CN106248599A - A kind of detection method of fungus polysaccharide - Google Patents
A kind of detection method of fungus polysaccharide Download PDFInfo
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- CN106248599A CN106248599A CN201610780376.8A CN201610780376A CN106248599A CN 106248599 A CN106248599 A CN 106248599A CN 201610780376 A CN201610780376 A CN 201610780376A CN 106248599 A CN106248599 A CN 106248599A
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Abstract
The detection method of a kind of fungus polysaccharide disclosed by the invention, first sample preparation obtains sample；Again with glucose solution as standard substance, preparing standard curve with sulphuric acid anthrone or Phenol-sulphate acid method, in standard curve, absorbance is vertical coordinate, and concentration is abscissa；Again with sulphuric acid anthrone or Phenol-sulphate acid method colour developing, use spectrophotometric determination absorbance, draw the concentration of anhydrous glucose the sample of correspondence according to the absorbance measured from standard curve；Polyoses content during computing obtains sample again；The present invention is easy and simple to handle, save the time, and detecting instrument requires low, can be all kinds of testing agency, manufacturer, the client test problems that solves to run on quality control, prevent the ginseng false pain product harm to market.
Description
Technical field
The invention belongs to health food or field of food, relate to the detection method of a kind of polysaccharide, be specifically related to a kind of fungus
The detection method of polysaccharide.
Technical background
The research of glucide has had the history of centuries, but for a long time, and people are to the understanding of glucide only
It is only a kind of energy substance or structural material, in-depth study is not had for its physiologically active.Along with the development of life sciences, sugar
The effect in high-level biosis of the class material increasingly comes into one's own, and the research of glucide has become the new burnt of scholars
Point.In recent years, Polyose extraction industry obtains high speed development, particularly in China, extracts with natural plant polyose and fungus polysaccharide
Polyose extraction industry for representing is in rank first.Polysaccharide extracting process obtains considerable sending out in the improvement of more than ten years
Exhibition, but the detection technique of polysaccharide never has the biggest progress.The most all do not have a kind of unified detection method can be the most accurate
Detect the content of polysaccharide in different extract, or have the content of the false phenomenon polysaccharide of ginseng.Trace it to its cause, be on the one hand various
Different material Different Extraction Method causes various polysaccharide properties different really, it is difficult to simple same process detects；Another
Aspect is also that the research to polysaccharide detection method is inadequate.Domestic conventional polysaccharide detection method mainly has Phenol sulfuric acid procedure, sulfur
Acid anthrone method, direct titrimetric method, 3,5-dinitrosalicylic acid system (DNS method), Orcinol method, indirect iodometric processes, efficient liquid phase
Chromatography etc..Phenol-sulphate acid method is mainly used in measuring total sugar (including all monosaccharide and all kinds of polysaccharide), when carrying out polysaccharide determination
Want first precipitate polysaccharides, remove the interference of monosaccharide.Sulphuric acid anthrone method is mainly used in measuring hexose and the polysaccharide containing hexose.Orcinol
Rule is mainly used in measuring pentose and the polysaccharide containing pentose, and domestic application is less.DNS method is for the mensuration of content of reducing sugar.Directly
Connecing titration measuring result may be higher, because the polysaccharide such as starch have interference effect to it.Syrups by HPLC polysaccharide needs
The standard specimen of polysaccharide, cost is high, the most oversize.
Chinese invention special 201310547868.9 discloses a kind of detection method containing ganoderan preparation, it is characterized in that
By the crude polysaccharides of preparation being carried out precipitate with ethanol extraction, then develop the color with phenol sulphuric acid after purification with basic cupric sulfate, according to ultraviolet-visible
The ganoderan of the colorimetric method for determining preparation of spectrophotography.This method can take the oligosaccharide such as the dextrin shadow to detection
Ring, but the interference of high molecular weight polysaccharide cannot be got rid of.Chinese invention patent 201210397189.3 relates to a kind of mixed with saccharide
The detection method of polyoses content in the Lycium barbarum polysaccharide extract of chaff interference, by molten for the Lycium barbarum polysaccharide extract mixed with saccharide chaff interference
In Xie Yushui, it is subsequently adding phosphate buffer, amylase, then with precipitate with ethanol, the starch impact on testing result can be discharged.This
Method can discharge the interference of starch, but does not differentiate effect for other polysaccharide.
