CN106279308A - The method utilizing the dreg separation 3-O-.alpha.-carboxyethyl-D-glucosamine. producing glutamic acid - Google Patents
The method utilizing the dreg separation 3-O-.alpha.-carboxyethyl-D-glucosamine. producing glutamic acid Download PDFInfo
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- CN106279308A CN106279308A CN201610565555.XA CN201610565555A CN106279308A CN 106279308 A CN106279308 A CN 106279308A CN 201610565555 A CN201610565555 A CN 201610565555A CN 106279308 A CN106279308 A CN 106279308A
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- carboxyethyl
- glucosamine
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- glutamic acid
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- 238000000034 method Methods 0.000 title claims abstract description 40
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 title claims abstract description 32
- 235000013922 glutamic acid Nutrition 0.000 title claims abstract description 32
- 239000004220 glutamic acid Substances 0.000 title claims abstract description 32
- 238000000926 separation method Methods 0.000 title claims abstract description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 25
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000000843 powder Substances 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000000605 extraction Methods 0.000 claims abstract description 19
- 239000000706 filtrate Substances 0.000 claims abstract description 16
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 14
- 238000003756 stirring Methods 0.000 claims abstract description 12
- 241000894006 Bacteria Species 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 239000002893 slag Substances 0.000 claims abstract description 7
- 239000012141 concentrate Substances 0.000 claims abstract description 5
- 238000001514 detection method Methods 0.000 claims description 4
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 claims description 2
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 abstract description 10
- 238000004440 column chromatography Methods 0.000 abstract description 6
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 11
- 229960004756 ethanol Drugs 0.000 description 9
- 238000004809 thin layer chromatography Methods 0.000 description 7
- 239000002253 acid Substances 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 4
- 230000001186 cumulative effect Effects 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 239000004519 grease Substances 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 2
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Natural products CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- -1 methanol-acetone-acetic acid-water-isopropanol Chemical group 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 229940086609 Lipase inhibitor Drugs 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000002481 ethanol extraction Methods 0.000 description 1
- 239000000469 ethanolic extract Substances 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000003808 methanol extraction Methods 0.000 description 1
- 239000000401 methanolic extract Substances 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 239000004223 monosodium glutamate Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000003334 potential effect Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 125000003944 tolyl group Chemical group 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
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- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of method utilizing the dreg producing glutamic acid to peel off 3-O-.alpha.-carboxyethyl-D-glucosamine., step is as follows: (1) takes glutamic acid wet bacteria slag, adds water and carries out supersound extraction, filter, obtain filtrate in dreg；(2) filtrate concentrates, and adds the ethanol solution of 8 12 times of volumes (ml/ml) in the filtrate after concentrating, and stirring is extracted, and filters, obtains white powder；(3) add water in white powder mixing, adds hydrochloric acid solution and hydrolyzes 35 hours under the conditions of 15 30 DEG C；(4) adding ether, separatory in hydrolyzed solution, solution is divided into three layers, separates and takes upper strata and middle level liquid, is concentrated to dryness, obtains 3-O-.alpha.-carboxyethyl-D-glucosamine. powder.The method using the present invention prepares 3-O-.alpha.-carboxyethyl-D-glucosamine., and the kind of required reagent is few, and quantity is little, reduces preparation cost, reduces the pollution to environment；And without steps such as decolouring, column chromatography for separation, simplify preparation process；The extraction ratio of 3-O-.alpha.-carboxyethyl-D-glucosamine. is high.
Description
Technical field
A kind of method that the present invention relates to dreg separation 3-O-.alpha.-carboxyethyl-D-glucosamine. utilizing and producing glutamic acid, belongs to food and chemical field.
Background technology
3-O-.alpha.-carboxyethyl-D-glucosamine. is the important component part of bacteria cell wall mucopeptide structure, is the biological marker of prokaryotic micro-organisms cell
Compound.The most frequently used use of 3-O-.alpha.-carboxyethyl-D-glucosamine. is as speculating the label of microorganism total amount in varying environment；It addition, 3-O-.alpha.-carboxyethyl-D-glucosamine. is solidifying
Collect bacterial lipase inhibitor plain, potential, and antitumor action has the biggest research application prospect.
