A fluorescence labeling method based on improving the thermal stability of soybean polysaccharide

Abstract

The present invention relates to the technical field of fluorescently labeled biological macromolecules, and provides a fluorescent labeling method based on improving the thermal stability of soybean polysaccharide, comprising the following steps: S1, dissolving soybean polysaccharide in a phosphate buffer solution to obtain a mixed solution; S2, sequentially adding tyramine and sodium cyanoborohydride to the mixed solution to obtain a first reaction solution; centrifuging the first reaction solution, taking the supernatant and performing freeze-drying to obtain a soybean polysaccharide-tyramine complex; S3, dissolving the soybean polysaccharide-tyramine complex in water, sequentially adding sodium bicarbonate and fluorescein isothiocyanate, continuously stirring in the dark to obtain a second reaction solution; adding absolute ethanol to the second reaction solution, centrifuging and discarding the supernatant; S4, taking the precipitate and performing freeze-drying to obtain fluorescently labeled soybean polysaccharide, while improving the thermal stability of soybean polysaccharide during labeling.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescently labeled biological macromolecules, and particularly to a fluorescent labeling method based on improving the thermal stability of soybean polysaccharide. Background Art

[0002] Soybean polysaccharide is a polysaccharide substance extracted from soybeans and their by-products (seed coats, soybean dregs, and soybean meal), and has various biological activities such as hypoglycemic, hypolipidemic, and regulating intestinal microecology. In addition, the soybean polysaccharide molecule contains hydrophilic sugar chains, glycoproteins, and uronic acid groups, making it have good emulsifying, thickening, and gelling properties. However, due to the large chemical molecular weight and complex structure of soybean polysaccharide, and the lack of fluorescent groups and ultraviolet absorption groups for detection, it is difficult to perform quantitative / qualitative analysis of soybean polysaccharide, which is not conducive to the tracking and positioning of soybean polysaccharide in the processes of food production and processing and related research.

[0003] However, the prior art lacks research on labeling soybean polysaccharide. At the same time, soybean polysaccharide has the problem of poor thermal stability and is prone to conformational changes such as aggregation during heat treatment, affecting the detection accuracy. Therefore, how to provide a method for labeling soybean polysaccharide and improving the thermal stability of soybean polysaccharide during labeling has become an urgent technical problem to be solved. Summary of the Invention

[0004] The purpose of the present invention is to provide a fluorescent labeling method based on improving the thermal stability of soybean polysaccharide and fluorescently labeled soybean polysaccharide to solve the technical problems in the prior art that lack a fluorescent labeling method based on improving the thermal stability of soybean polysaccharide and cannot improve the thermal stability of soybean polysaccharide during labeling.

[0005] To achieve the above purpose, according to one aspect of the present invention, there is provided a fluorescent labeling method based on improving the thermal stability of soybean polysaccharide. The labeling method includes the following steps: S1, dissolving soybean polysaccharide in a phosphate buffer solution to obtain a mixed solution; S2, sequentially adding tyramine and sodium cyanoborohydride to the mixed solution to obtain a first reaction solution; centrifuging the first reaction solution, taking the supernatant for freeze-drying to obtain a soybean polysaccharide-tyramine complex; S3, dissolving the soybean polysaccharide-tyramine complex in distilled water, sequentially adding sodium bicarbonate and fluorescein isothiocyanate, continuously stirring in the dark to obtain a second reaction solution; adding absolute ethanol to the second reaction solution, centrifuging and discarding the supernatant; S4, taking the precipitate for freeze-drying to obtain fluorescently labeled soybean polysaccharide.

[0006] Further, in S1, the soybean polysaccharide is soybean soluble polysaccharide, soybean seed coat polysaccharide, soybean dregs polysaccharide, or soybean meal polysaccharide.

[0007] Further, in S1, a phosphate buffer solution with a molar mass of 0.20 M was used to prepare a soy seed coat polysaccharide with a concentration of 2.0 wt% to 3.0 wt%. After continuously stirring at room temperature for 2 h, it was left at 4 °C for 12 h to obtain a mixed solution. If the concentration of the soy seed coat polysaccharide is lower than 2.0 wt%, the polysaccharide in the solution is too dispersed, which will affect the reaction rate with tyramine; if the concentration of the polysaccharide is higher than 3.0 wt%, the polysaccharide is prone to form excessive aggregates and lumps, which is not conducive to the full progress of the reaction. The experimental results show that the soy seed coat polysaccharide with a concentration range of 2.0 wt% to 3.0 wt% can be fully dissolved in the phosphate buffer solution and does not affect the subsequent chemical reactions.

[0008] Further, in S2, tyramine with a concentration of 2.0 wt% to 3.0 wt% was added to the mixed solution, and it was continuously stirred at 37 °C for 12 to 24 h, and then sodium cyanoborohydride was added. When the concentration of tyramine is lower than 2.0 wt%, there is unreacted soy seed coat polysaccharide in the mixed solution; when the concentration of tyramine exceeds 3.0 wt%, the excessive tyramine reacts with FITC, resulting in that part of the soy seed coat polysaccharide fails to react with sodium cyanoborohydride through tyramine, which is not conducive to the fluorescence labeling of the polysaccharide. Experiments show that tyramine with a concentration of 2.0 wt% to 3.0 wt% can successfully aminate the polysaccharide, which is conducive to the smooth progress of fluorescence labeling.

