Fluorescence labeling method based on improvement of thermal stability of soybean polysaccharide

Abstract

The invention relates to the technical field of fluorescence labeling biomacromolecules, and provides a fluorescence labeling method based on improvement of thermal stability of soybean polysaccharide, which comprises the following steps: S1, dissolving soybean polysaccharide in a phosphate buffer solution to obtain a mixed solution; s2, tyramine and sodium cyanoborohydride are sequentially added into the mixed solution, and a first reaction solution is obtained; the first reaction solution is subjected to centrifugal treatment, supernate is taken and subjected to freeze drying, and a soybean polysaccharide-tyramine compound is obtained; s3, dissolving the soybean polysaccharide-tyramine compound in water, sequentially adding sodium bicarbonate and fluorescein isothiocyanate, and continuously stirring in a dark place to obtain a second reaction solution; absolute ethyl alcohol is added into the second reaction solution, and supernate is discarded after centrifugal treatment; s4, the precipitate is taken and subjected to freeze drying, the fluorescently-labeled soybean polysaccharide is obtained, and the thermal stability of the soybean polysaccharide is improved while labeling is conducted.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescently labeled biomacromolecules, and in particular to a fluorescent labeling method based on improving the thermal stability of soybean polysaccharides. Background Art

[0002] Soybean polysaccharides are polysaccharides extracted from soybeans and their by-products (seed coat, dregs and soybean meal), which have multiple biological activities such as lowering blood sugar, lowering blood lipids, and regulating intestinal microecology. In addition, soybean polysaccharide molecules contain hydrophilic sugar chains, glycoproteins and uronic acid groups, which give them good emulsification, thickening and gel properties. However, due to the large chemical molecular weight and complex structure of soybean polysaccharides, and the lack of fluorescent groups and ultraviolet absorption groups that can be used for detection, there are difficulties in the quantitative / qualitative analysis of soybean polysaccharides, which is not conducive to the tracking and positioning of soybean polysaccharides in food production and processing and related research.

[0003] However, the existing technology lacks research on labeling soybean polysaccharides. At the same time, soybean polysaccharides have poor thermal stability and are prone to conformational changes such as aggregation during heat treatment, which affects detection accuracy. Therefore, how to provide a method for labeling soybean polysaccharides and improve the thermal stability of soybean polysaccharides while labeling has become a technical problem that urgently needs 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 polysaccharides and fluorescently labeled soybean polysaccharides, so as to solve the technical problems in the prior art of lacking a fluorescent labeling method based on improving the thermal stability of soybean polysaccharides and being unable to improve the thermal stability of soybean polysaccharides while labeling.

[0005] In order to achieve the above-mentioned purpose, according to one aspect of the present invention, a fluorescent labeling method based on improving the thermal stability of soybean polysaccharides is provided, and the labeling method comprises the following steps: S1, dissolving soybean polysaccharides 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, and continuously stirring in the dark to obtain a second reaction solution; adding anhydrous ethanol to the second reaction solution, and discarding the supernatant after centrifugation S4, taking the precipitate for freeze-drying to obtain fluorescently labeled soybean polysaccharides.

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

[0007] Furthermore, in S1, a phosphate buffer solution with a molar mass of 0.20M is used to prepare a soybean seed coat polysaccharide with a concentration of 2.0wt% to 3.0wt%, and after continuous stirring at room temperature for 2h, it is placed at 4°C for 12h to obtain a mixed solution. If the concentration of soybean seed coat polysaccharide is lower than 2.0wt%, 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 3.0wt%, the polysaccharide is prone to excessive aggregation and block formation, which is not conducive to the full progress of the reaction. The experimental results show that soybean seed coat polysaccharide with a concentration range of 2.0wt% to 3.0wt% can be fully dissolved in the phosphate buffer solution and does not affect the subsequent chemical reaction.

[0008] Further, in S2, tyramine with a concentration of 2.0wt% to 3.0wt% is added to the mixed solution, stirred continuously at 37°C for 12 to 24h, and then sodium cyanoborohydride is added. When the tyramine concentration is lower than 2.0wt%, unreacted soybean seed coat polysaccharide exists in the mixed solution; when the tyramine concentration exceeds 3.0wt%, the excess tyramine reacts with FITC, causing part of the soybean seed coat polysaccharide to fail to react with sodium cyanoborohydride through tyramine, which is not conducive to the fluorescent labeling of the polysaccharide. Experiments show that tyramine with a concentration of 2.0wt% to 3.0wt% can successfully aminize polysaccharides, which is conducive to the smooth progress of fluorescent labeling.

