阅读说明：本技术 一种监测蒸煮过程中谷物内部组分结构及分布的方法 (Method for monitoring internal component structure and distribution of grains in cooking process ) 是由 王莉 耿涵 于 2021-07-16 设计创作，主要内容包括：本发明公开了一种监测蒸煮过程中谷物内部组分结构及分布的方法,属于不同蒸煮程度谷物结构剖析技术领域。本发明所述的动态监测蒸煮过程中谷物内部组分分布变化的方法包括蒸煮取样、固定、冲洗、脱水、透明、浸蜡、包埋、切片、展片、染色、观察；所述蒸煮取样是将蒸煮到不同温度的谷物样品进行取样；所述浸蜡是在54～56℃之间进行；所述展片是采用镊子取切片面,展于含有体积百分比为5％～10％的乙醇溶液中,完全舒展后再展于42℃水中,捞片,选择平展的谷物切面样品黏附于载玻片上。本发明能用于比较不同品种谷物间的蒸煮过程变化,从微观角度解释各品种间的蒸煮食味品质差异,为谷物品质形成提供理论基础。(The invention discloses a method for monitoring internal component structure and distribution of grains in a cooking process, and belongs to the technical field of grain structure analysis of different cooking degrees. The method for dynamically monitoring the distribution change of the internal components of the grains in the cooking process comprises the steps of cooking sampling, fixing, washing, dehydrating, transparentizing, waxing, embedding, slicing, spreading, dyeing and observing; the cooking sampling is to sample grain samples cooked to different temperatures; the wax dipping is carried out at 54-56 ℃; the slide is obtained by taking a slide face by using tweezers, spreading the slide face in an ethanol solution containing 5-10% of volume percentage, spreading the slide face in water at 42 ℃ after complete spreading, fishing out the slide, and selecting a flat grain section sample to be adhered on a glass slide. The method can be used for comparing the change of the cooking process among different varieties of grains, explaining the difference of the cooking taste and quality among the varieties from a microscopic angle and providing a theoretical basis for the formation of the grain quality.)

1. A method for observing the microstructure change of a cross section in the grain cooking process is characterized by comprising the steps of cooking sampling, quenching, natural truncation and observation; wherein the cooking sampling is sampling of grain samples cooked to different temperatures; the quenching is carried out in liquid nitrogen immediately after sampling.

2. The method as claimed in claim 1, wherein the cooking in the cooking sampling is performed by washing the grains, soaking for 30-40 min at 20-30 ℃, cooking for 30-40 min, and stewing for 10-15 min.

3. Method according to claim 1 or 2, characterized in that the monitoring of the different temperatures in the cooking samples is carried out by inserting a thermocouple in the central area of the grain which starts to cook after soaking, observing the temperature shown on the display screen, and taking out samples of the grain every 10 ℃.

4. A method for dynamically monitoring the distribution change of internal components of grains in a cooking process is characterized by comprising the steps of cooking sampling, fixing, washing, dehydrating, transparentizing, waxing, embedding, slicing, spreading, dyeing and observing; the cooking sampling is to sample grain samples cooked to different temperatures; the wax dipping is carried out at the temperature of 54-56 ℃.

5. The method according to claim 4, wherein the waxing is carried out by immersing the transparent grains in low-temperature paraffin wax at 54-56 ℃ for 30 min.

6. The method of claim 4 or 5, wherein the slide is prepared by taking the section plane, spreading the section plane in an ethanol solution containing 5-10% by volume, spreading the section plane in water at 42 ℃ after complete stretching, taking the slide, and selecting a sample of the flattened grain section plane to adhere to the slide.

7. The method according to any one of claims 4 to 6, wherein the dehydration is carried out by immersing the fixed cereal grains in a gradient concentration of 30%, 50%, 70%, 90% and 100% ethanol solution for 4-6 min so as to reduce the moisture content to below 5%; or immediately after sampling, putting the sample in liquid nitrogen for quenching so that the moisture content is reduced to be below 5 percent.

8. The method according to any one of claims 4 to 7, wherein the slicing comprises demolding the embedded grains on a freezing table for 20 to 30min, and after demolding, putting the grains in a refrigerator at 4 ℃ for solidification.

9. The method according to any one of claims 4 to 8, wherein the transparency is to immerse the dehydrated grains in a mixed solution of 50% ethanol and 50% xylene for 20 to 30min, and then transfer the grains to 100% high-concentration xylene for 20 to 30 min; wherein "%" is volume percent.

10. The method for observing the change of the microstructure of the cross section of the grain in the cooking process as claimed in any one of claims 1 to 3 and the method for dynamically monitoring the change of the distribution of the internal components of the grain in the cooking process as claimed in claims 4 to 9 are applied to the field of food.

Technical Field

The invention relates to a method for monitoring internal component structure and distribution of grains in a cooking process, and belongs to the technical field of grain structure analysis of different cooking degrees.

