A method for simultaneously observing the distribution of starch granule-bound proteins and lipids

Abstract

The invention discloses a method for simultaneously observing the distribution of starch granule-bound proteins and lipids, including the steps of starch protein staining, starch lipid staining, centrifugation, cleaning, preparation of a double-stained starch granule suspension, and laser confocal microscopy observation, wherein the method is characterized in that: starch granule proteins and lipids are stained respectively with fluorescamine and Pro-Q Diamond, thereby fluorescently labeling proteins and lipids in starch granules at the same time; optically sectioning the sample with a laser confocal microscope, and obtaining the distribution of proteins and lipids in starch granules by dual-channel excitation and in-situ scanning imaging. The invention solves the problem that the distribution of starch granule-bound proteins and lipids cannot be observed simultaneously.

Description

Technical Field

[0001] The invention belongs to the technical field of starch granule staining and relates to a method for simultaneously observing the distribution of starch granule-bound proteins and lipids. Background Art

[0002] Although the content of starch granule-bound proteins and lipids is not high, they play an extremely important role in enhancing the integrity and rigidity of starch granule structure, as well as the physicochemical, functional and nutritional properties of starch. In view of this, revealing the distribution of starch granule-bound proteins and lipids in starch granules has always been a hot topic and frontier in the field of starch research.

[0003] At present, the research on starch binding lipids and proteins on starch structure, properties and functions is mainly to infer the role of starch binding lipids and proteins by comparing the changes in its structure, properties and functions before and after degreasing and deproteinization, and great progress has been made. With the continuous advancement of research on starch binding proteins and lipids, a more comprehensive and in-depth revelation of the functions of starch granule binding proteins and lipids has become the focus of attention. However, the revelation of the functions of starch binding proteins and lipids is not satisfactory. The main reason is that the distribution of starch granule binding proteins and lipids is observed by single staining, and the relative spatial position of starch binding proteins and lipids cannot be observed at the same time. When two fluorescent dyes are directly added to the staining system, the dyes used for fluorescent labeling of proteins and lipids have their own most suitable reaction conditions (such as temperature, pH, amount of fluorescent labeling dyes added, whether to avoid light, etc.), so the different reaction system conditions limit the simultaneous addition of the two dyes, and the physical or chemical reaction between the two fluorescent labeling dyes leads to fluorescence quenching of the dyes, or causes the labeling of lipids and proteins to be non-specific, making it difficult to reflect the relative spatial distribution of starch granule binding proteins and lipids in starch granules. When fluorescent dyes are used to indirectly mark lipids or proteins, the first-labeled fluorescent dye will produce corresponding steric hindrance, repulsion and other adverse effects on the subsequent binding of fluorescent dyes to starch after binding to starch granules, or fluorescence quenching occurs between the first-labeled dye and the subsequent fluorescent dye, and the previous fluorescent marker will be separated from the starch granules due to inconsistency with the previous reaction conditions when performing subsequent fluorescent labeling. Due to the problems shown by the fluorescent dyes labeled with starch granules and lipids in the process of dyeing reaction with starch granules, there is currently no relevant report or invention patent that can achieve the simultaneous observation of the relative spatial distribution of starch granule-bound proteins and lipids. Therefore, a method that can simultaneously observe the distribution of starch granule-bound proteins and lipids in starch granules is urgently needed. This method will provide technical support for comprehensively and deeply revealing the functions of starch granule-bound proteins and lipids in starch granule structure, and is of great significance for guiding the research on starch structure, properties and functions related to starch binding proteins and lipids, as well as the application of starch in the food industry. Summary of the invention

[0004] In order to overcome the shortcomings of the single staining method, the present invention provides a method for simultaneously observing the distribution of starch granule-bound proteins and lipids, which is of great significance for guiding the research on starch structure, properties and functions related to starch-bound proteins and lipids and the application of starch in the food industry.

[0005] The method of the present invention for simultaneously observing the distribution of starch granule-bound proteins and lipids comprises the steps of dyeing starch proteins, dyeing starch lipids, centrifuging, washing, preparing a double-dyed starch granule suspension and observing with a laser confocal microscope. The specific technical scheme is as follows:

[0006] The present invention provides a method for simultaneously observing the distribution of starch granule-bound proteins and lipids, the method comprising the following steps:

[0007] S1, using fluorescamine and Pro-Q Diamond to stain starch granule-bound proteins and lipids in sequence;

[0008] S2. Use laser confocal microscopy to perform optical sectioning of the sample, and obtain the distribution of protein and lipid in starch granules through dual-channel excitation and in-situ scanning imaging. At this time, green fluorescence represents the distribution of protein in starch granules, and red fluorescence represents the distribution of lipid.

[0009] In the present invention, the starch granules include wheat starch granules, rice starch granules, corn starch granules, and potato starch granules.

[0010] The wheat starch granules are one or a mixture of wheat type A and type B starch granules. The wheat type A and type B starch granules are separated by gravity sedimentation. Preferably, the separated wheat type A and type B starch granules are sieved through 100-300 meshes respectively.

[0011] The separation of wheat A-type and B-type starch granules includes the following steps:

[0012] A1. Mix starch and water, stir, let stand, and take the upper suspension;

[0013] A2, add water, repeat step A1 of stirring, letting stand, and taking the upper suspension;

[0014] A3. Repeat step A2 several times until the upper liquid is clear after standing; the precipitate is the type A starch granule sample;

[0015] A4. Mix the upper suspension obtained each time to obtain the B-type starch granule sample;

[0016] A5. The obtained A-type starch granule samples and B-type starch granule samples are filtered and dried respectively.

[0017] The staining method of the present invention is applicable not only to type A and type B starch granules, but also to samples mixed with type A and type B starch granules, so the separation of type A and type B starch granules will not affect the subsequent double staining reaction. Relying on the double staining method, it is found that the distribution of type A and type B starch granules bound proteins and lipids in starch granules is significantly different. The purity of the type A and type B wheat starch granules obtained by the separation will have a favorable effect on the subsequent observation of the distribution of double-stained starch granules bound proteins and lipids, and the size of the starch granules can be clearly seen by laser confocal microscopy to ensure the separation effect.

[0018] In step A1, 400-600 mL of water is added to every 10-30 g of starch and mixed.

