A method for observing the reaction sites of crosslinked starch

Abstract

The present invention discloses a method for observing the reaction sites of crosslinked starch, including the steps of preparation of crosslinked starch, silver nitrate staining, centrifugation, washing of the dye, centrifugation, UV reduction, preparation of a suspension of stained starch granules, and observation with a laser confocal microscope. Specifically, STMP crosslinked starch is prepared by a semi-dry method or POCl3 crosslinked starch is prepared by a wet method; silver nitrate is used to convert the anions of starch granules into silver salts; the silver ions of the silver-exchanged starch are reduced to silver atoms (Ag + →Ag ° ) by ultraviolet light; the sample is observed by optical sectioning with a laser confocal microscope, and the distribution of reaction sites in the starch granules is obtained by excitation and in-situ scanning imaging. The present invention provides a simple, effective, and direct method for observing the distribution of reaction sites of crosslinked starch.

Description

Technical Field

[0001] The present invention relates to a method for observing the reaction sites of cross-linked starch, belonging to the technical field of dyeing of cross-linked modified starch. Background Art

[0002] Due to some disadvantages of natural starch, such as poor solubility, intolerance to shear, and a high degree of aging, its application in industry is restricted. The chemical method is the most widely used method in the starch modification process. Through chemical modification, starches such as cross-linked, substituted, oxidized, and acid-hydrolyzed starches can be produced. Cross-linked starch refers to a substance with a multi-network structure in which hydroxyl groups in the original starch molecules form diester bonds or diether bonds with compounds containing n-functional groups (n≥2). Cross-linked starch is mainly used as a thickener and conditioner in sauces, soups, baked foods, and dairy products. However, with the further opening of the market, the requirement for food diversification has promoted the development of modified starches with specific functions as the main trend.

[0003] The functional properties of cross-linked starch are closely related to the cross-linking sites. Phosphate groups are bound to starch molecules through covalent bonds. If the intramolecular and intermolecular bond energies within the starch granules are enhanced, the granule stability is strengthened, a higher temperature is required for gelatinization, the peak viscosity decreases, and the viscosity peak temperature increases. If the cross-linking reaction only occurs on the surface of the starch granules, the solubility of the starch granules decreases, and the cross-linking of starch inhibits the dissolution of the soluble substances in the starch granules, enhances the mechanical strength of the starch granules, and enhances the viscosity of the modified starch paste. However, at present, there is a lack of revelation of the reaction sites of cross-linked starch, and more often it is expressed by the degree of cross-linking, but the characterization of the degree of cross-linking is not satisfactory. The existing methods all have limitations. For example, the viscosity method can only characterize starches with a relatively low degree of cross-linking because the addition of a higher amount of cross-linking agent will cause the starch to no longer show viscosity, and even within the detectable viscosity range, as the amount of cross-linking agent added increases, the viscosity of the starch first increases and then decreases, and its change is not linear; the sedimentation volume method is also a method for characterizing the degree of cross-linking, but for starches with a relatively low degree of cross-linking, the differences between samples are small; the national standard uses the method of phosphorus content to characterize the degree of cross-linking of cross-linked starch. Since the original starch itself contains free phosphorus that is easy to remove, the phosphorus content of cross-linked starch with a relatively low degree of cross-linking is lower than that of the original starch. How to more effectively clarify the cross-linking sites and the degree of cross-linking of cross-linked starch is an urgent problem to be solved at present.

[0004] During the cross-linking process, the starch granule structure ultimately determines the accessibility of individual starch molecules to the cross-linking agent, affects the cross-linking sites and the degree of cross-linking of starch, and thus affects the functional properties of starch. Therefore, the degree of cross-linking can be characterized by understanding the cross-linking reaction process within the starch granules. Summary of the Invention

[0005] The object of the present invention is to clarify the crosslinking reaction process within starch granules, provide a simple, effective method for directly observing the distribution of crosslinking reaction sites in crosslinked starch, and intuitively understand the degree of crosslinking of starch granules, which is of great significance for guiding the optimization of crosslinked starch processes and the research on functional properties, as well as the application of crosslinked starch.

[0006] The object of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a method for observing crosslinking reaction sites in crosslinked starch, including:

[0008] Preparation of crosslinked starch;

[0009] Silver nitrate staining: Using silver nitrate to convert anions in crosslinked starch granules into silver salts to obtain silver-exchanged starch granules;

[0010] UV reduction: Using ultraviolet light to reduce silver ions in silver-exchanged starch to silver atoms (Ag+ → Ag°);

[0011] Preparation of stained starch granule suspension: Adding the stained starch granules obtained by UV reduction to an aqueous sucrose solution so that the final concentration of starch granules is 4 - 6%, to prepare a stained starch granule suspension;

[0012] Observation with a laser confocal microscope: Using a laser confocal microscope to perform optical sectioning observation on the sample, and obtaining the distribution of reaction sites in starch granules through excitation and in-situ scanning imaging.

