A method of observing distribution of lipid bound to starch granules in alkaline environment

Abstract

The application discloses a method for observing distribution of starch granule combined with lipids in an alkaline environment, comprising the steps of staining of starch granule combined with lipids and laser confocal microscope observation, characterized in that: for the problem that conventional dyes are difficult to combine with starch in an alkaline environment, Nile red dye is selected to stain lipids of starch granule, so that fluorescence labeling of the lipids of the starch granule is completed; during the staining and cleaning process, an alkaline solution with the same pH is used as a diluent and a cleaning agent to avoid change of the pH of the solution during the staining process; and the sample is observed by using a laser confocal microscope, and distribution of the lipids of the starch granule in the alkaline environment is obtained by laser excitation and in-situ scanning imaging. The application solves the problem that conventional dyes are difficult to combine with lipids in an alkaline environment, keeps the pH of the alkaline environment unchanged during the staining and cleaning process, and realizes in-situ observation of the lipids of starch in the alkaline environment.

Description

Technical Field

[0001] This invention belongs to the food field, specifically relating to a method for observing the distribution of lipids bound to starch granules under alkaline conditions. Background Technology

[0002] Starch is the second largest renewable raw material in nature after cellulose. Due to its wide availability, complete biodegradability, renewability, and low cost, it is widely used in industries such as food, chemical, pharmaceutical, textile, and papermaking as a thickener, adhesive, stabilizer, or gelling agent. However, the industrial application of natural starch is somewhat limited by its inherent defects (poor resistance to heat treatment and shear stress, and poor processability). To meet industrial needs, natural starch is often modified through physical, chemical, or biological methods to improve its properties and functions. These modification methods often require an alkaline environment or directly use alkali as the modification method. The alkaline environment has a significant impact on the physicochemical properties of starch and the modification reaction, and has received increasing attention.

[0003] Starch granules are mainly composed of amylose and amylopectin, and also contain trace amounts of lipids, proteins, and minerals. Although present in low amounts, these trace components play a crucial role in the physicochemical, functional, and nutritional properties of starch. Lipids in starch granules can form complexes with amylose, enhancing the integrity and rigidity of the starch granule structure. Simultaneously, surface lipids may form a film on the starch granule surface, restricting the movement of molecules between the granule's interior and exterior environments. Many studies have also found that lipids play a significant role in the chemical modification of starch, influencing modification efficiency and the properties of modified starch. On the other hand, lipids undergo saponification in alkaline environments, leading to lipid degradation. The alkaline environment in starch chemical modification undoubtedly affects the lipids in starch, which is likely one of the effects of alkali in the chemical modification process. Therefore, it is necessary to investigate the changes in starch lipids under alkaline conditions.

[0004] Among numerous research methods, laser confocal microscopy is undoubtedly a highly intuitive and effective way to observe changes in the distribution of starch lipids. In recent years, many scholars have used confocal microscopy to study the distribution of proteins and lipids in starch granules, and have developed fluorescent dyes that specifically bind to lipids using Pro-Q Diamond reagent and Dil dye for starch lipid observation. However, when studying lipid distribution under alkaline conditions, the presence of a specific alkaline liquid environment places higher demands on lipid staining methods. There is an urgent need to find a fluorescent dye that can stably bind to starch lipids under alkaline conditions and to establish an effective method for observing the distribution of lipids bound to starch granules under alkaline conditions. Summary of the Invention

[0005] To meet the need for observing the distribution of starch-bound lipids under alkaline conditions, this invention provides a method for observing the distribution of starch-bound lipids in starch granules under alkaline conditions. This method is of great significance for guiding research on starch structure, properties, and functions related to starch modification and starch-bound lipids, as well as for the application of starch in the food industry.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] This invention relates to a method for observing the distribution of lipids bound to starch granules under alkaline conditions, comprising steps of staining for lipids bound to starch granules and observation using laser confocal microscopy.