[0019] In step A1, the stirring is performed by magnetic stirring, the stirring speed is 300-600 rpm, the stirring time is 10-50 min, and the standing time is 5-30 min.

[0020] In step A5, the filtration is performed by filtration using a Buchner funnel, and then filtration is performed several times using anhydrous ethanol.

[0021] In step A5, the drying is performed at 35-40° C. for 16-48 hours.

[0022] As a specific example, the separation is to mix 10-30g starch with 400-600mL water in a beaker, stir magnetically for 10-50min, and after standing for 5-30min, take 100-300mL of the upper suspension through a pipette, which is type B particles, add 100-300mL of water, repeat the above-mentioned water addition, magnetic stirring, standing and pipetting process until the upper liquid is clear after standing. Finally, the upper suspension obtained each time is mixed to obtain type B starch particles. Finally, the remaining precipitate in the beaker is collected as type A starch particles. The obtained wheat A and B type starch granule samples were filtered with a Buchner funnel respectively, and finally filtered several times with 50-200 mL of anhydrous ethanol. The filtered samples were placed in an iron plate and roughly crushed with a medicine spoon to spread them evenly on the sample plate with a thickness of less than 1 cm. They were placed in a 35-40°C oven and dried for 16-48 hours. After grinding in a mortar, they were sieved through a 100-300 mesh.

[0023] Preferably, in step A1, the wheat starch raw material used is extracted by alkali leaching, wherein the alkali is 0.1-0.3% sodium hydroxide solution.

[0024] Alkaline leaching extraction includes the following steps:

[0025] A1-1. Soak wheat seeds in water at 4-8°C overnight, pour out the water, mix the soaked seeds with 0.1-0.3% sodium hydroxide solution, and put them into a pulping machine for pulping. The pulping process lasts for 5-15 seconds each time. Repeat 3-5 times, with an interval of 5-10 seconds between two pulpings. Filter the starch slurry with a 100-300 mesh sieve, and repeat this step until there is no milky white starch on the upper layer.

[0026] A1-2. Place the starch slurry at 4-8°C for 10-18h, pour off the upper liquid, add water and stir to suspend the precipitated starch, centrifuge, scrape off the upper yellow protein, add water and vortex, centrifuge again, repeat this step until no yellow protein layer appears on the upper layer after centrifugation; add water to the starch from which the protein has been scraped off, stir, adjust the pH of the starch slurry to neutral with 0.4-0.6M hydrochloric acid, and then filter, dry, and sieve.

[0027] In step A1-2, the filtration is first performed using a Buchner funnel, and then filtered several times using anhydrous ethanol.

[0028] In step A1-2, the drying is carried out in a 35-40°C oven for 16-48 hours.

[0029] In step A1-2, the sieving is performed by grinding in a mortar and then passing through a 100-300 mesh sieve.

[0030] In the present invention, step S1, fluorescent amine staining includes: adding 0.1% (w / v) fluorescent amine staining solution and 0.1M borate buffer to the starch granule dry base sample, vortexing for 10-50s, and continuously shaking the reaction at 20-30°C for 1-2h; centrifuging the suspension to obtain a precipitate to obtain starch granules dyed with fluorescent amine; the fluorescent amine staining solution uses acetonitrile as a solvent. During the dyeing reaction, continuous shaking is required to make the starch granules evenly dispersed in the dye to ensure that all starch can contact the dyeing reagent, thereby ensuring the uniformity of the reaction.

[0031] As an embodiment, the fluorescein amine staining solution is prepared and used immediately.

[0032] Preferably, the usage ratio of the starch granule dry sample, the fluorescamine staining solution and the borate buffer is: 10-20 mg: 0.2-0.5 mL: 0.1-0.2 mL, more preferably 15 mg: 0.3 mL: 0.15 mL.

[0033] In the present invention, a specific example of starch protein staining is: weigh 10-20 mg of starch dry sample, add 0.2-0.5 mL 0.1% (w / v, acetonitrile as solvent) fluorescamine, and then add 0.1-0.2 mL borate buffer (0.05-0.25 M, pH 7-9); after vortexing for 10-50 seconds, continuously shake the reaction at 20-30°C for 1-2 hours, centrifuge the suspension for 2-8 minutes (3000-6000×g) after the reaction, discard the supernatant, wash the precipitate several times with distilled water to wash away the residual dye, thereby obtaining starch granules stained with fluorescamine.

[0034] In the present invention, in step S2, the Pro-Q Diamond solution is added to the starch granules dyed with fluorescent amine, vortexed for 10-50 seconds, and then continuously shaken at 20-30° C. for 1-2 hours, washed, and the residual dye is removed to obtain double-dyed starch granules. The residual dye is removed until the supernatant after vortexing and centrifugation has no obvious color.

[0035] Preferably, the ratio of the starch granule dry sample to the Pro-Q Diamond solution is 10-20 mg: 0.4-1 mL, more preferably 15 mg: 0.5 mL.

[0036] In the present invention, a specific example of starch lipid staining is: adding 0.4-1 mL of Pro-Q Diamond solution to the fluorescein amine-stained starch granules, vortexing for 10-50 seconds, continuously shaking the reaction at 20-30°C for 1-2 hours, washing the starch granules with distilled water several times to remove residual dye, thereby obtaining double-stained starch granules.

[0037] In the present invention, before step S3, a preparation step of a double-dyed starch granule suspension is also included, wherein the double-dyed starch granules are added to 40%-60% glycerol to make the final concentration of the starch granules 4-6%, and after vortexing for 10-50 seconds, 5-20 μl of the sample is drawn and dropped onto a glass slide, and evenly coated to form a thin layer.

