acid hydrolysis solution of brownish black, by acid hydrolysis solution concentrated evaporate to dryness (50 ~ 65 DEG C) on vacuum rotary evaporator, to fling to hydrochloric acid as far as possible, obtain muramic acid crude product;
Preferably, the described temperature by acid hydrolysis solution concentrated evaporate to dryness on vacuum rotary evaporator is 50 ~ 65 DEG C;
5) step 4) gained muramic acid crude product, dissolves with distilled water, adds chloroform, concuss rocks, and leaves standstill separatory, discards lower floor's organic phase (containing the impurity of lipophilicity and both sexes in organic phase), add chloroform extraction again 1 ~ 3 time, obtain upper solution;
Preferably, the consumption of described distilled water is 2 ~ 4 times of raw material bacterium slag amount, and chloroform consumption is 5 ~ 10 times of distilled water volume;
6) by step 5) gained upper solution evaporation volatilize, with methyl alcohol dissolve, add activated charcoal mixing and absorption 1 ~ 3 time, become colorless to solution transparent, obtain muramic acid solution, concentrate drying, obtain muramic acid;
Preferably, described activated charcoal selects model 303 craboraffin, and each activated carbon dosage is 0.5 ~ 1.5 times of raw material bacterium slag amount.
A thin-layer chromatography scanning detection method for muramic acid, step is as follows:
1) get detected sample liquid (i.e. said method prepare muramic acid solution), point sample on High Performance Thin silica gel plate, meanwhile, by the titer point sample of variable concentrations in same High Performance Thin silica gel plate; Developping agent adds in expansion cylinder and carries out pre-equilibration, after High Performance Thin silica gel plate put into expansion cylinder carry out second outspread, taking-up is dried;
The GF254 High Performance Thin silica gel plate of 10cm × 10cm selected by described High Performance Thin silica gel plate;
The point sample amount of described detected sample liquid is 10 μ L/ points;
Described titer is the titer of 5 kinds of variable concentrations, and concentration is respectively 2.0,3.0,4.0,5.0,6.0mg/ml, and point sample amount is 1 μ L/ point;
Described developping agent is methanol-acetone-acetic acid-water-isopropyl alcohol mixed liquor, and wherein, the volume ratio of methyl alcohol, acetone, acetic acid, water, isopropyl alcohol is 4 ~ 6:2 ~ 4:0.5 ~ 1:0.5 ~ 1:0.5 ~ 2;
The temperature of described developping agent pre-equilibration and silica gel plate expansion process is 25 DEG C, 20 minutes, second outspread, and each exhibition is apart from 8cm (being all from initial point meter) at every turn.
2) by mass concentration be 1% ethanol solution of ninhydrin be sprayed onto and High Performance Thin silica gel plate carry out baking dyeing;
Further, the baking temperature of described dyeing is 110 DEG C, time 5min;
3) utilize thin-layer chromatogram scanner to carry out thin-layer chromatography scanning, obtain muramic acid Rf (Rf value) and integrating peak areas value; Simultaneously, the regression equation calculating the relation of titer quality and integrating peak areas value (is calculated by thin layer chromatograph management software, for conventional means), then the integrating peak areas value of liquid calculates the mass concentration of muramic acid in sample liquid per sample.
The present invention, when carrying out thin-layer chromatography, for obtaining effective effect, having carried out many explorations to developping agent, and having utilized different agent combination: acetone-water; Isopropyl alcohol-glacial acetic acid-water; Normal butyl alcohol-toluene-glacial acetic acid-water; Normal butyl alcohol-glacial acetic acid-isopropyl alcohol; Normal butyl alcohol-toluene-glacial acetic acid-water; Isopropyl alcohol: acetic acid: water: methyl alcohol and methyl alcohol: acetone: acetic acid: water: isopropyl alcohol etc.Each combination, all carry out ratio debugging repeatedly, finally, find with methanol-acetone-acetic acid-water-isopropyl alcohol, effect is launched better, with the volume ratio of methyl alcohol, acetone, acetic acid, water, isopropyl alcohol for best during 5:3:1:1:1 when volume ratio is developping agent and the expansion temperature 25 DEG C of 4 ~ 6:2 ~ 4:0.5 ~ 1:0.5 ~ 1:0.5 ~ 2 proportioning.