[0009] Further, in S2, sodium cyanoborohydride with a concentration of 0.5 wt% to 1.5 wt% was added, and it was continuously stirred at 37 °C for 12 to 36 h to obtain a first reaction solution. When the concentration of sodium cyanoborohydride is lower than 0.5 wt%, there is excessive soy seed coat polysaccharide connected to tyramine in the solution, and this part of the polysaccharide cannot undergo a chemical reaction with FITC, thereby reducing the fluorescence labeling degree of the polysaccharide; when the concentration of sodium cyanoborohydride is higher than 1.5 wt%, there is excessive sodium cyanoborohydride in the solution, which affects the Brownian motion of the polysaccharide and FITC, reducing the chemical reaction rate. Experiments prove that when the concentration range of sodium cyanoborohydride is 0.5 wt% to 1.5 wt%, the aminated polysaccharide can fully react with FITC to complete the fluorescence labeling of the polysaccharide.

[0010] Further, in S2, when centrifuging the first reaction solution, the parameters were set as:

[0011] The centrifugation rate was 4000 r / min, and the centrifugation time was 10 min. Centrifuging at a rate of 4000 r / min for 10 min can fully remove the unreacted small molecule substances and obtain the aminated polysaccharide.

[0012] Further, in S3, first add 0.5 M sodium bicarbonate to adjust the pH value to 7.0 - 9.0. Using 0.5 M sodium bicarbonate to adjust the solution to a slightly alkaline environment can avoid the aggregation of soybean seed coat polysaccharides under acidic conditions, which is not conducive to the full reaction of polysaccharides with FITC. If the pH of the solution exceeds 9.0, there are too many hydroxide ions in the solution, and the chemical structure of the polysaccharides is damaged, and they cannot react fully with FITC, resulting in too low fluorescence labeling degree and difficult fluorescence labeling. Experiments have confirmed that using 0.5 M sodium bicarbonate to adjust the pH value to 7.0 - 9.0 can provide a slightly alkaline environment for the solution and enable FITC to react fully with soybean seed coat polysaccharides.

[0013] Further, in S3, fluorescein isothiocyanate with a concentration of 0.2 wt% - 0.3 wt% should be added to the reaction solution. When the concentration of fluorescein isothiocyanate is lower than 0.2 wt%, there are excessive unlabeled polysaccharides in the solution. When the concentration of fluorescein isothiocyanate is higher than 0.3 wt%, the excessive free fluorescein isothiocyanate in the solution will be misinterpreted as labeled polysaccharides, thus interfering with the judgment of experimental results. Experimental results show that fluorescein isothiocyanate with a concentration of 0.2 wt% - 0.3 wt% can successfully achieve the fluorescence labeling of soybean seed coat polysaccharides, and at the same time, less free fluorescein is produced.

[0014] Further, in S3, the reaction temperature of the reaction solution after adding fluorescein isothiocyanate should be maintained at 30 - 40 °C. When the reaction solution temperature is lower than 30 °C, the solubility of fluorescein isothiocyanate is relatively low, which is not conducive to the chemical reaction between fluorescein and polysaccharides. When the reaction solution temperature is higher than 40 °C, the chemical bonds of polysaccharides are prone to breakage and the structure is damaged, resulting in the inability of polysaccharides to react chemically with fluorescein. Experimental results show that when the reaction solution temperature is in the range of 30 - 40 °C, the fluorescence labeling of polysaccharides can be completed.

[0015] Further, in S3, continuously stir in the dark for 24 h after adding fluorescein isothiocyanate. In this way, by limiting the time of continuous stirring in the dark, the reaction between polysaccharides and fluorescein can be completed, and the fluorescence labeling of polysaccharides can be achieved.

[0016] Further, in S3, three times the amount of absolute ethanol is added to the second reaction solution, and centrifugation is performed at a centrifugation rate of 3500 - 4000 r / min for 10 - 20 min. When the centrifugation time is less than 10 min or the rotation speed is less than 3500 r / min, excessive small molecules in the solution are not fully removed, resulting in impure polysaccharides after fluorescence labeling; when the centrifugation time is more than 20 min or the rotation speed is more than 4000 r / min, macromolecules in the solution may be removed, thereby reducing the fluorescence labeling degree of the polysaccharides and causing inaccurate experimental results. Experimental results show that centrifugation at a centrifugation rate of 3500 - 4000 r / min for 10 - 20 min can fully remove unreacted substances and obtain fluorescently labeled soybean seed coat polysaccharides.

[0017] Further, between S3 and S4, the following steps are also included: S31, taking the precipitate, redissolving it in water, adding absolute ethanol, and discarding the supernatant after centrifugation; S32, repeating S31 at least twice. In this way, by increasing the number of times of step S31, substances such as free fluorescein can be effectively removed.

[0018] According to another aspect of the present invention, there is provided a fluorescently labeled soybean seed coat polysaccharide, which is prepared by using the above fluorescent labeling method based on improving the thermal stability of soybean polysaccharides.

[0019] Applying the fluorescent labeling method based on improving the thermal stability of soybean polysaccharides provided by the present invention improves the thermal stability of soybean seed coat polysaccharides during labeling, which is beneficial for the tracking and positioning of soybean seed coat polysaccharides in the processes of food production and processing, etc. Applying the fluorescently labeled soybean seed coat polysaccharide provided by the present invention can play a role in fields with high requirements for thermal stability.

[0020] Based on the implementation manners provided in the above aspects of the present application, further combinations can be made to provide more implementation manners. Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly introduced below. Obviously, the following drawings are only some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings without creative efforts based on these drawings.