[0038] In the present invention, in step S3, the prepared slide is placed above the objective lens to which cedar oil has been dropped in advance, and a diode (405 nm) and DPSS (561 nm) laser are operated at powers of 50%-100% and 10%-30% respectively for excitation; and the emitted light is detected at interval wavelengths of 414-483 nm and 570-649 nm.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1) Compared with traditional single staining, by using two fluorescent dyes to simultaneously label starch granule-bound proteins and lipids, dual-channel excitation can simultaneously reveal the relative spatial positions of starch granule-bound proteins and lipids in starch granules, which is more systematic and in-depth;

[0041] 2) The simultaneous observation of starch granule-bound proteins and lipids provides technical support for revealing the interaction between proteins and lipids in starch granules and the functions of proteins and lipids in starch biosynthesis;

[0042] 3) The simultaneous observation of starch granule-bound proteins and lipids can provide guidance for the study of starch structure, properties (rheology, thermodynamics, texture, etc.) and functions (thickening, emulsification, etc.) related to starch-bound proteins and lipids, as well as the application of starch in the food industry;

[0043] 4) This double staining method is simple, efficient and reproducible, and can also be extended to the analysis of the relative spatial positions of granule-bound proteins and lipids in other starch granules (rice, corn, potato starch, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0045] Figure 1 The pictures of the distribution of wheat starch granule binding protein observed in Comparative Example 1; wherein (a) is a picture of the distribution of wheat A-type starch granule binding protein, and (b) is a picture of the distribution of wheat B-type starch granule binding protein;

[0046] Figure 2 The pictures of the lipid distribution of wheat starch granules observed in Comparative Example 2; wherein (a) is a picture of the lipid distribution of wheat A-type starch granules, and (b) is a picture of the lipid distribution of wheat B-type starch granules;

[0047] Figure 3 The pictures of wheat starch granule binding protein and lipid distribution observed in Example 1; wherein (a) is a picture of wheat A-type starch granule binding protein and lipid distribution, and (b) is a picture of wheat B-type starch granule binding protein and lipid distribution;

[0048] Figure 4 The pictures of wheat starch granule binding protein and lipid distribution observed in Example 2; wherein (a) is a picture of wheat A-type starch granule binding protein and lipid distribution, and (b) is a picture of wheat B-type starch granule binding protein and lipid distribution;

[0049] Figure 5These are pictures showing the distribution of wheat starch granule-bound proteins and lipids observed in Example 3; wherein (a) is a picture showing the distribution of wheat A-type starch granule-bound proteins and lipids, and (b) is a picture showing the distribution of wheat B-type starch granule-bound proteins and lipids. DETAILED DESCRIPTION

[0050] The present invention is fully described below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, without departing from the inventive concept, corresponding improvements can also be made, all of which belong to the protection scope of the present invention.

[0051] In order to overcome the shortcomings of the single staining method, the present invention provides a method for simultaneously observing the distribution of starch granule-bound proteins and lipids, which is of great significance for guiding the research on starch structure, properties and functions related to starch-bound proteins and lipids, as well as the application of starch in the food industry. The specific example technical scheme is as follows:

[0052] A method for simultaneously observing the distribution of proteins and lipids bound to wheat starch granules, comprising the steps of separation of wheat A-type and B-type starch granules, staining of starch proteins, staining of starch lipids, centrifugation, washing, preparation of a suspension of double-stained starch granules and observation using a laser confocal microscope, specifically comprising: using a gravity sedimentation method to separate wheat A-type and B-type starch granules; using fluorescein amine and Pro-Q Diamond (phosphorylated protein gel staining reagent) to stain starch granule proteins and lipids respectively; using a laser confocal microscope to optically section the sample, and obtaining distribution imaging of proteins and lipids in wheat starch granules through dual-channel excitation and in-situ scanning.

[0053] The wheat starch raw material used in the present invention is extracted by an alkali leaching method. Wheat seeds are placed in an appropriate amount of water and soaked overnight at 5°C. The water is poured out, and the soaked seeds are mixed with a 0.2% sodium hydroxide solution, and then placed in a pulping machine for pulping. The pulping is run for 10 seconds each time. After repeating 5 times, continuous operation may damage starch particles, so a 5-second interval is required between two pulpings. The starch slurry is filtered with a 200-mesh sieve, and this step is repeated until there is no milky white starch in the upper layer. After placing the starch slurry in an 8°C cold storage for 12 hours, pour off the upper liquid, add appropriate amount of water and stir continuously to suspend the precipitated starch, pour it into a 50mL centrifuge tube, centrifuge for 15 minutes (3000-5000g), scrape off the upper yellow protein with a weighing spoon, add appropriate amount of water and vortex and centrifuge, repeat this step until no yellow protein layer appears on the upper layer after centrifugation, add appropriate amount of water to the starch with the protein scraped off and stir, adjust the pH of the starch slurry to neutral with 0.6M hydrochloric acid, then filter with a Buchner funnel, and then filter with appropriate amount of anhydrous ethanol several times, put it in a 40°C oven to dry for 24 hours, grind it in a mortar and pass it through a 200-mesh sieve.

[0054] Comparative Example 1

[0055] (1) Separation of wheat type A and B starch granules: 20 g starch was mixed with 500 mL water in a beaker, magnetically stirred for 30 min, and after standing for 10 min, 200 mL of the upper suspension was removed by pipette. The suspension was type B granules. 200 mL water was added, and the above water addition, magnetic stirring, standing and pipetting processes were repeated until the upper liquid was clear after standing. Finally, the upper suspension obtained each time was mixed to obtain type B starch granules. Finally, the remaining precipitate in the beaker was collected as type A starch granules. The obtained wheat type A and B starch granule samples were filtered with a Buchner funnel, and finally filtered with 100 mL of anhydrous ethanol for 3 times. The filtered samples were placed in an iron plate and roughly crushed with a medicine spoon to make it flat on the sample plate with a thickness of less than 1 cm. They were placed in a 37°C oven and dried for 24 h. After grinding in a mortar, they were sieved through a 200-mesh sieve.

[0056] (2) Staining of starch protein: 15 mg of starch dry sample was accurately weighed and added to a 2 mL centrifuge tube containing 0.3 mL of 0.1% (w / v, acetonitrile as solvent) fluoresceinamine, and then 0.15 mL of borate buffer (0.1 M, pH 8.0) was added to the centrifuge tube. After vortexing for 15 seconds, the reaction was continuously shaken at 25°C for 1 hour. After the reaction, the suspension was centrifuged for 3 minutes (5000g), the supernatant was discarded, and the precipitate was washed 5 times with distilled water to wash away the residual dye, thereby obtaining dyed starch granules.

[0057] (3) Preparation of dyed starch granule suspension: Add dyed starch granules to 50% glycerol to make the final concentration of starch granules 5%. After vortexing for 15 seconds, pipette 10 μl of sample and drop it onto a glass slide. Use a pipette tip to evenly spread the droplet on the glass slide to form a thin layer and leave it for 5 minutes.

