CN1220876C - Method for detecting active polysaccharide content in ash tree flower polysaccharide - Google Patents
Method for detecting active polysaccharide content in ash tree flower polysaccharide Download PDFInfo
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- CN1220876C CN1220876C CN 03138964 CN03138964A CN1220876C CN 1220876 C CN1220876 C CN 1220876C CN 03138964 CN03138964 CN 03138964 CN 03138964 A CN03138964 A CN 03138964A CN 1220876 C CN1220876 C CN 1220876C
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Abstract
The present invention discloses a method for measuring the content of water soluble polysaccharide with biological activity in a product of grifola frondosa polysaccharide, which comprises the steps: fatsoluble components are removed; soluble polysaccharide interfering substances are removed; a sulphuric acid-phenol method is used for measuring the content of active polysaccharide, etc. The method of the present invention has the advantages of strong operability, stable and reliable measuring result, small variation coefficient of the measuring result, small error, high reproduction, etc.
Description
(1) technical field
The present invention relates to the assay method of grifolan extract, relate in particular to the method for quantitatively determining of the water-soluble polysaccharide content of biologically active in the grifola frondosus deep fermentation liquid polyoses extract.
(2) background technology
Active component polysaccharides and non-active ingredient starch have mainly been comprised in the grifola frondosus deep fermentation liquid polyoses extract; Wherein, the quantitative measurement of active component polysaccharides content determines it is very necessary for the quality control of grifola frondosus active polysaccharide product and polysaccharide product producing process.Yet, owing to the existence of non-active ingredient starch in the biological polyoses extract is difficult to carry out the control of target level of product quality.Still do not have desirable conventional method of analysis at present about active component polysaccharides Determination on content in the grifolan, just adopt " assay method of GB/T 15672-1995 edible fungi total sugar content " to measure; The national standard that does not have grifolan to measure at present used method does not have the assay method of edible fungi or plant polyose can supply to use for reference yet.Therefore, the mensuration of bioactive polysaccharide in the grifolan product all is the assay method of continuing to use crude polysaccharides at present.In the crude polysaccharides measurement result, in grifolan the content of the water-soluble polysaccharide of biologically active, also comprised non-active ingredient polysaccharide-contents of starch in the grifolan, thereby measurement result can't be applied to check, the supervision of product quality, makes that the accurate detection of the content of grifola frondosus active polysaccharide has produced very big uncertainty in the quality control, production run of grifola frondosus active polysaccharide product.Therefore, the quantitative measurement of active component polysaccharides content has become key technical problem in its product development, production and the quality control in the grifola frondosus active polysaccharide product.And how further existing method " assay method of GB/T 15672-1995 edible fungi total sugar content " existing deficiency and improvement and perfect have become institute's problem demanding prompt solution in the method for quantitatively determining of active component polysaccharides content in the present grifolan product.
(3) summary of the invention
Purpose of the present invention promptly is at the existing deficiency of active component polysaccharides content assaying method in the above-mentioned grifolan product, and the method for active component polysaccharides content in a kind of accurate mensuration grifolan product is provided.The water-soluble polysaccharide Determination on content method of biologically active in the grifolan product involved in the present invention, have workable, measurement result accurately, stable, reliable, error is little, reappearance is high, advantage such as can measure at general Routine Test Lab.