[0013] As an embodiment, the crosslinked starch includes STMP crosslinked starch or POCl3 crosslinked starch.

[0014] As an embodiment, STMP crosslinked starch is prepared by a semi-dry method; specifically including: adjusting the pH value of the starch aqueous solution to 10, slowly dropping a mixed solution of STMP and Na2SO4, and continuously reacting the reaction mixture at a pH of 10 - 11 at room temperature for 1 - 2 hours; drying at 40 - 50°C until the moisture content is below 10 wt.%, and drying at 130 - 150°C for 2 - 3 hours; redissolving in deionized water, adjusting the pH to 6.5 to terminate the reaction, and washing with deionized water to obtain STMP crosslinked starch. The mass ratio of STMP, sodium sulfate, and starch is 0.05 - 0.4:1:20.

[0015] As an embodiment, wet method is used to prepare POCl3 cross-linked starch; specifically, it includes: adjusting the pH value of the starch aqueous solution to 10 - 11.3, slowly dropping the POCl3 solution, maintaining the pH value of the slurry at 11.3 and continuously reacting for 1 - 2 hours; adjusting the pH to 6.5 to terminate the reaction, washing with deionized water and then with absolute ethanol, and subjecting the obtained starch suspension to vacuum filtration and air drying at room temperature to obtain POCl3 cross-linked starch. The concentration of the starch aqueous solution is 0.5 g / mL; the dosage ratio of the starch to the POCl3 solution for cross-linking is 20 g:0.067 - 6.667 mL.

[0016] As an embodiment, in the silver nitrate staining, the dry basis sample of STMP cross-linked starch or POCl3 cross-linked starch is added to a 0.25 - 0.5 M silver nitrate solution, centrifugally vortexed for 10 - 15 s, and then continuously shaken and reacted at 25 - 35 °C for 48 - 72 h. After the reaction, the suspension is centrifuged for 10 - 15 min (3000 - 6000×g), and the supernatant is discarded; the residual dye is washed away with distilled water, and suction filtration is carried out with 85% ethanol, followed by air drying to obtain silver-exchanged starch particles. During the staining reaction process, continuous shaking is required to uniformly disperse the starch particles in the silver nitrate solution to ensure that all the starch can contact with silver nitrate, thus ensuring the uniformity of the reaction.

[0017] As an embodiment, the dosage ratio of the dry basis sample of the cross-linked starch to the silver nitrate solution is 0.2 - 0.5 g:20 mL.

[0018] As an embodiment, before suction filtration with 85% ethanol, the residual silver nitrate is removed with distilled water, continuously shaken for 30 - 60 min, centrifuged, and the supernatant is discarded.

[0019] As an embodiment, the silver nitrate staining step is repeated 2 - 3 times.

[0020] As an embodiment, the entire process of silver nitrate staining is carried out under light-proof conditions.

[0021] As an embodiment, for UV reduction, the silver-exchanged starch is placed under UV 254 nm for more than 48 h to completely reduce the silver ions in the starch particles to silver atoms.

[0022] As an embodiment, in the preparation of the starch particle suspension, the concentration of the sucrose aqueous solution is 20 wt.% - 30 wt.%.

[0023] As an embodiment, after the starch particle suspension is vortexed for 10 - 50 s, 5 - 20 μL of the sample is aspirated and dropped onto a glass slide, and the liquid droplet is evenly spread on the glass slide with a pipette tip and waited to be observed.

[0024] As an implementation example, a glass slide coated with a starch granule suspension is placed above an objective lens (100×) pre-dropped with cedarwood oil, and excited using an Argon (488 nm) laser operating at 80%-100% power respectively; the beam splitter is RT15 / 85, and signals reflected from the excitation light of the material are collected at wavelength intervals of 485 - 506 nm.

[0025] In some embodiments, a method for observing the reaction sites of STMP cross-linked starch is provided, including the following steps:

[0026] (1) Preparation of STMP cross-linked starch: First, sodium trimetaphosphate (STMP, 0.05 - 0.4 g) and sodium sulfate (Na2SO4, 1 g) are dissolved in 5 mL of deionized water, and 20 g of starch is dispersed in 35 mL of deionized water and stirred evenly at room temperature. The pH value of the solution is adjusted to 10 with 1 M NaOH, and then the mixed solution of STMP and Na2SO4 is slowly added dropwise. The reaction mixture continues to react for 1 hour at pH 10 at room temperature. The mixed solution is poured into a glass petri dish and dried in a forced-air drying oven at 40℃ until the water content is below 10%. It is baked at 130℃ for two hours, then redissolved in 40 mL of deionized water, and the pH is adjusted to 6.5 with hydrochloric acid to terminate the reaction. After repeated washing three times with deionized water and drying, STMP cross-linked starch with different degrees is obtained.