[0058] (4) Laser confocal microscopy observation: The prepared slide was placed on the objective lens on which cedar oil was previously added, and the DPSS (561 nm) laser was operated at 20% power for excitation. The emitted light was detected at intervals of 570-649 nm.

[0059] (5) Implementation effect Figure 1 As shown, by staining the protein, the green fluorescence shows the distribution of protein in the starch granules.

[0060] Comparative Example 2

[0061] (1) Separation of wheat type A and B starch granules: 20 g starch was mixed with 500 mL water in a beaker, magnetically stirred for 30 min, and after standing for 10 min, 200 mL of the upper suspension was removed by pipette. The suspension was type B granules. 200 mL water was added, and the above water addition, magnetic stirring, standing and pipetting processes were repeated until the upper liquid was clear after standing. Finally, the upper suspension obtained each time was mixed to obtain type B starch granules. Finally, the remaining precipitate in the beaker was collected as type A starch granules. The obtained wheat type A and B starch granule samples were filtered with a Buchner funnel, and finally filtered with 100 mL of anhydrous ethanol for 3 times. The filtered samples were placed in an iron plate and roughly crushed with a medicine spoon to make it flat on the sample plate with a thickness of less than 1 cm. They were placed in a 37°C oven and dried for 24 h. After grinding in a mortar, they were sieved through a 200-mesh sieve.

[0062] (2) Starch lipid staining: 15 mg of dry starch sample was accurately weighed and added to a centrifuge tube. 0.5 mL of Pro-Q Diamond solution was added and vortexed for 15 seconds. The mixture was continuously shaken at 25°C for 1 hour. The starch granules were washed 5 times with distilled water to remove the residual dye, thereby obtaining dyed starch granules.

[0063] (3) Preparation of dyed starch granule suspension: Add dyed starch granules to 50% glycerol to make the final concentration of starch granules 5%. After vortexing for 15 seconds, pipette 10 μl of sample and drop it onto a glass slide. Use a pipette tip to evenly spread the droplet on the glass slide to form a thin layer and leave it for 5 minutes.

[0064] (4) Laser confocal microscopy observation: The prepared slide was placed on the objective lens on which cedar oil had been dropped in advance, and a diode (405 nm) laser was used at 100% power for excitation, and the emitted light was detected at intervals of 414-483 nm.

[0065] (5) Implementation effect Figure 2 As shown, by staining lipids, red fluorescence shows the distribution of lipids in starch granules.

[0066] Example 1

[0067] (1) Separation of wheat type A and B starch granules: 20 g starch was mixed with 500 mL water in a beaker, magnetically stirred for 30 min, and after standing for 10 min, 200 mL of the upper suspension was removed by pipette. The suspension was type B granules. 200 mL water was added, and the above water addition, magnetic stirring, standing and pipetting processes were repeated until the upper liquid was clear after standing. Finally, the upper suspension obtained each time was mixed to obtain type B starch granules. Finally, the remaining precipitate in the beaker was collected as type A starch granules. The obtained wheat type A and B starch granule samples were filtered with a Buchner funnel, and finally filtered with 100 mL of anhydrous ethanol for 3 times. The filtered samples were placed in an iron plate and roughly crushed with a medicine spoon to make it flat on the sample plate with a thickness of less than 1 cm. They were placed in a 37°C oven and dried for 24 h. After grinding in a mortar, they were sieved through a 200-mesh sieve.

[0068] (2) Staining of starch protein: Type A and type B starch granules were respectively subjected to the staining operation, specifically, 15 mg of starch dry sample was accurately weighed and added to a 2 mL centrifuge tube containing 0.1 mL of 0.1% (w / v, acetonitrile as solvent) fluoresceinamine, and then 0.15 mL of borate buffer (0.1 M, pH 8.0) was added to the centrifuge tube. After vortexing for 15 seconds, the reaction was continuously shaken at 25° C. for 1 hour. After the reaction, the suspension was centrifuged for 3 minutes (5000 g), the supernatant was discarded, and the precipitate was washed 5 times with distilled water to wash away the residual dye, thereby obtaining dyed starch granules.

[0069] (3) Starch lipid staining: 0.5 mL of Pro-Q Diamond solution was added to the centrifuge tube containing the above-mentioned fluorescent amine-stained starch granules. After vortexing for 15 seconds, the reaction was continuously shaken at 25°C for 1 hour. The starch granules were washed 5 times with distilled water to remove the residual dye, thereby obtaining double-stained starch granules.

[0070] (4) Preparation of double-dyed starch granule suspension: Add double-dyed starch granules to 50% glycerol to make the final concentration of starch granules 5%. After vortexing for 15 seconds, draw 10 μl of sample and drop it onto a glass slide. Use a pipette to spread the droplet evenly on the glass slide to form a thin layer and leave it for 5 minutes.

[0071] (5) Laser confocal microscopy observation: The prepared slide was placed on the objective lens to which cedar oil had been added in advance, and the laser was excited by using a diode (405 nm) and DPSS (561 nm) laser at 100% and 20% power, respectively. The emitted light was detected at intervals of 414-483 nm and 570-649 nm. The green fluorescence represents the distribution of proteins in the wheat starch granules, and the red fluorescence represents the distribution of lipids.

[0072] (6) Implementation effect Figure 3 As shown, the distribution of proteins and lipids in starch granules is simultaneously displayed, but the fluorescence is not clear.

[0073] Example 2

[0074] (1) Separation of wheat type A and B starch granules: 20 g starch was mixed with 500 mL water in a beaker, magnetically stirred for 30 min, and after standing for 10 min, 200 mL of the upper suspension was removed by pipette. The suspension was type B granules. 200 mL water was added, and the above water addition, magnetic stirring, standing and pipetting processes were repeated until the upper liquid was clear after standing. Finally, the upper suspension obtained each time was mixed to obtain type B starch granules. Finally, the remaining precipitate in the beaker was collected as type A starch granules. The obtained wheat type A and B starch granule samples were filtered with a Buchner funnel, and finally filtered with 100 mL of anhydrous ethanol for 3 times. The filtered samples were placed in an iron plate and roughly crushed with a medicine spoon to make it flat on the sample plate with a thickness of less than 1 cm. They were placed in a 37°C oven and dried for 24 h. After grinding in a mortar, they were sieved through a 200-mesh sieve.