[0022] Figure 1 It is the regression equation curve of FITC in the present invention;

[0023] Figure 2 It is the fluorescence spectrogram of FTSHP labeled with different FITC concentrations in the present invention;

[0024] Figure 3 These are the ultraviolet spectra of SHP and TSHP in the present invention;

[0025] Figure 4 These are the ultraviolet spectra of FTSHP labeled with different FITC concentrations in the present invention.

[0026] Figure 5 These are the infrared spectra of fluorescently labeled SHP, TSHP, and FTSHP labeled with different FITC concentrations in the present invention;

[0027] Figure 6 From left to right in are the supernatant liquids obtained after adding anhydrous ethanol three times to the second reaction solution and centrifuging in an optional embodiment of the present invention;

[0028] Figure 7 These are the fluorescence micrographs of FTSHP labeled with different FITC concentrations in the present invention, where Figure 7 A is the fluorescence image of FTSHP obtained by labeling polysaccharide with 2.0 wt% FITC, Figure 7 B is the fluorescence image of FTSHP obtained by labeling polysaccharide with 2.5 wt% FITC, Figure 7 C is the fluorescence image of FTSHP obtained by labeling polysaccharide with 3.0 wt% FITC, Figure 7 D is the fluorescence image of FTSHP obtained by labeling polysaccharide with 3.5 wt% FITC;

[0029] Figure 8 These are the graphs showing the evolution of the interfacial tension of SHP, TSHP, and FTSHP labeled with different FITC concentrations over time in the present invention;

[0030] Figure 9 These are the differential scanning heat flow diagrams of SHP, TSHP, and FTSHP labeled with different FITC concentrations in the present invention. Detailed implementation manners

[0031] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention. If not specifically specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0032] Radioisotope labeling method is often used to label polysaccharides. However, due to the harm that radioisotopes can cause to human health, it is not convenient to use. Fluorescent labeling technology, on the other hand, has been widely used in the field of life sciences and other fields due to its advantages such as strong specificity, high sensitivity, and flexible labeling methods. As a non-radioactive technology, fluorescent labeling technology uses the covalent binding of fluorescent substances to target molecular groups to complete the qualitative and quantitative research of target substances. The present invention provides a fluorescent labeling method for soybean polysaccharides, explores the properties and action mechanisms of soybean polysaccharides, and at the same time improves the emulsifying ability and thermal stability of soybean polysaccharides, providing solutions for related research in the fields of water quality monitoring, food monitoring, biological mechanism research of polysaccharides, and pharmacokinetics of soybean polysaccharides.

[0033] An alternative embodiment of the present invention provides a fluorescent labeling method based on improving the thermal stability of soybean polysaccharides, comprising the following steps:

[0034] Step 1: Weigh soybean polysaccharides and dissolve them in 0.2M phosphate buffer solution. After continuously stirring at room temperature for 2 h, place them at 4 °C for 12 h for sufficient hydration.

[0035] Step 2: Add tyramine to the solution obtained in Step 1. After continuously stirring the mixture at 37 °C, add sodium cyanoborohydride, and continuously stir at 37 °C. Then centrifuge the reaction solution at 4000 r / min for 10 min, and take the supernatant for freeze-drying to obtain the soybean polysaccharide-tyramine sample (TSHP).

[0036] Step 3: Dissolve the complex obtained in Step 2 in distilled water, adjust the pH of the solution with 0.5M sodium bicarbonate, and then add fluorescein isothiocyanate (FITC) and continuously stir in the dark for 24 h. After the reaction is completed, add three times the amount of absolute ethanol to the reaction solution and centrifuge. Discard the supernatant, dissolve the precipitate in water, and then centrifuge and precipitate with absolute ethanol three times. Finally, freeze-dry the precipitate to obtain the fluorescently labeled soybean polysaccharide.

[0037] Further, in Step 1, the concentration of soy polysaccharide is 2.0 wt% to 3.0 wt%. In Step 2, the concentration of added tyramine is 2.0 wt% to 3.0 wt%. In Step 2, the mixture is continuously stirred at 37°C, and the continuous stirring time is 12 to 24 h. In Step 2, the concentration of added sodium cyanoborohydride is 0.5 wt% to 1.5 wt%. In Step 2, it is continuously stirred at 37°C, and the continuous stirring time is 12 to 36 h. In Step 3, the pH of the solution is adjusted to 7.0 to 9.0 with 0.5 M sodium bicarbonate. In Step 3, the addition amount of FITC is 0.2 wt% to 0.3 wt%. In Step 3, FITC is further added and continuously stirred in the dark for 24 h, and the reaction temperature is 30 to 40°C. In Step 3, the centrifugation rate of adding three times absolute ethanol to the reaction solution is 3500 to 4000 r / min. In Step 3, the reaction solution is centrifuged for 10 to 20 min.

[0038] The following is a detailed description of specific embodiments of the present invention:

[0039] Example 1

[0040] Fluorescent labeling of soybean seed coat polysaccharide

[0041] (1) First, a 2.00 wt% SHP solution is prepared with 0.20 M phosphate buffer solution as the solvent. After continuously stirring at room temperature for 2 h, it is placed at 4°C for 12 h for sufficient hydration.

[0042] (2) Secondly, 2.00 wt% tyramine is added to the solution obtained in step (1), stirred at 37°C for 12 h for sufficient reaction, then 0.50 wt% sodium cyanoborohydride is added and the reaction continues at 37°C for 36 h. After that, the reaction solution is centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant is freeze-dried to obtain TSHP.

