CN112683888A - Method for detecting polymerization degree of sodium alginate oligosaccharide - Google Patents
Method for detecting polymerization degree of sodium alginate oligosaccharide Download PDFInfo
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Abstract
The invention provides a method for detecting the polymerization degree of sodium alginate oligosaccharide, which utilizes an absorbance method to measure the average polymerization degree of the sodium alginate oligosaccharide, can directly obtain the polymerization degree of the oligosaccharide through the absorbance value after correcting the error through NMR, greatly simplifies the detection process of the average polymerization degree of the seaweed oligosaccharide, and is suitable for judging the average polymerization degree in the industrial production, separation and purification processes of the seaweed oligosaccharide.
Description
Technical Field
The invention belongs to the technical field of saccharide analysis, and particularly relates to a method for measuring the polymerization degree of sodium alginate oligosaccharides.
Background
Oligosaccharides are a general term for a group of saccharides having a straight chain or a branched chain formed by connecting 2 to 30 monosaccharide units by glycosidic linkages, and may be called oligosaccharides, such as alginate oligosaccharides, chitosan oligosaccharides, pectin oligosaccharides, and the like. Oligosaccharides such as sucrose, maltose, lactose, etc., which are commonly consumed by humans, are only additives used in food processing and are generally not considered to have a specific regulatory function. However, oligosaccharides used in agriculture are oligosaccharides having a specific functional effect or effects, such as algal oligosaccharides. Sodium alginate, an anionic linear saccharide, is composed of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). The natural alginate polysaccharide has three M-block, G-block and MG-block, and the alginate oligosaccharide is mainly obtained by alginate lyase through beta-elimination reaction, and can be used as natural fertilizer, growth regulator, food additive, etc. Many properties of oligosaccharides are closely related to molecular mass, and in particular their physiological activity, is expressed only within a certain range of molecular mass, and it is therefore important to accurately determine the relative average molecular mass of oligosaccharides. The polymerization degree is another embodiment form of molecular mass, and compared with methods such as an optical rotation method, an ultracentrifugation method, a high-pressure electrophoresis method, a gel chromatography method, a mass spectrometry method, a nuclear magnetic method and the like, the methods for measuring the polymerization degree have larger errors or are complicated to operate, the polymerization degrees measured by the mass spectrometry method and the nuclear magnetic method are more accurate, but related structure information of sugar needs to be known in advance, and the detection cost is more expensive. Therefore, it is important to find a fast, simple and accurate method for detecting the polymerization degree of oligosaccharides.
Disclosure of Invention
The invention aims to: the method for conveniently, quickly and accurately measuring the average polymerization degree of the algal oligosaccharides is simple to operate and has low requirements on instruments.
The invention utilizes an absorbance method to measure the average polymerization degree of the sodium alginate oligosaccharide, and under the conventional condition, the formula of the average polymerization degree is as follows: average polymerization degree is 1/percentage content of reducing sugar; however, monosaccharide of sodium alginate is difficult to obtain and expensive, so that the standard curve and a sample lack an accurate corresponding relation; after the error is corrected by NMR, the method can directly obtain the polymerization degree of the oligosaccharide through the absorbance value, greatly simplifies the detection process of the average polymerization degree of the algal oligosaccharide, and is suitable for judging the average polymerization degree in the industrial production, separation and purification processes of the algal oligosaccharide.
The invention provides a method for detecting the polymerization degree of sodium alginate oligosaccharide, which directly evaluates the average polymerization degree of sodium alginate by using an absorbance value method;
the method comprises the following steps:
(1) preparation of DNS (3, 5-dinitrosalicylic acid) reagent: adding 45.5g of potassium sodium tartrate into 125ml of hot water for dissolving, and adding 1.575g of DNS and 65.5ml of 2mol/L NaOH; then adding 1.25g of redistilled phenol and 1.25g of sodium sulfite, stirring for dissolving, cooling, adding distilled water to constant volume of 250ml, storing in a brown bottle, and standing for 7 days for later use;
(2) a series of oligosaccharide solutions of specific concentrations and solutions required for the standard curve were prepared: the DNS method is adopted, and the determination method and the steps are as follows: (1) drawing a standard curve: preparing glucose solutions with different concentrations, and measuring the absorbance values of the glucose solutions at 540nm to obtain different glucose and absorbance curve graphs as standard curve graphs;
(3) preparation of a series of oligosaccharide solutions: preparing sodium alginate oligosaccharide solutions to be detected with different concentrations, wherein the concentration of the sodium alginate oligosaccharide solution is within the linear range of the standard curve;
(4) taking the sodium alginate oligosaccharide solution in the step (3), adding the DNS reagent, heating at 100 ℃ for 5-10min, and measuring the absorbance value at 540 nm; calculating the concentration of reducing sugar according to a standard curve, and then calculating the percentage content of the reducing sugar according to the amount of the added sodium alginate oligosaccharide to be detected;
(5) and obtaining the polymerization degree of the sodium alginate oligosaccharide to be detected through a relation curve of the percentage content of reducing sugar and the polymerization degree.