[0008] S1. The starch treated in an alkaline environment is diluted to a suitable concentration with an alkaline solution, and Nile Red dye is selected to stain the lipids of the starch granules, thereby completing the fluorescent labeling of the lipids of the starch granules. Throughout the staining process, an alkaline solution is used as the reaction dispersion system to ensure that the starch granules are in the alkaline pH environment required for the experiment and to avoid changes in the pH of the solution during the staining process.

[0009] S2. The sample obtained in step S1 is observed using a laser confocal microscope. The distribution of lipids in starch granules under alkaline conditions is obtained through laser excitation and in-situ scanning imaging.

[0010] As one implementation, the starch is selected from corn starch, rice starch, potato starch, glutinous rice starch, wheat starch, or konjac starch.

[0011] As one implementation scheme, the alkali used includes, but is not limited to, sodium hydroxide, potassium hydroxide, and calcium hydroxide.

[0012] As one implementation method, continuous shaking during the dyeing reaction ensures that the starch granules are evenly dispersed in the dyeing agent. This is to ensure that all the starch comes into contact with the dyeing agent, thereby guaranteeing the uniformity of the reaction.

[0013] As one implementation method, step S1 includes the following steps:

[0014] S1-1, Add starch to alkaline solution and adjust to an alkaline environment of pH 7-14 according to the required experimental conditions to prepare starch emulsion;

[0015] S1-2, the starch emulsion is diluted with an alkaline solution of the same pH as the starch emulsion, so that the starch concentration after dilution is 10-40 mg / L, to obtain a dilute starch solution with the same pH as the starch emulsion; the alkaline solution is the same alkaline reagent used in step S1-1.

[0016] S1-3, mix a dilute starch solution with 1-5 mg / mL Nile Red ethanol staining solution at a ratio of 20-500:1, and shake the mixture at room temperature for 3-5 minutes to ensure that Nile Red is in full contact with starch particles and to label starch-bound lipids.

[0017] S1-4, centrifuge the reaction system for 5-20 min (5000-15000×g), discard the supernatant, and wash the precipitate with the alkaline solution described in step S1-2 to remove residual dye.

[0018] As one implementation method, the Nile Red ethanol dye solution is prepared fresh for each use; the ethanol used is 100% pure ethanol.

[0019] In some embodiments, in steps S1-3, the starch dilute solution is mixed with 1-5 mg / mL Nile red ethanol dye solution at a ratio of 100:1.

[0020] In some embodiments, in steps S1-4, the reaction system is centrifuged for 5 min (5000-15000×g).

[0021] As one implementation, the purpose of starch dilution in step S1-2 is to ensure that the starch concentration is appropriate during observation. This step can also be moved after step S1-3.

[0022] In one implementation, steps S1-4 involve washing away residual dye until the supernatant after vortexing and centrifugation is colorless. In some implementation examples, the washing is repeated 3-5 times.

[0023] As one implementation method, step S2 includes the following steps:

[0024] S2-1, Add 0.5-2 mL of the alkaline solution described in step S1-2 to the starch granule precipitate, vortex, and then drop 5-20 μL of sample onto a glass slide and spread it evenly.

[0025] S2-2, the prepared glass slide is placed above the objective lens on which cedar oil has been added beforehand, and excited with a laser; its emitted light is detected at wavelength intervals of 570-649nm.

[0026] In some embodiments, in S2-1, 1 mL of the alkaline solution described in step S1-2 is added to the starch granule precipitate.

[0027] As one implementation, the excitation is performed using a DPSS (561nm) laser operating at 1-4% power.