[0027] (2) Staining with silver nitrate: Weigh 0.2 - 0.5 g of the starch dry basis sample and add it to a 50 mL centrifuge tube containing 20 mL of 0.5 M silver nitrate solution. After vortexing for 10 s, continuously shake and react at 25℃ for 48 h. After the reaction, centrifuge the suspension for 10 - 15 min (3000 - 6000×g), and discard the supernatant; wash away the remaining dye with distilled water, filter by suction with 85% ethanol, and air-dry to obtain starch granules after silver exchange.

[0028] (3) Reduction with ultraviolet lamp: Place the starch under UV 254 nm to completely reduce the silver ions in the starch granules to silver atoms.

[0029] (4) Preparation of starch granule suspension: Add starch granules to an aqueous sucrose solution so that the final concentration of the starch granules is 4 - 6%. After vortexing for 10 - 50 s, pipette 5 - 20 μL of the sample and drop it onto a glass slide, and spread the liquid drop evenly on the glass slide with a pipette tip and wait for observation.

[0030] (5) Observation with a laser confocal microscope: Place the prepared glass slide above an objective lens (100×) pre-dropped with cedarwood oil, and use an Argon (488 nm) laser to operate at 80% power respectively for excitation.

[0031] In some embodiments, a method for observing the reaction sites of POCl3 cross-linked starch is provided, including the following steps:

[0032] (1) Preparation of POCl3 cross-linked starch: Pipette 0.182 mL of commercially available phosphorus oxychloride (POCl3) solution and dissolve it in 9.818 mL of 1,4-dioxane solution to prepare a POCl3 solution for cross-linking reaction. Disperse 20 g of starch in 40 mL of deionized water and stir evenly at room temperature. Adjust the pH value of the solution to 11.3 with 1 M NaOH, slowly dropwise add the prepared 0.067 - 6.667 mL of POCl3 solution, and react for 1 h under the condition that the pH value of the slurry is always maintained at 11.3. Adjust the pH value of the solution to 6.5 with diluted hydrochloric acid to terminate the reaction. Centrifuge to discard the supernatant, wash three times with deionized water and once with absolute ethanol. Pour the starch suspension into a Buchner funnel and perform vacuum filtration with the Buchner funnel. Place the filter cake to dry at room temperature to obtain POCl3 cross-linked starch.

[0033] (2) Staining with silver nitrate: Weigh 0.2 - 0.5 g of starch dry basis sample and add it to a 50 mL centrifuge tube containing 20 mL of 0.5 M silver nitrate solution. After vortexing for 10 s, continuously shake and react at 25 °C for 48 h. After the reaction, centrifuge the suspension for 10 - 15 min (3000 - 6000×g) and discard the supernatant; Wash away the residual dye with distilled water, perform suction filtration with 85% ethanol, and dry to obtain silver-exchanged starch granules.

[0034] (3) Reduction with ultraviolet lamp: Place the starch under UV 254 nm to completely reduce the silver ions in the starch granules to silver atoms.

[0035] (4) Preparation of starch granule suspension: Add the starch granules to an aqueous sucrose solution so that the final concentration of the starch granules is 4 - 6%. After vortexing for 10 - 50 s, pipette 5 - 20 μL of the sample and drop it onto a glass slide, and use a pipette tip to spread the liquid drop evenly on the glass slide and wait for observation.

[0036] (5) Observation with a laser confocal microscope: Place the prepared glass slide above an objective lens (100 times) pre-dropped with cedar oil, and use an Argon (488 nm) laser to work at 80% power respectively for excitation.

[0037] The cross-linked starch granules of the present invention carry anionic groups. By using a silver nitrate exchange reaction, the anionic groups in the cross-linked starch granules are converted into silver salts. The silver ions of the silver-exchanged starch are reduced to silver atoms by ultraviolet light (Ag+ → Ag°). The sample is observed by optical sectioning with a laser confocal microscope, and the silver atoms are located by excitation and in-situ scanning imaging, so as to obtain the distribution of reaction sites in the cross-linked starch granules.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1) By adopting the silver nitrate staining combined with laser confocal microscopy scanning technology, the anionic sites in cross-linked starch can be directly imaged.

[0040] 2) The present invention provides a simple, effective and direct method for observing the distribution of reaction sites of STMP cross-linked starch, and finds that the STMP cross-linking reaction first occurs on the surface of starch granules and finally reaches the channels and interior of starch granules, and the reaction sites on the surface are more numerous and denser.