Based on the above technical solution, preferably, the equation y of the relationship between the percentage content of raw sugar and the polymerization degree in step (5) is 1.085 x-0.136; the equation is obtained by fitting the polymerization degree and the reducing sugar content which are measured through nuclear magnetism; x represents 100/DP (DP represents the degree of polymerization), y represents the concentration of reducing sugar;
based on the technical scheme, preferably, the sodium alginate oligosaccharide is alginic acid oligosaccharide and seaweed oligosaccharide, and the polymerization degree is 2-30.
The sodium alginate oligosaccharide is obtained by treating with alginate lyase for different time, and through beta-elimination reaction, reducing sugar is generated at the end group position.
The determination method of the invention can also be called as a colorimetric method, namely a DNS method, namely, after 3, 5-dinitrosalicylic acid (DNS) and reducing sugar are heated together, a color reaction is carried out under an alkaline condition, and a curve of the concentration and the absorbance of the reducing sugar is obtained
Based on the technical scheme, preferably, the sodium alginate oligosaccharide is formed by mixing and arranging beta-D-mannuronic acid (M) and alpha-L-guluronic acid (alpha-L-guluronic acid, G); the structural formulas of the beta-D-mannuronic acid (beta-D-mannuronic acid, M) and the alpha-L-guluronic acid (alpha-L-guluronic acid, G) are as follows:
based on the technical scheme, preferably, the structural formula of the sodium alginate oligosaccharide is as follows:
advantageous effects
(1) Under conventional conditions, the average degree of polymerization is given by the formula: average polymerization degree is 1/percentage content of reducing sugar; however, monosaccharide of sodium alginate is difficult to obtain and expensive, so that the standard curve and a sample lack an accurate corresponding relation; after the error is corrected by NMR, the method can directly obtain the polymerization degree of the oligosaccharide through the absorbance value, greatly simplifies the detection process of the average polymerization degree of the algal oligosaccharide, and is suitable for judging the average polymerization degree in the industrial production, separation and purification processes of the algal oligosaccharide.
(2) Under conventional conditions, DNS method is usually used for detecting the amount of reducing sugar generated to evaluate the activity of hydrolase, and by the method, the degree of polymerization of the product can be further calculated, thereby providing convenience for subsequent experimental arrangement.
Drawings
Fig. 1 is a DNS diagram of the present invention.
FIG. 2 is a graph of a reducing sugar standard.
FIG. 3 is an NMR spectrum of algal oligosaccharides.
FIG. 4 is a graph showing the relationship between the reducing sugar content and the degree of polymerization.
FIG. 5 is a graph of ESI-MS analysis of algal oligosaccharides.
Detailed Description
The technical solution of the present invention is further described with reference to specific embodiments, the conditions of the high resolution nmr spectrometer used in the examples: pulse program zg30, test temperature 70 ℃, scan width 5000HZ, scan delay 2s, scan number 64. ESI-MS used was negative ion detection mode with an Agilent 1290Infinity ultra performance liquid chromatography DAD UV detector. The method used in The present invention for NMR detection of polymerization degree is described in The references of The Catalytic Activities of The Bifunctional Azotobacter virelandii Mannulon C-5-epiferase and Alginate Lyase AlgE7 basic origin from The Same Active Site in The Enzyme; THE Je JOURNAL OF BIOLOGICAL CHEMISTRY Vol.276, No.34, Issue OF August 24, pp.31542-31550,2001.