[0028] The dye used in this invention is Nile Red. While it is generally accepted in the art that Nile Red can be used for lipid staining, its use for starch and lipid staining is rare; it is more commonly used for cell staining. Furthermore, most literature mentioning Nile Red staining conditions uses neutral aqueous solutions, and there are no clear reports of using Nile Red for staining at alkaline pH conditions.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The method of observing the distribution of starch granules bound to lipids in an alkaline environment according to the present invention is carried out while maintaining the pH of the system constant throughout the staining process. This achieves in-situ staining in an alkaline environment, avoids the influence of pH changes on lipid distribution, and allows for better observation of the distribution of starch lipids in an alkaline environment. Attached Figure Description

[0031] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0032] Figure 1 Images showing the lipid distribution of starch granules under alkaline conditions observed in Example 1;

[0033] Figure 2 Images showing the lipid distribution of starch granules under alkaline conditions observed in Example 2;

[0034] Figure 3 Images showing the lipid distribution of starch granules under alkaline conditions observed in Example 3;

[0035] Figure 4 Images showing the lipid distribution of starch granules under alkaline conditions observed in Example 4;

[0036] Figure 5 This is an image showing the lipid distribution of starch granules under alkaline conditions observed in Comparative Example 1. Detailed Implementation

[0037] The present invention will now be described with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0038] Example 1

[0039] This embodiment relates to a method for observing the distribution of lipids bound to starch granules under alkaline conditions, comprising the following steps:

[0040] 1. Add 40% starch aqueous solution to 0.1 mol / L sodium hydroxide solution and adjust to an alkaline environment of pH 8.5 according to the required experimental conditions to prepare starch emulsion. The pH of the starch emulsion after the reaction is measured to be 8.47.

[0041] 2. Dilute the obtained starch emulsion with a sodium hydroxide solution at pH 8.5 to make the starch concentration at 20 mg / mL, thus obtaining a dilute starch solution at pH 8.5;

[0042] 3. Mix the dilute starch solution with 2 mg / mL Nile red ethanol staining solution at a ratio of 100:1, and shake the mixture at room temperature for 3 min to ensure that the Nile red comes into full contact with the starch particles and to label the starch-bound lipids.

[0043] 4. Centrifuge the reaction system for 5 min (10000×g), discard the supernatant, and wash the precipitate with sodium hydroxide solution at pH 8.5. Repeat 5 times to wash away the residual dye and obtain starch granule precipitate.

[0044] 5. Add 1 mL of pH 8.5 sodium hydroxide solution to the starch granule precipitate, vortex, and then add 10 μL of sample onto a glass slide. Spread the droplet evenly on the glass slide using a pipette tip.

[0045] 6. Place the prepared glass slide above the objective lens on which cedar oil has been pre-dropped, and excite it using a DPSS (561nm) laser operating at 2% power. Detect the emitted light at wavelength intervals of 570-649nm.

[0046] Implementation results are as follows Figure 1 As shown, the distribution of lipids bound to starch granules under alkaline conditions can be clearly demonstrated.

[0047] Example 2

[0048] This embodiment relates to a method for observing the distribution of lipids bound to starch granules under alkaline conditions, comprising the following steps:

[0049] 1. Add 40% starch aqueous solution to 0.1 mol / L sodium hydroxide solution and adjust to an alkaline environment of pH 10.0 according to the required experimental conditions to prepare starch emulsion. The pH of the starch emulsion after the reaction is measured to be 10.03.

[0050] 2. Dilute the obtained starch emulsion with a sodium hydroxide solution at pH 10.0 to make the starch concentration at 20 mg / mL, thus obtaining a dilute starch solution at pH 10.0;

[0051] 3. Mix the dilute starch solution with 2 mg / mL Nile red ethanol staining solution at a ratio of 100:1, and shake the mixture at room temperature for 3 min to ensure that the Nile red comes into full contact with the starch particles and to label the starch-bound lipids.

[0052] 4. Centrifuge the reaction system for 5 min (10000×g), discard the supernatant, and wash the precipitate with sodium hydroxide solution at pH 10.0. Repeat 5 times to wash away the residual dye and obtain starch granule precipitate.

[0053] 5. Add 1 mL of sodium hydroxide solution of the same pH to the starch granule precipitate, vortex, and then add 10 μL of sample onto a glass slide. Use a pipette tip to spread the droplet evenly on the glass slide.