Summary of the invention
The technical problem to be solved is for the deficiencies in the prior art, it is provided that the detection side of a kind of fungus polysaccharide
Method, the method can accurately detect the content of fungus polysaccharide in fungus, and operation sequence is easy, rapidly.
In order to solve the problems referred to above, the detection method of a kind of fungus polysaccharide of the present invention, comprise the steps:
A, sample preparation
(1) the fungus polysaccharide sample weighing 0.1-1g is placed in 100ml or 250ml volumetric flask, adds water and is heated to 80 DEG C, super
Sound wave 30min makes fungus polysaccharide sample dissolve, then constant volume；
(2) take solution 5-10ml in step (1), put in centrifuge tube, add ethanol 100ml, standing 10-20min after shaking up, then with
The rotating speed of 4000r/min is centrifuged 10-20min, abandoning supernatant, is repeated 3 times, and is precipitated thing；
(3) taking precipitate, adds 85 DEG C of hot water dissolvings, is placed in beaker, precipitate: hot water is 1:1.35, by weight, and
Add lichen enzyme or cellulase 0.1ml, use immersion method water-bath 1h at a temperature of 40 DEG C to obtain mixed liquor, mixed liquor is taken
Go out and be heated to boiling, keeping boiling 10min, adjusting the temperature to 40 DEG C and add 0.1ml 1,4 beta-glucanase, then addition β-Portugal is gathered
The mixed liquor water-bath 1h post-heating of carbohydrase is to boiling, and keeps the 10min that seethes with excitement, then water bath method obtains sample semi-finished product, sample
The water content of semi-finished product is 10%；
(4) add sample semi-finished product in 5-10ml water dissolution beaker, then divide addition ethanol 70-75ml to add second with flow velocity 10ml/ again
Alcohol 70-75ml, stands 10-20min, is centrifuged 10-20min with the rotating speed of 4000r/minr, discard precipitation after shaking up, supernatant is used
Ethanol is settled to 25ml, obtains sample；
B, with glucose solution as standard substance, prepare standard curve with sulfuric acid-anthrone or sulfuric acid-phynol method, in standard curve
Absorbance is vertical coordinate, and concentration is abscissa；
C, taking 1-2ml sample and be evaporated, add water 2ml, develops the color with sulfuric acid-anthrone or sulfuric acid-phynol method, inhales with spectrophotometric determination
Luminosity, draws the concentration of anhydrous glucose the sample of correspondence according to the absorbance measured from standard curve；
D, it is calculated polyoses content in sample according to below equation；
。
The detection method of above-mentioned fungus polysaccharide, wherein, described in described step (1), the power of ultrasound wave is 380KW.
The detection method of above-mentioned fungus polysaccharide, wherein, the concentration quality hundred of ethanol in described step (1) and step (4)
Proportion by subtraction concentration is 95%.
Beneficial effects of the present invention:
The glucide pair that the present invention is purified by water for the first time when sample preparation and eliminates various monosaccharide, oligosaccharide equimolecular quantity is little
The interference of testing result, has good effect, then with having β-glycosidic bond for the glucose added, fructose, dextrin etc.
The polysaccharide extracted is decomposed by narrow spectrum enzyme, and monosaccharide is resolved in the fungus polysaccharide with β-glycosidic bond, and have α-
The starch of glycosidic bond etc. ginseng pseudocomponent cannot be decomposed, last precipitate with ethanol remove heteropolysaccharide, the monosaccharide determination of color obtained its
Content, then become glucosan with the coefficient conversion of 0.9, i.e. obtain the content of fungus polysaccharide in sample.