At present, the 3-O-.alpha.-carboxyethyl-D-glucosamine. on market is expensive, and the market demand potential is big.For the preparation of 3-O-.alpha.-carboxyethyl-D-glucosamine., can pass through
Prepared by the method for chemosynthesis, but the cost intensive of chemosynthesis, easily cause the pollution of environment.It is therefore desirable to develop new
The stripping means of 3-O-.alpha.-carboxyethyl-D-glucosamine..
Glutamic acid dreg is the principal by product of monosodium glutamate industry, and the glutamic acid dreg that China produces every year reaches million tons, right
In the recycling of glutamic acid dreg, the most as feedstuff and Fertilizer application, produced value is relatively low.
The dreg utilizing production glutamic acid is prepared 3-O-.alpha.-carboxyethyl-D-glucosamine. and is studied, in preparation and the inspection of 3-O-.alpha.-carboxyethyl-D-glucosamine. by Jiang Xinying etc.
Achieve relatively much progress in survey method, but its step extracting 3-O-.alpha.-carboxyethyl-D-glucosamine. include: wash, stir cracking, grease removal, acidolysis, extraction,
Decolouring, concentration extraction, HPTLC detection, column chromatography for separation and purity detecting, whole extraction process steps is many, and expending substantial amounts of has
Machine reagent, such as chloroform, trichloroacetic acid etc., not only increases the Financial cost of extraction, also pollutes the environment if discharging these reagent.
The extraction process of 3-O-.alpha.-carboxyethyl-D-glucosamine. is improved by Jiang Xinying etc. further, and it is extensive that the extraction process after improvement includes being suitable for factory
Extract and two kinds of extraction processes of production of applicable small lot 3-O-.alpha.-carboxyethyl-D-glucosamine..Wherein, the technological process that applicable factory extracts on a large scale is:
Acidolysis, decolouring, methanol/ethanol extraction, HPTLC detection, column chromatography for separation and purity detecting；But this technological process is will to produce paddy
The dreg of propylhomoserin directly carries out acidolysis, if carrying out commercial production, the amount of the acid solution of required addition is too big, and production cost is the highest.
Being suitable for the technological process that small lot 3-O-.alpha.-carboxyethyl-D-glucosamine. extracts is: ultrasonic treatment, centrifugal, acidolysis, decolouring, methanol/ethanol extract,
HPTLC detection, column chromatography for separation and purity detecting；But the dreg producing glutamic acid is directly used supercritical ultrasonics technology to split by this technique
Solving, can produce the impurity such as lipid, need to be purified by operations such as decolouring, column chromatography for separation, preparation process is the most comparatively laborious.
Summary of the invention
For above-mentioned prior art, it is an object of the invention to provide a kind of dreg utilizing production glutamic acid and prepare 3-O-.alpha.-carboxyethyl-D-glucosamine.
Method.
For achieving the above object, the present invention uses following technical proposals:
A kind of method utilizing the dreg separation 3-O-.alpha.-carboxyethyl-D-glucosamine. producing glutamic acid, step is as follows:
(1) take glutamic acid wet bacteria slag, in dreg, add water carry out supersound extraction, filter, obtain filtrate；
(2) filtrate concentrates, and adds the ethanol solution of 8-12 times of volume (ml/ml) in the filtrate after concentrating, and stirring is extracted,
Filter, obtain white powder；
(3) add water in white powder mixing, adds hydrochloric acid solution and hydrolyzes 3-5 hour under the conditions of 15-30 DEG C；
(4) adding ether, separatory in hydrolyzed solution, solution is divided into three layers, separates and takes upper strata and middle level liquid, is concentrated into
Dry, obtain 3-O-.alpha.-carboxyethyl-D-glucosamine. powder.
In step (1), the water content of described glutamic acid wet bacteria slag is 40-60% (mass percent).