[0054] 6. Place the prepared glass slide above the objective lens on which cedar oil has been pre-dropped, and excite it using a DPSS (561nm) laser operating at 2% power. Detect the emitted light at wavelength intervals of 570-649nm.

[0055] Implementation results are as follows Figure 2 As shown, the distribution of lipids bound to starch granules under alkaline conditions can be clearly demonstrated.

[0056] Example 3

[0057] This embodiment relates to a method for observing the distribution of lipids bound to starch granules under alkaline conditions, comprising the following steps:

[0058] 1. Add 40% starch aqueous solution to 0.1 mol / L sodium hydroxide solution and adjust to an alkaline environment of pH 11.5 according to the required experimental conditions to prepare starch emulsion. The pH of the starch emulsion after the reaction is measured to be 11.48.

[0059] 2. Dilute the obtained starch emulsion with a sodium hydroxide solution at pH 11.5 to make the starch concentration at 20 mg / mL, thus obtaining a dilute starch solution at pH 11.5;

[0060] 3. Mix the dilute starch solution with 2 mg / mL Nile red ethanol staining solution at a ratio of 100:1, and shake the mixture at room temperature for 3 min to ensure that the Nile red comes into full contact with the starch particles and to label the starch-bound lipids.

[0061] 4. Centrifuge the reaction system for 5 min (10000×g), discard the supernatant, and wash the precipitate with sodium hydroxide solution at pH 11.5. Repeat 5 times to wash away the residual dye and obtain starch granule precipitate.

[0062] 5. Add 1 mL of pH 11.5 sodium hydroxide solution to the starch granule precipitate, vortex, and then add 10 μL of sample onto a glass slide. Spread the droplet evenly on the glass slide using a pipette tip.

[0063] 6. Place the prepared glass slide above the objective lens on which cedar oil has been pre-dropped, and excite it using a DPSS (561nm) laser operating at 2% power. Detect the emitted light at wavelength intervals of 570-649nm.

[0064] Implementation results are as follows Figure 3 As shown, the distribution of lipids bound to starch granules under alkaline conditions can be clearly demonstrated.

[0065] Example 4

[0066] This embodiment relates to a method for observing the distribution of lipids bound to starch granules under alkaline conditions, comprising the following steps:

[0067] 1. Add 40% starch aqueous solution to 0.1 mol / L sodium hydroxide solution and adjust to an alkaline environment of pH 11.5 according to the required experimental conditions to prepare starch emulsion. The pH of the starch emulsion after the reaction is measured to be 11.48.

[0068] 2. Mix starch solution with 2 mg / mL Nile red ethanol staining solution at a ratio of 100:1, and shake at room temperature for 3 min to ensure that Nile red comes into full contact with starch particles and to label starch-bound lipids;

[0069] 3. Dilute the obtained starch emulsion with a sodium hydroxide solution at pH 11.5 to make the starch concentration at 20 mg / mL, thus obtaining a dilute starch solution at pH 11.5;

[0070] 4. Centrifuge the reaction system for 5 min (10000×g), discard the supernatant, and wash the precipitate with sodium hydroxide solution at pH 11.5. Repeat 5 times to wash away the residual dye and obtain starch granule precipitate.

[0071] 5. Add 1 mL of pH 11.5 sodium hydroxide solution to the starch granule precipitate, vortex, and then add 10 μL of sample onto a glass slide. Spread the droplet evenly on the glass slide using a pipette tip.

[0072] 6. Place the prepared glass slide above the objective lens on which cedar oil has been pre-dropped, and excite it using a DPSS (561nm) laser operating at 2% power. Detect the emitted light at wavelength intervals of 570-649nm.

[0073] Implementation results are as follows Figure 4As shown, the distribution of lipids bound to starch granules under alkaline conditions can be clearly demonstrated.

