CN103901031A - Method for rapid and high-flux determination of polysaccharide content based on sulfuric acid and phenol - Google Patents
Method for rapid and high-flux determination of polysaccharide content based on sulfuric acid and phenol Download PDFInfo
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Abstract
The invention discloses a method for rapid and high-flux determination of polysaccharide content based on sulfuric acid and phenol. The method is characterized by comprising the following steps: simultaneously adding polysaccharide determination liquid and concentrated sulfuric acid into a micro-pore plate; hydrolyzing a polysaccharide into a monosaccharide and rapidly dehydrating to form a furfural derivative; continually adding phenol into the micro-pore plate and enabling phenol and the furfural derivative to react to generate orange-yellow liquid, wherein the reaction temperature is 90 DEG C and the time is 23 minutes; after the reaction, carrying out cooling treatment for 5 minutes; determining the absorbance value of the polysaccharide in a micro-plate reader under 490nm; taking a glucose solution as a standard solution and drawing a standard curve; substituting the absorbance value of the polysaccharide into the standard curve to calculate the content of the polysaccharide. The method has the beneficial effects that the method is simple, rapid, flexible and good in repeatability; the color is lasting and one standard curve is drawn for each type of sugar; with regard to a heteropolysaccharide, the total amount can be measured one time under the condition of no need of exquisitely dividing each type of the monosaccharide; the method can be used for determining methylated sugar, pentose, oligosaccharides and the polysaccharides.
Description
Technical field
The present invention relates to a kind of measurement of the polysaccharide content method, be specifically related to a kind of method of measuring polyoses content based on phenol sulfuric acid fast high-flux, belong to technical field of analytical chemistry.
Background technology
Sugar is the important component that forms biological cell tissue, is one of material base of life.Sugar and Nutrition and Metabolism, eucaryotic cell structure, antiviral, to strengthen immunity, substance operation etc. closely related, and human body must absorb enough sugar to maintain the normal operation of every physiological function from food.Therefore, sugared content is one of most important nutritive index of many food.
The method of determination of polysaccharide has a lot:
One class is to utilize the reductibility of polysaccharide, measures reductibility polysaccharide, and method has: 3,5-dinitro-salicylate (DNS) colourimetry and Somogyi-Nelson method.
Another kind of assay method, also be the most general method of applying at present, furfural condensation development process, it mainly utilizes polysaccharide to dewater under strong acidic condition and generates furfural or derivatives thereof, then with phenols (or amine) compound condensation, generate and have this character of coloring matter of specific absorption wavelength to measure.These class methods have orcin-sulfuric acid (hydrochloric acid) method, phenolsulfuric acid method and By Anthrone Sulphuric acid method.
As can be seen here, tradition survey polysaccharide method is carried out in color comparison tube, need to add a large amount of glucose standard solution and phenol reagent, and not only detection efficiency is low, reagent loss is many, error is large, and environmental pollution is also larger.
Summary of the invention
For solving the deficiencies in the prior art, the object of the present invention is to provide a kind of based on phenol sulfuric acid, can realize fast, the method for high throughput assay polyoses content.
In order to realize above-mentioned target, the present invention adopts following technical scheme:
A method of measuring polyoses content based on phenol sulfuric acid fast high-flux, is characterized in that, comprises the following steps:
(1) polysaccharide determination liquid and the concentrated sulphuric acid are added in microwell plate simultaneously, under the effect of the concentrated sulphuric acid, polysaccharide hydrolysis becomes monose and dewaters rapidly to form furfural derivatives;
(2) in microwell plate, continue to add phenol, phenol reacts with furfural derivatives and generates orange-yellow liquid;
(3) in microplate reader, under 490nm, measure the absorbance value of polysaccharide;
(4) taking glucose solution as titer, drawing standard curve;
(5) absorbance value of polysaccharide is brought into and in typical curve, calculated polyoses content.
The aforesaid method of measuring polyoses content based on phenol sulfuric acid fast high-flux, is characterized in that, the concentration of the aforementioned concentrated sulphuric acid is 98%, and the concentration of aforementioned phenol is 4.5%.
The aforesaid method of measuring polyoses content based on phenol sulfuric acid fast high-flux, is characterized in that, the volume ratio of aforementioned polysaccharide determination liquid, the concentrated sulphuric acid, phenol is 5:15:3.
Aforesaidly measure the method for polyoses content based on phenol sulfuric acid fast high-flux, it is characterized in that, in step (2), the temperature that phenol reacts with furfural derivatives is that 90 DEG C, reaction time are 23min, carries out 5min cooling processing after having reacted again.