[0075] (2) Staining of starch protein: Type A and type B starch granules were respectively subjected to the staining operation, specifically, 15 mg of starch dry sample was accurately weighed and added to a 2 mL centrifuge tube containing 0.3 mL of 0.1% (w / v, acetonitrile as solvent) fluoresceinamine, and then 0.15 mL of borate buffer (0.1 M, pH 8.0) was added to the centrifuge tube. After vortexing for 15 seconds, the reaction was continuously shaken at 25° C. for 1 hour. After the reaction, the suspension was centrifuged for 3 minutes (5000×g), the supernatant was discarded, and the precipitate was washed 5 times with distilled water to wash away the residual dye, thereby obtaining dyed starch granules.

[0076] (3) Starch lipid staining: 0.5 mL of Pro-Q Diamond solution was added to the centrifuge tube containing the above-mentioned fluorescent amine-stained starch granules. After vortexing for 15 seconds, the reaction was continuously shaken at 25°C for 1 hour. The starch granules were washed 5 times with distilled water to remove the residual dye, thereby obtaining double-stained starch granules.

[0077] (4) Preparation of double-dyed starch granule suspension: Add double-dyed starch granules to 50% glycerol to make the final concentration of starch granules 5%. After vortexing for 15 seconds, draw 10 μl of sample and drop it onto a glass slide. Use a pipette to spread the droplet evenly on the glass slide to form a thin layer and leave it for 5 minutes.

[0078] (5) Laser confocal microscopy observation: The prepared slide was placed on the objective lens to which cedar oil had been added in advance, and the laser was excited by using a diode (405 nm) and DPSS (561 nm) laser at 100% and 20% power, respectively. The emitted light was detected at intervals of 414-483 nm and 570-649 nm. The green fluorescence represents the distribution of proteins in the wheat starch granules, and the red fluorescence represents the distribution of lipids.

[0079] (6) Implementation effect Figure 4 As shown, the distribution of proteins and lipids in starch granules is simultaneously displayed, with clear fluorescence and high image quality.

[0080] Example 3

[0081] (1) Separation of wheat type A and B starch granules: 20 g starch was mixed with 500 mL water in a beaker, magnetically stirred for 30 min, and after standing for 10 min, 200 mL of the upper suspension was removed by pipette. The suspension was type B granules. 200 mL water was added, and the above water addition, magnetic stirring, standing and pipetting processes were repeated until the upper liquid was clear after standing. Finally, the upper suspension obtained each time was mixed to obtain type B starch granules. Finally, the remaining precipitate in the beaker was collected as type A starch granules. The obtained wheat type A and B starch granule samples were filtered with a Buchner funnel, and finally filtered with 100 mL of anhydrous ethanol for 3 times. The filtered samples were placed in an iron plate and roughly crushed with a medicine spoon to make it flat on the sample plate with a thickness of less than 1 cm. They were placed in a 37°C oven and dried for 24 h. After grinding in a mortar, they were sieved through a 200-mesh sieve.

[0082] (2) Staining of starch protein: 15 mg of starch dry sample was accurately weighed and added to a 2 mL centrifuge tube containing 0.6 mL of 0.1% (w / v, acetonitrile as solvent) fluoresceinamine, and then 0.15 mL of borate buffer (0.1 M, pH 8.0) was added to the centrifuge tube. After vortexing for 15 seconds, the reaction was continuously shaken at 25°C for 1 hour. After the reaction, the suspension was centrifuged for 3 minutes (5000×g), the supernatant was discarded, and the precipitate was washed 5 times with distilled water to wash away the residual dye, thereby obtaining dyed starch granules.

[0083] (3) Starch lipid staining: 0.5 mL of Pro-Q Diamond solution was added to the centrifuge tube of the starch granules stained with fluorescent amine. After vortexing for 15 seconds, the mixture was continuously shaken at 25°C for 1 hour. The starch granules were washed 5 times with distilled water to remove the residual dye, thereby obtaining double-stained starch granules.

[0084] (4) Preparation of double-dyed starch granule suspension: Add double-dyed starch granules to 50% glycerol to make the final concentration of starch granules 5%. After vortexing for 15 seconds, draw 10 μl of sample and drop it onto a glass slide. Use a pipette to spread the droplet evenly on the glass slide to form a thin layer and leave it for 5 minutes.

[0085] (5) Laser confocal microscopy observation: The prepared slide was placed on the objective lens to which cedar oil had been added in advance, and the laser was excited by using a diode (405 nm) and DPSS (561 nm) laser at 100% and 20% power, respectively. The emitted light was detected at intervals of 414-483 nm and 570-649 nm. The green fluorescence represents the distribution of proteins in the wheat starch granules, and the red fluorescence represents the distribution of lipids.

[0086] (6) Implementation effect Figure 5 As shown, the distribution of proteins and lipids in starch granules is displayed at the same time, and the picture has double images.

[0087] Example 4

[0088] (1) Separation of wheat type A and B starch granules: 20 g starch was mixed with 500 mL water in a beaker, magnetically stirred for 30 min, and after standing for 10 min, 200 mL of the upper suspension was removed by pipette. The suspension was type B granules. 200 mL water was added, and the above water addition, magnetic stirring, standing and pipetting processes were repeated until the upper liquid was clear after standing. Finally, the upper suspension obtained each time was mixed to obtain type B starch granules. Finally, the remaining precipitate in the beaker was collected as type A starch granules. The obtained wheat type A and B starch granule samples were filtered with a Buchner funnel, and finally filtered with 100 mL of anhydrous ethanol for 3 times. The filtered samples were placed in an iron plate and roughly crushed with a medicine spoon to make it flat on the sample plate with a thickness of less than 1 cm. They were placed in a 37°C oven and dried for 24 h. After grinding in a mortar, they were sieved through a 200-mesh sieve.