The method extracting muramic acid from corynebacterium glutamicum bacterium slag of the present invention, eliminates the impurity in bacterium slag, obtains the muramic acid of purifying.The thin-layer chromatography detection method of muramic acid of the present invention, has the advantages such as simple, quick, accurate, reliable, stable.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1 is extracted muramic acid and is carried out thin-layer chromatography detection
Extract: get fresh corynebacterium glutamicum bacterium slag (raw material bacterium slag) 5 grams, be placed in 150ml beaker, first at room temperature repeatedly wash bacterium slag with the physiological saline of 0.9%, removing foreign odor taste, filter, obtain filter residue, proceed in 200ml triangular flask, add the solution of trichloroacetic acid of 25ml10%, by constant temperature blender with magnetic force at 55 DEG C, stirring 30min is carried out under suitable rotating speed, with cracking thalline, remove LTA, filter, removing filtrate, with distilled water washing residue to wash away trichloroacetic acid, then 50ml methyl alcohol is used successively, 50ml methanol-chloroform mixed liquor (methyl alcohol and chloroform by volume 1:1 mix), the degreasing of 50ml chloroform, each 2 times, obtain degreasing filter residue, add the hydrochloric acid 50ml of 6mol/L, at water-bath 95 DEG C heating acidolysis 5h, obtain the acid hydrolysis solution of brownish black, by the acid hydrolysis solution of brownish black at the concentrated 50 DEG C of evaporates to dryness of Rotary Evaporators, to fling to hydrochloric acid as far as possible, dissolve with 20ml tri-distilled water, add 100ml chloroform, concuss rocks, and leaves standstill separatory, except the impurity of sub-cloud lipophilicity and both sexes, add chloroform extraction again 2 times, each chloroform consumption is 100ml, obtains upper solution, evaporation volatilizes, and dissolves with the methyl alcohol of 30ml, adds 5g303 craboraffin mixing and absorption 3 times, becomes colorless transparent to solution, obtain muramic acid solution.
Detect: get muramic acid solution 1ml, dilute 15 times, obtain sample liquid.The titer of preparation muramic acid, concentration is respectively: 0.2,0.3,0.4,0.5,0.6mg/ml.Sample liquid (10 μ L/ point) and titer (1 μ L/ point) are put on same GF254 High Performance Thin silica gel plate (10cm × 10cm).Developping agent is methanol-acetone-glacial acetic acid-water-isopropyl alcohol mixed liquor, and wherein, the volume ratio of methyl alcohol, acetone, acetic acid, water, isopropyl alcohol is 5:3:1:1:1.Developping agent is added expansion cylinder (10cm × 12cm × 15cm) pre-equilibration 20 minutes at 25 DEG C.Expansion cylinder put into by High Performance Thin plate, launches at 25 DEG C, and silica gel plate plate, apart from when reaching 8cm, takes out and dries by exhibition.And then put back to expansion cylinder secondary (from initial point meter) and launch 8cm, after launching to terminate, silica gel plate plate is taken out and dries.With the ethanol solution of ninhydrin spray plate dyeing that mass concentration is 1%, toast 5 minutes at being then placed in 110 DEG C.Sample liquid and point corresponding to standard items dye redness.With camag3 type thin-layer chromatogram scanner, scan with the absorbing wavelength of 492nm, obtain integrating peak areas value and muramic acid migration value Rf is 0.84, the regression equation of the relation of thin-layer chromatogram scanner software winCATS1.4.1 statistical computation integrating peak areas value and titer quality, correlativity Y=16.854X+4572.2, r=0.9991RSD=1.53%.After testing and calculate, obtaining muramic acid content in corynebacterium glutamicum bacterium slag is 1.10g/100g.
