CN103901031A - Method for rapid and high-flux determination of polysaccharide content based on sulfuric acid and phenol - Google Patents

Abstract

The invention discloses a method for fast and high-throughput determination of polysaccharide content based on sulfuric acid phenol, which is characterized in that it comprises the following steps: adding polysaccharide measurement solution and concentrated sulfuric acid to a microporous plate at the same time, polysaccharides are hydrolyzed into monosaccharides and rapidly dehydrated Furfural derivatives are formed; phenol continues to be added to the microplate, and phenol and furfural derivatives react to form an orange-yellow liquid. The reaction temperature is 90°C and the time is 23 minutes. Determination of the light absorption value of the polysaccharide; using glucose solution as the standard solution, draw a standard curve, and bring the light absorption value of the polysaccharide into the standard curve to calculate the polysaccharide content. The advantages of the present invention are: the method is simple, fast, sensitive, and repeatable, and the color is durable, and only one standard curve is prepared for each type of sugar; for heteropolysaccharides, it is not necessary to carefully divide various monosaccharides. The total amount is measured at one time; it can be used for the determination of methylated sugars, pentoses, oligosaccharides and polysaccharides.

Description

A rapid and high-throughput method for the determination of polysaccharide content based on phenol sulfate

technical field

The invention relates to a method for determining polysaccharide content, in particular to a method for rapidly and high-throughput determining polysaccharide content based on phenol sulfate, and belongs to the technical field of analytical chemistry. the

Background technique

Sugar is an important component of the cell tissue of organisms and one of the material basis of life. Sugar is closely related to nutrient metabolism, cell structure, anti-virus, immune enhancement, material movement, etc. The human body must take in enough sugar from food to maintain the normal operation of various physiological functions. Therefore, sugar content is one of the most important nutritional indicators of many foods. the

There are many methods for the determination of polysaccharide content:

One is to use the reducing property of polysaccharides to determine reducing polysaccharides. The methods include: 3,5-dinitrosalicylate (DNS) colorimetric method and Somogyi-Nelson method. the

Another type of determination method is also the most commonly used method at present, the furfural condensation chromogenic method, which mainly uses polysaccharides to dehydrate under strong acidic conditions to generate furfural or its derivatives, and then condenses with phenolic (or amine) compounds to form The property of colored substances with specific absorption wavelengths is measured. Such methods include orcinol-sulfuric acid (hydrochloric acid) method, phenol-sulfuric acid method and anthrone-sulfuric acid method. the

It can be seen that the traditional method of measuring polysaccharides is carried out in a colorimetric tube, which requires the addition of a large amount of glucose standard solution and phenol reagent. Not only the detection efficiency is low, the reagent loss is large, the error is large, and the environmental pollution is also large. the

Contents of the invention

In order to solve the deficiencies in the prior art, the object of the present invention is to provide a method based on phenol sulfate, which can realize fast and high-throughput determination of polysaccharide content. the

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for fast high-throughput determination of polysaccharide content based on phenol sulfate, is characterized in that, comprises the following steps:

(1) Add the polysaccharide measurement solution and concentrated sulfuric acid to the microporous plate at the same time, and under the action of concentrated sulfuric acid, the polysaccharide is hydrolyzed into monosaccharides and rapidly dehydrated to form furfural derivatives;

(2) Continue to add phenol to the microwell plate, and the phenol reacts with furfural derivatives to form an orange-yellow liquid;

(3) Measure the light absorption value of the polysaccharide at 490nm in a microplate reader;

(4) Draw a standard curve with glucose solution as the standard solution;

(5) Bring the light absorption value of the polysaccharide into the standard curve to calculate the polysaccharide content. the

The aforementioned method for rapid and high-throughput determination of polysaccharide content based on sulfuric acid phenol is characterized in that the concentration of the aforementioned concentrated sulfuric acid is 98%, and the aforementioned phenol concentration is 4.5%. the

The aforementioned method for rapid and high-throughput determination of polysaccharide content based on sulfuric acid phenol is characterized in that the volume ratio of the aforementioned polysaccharide measurement solution, concentrated sulfuric acid, and phenol is 5:15:3. the

The aforementioned method for rapid and high-throughput determination of polysaccharide content based on phenol sulfate is characterized in that in step (2), the temperature of the reaction between phenol and furfural derivatives is 90°C, the reaction time is 23 minutes, and cooling is carried out for 5 minutes after the reaction is completed. deal with. the