[0041] 3) It can be known from the present invention that the reaction sites of POCl3 cross-linked starch are first densely distributed on the surface and channels of starch granules and finally reach the interior of the granules, and the reaction sites are more distributed on the surface and channels of starch and less distributed in the interior of the granules.

[0042] 4) By understanding the distribution of reaction sites on starch granules, the present invention can intuitively understand the cross-linking degree of starch granules, which is helpful for guiding the optimization of cross-linked starch process and its application. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Other features, objects and advantages of the present invention will become more apparent by reading the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0044] Figure 1 It is a picture of the reaction sites of cross-linked starch, where (a) is the picture of observing the reaction sites of STMP cross-linked starch in Example 1, and (b) is the picture of observing the reaction sites of POCl3 cross-linked starch in Example 3;

[0045] Figure 2 It is the picture of observing the reaction sites of STMP cross-linked starch in Comparative Example 1;

[0046] Figure 3 It is the picture of observing the reaction sites of STMP cross-linked starch in Comparative Example 3.

[0047] Figure 4 It is the picture of observing the reaction sites of STMP cross-linked starch in Comparative Example 5;

[0048] Figure 5 It is the picture of observing the reaction sites of STMP cross-linked starch in Example 2;

[0049] Figure 6 It is the picture of observing the reaction sites of POCl3 cross-linked starch in Comparative Example 6;

[0050] Figure 7 It is the picture of observing the reaction sites of POCl3 cross-linked starch in Comparative Example 8;

[0051] Figure 8 It is a picture for observing the reaction sites of POCl3 crosslinked starch in Comparative Example 10;

[0052] Figure 9 It is a picture for observing the reaction sites of POCl3 crosslinked starch in Example 4. Detailed implementation manners

[0053] The present invention will be described completely in combination with specific embodiments below. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, corresponding improvements can also be made, which all belong to the protection scope of the present invention.

[0054] In order to clarify the crosslinking reaction process inside starch granules, the present invention provides a method for directly observing the distribution of crosslinked starch reaction sites, which is of great significance for guiding the optimization of crosslinked starch technology, the research of functional properties and the application of crosslinked starch.

[0055] The technical solution adopted by the present invention is as follows:

[0056] A method for observing crosslinked starch reaction sites includes the steps of preparation of crosslinked starch, silver nitrate staining, centrifugation, washing the dye, centrifugation, UV reduction, preparation of stained starch granule suspension and observation with a laser confocal microscope. Specifically, silver nitrate is used to convert anions of starch granules into silver salts respectively; silver ions of silver-exchanged starch are reduced to silver atoms by ultraviolet light (Ag+→Ag°); the sample is observed by optical sectioning with a laser confocal microscope, and the distribution of reaction sites in starch granules is obtained through excitation and in-situ scanning imaging.

[0057] Example 1

[0058] Step 1. Preparation of STMP crosslinked starch: First, sodium trimetaphosphate (STMP, 0.05 g) and sodium sulfate (Na2SO4, 1 g) are dissolved in 5 mL of deionized water; 20 g of corn starch is dispersed in 35 mL of deionized water and stirred evenly at room temperature. The pH value of the corn starch aqueous solution is adjusted to 10 with 1 M NaOH, and then the mixed solution of STMP and Na2SO4 is slowly added dropwise. The reaction mixture continues to react for 1 hour in an environment with a pH of 10 at room temperature. The mixed solution is poured into a glass petri dish and dried in a blast drying oven at 40°C until the water content is below 10%. It is baked at a high temperature of 130°C for two hours, and then redissolved in 40 mL of deionized water. The pH is adjusted to 6.5 with hydrochloric acid to terminate the reaction, and it is washed three times repeatedly with deionized water and then dried to obtain STMP crosslinked starch.

[0059] Step 2: Staining with silver nitrate: Weigh 0.2 g of the dry starch sample and add it to a 50 mL centrifuge tube containing 20 mL of 0.5 M silver nitrate solution. After vortexing for 10 s, continuously shake the reaction at 25 °C for 48 h. After the reaction, centrifuge the suspension for 10 min (3000 - 6000×g) and discard the supernatant; repeat the silver nitrate staining step 2 times. Use distilled water to remove the residual silver nitrate, continuously shake for 30 min, centrifuge, and discard the supernatant. Filter with 85% ethanol and air dry to obtain silver-exchanged starch granules. The whole process must be carried out in the dark.

[0060] Step 3: Reduction with ultraviolet lamp: Place the starch under UV 254 nm for more than 48 h to completely reduce the silver ions in the starch granules to silver atoms.

[0061] Step 4: Preparation of starch granule suspension: Add the starch granules to a 25% sucrose aqueous solution so that the final concentration of the starch granules is 5%. After vortexing for 30 s, pipette 10 μL of the sample and drop it onto a glass slide, and use a pipette tip to spread the liquid drop evenly on the glass slide and wait for observation.