Example 1
(1) Preparation of DNS (3, 5-dinitrosalicylic acid) reagent: adding 45.5g of potassium sodium tartrate into 125ml of hot water for dissolving, and adding 1.575g of DNS and 65.5ml of 2mol/L NaOH; then adding 1.25g of redistilled phenol and 1.25g of sodium sulfite, stirring for dissolving, cooling, adding distilled water to constant volume of 250ml, storing in a brown bottle, and standing for 7 days for later use;
(2) drawing a standard curve: preparing glucose solutions with different concentrations, and measuring absorbance values of the glucose solutions at 540nm by using a DNS method to obtain different glucose concentration and absorbance curve graphs serving as standard curve graphs; as shown in fig. 4: the standard curve is that y is 0.16x-0.02
(3) Preparation of a series of oligosaccharide solutions: taking oligomeric sodium alginate, wherein the oligomeric sodium alginate is a mixed fragment of mannuronic acid and guluronic acid, and preparing into a solution with the concentration of 1 mg/ml;
(4) transferring 128 μ l of the solution in a test tube, adding 96 μ l of DNS reagent, developing at 100 deg.C for 10min, taking out to normal temperature of 25 deg.C, adding 1.376ml of primary water, sucking 200 μ l of the solution, placing into a cuvette, and measuring absorbance of reducing sugar at 540 nm. The percentage of reducing sugars was calculated to be 10.96% by standard curve,
(5) substituting equation y to 1.085x-0.136 gives a corresponding average degree of polymerization of 9.67.
The average degree of polymerization was 9.8 as determined by NMR.
Example 2
Other steps of the invention are the same as example 1
(3) Taking oligomeric sodium alginate, which are guluronic acid segments, and preparing into a solution with the concentration of 1 mg/ml;
(4) transferring 128 μ l of the solution in a test tube, adding 96 μ l of DNS reagent, developing at 100 deg.C for 10min, taking out to normal temperature of 25 deg.C, adding 1.376ml of primary water, sucking 200 μ l of the solution, placing into a cuvette, and measuring absorbance of reducing sugar at 540 nm. The percentage of reducing sugars was calculated to be 10.96% by standard curve,
(5) substituting equation y for 1.085x-0.136 yields a corresponding average degree of polymerization of 9.67; the average degree of polymerization was 9.8 by NMR.
Example 3
Other steps of the invention are the same as example 1
(3) Taking oligomeric sodium alginate as a mixed fragment of mannuronic acid and guluronic acid, and preparing into a solution with the concentration of 1 mg/ml;
(4) transferring 128 μ l of the solution in a test tube, adding 96 μ l of DNS reagent, developing at 100 deg.C for 10min, taking out to normal temperature of 25 deg.C, adding 1.376ml of primary water, sucking 200 μ l of the solution, placing into a cuvette, and measuring absorbance of reducing sugar at 540 nm. The percentage of reducing sugars was calculated to be 29.57% by standard curve,
(5) substituting equation y for 1.085x-0.136 to obtain the corresponding average degree of polymerization of 3.61; the average degree of polymerization was 3.6 by NMR. The detection is carried out again by ESI-MS, as shown in FIG. 5, it can be seen that the oligosaccharide mixed liquid component contains disaccharide, trisaccharide and tetrasaccharide, and the content of disaccharide is slightly higher than that of tetrasaccharide, and it is known that the average degree of polymerization is between 3 and 4, which is consistent with the degree of polymerization range measured in the invention.
FIG. 1 is a schematic diagram of DNS in accordance with the present invention, wherein M and G can form reducing sugar in aqueous solution, and DNS can change color with reducing sugar under alkaline heating.
Fig. 3 is a graph of NMR spectroscopy analysis of algal oligosaccharides, from which it can be seen that H under different chemical environments corresponds to different peak shifts and H of different contents corresponds to different peak areas, so that the information can be substituted into the formula DPn ═ IG-5+IM-1+(IGred+IMred)ⅹ2]/[IGred+IMred]。
Example 4
Taking 5mg of a series of seaweed oligosaccharides in 0.5ml D2In O, at 70 ℃1And H NMR detection. And analyzing the obtained nuclear magnetic peak maps one by one, and calculating to obtain the polymerization degree of the corresponding oligosaccharide. Fitting the polymerization degree with the content of reducing sugar corresponding to the oligosaccharide to obtain a linear equation, as shown in FIG. 4.