Background

The section technology is a basic experimental method for researching and observing the internal microstructures of animals and plants in biology and histology, and provides a basic technology for histochemical staining, immunohistochemical research and in-situ hybridization of the animals and plants. However, the images displayed by the sections are planar images and the magnification of the optical microscope is limited, so that the overall shape, three-dimensional shape and subcellular structure of the plant tissue are difficult to further observe.

Scanning electron microscopes are used for modulating and imaging various physical signals excited by a fine focused electron beam when scanning on the surface of a sample, and are widely used for observing microstructures in the subjects of biology, medicine, metallurgy and the like. The scanning electron microscope has large depth of field, imaging stereoscopic impression and high resolution, and is suitable for observing and analyzing the surface of a rough grain sample. The laser scanning confocal microscope is additionally provided with a laser scanning device on the basis of fluorescent microscope imaging, and a computer is used for image processing, so that a fluorescent image of a microstructure in a cell or tissue is obtained. Paraffin section, electron microscope scanning observation technology and laser confocal observation technology for plant tissues tend to be mature, and tissue profiling of different temperature sections in the grain cooking process is not common in dynamic monitoring.

Grains are used as main grain crops of people in the world, the composition and structural characteristics of grains are determined by the factors such as variety, production area, planting, harvesting, storage, processing and the like of the grains, and the process of cooking into fresh cooked food is the release process of final cooking quality. With the intensive research on the quality of grains, the relationship between the change of components and structures and the quality characteristics in the grain cooking process becomes a new hotspot of research. The non-invasive temperature measurement method can accurately control the cooking process, fully know the core temperature of the cooking process, and avoid the defect of inaccurate temperature measurement caused by the invasive method, such as infrared sensing, thermocouple probe temperature measurement and other approaches. While infrared sensing has limited penetration capability and is commonly used to monitor surface temperature. The thermocouple directly measures temperature, converts the temperature signal into a thermal electromotive force signal, converts the thermal electromotive force signal into the temperature of a measured medium through the electric instrument, generally comprises main parts such as a thermode, an insulating sleeve protection tube, a junction box, a display instrument and a recording instrument, is high in measurement accuracy, is not influenced by an intermediate medium, and is simple in structure and convenient to use.

The grain kernel growth process generally goes through the initial grain filling stage, the milk stage, the wax stage, the yellow stage and the full stage. As the kernel grows, the main nutrients in the kernel are accumulated continuously, and the endosperm gradually develops from a liquid state to a solid state with compact texture. Conventional grain texture analysis is mainly from early grain samples in grain filling due to the high moisture content and soft texture of grain kernels in the early stages of grain filling. As the moisture content inside the kernel decreases, the accumulation of starch and protein increases and the kernel structure becomes tight and difficult to cut.

The commonly used current slicing methods include paraffin section, resin-embedded section, microtome section, etc., such as: patent CN105699142A discloses a paraffin slicing method for wheat grains, and patent CN1043746019A discloses a resin slicing method for mature grains. The mature grain has high starch content, compact internal tissue and low water content, and the premise of obtaining complete slices is to improve the toughness of grains and reduce the brittleness. The traditional paraffin slicing method has long time and can lead the structure of a grain sample to be hard after dehydration for many times, the grain sample is easy to break when being sliced, and grain kernel slices with moderate hardness and complete structure can not be obtained. After the resin slices are embedded, the tissue structure is hard and difficult to cut, the operation difficulty is high, the requirement on a slicing machine is high, and the cost is high.

Disclosure of Invention

[ problem ] to

Paraffin section, electron microscope scanning observation technology and laser confocal observation technology for plant tissues tend to be mature, and tissue profiling of different temperature sections in the grain cooking process is not common in dynamic monitoring.

[ solution ]

In order to solve the problems, the invention provides a method for dynamically monitoring the structure and distribution change of internal components of grains in the cooking process, which not only expands a new method for researching the quality of grains, improves the efficiency of grain slicing, preserves the structure and distribution state of the internal components of grains, obtains smoother slices by ethanol-assisted slice spreading, realizes the distinguishing and distinguishing between the components by different fluorescent dyeing methods, and provides a new idea for identifying the fine structure of grains.

The first purpose of the invention is to provide a method for observing the change of the microstructure of the cross section in the grain cooking process, which comprises the steps of cooking sampling, quenching, natural truncation and observation; wherein the cooking sampling is sampling of grain samples cooked to different temperatures; the quenching is carried out in liquid nitrogen immediately after sampling.

In one embodiment of the invention, the cooking sampling comprises the steps of elutriating grains, soaking for 30-40 min at 20-30 ℃, cooking for 30-40 min and stewing for 10-15 min.

In one embodiment of the invention, the monitoring of different temperatures in the cooking sampling is carried out by inserting a thermocouple into the central area of the grain which starts to be cooked after soaking, observing the temperature shown by a display screen, and taking out the grain samples every 10 ℃.

In one embodiment of the present invention, the cereal is a cereal with a starch content of more than 40%, and includes rice, corn, wheat, quinoa, highland barley, soybean, mung bean, red bean, and the like.