Usefulness of the present invention is: the principle that the present invention is based on the quantitative carbohydrates of phenol sulfuric acid, polysaccharide is under the effect of the concentrated sulphuric acid, first be hydrolyzed into monose and dewater rapidly and form furfural derivatives, then phenol generates orange-yellow chemicals with furfural derivatives again, within the specific limits, the depth of color is directly proportional to sugared content, can be for the mensuration of methylated sugar, pentose and oligosaccharide kind and polysaccharide, and method is simple, quick, sensitive, reproducible, color is lasting, and every kind of sugar is only made to a typical curve; For heteroglycan, analysis result can be according to the when correction coefficient correction calculation in addition of the typical curve of key component monose of the composition of each monose, in the situation that there is no need the various monose of careful division, adopt detection method of the present invention can once measure total amount, save many troubles, there is special using value; Have quick, high-throughout characteristic, in microwell plate, carry out, addition many due to micropore plate hole and that need is little, so can measure the polyoses content of multiple samples simultaneously.
Brief description of the drawings
Fig. 1 is the affect figure of phenol concentration on accuracy of measurement;
Fig. 2 is the affect figure of reaction time on accuracy of measurement;
Fig. 3 is the affect figure of temperature of reaction on accuracy of measurement;
Fig. 4 is phenol concentration and the affect figure of reaction time reciprocation on accuracy;
Fig. 5 is phenol concentration and the affect figure of temperature of reaction reciprocation on accuracy;
Fig. 6 is the glucose canonical plotting based under Microdilution plate method;
Fig. 7 is the glucose canonical plotting based under test tube colourimetry.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is done to concrete introduction.
400 μ g/mL glucose solutions: accurately take 0.0400g anhydrous dextrose and be dissolved in distilled water, be settled to 100mL.
The concentrated sulphuric acid: concentration is 98%.
4.5% phenol: accurately take 0.4500g phenol and be dissolved in distilled water, be settled to 10mL.
Drawing standard curve
Using 400 μ g/mL glucose solutions as standard solution.
Detailed operating process is:
(1), in 96 hole ELISA Plate, 5 groups of parallel application of samples, get glucose mother liquid 0,5,10,15,20,25,30,35,40,45,50 μ L for every group respectively, add and reenter deionized water and supply 50 μ L, in each like this micropore, the concentration of glucose is respectively 0,40,80,120,160,200,240,280,320,360,400 μ g/mL.
(2), add the concentrated sulphuric acid (concentration is 98%) 150 μ L, phenol reagent (concentration is 4.5%) 30 μ L, after mixing, in 90 DEG C of water-baths, hatch 23min, after heating, be cooled to rapidly room temperature, after 5min by microplate reader at 490nm place mensuration absorbance value.
The glucose solution of each concentration repeats to read absorbance value 5 times, gets the average of five parallel absorbance values, can reject at the most two and differ larger data with mean value under individual cases.The data that microplate reader is measured are in table 1
The tables of data that table 1 microplate reader is measured
(3), taking glucose solution concentration as horizontal ordinate, absorbance value is ordinate drawing standard curve, as shown in Figure 6, the equation of this typical curve is Y=0.00281X+0.00989, R
2=0.9992, RSD=0.0169, wherein x represents the concentration (μ g/mL) of glucose, Y represents absorbance value.
In addition, as shown in Figure 6, in the time that phenol reagent concentration is 4.5%, concentration of glucose gradient is within the scope of 0-400 μ g/mL, and significantly (linear equation slope value is larger) of absorbance value gradient disparities at 490nm place, linear relationship is good.
The R of the mark song of making
2rSD value between value and parallel value is as the standard of weighing result:
(1) R
2larger, the correlativity of the point on typical curve is better, and the accuracy of experimental result is higher; Contrary R
2less, represent in experimental implementation that error is large or method design itself is unreasonable, thus the good method of designing, its R
2must greatly and get over close to 1.
(2) RSD value representation each otherness between parallel, each condition of each parallel reactor is the same, but there is the method poor stability that difference shows there is systematic error or sets up in result, a good method must be stable under identical condition determination, RSD is larger, method is more unstable, therefore can be used as an index of corresponding analysis.
The table song that adopts method of the present invention to draw out, its R
2value=0.9992, RSD=0.0169, as can be seen here, adopts the accuracy of the experimental result that method of the present invention obtains higher, stability is better.
Fig. 1 is the affect figure of phenol concentration on accuracy of measurement.