[0009] Furthermore, in S2, sodium cyanoborohydride is added at a concentration of 0.5wt% to 1.5wt%, and stirred continuously at 37°C for 12 to 36h to obtain a first reaction solution. When the concentration of sodium cyanoborohydride is lower than 0.5wt%, there is an excess of soybean seed coat polysaccharide connected to tyramine in the solution, and this part of the polysaccharide cannot react chemically with FITC, thereby reducing the fluorescent labeling degree of the polysaccharide; when the concentration of sodium cyanoborohydride is higher than 1.5wt%, the excess sodium cyanoborohydride in the solution affects the Brownian motion of the polysaccharide and FITC, and reduces the chemical reaction rate. Experiments have shown that when the concentration of sodium cyanoborohydride is in the range of 0.5wt% to 1.5wt%, the polysaccharide can fully react with FITC after amination to complete the fluorescent labeling of the polysaccharide.

[0010] Further, in S2, when the first reaction solution is centrifuged, the parameters are set as:

[0011] The centrifugal speed is 4000 r / min and the centrifugal time is 10 min. Centrifugation at a speed of 4000 r / min for 10 min can fully remove the small molecular substances that have not fully reacted and obtain the aminated polysaccharide.

[0012] Furthermore, in S3, 0.5M sodium bicarbonate is first added to adjust the pH value to 7.0-9.0. 0.5M sodium bicarbonate is used to adjust the solution to a slightly alkaline environment to 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 solution pH exceeds 9.0, there are too many hydroxides in the solution, the chemical structure of the polysaccharide is destroyed, and it cannot fully react with FITC, resulting in low fluorescence labeling, and fluorescence labeling is not easy to carry out. Experiments have confirmed that using 0.5M sodium bicarbonate to adjust the pH value to 7.0-9.0 provides a slightly alkaline environment for the solution, allowing FITC to fully react with soybean seed coat polysaccharides.

[0013] Furthermore, in S3, fluorescein isothiocyanate at a concentration of 0.2wt% to 0.3wt% should be added to the reaction solution. When the concentration of fluorescein isothiocyanate is lower than 0.2wt%, there is an excess of unlabeled polysaccharides in the solution; when the concentration of fluorescein isothiocyanate is higher than 0.3wt%, the excess free fluorescein in the solution will be mistaken for successfully labeled polysaccharides, thereby interfering with the judgment of the experimental results. The experimental results show that fluorescein isothiocyanate at a concentration of 0.2wt% to 0.3wt% can successfully achieve fluorescent labeling of soybean seed coat polysaccharides, while producing less free fluorescein.

[0014] Furthermore, in S3, the reaction temperature after adding fluorescein isothiocyanate to the reaction solution should be maintained at 30-40°C. When the temperature of the reaction solution is lower than 30°C, the solubility of fluorescein isothiocyanate is low, which is not conducive to the chemical reaction between fluorescein and polysaccharide; when the temperature of the reaction solution is higher than 40°C, the chemical bonds of the polysaccharide are prone to break and the structure is destroyed, resulting in the inability of the polysaccharide to react chemically with fluorescein. The experimental results show that when the temperature of the reaction solution is within the range of 30-40°C, the fluorescent labeling of polysaccharides can be completed.

[0015] Furthermore, in S3, after adding fluorescein isothiocyanate, stirring is continued for 24 hours in the dark. In this way, by limiting the time of continuous stirring in the dark, the polysaccharide and fluorescein can react completely, thereby completing the fluorescent labeling of the polysaccharide.

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

[0017] Furthermore, between S3 and S4, the following steps are included: S31, taking the precipitate, adding water to dissolve it, adding anhydrous ethanol, centrifuging it and discarding the supernatant; S32, repeating S31 at least twice. In this way, by increasing the number of steps S31, free substances such as fluorescein can be effectively removed.

[0018] According to another aspect of the present invention, a fluorescently labeled soybean testa polysaccharide is provided. The fluorescently labeled soybean testa polysaccharide is prepared by using the fluorescent labeling method based on improving the thermal stability of soybean polysaccharide.

