Method for accurate measurement of wheat starch granule size using scanning electron microscopy with effective dispersion of starch granules

Abstract

The application belongs to the technical field of analysis and determination, and discloses a method for effectively dispersing starch particles for accurately measuring the size of wheat starch particles by using a scanning electron microscope, which comprises the following steps: weighing the extracted wheat starch, dissolving the wheat starch in pure water to prepare a wheat starch mother liquor; dissolving the wheat starch mother liquor in a Tween solution to obtain a wheat starch suspension; sucking the wheat starch suspension and dropping it on a silicon wafer to dry in an oven; spraying gold on the surface of the silicon wafer by using an ion sputtering instrument; and observing and measuring the silicon wafer by using a scanning electron microscope after the spraying. The determination result of the method is more accurate, and the phenomenon of inaccurate measurement result caused by the aggregation or incomplete exposure of starch particles can be avoided.

Description

Technical Field

[0001] This invention belongs to the field of analytical measurement technology and relates to a method for effectively dispersing starch granules for accurate measurement of wheat starch particle size using a scanning electron microscope. Background Technology

[0002] Wheat (Triticum aestivum L.) is one of the world's three major grains, and its cultivated yield, planting area, and distribution range are among the largest of all food crops globally. Currently, the global annual planting area exceeds 2.2 million hectares. 2 With an annual output of nearly 750 million tons, wheat accounts for one-third of the world's total grain production. In recent years, with the improvement of people's living standards, while pursuing high yields, high quality has also become crucial for wheat production. The main component of wheat is starch, which plays a key role in the quality and functional characteristics of wheat. Wheat starch is affected by the growth environment, and its structure and properties, in turn, affect wheat quality. In the study of starch structure and properties, the research on starch morphology and types is fundamental.

[0003] Conventional methods for observing wheat starch involve transversely slicing mature wheat grains, attaching them to conductive adhesive, sputtering gold, and then observing them under a scanning electron microscope; alternatively, extracting wheat starch is directly sprinkled onto conductive adhesive, sputtered with gold, and then observed; another method involves dropping starch in ethanol or an aqueous solution onto a silicon wafer, drying it, and then sputtering gold for observation. Wheat starch has a spherical, biconvex lens-like morphology with smooth edges. For direct characterization of starch granule morphology and accurate particle size measurement, the convex surface needs to be laid flat on a conductive medium. However, all of the above methods have drawbacks:

[0004] 1. In the method of observing wheat grains by cutting them open, the cross-section of wheat grains is easily damaged by the blade, resulting in a "smoothing" phenomenon where the overall structure is not prominent. There is also insufficient exposure of starch granules, making it impossible to fully observe the starch morphology and measure its size.

[0005] 2. When the extracted wheat starch granules are directly sprinkled onto the conductive adhesive for observation, the wheat starch granules will appear to aggregate and cluster. Although individual starch granules can be found, the background of the conductive adhesive is not clean, which affects the overall imaging effect.

[0006] 3. The method of observing starch particles by dispersing them with ethanol or water is not suitable for dispersing starch because the surface tension of ethanol and water is not suitable for dispersing starch. During the drying process of the droplets, starch particles will accumulate or become obliquely embedded, making it impossible to directly characterize the morphology and measure the true particle size.

[0007] In the above method, it is difficult to obtain images of three types of starch, A, B and C, that are uniformly dispersed and have complete morphology in a single scanning electron microscope image, which affects the effective determination of wheat starch particle size. Summary of the Invention

[0008] This invention provides a method for altering the surface tension of starch droplets, causing the biconvex surfaces of starch granules to be uniformly dispersed on a silicon wafer, thus avoiding the embedding or stacking of starch granules. Starch thin films prepared using this method can be observed under a scanning electron microscope to simultaneously obtain clear images of the biconvex surfaces of types A, B, and C starch, and the starch particle size can be accurately measured.