The bent corresponding table of mark under the different phenol concentration of table 2
| Phenol concentration | Typical curve | R 2 |
[0048]?
| 3.0% | y=0.0081x+0.0908 | 0.9323 |
| 3.5% | y=0.0066x+0.0660 | 0.9767 |
| 4.0% | y=0.0068x+0.0650 | 0.9534 |
| 4.5% | y=0.0092x+0.0522 | 0.9897 |
| 5.0% | y=0.0095x+0.0122 | 0.9940 |
| 5.5% | y=0.0084x+0.0698 | 0.9839 |
| 6.0% | y=0.0096x+0.0260 | 0.9935 |
As can be seen here, when phenol concentration is 5%, linearly dependent coefficient maximum, accuracy is best.
Fig. 2 is the affect figure of reaction time on accuracy of measurement.
The bent corresponding table of mark under the table 3 differential responses time
| Reaction time | Typical curve | R 2 |
| 5min | y=0.0024x+0.0471 | 0.6363 |
| 10min | y=0.0038x+0.0483 | 0.8691 |
| 15min | y=0.0058x+0.0395 | 0.9311 |
| 20min | y=0.0080x+0.0365 | 0.9907 |
| 25min | y=0.0101x+0.0420 | 0.9838 |
| 30min | y=0.0111x+0.0484 | 0.9872 |
As can be seen here, the reaction time is linearly dependent coefficient maximum in the time of 20min, and therefore selecting the reaction time is 20min the best.
Fig. 3 is the affect figure of temperature of reaction on accuracy of measurement.
The bent corresponding table of mark at table 4 differential responses temperature
| Temperature of reaction | Typical curve | R 2 |
[0057]?
| 80℃ | y=0.0063x+0.0465 | 0.9764 |
| 85℃ | y=0.0095x+0.0122 | 0.9930 |
| 90℃ | y=0.0080x+0.0365 | 0.9907 |
| 95℃ | y=0.0110x+0.1008 | 0.9797 |
As can be seen here, temperature of reaction is linearly dependent coefficient maximum in the time of 85 DEG C, therefore selects 85 DEG C of the bests of temperature.
Fig. 4 is phenol concentration and the affect figure of reaction time reciprocation on accuracy.
As can be seen here, the interaction in phenol concentration and reaction time is remarkable on the impact of accuracy of measurement.
Fig. 5 is phenol concentration and the affect figure of temperature of reaction reciprocation on accuracy.
As can be seen here, the interaction of phenol concentration and temperature of reaction is remarkable on the impact of accuracy of measurement.
In sum, method of the present invention, its best parameter combinations is: 90 DEG C of temperature of reaction, reaction time 23min, phenol concentration 4.5%.
Glucose typical curve based under test tube colourimetry is shown in Fig. 7.As seen from the figure, phenol reagent concentration is 6%, concentration of glucose gradient within the scope of 0-300 μ g/mL, significantly (linear equation slope value is larger) of absorbance value gradient disparities at 490nm place, linear relationship is good.
Embodiment 1 measures the content of Microbial exopolysaccharides
Detailed mensuration process is:
(1), accurately take purity and be 55.7% exocellular polysaccharide 0.0200g, be dissolved in 100mL deionized water, now the concentration of exocellular polysaccharide solution is 111.4 μ g/mL.
(2), get 50 μ L exocellular polysaccharide solution and add in microwell plate, then add the concentrated sulphuric acid (98%) 150 μ L, phenol reagent (4.5%) 30 μ L, after mixing, in 90 DEG C of water-baths, hatch 23min.
(3), heating after be cooled to rapidly room temperature, after 5min with microplate reader 490nm place measure absorbance value, mensuration establish three groups parallel.
(4), absorbance value is brought in typical curve into the concentration of calculating Microbial exopolysaccharides, result of calculation: the concentration of Microbial exopolysaccharides is 111.21 μ g/mL.
Embodiment 2 measures the content of shitosan
Detailed mensuration process is:
(1), accurately take shitosan (analyzing pure) 0.0200g, be dissolved in 100mL deionized water, now the concentration of chitosan solution is 200 μ g/mL.
(2), get 50 μ L chitosan solutions and add in microwell plate, then add the concentrated sulphuric acid (98%) 150 μ L, phenol reagent (4.5%) 30 μ L, after mixing, in 90 DEG C of water-baths, hatch 23min.
(3), heating after be cooled to rapidly room temperature, after 5min with microplate reader 490nm place measure absorbance value, mensuration establish three groups parallel.
(4), absorbance value is brought in typical curve into the concentration of calculating shitosan, result of calculation: the concentration of shitosan is 200.42 μ g/mL.
Embodiment 3 measures the content of LBP-X
Detailed mensuration process is:
(1), accurately take LBP-X (purity is 67%) 0.0200g, be dissolved in 100mL deionized water, now the concentration of LBP-X solution is 134 μ g/mL.