[0089] (2) Staining of starch protein: 15 mg of dry starch sample was accurately weighed and added to a 2 mL centrifuge tube containing 0.5 mL of sodium borate solution (0.1 M, pH 9.0), and then 0.2 mL of potassium cyanide (10 M) and CBQCA (10 M) were added to the centrifuge tube. After vortexing for 15 seconds, the reaction was continuously shaken at 25°C for 2.5 hours. After the reaction, the suspension was centrifuged for 3 minutes (5000 g), the supernatant was discarded, and the precipitate was washed 5 times with distilled water to wash away the residual dye, thereby obtaining dyed starch particles.

[0090] (3) Starch lipid staining: 0.5 mL of Pro-QDiamond solution was added to the centrifuge tube containing the CBQCA-stained starch granules. The mixture was vortexed for 15 s and then continuously shaken at 25 °C for 1 h. The starch granules were washed 5 times with distilled water to remove the residual dye, thereby obtaining double-stained starch granules.

[0091] (4) Preparation of double-dyed starch granule suspension: Add double-dyed starch granules to 50% glycerol to make the final concentration of starch granules 5%. After vortexing for 15 seconds, draw 10 μl of sample and drop it onto a glass slide. Use a pipette to spread the droplet evenly on the glass slide to form a thin layer and leave it for 5 minutes.

[0092] (5) Laser confocal microscopy observation: The prepared slide was placed on the objective lens to which cedar oil had been dropped in advance, and the laser was excited with a diode (405 nm) and an argon ion (488 nm) laser at 100% and 25% power, respectively. The emitted light was detected at intervals of 414-483 nm and 500-590 nm.

[0093] (6) The lipid staining in the implementation effect picture is not clear. Further adjustment of the dosage ratio of the dye combination cannot achieve the purpose of the present invention. This may be due to the steric hindrance and repulsion generated after the reaction of CBQCA with starch granule binding protein, which hinders the subsequent reaction of Pro-Q Diamond to starch granules, or because the CBQCA dye causes fluorescence quenching of the Pro-Q Diamond dye.

[0094] Example 5

[0095] (1) Separation of wheat type A and B starch granules: 20 g starch was mixed with 500 mL water in a beaker, magnetically stirred for 30 min, and after standing for 10 min, 200 mL of the upper suspension was removed by pipette. The suspension was type B granules. 200 mL water was added, and the above water addition, magnetic stirring, standing and pipetting processes were repeated until the upper liquid was clear after standing. Finally, the upper suspension obtained each time was mixed to obtain type B starch granules. Finally, the remaining precipitate in the beaker was collected as type A starch granules. The obtained wheat type A and B starch granule samples were filtered with a Buchner funnel, and finally filtered with 100 mL of anhydrous ethanol for 3 times. The filtered samples were placed in an iron plate and roughly crushed with a medicine spoon to make it flat on the sample plate with a thickness of less than 1 cm. They were placed in a 37°C oven and dried for 24 h. After grinding in a mortar, they were sieved through a 200-mesh sieve.

[0096] (2) Staining of starch protein: 15 mg of starch dry sample was accurately weighed and added to a 2 mL centrifuge tube containing 0.5 mL of FITC solution (0.1%, solvent: DMSO), and then 0.5 mL of sodium acetate buffer solution (10 mg / mL, pH 5.5) was added to the centrifuge tube. After vortexing for 15 seconds, the mixture was continuously shaken at 25°C and reacted for 2 hours in the dark. After the reaction, the suspension was centrifuged for 3 minutes (5000 g), the supernatant was discarded, and the precipitate was washed 5 times with distilled water to remove the residual dye, thereby obtaining dyed starch particles.

[0097] (3) Starch lipid staining: 0.5 mL of Pro-Q Diamond solution was added to the centrifuge tube containing the FITC-stained starch granules. The mixture was vortexed for 15 seconds and then continuously shaken at 25°C for 1 hour. The starch granules were washed 5 times with distilled water to remove the residual dye, thereby obtaining double-stained starch granules.

[0098] (4) Preparation of double-dyed starch granule suspension: Add double-dyed starch granules to 50% glycerol to make the final concentration of starch granules 5%. After vortexing for 15 seconds, draw 10 μl of sample and drop it onto a glass slide. Use a pipette to spread the droplet evenly on the glass slide to form a thin layer and leave it for 5 minutes.

[0099] (5) Laser confocal microscopy observation: The prepared slide was placed on the objective lens to which cedar oil had been dropped in advance, and the laser was excited by a diode (405 nm) and an argon ion (488 nm) laser at 100% and 10% power, respectively. The emitted light was detected at intervals of 414-483 nm and 500-560 nm.

[0100] (6) The protein staining in the implementation effect picture is not clear. Further adjustment of the dosage ratio of the dye combination cannot achieve the purpose of the present invention. This may be due to the difference between the reaction conditions of the subsequent Pro-Q Diamond staining system and the previous FITC staining system, which caused the protein already labeled with the fluorescent dye to be separated by the dye, or it may be due to the fluorescence quenching of the FITC dye by the Pro-Q Diamond dye.

[0101] Example 6

[0102] (1) Separation of wheat type A and B starch granules: 20 g starch was mixed with 500 mL water in a beaker, magnetically stirred for 30 min, and after standing for 10 min, 200 mL of the upper suspension was removed by pipette. The suspension was type B granules. 200 mL water was added, and the above water addition, magnetic stirring, standing and pipetting processes were repeated until the upper liquid was clear after standing. Finally, the upper suspension obtained each time was mixed to obtain type B starch granules. Finally, the remaining precipitate in the beaker was collected as type A starch granules. The obtained wheat type A and B starch granule samples were filtered with a Buchner funnel, and finally filtered with 100 mL of anhydrous ethanol for 3 times. The filtered samples were placed in an iron plate and roughly crushed with a medicine spoon to make it flat on the sample plate with a thickness of less than 1 cm. They were placed in a 37°C oven and dried for 24 h. After grinding in a mortar, they were sieved through a 200-mesh sieve.

[0103] (2) Staining of starch protein: 15 mg of starch dry sample was accurately weighed and added to a 2 mL centrifuge tube containing 0.3 mL of 0.1% (w / v, acetonitrile as solvent) fluoresceinamine, and then 0.15 mL of borate buffer (0.1 M, pH 8.0) was added to the centrifuge tube. After vortexing for 15 seconds, the reaction was continuously shaken at 25°C for 1 hour. After the reaction, the suspension was centrifuged for 3 minutes (5000g), the supernatant was discarded, and the precipitate was washed 5 times with distilled water to wash away the residual dye, thereby obtaining dyed starch granules.