The assay method of the water-soluble polysaccharide of biologically active in the grifolan product of the present invention, form by following steps:
1) removes liposoluble constituent: get the polysaccharide sample to be measured that oven dry grinds, divide another name 0.5~2 to restrain in 6 Centrifuge Cups, add the 30ml ether respectively, fully vibration is 5~15 minutes, centrifugal 5~10 minutes with 4000~5000 rev/mins, abandoning supernatant adds the 30ml ether again, and so repeated centrifugation is 3 times;
2) remove the soluble saccharide interfering material: the 80% ethanol 30ml that adds 50~60 ℃ in the Centrifuge Cup after above-mentioned abandoning supernatant, fully vibration is 5~15 minutes, with 4000~5000 rev/mins centrifugal 5~10 minutes, abandoning supernatant, so repeated centrifugation is 3 times; And then add 40~50 ℃ of distilled water 30ml in the cup after centrifugal, vibration is 60 minutes in 40~50 ℃ of waters bath with thermostatic control, with 4000~5000 rev/mins centrifugal 5~10 minutes, collect supernatant, with the method repeated centrifugation 3 times, supernatant all is collected in the volumetric flask again; Be settled to 100~250ml, as the special solution of surveying of active polysaccharide;
3) sulfuric acid-phynol method is measured active polysaccharide content: draw polysaccharide solution 5~25ml to be measured, with distilled water diluting to 50~300ml, draw this dilution and decide solution 1~2ml, be added on respectively in 6 25ml color comparison tubes, add successively distilled water mend to cumulative volume be 2.0ml, add 5% phenol 1.0ml more respectively, concentrated sulphuric acid 5.0ml, put into boiling water bath then rapidly 12~18 minutes, be cooled to 25~30 ℃ more rapidly, be settled to 10ml with distilled water, shake up, the cooling back is a reference with the blank sample, and under the 485nm wavelength, the 1cm cuvette is measured optical density, draw the quality of polysaccharide in the liquid to be measured according to typical curve, the Mass Calculation by polysaccharide in the solution to be measured goes out active polysaccharide content in the grifola frondosus polyose then.
Wherein, above-mentioned steps 1) or 2) in add that duration of oscillation is 8~12 minutes behind ether or the ethanol.
Wherein, above-mentioned steps 2) temperature of described 80% ethanol is 55~58 ℃.
Wherein, above-mentioned steps 2) distilled water of described adding and the temperature of water bath with thermostatic control be 45~50 ℃.
Wherein, above-mentioned steps 3) consumption of described polysaccharide solution to be measured is 5~15ml.
Wherein, above-mentioned steps 3) final volume of described polysaccharide solution to be measured after with distilled water diluting be 50~250ml.
Wherein, above-mentioned steps 3) the described processing time in boiling water bath is 15 minutes.
The water-soluble polysaccharide Determination on content method of biologically active in the grifolan product of the present invention, have workable, measurement result is reliable and stable, the coefficient of variation of measurement result is little, error is little, the reappearance advantages of higher.Experiment shows, per sample in the content difference of water-soluble polysaccharide, the coefficient of variation is generally in 5%~10% scope.Can satisfy Routine Test Lab to the requirement of analytical test and relevant enterprise to the controllable quality requirement.
The present invention is further illustrated below in conjunction with embodiment.
(4) embodiment
Embodiment 1:
1) removes liposoluble constituent: get the active polysaccharide sample to be measured (being numbered No. 1) that oven dry grinds, quality is respectively 1.0727 grams, 1.1028 grams, 1.1509 grams, 0.948 gram, 1.1813 grams, 1.0481 grams, be put in respectively in 6 Centrifuge Cups, add the 30ml ether respectively, fully vibration is 12 minutes, with 4500 rev/mins centrifugal 8 minutes, abandoning supernatant, add the 30ml ether again, so repeated centrifugation is 3 times;
2) remove the soluble saccharide interfering material: add 58 ℃ 80% ethanol 30ml in the Centrifuge Cup after above-mentioned abandoning supernatant, fully vibrated 15 minutes, with 5000 rev/mins centrifugal 10 minutes, abandoning supernatant, so repeated centrifugation is 3 times; And then add 50 ℃ of distilled water 30ml in the Centrifuge Cup after abandoning supernatant, vibration is 60 minutes in 50 ℃ of waters bath with thermostatic control, with 5000 rev/mins centrifugal 5 minutes, collect supernatant, with the method repeated centrifugation 3 times, supernatant all is collected in the volumetric flask again; Be settled to 100ml, as active polysaccharide solution (V to be measured
1);
3) sulfuric acid-phynol method is measured active polysaccharide content:
3.1 the drafting of typical curve:
3.1.1 the configuration of glucosan standard reserving solution: the glucosan 0.5000g that accurately take by weighing relative molecular mass T500 (500,000 unit), in baking oven, has dried to constant weight, be settled to 50ml behind the dissolved in distilled water, mixing is placed in the refrigerator standby.
3.1.2 the glucosan standard is used the configuration of liquid: accurately draw glucosan standard reserving solution 1.0ml in the 100ml volumetric flask, be diluted to scale, mixing is placed in the refrigerator standby.