In step (1), the condition of supersound extraction is: 20kHz, ultrasonic power 700～900W, ultrasonic 2 seconds gaps 2 seconds, super
Sound 4-6 time, each 0.5-1h.The purpose of supersound extraction is by broken wall treatment；The power of supersound extraction, extraction time and ultrasonic
Time is the principal element affecting broken wall treatment effect, and the present invention is by the optimization to ultrasonic process conditions so that it is ensureing relatively
On the premise of good shell-broken effect, reduce again energy consumption.
In step (1), the addition of water is that the 8-12 times of volume (g/ml) of glutamic acid dreg dry weight is (in i.e. every gram dry dreg
Add water 8-12ml).
In step (2), concentrate the filtrate to the 45-55% of glutamic acid dreg dry weight, preferably 50%.
In step (2), the percentage by volume of described ethanol solution is 90-100%.Use the mesh that it is extracted by ethanol solution
Be grease removal.
In step (2), the time that stirring is extracted is 0.5-1 hour.
In step (3), white powder add water mixing after the 45-55% that quality is glutamic acid dreg dry weight, be preferably
50%.
In step (3), the volume fraction of hydrochloric acid solution is 50% (v/v, i.e. concentrated hydrochloric acid and water press 1:1 proportioning).
In step (4), the addition of ether and the volume ratio of hydrolyzed solution are 1:1.Present invention firstly discovers that and utilize ether pair
Hydrolyzed solution after acid hydrolysis carries out separatory process, can be separated with other materials by object 3-O-.alpha.-carboxyethyl-D-glucosamine., simplify greatly
The purification procedures of 3-O-.alpha.-carboxyethyl-D-glucosamine..
The 3-O-.alpha.-carboxyethyl-D-glucosamine. using the method for thin layer chromatography Scanning Detction to prepare the present invention detects, by the 3-O-.alpha.-carboxyethyl-D-glucosamine. of preparation
Crystallize 0.3 milligram to be dissolved in 1 ml methanol solution as sample liquid.The titer of preparation 3-O-.alpha.-carboxyethyl-D-glucosamine. is respectively 0.1, and 0.2,
0.3,0.4,0.5,0.6 mg/ml.By efficient at same GF254 to sample liquid (1 μ l/ point) and titer (1 μ l/ point) point
On thin-layer silicon offset plate (10cm*10cm), developing solvent is methanol-acetone-acetic acid-water-isopropanol (5:3:1:1:1, volume/volume)
Mixed liquor.Start to spread out after the most saturated 25 minutes, Zhan Cheng 8 centimetres, silica gel plate is taken out and dries.By mass concentration it is
1% ethanol solution of ninhydrin spray plate dyeing, plate is placed on 110 degree and bakes 5 minutes.Sample liquid point and standard substance liquid point all dye redness,
Rf value is 0.84, scans at 492nm with CAMAG3 thin-layer chromatogram scanner.With the WINCATS1.4.1 software statistics peak area of this instrument
Integrated value, regression equation calculation according to titer quality.Detect and calculate the content of 3-O-.alpha.-carboxyethyl-D-glucosamine..
Beneficial effects of the present invention:
(1) using the method for the present invention to prepare 3-O-.alpha.-carboxyethyl-D-glucosamine., the kind of required reagent is few, and quantity is little, reduces and is prepared as
This, reduce the pollution to environment；And without steps such as decolouring, column chromatography for separation, simplify preparation process；Carrying of 3-O-.alpha.-carboxyethyl-D-glucosamine.
The rate that takes is high, and using the inventive method to extract the 3-O-.alpha.-carboxyethyl-D-glucosamine. obtained is that more than the 1.2% of dreg quality done by glutamic acid, and prepare
The purity of 3-O-.alpha.-carboxyethyl-D-glucosamine. is high.