[0009] This invention provides a method for effectively dispersing starch granules for accurate measurement of wheat starch particle size using a scanning electron microscope, comprising the following steps:

[0010] S1 Weigh the extracted wheat starch and dissolve it in pure water to prepare wheat starch mother liquor;

[0011] S2 dissolves wheat starch mother liquor in Tween solution to obtain wheat starch suspension;

[0012] S3 picks up a wheat starch suspension and drops it onto a silicon wafer, then dries it in an oven;

[0013] S4 was sprayed with gold using an ion sputtering apparatus.

[0014] After S5 is sputter-coated with gold, it is observed and measured using a scanning electron microscope.

[0015] Specifically, the concentration of the starch suspension mother liquor in S1 is 0.05 g / ml-1.0 g / ml, preferably 0.10 g / ml-0.5 g / ml, such as 0.2 g / ml.

[0016] In a specific embodiment, the wheat starch suspension in S2 is diluted 10-30 times relative to the wheat starch mother liquor, preferably 15-25 times, such as 20 times.

[0017] In a preferred embodiment, the Tween is Tween 20, and the concentration of the Tween solution is 0.001%-0.002%.

[0018] Specifically, in S3, a starch thin layer is formed by drying at 35-40℃ for 5-15 minutes.

[0019] In a specific embodiment, during operation S3, 9-12 μL of wheat starch suspension is taken and dropped onto a 0.4-1 cm medium. 2 On the silicon wafer.

[0020] In a specific embodiment, when gold is sputtered onto the starch in S4, the vacuum degree is 3.0-5.0 Pa; the ion current is 20-30 mA; and the sputtering time is 15-45 s, forming a 10-20 nm conductive thin layer on the surface of the starch thin layer.

[0021] Preferably, the working distance is 8-10mm; the magnification is 300-500X; and the voltage is 3-5KV.

[0022] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0023] By altering the surface tension of starch droplets using Tween 20, multiple diffusion spots are formed within the starch-spreading area. At the edges of these diffusion spots, a monolayer of starch granules can be formed, with all starch particles in this monolayer dispersed with their biconvex surfaces facing upwards, without any embedding or overlapping. Starch thin layers prepared using this method can be observed under a scanning electron microscope, simultaneously revealing clear biconvex surfaces of starch of types A, B, and C, and allowing for accurate measurement of starch particle size.

[0024] The choice of Tween20 in this invention was made after careful consideration and experimental verification. Tween is a surfactant that can alter the surface tension of a solution, playing a role in the uniform dispersion of solutes. Tween20 has the shortest fatty acid chain, the lowest hydrophobicity, and the strongest hydrophilicity, thus exhibiting the strongest surface activity and the greatest ability to alter the surface tension of a solution within its family. Based on Tween20's ability to alter surface tension, this invention forms a thin starch layer at the edge of the starch aggregation area during the starch solution drying process, allowing the biconvex surface of the starch to face upwards. However, to simultaneously obtain three types of starch (A, B, and C) in a single image, it is necessary to ensure a sufficient number of starch granules within the field of view. Attached Figure Description

[0025] Figure 1 Image showing the dispersion of 0.002% Tween20 starch solution according to the present invention (Example 1);

[0026] Figure 2 The morphology of starch granules in a cross-section of wheat grains (Comparative Example 1);

[0027] Figure 3 The image obtained by directly sprinkling the extracted starch onto conductive adhesive (Comparative Example 2);

[0028] Figure 4 Image showing the dispersion of starch in aqueous solution (Comparative Example 3);

[0029] Figure 5 Image of starch dispersion in ethanol solution (Comparative Example 4);

[0030] Figure 6 Image showing dispersion of 0.01% Tween20 starch solution (Control Example 5);

[0031] Figure 7 Image showing dispersion of 0.1% Tween20 starch solution (Control Example 5);

[0032] Figure 8Image showing the dispersion of a 0.001% Tween20 starch solution;

[0033] Figure 9 Image showing the dispersion of a 0.0005% Tween20 starch solution. Detailed Implementation

[0034] The present invention will be described below through specific embodiments in order to better understand the inventors, but this does not constitute a limitation on the present invention.