The benefits of the present invention are: the present invention is based on the principle of quantitative carbohydrates with phenol sulfate. Under the action of concentrated sulfuric acid, polysaccharides are first hydrolyzed into monosaccharides and rapidly dehydrated to form furfural derivatives, and then phenol and furfural derivatives form orange yellow Chemicals, within a certain range, the color depth is proportional to the sugar content, can be used for the determination of methylated sugars, pentoses, oligosaccharides and polysaccharides, and the method is simple, fast, sensitive and reproducible Well, the color is long-lasting, and only one standard curve is made for each sugar; for heteropolysaccharides, the analysis results can be corrected and calculated according to the composition ratio of each monosaccharide and the calibration coefficient of the standard curve of the main component monosaccharide, and it is not necessary to divide carefully In the case of various monosaccharides, the detection method of the present invention can be used to measure the total amount at one time, which saves a lot of trouble and has special application value; it has the characteristics of fast and high throughput, and it is carried out in a microwell plate. The orifice plate has many holes and requires a small amount of addition, so the polysaccharide content of multiple samples can be determined at the same time. the

Description of drawings

Fig. 1 is the figure of influence of phenol concentration on measurement accuracy;

Fig. 2 is the impact figure of reaction time on measurement accuracy;

Fig. 3 is the impact figure of reaction temperature on measurement accuracy;

Fig. 4 is the figure of influence of phenol concentration and reaction time interaction on accuracy;

Fig. 5 is the figure of influence of phenol concentration and reaction temperature interaction on accuracy;

Fig. 6 is based on the glucose standard curve figure under the microplate method;

Fig. 7 is a glucose standard curve diagram based on the test tube colorimetry method. the

Detailed ways

The present invention will be specifically introduced below in conjunction with the accompanying drawings and specific embodiments. the

400μg/mL glucose solution: Accurately weigh 0.0400g of anhydrous glucose, dissolve in distilled water, and dilute to 100mL. the

Concentrated sulfuric acid: the concentration is 98%. the

4.5% phenol: Accurately weigh 0.4500g phenol, dissolve in distilled water, and dilute to 10mL. the

draw standard curve

A 400 μg/mL glucose solution was used as the standard solution. the

The detailed operation process is:

(1) On a 96-well ELISA plate, add 5 groups of samples in parallel, take 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μL of glucose mother solution for each group, add deionized Make up 50 μL of water so that the concentration of glucose in each microwell is 0, 40, 80, 120, 160, 200, 240, 280, 320, 360, 400 μg/mL. the

(2) Add 150 μL of concentrated sulfuric acid (concentration: 98%) and 30 μL phenol reagent (concentration: 4.5%), mix well and incubate in a water bath at 90°C for 23 minutes, cool to room temperature quickly after heating, and use a microplate reader after 5 minutes Absorbance values were measured at 490 nm. the

For each concentration of glucose solution, read the light absorption value repeatedly 5 times, take the average of five parallel light absorption values, and in individual cases, at most two data with a large difference from the average value can be eliminated. The data measured by microplate reader are shown in Table 1

Table 1 Data table for microplate reader determination

(3) Draw a standard curve with the glucose solution concentration as the abscissa and the light absorption value as the ordinate, as shown in Figure 6, the equation of the standard curve is Y=0.00281X+0.00989, R ² =0.9992, RSD=0.0169, Where x represents the concentration of glucose (μg/mL), and Y represents the light absorption value.

In addition, it can be seen from Figure 6 that when the concentration of phenol reagent is 4.5%, the concentration gradient of glucose is in the range of 0-400μg/mL, and the gradient of light absorption value at 490nm is significantly different (the slope value of the linear equation is large), and the linear relationship is good . the

The RSD value between the ^R2 value of the bracket and the parallel value is used as the standard for measuring the results:

(1) The larger the R ² , the better the correlation of the points on the standard curve, and the higher the accuracy of the experimental results; on the contrary, the smaller the R ² , it means that the error in the experimental operation is large or the design of the method itself is unreasonable, so the designed For a good method, its ^R2 must be larger and closer to 1.