[0062] Step 5: Observation with a laser confocal microscope: Place the prepared glass slide above the objective lens (100×) pre-dropped with cedar oil, and use an Argon (488 nm) laser to work at 80% power respectively for excitation. The beam splitter is RT15 / 85, and the signal reflected from the excitation light of the material is collected at the interval wavelength of 485 - 506 nm.

[0063] Implementation effect: As Figure 1 (a) shows the distribution of reaction sites in the starch granules, with clear fluorescence and high picture quality. The reaction sites of STMP cross-linked starch are only on the surface of the starch granules and not inside the granules.

[0064] Comparative Example 1

[0065] This comparative example relates to a method for observing the reaction sites of STMP cross-linked starch. The method is the same as that in Example 1, except that;

[0066] Step 2: Staining with silver nitrate: Weigh 0.2 g of the dry starch sample and add it to a 50 mL centrifuge tube containing 20 mL of 0.5 M silver nitrate solution. After vortexing for 10 s, continuously shake the reaction at 25 °C for 48 h. After the reaction, centrifuge the suspension for 10 min (3000 - 6000×g) and discard the supernatant; repeat the silver nitrate staining step 2 times. Use distilled water to remove the residual silver nitrate, continuously shake for 30 min, centrifuge, and discard the supernatant. Filter with 85% ethanol and air dry to obtain silver-exchanged starch granules. The whole process was not carried out in the dark.

[0067] Implementation effect: As Figure 2As shown, the fluorescence of the starch granules is not clear and the intensity is too low to observe the reaction sites on the starch granules because silver nitrate decomposes upon exposure to light and the exchange reaction cannot proceed.

[0068] Comparative Example 2

[0069] This comparative example relates to a method for observing the reaction sites of STMP cross-linked starch. The method is the same as that in Example 1, except that;

[0070] Step 2, Staining with silver nitrate: Weigh 0.2 g of the dry starch sample and add it to a 50 mL centrifuge tube containing 20 mL of 0.5 M silver nitrate solution. After vortexing for 10 s, continuously shake and react at 25 °C for 48 h. After the reaction, centrifuge the suspension for 10 min (3000 - 6000 × g), and discard the supernatant. Remove the residual silver nitrate with distilled water, which requires continuous shaking for 30 min, centrifugation, and discard the supernatant. Filter with 85% ethanol and air dry to obtain the starch granules after silver exchange. The whole process must be carried out in the dark.

[0071] Implementation effect: The fluorescence of the starch granules is not clear and the intensity is too low to observe the reaction sites on the starch granules because the silver nitrate exchange reaction is incomplete.

[0072] Comparative Example 3

[0073] This comparative example relates to a method for observing the reaction sites of STMP cross-linked starch. The method is the same as that in Example 1, except that;

[0074] Step 2, Staining with silver nitrate: Weigh 0.2 g of the dry starch sample and add it to a 50 mL centrifuge tube containing 20 mL of 0.5 M silver nitrate solution. After vortexing for 10 s, continuously shake and react at 25 °C for 48 h. After the reaction, centrifuge the suspension for 10 min (3000 - 6000 × g), and discard the supernatant; repeat the silver nitrate staining step 2 times. Filter with 85% ethanol and air dry to obtain the starch granules after silver exchange. The whole process must be carried out in the dark.

[0075] Implementation effect: As Figure 3 shown, the background value is too high to clearly observe the reaction sites on the starch granules because the excess dye silver nitrate is not washed with distilled water.

[0076] Comparative Example 4

[0077] This comparative example relates to a method for observing the reaction sites of STMP cross-linked starch. The method is the same as that in Example 1, except that;

[0078] Step 3, Reduction with ultraviolet lamp: Place the starch under UV 254 nm for 24 h to reduce the silver ions in the starch granules to silver atoms.

[0079] Implementation effect: The fluorescence of starch granules is not clear and the intensity is too low. Reaction sites cannot be observed on starch granules because silver ions in the starch granules are not completely reduced to silver atoms.

[0080] Comparative Example 5

[0081] This comparative example relates to a method for observing reaction sites of STMP cross-linked starch. The method is the same as that in Example 1, except that;

[0082] Step 5. Observation with a laser confocal microscope: Place the prepared glass slide above the objective lens (100 times) pre-dropped with cedar oil, and use an Argon (488 nm) laser to work at a power of 80% respectively for excitation, and collect the excitation signal at an interval wavelength of 485 - 506 nm.

[0083] Implementation effect: As Figure 4 shown, it is difficult to observe the fluorescence on starch granules and the reaction sites cannot be identified because the beam splitter needs to be set to RT15 / 85 to receive the reflected laser rather than the fluorescence excited by the sample.