[0074] Comparative Example 1

[0075] This comparative example relates to a method for observing the distribution of lipids bound to starch granules under alkaline conditions, comprising the following steps:

[0076] 1. Add 40% starch aqueous solution to 0.1 mol / L sodium hydroxide solution and adjust to an alkaline environment of pH 11.5 according to the required experimental conditions to prepare starch emulsion. The pH of the starch emulsion after the reaction is measured to be 11.48.

[0077] 2. Dilute the obtained starch emulsion with sodium hydroxide solution at pH 11.5 to make the starch concentration at 20 mg / mL, thus obtaining a starch dilute solution at pH 11.48.

[0078] 3. Mix the dilute starch solution with Dil dye at a ratio of 100:1, and shake the mixture at 37°C for 2 hours to ensure that the Dil dye is in full contact with the starch particles and to label the starch-bound lipids.

[0079] 4. Centrifuge the reaction system for 5 min (10000×g), discard the supernatant, and wash the precipitate with sodium hydroxide solution at pH 11.5. Repeat 5 times to wash away the residual dye and obtain starch granule precipitate.

[0080] 5. Add 1 mL of pH 11.5 sodium hydroxide solution to the starch granule precipitate, vortex, and then add 10 μL of sample onto a glass slide. Spread the droplet evenly on the glass slide using a pipette tip.

[0081] 6. Place the prepared glass slide above the objective lens on which cedar oil has been pre-dropped, and excite it using a DPSS (561nm) laser operating at 2% power. Detect the emitted light at wavelength intervals of 570-649nm.

[0082] Implementation results are as follows Figure 5 As shown, the staining effect is not ideal, the image is not clear, and only some particles are stained, and the internal lipid distribution cannot be displayed.

[0083] Comparative Example 2

[0084] This comparative example relates to a method for observing the distribution of lipids bound to starch granules under alkaline conditions, comprising the following steps:

[0085] 1. Add 40% starch aqueous solution to 0.1 mol / L sodium hydroxide solution and adjust to an alkaline environment of pH 11.5 according to the required experimental conditions to prepare starch emulsion. The pH of the starch emulsion after the reaction is measured to be 11.48.

[0086] 2. Dilute the obtained starch emulsion with a sodium hydroxide solution at pH 11.5 to make the starch concentration at 20 mg / L, thus obtaining a dilute starch solution at pH 11.5;

[0087] 3. Mix the dilute starch solution with Pro-Q Diamond dye in an appropriate ratio. Since the Pro-Q Diamond dye reaction requires starch to react in the Pro-Q Diamond dye system, and the reaction pH is determined by the Pro-Q Diamond dye, it is impossible to guarantee that the starch is at the required alkaline pH, so the experiment cannot be carried out.

[0088] In the above embodiments and comparative examples, the experiment in Comparative Example 2 could not guarantee that the starch was in the required pH environment, as in Comparative Example 1. Figure 5 As shown, the Dil staining effect was poor, the image was unclear, and only some particles were stained, and the internal lipid distribution could not be displayed.

[0089] This invention reveals that for Pro-Q Diamond reagent to function effectively, starch must be mixed with it alone without the addition of any external solvent or liquid. It can only bind to lipids within the reagent's own environment. This necessitates that starch be in powder form for staining in its liquid environment, making it unsuitable for staining starch emulsions. Furthermore, an alkaline environment may cause changes in the fluorescent or lipid-binding groups of the Dil dye, altering the photophysical properties and lipid-binding characteristics of the staining agent, thus affecting lipid fluorescence staining results and failing to meet the requirements for in-situ observation of starch-bound lipid distribution in an alkaline environment. Additionally, current lipid staining processes use water as a solvent during both staining and washing, which can lead to pH changes when staining lipids in alkaline solutions, affecting experimental results.