The present invention is easy and simple to handle, save the time, and detecting instrument requires low, can be all kinds of testing agency, manufacturer, client's solution
The test problems certainly run on quality control, prevents from joining the harm to market of the false pain product.
Detailed description of the invention
Embodiment 1
Mixed with the detection of Grifola frondosa polyoses content in the grifolan extract of dextrin:
A, sample preparation
(1) weigh the grifolan extract 0.2g mixed with dextrin, put in 100ml volumetric flask, add water and be heated to 80 DEG C, with super
Sound wave 30min makes to dissolve mixed with the grifolan extract of dextrin, then constant volume constant volume；
(2) take solution 5ml in step (1), put in centrifuge tube, add 95% ethanol 100ml, standing 10min after shaking up, then with
The rotating speed of 4000r/min is centrifuged 10min, abandoning supernatant, is repeated 3 times, and is precipitated thing；
(3) taking precipitate, adds 85 DEG C of hot water dissolvings, is placed in beaker, precipitate: hot water is 1:1.35, by weight, adds
Enter lichen enzyme 0.1ml, use immersion method water-bath 1h at a temperature of 40 DEG C to obtain mixed liquor, mixed liquor is taken out and is heated to boiling
Rise, keep boiling 10min, adjust the temperature to 40 DEG C and add 0.1ml 1,4 beta-glucanase, then the mixed liquor of 1,4 beta-glucanase will be added
Water-bath 1h post-heating is to boiling, and keeps the 10min that seethes with excitement, then water bath method obtains sample semi-finished product, sample semi-finished product aqueous
Amount is 10%；
(3) add semi-finished product in 5ml water dissolution beaker, then divide 95% ethanol 75ml with flow velocity 10ml/, after shaking up, stand 10min, with
The rotating speed of 4000r/minr is centrifuged 10-20min, discards precipitation, and supernatant ethanol is settled to 25ml, obtains sample；
B, with glucose solution as standard substance, prepare standard curve with sulfuric acid-anthrone or sulfuric acid-phynol method, in standard curve
Absorbance is vertical coordinate, and concentration is abscissa；Take anhydrous glucose to add water and make every lml containing the glucose solution of 0.12mg, with
Glucose solution is standard substance.Take glucose solution 0. 2,0. 4,0. 6,0.8,1.0,1.2ml, put 10ml tool plug respectively
In test tube, respectively add water to 2.0ml, add blue vitriol solution and (weigh ketone or phenol 0.lg, add sulphuric acid 100ml and make dissolving, shake
Even) 6ml, shake up, after placing 15 minutes, put in ice bath and cool down 5 minutes, take out, develop the color with sulfuric acid-anthrone or sulfuric acid-phynol method,
Measuring absorbance at 623nm wavelength, with absorbance as vertical coordinate, concentration is abscissa, obtains standard curve: y=
5.27206x-0.00566, R2=0.99895.Ice bath i.e. has outside plug test tube places the most a little containers loading ice again, rises cold
But act on.
C, taking 1ml sample and be evaporated, add water 2ml, develops the color with sulfuric acid-anthrone or sulfuric acid-phynol method, uses spectrophotometric determination
Absorbance, measuring absorbance is 0.2568, draws anhydrous grape the sample of correspondence according to the absorbance measured from standard curve
The concentration of sugar；Reading the content of anhydrous glucose in sample from standard curve is 0.0498mg/mL.
D, it is calculated polyoses content in sample according to below equation；It is calculated the ash mixed with dextrin according to below equation
In tree flower polyoses extract, Grifola frondosa polyoses content is 13.13%.