Embodiment 2 is extracted muramic acid and is carried out thin-layer chromatography detection
Extract: get fresh corynebacterium glutamicum bacterium slag (raw material bacterium slag) 5 grams, be placed in 150ml beaker, first at room temperature repeatedly wash bacterium slag with the physiological saline of 0.9%, removing foreign odor taste, filter, obtain filter residue, proceed in 200ml triangular flask, add the solution of trichloroacetic acid of 15ml10%, by constant temperature blender with magnetic force at 45 DEG C, stirring 15 minutes is carried out under suitable rotating speed, with cracking thalline, remove LTA, filter, removing filtrate, with distilled water washing residue to wash away trichloroacetic acid, then 10ml methyl alcohol 10ml methanol-chloroform mixed liquor (methyl alcohol and chloroform by volume 1:1 mix) is used successively, the degreasing of 10ml chloroform, each 2 times, obtain degreasing filter residue, add the hydrochloric acid 20ml of 5mol/L, at water-bath 90 DEG C heating acidolysis 4h, obtain the acid hydrolysis solution of brownish black, by the acid hydrolysis solution of brownish black at the concentrated 50 DEG C of evaporates to dryness of Rotary Evaporators, to fling to hydrochloric acid as far as possible, dissolve with 10ml tri-distilled water, add 50ml chloroform, concuss rocks, and leaves standstill separatory, except the impurity of sub-cloud lipophilicity and both sexes, add chloroform extraction again 2 times, each chloroform consumption is 50ml, obtains upper solution, evaporation volatilizes, and dissolves with the methyl alcohol of 30ml, adds 2.5g303 craboraffin mixing and absorption 3 times, becomes colorless transparent to solution, obtain muramic acid solution.
Detect: get muramic acid solution 1ml, dilute 15 times, obtain sample liquid.The titer of preparation muramic acid, concentration is respectively: 0.2,0.3,0.4,0.5,0.6mg/ml.Sample liquid (10 μ L/ point) and titer (1 μ L/ point) are put on same GF254 High Performance Thin silica gel plate (10cm × 10cm).Developping agent is methanol-acetone-glacial acetic acid-water-isopropyl alcohol mixed liquor, and wherein, the volume ratio of methyl alcohol, acetone, acetic acid, water, isopropyl alcohol is 4:2:0.5:0.5:0.5.Developping agent is added expansion cylinder (10cm × 12cm × 15cm) pre-equilibration 20 minutes at 25 DEG C.Expansion cylinder put into by High Performance Thin plate, launches at 25 DEG C, and silica gel plate plate, apart from when reaching 8cm, takes out and dries by exhibition.And then put back to expansion cylinder secondary (from initial point meter) and launch 8cm, after launching to terminate, silica gel plate plate is taken out and dries.With the ethanol solution of ninhydrin spray plate dyeing that mass concentration is 1%, toast 5 minutes at being then placed in 110 DEG C.Sample liquid and point corresponding to standard items dye redness.With camag3 type thin-layer chromatogram scanner, scan with the absorbing wavelength of 492nm, obtain integrating peak areas value and muramic acid migration value Rf is 0.84, the regression equation of the relation of thin-layer chromatogram scanner software winCATS1.4.1 statistical computation integrating peak areas value and titer quality, correlativity Y=4113.9915+30.0484*X, r=0.99953RSD=3.71%.After testing and calculate, obtaining muramic acid content in corynebacterium glutamicum bacterium slag is 1.16g/100g.
Embodiment 3 is extracted muramic acid and is carried out thin-layer chromatography detection
Extract: get fresh corynebacterium glutamicum bacterium slag (raw material bacterium slag) 5 grams, be placed in 150ml beaker, first at room temperature repeatedly wash bacterium slag with the physiological saline of 0.9%, removing foreign odor taste, filter, obtain filter residue, proceed in 200ml triangular flask, add the solution of trichloroacetic acid of 20ml10%, by constant temperature blender with magnetic force at 60 DEG C, stirring 1 hour is carried out under suitable rotating speed, with cracking thalline, remove LTA, filter, removing filtrate, with distilled water washing residue 2 times to wash away trichloroacetic acid, then 30ml methyl alcohol is used successively, 30ml methanol-chloroform mixed liquor (methyl alcohol and chloroform by volume 1:1 mix), the degreasing of 30ml chloroform, each 2 times, obtain degreasing filter residue, add the hydrochloric acid 30ml of 8mol/L, at water-bath 100 DEG C heating acidolysis 8h, obtain the acid hydrolysis solution of brownish black, by the acid hydrolysis solution of brownish black at the concentrated 65 DEG C of evaporates to dryness of Rotary Evaporators, to fling to hydrochloric acid as far as possible, dissolve with 15ml tri-distilled water, add 90ml chloroform, concuss rocks, and leaves standstill separatory, except the impurity of sub-cloud lipophilicity and both sexes, add chloroform extraction again 2 times, each chloroform consumption is 90ml, obtains upper solution, evaporation volatilizes, and dissolves with the methyl alcohol of 20ml, adds 7.5g303 craboraffin mixing and absorption 3 times, becomes colorless transparent to solution, obtain muramic acid solution.