3.1.3 the drafting of typical curve: accurately draw the glucosan standard respectively and use liquid 0ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml in 6 25ml color comparison tubes, add successively distilled water mend to cumulative volume be 2.0ml, add 5% phenol 1.0ml more respectively, concentrated sulphuric acid 5.0ml, put into boiling water bath 15min then rapidly, be cooled to 27 ℃ more rapidly.Be settled to 10ml with distilled water, shake up, cool off the back and under the 485nm wavelength, measure optical density.With the standard blank is reference, and optical density is an ordinate, and glucosan concentration is horizontal ordinate, the drawing standard curve.
3.2 active polysaccharide Determination on content:
Draw polysaccharide solution 5ml to be measured (V
2), with distilled water diluting to 50ml (V
3), draw this dilution and decide solution 2ml (V
4), be added on respectively in 6 25ml color comparison tubes, add successively distilled water mend to cumulative volume be 2.0ml, add 5% phenol 1.0ml more respectively, concentrated sulphuric acid 5.0ml puts into boiling water bath 15min then rapidly, is cooled to 25 ℃ more rapidly, is settled to 10ml (V with distilled water
5), shaking up, cool off the back is reference with the blank sample, under the 485nm wavelength, the 1cm cuvette is measured optical density, draws the quality (A) of polysaccharide in the liquid to be measured according to typical curve.
Do the reagent blank test by Same Way simultaneously.
3.3 the result calculates: active polysaccharide %=(A-A
0)/m*n*100
In the formula: active polysaccharide content is in glucosan,
A---survey the quality of polysaccharide in the liquid, μ g;
A
0---the quality of polysaccharide in the blank solution product liquid, μ g;
M---sample quality, μ g;
N---extension rate.Among the embodiment n=100/5*50/2*10=5000
Grifola frondosus active polysaccharide Determination on content embodiment analysis result sees attached list 1:
Subordinate list 1: grifola frondosus active polysaccharide Determination on content embodiment analysis result | |||||||||
Embodiment 1 (C=1mg/ml) | |||||||||
Sample number into spectrum | Repeat | m(g) | V 1 (ml) | V 2 (ml) | V 3 (ml) | V 4 (ml) | V 5 (ml) | A-A 0 (μg) | Active polysaccharide % |
No. 1 (grifola frondosus crude polysaccharides 2N) | 1 | 1.0727 | 100 | 5 | 50 | 2 | 10 | 49.04 | 22.86 |
2 | 1.1028 | 100 | 5 | 50 | 2 | 10 | 48.16 | 21.84 | |
3 | 1.1509 | 100 | 5 | 50 | 2 | 10 | 48.52 | 21.08 | |
4 | 0.948 | 100 | 5 | 50 | 2 | 10 | 49.00 | 25.84 | |
5 | 1.1813 | 100 | 5 | 50 | 2 | 10 | 48.51 | 20.53 | |
6 | 1.0481 | 100 | 5 | 50 | 2 | 10 | 47.52 | 22.67 | |
On average | 22.47 | ||||||||
Standard deviation STDEV | 1.880 | ||||||||
Coefficient of variation CV% | 8.365 | ||||||||
Mean standard deviation | 0.7674 | ||||||||
Average ± mean standard deviation | 22.47±0.77 |
Embodiment 2:
1) removes liposoluble constituent: get the active polysaccharide sample to be measured (being numbered No. 2) that oven dry grinds, quality is respectively 0.9368 gram, 0.9465 gram, 0.9254 gram, 1.0136 grams, 0.8831 gram, 0.8177 gram, be put in respectively in 6 Centrifuge Cups, add the 30ml ether respectively, fully vibration is 8 minutes, with 5000 rev/mins centrifugal 10 minutes, abandoning supernatant, add the 30ml ether again, so repeated centrifugation is 3 times;
2) remove the soluble saccharide interfering material: add 55 ℃ 80% ethanol 30ml in the Centrifuge Cup after above-mentioned abandoning supernatant, fully vibrated 13 minutes, with 4000 rev/mins centrifugal 10 minutes, abandoning supernatant, so repeated centrifugation is 3 times; And then add 40 ℃ of distilled water 30ml in the Centrifuge Cup after abandoning supernatant, vibration is 60 minutes in 45 ℃ of waters bath with thermostatic control, with 5000 rev/mins centrifugal 5 minutes, collect supernatant, with the method repeated centrifugation 3 times, supernatant all is collected in the volumetric flask again; Be settled to 250ml, as active polysaccharide solution (V to be measured
1);
3) sulfuric acid-phynol method is measured active polysaccharide content:
3.1 the drafting of typical curve:
3.1.1 the configuration of glucosan standard reserving solution: the glucosan 0.5000g that accurately take by weighing relative molecular mass T500 (500,000 unit), in baking oven, has dried to constant weight, be settled to 50ml behind the dissolved in distilled water, mixing is placed in the refrigerator standby.