[0084] Example 2

[0085] This example relates to a method for observing reaction sites of STMP cross-linked starch. The method is the same as that in Example 1, except that;

[0086] Step 1. Preparation of STMP cross-linked starch: First, dissolve sodium trimetaphosphate (STMP, 0.4 g) and sodium sulfate (Na2SO4, 1 g) in 5 mL of deionized water, disperse 20 g of corn starch in 35 mL of deionized water, and stir evenly at room temperature. Adjust the pH value of the solution to 10 with 1 M NaOH, then slowly drop the mixed solution of STMP and Na2SO4. The reaction mixture continues to react for 1 hour in an environment with a pH of 10 at room temperature. Pour the mixed solution into a glass petri dish, dry it in a forced-air drying oven at 40 °C until the water content is below 10%, bake it at a high temperature of 130 °C for two hours, then redissolve it in 40 mL of deionized water, adjust the pH to 6.5 with hydrochloric acid to terminate the reaction, and repeat washing three times with deionized water and then dry it to obtain STMP cross-linked starch.

[0087] Implementation effect: As Figure 5 shown, the distribution of reaction sites in starch granules is shown, the fluorescence is clear, and the picture quality is high. In the preparation of STMP cross-linked starch in this example, the addition amount of the cross-linking reagent is more. In Figure 5It can be directly observed that there are more fluorescence reaction sites on the surface of starch granules, and fluorescence can also be observed in a small amount of channels and the interior of starch granules, indicating that the cross-linking process is faster and the degree of cross-linking is higher than that in Example 1. The cross-linking reaction first occurs on the surface of starch granules and finally reaches the channels and interior of starch granules, further indicating that the cross-linking reaction process inside the granules can be intuitively understood in the present invention, and the characterization of the degree of cross-linking can be realized.

[0088] Example 3

[0089] Step 1: Preparation of POCl3 cross-linked starch: First, 0.182 mL of commercially available phosphorus oxychloride (POCl3) solution was sucked and dissolved in 9.818 mL of 1,4-dioxane solution to prepare a POCl3 solution for cross-linking. 20 g of corn starch was dispersed in 40 mL of deionized water and stirred evenly at room temperature. The pH value of the solution was adjusted to 11.3 with 1 M NaOH, and 0.067 mL of the prepared POCl3 solution was slowly added dropwise, and the slurry was reacted for 1 h under the condition that the pH value was always maintained at 11.3. The pH value of the solution was adjusted to 6.5 with diluted hydrochloric acid to terminate the reaction. The supernatant was discarded by centrifugation, and the precipitate was washed three times with deionized water and once with absolute ethanol. The starch suspension was poured into a Buchner funnel and filtered under reduced pressure with the Buchner funnel. The filter cake was placed at room temperature to dry, and thus POCl3 cross-linked starch was prepared.

[0090] Step 2: Staining with silver nitrate: 0.2 g of starch dry basis sample was weighed and added to a 50 mL centrifuge tube containing 20 mL of 0.5 M silver nitrate solution. After vortexing for 10 s, it was continuously shaken and reacted at 25 °C for 48 h. After the reaction, the suspension was centrifuged for 15 min (3000 - 6000×g), and the supernatant was discarded; the silver nitrate staining step was repeated 3 times. Residual silver nitrate was removed with distilled water, and it was continuously shaken for 60 min, centrifuged, and the supernatant was discarded. It was filtered with 85% ethanol and dried to obtain starch granules after silver exchange. The whole process must be carried out in the dark.

[0091] Step 3: Reduction under ultraviolet lamp: The starch was placed under UV 254 nm for more than 48 h to completely reduce the silver ions in the starch granules to silver atoms.

[0092] Step 4: Preparation of starch granule suspension: The starch granules were added to 30% sucrose aqueous solution so that the final concentration of the starch granules was 6%. After vortexing for 50 s, 20 μL of the sample was sucked and dropped onto a glass slide, and the droplet was evenly spread on the glass slide with a pipette tip and waited to be observed.

[0093] Step 5: Observation with a laser confocal microscope: Place the prepared glass slide above the objective lens (100x) with cedarwood oil pre-dropped. Use the Argon (488 nm) laser to work at 80% power for excitation. The beam splitter is RT15 / 85, and the signal reflected from the excitation light of the material is collected at the interval wavelength of 485 - 506 nm.

[0094] Implementation effect: As Figure 1 (b) shows, presenting the distribution of reaction sites in starch granules, with clear fluorescence and high picture quality. The reaction sites of POCl₃ cross-linked starch can be observed on the surface and channels of starch granules, and the fluorescence sites are dense, but the reaction sites cannot be observed inside the starch granules.