Detect: get muramic acid solution 1ml, dilute 15 times, obtain sample liquid.The titer of preparation muramic acid, concentration is respectively: 0.2,0.3,0.4,0.5,0.6mg/ml.Sample liquid (10 μ L/ point) and titer (1 μ L/ point) are put on same GF254 High Performance Thin silica gel plate (10cm × 10cm).Developping agent is methanol-acetone-glacial acetic acid-water-isopropyl alcohol mixed liquor, and wherein, the volume ratio of methyl alcohol, acetone, acetic acid, water, isopropyl alcohol is 6:4:0.9:0.8:2.Developping agent is added expansion cylinder (10cm × 12cm × 15cm) pre-equilibration 20 minutes at 25 DEG C.Expansion cylinder put into by High Performance Thin plate, launches at 25 DEG C, and silica gel plate plate, apart from when reaching 8cm, takes out and dries by exhibition.And then put back to expansion cylinder secondary (from initial point meter) and launch 8cm, after launching to terminate, silica gel plate plate is taken out and dries.With the ninhydrin ethanolic solution spray plate dyeing that mass concentration is 1%, toast 5 minutes at being then placed in 110 DEG C.Sample liquid and point corresponding to standard items dye redness.With camag3 type thin-layer chromatogram scanner, scan with the absorbing wavelength of 492nm, obtain integrating peak areas value and muramic acid migration value Rf is 0.84, use gained relation regression equation in embodiment 1, after testing and calculate, obtaining muramic acid content in corynebacterium glutamicum bacterium slag is 1.13g/100g.
Claims (8)
1. a thin-layer chromatography scanning detection method for muramic acid, is characterized in that: step is as follows:
1) get detected sample liquid, point sample on High Performance Thin silica gel plate, meanwhile, by the titer point sample of variable concentrations in same High Performance Thin silica gel plate; Developping agent adds in expansion cylinder and carries out pre-equilibration, after High Performance Thin silica gel plate put into expansion cylinder carry out second outspread, taking-up is dried;
Described developping agent is methanol-acetone-acetic acid-water-isopropyl alcohol mixed liquor, and wherein, the volume ratio of methyl alcohol, acetone, acetic acid, water, isopropyl alcohol is 4 ~ 6:2 ~ 4:0.5 ~ 1:0.5 ~ 1:0.5 ~ 2;
2) by mass concentration be 1% ethanol solution of ninhydrin be sprayed onto and High Performance Thin silica gel plate carry out baking dyeing;
3) utilize thin-layer chromatogram scanner to carry out thin-layer chromatography scanning, obtain muramic acid Rf value and integrating peak areas value; Meanwhile, calculate the regression equation of the relation of titer quality and integrating peak areas value, then the integrating peak areas value of liquid calculates the mass concentration of muramic acid in sample liquid per sample;
Described sample liquid prepares by the following method:
1) get fresh corynebacterium glutamicum raw material bacterium slag, first at room temperature repeatedly wash bacterium slag with the physiological saline that mass concentration is 0.9%, to remove foreign odor taste and lyotrope matter, filter, obtain filter residue;
2) step 1) gained filter residue, the mass concentration adding raw material bacterium slag amount 3 ~ 5 times is the solution of trichloroacetic acid of 10%, at temperature 45 ~ 60 DEG C, stirs 15min ~ 1h, filters, obtain filter residue;
3) by step 2) the distilled water washing 1 ~ 2 time of gained filter residue, to remove trichloroacetic acid, then use the methyl alcohol of filter residue quality 2 ~ 10 times, methanol-chloroform mixed liquor, chloroform degreasing successively, each 2 times; Obtain degreasing filter residue;
4) to step 3) add the hydrochloric acid that concentration is 5 ~ 8mol/L in gained degreasing filter residue, add 4 ~ 10 milliliters of hydrochloric acid by every gram of raw material filter residue, be placed in water-bath, acidolysis 4 ~ 8h at temperature 90 ~ 100 DEG C; After acidolysis, obtain the acid hydrolysis solution of brownish black, acid hydrolysis solution is concentrated evaporate to dryness, obtain muramic acid crude product;
5) step 4) gained muramic acid crude product, dissolve with distilled water, add chloroform, concuss rocks, and leaves standstill separatory, discards lower floor's organic phase, then add chloroform extraction 1 ~ 3 time, obtain upper solution;
6) by step 5) gained upper solution evaporation volatilize, with methyl alcohol dissolve, add activated charcoal mixing and absorption 1 ~ 3 time, become colorless to solution transparent, obtain sample liquid.