[0043] (3) Finally, the complex obtained in step (2) is dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, then 0.25 wt% FITC is added and reacted in the dark at 37°C for 24 h. Three times absolute ethanol is added to the reaction solution and centrifuged at 3500 r / min for 10 min. The supernatant is removed by centrifugation, the precipitate is redissolved in water, and then absolute ethanol is added for centrifugal precipitation twice. Finally, the precipitate is freeze-dried to obtain FTSHP.

[0044] Example 2

[0045] Fluorescent labeling of soybean seed coat polysaccharide

[0046] (1) First, a 2.50 wt% SHP solution is prepared with 0.20 M phosphate buffer solution as the solvent. After continuously stirring at room temperature for 2 h, it is placed at 4°C for 12 h for sufficient hydration.

[0047] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir for 12 h at 37 °C to allow full reaction, then add 0.50 wt% sodium cyanoborohydride and continue to react at 37 °C for 36 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0048] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.25 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the volume of absolute ethanol to the reaction solution and centrifuge at 3500 r / min for 10 min. Discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge and precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0049] Example 3

[0050] Fluorescent labeling of soybean seed coat polysaccharide

[0051] (1) First, prepare a 3.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0052] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir for 12 h at 37 °C to allow full reaction, then add 0.50 wt% sodium cyanoborohydride and continue to react at 37 °C for 36 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0053] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.25 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the volume of absolute ethanol to the reaction solution and centrifuge at 3500 r / min for 10 min. Discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge and precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0054] Example 4

[0055] Fluorescent labeling of soybean seed coat polysaccharide

[0056] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0057] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir for 18 h at 37 °C to allow sufficient reaction, then add 0.50 wt% sodium cyanoborohydride and continue to react at 37 °C for 36 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0058] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.25 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the volume of absolute ethanol to the reaction solution and centrifuge at 3500 r / min for 10 min. Discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge to precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0059] Example 5

[0060] Fluorescent labeling of soybean seed coat polysaccharide

[0061] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0062] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir for 24 h at 37 °C to allow sufficient reaction, then add 0.50 wt% sodium cyanoborohydride and continue to react at 37 °C for 36 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0063] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.25 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the volume of absolute ethanol to the reaction solution and centrifuge at 3500 r / min for 10 min. Discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge to precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0064] Example 6

[0065] Fluorescent labeling of soybean seed coat polysaccharide

[0066] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0067] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir for 18 h at 37 °C to allow sufficient reaction, then add 0.75 wt% sodium cyanoborohydride and continue to react at 37 °C for 36 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, lyophilize the supernatant to obtain TSHP;

[0068] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.25 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the volume of absolute ethanol to the reaction solution, centrifuge at 3500 r / min for 10 min, discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge to precipitate twice. Finally, lyophilize the precipitate to obtain FTSHP.

[0069] Example 7

[0070] Fluorescent labeling of soybean seed coat polysaccharide

[0071] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0072] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir for 18 h at 37 °C to allow sufficient reaction, then add 1.50 wt% sodium cyanoborohydride and continue to react at 37 °C for 36 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, lyophilize the supernatant to obtain TSHP;

[0073] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.25 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the volume of absolute ethanol to the reaction solution, centrifuge at 3500 r / min for 10 min, discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge to precipitate twice. Finally, lyophilize the precipitate to obtain FTSHP.

[0074] Example 8

[0075] Fluorescent labeling of soybean seed coat polysaccharide

[0076] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0077] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir for 18 h at 37 °C to allow sufficient reaction, then add 0.75 wt% sodium cyanoborohydride and continue to react at 37 °C for 24 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0078] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.25 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the volume of absolute ethanol to the reaction solution, centrifuge at 3500 r / min for 10 min, discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge to precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0079] Example 9

[0080] Fluorescent labeling of soybean seed coat polysaccharide

[0081] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0082] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir for 36 h at 37 °C to allow sufficient reaction, then add 0.75 wt% sodium cyanoborohydride and continue to react at 37 °C for 36 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0083] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.25 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the volume of absolute ethanol to the reaction solution, centrifuge at 3500 r / min for 10 min, discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge to precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0084] Example 10

[0085] Fluorescent labeling of soybean seed coat polysaccharide

[0086] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0087] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir for 18 h at 37 °C to allow sufficient reaction, then add 0.75 wt% sodium cyanoborohydride and continue to react at 37 °C for 24 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0088] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 7 with 0.50 M sodium bicarbonate, then add 0.25 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the volume of absolute ethanol to the reaction solution and centrifuge at 3500 r / min for 10 min. Discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge to precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0089] Example 11

[0090] Fluorescent labeling of soybean seed coat polysaccharide

[0091] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0092] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir for 18 h at 37 °C to allow sufficient reaction, then add 0.75 wt% sodium cyanoborohydride and continue to react at 37 °C for 24 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0093] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 9 with 0.50 M sodium bicarbonate, then add 0.25 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the volume of absolute ethanol to the reaction solution and centrifuge at 3500 r / min for 10 min. Discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge to precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0094] Example 12