。
Embodiment 2 is mixed with the detection of ganoderma polyoses content in the ganoderan extract of starch
A, sample preparation
(1) weigh the ganoderan extract 0.2g mixed with starch, put in 250ml volumetric flask, add water and be heated to 80 DEG C, ultrasound wave
30min makes to dissolve mixed with the ganoderan extract of starch, then constant volume；
(2) take solution 5ml in step (1), put in centrifuge tube, add 95% ethanol 100ml, standing 10min after shaking up, then with
The rotating speed of 4000r/min is centrifuged 10-20min, abandoning supernatant, is repeated 3 times, and is precipitated thing；
(3) taking precipitate, adds 85 DEG C of hot water dissolvings, is placed in beaker, precipitate: hot water is 1:1.35, by weight, and
Add lichen enzyme or cellulase 0.1ml, use immersion method water-bath 1h at a temperature of 40 DEG C to obtain mixed liquor, mixed liquor is taken
Go out and be heated to boiling, keeping boiling 10min, adjusting the temperature to 40 DEG C and add 0.1ml 1,4 beta-glucanase, then addition β-Portugal is gathered
The mixed liquor water-bath 1h post-heating of carbohydrase is to boiling, and keeps the 10min that seethes with excitement, then water bath method obtains sample semi-finished product, sample
The water content of semi-finished product is 10%；
(4) add sample semi-finished product in 5ml water dissolution beaker, add 95% ethanol 75ml, stand 10min after shaking up, with 4000r/
The rotating speed of minr is centrifuged 10-20min, discards precipitation, and supernatant ethanol is settled to 25ml, obtains sample；
B, with glucose solution as standard substance, prepare standard curve with sulfuric acid-anthrone or sulfuric acid-phynol method, in standard curve
Absorbance is vertical coordinate, and concentration is abscissa；Take anhydrous glucose to add water and make every lml containing the glucose solution of 0.12mg, with
Glucose solution is standard substance.Take glucose solution 0. 2,0. 4,0. 6,0.8,1.0,1.2ml, put 10ml tool plug respectively
In test tube, respectively add water to 2.0ml, add blue vitriol solution and (weigh ketone or phenol 0.lg, add sulphuric acid 100ml and make dissolving, shake
Even) 6ml, shake up, after placing 15 minutes, put in ice bath and cool down 5 minutes, take out, develop the color with sulfuric acid-anthrone or sulfuric acid-phynol method,
Measuring absorbance at 623nm wavelength, with absorbance as vertical coordinate, concentration is abscissa, obtains standard curve: y=
5.27206x-0.00566, R2=0.99895.Ice bath i.e. has outside plug test tube places the most a little containers loading ice again, rises cold
But act on.
C, taking 1ml sample and be evaporated, add water 2ml, develops the color with sulfuric acid-anthrone or sulfuric acid-phynol method, uses spectrophotometric determination
Absorbance, measuring absorbance is 0.2849, draws anhydrous grape the sample of correspondence according to the absorbance measured from standard curve
The concentration of sugar；Reading the content of anhydrous glucose in sample from standard curve is 0.0551mg/mL.
Take above-mentioned test sample 1ml, be evaporated, add water to 2ml, from " the most accurate addition blue vitriol solution 6ml ", with
Method operates, and measuring absorbance is 0.2849, reads the content of anhydrous glucose in need testing solution and be from standard curve
0.0551mg/mL。
D, it is calculated polyoses content in sample according to below equation；It is calculated the ash mixed with dextrin according to below equation
In tree flower polyoses extract, Grifola frondosa polyoses content is 34.22%.
。
This detection method can differentiate adulterated and inferior fungus polysaccharide product conventional on the market.
Research shows, many fungus polysaccharides are mainly (1 → 3) glycosidic bond and connect configuration, and with a small amount of 1 → 6
The structure that chain is bonded, fungus polysaccharide is to be connected the poly-polysaccharide in Portugal by D-Glucose unit by β-(1 → 3) glycosidic bond, and it is main
Configuration characteristic is the framing structure of (1 → 3) β-D-linearly connected.
Cellulase (cellulase), EC 3.2.1.4, it is widely present in the organism of nature.Antibacterial, fungus,
Cellulase can be produced in animal body etc..The cellulase being generally used for producing comes from fungus, has Trichoderma spp. than more typical
Belong to (Trichoderma), aspergillus (Aspergillus) and Penicillium (Penicillium).Cellulase in food service industry and
Environmental industry is all widely used.When carrying out alcohol fermentation, the interpolation of cellulase can increase the utilization rate of raw material, and right
Vinosity has promoted.