(2), get 50 μ L LBP-X solution and add in microwell plate, then add the concentrated sulphuric acid (98%) 150 μ L, phenol reagent (4.5%) 30 μ L, after mixing, in 90 DEG C of water-baths, hatch 23min.
(3), heating after be cooled to rapidly room temperature, after 5min with microplate reader 490nm place measure absorbance value, mensuration establish three groups parallel.
(4), absorbance value is brought in typical curve into the concentration of calculating shitosan, result of calculation: the concentration of shitosan is 133.81 μ g/mL.
In addition, measure respectively the content of mentioned microorganism exocellular polysaccharide, shitosan and LBP-X by traditional sulfuric acid-phynol color comparison tube method, and compare with the result that sulfuric acid-phynol Microdilution plate method of the present invention is measured, relatively the results are shown in following table:
The comparison of table 5 determination of polysaccharide result
| ? | Microbial exopolysaccharides | Shitosan | LBP-X |
| Microdilution plate method | 111.21μg/mL | 200.42μg/mL | 133.81μg/mL |
| Color comparison tube method | 110.86μg/mL | 198.67μg/mL | 134.92μg/mL |
As can be seen here, with the concentration content that the conventional color comparison tube method of sulfuric acid-phynol Microdilution plate method and sulfuric acid-phynol records known sample be more or less the same (the former error is less), but the former only need add 50 μ L(230 μ L systems) sample, and the latter need to add 2.5mL(10mL system) sample, not only the consumption of sample and reagent is few for the former, and adopt microwell plate can survey many group samples simultaneously, there is quick and high-throughout advantage.
Recovery experiment
, add standard specimen concentration and be 100 μ g/mL respectively as adding standard specimen using fructose, wood sugar, sucrose and soluble starch, addition 25 μ L, glucose concentration of standard solution 200 μ g/mL, addition 25 μ L.5 groups parallel, and 90 DEG C of heating 23min, bring result into following formula calculate recovery rate.
Experimental result is in table 6.
Table 6 recovery testu and result
| Add standard specimen | Fructose | Wood sugar | Sucrose | Soluble starch |
| (μ g) to add scalar | 2.5 | 2.5 | 2.5 | 2.5 |
| (μ g) for glucose content | 5 | 5 | 5 | 5 |
| Absorbance value | 0.432008 | 0.43094 | 0.432177 | 0.432275 |
| Bringing mark song into records content (μ g) | 7.511 | 7.492 | 7.514 | 7.516 |
| Recovery of standard addition (%) | 100.44 | 99.68 | 100.56 | 100.63 |
As can be seen here, the recovery that recovery testu result calculates is all between 99-101%, illustrate under the condition drawing after optimizing by response surface, do typical curve comparatively accurate, the certain accuracy of method tool of setting up, can be used for rapid, high volume measure polyoses content, there is certain practical advice meaning.
Method of the present invention, its detection is limited to 0-400 μ g/L, and polyoses content has the good recovery within the scope of this.
Method of the present invention can be for the mensuration of methylated sugar, pentose and oligosaccharide kind and polysaccharide, and the method is simple, quick, sensitive, reproducible, and color is lasting, and every kind of sugar is only made to a typical curve.
For heteroglycan, analysis result can be according to the when correction coefficient correction calculation in addition of the typical curve of key component monose of the composition of each monose.
Heteroglycan: 20% fructose, 40% shitosan, 40% LBP-X (purity 67%).
Three kinds of sugar Specification Curve of Increasing process is separately as follows:
1, preparing standard solution
Accurately take respectively fructose 0.0400g, shitosan 0.0400g, LBP-X 0.0600g, be dissolved in respectively in distilled water, be settled to separately 100mL, be configured to 400 μ g/mL solution, as the standard solution of typical curve separately.
2, mensuration process
(1) in 96 hole ELISA Plate, 3 groups of parallel application of samples, sugaring mother liquor 0,5,10,15,20,25,30,35,40,45,50 μ L respectively, add and reenter deionized water and supply 50 μ L, in each like this micropore, the concentration of sugar is respectively 0,40,80,120,160,200,240,280,320,360,400 μ g/mL.
(2) add the concentrated sulphuric acid (concentration is 98%) 150 μ L, phenol reagent (concentration is 4.5%) 30 μ L, after mixing 90
in bath, hatch 23min, after heating, be cooled to rapidly room temperature, after 5min, measure absorbance value by microplate reader at 490nm place.
3, drawing standard curve
Fructose: Y=0.00285x+0.01134R
2=0.9989, the correction coefficient of glucose typical curve relatively: 0.986.