[0095] Fluorescent labeling of soybean seed coat polysaccharide

[0096] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0097] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir at 37 °C for 18 h to allow sufficient reaction, then add 0.75 wt% sodium cyanoborohydride and continue to react at 37 °C for 24 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0098] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.27 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the volume of absolute ethanol to the reaction solution, centrifuge at 3500 r / min for 10 min, discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge to precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0099] Example 13

[0100] Fluorescent labeling of soybean seed coat polysaccharide

[0101] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0102] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir at 37 °C for 18 h to allow sufficient reaction, then add 0.75 wt% sodium cyanoborohydride and continue to react at 37 °C for 24 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0103] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.30 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the volume of absolute ethanol to the reaction solution, centrifuge at 3500 r / min for 10 min, discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge to precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0104] Example 14

[0105] Fluorescent labeling of soybean seed coat polysaccharide

[0106] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0107] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir at 37 °C for 18 h to allow sufficient reaction, then add 0.75 wt% sodium cyanoborohydride and continue to react at 37 °C for 24 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0108] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.30 wt% FITC and react in the dark at 30 °C for 24 h. Add three times the volume of absolute ethanol to the reaction solution and centrifuge at 3500 r / min for 10 min. Discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge to precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0109] Example 15

[0110] Fluorescent labeling of soybean seed coat polysaccharide

[0111] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0112] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir at 37 °C for 18 h to allow sufficient reaction, then add 0.75 wt% sodium cyanoborohydride and continue to react at 37 °C for 24 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0113] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.30 wt% FITC and react in the dark at 40 °C for 24 h. Add three times the volume of absolute ethanol to the reaction solution and centrifuge at 3500 r / min for 10 min. Discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge to precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0114] Example 16

[0115] Fluorescent labeling of soybean seed coat polysaccharide

[0116] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0117] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir for 18 h at 37 °C to allow full reaction, then add 0.75 wt% sodium cyanoborohydride and continue to react at 37 °C for 24 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0118] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.30 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the amount of absolute ethanol to the reaction solution and centrifuge at 3700 r / min for 10 min. Discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge and precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0119] Example 17

[0120] Fluorescent labeling of soybean seed coat polysaccharide

[0121] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0122] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir for 18 h at 37 °C to allow full reaction, then add 0.75 wt% sodium cyanoborohydride and continue to react at 37 °C for 24 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0123] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.30 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the amount of absolute ethanol to the reaction solution and centrifuge at 4000 r / min for 10 min. Discard the supernatant after centrifugation, redissolve the precipitate in water, add absolute ethanol again and centrifuge and precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0124] Example 18

[0125] Fluorescent labeling of soybean seed coat polysaccharide

[0126] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0127] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir for 18 h at 37 °C to allow sufficient reaction, then add 0.75 wt% sodium cyanoborohydride and continue to react at 37 °C for 24 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0128] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.30 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the amount of absolute ethanol to the reaction solution and centrifuge at 3700 r / min for 15 min. Centrifuge to remove the supernatant, redissolve the precipitate in water, add absolute ethanol again and centrifuge and precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0129] Example 19

[0130] Fluorescent labeling of soybean seed coat polysaccharide

[0131] (1) First, prepare a 2.00 wt% SHP solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0132] (2) Secondly, add 2.00 wt% tyramine to the solution obtained in step (1), stir for 18 h at 37 °C to allow sufficient reaction, then add 0.75 wt% sodium cyanoborohydride and continue to react at 37 °C for 24 h. After that, centrifuge the reaction solution at 4000 r / min for 10 min. After centrifugation, freeze-dry the supernatant to obtain TSHP;

[0133] (3) Finally, dissolve the complex obtained in step (2) in distilled water, adjust the pH to 8 with 0.50 M sodium bicarbonate, then add 0.30 wt% FITC and react in the dark at 37 °C for 24 h. Add three times the amount of absolute ethanol to the reaction solution and centrifuge at 3700 r / min for 20 min. Centrifuge to remove the supernatant, redissolve the precipitate in water, add absolute ethanol again and centrifuge and precipitate twice. Finally, freeze-dry the precipitate to obtain FTSHP.

[0134] Example 20

[0135] Fluorescent labeling of soybean soluble polysaccharide

[0136] (1) First, prepare a 2.00 wt% soybean soluble polysaccharide solution using 0.20 M phosphate buffer solution as the solvent. Continuously stir at room temperature for 2 h, then place at 4 °C for 12 h for sufficient hydration;

[0137] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1), and the mixture was stirred at 37 °C for 12 h to allow sufficient reaction. Subsequently, 0.75 wt% sodium cyanoborohydride was added and the reaction continued at 37 °C for 24 h. After that, the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0138] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate. Subsequently, 0.30 wt% FITC was added and the reaction was carried out in the dark at 40 °C for 24 h. Three times the volume of absolute ethanol was added to the reaction solution, and it was centrifuged at 3700 r / min for 20 min. The supernatant was removed by centrifugation, the precipitate was redissolved in water, and then absolute ethanol was added for centrifugal precipitation twice. Finally, the precipitate was freeze-dried to obtain the fluorescently labeled soybean soluble polysaccharide.