[0035] Example 1

[0036] Weigh 0.01 g of extracted wheat starch and dissolve it in 50 μL of pure water to prepare a starch suspension stock solution. Dilute the stock solution with 0.002% Tween 20 at a ratio of 20:1 to prepare a starch suspension. Pipette 10 μL and drop it into a 0.5 cm... 2 The silicon wafer was dried in an oven at 37°C for 10 minutes. After removal, it was sputtered with gold using an ion sputtering apparatus under the following conditions: vacuum degree of 3.0-5.0 Pa; ion current of 30 mA; sputtering time of 30 s, forming a 15 nm conductive thin layer on the surface of the starch granules.

[0037] The prepared starch thin layer was placed in a scanning electron microscope (SEM) with a working distance of 9.37 mm and a voltage of 3 kV for observation. Measurements were performed using the built-in measurement tools of the SEM software. The observed starch granule morphology and particle size are shown below. Figure 1 As can be seen from the figure, the biconvex surface of wheat starch lies flat on the silicon wafer, and there are no stacking effects between the starch granules, making it easy to measure.

[0038] Compare with Example 1

[0039] Wheat grains were cross-sectioned using a blade to prepare their cross-sections. The wheat grains were then adhered to conductive tape, sputter-coated with gold, and observed and measured under a scanning electron microscope (SEM) (gold sputtering and SEM parameters were the same as in the previous example). The observed starch granule morphology and particle size are as follows: Figure 2 As can be seen from the figure, the wheat starch granules are stuck together, making effective measurement difficult. This comparative example demonstrates that directly cutting wheat grains with a blade to measure starch granule size is not an effective method.

[0040] Compare with Example 2

[0041] The extracted starch granules were evenly sprinkled onto a T-shaped stage coated with conductive adhesive, sputtered with gold, and then observed and measured under a scanning electron microscope (SEM) (the sputtering and SEM parameters were the same as in the previous example). The observed morphology and particle size of the starch granules are as follows: Figure 3As can be seen from the figure, the wheat starch granules are aggregated, the biconvex surface fails to lie flat on the silicon wafer, and there are granules embedded between each other, while the granules also stack against each other, affecting the measurement of wheat starch granules. This comparative example shows that directly sprinkling starch granules onto conductive adhesive to measure starch particle size is not an effective method.

[0042] Compare with Example 3

[0043] 0.01 g of extracted wheat starch was weighed and dissolved in 50 μL of pure water to prepare a starch suspension mother liquor. The mother liquor was diluted with pure water at a ratio of 20:1, and 10 μL was dropped onto a silicon wafer. The wafer was then dried in a 37°C oven for 10 min. After drying, gold was sputtered onto the wafer, and the microscopy (Gold sputtering and SEM parameters were the same as in the previous example) was used for observation and measurement. The observed starch granule morphology and particle size are as follows: Figure 4 .from Figure 4 As can be seen, although the dispersion of wheat grains was better than that of Control Example 1, the same phenomenon still existed: wheat starch grains aggregated, the biconvex surfaces failed to lie flat on the silicon wafer, and grains were embedded between each other. Furthermore, the grains stacked together, making it difficult to simultaneously display the biconvex morphology of types A, B, and C starch in a single image, thus affecting the measurement of wheat starch grains. This control example demonstrates that the method of measuring starch grain size using water-dispersed starch grains is not effective.