(2) The RSD value represents the difference between each parallel. The conditions of each parallel reaction are the same, but the difference in the results indicates that there is a systematic error or the stability of the established method is poor. A good method must be in the same Stable under the assay conditions, the larger the RSD, the less stable the method and thus can be used as an indicator of the corresponding analysis. the

The surface curve drawn by the method of the present invention has an R ^value of 0.9992 and RSD=0.0169. It can be seen that the experimental results obtained by the method of the present invention have higher accuracy and better stability.

Figure 1 is a graph showing the influence of phenol concentration on the measurement accuracy. the

Table 2 The corresponding table of standard music under different phenol concentrations

Phenol concentration standard curve line 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

It can be seen that when the concentration of phenol is 5%, the linear correlation coefficient is the largest and the accuracy is the best. the

Fig. 2 is a graph showing the influence of reaction time on measurement accuracy. the

Table 3 Correspondence table of standard music under different reaction times

Reaction time standard curve line 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

It can be seen that the linear correlation coefficient is the largest when the reaction time is 20 minutes, so it is best to choose the reaction time as 20 minutes. the

Fig. 3 is a graph showing the influence of reaction temperature on measurement accuracy. the

Table 4 The standard song correspondence table under different reaction temperatures

temperature reflex standard curve line R ²

[0057] 80°C y=0.0063x+0.0465 0.9764 85°C y=0.0095x+0.0122 0.9930 90°C y=0.0080x+0.0365 0.9907 95°C y=0.0110x+0.1008 0.9797

It can be seen that the linear correlation coefficient is the largest when the reaction temperature is 85°C, so the optimum temperature is 85°C. the

Figure 4 is a graph showing the influence of the interaction between phenol concentration and reaction time on accuracy. the

It can be seen that the interaction between phenol concentration and reaction time has a significant impact on the determination accuracy. the

Fig. 5 is a diagram showing the influence of the interaction of phenol concentration and reaction temperature on the accuracy. the

It can be seen that the interaction between phenol concentration and reaction temperature has a significant impact on the measurement accuracy. the

In summary, the method of the present invention has the best combination of parameters: reaction temperature 90°C, reaction time 23 minutes, phenol concentration 4.5%. the

The glucose standard curve based on the test tube colorimetry is shown in Figure 7. It can be seen from the figure that when the concentration of phenol reagent is 6%, and the concentration gradient of glucose is in the range of 0-300μg/mL, the gradient of light absorption value at 490nm is significantly different (the slope value of the linear equation is large), and the linear relationship is good. the

Embodiment 1 measures the content of microbial exopolysaccharide

The detailed measurement process is:

(1) Accurately weigh 0.0200 g of exopolysaccharide with a purity of 55.7%, and dissolve it in 100 mL of deionized water. At this time, the concentration of the exopolysaccharide solution is 111.4 μg/mL. the

(2) Add 50 μL of exopolysaccharide solution into a microwell plate, then add 150 μL of concentrated sulfuric acid (98%), 30 μL of phenol reagent (4.5%), mix well and incubate in a 90°C water bath for 23 minutes. the

(3) Cool down to room temperature quickly after heating, and measure the light absorption value at 490nm with a microplate reader after 5 minutes, and set up three parallel groups for measurement. the

(4) Bring the light absorption value into the standard curve to calculate the concentration of microbial exopolysaccharide. The calculation result: the concentration of microbial exopolysaccharide is 111.21 μg/mL. the

Embodiment 2 measures the content of chitosan

The detailed measurement process is:

(1) Accurately weigh 0.0200 g of chitosan (analytically pure), dissolve it in 100 mL of deionized water, and the concentration of the chitosan solution at this time is 200 μg/mL. the

(2) Add 50 μL of chitosan solution into a microwell plate, then add 150 μL of concentrated sulfuric acid (98%) and 30 μL of phenol reagent (4.5%), mix well and incubate in a 90°C water bath for 23 minutes. the

(3) Cool down to room temperature quickly after heating, and measure the light absorption value at 490nm with a microplate reader after 5 minutes, and set up three parallel groups for measurement. the

(4) Bring the light absorption value into the standard curve to calculate the concentration of chitosan. The calculation result: the concentration of chitosan is 200.42 μg/mL. the

Embodiment 3 measures the content of Lycium barbarum polysaccharide

The detailed measurement process is:

(1) Accurately weigh 0.0200 g of Lycium barbarum polysaccharide (purity: 67%) and dissolve it in 100 mL of deionized water. At this time, the concentration of Lycium barbarum polysaccharide solution is 134 μg/mL. the

(2) Add 50 μL of Lycium barbarum polysaccharide solution into the microwell plate, then add 150 μL of concentrated sulfuric acid (98%), 30 μL of phenol reagent (4.5%), mix well and incubate in a 90°C water bath for 23 minutes. the

(3) Cool down to room temperature quickly after heating, and measure the light absorption value at 490nm with a microplate reader after 5 minutes, and set up three parallel groups for measurement. the

(4) Bring the light absorption value into the standard curve to calculate the concentration of chitosan. The calculation result: the concentration of chitosan is 133.81 μg/mL. the

In addition, the traditional sulfuric acid-phenol colorimetric tube method was used to measure the content of the above-mentioned microbial exopolysaccharides, chitosan and Lycium barbarum polysaccharide respectively, and compared with the results measured by the sulfuric acid-phenol microplate method of the present invention, the compared results See the table below:

Table 5 Polysaccharide Content Determination Results Comparison

the microbial exopolysaccharide Chitosan Lycium barbarum polysaccharide microplate method 111.21μg/mL 200.42μg/mL 133.81μg/mL Colorimetric tube method 110.86μg/mL 198.67μg/mL 134.92μg/mL

It can be seen that there is little difference in the concentration of known samples measured by the sulfuric acid-phenol microplate method and the sulfuric acid-phenol conventional colorimetric tube method (the error of the former is smaller), but the former only needs to add 50 μL (230 μL system) The latter needs to add 2.5mL (10mL system) samples, while the former not only consumes less samples and reagents, but also can measure multiple groups of samples at the same time by using a microplate, which has the advantages of rapidity and high throughput. the

Recovery experiment

Fructose, xylose, sucrose and soluble starch were used as standard samples respectively. The concentration of the standard sample was 100 μg/mL, and the addition amount was 25 μL. The concentration of the glucose standard solution was 200 μg/mL, and the addition amount was 25 μL. Five groups were parallelized, heated at 90°C for 23 minutes, and the results were entered into the following formula to calculate the recovery rate. the

The experimental results are shown in Table 6. the

Table 6 Standard addition recovery experiment and results

Add standard fructose Xylose sucrose soluble starch Scaling amount (μg) 2.5 2.5 2.5 2.5 Glucose content (μg) 5 5 5 5 light absorption value 0.432008 0.43094 0.432177 0.432275 Bring into the standard curve to measure the content (μg) 7.511 7.492 7.514 7.516 Spike recovery (%) 100.44 99.68 100.56 100.63

It can be seen that the recoveries calculated from the results of the standard addition recovery experiment are all between 99-101%, indicating that the standard curve made under the conditions obtained after the response surface optimization is relatively accurate, and the established method has certain accuracy. It can be used to quickly determine the content of polysaccharides in large quantities, and has certain practical guiding significance. the

The detection limit of the method of the invention is 0-400 μg/L, and the polysaccharide content has a better recovery rate within this range. the

The method of the present invention can be used for the determination of methylated sugars, pentoses, oligosaccharides and polysaccharides, and the method is simple, fast, sensitive, reproducible, and the color is durable, and only one standard is prepared for each sugar curve. the

For heteropolysaccharides, the analysis results can be corrected and calculated according to the composition ratio of each monosaccharide and the calibration coefficient of the standard curve of the main component monosaccharide. the

Example 4

Heteropolysaccharides: 20% fructose, 40% chitosan, 40% wolfberry polysaccharide (purity 67%). the

The respective standard curve drawing process of the three sugars is as follows:

1. Prepare standard solution

Accurately weigh 0.0400g of fructose, 0.0400g of chitosan, and 0.0600g of Lycium barbarum polysaccharide, respectively, dissolve them in distilled water, adjust the volume to 100mL, and prepare 400μg/mL solutions as standard solutions for their respective standard curves. the

2. Measurement process

(1) On a 96-well ELISA plate, add 3 groups of samples in parallel, add sugar mother solution 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μL, add deionized water to make up 50 μL, In this way, the concentration of sugar in each microwell is 0, 40, 80, 120, 160, 200, 240, 280, 320, 360, 400 μg/mL, respectively. the

浴中孵育23min，加热完毕后迅速冷却至室温，5min后用酶标仪在490nm处测定光吸收值。  (2) Add 150 μL of concentrated sulfuric acid (concentration of 98%) and 30 μL of phenol reagent (concentration of 4.5%), mix well at 90

Incubate in the bath for 23min, cool down to room temperature rapidly after heating, and measure the light absorption value at 490nm with a microplate reader after 5min.