[0139] Example 21

[0140] Fluorescent labeling of okara polysaccharide

[0141] (1) First, a 2.50 wt% okara polysaccharide solution was prepared using 0.20 M phosphate buffer solution as the solvent. After continuous stirring at room temperature for 2 h, it was placed at 4 °C for 12 h for sufficient hydration;

[0142] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1), and the mixture was stirred at 37 °C for 12 h to allow sufficient reaction. Subsequently, 0.75 wt% sodium cyanoborohydride was added and the reaction continued at 37 °C for 24 h. After that, the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0143] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 9 with 0.50 M sodium bicarbonate. Subsequently, 0.30 wt% FITC was added and the reaction was carried out in the dark at 40 °C for 24 h. Three times the volume of absolute ethanol was added to the reaction solution, and it was centrifuged at 4000 r / min for 20 min. The supernatant was removed by centrifugation, the precipitate was redissolved in water, and then absolute ethanol was added for centrifugal precipitation twice. Finally, the precipitate was freeze-dried to obtain the fluorescently labeled okara polysaccharide.

[0144] Example 22

[0145] Fluorescent labeling of soybean meal polysaccharide

[0146] (1) First, a 2.00 wt% soybean meal polysaccharide solution was prepared using 0.20 M phosphate buffer solution as the solvent. After continuous stirring at room temperature for 2 h, it was placed at 4 °C for 12 h for sufficient hydration;

[0147] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1), and the mixture was stirred at 37 °C for 18 h to allow sufficient reaction. Subsequently, 1.5 wt% sodium cyanoborohydride was added, and the reaction was continued at 37 °C for 24 h. After that, the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0148] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, and the pH was adjusted to 7 with 0.50 M sodium bicarbonate. Subsequently, 0.30 wt% FITC was added, and the reaction was carried out in the dark at 40 °C for 24 h. Three times the volume of absolute ethanol was added to the reaction solution, and the mixture was centrifuged at 3700 r / min for 15 min. The supernatant was removed by centrifugation, the precipitate was redissolved in water, and then centrifuged and precipitated twice with absolute ethanol. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0149] Comparisons among Examples 1, 2, and 3 showed that adding 2.00 wt% of soybean seed coat polysaccharide to the mixed solution could react sufficiently with tyramine.

[0150] Comparisons among Examples 1, 4, and 5 showed that when 2.00 wt% tyramine was added to the mixed solution and the optimal reaction time at 37 °C was 18 h, the polysaccharide could react sufficiently with tyramine at this time, and the labeling degree of the fluorescent polysaccharide was high.

[0151] Comparisons among Examples 4, 6, and 7 showed that when the addition amounts of both soybean seed coat polysaccharide and tyramine were 2.00 wt%, the optimal addition amount of sodium cyanoborohydride should be 0.75 wt%, and at this time, the soybean seed coat polysaccharide was successfully aminated.

[0152] Comparisons among Examples 6, 8, and 9 showed that when 0.75 wt% sodium cyanoborohydride was added to the reaction solution and the optimal reaction time at 37 °C was 24 h, the polysaccharide linked to tyramine could react sufficiently with sodium cyanoborohydride.

[0153] Comparisons among Examples 8, 10, and 11 showed that the solution should be adjusted to pH 8.0 with 0.50 M sodium bicarbonate so that FITC could react sufficiently with the aminated polysaccharide, and the resulting polysaccharide had a high fluorescence labeling degree.

[0154] Comparisons among Examples 8, 12, and 13 showed that when 0.30 wt% FITC was added to the reaction solution, the resulting fluorescently labeled polysaccharide had a high fluorescence intensity and good thermal stability, as Figure 2 and 9 shown.

[0155] Comparisons among Examples 13, 14, and 15 showed that when 0.30 wt% FITC was added to the reaction solution, the reaction temperature of the system should be maintained at 37 °C at this time, and the resulting fluorescently labeled polysaccharide had a strong fluorescence signal, as Figure 7 shown.

[0156] Comparisons among Examples 13, 16 and 17 show that when three times of absolute ethanol is added to the reaction solution and centrifuged at a rate of 3700 r / min, free small molecules in the system can be sufficiently removed.

[0157] Comparisons among Examples 16, 18 and 19 show that when three times of absolute ethanol is added to the reaction solution and centrifuged for 15 min, free fluorescein in the system can be removed.

[0158] Examples 1-19 show that when the addition amounts of polysaccharide and tyramine are both 2.00 wt%, and the reaction is carried out at 37 °C for 18 h, 0.75 wt% of sodium cyanoborohydride is added to the obtained reaction solution, after reacting at 37 °C for 24 h, centrifuged at 4000 r / min for 10 min, and the supernatant is taken for freeze-drying. The complex is dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, 0.30 wt% of FITC is added to the solution, reacted at 37 °C in the dark for 24 h, three times of absolute ethanol is added to the reaction solution and centrifuged at 3700 r / min for 15 min, the precipitate is redissolved with water, and then centrifuged and precipitated twice with absolute ethanol, and finally the precipitate is freeze-dried to obtain FTSHP. The obtained polysaccharide has strong fluorescence intensity and its thermal stability is significantly improved.

[0159] Examples 19-22 show that the fluorescence labeling method of the present invention is applicable to some other soybean polysaccharides, and no examples are given one by one here.

[0160] In the present application, the process parameters in the fluorescence labeling method can be adjusted according to actual needs.

[0161] Drawing of the FITC standard curve: Accurately weigh FITC and prepare a 1.0 mg / mL solution with phosphate buffer (pH 7.4), and perform gradient dilution with phosphate buffer (pH 7.4) to prepare FITC solutions with concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0 μg / mL respectively, using phosphate buffer (pH 7.4) as the blank control. At an excitation wavelength of 490 nm and a wavelength range of 510-700 nm, the fluorescence intensity of each sample is measured by a fluorescence spectrophotometer. The experimental results show that the regression equation of FITC is y = 562.86x - 77.50 (R 2 = 0.9907), as Figure 1 shown.