[0104] (3) Starch lipid staining: 0.5 mL of sodium acetate buffer solution (10 mg / mL, pH 5.5) was added to the centrifuge tube containing the dyed starch granules. After vortexing for 15 seconds, 0.5 mL of Nile red (0.1%, ethanol as solvent) was added. The mixture was continuously shaken at 25°C for 2 hours. The starch granules were washed 5 times with distilled water to remove the residual dye, thereby obtaining double-stained starch granules.

[0105] (4) Preparation of double-dyed starch granule suspension: Add double-dyed starch granules to 50% glycerol to make the final concentration of starch granules 5%. After vortexing for 15 seconds, draw 10 μl of sample and drop it onto a glass slide. Use a pipette to spread the droplet evenly on the glass slide to form a thin layer and leave it for 5 minutes.

[0106] (5) Laser confocal microscopy observation: The prepared slide was placed on the objective lens to which cedar oil had been dropped in advance, and excited with argon ion (514 nm) and DPSS (561 nm) lasers at 50% and 20% power, respectively. The emitted light was detected at intervals of 558-640 nm and 570-649 nm.

[0107] (6) The lipid staining in the implementation effect picture is not clear. Further adjustment of the dosage ratio of the dye combination cannot achieve the purpose of the present invention. This may be due to the steric hindrance and repulsion generated after the reaction of fluorescent amine with starch granule binding protein, which hinders the subsequent reaction of Nile red to starch granules, or due to the fluorescence quenching of Nile red dye by fluorescent amine dye.

[0108] Example 7

[0109] (1) Separation of wheat type A and B starch granules: 20 g starch was mixed with 500 mL water in a beaker, magnetically stirred for 30 min, and after standing for 10 min, 200 mL of the upper suspension was removed by pipette. The suspension was type B granules. 200 mL water was added, and the above water addition, magnetic stirring, standing and pipetting processes were repeated until the upper liquid was clear after standing. Finally, the upper suspension obtained each time was mixed to obtain type B starch granules. Finally, the remaining precipitate in the beaker was collected as type A starch granules. The obtained wheat type A and B starch granule samples were filtered with a Buchner funnel, and finally filtered with 100 mL of anhydrous ethanol for 3 times. The filtered samples were placed in an iron plate and roughly crushed with a medicine spoon to make it flat on the sample plate with a thickness of less than 1 cm. They were placed in a 37°C oven and dried for 24 h. After grinding in a mortar, they were sieved through a 200-mesh sieve.

[0110] (2) Staining of starch protein: 15 mg of dry starch sample was accurately weighed and added to a 2 mL centrifuge tube containing 0.5 mL of sodium borate solution (0.1 M, pH 9.0), and then 0.2 mL of potassium cyanide (10 M) and CBQCA (10 M) were added to the centrifuge tube. After vortexing for 15 seconds, the reaction was continuously shaken at 25°C for 2.5 hours. After the reaction, the suspension was centrifuged for 3 minutes (5000 g), the supernatant was discarded, and the precipitate was washed 5 times with distilled water to wash away the residual dye, thereby obtaining dyed starch particles.

[0111] (3) Starch lipid staining: 0.5 mL of sodium acetate buffer solution (10 mg / mL, pH 5.5) was added to the centrifuge tube containing the dyed starch granules. After vortexing for 15 seconds, 0.5 mL of Nile red (0.1%, ethanol as solvent) was added. The mixture was continuously shaken at 25°C for 2 hours. The starch granules were washed 5 times with distilled water to remove the residual dye, thereby obtaining double-stained starch granules.

[0112] (4) Preparation of double-dyed starch granule suspension: Add double-dyed starch granules to 50% glycerol to make the final concentration of starch granules 5%. After vortexing for 15 seconds, draw 10 μl of sample and drop it onto a glass slide. Use a pipette to spread the droplet evenly on the glass slide to form a thin layer and leave it for 5 minutes.

[0113] (5) Laser confocal microscopy observation: The prepared slide was placed on the objective lens on which cedar oil had been dropped in advance, and excited with an argon ion (514 and 488 nm) laser at 50% and 25% power, respectively. The emitted light was detected at intervals of 558-640 nm and 500-590 nm.

[0114] (6) The protein staining in the implementation effect picture is not clear. Further adjustment of the dosage ratio of the dye combination cannot achieve the purpose of the present invention. This may be due to the pH of the subsequent Nile red staining system being inconsistent with the previous CBQCA staining system, which causes the protein already labeled with the fluorescent dye to be separated by the dye, or it may be due to the fluorescence quenching of the CBQCA dye by the Nile red dye.

[0115] Example 8

[0116] (1) Separation of wheat type A and B starch granules: 20 g starch was mixed with 500 mL water in a beaker, magnetically stirred for 30 min, and after standing for 10 min, 200 mL of the upper suspension was removed by pipette. The suspension was type B granules. 200 mL water was added, and the above water addition, magnetic stirring, standing and pipetting processes were repeated until the upper liquid was clear after standing. Finally, the upper suspension obtained each time was mixed to obtain type B starch granules. Finally, the remaining precipitate in the beaker was collected as type A starch granules. The obtained wheat type A and B starch granule samples were filtered with a Buchner funnel, and finally filtered with 100 mL of anhydrous ethanol for 3 times. The filtered samples were placed in an iron plate and roughly crushed with a medicine spoon to make it flat on the sample plate with a thickness of less than 1 cm. They were placed in a 37°C oven and dried for 24 h. After grinding in a mortar, they were sieved through a 200-mesh sieve.

[0117] (2) Staining of starch protein: 15 mg of starch dry sample was accurately weighed and added to a 2 mL centrifuge tube containing 0.5 mL of FITC solution (0.1%, solvent: DMSO), and then 0.5 mL of sodium acetate buffer solution (10 mg / mL, pH 5.5) was added to the centrifuge tube. After vortexing for 15 seconds, the mixture was continuously shaken at 25°C and reacted for 2 hours in the dark. After the reaction, the suspension was centrifuged for 3 minutes (5000 g), the supernatant was discarded, and the precipitate was washed 5 times with distilled water to remove the residual dye, thereby obtaining dyed starch particles.