[0019] The fluorescent labeling method based on improving the thermal stability of soybean polysaccharides provided by the present invention is used to improve the thermal stability of soybean seed coat polysaccharides while labeling, thereby facilitating the tracking and positioning of soybean seed coat polysaccharides in food production and processing. The fluorescently labeled soybean seed coat polysaccharides provided by the present invention can be used in fields with high requirements for thermal stability.

[0020] Based on the implementations provided in the above aspects, this application can also be further combined to provide more implementations. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

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

[0023] Figure 2 The fluorescence spectra of FTSHP labeled with different FITC concentrations in the present invention are shown in FIG.

[0024] Figure 3 It is the ultraviolet spectrum of SHP and TSHP in the present invention;

[0025] Figure 4 It is the ultraviolet spectrum diagram of FTSHP labeled with different FITC concentrations in the present invention.

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

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

[0028] Figure 7 The fluorescence micrographs of FTSHP labeled with different FITC concentrations in the present invention are shown in FIG. Figure 7 A is the fluorescence image of FTSHP obtained by 2.0wt% FITC labeled polysaccharide. Figure 7 B is the fluorescence image of FTSHP obtained by 2.5wt% FITC labeled polysaccharide. Figure 7 C is the fluorescence image of FTSHP obtained by 3.0wt% FITC labeled polysaccharide. Figure 7 D is the fluorescence image of FTSHP obtained by 3.5 wt % FITC-labeled polysaccharide;

[0029] Figure 8 It is a graph showing the evolution of the interfacial tension of SHP, TSHP and FTSHP labeled with different FITC concentrations over time in the present invention;

[0030] Fig. 9 The differential scanning calorimetry diagrams of SHP, TSHP and FTSHP labeled with different FITC concentrations in the present invention are shown. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0032] Radioisotope labeling is often used to label polysaccharides, but since radioisotopes can cause harm to human health, it is not convenient to use. Fluorescent labeling technology has been widely used in fields such as life sciences 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 bonding of fluorescent substances and target molecular groups to complete qualitative and quantitative studies of target substances. The present invention provides a fluorescent labeling method for soybean polysaccharides to explore the properties and mechanism of action of soybean polysaccharides, while improving the emulsification ability and thermal stability of soybean polysaccharides, providing solutions for related research on soybean polysaccharides in the fields of water quality monitoring, food monitoring, and research on the biological mechanism and pharmacokinetics of polysaccharides.

[0033] An optional 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 polysaccharide and dissolve it in 0.2 M phosphate buffer solution. Stir continuously at room temperature for 2 hours, and then place it at 4°C for 12 hours to fully hydrate.

[0035] Step 2: Add tyramine to the solution obtained in step 1, stir the mixture continuously at 37° C., then add sodium cyanoborohydride, stir the mixture continuously at 37° C., centrifuge the reaction solution at 4000 r / min for 10 min, and freeze-dry the supernatant to obtain a 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 0.5 M sodium bicarbonate, add fluorescein isothiocyanate (FITC) and stir continuously for 24 hours in a dark place; after the reaction is completed, add three times of anhydrous ethanol to the reaction solution and centrifuge, discard the supernatant, re-dissolve the precipitate in water, centrifuge and precipitate three times with anhydrous ethanol, and finally freeze-dry the precipitate to obtain fluorescently labeled soybean polysaccharide.

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

[0038] The specific embodiments of the present invention are described in detail below:

[0039] Example 1

[0040] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0041] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0042] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 12 h at 37° C. to allow for sufficient reaction, followed by the addition of 0.50 wt% sodium cyanoborohydride and continued reaction at 37° C. for 36 h. The reaction solution was centrifuged at 4000 r / min for 10 min, and the supernatant was freeze-dried to obtain TSHP.

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

[0044] Example 2

[0045] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0046] (1) First, a 2.50 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0047] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 12 h at 37° C. to allow for sufficient reaction, followed by the addition of 0.50 wt% sodium cyanoborohydride and continued reaction at 37° C. for 36 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0048] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.25 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3500 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0049] Example 3

[0050] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0051] (1) First, a 3.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0052] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 12 h at 37° C. to allow for sufficient reaction, followed by the addition of 0.50 wt% sodium cyanoborohydride and continued reaction at 37° C. for 36 h. The reaction solution was centrifuged at 4000 r / min for 10 min, and the supernatant was freeze-dried to obtain TSHP.