(2) method utilizing the dreg producing glutamic acid to prepare 3-O-.alpha.-carboxyethyl-D-glucosamine. of the present invention, first enters the dreg of glutamic acid
Row ultrasonication processes, and first filtrate is concentrated, add ethanol solution and carry out grease removal process, filter after grease removal after broken wall treatment
To white powder acid adding again carry out acidolysis, greatly reduce the consumption of acid hemolysis process acid solution, after acidolysis, utilize ether to hydrolysis
Liquid carries out separatory process, can be separated with other materials by object 3-O-.alpha.-carboxyethyl-D-glucosamine., greatly simplify the separation of 3-O-.alpha.-carboxyethyl-D-glucosamine.
Purification step.Above-mentioned each processing step is an organic whole, conditional, is effectively simplified from the dreg producing glutamic acid
The middle technological process preparing 3-O-.alpha.-carboxyethyl-D-glucosamine., and improve the extraction ratio of 3-O-.alpha.-carboxyethyl-D-glucosamine..
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated, it should explanation, following embodiment merely to
Explain the present invention, its content is not defined.
Embodiment 1: utilize the dreg separation 3-O-.alpha.-carboxyethyl-D-glucosamine. producing glutamic acid
Specifically comprise the following steps that
(1) the wet bacteria slag 120g (dreg dry weight is 48g) of glutamic acid is produced.The cumulative volume is made to be after the dispersed with stirring that adds water is good
500ml.Ultrasonic 0.5h under the conditions of 20kHz, 700w, ultrasonic 2 seconds gaps 2 seconds, filters to get filtrate.Filtering residue adds water more than repetition
Process 4 times, ultrasound filtration 5 times, merges 5 filtrate the most about 1000ml altogether.
(2) filter vacuum of merging is concentrated into 20ml, adds 95% ethanol 200ml stirring and extract 0.5h, static, isolate
Supernatant.Remainder adds the stirring of 200ml 95% ethanol again and extracts 0.5h, filters to obtain 5g white dry powder.
(3) adding water in 5g white dry powder, it is 20ml that mixing makes cumulative volume, then is added thereto to 20ml 50% (volume integral
Number, v/v) HCl, ordinary-temp hydrolysis 4h under magnetic agitation.
(4) in hydrolyzed solution, 40ml ether is added.Pouring in separatory funnel, shake up, stand and be divided into three layers, upper strata is transparent micro-
Huang, middle level is white slimy, lower floor's brownish red.Identified upper strata and middle level are 3-O-.alpha.-carboxyethyl-D-glucosamine., separate and take out upper strata and middle level liquid,
Through rotary evaporation in vacuo to dry, obtain white 3-O-.alpha.-carboxyethyl-D-glucosamine. powder 0.62g.
The 3-O-.alpha.-carboxyethyl-D-glucosamine. using the method for thin layer chromatography Scanning Detction to prepare the present embodiment detects, by the cell wall of preparation
Acid crystal 0.3 milligram is dissolved in 1 ml methanol solution as sample liquid.The titer of preparation 3-O-.alpha.-carboxyethyl-D-glucosamine. is respectively 0.1, and 0.2,
0.3,0.4,0.5,0.6 mg/ml.By efficient at same GF254 to sample liquid (1 μ l/ point) and titer (1 μ l/ point) point
On thin-layer silicon offset plate (10cm*10cm), developing solvent is methanol-acetone-acetic acid-water-isopropanol (5:3:1:1:1, volume/volume)
Mixed liquor.Start to spread out after the most saturated 25 minutes, Zhan Cheng 8 centimetres, silica gel plate is taken out and dries.By mass concentration it is
1% ethanol solution of ninhydrin spray plate dyeing, plate is placed on 110 degree and bakes 5 minutes.Sample liquid point and standard substance liquid point all dye redness,
Rf value is 0.84, illustrates that white powder prepared by the present embodiment is 3-O-.alpha.-carboxyethyl-D-glucosamine..
Scan at 492nm with CAMAG3 thin-layer chromatogram scanner.Amass with the WINCATS1.4.1 software statistics peak area of this instrument
Score value, regression equation calculation according to titer quality.Detect and calculate the content of 3-O-.alpha.-carboxyethyl-D-glucosamine..Be computed, prepared by the present embodiment
The purity of 3-O-.alpha.-carboxyethyl-D-glucosamine. be 98.2%, gained 3-O-.alpha.-carboxyethyl-D-glucosamine. is dry dreg recuperation 1.27%.