3. Draw a standard curve

Fructose: Y=0.00285x+0.01134R ² =0.9989, correction coefficient relative to glucose standard curve: 0.986.

Chitosan: Y=0.00283x+0.00944R ² =0.9982, correction coefficient relative to glucose standard curve: 0.991.

Lycium barbarum polysaccharide: Y=0.00287x+0.00824R ² =0.9985, the correction coefficient relative to the glucose standard curve: 0.981.

Determination of heteropolysaccharide content:

(1) Take 0.004g, 0.008g, and 0.008g of fructose, chitosan, and wolfberry polysaccharide (67%) respectively, and then dissolve them in 100mL of deionized water. The heteropolysaccharide concentration was 173.6 μg/mL. the

浴中孵育23min。  (2) Take 50 μL of heteropolysaccharide solution and add it to the microwell plate, then add 150 μL of concentrated sulfuric acid (98%), 30 μL of phenol reagent (4.5%), and mix well at 90

Incubate in the bath for 23min.

(3) Cool down to room temperature quickly after heating, and measure the light absorption value at 490nm with a microplate reader after 5 minutes, and set up three parallel groups for measurement. the

(4) Bring the light absorption value into the standard curve to calculate the concentration of the heteropolysaccharide, and the calculation result: the concentration of the heteropolysaccharide is 172.5 μg/mL. the

The calculation process is as follows:

After multiplying the contents of the three polysaccharides by their respective correction coefficients, the concentrations of the three polysaccharides were 39.44, 79.28, and 52.58 μg/mL. The total concentration was 171.30 μg/mL. the

Conclusion: the method of the present invention is used to determine the concentration and content of heteropolysaccharides, and the recovery rate of the content calculated after correction of each polysaccharide after the individual determination reaches 99.3%. the

It can be seen that, under the situation that there is no need to carefully divide various monosaccharides, the total amount can be measured at one time by the method of the present invention, which saves a lot of trouble, and therefore has special application value. the

Different sugars and phenol reagents have different color depths, fructose has the deepest color, glucose is the second, galactose and mannose are lighter, and five-carbon sugar is lighter. Different proportions of sugars cause errors, but when measuring a single sugar, such errors can be avoided. the

It should be noted that the above-mentioned embodiments do not limit the present invention in any form, and all technical solutions obtained by means of equivalent replacement or equivalent transformation all fall within the protection scope of the present invention. the

Claims (4)

1. The method for the fast high-throughput determination of polysaccharide content based on phenol sulfate, is characterized in that, comprises the following steps: (1) Add polysaccharide measurement solution and concentrated sulfuric acid to the microwell plate at the same time, under the action of concentrated sulfuric acid, polysaccharides are hydrolyzed into monosaccharides and rapidly dehydrated to form furfural derivatives; (2) Continue to add phenol to the microwell plate, and the phenol reacts with furfural derivatives to form an orange-yellow liquid; (3) Measure the light absorption value of the polysaccharide at 490nm in a microplate reader; (4) Draw a standard curve with glucose solution as the standard solution; (5) Bring the light absorption value of the polysaccharide into the standard curve to calculate the polysaccharide content. 2. the method for fast high-throughput determination of polysaccharide content based on sulfuric acid phenol according to claim 1, is characterized in that, the concentration of described vitriol oil is 98%, and the concentration of described phenol is 4.5%. 3. the method for fast high-throughput determination of polysaccharide content based on sulfuric acid phenol according to claim 2, is characterized in that, the volume ratio of described polysaccharide measurement liquid, vitriol oil, phenol is 5:15:3. 4. The method for rapid and high-throughput determination of polysaccharide content based on phenol sulfate according to claim 3, characterized in that, in step (2), the reaction temperature of phenol and furfural derivatives is 90°C, and the reaction time is 23min. After the reaction is completed, carry out cooling treatment for 5 min.

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