[0118] (3) Starch lipid staining: 0.5 mL of sodium acetate buffer solution (10 mg / mL, pH 5.5) was added to the centrifuge tube containing the dyed starch granules. After vortexing for 15 seconds, 0.5 mL of Nile red (0.1%, ethanol as solvent) was added. The mixture was continuously shaken at 25°C for 2 hours. The starch granules were washed 5 times with distilled water to remove the residual dye, thereby obtaining double-stained starch granules.

[0119] (4) Preparation of double-dyed starch granule suspension: Add double-dyed starch granules to 50% glycerol to make the final concentration of starch granules 5%. After vortexing for 15 seconds, draw 10 μl of sample and drop it onto a glass slide. Use a pipette to spread the droplet evenly on the glass slide to form a thin layer and leave it for 5 minutes.

[0120] (5) Laser confocal microscopy observation: The prepared slide was placed on the objective lens with cedar oil added in advance, and excited with argon ion (514 and 488 nm) lasers at 50% and 10% power, respectively. The emitted light was detected at intervals of 558-640 nm and 500-560 nm.

[0121] (6) The lipid and protein staining in the implementation effect pictures is not clear. Further adjustment of the dosage ratio of the dye combination cannot achieve the purpose of the present invention. This may be due to the steric hindrance and repulsion generated after the reaction of FITC with starch granule binding protein, which hinders the subsequent reaction of Nile red to starch granules, or due to the difference between the reaction conditions of the subsequent Nile red staining system and the previous FITC staining system, resulting in the separation of the protein already labeled with the fluorescent dye from the dye, or due to the mutual fluorescence quenching of the Nile red dye and the FITC dye.

[0122] By comparing the above implementation cases, the wheat starch granule binding protein and lipid distribution were clearly observed in the examples, especially in Example 2.

[0123] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for simultaneously observing the distribution of starch granule-bound proteins and lipids, characterized in that: The method comprises the following steps: S1, using fluorescamine and Pro-Q Diamond to stain starch granule-bound proteins and lipids in turn; Fluorescein staining includes: adding 0.1% (w / v) fluorescein staining solution and borate buffer to a starch granule dry sample, vortexing for 10-50 seconds, and continuously shaking at 20-30° C. for 1-2 hours; centrifuging the suspension to obtain a precipitate to obtain starch granules stained with fluorescein; the fluorescein staining solution uses acetonitrile as a solvent; Add Pro-Q Diamond solution to the starch granules stained with fluorescamine, vortex for 10-50 seconds, and then continuously shake at 20-30°C for 1-2 hours, wash, remove the residual dye, and obtain double-stained starch granules; The usage ratio of the starch granule dry sample, the fluorescent amine staining solution and the borate buffer solution is: 10-20 mg: 0.2-0.5 mL: 0.1-0.2 mL; the usage ratio of the starch granule dry sample and the Pro-Q Diamond solution is 10-20 mg: 0.4-1 mL; S2. Use laser confocal microscopy to perform optical sectioning of the sample, and obtain the distribution of protein and lipid in starch granules through dual-channel excitation and in situ scanning imaging.

2. The method for simultaneously observing the distribution of starch granule-bound proteins and lipids according to claim 1, characterized in that: In step S1, the starch granules include wheat starch granules, rice starch granules, corn starch granules, and potato starch granules.

3. The method for simultaneously observing the distribution of starch granule-bound proteins and lipids according to claim 2, characterized in that: The wheat starch granules are one or a mixture of wheat type A and type B starch granules; the separation of wheat type A and type B starch granules comprises the following steps: A1. Mix starch and water, stir, let stand, and take the upper suspension; A2, add water, repeat step A1 of stirring, letting stand, and taking the upper suspension; A3. Repeat step A2 several times until the upper liquid is clear after standing; the precipitate is the type A starch granule sample; A4. Mix the upper suspension obtained each time to obtain the B-type starch granule sample; A5. The obtained A-type starch granule samples and B-type starch granule samples are filtered and dried respectively.

4. The method for simultaneously observing the distribution of starch granule-bound proteins and lipids according to claim 3, characterized in that: In step A1, 400-600 mL of water is added to every 10-30 g of starch for mixing; the stirring is performed by magnetic stirring, the stirring speed is 300-600 rpm, the stirring time is 10-50 min, and the standing time is 5-30 min.

5. The method for simultaneously observing the distribution of starch granule-bound proteins and lipids according to claim 3, characterized in that: In step A1, the wheat starch raw material used is extracted by alkali leaching; the extraction comprises the following steps: A1-1. Soak wheat seeds in water at 4-8°C overnight, pour out the water, mix the soaked seeds with 0.1-0.3% sodium hydroxide, and beat them. Each time the beating lasts for 5-15 seconds. Repeat 3-5 times, with an interval of 5-10 seconds between two beatings. Filter the starch slurry with a 100-300 mesh sieve, and repeat this step until there is no milky white starch on the upper layer. A1-2. Place the starch slurry at 4-8°C for 10-18h, pour off the upper liquid, add water and stir to suspend the precipitated starch, centrifuge, scrape off the upper yellow protein, add water and vortex, centrifuge again, repeat this step until no yellow protein layer appears on the upper layer after centrifugation; add water to the starch from which the protein has been scraped off, stir, adjust the pH of the starch slurry to neutral with 0.4-0.6M hydrochloric acid, and then filter, dry, and sieve.

6. The method for simultaneously observing the distribution of starch granule-bound proteins and lipids according to claim 1, characterized in that: Before step S2, the method also includes the step of preparing a suspension of double-dyed starch particles, adding the double-dyed starch particles into 40%-60% glycerol to make the final concentration of the starch particles 4-6%, vortexing for 10-50 seconds, and then aspirating 5-20 μl of the sample and dropping it onto a glass slide, and coating it evenly to form a thin layer.

7. The method for simultaneously observing the distribution of starch granule-bound proteins and lipids according to claim 6, characterized in that: In step S2, the prepared glass slide is placed above the objective lens onto which cedar oil is previously dropped, and the diode and DPSS lasers are operated at powers of 50%-100% and 10%-30% respectively for excitation; and the emitted light is detected at interval wavelengths of 414-483nm and 570-649nm.

Images