Embodiment 2: utilize the dreg separation 3-O-.alpha.-carboxyethyl-D-glucosamine. producing glutamic acid
Specifically comprise the following steps that
(1) take and produce the wet bacteria slag 150g (dreg dry weight is 60g) of glutamic acid, after the dispersed with stirring that adds water is good, make the cumulative volume be
600ml, ultrasonic 0.5h under the conditions of 20kHz, 900w, ultrasonic 2 seconds gaps 2 seconds, filter to get filtrate.Filtering residue adds water more than repetition
Process 4 times, altogether ultrasound filtration 5 times, merging filtrate 1200ml altogether.
(2) filter vacuum of merging being concentrated into 30ml, add dehydrated alcohol 300ml, 0.5h is extracted in stirring, stands, pours out
Supernatant, remainder adds the stirring of 300ml ethanol again and extracts 0.5h, filters to obtain 5.5g white dry powder.
(3) adding water in white dry powder, it is 30ml that mixing makes cumulative volume, then is added thereto to 30ml 50% (volume integral
Number, v/v) HCl, ordinary-temp hydrolysis 4h under magnetic agitation.
(4) in hydrolyzed solution, add 60ml ether, pour in separatory funnel, shake up, stand and divide three layers, separate and take out upper strata
With middle level liquid.Through rotary evaporation in vacuo to dry, obtain white 3-O-.alpha.-carboxyethyl-D-glucosamine. powder 0.74g.
The 3-O-.alpha.-carboxyethyl-D-glucosamine. using the method for thin layer chromatography Scanning Detction to prepare the present embodiment detects, and result shows this reality
The purity executing 3-O-.alpha.-carboxyethyl-D-glucosamine. prepared by example is 97.4%, and gained 3-O-.alpha.-carboxyethyl-D-glucosamine. is the 1.20% of dry dreg weight.
Embodiment 3: utilize the dreg separation 3-O-.alpha.-carboxyethyl-D-glucosamine. producing glutamic acid
Ultrasonic power used is adjusted to 800w, and remaining operation, with embodiment 1, obtains white 3-O-.alpha.-carboxyethyl-D-glucosamine. powder 0.65g.
The 3-O-.alpha.-carboxyethyl-D-glucosamine. using the method for thin layer chromatography Scanning Detction to prepare the present embodiment detects, and result shows this reality
The purity executing 3-O-.alpha.-carboxyethyl-D-glucosamine. prepared by example is 97.8%, and gained 3-O-.alpha.-carboxyethyl-D-glucosamine. is the 1.32% of dry dreg weight.
Comparative example 1:
Ultrasonic power used is adjusted to 450w, and remaining operation, with embodiment 1, the 3-O-.alpha.-carboxyethyl-D-glucosamine. powder prepared, is weighed.
Gained 3-O-.alpha.-carboxyethyl-D-glucosamine. is 0.53g.
The 3-O-.alpha.-carboxyethyl-D-glucosamine. using the method for thin layer chromatography Scanning Detction to prepare the present embodiment detects, and result shows this reality
The purity executing 3-O-.alpha.-carboxyethyl-D-glucosamine. prepared by example is 96.8%, and gained 3-O-.alpha.-carboxyethyl-D-glucosamine. is the 1.07% of dry dreg weight.
Comparative example 2:
Ether in embodiment 1 step (4) is replaced with dichloromethane, and remaining operation, with embodiment 1, is examined through thin layer chromatography
Survey, non-isolated 3-O-.alpha.-carboxyethyl-D-glucosamine..
Comparative example 3:
Ether in embodiment 1 step (4) is replaced with toluene, and remaining operation, with embodiment 1, detects through thin layer chromatography,
Non-isolated 3-O-.alpha.-carboxyethyl-D-glucosamine..