3.1.2 the glucosan standard is used the configuration of liquid: accurately draw glucosan standard reserving solution 1.0ml in the 100ml volumetric flask, be diluted to scale, mixing is placed in the refrigerator standby.
3.1.3 the drafting of typical curve: accurately draw the glucosan standard respectively and use liquid 0ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml in 6 25ml color comparison tubes, add successively distilled water mend to cumulative volume be 2.0ml, add 5% phenol 1.0ml more respectively, concentrated sulphuric acid 5.0ml, put into boiling water bath 15min then rapidly, be cooled to 27 ℃ more rapidly.Be settled to 10ml with distilled water, shake up, cool off the back and under the 485nm wavelength, measure optical density.With the standard blank is reference, and optical density is an ordinate, and glucosan concentration is horizontal ordinate, the drawing standard curve.
3.2 active polysaccharide Determination on content:
Draw polysaccharide solution 15ml to be measured (V
2), with distilled water diluting to 50ml (V
3), draw this dilution and decide solution 1ml (V
4), be added on respectively in 6 25ml color comparison tubes, add successively distilled water mend to cumulative volume be 2.0ml, add 5% phenol 1.0ml more respectively, concentrated sulphuric acid 5.0ml puts into boiling water bath 15min then rapidly, is cooled to 25 ℃ more rapidly, is settled to 10ml (V with distilled water
5), shaking up, cool off the back is reference with the blank sample, under the 485nm wavelength, the 1cm cuvette is measured optical density, draws the quality (A) of polysaccharide in the liquid to be measured according to typical curve.
Do the reagent blank test by Same Way simultaneously.
3.3 the result calculates: active polysaccharide %=(A-A
0)/m*n*100
In the formula: active polysaccharide content is in glucosan,
A---survey the quality of polysaccharide in the liquid, μ g;
A
0---the quality of polysaccharide in the blank solution product liquid, μ g;
M---sample quality, μ g;
N---extension rate.Among the embodiment n=100/5*50/2*10=5000
Grifola frondosus active polysaccharide Determination on content embodiment analysis result sees attached list 2:
Subordinate list 2: grifola frondosus active polysaccharide Determination on content embodiment analysis result | |||||||||
Embodiment 2 (C=1mg/ml) | |||||||||
Sample number into spectrum | Repeat | m(g) | V 1 (ml) | V 2 (ml) | V 3 (ml) | V 4 (ml) | V 5 (ml) | A-A 0 (μg) | Active polysaccharide % |
No. 2 (grifolan) | 1 | 0.9368 | 250 | 15 | 50 | 1 | 10 | 37.77 | 10.08 |
2 | 0.9465 | 250 | 15 | 50 | 1 | 10 | 39.2 | 10.35 | |
3 | 0.9254 | 250 | 15 | 50 | 1 | 10 | 38.98 | 10.53 | |
4 | 1.0136 | 250 | 15 | 50 | 1 | 10 | 41.8 | 10.31 | |
5 | 0.8831 | 250 | 15 | 50 | 1 | 10 | 37.79 | 10.70 | |
6 | 0.8177 | 250 | 15 | 50 | 1 | 10 | 35.34 | 10.80 | |
On average | 10.46 | ||||||||
Standard deviation STDEV | 0.268 | ||||||||
Coefficient of variation CV% | 2.561 | ||||||||
Mean standard deviation | 0.1094 | ||||||||
Average ± mean standard deviation | 10.46±0.11 |
Claims (7)
1. active polysaccharide Determination on content method in the grifolan product, form by following steps:
1) removes liposoluble constituent: get the polysaccharide sample to be measured that oven dry grinds, divide another name 0.5~2 to restrain in 6 Centrifuge Cups, add the 30ml ether respectively, fully vibration is 5~15 minutes, centrifugal 5~10 minutes with 4000~5000 rev/mins, abandoning supernatant adds the 30ml ether again, and so repeated centrifugation is 3 times;