2. the thin-layer chromatography scanning detection method of muramic acid according to claim 1, is characterized in that: GF254 High Performance Thin silica gel plate selected by described High Performance Thin silica gel plate.
3. the thin-layer chromatography scanning detection method of muramic acid according to claim 1, is characterized in that: described titer is the titer of 5 kinds of variable concentrations, and concentration is respectively 2.0,3.0,4.0,5.0,6.0mg/ml.
4. the thin-layer chromatography scanning detection method of muramic acid according to claim 1, is characterized in that: the volume ratio of described methyl alcohol, acetone, acetic acid, water, isopropyl alcohol is 5:3:1:1:1.
5. the thin-layer chromatography scanning detection method of muramic acid according to claim 1, is characterized in that: the temperature of described developping agent pre-equilibration and silica gel plate expansion process is 25 DEG C, time 20min.
6. the thin-layer chromatography scanning detection method of muramic acid according to claim 1, is characterized in that: the baking temperature of described baking dyeing is 110 DEG C, time 5min.
7. from corynebacterium glutamicum bacterium slag, extract a method for muramic acid, it is characterized in that: step is as follows:
1) get fresh corynebacterium glutamicum raw material bacterium slag, first at room temperature repeatedly wash bacterium slag with the physiological saline that mass concentration is 0.9%, to remove foreign odor taste and lyotrope matter, filter, obtain filter residue;
2) step 1) gained filter residue, the mass concentration adding raw material filter residue quality 3 ~ 5 times be the solution of trichloroacetic acid of 10% at temperature 45 ~ 60 DEG C, stir 15min ~ 1h, filter, obtain filter residue;
3) by step 2) the distilled water washing 1 ~ 2 time of gained filter residue, to remove trichloroacetic acid, then use the methyl alcohol of raw material bacterium slag amount 2 ~ 10 times, methanol-chloroform mixed liquor, chloroform degreasing successively, each 2 times; Obtain degreasing filter residue;
4) to step 3) add the hydrochloric acid that concentration is 5 ~ 8mol/L in gained degreasing filter residue, add 4 ~ 10 milliliters of hydrochloric acid by every 1 gram of raw material bacterium slag, be placed in water-bath, acidolysis 4 ~ 8h at temperature 90 ~ 100 DEG C; After acidolysis, obtain the acid hydrolysis solution of brownish black, acid hydrolysis solution is concentrated evaporate to dryness, obtain muramic acid crude product;
5) step 4) gained muramic acid crude product, dissolve with distilled water, add chloroform, concuss rocks, and leaves standstill separatory, discards lower floor's organic phase, then add chloroform extraction 1 ~ 3 time, obtain upper solution;
6) by step 5) gained upper solution evaporation volatilize, with methyl alcohol dissolve, add activated charcoal mixing and absorption 1 ~ 3 time, become colorless to solution transparent, obtain muramic acid solution, concentrate drying, obtain muramic acid.
8. the method extracting muramic acid from corynebacterium glutamicum bacterium slag according to claim 7, is characterized in that: described activated charcoal selects model 303 craboraffin.
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| CN106188168B (en) * | 2016-07-18 | 2018-10-02 | 山东师范大学 | The method for preparing aminoglucose hydrochloride using the bacteria residue of production glutamic acid |
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