Lichen enzyme (laminarinase), EC 3.2.1.6, EC 3.2.1.39.Specific can act on β type glucosides
Key, makes fungus polysaccharide be decomposed into monosaccharide.The use of two kinds of enzymes can decompose cell wall well to fungal cell wall generation effect.
Examples detailed above is the further description to the present invention, but does not means that any limitation of the invention.Not
In the case of departing from the above-mentioned thought of the present invention, the various replacement sides made according to ordinary skill knowledge and conventional means
Formula or change, within being all contained in the present invention.
Claims (3)
1. the detection method of a fungus polysaccharide, it is characterised in that comprise the steps:
A, sample preparation
(1) the fungus polysaccharide sample weighing 0.1-1g is placed in 100ml or 250ml volumetric flask, adds water and is heated to 80 DEG C, super
Sound wave 30min makes fungus polysaccharide sample dissolve, then constant volume；
(2) take solution 5-10ml in step (1), put in centrifuge tube, add ethanol 100ml, standing 10-20min after shaking up, then with
The rotating speed of 4000r/min is centrifuged 10-20min, abandoning supernatant, is repeated 3 times, and is precipitated thing；
(3) taking precipitate, adds 85 DEG C of hot water dissolvings, is placed in beaker, precipitate: hot water is 1:1.35, by weight, and
Add lichen enzyme or cellulase 0.1ml, use immersion method water-bath 1h at a temperature of 40 DEG C to obtain mixed liquor, mixed liquor is taken
Go out and be heated to boiling, keeping boiling 10min, adjusting the temperature to 40 DEG C and add 0.1ml 1,4 beta-glucanase, then addition β-Portugal is gathered
The mixed liquor water-bath 1h post-heating of carbohydrase is to boiling, and keeps the 10min that seethes with excitement, then water bath method obtains sample semi-finished product, sample
The water content of semi-finished product is 10%；
(4) add sample semi-finished product in 5-10ml water dissolution beaker, then divide addition ethanol 70-75ml with flow velocity 10ml/, quiet after shaking up
Putting 10-20min, be centrifuged 10-20min with the rotating speed of 4000r/minr, discard precipitation, supernatant ethanol is settled to 25ml,
To sample；
B, with glucose solution as standard substance, prepare standard curve with sulfuric acid-anthrone or sulfuric acid-phynol method, in standard curve
Absorbance is vertical coordinate, and concentration is abscissa；
C, taking 1-2ml sample and be evaporated, add water 2ml, develops the color with sulfuric acid-anthrone or sulfuric acid-phynol method, inhales with spectrophotometric determination
Luminosity, draws the concentration of anhydrous glucose the sample of correspondence according to the absorbance measured from standard curve；
D, it is calculated polyoses content in sample according to below equation；
。
2. the detection method of fungus polysaccharide as claimed in claim 1, it is characterised in that ultrasound wave described in described step (1)
Power be 380Kw.
3. the detection method of fungus polysaccharide as claimed in claim 1, wherein, in described step (1) and step (4), ethanol is dense
Degree mass percent concentration is 95%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107132149A (en) * | 2017-04-12 | 2017-09-05 | 广西大学 | A kind of method of quick specific detection curdlan content |
| CN112485421A (en) * | 2020-11-09 | 2021-03-12 | 江苏江大源生态生物科技股份有限公司 | Test method for propolis anti-tumor effect |
| CN114166771A (en) * | 2021-11-25 | 2022-03-11 | 仙芝科技(福建)股份有限公司 | Method for determining content of beta-glucan in ganoderma lucidum |
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| CN114166771A (en) * | 2021-11-25 | 2022-03-11 | 仙芝科技(福建)股份有限公司 | Method for determining content of beta-glucan in ganoderma lucidum |
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