[0053] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.25 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3500 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0054] Example 4

[0055] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0056] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0057] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred at 37°C for 18 h to fully react, and then 0.50 wt% sodium cyanoborohydride was added and the reaction was continued at 37°C for 36 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0058] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.25 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3500 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0059] Example 5

[0060] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0061] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0062] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred at 37°C for 24 h to fully react, followed by adding 0.50 wt% sodium cyanoborohydride and continuing the reaction at 37°C for 36 h, and then the reaction solution was centrifuged at 4000 r / min for 10 min, and the supernatant was freeze-dried to obtain TSHP;

[0063] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.25 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3500 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0064] Example 6

[0065] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0066] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0067] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 18 h at 37° C. to allow for sufficient reaction, followed by the addition of 0.75 wt% sodium cyanoborohydride and continued reaction at 37° C. for 36 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0068] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.25 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3500 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0069] Example 7

[0070] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0071] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0072] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 18 h at 37° C. to allow it to react fully, and then 1.50 wt% sodium cyanoborohydride was added and continued to react at 37° C. for 36 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0073] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.25 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3500 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0074] Example 8

[0075] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0076] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0077] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 18 h at 37° C. to allow for sufficient reaction, followed by the addition of 0.75 wt% sodium cyanoborohydride and continued reaction at 37° C. for 24 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0078] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.25 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3500 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0079] Example 9

[0080] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0081] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0082] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred at 37°C for 36 h to fully react, and then 0.75 wt% sodium cyanoborohydride was added and the reaction was continued at 37°C for 36 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0083] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.25 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3500 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0084] Example 10

[0085] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0086] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0087] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 18 h at 37° C. to allow for sufficient reaction, followed by the addition of 0.75 wt% sodium cyanoborohydride and continued reaction at 37° C. for 24 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0088] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 7 with 0.50 M sodium bicarbonate, and then 0.25 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3500 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0089] Embodiment 11

[0090] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0091] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0092] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 18 h at 37° C. to allow for sufficient reaction, followed by the addition of 0.75 wt% sodium cyanoborohydride and continued reaction at 37° C. for 24 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0093] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 9 with 0.50 M sodium bicarbonate, and then 0.25 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3500 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0094] Example 12

[0095] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0096] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0097] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 18 h at 37° C. to allow for sufficient reaction, followed by the addition of 0.75 wt% sodium cyanoborohydride and continued reaction at 37° C. for 24 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0098] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.27 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3500 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0099] Embodiment 13

[0100] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0101] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0102] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 18 h at 37° C. to allow for sufficient reaction, followed by the addition of 0.75 wt% sodium cyanoborohydride and continued reaction at 37° C. for 24 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0103] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.30 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3500 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0104] Embodiment 14

[0105] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0106] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0107] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 18 h at 37° C. to allow for sufficient reaction, followed by the addition of 0.75 wt% sodium cyanoborohydride and continued reaction at 37° C. for 24 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0108] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.30 wt % FITC was added to react at 30° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3500 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0109] Embodiment 15

[0110] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0111] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0112] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 18 h at 37° C. to allow for sufficient reaction, followed by the addition of 0.75 wt% sodium cyanoborohydride and continued reaction at 37° C. for 24 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0113] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.30 wt % FITC was added to react at 40° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3500 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0114] Example 16

[0115] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0116] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0117] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 18 h at 37° C. to allow for sufficient reaction, followed by the addition of 0.75 wt% sodium cyanoborohydride and continued reaction at 37° C. for 24 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0118] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.30 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3700 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0119] Embodiment 17

[0120] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0121] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0122] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 18 h at 37° C. to allow for sufficient reaction, followed by the addition of 0.75 wt% sodium cyanoborohydride and continued reaction at 37° C. for 24 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0123] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.30 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 4000 r / min for 10 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0124] Embodiment 18

[0125] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0126] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0127] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 18 h at 37° C. to allow for sufficient reaction, followed by the addition of 0.75 wt% sodium cyanoborohydride and continued reaction at 37° C. for 24 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0128] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.30 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3700 r / min for 15 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0129] Embodiment 19