Comparative example 4:
Use the method for embodiment 2 in patent CN103954726A to carry out the extraction of 3-O-.alpha.-carboxyethyl-D-glucosamine., take fresh glutamic acid bar-shaped
Bacillus dreg 120 grams (water content is 60%, dreg dry weight 48 grams), carries out extraction and the inspection of 3-O-.alpha.-carboxyethyl-D-glucosamine. as described in Example 2
Survey (amount of reaction reagent doubles by corresponding mass ratio), after testing and calculate, being extracted the 3-O-.alpha.-carboxyethyl-D-glucosamine. obtained is paddy ammonia
The 1.09% of acid corynebacterium dreg dry weight.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify,
All should be the substitute mode of equivalence, within being included in protection scope of the present invention.
Claims (10)
1. the method utilizing the dreg separation 3-O-.alpha.-carboxyethyl-D-glucosamine. producing glutamic acid, it is characterised in that step is as follows:
(1) take glutamic acid wet bacteria slag, in dreg, add water carry out supersound extraction, filter, obtain filtrate；
(2) filtrate concentrates, and adds the ethanol solution of 8-12 times of volume (ml/ml) in the filtrate after concentrating, and stirring is extracted, mistake
Filter, obtains white powder；
(3) add water in white powder mixing, adds hydrochloric acid solution and hydrolyzes 3-5 hour under the conditions of 15-30 DEG C；
(4) adding ether, separatory in hydrolyzed solution, solution is divided into three layers, separates and takes upper strata and middle level liquid, is concentrated to dryness, i.e.
Obtain 3-O-.alpha.-carboxyethyl-D-glucosamine. powder.
2. the method for claim 1, it is characterised in that in step (1), the water content of described glutamic acid wet bacteria slag is
40-60%.
3. the method for claim 1, it is characterised in that in step (1), the condition of supersound extraction is: 20kHz, ultrasonic
Power 700～900W, ultrasonic 2 seconds gaps 2 seconds, ultrasonic 4-6 time, each 0.5-1h.
4. the method for claim 1, it is characterised in that in step (1), the addition of water is glutamic acid dreg dry weight
8-12 times of volume (g/ml).
5. the method for claim 1, it is characterised in that in step (2), concentrates the filtrate to glutamic acid dreg dry weight
45-55%, preferably 50%.
6. detection method as claimed in claim 1, it is characterised in that in step (2), the percentage by volume of described ethanol solution
For 90-100%.
7. method as claimed in claim 6, it is characterised in that in step (2), the time that stirring is extracted is 0.5-1 hour.
8. the method for claim 1, it is characterised in that in step (3), white powder add water mixing after quality be paddy
The 45-55% of propylhomoserin dreg dry weight, preferably 50%.
9. the method described in claim 1, it is characterised in that in step (3), the volume fraction of hydrochloric acid solution is 50%.
10. the method described in claim 1, it is characterised in that in step (4), the addition of ether and the volume ratio of hydrolyzed solution
For 1:1.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5215894A (en) * | 1975-07-29 | 1977-02-05 | Yuichi Yamamura | Process for preparing a substance possessing biological actions |
| WO2006027561A1 (en) * | 2004-09-06 | 2006-03-16 | The Scottish Association For Marine Science | Production and use of surface active agents |
| CN103954726A (en) * | 2014-05-06 | 2014-07-30 | 山东师范大学 | Method for extracting muramic acid from corynebacterium glutamicum dregs and detection method |
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2016
- 2016-07-18 CN CN201610565555.XA patent/CN106279308B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5215894A (en) * | 1975-07-29 | 1977-02-05 | Yuichi Yamamura | Process for preparing a substance possessing biological actions |
| WO2006027561A1 (en) * | 2004-09-06 | 2006-03-16 | The Scottish Association For Marine Science | Production and use of surface active agents |
| CN103954726A (en) * | 2014-05-06 | 2014-07-30 | 山东师范大学 | Method for extracting muramic acid from corynebacterium glutamicum dregs and detection method |
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