[0090] This invention reveals that Nile Red dye maintains good stability under alkaline conditions, can stably bind to lipids, and exhibits high quantum yield fluorescence. It can be used for lipid staining and identification under alkaline conditions. Based on this, a method for staining and identifying the distribution of starch lipids while maintaining a constant pH in an alkaline environment was established. In the other embodiments, it can be seen that Nile Red dye can clearly show the lipid distribution in starch granules under alkaline conditions. In summary, the method for observing the distribution of lipids bound to starch granules under alkaline conditions provided by this invention enables in-situ observation of lipids bound to starch granules under alkaline conditions.

[0091] Obviously, the above embodiments are not all embodiments of the present invention. The present invention is not limited to the specific implementation methods described above. Those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the substantive content of the present invention.

Claims

1. A method for observing the distribution of lipids bound to starch granules under alkaline conditions, comprising steps of staining for lipids bound to starch granules and observation using laser confocal microscopy, characterized in that, S1. The starch treated in an alkaline environment is diluted with an alkaline solution, and Nile Red dye is selected to stain the lipids of the starch granules, thereby completing the fluorescent labeling of the lipids of the starch granules. Throughout the staining process, an alkaline solution is used as the reaction dispersion system to ensure that the starch granules are in the alkaline pH environment required for the experiment and to avoid changes in the pH of the solution during the staining process. In the process of treating starch in an alkaline environment, an aqueous starch solution is added to an alkaline solution, and the pH is adjusted to 7-14 according to the required experimental conditions to prepare a starch emulsion. When diluting with an alkaline solution, the starch emulsion is diluted with an alkaline solution of the same pH as the starch emulsion, so that the starch concentration after dilution is 10-40 mg / L, to obtain a dilute starch solution with the same pH as the starch emulsion; the alkaline solution is the same as the alkaline solution used to treat starch in an alkaline environment; When staining starch granules with Nile Red dye, a dilute starch solution is mixed with 1-5 mg / mL Nile Red ethanol dye solution at a ratio of 20-500:

1. The mixture is shaken at room temperature for 3-5 minutes to ensure that Nile Red is in full contact with the starch granules and to label the starch-bound lipids. Afterward, the reaction system is centrifuged for 5-20 minutes, the supernatant is discarded, and the starch granules are precipitated and washed with the alkaline solution to remove residual dye. S2. Add 0.5-2 mL of the alkaline solution described in step S1 to the starch granule precipitate, vortex, and drop 5-20 μL of sample onto a glass slide, spreading it evenly. Observe the sample using a laser confocal microscope, and obtain the distribution of lipids in the starch granules under alkaline conditions through laser excitation and in-situ scanning imaging.

2. The method for observing the distribution of lipids bound to starch granules under alkaline conditions according to claim 1, characterized in that, The starch is selected from corn starch, rice starch, potato starch, glutinous rice starch, wheat starch, or konjac starch.

3. The method for observing the distribution of lipids bound to starch granules under alkaline conditions according to claim 1, characterized in that, The alkaline solution is a sodium hydroxide solution.

4. The method for observing the distribution of lipids bound to starch granules under alkaline conditions according to claim 1, characterized in that, During the dyeing reaction, continuous shaking ensures that the starch granules are evenly dispersed in the dye.

5. The method for observing the distribution of lipids bound to starch granules under alkaline conditions according to claim 1, characterized in that, The Nile Red ethanol dye solution is prepared fresh for each use; the ethanol used is 100% pure ethanol.

6. The method for observing the distribution of lipids bound to starch granules under alkaline conditions according to claim 1, characterized in that, In step S1, wash away any residual dye until the supernatant after vortexing and centrifugation has no obvious color.

7. The method for observing the distribution of lipids bound to starch granules under alkaline conditions according to claim 1, characterized in that, In step S2, the observation of the sample using a laser confocal microscope includes the following steps: placing the prepared glass slide above the objective lens on which cedar oil has been added in advance, and exciting it with a laser; detecting its emitted light at wavelength intervals of 570-649 nm.

8. The method for observing the distribution of lipids bound to starch granules under alkaline conditions according to claim 7, characterized in that, The excitation was performed using a DPSS and a 561 nm laser operating at 1-4% power, respectively.

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