[0130] Fluorescence Labeling of Soybean Seed Coat Polysaccharide

[0131] (1) First, a 2.00 wt% SHP solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0132] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 18 h at 37° C. to allow for sufficient reaction, followed by the addition of 0.75 wt% sodium cyanoborohydride and continued reaction at 37° C. for 24 h, and the reaction solution was centrifuged at 4000 r / min for 10 min. After centrifugation, the supernatant was freeze-dried to obtain TSHP;

[0133] (3) Finally, the complex obtained in step (2) was dissolved in distilled water, adjusted to pH 8 with 0.50 M sodium bicarbonate, and then 0.30 wt % FITC was added to react at 37° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3700 r / min for 20 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0134] Embodiment 20

[0135] Fluorescence Labeling of Soluble Polysaccharides from Soybean

[0136] (1) First, a 2.00 wt% soybean soluble polysaccharide solution was prepared using 0.20 M phosphate buffer solution as solvent, stirred continuously at room temperature for 2 h, and then placed at 4° C. for 12 h to allow for sufficient hydration;

[0137] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 12 h at 37° C. to allow it to react fully, and then 0.75 wt% sodium cyanoborohydride was added and continued to react at 37° C. for 24 h, and 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, and then 0.30 wt % FITC was added to react at 40° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3700 r / min for 20 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain fluorescently labeled soybean soluble polysaccharide.

[0139] Embodiment 21

[0140] Fluorescence labeling of okara polysaccharides

[0141] (1) First, a 2.50 wt% okara polysaccharide solution was prepared using 0.20 M phosphate buffer solution as a solvent, stirred continuously at room temperature for 2 h, and then placed at 4 °C for 12 h to allow for sufficient hydration;

[0142] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 12 h at 37° C. to allow it to react fully, and then 0.75 wt% sodium cyanoborohydride was added and continued to react at 37° C. for 24 h, and 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, and then 0.30 wt % FITC was added to react at 40° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 4000 r / min for 20 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain fluorescently labeled okara polysaccharide.

[0144] Embodiment 22

[0145] Fluorescence labeling of soybean meal polysaccharides

[0146] (1) First, a 2.00 wt% soybean meal polysaccharide solution was prepared using 0.20 M phosphate buffer solution as a solvent, stirred continuously at room temperature for 2 h, and then placed at 4 ° C for 12 h to fully hydrate;

[0147] (2) Secondly, 2.00 wt% tyramine was added to the solution obtained in step (1) and stirred for 18 h at 37° C. to allow for sufficient reaction, followed by the addition of 1.5 wt% sodium cyanoborohydride and continued reaction at 37° C. for 24 h, and 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, adjusted to pH 7 with 0.50 M sodium bicarbonate, and then 0.30 wt % FITC was added to react at 40° C. in the dark for 24 h. Three times of anhydrous ethanol was added to the reaction solution and centrifuged at 3700 r / min for 15 min. The supernatant was removed by centrifugation, the precipitate was re-dissolved in water, and anhydrous ethanol was added to centrifuge and precipitated twice. Finally, the precipitate was freeze-dried to obtain FTSHP.

[0149] Comparison of Examples 1, 2 and 3 shows that adding 2.00 wt% of soybean hull polysaccharide to the mixed solution can fully react with tyramine.

[0150] Comparison of Examples 1, 4 and 5 shows that when 2.00 wt% tyramine is added to the mixed solution, the optimal reaction time at 37° C. is 18 h, at which time the polysaccharide can fully react with tyramine and the labeling degree of the fluorescent polysaccharide is high.

[0151] Comparison of Examples 4, 6 and 7 shows that when the addition amount of soybean seed coat polysaccharide and the addition amount of tyramine are both 2.00 wt%, the optimum addition amount of sodium cyanoborohydride should be 0.75 wt%, at which time soybean seed coat polysaccharide is successfully aminated.

[0152] Comparison of Examples 6, 8 and 9 shows that the optimum reaction time for adding 0.75 wt% sodium cyanoborohydride to the reaction solution at 37° C. is 24 h, and the polysaccharide linked to tyramine can fully react with sodium cyanoborohydride.

[0153] Comparison of Examples 8, 10 and 11 shows that the solution should be adjusted to pH 8.0 with 0.50 M sodium bicarbonate, FITC fully reacts with the aminated polysaccharide, and the obtained polysaccharide has a high degree of fluorescent labeling.