[0095] Comparative Example 6

[0096] This comparative example relates to a method for observing the reaction sites of POCl₃ cross-linked starch. The method is the same as that in Example 3, except that;

[0097] Step 3: Staining with silver nitrate: Weigh 0.2 g of starch dry basis sample and add it to a 50 mL centrifuge tube containing 20 mL of 0.5 M silver nitrate solution. After vortexing for 10 s, continuously shake and react at 25 °C for 48 h. After the reaction, centrifuge the suspension for 15 min (3000 - 6000×g), and discard the supernatant; repeat the silver nitrate staining step 3 times. Remove the residual silver nitrate with distilled water, which requires continuous shaking for 60 min, centrifugation, and discarding the supernatant. Filter with 85% ethanol and air dry to obtain starch granules after silver exchange. The whole process is not protected from light.

[0098] Implementation effect: As Figure 6 shown, the fluorescence of starch granules is not clear and the intensity is too low to observe the reaction sites in starch granules because silver nitrate decomposes upon exposure to light and cannot undergo the exchange reaction.

[0099] Comparative Example 7

[0100] This comparative example relates to a method for observing the reaction sites of POCl₃ cross-linked starch. The method is the same as that in Example 3, except that;

[0101] Step 3: Staining with silver nitrate: Weigh 0.2 g of starch dry basis sample and add it to a 50 mL centrifuge tube containing 20 mL of 0.5 M silver nitrate solution. After vortexing for 10 s, continuously shake and react at 25 °C for 48 h. After the reaction, centrifuge the suspension for 15 min (3000 - 6000×g), and discard the supernatant. Remove the residual silver nitrate with distilled water, which requires continuous shaking for 60 min, centrifugation, and discarding the supernatant. Filter with 85% ethanol and air dry to obtain starch granules after silver exchange. The whole process must be protected from light.

[0102] Implementation effect: The fluorescence of starch granules is not clear and the intensity is too low. Reaction sites cannot be observed on the starch granules because the exchange reaction of silver nitrate is incomplete.

[0103] Comparative Example 8

[0104] This comparative example relates to a method for observing the reaction sites of POCl3 cross-linked starch. The method is the same as that of Example 3, except that;

[0105] Step 3: Staining with silver nitrate: Weigh 0.2 g of the dry starch sample and add it to a 50 mL centrifuge tube containing 20 mL of 0.5 M silver nitrate solution. After vortexing for 10 s, continuously shake and react at 25 °C for 48 h. After the reaction, centrifuge the suspension for 15 min (3000 - 6000 × g), and discard the supernatant; repeat the silver nitrate staining step 3 times. Filter with 85% ethanol and air dry to obtain silver-exchanged starch granules. The whole process must be carried out in the dark.

[0106] Implementation effect: As Figure 7 shown, the background value is too high, and reaction sites cannot be clearly observed on the starch granules because the excess dye silver nitrate is not washed with distilled water.

[0107] Comparative Example 9

[0108] This comparative example relates to a method for observing the reaction sites of POCl3 cross-linked starch. The method is the same as that of Example 3, except that;

[0109] Step 4: Reduction with ultraviolet lamp: Place the starch under UV 254 nm for 24 h to reduce the silver ions in the starch granules to silver atoms.

[0110] Implementation effect: The fluorescence of starch granules is not clear and the intensity is too low. Reaction sites cannot be observed on the starch granules because the silver ions in the starch granules are not completely reduced to silver atoms.

[0111] Comparative Example 10

[0112] This comparative example relates to a method for observing the reaction sites of POCl3 cross-linked starch. The method is the same as that of Example 3, except that;

[0113] Step 6: Observation with a laser confocal microscope: Place the prepared glass slide above the objective lens (100 times) with cedar oil dropped in advance, and use an Argon (488 nm) laser to work at 80% power respectively for excitation, and collect the excitation signal at an interval wavelength of 485 - 506 nm.

[0114] Implementation effect: As Figure 8As shown, it is difficult to observe the fluorescence on the starch granules and identify the reaction sites because the beam splitter needs to be set to RT15 / 85 to receive the reflected laser rather than the fluorescence excited by the sample.

[0115] Example 4

[0116] This example relates to a method for observing the reaction sites of POCl3 cross-linked starch. The method is the same as that in Example 3, except that:

[0117] Step 1: Preparation of POCl3 cross-linked starch: First, absorb 0.182 mL of commercially available phosphorus oxychloride (POCl3) solution and dissolve it in 9.818 mL of 1,4-dioxane solution to prepare a POCl3 solution for cross-linking. Disperse 20 g of corn starch in 40 mL of deionized water and stir evenly at room temperature. Adjust the pH value of the solution to 11.3 with 1 M NaOH, slowly dropwise add 6.667 mL of the prepared POCl3 solution, and react for 1 h under the condition that the pH value of the slurry is always maintained at 11.3. Adjust the pH value of the solution to 6.5 with diluted hydrochloric acid to terminate the reaction. Centrifuge to discard the supernatant, wash three times with deionized water and once with absolute ethanol. Pour the starch suspension into a Buchner funnel and filter under reduced pressure with the Buchner funnel. Place the filter cake to dry at room temperature. Then grind the dried filter cake and pass it through a sieve with a pore size of 0.4 mm to obtain POCl3 cross-linked starch.