[0044] Compare with Example 4

[0045] 0.01 g of extracted wheat starch was weighed and dissolved in 50 μL of pure water to prepare a starch suspension mother liquor. The mother liquor was diluted with anhydrous ethanol at a ratio of 20:1, and 10 μL was dropped onto a silicon wafer. The wafer was then dried in a 37°C oven for 10 min. After drying, gold was sputtered onto the wafer, and the microscopy (Gold sputtering and SEM parameters were the same as in the previous example) was used for observation and measurement. The observed starch granule morphology and particle size are as follows: Figure 5 As can be seen from the figure, although the wheat grain dispersion was better than in Control Example 2, the grains were still aggregated. The biconvex surface failed to lie flat on the silicon wafer, and some grains were embedded between each other, while the grains also stacked against each other, which seriously affected the measurement of wheat starch grains. This control example shows that the method of using ethanol to disperse starch grains is not effective for measuring starch grain size.

[0046] Compare with Example 5

[0047] Following the method described in Example 1, starch suspensions were prepared using 0.01% and 0.1% Tween 20, respectively, and then observed using scanning electron microscopy. The observed starch granule morphology and particle size are shown below. Figure 6 and Figure 7As shown, it was found that as the Tween20 concentration gradually increased, the number of starch granules with biconvex surfaces horizontally spread on the silicon wafer decreased, and the area of ​​starch aggregation regions formed under the same field of view became smaller and more dispersed, making it impossible to simultaneously present the three types of wheat starch, A, B, and C, in the same image.

[0048] Based on Example 1 and Comparative Example 5, it can be found that the starch aggregates formed by the higher concentration of Tween20 (0.1%) are small and dispersed, with fewer starch particles in the same field of view and severe background contamination; the aggregate area and dispersion of Tween20 (0.01%) diluted 10 times are improved compared with 0.1% Tween20, but are still relatively dispersed.

[0049] The inventors further tested the following experiment: the aggregated area formed by Tween20 diluted 50 times (0.002%) was moderate, ensuring the amount of starch while allowing the starch to spread evenly on the silicon wafer with its biconvex surface facing upwards. The dispersion effect of Tween20 was particularly noticeable at the diffusion spots formed at the edges of the aggregated areas. Further experiments were conducted using 0.001% (100 times) and 0.0005% (200 times) Tween20, and the resulting aggregated area was found to be moderate, ensuring the amount of starch while allowing the starch to spread evenly on the silicon wafer with its biconvex surface facing upwards. However, the area of ​​the diffusion spots formed at the edges of the aggregated areas gradually decreased (see [see details]). Figure 8 and Figure 9 Therefore, the optimal concentration of Tween solution is determined to be 0.001%-0.002%.

Claims

1. A method for accurate measurement of wheat starch granule size by scanning electron microscopy with efficient dispersion of starch granules, characterized in that, The method comprises the following steps: S1: taking the extracted wheat starch and dissolving it in pure water to prepare a wheat starch mother liquor; S2: dissolving the wheat starch mother liquor in a Tween solution to obtain a wheat starch suspension; S3: taking the wheat starch suspension and dropping it on a silicon wafer for drying in an oven; S4: using an ion sputtering instrument to spray gold on the surface; S5: observing and measuring after spraying gold using a scanning electron microscope; The concentration of the starch suspension mother liquor in S1 is 0.10g / ml-0.5g / ml; The wheat starch suspension in S2 is diluted 15-25 times relative to the wheat starch mother liquor; the Tween is Tween20, and the concentration of the Tween solution is 0.001%-0.002%; S3 is dried at 35-40°C to form a thin layer of starch, and the drying time is 5-15 min; 9-12 ul of wheat starch suspension is dropped on 0.4-1 cm 2 on a silicon wafer; In S4, when the ion sputtering instrument is used to spray gold, the vacuum degree is 3.0-5.0Pa; the ion current is 20-30mA; the sputtering time is 15-45S, and a 10-20nm conductive thin layer is formed on the surface of the starch thin layer; in S4, when the scanning electron microscope is used for observation, the working distance is 8-10mm; the magnification is 300-500X; and a voltage of 3-5KV is selected.

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