[0162] Accurately weigh FTSHP and prepare a 2 mg / mL solution with phosphate buffer (pH 7.4). Under the conditions of an excitation wavelength of 490 nm and a scanning frequency of 2400 nm / min, use a fluorescence spectrophotometer to measure the fluorescence spectrogram with a scanning range of 510-600 nm, as Figure 2As shown, as the FITC concentration increased from 0.20 wt% to 0.35 wt%, a significant increase in the fluorescence intensity of FTSHP was observed. The fluorescence intensity values of FTSHP measured at FITC concentrations of 0.20 wt%, 0.25 wt%, 0.30 wt%, and 0.35 wt% were respectively substituted into the FITC regression equation, and the fluorescence substitution degrees of the corresponding FITC in FTSHPs were calculated to be 1.51%, 1.58%, 1.63%, and 1.67%, respectively.

[0163] To further explore whether the fluorescence labeling reaction was completed, ultraviolet spectral scanning was performed using a UV-visible spectrophotometer. Before measurement, SHP, TSHP, and FTSHP samples were dissolved in water to prepare solutions with a concentration of 2.0 mg / mL. The scanning range of the ultraviolet spectrum was 210 - 700 nm, and the scanning rate was 50 nm / min. The obtained ultraviolet spectra are as Figure 3 and Figure 4 shown. In Figure 3 , compared with SHP, TSHP showed a new characteristic peak corresponding to tyramine at 274 nm, indicating the successful amination of the polysaccharide, that is, the synthesis of TSHP was completed. In Figure 4 , FTSHP showed a new characteristic peak corresponding to FITC at 495 nm, which proved the successful binding of SHP and FITC through a chemical reaction.

[0164] An appropriate amount of KBr was respectively mixed with the freeze-dried powders of SHP, TSHP, and FTSHP at a mass ratio of 1:100, ground into a powder, pressed into a tablet, and placed in a sample cell. The Fourier transform infrared spectrometer was used to scan in the spectral range of 4000 - 400 cm -1 with a resolution of 4 cm -1 , and the obtained infrared spectra are as Figure 5 shown. In the infrared spectrum, the peak intensity of the hydroxyl group in FTSHP gradually decreased with the increase of the FITC content, which may be due to the weakening of the hydrogen bond interaction between intramolecular hydroxyl groups. In addition, the content of ester (COO-R) gradually increased with the increase of the FITC concentration, while the content of free carboxyl group (COO-) decreased. The weakening of the carboxyl group intensity at 1613 cm -1 in the FTSHP spectrum indicated that each TSHP reacted with the FITC molecule during the electrostatic complexation process. As a linker between SHP and FITC, tyramine can not only successfully aminate the reducing end of SHP, but also promote the successful connection of the intermediate product TSHP with the isothiocyanate group of FITC through a nucleophilic reaction under weak alkaline conditions, thus realizing the fluorescence labeling of SHP.

[0165] Figure 6From left to right are the supernatant liquids obtained after adding anhydrous ethanol to the reaction solution containing FITC three times and centrifuging. The supernatant liquids obtained from the three centrifugations gradually became clear and transparent, indicating that the free FITC in the reaction solution was successfully removed.

[0166] Prepare a 5 mg / mL FTSHP solution with phosphate buffer (pH 7.4). Take 2 mL of the FTSHP solution and spread it evenly on a glass slide, cover it with a coverslip, invert it on the stage, and observe it under a 40x objective lens using an inverted fluorescence microscope. The obtained FTSHP fluorescence images are as Figure 7 shown. Green fluorescence can be observed in all FTSHPs images. Among them, the fluorescence signal of 0.20 wt% FTSHP is relatively weak, probably because the FITC content in FTSHP is relatively low at this time. On the contrary, the fluorescence signal of FTSHP with a higher FITC concentration is strong.

[0167] The interfacial tension of the sample at the oil-water interface was recorded using an optical contact angle meter. Before the formal test, the densities of the sample and the oil phase were detected using a portable densitometer. First, 10 μL of the sample solution was dropped into a syringe and then dropped into soybean oil. The interfacial tension of the sample was measured from 0 to 10800 s, and the change in interfacial tension with time is as Figure 8 shown. The interfacial adsorption experiment shows that the interfacial tension of the polysaccharide after fluorescence labeling is significantly lower than that of the pure polysaccharide. Among them, 0.30 wt% FITC shows the best interfacial tension and has a strong ability to reduce the interfacial activity.

[0168] The thermal stability of the polysaccharide after fluorescence labeling was investigated by differential scanning calorimetry. First, 5.0 - 10.0 mg of the sample was placed in a sealed aluminum pan and heated from 40 °C to 250 °C at a rate of 10 °C / min under nitrogen conditions, and the nitrogen flow rate was 30 mL / min. A sealed empty aluminum pan was used as a standard. The DSC curves of SHP, TSHP, and FTSHP are as Figure 9 shown, and the DSC thermal spectrum parameters are shown in Table 1.