[0118] Implementation effect: As Figure 9 shown, it shows the distribution of reaction sites in the starch granules with clear fluorescence and high picture quality. In the preparation of POCl3 cross-linked starch in this example, more cross-linking reagent is added. In Figure 9 it can be directly observed that there are more fluorescence reaction sites on the surface and channels of the starch, and fluorescence can also be observed inside the starch granules, indicating that the cross-linking process is faster and the cross-linking degree is higher than that in Example 1. The cross-linking reaction first occurs on the surface and channels of the starch granules and finally in the granules, further indicating that the present invention can intuitively understand the cross-linking reaction sites and cross-linking reaction process in the granules and can realize the characterization of the cross-linking degree.

[0119] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention.

Claims

1. A method for observing the reaction sites of cross-linked starch, characterized in that, Including: Preparation of cross-linked starch; the cross-linked starch includes STMP cross-linked starch or POCl3 cross-linked starch; Silver nitrate staining: Using silver nitrate to convert the anions of cross-linked starch granules into silver salts to obtain silver-exchanged starch granules; Including adding a dry basis sample of cross-linked starch to a 0.25 - 0.5 M silver nitrate solution, centrifuging and vortexing for 10 - 15 s, continuously shaking and reacting at 25 - 35 °C for 48 - 72 h. After the reaction, centrifuge the suspension, with the centrifugation parameters being 3000 - 6000 × g for 10 - 15 min, and discard the supernatant; wash away the residual dye with distilled water, filter with 85% ethanol, and dry to obtain silver-exchanged starch granules; The entire process of silver nitrate staining is carried out under light-proof conditions; UV reduction: Using ultraviolet light to reduce the silver ions in the silver-exchanged starch to silver atoms to obtain stained starch granules; including placing the silver-exchanged starch under UV 254 nm for more than 48 h to completely reduce the silver ions in the starch granules to silver atoms; Preparation of stained starch granule suspension: Adding the stained starch granules obtained by UV reduction to a sucrose aqueous solution so that the final concentration of the starch granules is 4 - 6% to prepare a stained starch granule suspension; Observation with a laser confocal microscope: Using a laser confocal microscope to perform optical sectioning observation on the sample, and obtaining the distribution of reaction sites in the starch granules through excitation and in-situ scanning imaging; including placing the glass slide coated with the starch granule suspension above the objective lens where cedar oil has been previously dropped, working with a 488 nm Argon laser at a power of 80% - 100% for excitation; the beam splitter is RT15 / 85, and collecting the signal reflected by the excitation light of the material at an interval wavelength of 485 - 506 nm.

2. The method for observing the reaction sites of crosslinked starch according to claim 1, wherein Preparing STMP cross-linked starch by the semi-dry method; specifically including: adjusting the pH value of the starch aqueous solution to 10, slowly dropping a mixed solution of STMP and Na2SO4, and continuously reacting the reaction mixture at a pH of 10 - 11 at room temperature for 1 - 2 hours; drying at 40 - 50 °C until the water content is below 10 wt.%, and drying at 130 - 150 °C for 2 - 3 hours; redissolving in deionized water, adjusting the pH to 6.5 to terminate the reaction, and washing with deionized water to obtain STMP cross-linked starch.

3. The method for observing cross-linking starch reaction sites according to claim 1, wherein Preparing POCl3 cross-linked starch by the wet method; specifically including: adjusting the pH value of the starch aqueous solution to 10 - 11.3, slowly dropping the POCl3 solution, and maintaining the pH value of the slurry at 11.3 for continuous reaction for 1 - 2 hours; adjusting the pH to 6.5 to terminate the reaction, washing with deionized water, washing with absolute ethanol, and subjecting the obtained starch suspension to reduced pressure filtration and drying at room temperature to obtain POCl3 cross-linked starch.

4. The method for observing the reaction sites of crosslinked starch according to claim 1, characterized in that, The dosage ratio of the dry basis sample of the cross-linked starch to the silver nitrate solution is 0.2 - 0.5 g:20 mL.

5. The method for observing the reaction sites of crosslinked starch according to claim 1, characterized in that, In the preparation of the starch granule suspension, the concentration of the sucrose aqueous solution is 20 wt.% - 30 wt.%.

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