[0154] Comparison of Examples 8, 12 and 13 shows that when 0.30 wt% FITC is added to the reaction solution, the obtained fluorescently labeled polysaccharide has a higher fluorescence intensity and better thermal stability. Figure 2 and 9 shown.

[0155] Comparison of Examples 13, 14 and 15 shows that when 0.30 wt % FITC is added to the reaction solution, the reaction temperature of the system should be maintained at 37° C., and the fluorescent signal of the obtained fluorescently labeled polysaccharide is strong. Figure 7 shown.

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

[0157] Comparison of Examples 16, 18 and 19 shows that when three times anhydrous ethanol is added to the reaction solution and centrifuged for 15 minutes, the free fluorescein in the system can be removed.

[0158] Example 1-19 shows that when the amount of polysaccharide and tyramine added is 2.00wt%, and the reaction is carried out at 37℃ for 18h, 0.75wt% sodium cyanoborohydride is added to the obtained reaction solution, and the reaction is carried out at 37℃ for 24h, and the reaction is centrifuged at 4000r / min for 10min, and the supernatant is freeze-dried. The complex is dissolved in distilled water, adjusted to pH 8 with 0.50M sodium bicarbonate, 0.30wt% FITC is added to the solution, and the reaction is carried out at 37℃ for 24h in the dark, and three times of anhydrous ethanol is added to the reaction solution and centrifuged at 3700r / min for 15min, the precipitate is redissolved in water, and anhydrous ethanol is added to centrifuge and precipitate twice, and finally the precipitate is freeze-dried to obtain FTSHP. The obtained polysaccharide has a large fluorescence intensity and its thermal stability is significantly improved.

[0159] Examples 19-22 show that the fluorescent labeling method of the present invention is applicable to some other soybean polysaccharides, which are not listed one by one here.

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

[0161] Drawing of FITC standard curve: FITC was accurately weighed and prepared into a 1.0 mg / mL solution with phosphate buffer (pH 7.4), and gradient dilution was performed 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. Phosphate buffer (pH 7.4) was used as a blank control. The fluorescence intensity of each sample was measured by a fluorescence spectrophotometer at an excitation wavelength of 490 nm and a wavelength range of 510-700 nm. The experimental results showed that the regression equation of FITC was y=562.86x-77.50(R 2 =0.9907), such as Figure 1 shown.

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

[0163] In order to further explore whether the fluorescent labeling reaction is complete, a UV spectrophotometer was used to perform UV spectrum scanning. Before measurement, SHP, TSHP and FTSHP samples were dissolved in water to prepare a solution with a concentration of 2.0 mg / mL. The scanning range of the UV spectrum was 210-700 nm, and the scanning rate was 50 nm / min. The obtained UV spectrum is as follows: Figure 3 and Figure 4 As shown. Figure 3 Compared with SHP, TSHP showed a new characteristic peak corresponding to tyramine at 274 nm, which indicated the successful amination of polysaccharide, that is, the completion of TSHP synthesis. Figure 4 In the experiment, a new characteristic peak corresponding to FITC appeared at 495 nm in FTSHP, which proved the successful combination of SHP and FITC through chemical reaction.

[0164] Take appropriate amount of KBr and freeze-dried powder of SHP, TSHP and FTSHP at a mass ratio of 1:100, grind into powder tablets and place them in the sample tank. Use Fourier transform infrared spectrometer at 4000-400cm -1 Scanning within the spectral range with a resolution of 4cm -1 The infrared spectrum obtained is as follows Figure 5 As shown in the figure. In the infrared spectrum, the peak intensity of the hydroxyl group in FTSHP gradually decreases with the increase of FITC content, which may be due to the weakening of the hydrogen bonding interaction between the hydroxyl groups in the molecule. In addition, the ester (COO-R) content gradually increases with the increase of FITC concentration, while the free carboxyl (COO-) content decreases. -1 The intensity of the carboxyl group at the end of the TSHP was weakened, indicating that each TSHP reacted with the FITC molecule during the electrostatic complexation process. Tyramine, as a linker between SHP and FITC, not only successfully aminized the reducing end of SHP, but also promoted the successful connection of the intermediate product TSHP with the isothiocyanate group of FITC through a nucleophilic reaction under weak alkaline conditions, thereby achieving fluorescent labeling of SHP.