[0169] Table 1

[0170]

[0171]

[0172] According to Figure 9The results in Table 1 show that the first not obvious endothermic peak appears in the range of 130 °C to 140 °C in all samples, which may be due to the loss of bound water and the glass transition of the samples, while the second main endothermic peak appears between 140 °C and 160 °C, at which time the skeleton in the polymer may have carbonized. Specifically, the SHP curve has an obvious peak at 152.08 °C, and its enthalpy value is 203.00 J / g. The curve of TSHP has a peak in the range of 130 °C to 140 °C, and its enthalpy value is 123.30 J / mg. When the concentration of FITC is in the range of 0.20 wt% to 0.35 wt%, the enthalpies of FTSHP are all less than 100 J / g (60.53 J / g, 72.49 J / g, 53.79 J / g, and 79.03 J / g respectively). With the increase of the FITC concentration, the enthalpy value gradually decreases and is lower than the enthalpies of SHP and TSHP. Among them, 0.30 wt% FTSHP has the highest thermal stability, which indicates that adding FITC is beneficial to improving the thermal stability of polysaccharides. Thermodynamic experiments show that the enthalpy value of FTSHPs is lower than that of SHP, which proves that the thermal stability of the polysaccharide after fluorescence labeling has been significantly improved.

[0173] An alternative embodiment of the present invention provides a method for fluorescently labeling soybean seed coat polysaccharide by using tyramine as an intermediate to connect soybean seed coat polysaccharide and fluorescein. First, the reducing property at the end of soybean seed coat polysaccharide SHP is used to carry out a reductive amination reaction with tyramine, and unreacted molecules such as tyramine are removed by centrifugation to obtain a soybean seed coat polysaccharide-tyramine complex TSHP. Secondly, the primary amino group in the complex TSHP reacts with the isothiocyanato group of fluorescein isothiocyanate FITC to form a thiourea bond, and FITC undergoes a nucleophilic reaction with SHP. Then, the reaction solution is centrifuged and precipitated three times to remove substances such as free fluorescein. After the precipitate is freeze-dried, the fluorescently labeled soybean seed coat polysaccharide complex FTSHP is obtained.

[0174] In addition, an alternative embodiment of the present invention establishes a fluorescence standard curve of fluorescein FITC and uses it to calculate the fluorescence substitution degree of the FTSHP complex to verify the labeling result. The results show that a fluorescent group that can be used for detection is successfully imparted to SHP, realizing the in-situ visualization of polysaccharides.

[0175] The labeling method of soybean seed coat polysaccharide provided by the present invention has the advantages of easy availability of reaction products, mild reaction conditions, low energy consumption, simple and easy operation, etc. It realizes the directional tracking of soybean seed coat polysaccharide, and at the same time improves the emulsifying ability and thermal stability of soybean seed coat polysaccharide, providing a reference for studying the functions and application mechanisms related to polysaccharides.

[0176] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the said claims.

Claims

1. Application of a fluorescent labeling method in improving the thermal stability of soybean polysaccharides, characterized in that: The marking method comprises the following steps: S1, dissolving soybean polysaccharide in phosphate buffer solution to obtain a mixed solution; S2, sequentially adding tyramine and sodium cyanoborohydride to the mixed solution to obtain a first reaction solution; centrifuging the first reaction solution, taking the supernatant and freeze-drying it to obtain a soybean polysaccharide-tyramine complex; S3, dissolving the soybean polysaccharide-tyramine complex in distilled water, adding sodium bicarbonate and fluorescein isothiocyanate in sequence, and stirring continuously in the dark to obtain a second reaction solution; adding anhydrous ethanol to the second reaction solution, and discarding the supernatant after centrifugation; In the S3, 0.5 M sodium bicarbonate is first added to adjust the pH value to 7.0-9.0, and then 0.2 wt%-0.3 wt% of fluorescein isothiocyanate is added, and the mixture is stirred continuously for 24 h in the dark at a reaction temperature of 30-40° C.; S4, taking the precipitate and freeze-drying it to obtain fluorescently labeled soybean polysaccharide.

2. The use according to claim 1, characterized in that: In S1, the soybean polysaccharide is soybean soluble polysaccharide, soybean seed coat polysaccharide, soybean dregs polysaccharide or soybean meal polysaccharide.

3. The use according to claim 1, characterized in that: In the S1, a phosphate buffer solution with a molar mass of 0.20 M is used to prepare soybean seed coat polysaccharide with a concentration of 2.0 wt% to 3.0 wt%, and after continuous stirring at room temperature for 2 h, the mixture is placed at 4° C. for 12 h to obtain the mixed solution.

4. The use according to claim 1, characterized in that: In S2, tyramine with a concentration of 2.0 wt% to 3.0 wt% is added to the mixed solution, stirred continuously at 37° C. for 12 to 24 h, and then sodium cyanoborohydride is added.

5. The use according to claim 1, characterized in that: In the S2, sodium cyanoborohydride with a concentration of 0.5 wt% to 1.5 wt% is added, and the mixture is continuously stirred at 37° C. for 12 to 36 hours to obtain the first reaction solution.

6. The use according to claim 1, characterized in that: In S2, when the first reaction solution is centrifuged, the parameters are set as follows: the centrifugal speed is 4000 r / min, and the centrifugal time is 10 min.

7. The use according to claim 1, characterized in that: In S3, three times of anhydrous ethanol is added to the second reaction solution, and the solution is centrifuged at a centrifugal speed of 3500-4000 r / min for 10-20 min.

8. The use according to claim 1, characterized in that: Between S3 and S4, the following steps are also included: S31, taking the precipitate, adding water to re-dissolve it, adding anhydrous ethanol, centrifuging it and discarding the supernatant; S32, repeat S31 at least twice.

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