Shitosan: Y=0.00283x+0.00944R
2=0.9982, the correction coefficient of glucose typical curve relatively: 0.991.
LBP-X: Y=0.00287x+0.00824R
2=0.9985, the correction coefficient of glucose typical curve relatively: 0.981.
Measure heteroglycan content:
(1) get respectively fructose, shitosan, LBP-X (67%) 0.004g, 0.008g, 0.008g, be then dissolved in the deionized water of 100mL, three kinds of sugared content 40,80,53.6 μ g/mL respectively, total heteroglycan concentration is 173.6 μ g/mL.
(2), get 50 μ L heteroglycan solution and add in microwell plate, then add the concentrated sulphuric acid (98%) 150 μ L, phenol reagent (4.5%) 30 μ L, after mixing 90
in bath, hatch 23min.
(3), heating after be cooled to rapidly room temperature, after 5min with microplate reader 490nm place measure absorbance value, mensuration establish three groups parallel.
(4), absorbance value is brought in typical curve into the concentration of calculating heteroglycan, result of calculation: the concentration of heteroglycan is 172.5 μ g/mL.
Computation process is as follows:
Three kinds of polyoses contents are multiplied by respectively separately after correction coefficient, and three kinds of polysaccharide concentrations are 39.44,79.28,52.58 μ g/mL in proper order.Total concentration is 171.30 μ g/mL.
Conclusion: reach 99.3% by the content recovery calculating after proofreading and correct after measuring heteroglycan concentration content and the independent mensuration of each polysaccharide by method of the present invention.
As can be seen here, in the situation that there is no need the various monose of careful division, can once measure total amount by method of the present invention, save many troubles, therefore, have special using value.
Different from the colour developing degree of depth of phenol reagent with different carbohydrates, fructose colour developing is the darkest, glucose takes second place, galactose, mannose are more shallow, pentose colour developing is more shallow, therefore while measuring sugared potpourri, often because the ratio difference of different carbohydrates causes error, but while measuring single carbohydrate, can avoid this kind of error.
It should be noted that, above-described embodiment does not limit the present invention in any form, and all employings are equal to replaces or technical scheme that the mode of equivalent transformation obtains, all drops in protection scope of the present invention.
Claims (4)
1. the method for measuring polyoses content based on phenol sulfuric acid fast high-flux, is characterized in that, comprises the following steps:
(1) polysaccharide determination liquid and the concentrated sulphuric acid are added in microwell plate simultaneously, under the effect of the concentrated sulphuric acid, polysaccharide hydrolysis becomes monose and dewaters rapidly to form furfural derivatives;
(2) in microwell plate, continue to add phenol, phenol reacts with furfural derivatives and generates orange-yellow liquid;
(3) in microplate reader, under 490nm, measure the absorbance value of polysaccharide;
(4) taking glucose solution as titer, drawing standard curve;
(5) absorbance value of polysaccharide is brought into and in typical curve, calculated polyoses content.
2. the method for measuring polyoses content based on phenol sulfuric acid fast high-flux according to claim 1, is characterized in that, the concentration of the described concentrated sulphuric acid is 98%, and the concentration of described phenol is 4.5%.
3. the method for measuring polyoses content based on phenol sulfuric acid fast high-flux according to claim 2, is characterized in that, the volume ratio of described polysaccharide determination liquid, the concentrated sulphuric acid, phenol is 5:15:3.
4. the method for measuring polyoses content based on phenol sulfuric acid fast high-flux according to claim 3, it is characterized in that, in step (2), the temperature that phenol reacts with furfural derivatives is that 90 DEG C, reaction time are 23min, carries out 5min cooling processing after having reacted again.
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| CN113607779A (en) * | 2021-07-08 | 2021-11-05 | 武汉轻工大学 | Method for detecting polysaccharide concentration |
| CN116875657A (en) * | 2023-04-14 | 2023-10-13 | 上海腾瑞制药股份有限公司 | Quantitative analysis method for glycoprotein of recombinant batroxobin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113607779A (en) * | 2021-07-08 | 2021-11-05 | 武汉轻工大学 | Method for detecting polysaccharide concentration |
| CN116875657A (en) * | 2023-04-14 | 2023-10-13 | 上海腾瑞制药股份有限公司 | Quantitative analysis method for glycoprotein of recombinant batroxobin |
| CN116875657B (en) * | 2023-04-14 | 2024-03-22 | 上海腾瑞制药股份有限公司 | Quantitative analysis method for glycoprotein of recombinant batroxobin |
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Application publication date: 20140702 |