Claims (5)
1. A method for detecting the polymerization degree of sodium alginate oligosaccharide is characterized in that: the method comprises the following steps:
(1) preparation of DNS (3, 5-dinitrosalicylic acid) reagent: dissolving sodium potassium tartrate in water at 70 ℃, and then adding DNS and NaOH solution; adding redistilled phenol and sodium sulfite, and stirring for dissolving to obtain the DNS reagent; the concentration of the NaOH solution is 2 mol/L; in the DNS reagent, the ratio of potassium to sodium tartrate (w/v) is 18.2%; the percentage of DNS (w/v) is 0.63%; the ratio of NaOH solution (v/v) is 26.2%; the proportion (w/v) of redistilled phenol was 0.5%; the proportion of sodium sulfite (w/v) is 0.5 percent;
(2) drawing a standard curve: preparing glucose solutions with different concentrations, and measuring the absorbance values of the glucose solutions at 540nm to obtain different glucose and absorbance curve graphs as standard curve graphs;
(3) preparing sodium alginate oligosaccharide solutions to be detected with different concentrations, wherein the concentration of the sodium alginate oligosaccharide solution is within the linear range of the standard curve;
(4) taking the sodium alginate oligosaccharide solution in the step (3), adding the DNS reagent, carrying out water bath at 100 ℃ for 5-10min, and measuring the absorbance value at 540 nm; calculating the concentration of reducing sugar according to a standard curve, and then calculating the percentage content of the reducing sugar according to the amount of the added sodium alginate oligosaccharide to be detected;
(5) and obtaining the polymerization degree of the sodium alginate oligosaccharide to be detected through a relation curve of the percentage content of reducing sugar and the polymerization degree.
2. The method according to claim 1, wherein the percentage of the raw sugar in step (5) is 1.085 x-0.136; the equation is obtained by fitting the polymerization degree and the reducing sugar content which are measured through nuclear magnetism; x represents 100/DP (DP represents the degree of polymerization) and y represents the reducing sugar concentration.
3. The method for measuring according to claim 1, wherein: the sodium alginate oligosaccharide is seaweed oligosaccharide, and the polymerization degree of the sodium alginate oligosaccharide is 2-30.
4. The method for measuring according to claim 1, wherein: the sodium alginate oligosaccharide is formed by mixing and arranging beta-D-mannuronic acid (M) and alpha-L-guluronic acid (alpha-L-guluronic acid, G); the structural formulas of the beta-D-mannuronic acid (beta-D-mannuronic acid, M) and the alpha-L-guluronic acid (alpha-L-guluronic acid, G) are as follows:
5. an assay as claimed in claim 4, wherein the sodium alginate oligosaccharide has the following structural formula:
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| CN102608046A (en) * | 2012-03-05 | 2012-07-25 | 熊鹏 | Simple method for measuring average polymerization degree of xylo-oligosaccharide |
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| CN102608046A (en) * | 2012-03-05 | 2012-07-25 | 熊鹏 | Simple method for measuring average polymerization degree of xylo-oligosaccharide |
Non-Patent Citations (5)
| Title |
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| BRITT IREN GLÆRUM SVANEM ET.AL: "The Catalytic Activities of the Bifunctional Azotobacter vinelandii Mannuronan C-5-Epimerase and Alginate Lyase AlgE7 Probably Originate from the Same Active Site in the Enzyme", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| FARIDEH GOURANLOU ET.AL: "Determination of Average Degree of Polymerization and Average Number of Molecular Weightfor Hydroxy Terminated Polybutadiene Polymer using 1H NMR", 《ASIAN JOURNAL OF CHEMISTRY》 * |
| LIN MA ET.AL: "Smart Assembly Behaviors of Hydroxypropylcellulose-graft-poly(4-vinylpyridine) Copolymers in Aqueous Solution by Thermo and pH Stimuli", 《LANGMUIR》 * |
| MING SUN ET.AL: "A simple method to calculate the degree of polymerization of alginate oligosaccharides and low molecular weight alginates", 《CARBOHYDRATE RESEARCH》 * |
| 王宗继等: "壳寡糖分析检测方法的研究进展", 《食品安全质量检测学报》 * |
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