2) remove the soluble saccharide interfering material: the 80% ethanol 30ml that adds 50~60 ℃ in the Centrifuge Cup after above-mentioned abandoning supernatant, fully vibration is 5~15 minutes, with 4000~5000 rev/mins centrifugal 5~10 minutes, abandoning supernatant, so repeated centrifugation is 3 times; And then add 40~50 ℃ of distilled water 30ml in the Centrifuge Cup after abandoning supernatant, vibration is 60 minutes in 40~50 ℃ of waters bath with thermostatic control, with 4000~5000 rev/mins centrifugal 5~10 minutes, collect supernatant, with the method repeated centrifugation 3 times, supernatant all is collected in the volumetric flask again; Be settled to 100~250ml, as active polysaccharide solution to be measured;
3) sulfuric acid-phynol method is measured active polysaccharide content: draw polysaccharide solution 5~25ml to be measured, with distilled water diluting to 50~300ml, draw this dilution and decide solution 1~2ml, be added on respectively in 6 25ml color comparison tubes, add successively distilled water mend to cumulative volume be 2.0ml, add 5% phenol 1.0ml more respectively, concentrated sulphuric acid 5.0ml, put into boiling water bath then rapidly 12~18 minutes, be cooled to 25~30 ℃ more rapidly, be settled to 10ml with distilled water, shake up, the cooling back is a reference with the blank sample, and under the 485nm wavelength, the 1cm cuvette is measured optical density, draw the quality of polysaccharide in the liquid to be measured according to typical curve, the Mass Calculation by polysaccharide in the solution to be measured goes out active polysaccharide content in the grifola frondosus polyose then.
2. active polysaccharide Determination on content method is characterized in that in the grifolan product as claimed in claim 1, step 1) or 2) in add that duration of oscillation is 8~12 minutes behind ether or the ethanol.
3. active polysaccharide Determination on content method is characterized in that step 2 in the grifolan product as claimed in claim 1) temperature of described 80% ethanol is 55~58 ℃.
4. active polysaccharide Determination on content method is characterized in that step 2 in the grifolan product as claimed in claim 1) distilled water of described adding and the temperature of water bath with thermostatic control be 45~50 ℃.
5. active polysaccharide Determination on content method is characterized in that in the grifolan product as claimed in claim 1, and the consumption of the described polysaccharide of step 3) solution to be measured is 5~15ml.
6. active polysaccharide Determination on content method is characterized in that in the grifolan product as claimed in claim 1, and the final volume of the described polysaccharide of step 3) solution to be measured after with distilled water diluting is 50~250ml.
7. active polysaccharide Determination on content method is characterized in that in the grifolan product as claimed in claim 1, and the described processing time in boiling water bath of step 3) is 15 minutes.
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CN100567956C (en) * | 2005-11-30 | 2009-12-09 | 广东省农业科学院农业生物技术研究所 | A kind of lichee polysaccharide Determination on content method |
CN101441202B (en) * | 2008-12-29 | 2011-08-10 | 河南工业大学 | Method for detecting fungal polysaccharide |
CN101532949B (en) * | 2009-04-23 | 2011-04-13 | 云南省轻工业科学研究所 | Method for real-time detection of micro total sugar in water body |
CN102252978B (en) * | 2010-12-24 | 2016-03-30 | 天津天狮生物发展有限公司 | The assay method of Thick many candies content in a kind of cordyceps mycelia |
CN102519891A (en) * | 2011-12-06 | 2012-06-27 | 上海景峰制药有限公司 | Method for detecting residual sugar in sodium hyaluronate fermentation broth |
CN102788834B (en) * | 2012-08-29 | 2015-06-03 | 浙江农林大学 | Method for quickly measuring soluble sugar in fruit |
CN109374549A (en) * | 2018-11-14 | 2019-02-22 | 遵义市精科信检测有限公司 | A kind of detection method of function of health food composition |
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