[0165] Figure 6From left to right in the figure are the supernatants obtained after adding anhydrous ethanol three times to the reaction solution containing FITC and centrifuging. The supernatants obtained after three centrifugations gradually become clear and transparent, indicating that the free FITC in the reaction solution has been successfully removed.

[0166] A 5 mg / mL FTSHP solution was prepared with phosphate buffer (pH 7.4), and 2 mL of FTSHP solution was spread on a glass slide, covered with a cover slip, and inverted on a stage. The FTSHP fluorescence image was obtained under a 40x objective lens using an inverted fluorescence microscope. Figure 7 As shown. Green fluorescence can be observed in all FTSHPs images. Among them, the fluorescence signal of 0.20wt% FTSHP is weak, which may be due to the low FITC content in FTSHP at this time. On the contrary, the fluorescence signal of FTSHP with 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 density of the sample and the oil phase was measured using a portable density meter. First, 10 μL of the sample solution was dripped into a syringe and then dripped into the soybean oil. The interfacial tension of the sample was measured from 0 to 10800 s. The change of interfacial tension with time is shown in Figure 2. Figure 8 The interfacial adsorption experiment showed that the interfacial tension of the fluorescently labeled polysaccharide was significantly lower than that of the pure polysaccharide, among which 0.30wt% FITC showed the best interfacial tension and had a strong ability to reduce interfacial activity.

[0168] The thermal stability of the fluorescently labeled polysaccharides was investigated by differential 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. The nitrogen flow rate was 30 mL / min, and a sealed empty aluminum pan was used as a standard. The DSC curves of SHP, TSHP, and FTSHP are shown in Fig. 9 The DSC thermal spectrum parameters are shown in Table 1.

[0169] Table 1

[0170]

[0171]

[0172] according to Fig. 9The results in Table 1 show that the first inconspicuous endothermic peak appears in the range of 130℃ to 140℃ in all samples, which may be due to the loss of bound water and the glass transition of the sample, while the second main endothermic peak appears between 140℃ and 160℃, when the skeleton in the polymer may be carbonized. Specifically, the SHP curve has an obvious peak at 152.08℃, and its enthalpy value is 203.00J / g. The TSHP curve has a peak in the range of 130℃ to 140℃, and its enthalpy value is 123.30J / mg. When the concentration of FITC is in the range of 0.20wt% to 0.35wt%, the FTSHP enthalpy is less than 100J / g (60.53J / g, 72.49J / g, 53.79J / g and 79.03J / g, respectively). As the concentration of FITC increases, the enthalpy value gradually decreases and is lower than that of SHP and TSHP. Among them, 0.30wt% FTSHP has the highest thermal stability, which indicates that the addition of FITC is beneficial to improve the thermal stability of polysaccharides. Thermodynamic experiments show that the enthalpy value of FTSHPs is lower than that of SHP, proving that the thermal stability of polysaccharides after fluorescent labeling has been significantly improved.

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

[0174] In addition, an optional embodiment of the present invention established a fluorescein FITC fluorescence standard curve, and used it to calculate the fluorescence substitution degree of the FTSHP complex to verify the labeling results. The results showed that SHP was successfully endowed with a fluorescent group that could be used for detection, achieving in situ visualization of the polysaccharide.

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

[0176] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A fluorescent labeling method based on 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; S4, taking the precipitate and freeze-drying it to obtain fluorescently labeled soybean polysaccharide.

2. The fluorescent labeling method 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 marking method 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 hours, the mixture is placed at 4° C. for 12 hours to obtain the mixed solution.

4. The fluorescent labeling method 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 fluorescent labeling method 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 stirred continuously at 37° C. for 12 to 36 hours to obtain the first reaction solution.

6. The fluorescent labeling method 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 fluorescent labeling method according to claim 1, characterized in that: 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, and the reaction temperature is 30-40°C.

8. The fluorescent labeling method 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 rate of 3500-4000 r / min for 10-20 min.

9. The fluorescent labeling method 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.

10. A fluorescently labeled soybean polysaccharide, characterized in that: The fluorescently labeled soybean polysaccharide is prepared by using the fluorescent labeling method based on improving the thermal stability of soybean polysaccharide according to any one of claims 1 to claim 9.

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