In one embodiment of the invention, the cooked sample is selected from a full grain and full grain.

In one embodiment of the invention, the purpose of the quenching is to dry the moisture on the surface of the grain.

In one embodiment of the present invention, the natural cutting is to use two tweezers to clamp the quenched grain, break the grain at the middle part to obtain a grain section, and stick the section on the conductive adhesive.

In one embodiment of the invention, the observation is that the cross section of the grain is subjected to gold spraying treatment by an ion sputtering instrument, and the grain is placed into a scanning electron microscope to observe the component microstructure of the grain at different cooking temperatures.

The second purpose of the invention is to provide a method for dynamically monitoring the distribution change of internal components of grains in the cooking process, which comprises the steps of cooking sampling, fixing, washing, dehydrating, transparentizing, waxing, embedding, slicing, spreading, dyeing and observing; the cooking sampling is to sample grain samples cooked to different temperatures; the wax dipping is carried out at the temperature of 54-56 ℃.

In one embodiment of the invention, the cooking in the cooking sampling is to wash the grains, soak the grains for 30-40 min at 20-30 ℃, cook the grains for 30-40 min and stew the grains for 10-15 min.

In one embodiment of the invention, the monitoring of different temperatures in the cooking sampling is carried out by inserting a thermocouple into the central area of the grain which starts to be cooked after soaking, observing the temperature shown by a display screen, and taking out the grain samples every 10 ℃.

In one embodiment of the present invention, the cereal is a cereal with a starch content of more than 40%, and includes rice, corn, wheat, quinoa, highland barley, soybean, mung bean, red bean, and the like.

In one embodiment of the invention, the fixing is that the sampled grains are soaked in a fixing solution and placed in an environment at 25 ℃ for 24 hours, and the fixing solution is properly updated by observing the state of the grains; the fixing solution is prepared by one or more mixed solutions of glutaraldehyde and paraformaldehyde.

In one embodiment of the invention, the flushing is performed with water.

In one embodiment of the invention, the dehydration is to immerse the fixed cereal grains in ethanol solution with gradient concentration of 30%, 50%, 70%, 90% and 100% for 4-6 min, so as to reduce the water content to below 5%; or, immediately after the fixed sampling flushing, putting the fixed sampling into liquid nitrogen for quenching, so that the moisture content is reduced to be below 5 percent.

In one embodiment of the invention, the transparent step is that the dehydrated grains are soaked in a mixed solution of 50% ethanol and 50% xylene for 20-30 min, and then are transferred to 100% high-concentration xylene for soaking for 20-30 min; wherein "%" is volume percent.

In one embodiment of the invention, the wax dipping is to dip the transparent grains into low-temperature paraffin wax at 54-56 ℃ for 30 min.

In one embodiment of the invention, the embedding is carried out by putting the grain after being waxed into a high-temperature tank of an embedding instrument, and pouring liquid paraffin into the high-temperature tank at the temperature of 58-60 ℃ for embedding.

In one embodiment of the invention, the slicing is to demould the embedded grains on a freezing table for 20-30 min, and after demould, put the grains into a refrigerator at 4 ℃ for solidification so as to facilitate slicing. And carrying out ultramicro sectioning according to the required thickness by using a section cutter, wherein the section cutter is any one of a freezing section cutter, a paraffin section cutter and an ultramicro section cutter.

In one embodiment of the invention, the slide is obtained by taking the slide surface by using tweezers, spreading the slide surface in an ethanol solution containing 5-10% by volume, spreading the slide surface in water at 42 ℃ after complete spreading, fishing the slide, and selecting a flat grain section sample to be adhered to a glass slide.

In one embodiment of the invention, the sheeting is followed by baking, in particular, the glass slide with the cereal flakes attached is baked overnight in a 37 ℃ oven.

In one embodiment of the invention, the dyeing is fluorescent dyeing, the fluorescent dyeing comprises single dyeing and double dyeing, the single dyeing is fluorescein isothiocyanate, methyl orange, rhodamine, methyl red and nile red dyes which are prepared at the concentration of 0.2-4 mg/mL, and the single dyeing is used for dyeing starch, protein and fat in the grains respectively; wherein the starch is stained with fluorescein isothiocyanate or methyl orange; proteins were stained with rhodamine or methyl red; fat was stained with nile red. Washing with water for 1-2 times after dyeing, preparing a dye, and carrying out light-proof treatment in the dye storage, dyeing and washing processes; the re-dyeing is carried out by adopting a compound dyeing solution with the concentration of 0.2-4 mg/mL, and the compound dyeing solution contains two or more of rhodamine, nile red, fluorescein isothiocyanate or methyl orange; wherein the starch is stained with fluorescein isothiocyanate or methyl orange; proteins were stained with rhodamine or methyl red; staining fat with nile red; when the dye is used for dyeing starch and protein, the concentration of the starch dye is 10-20 times higher than that of the protein dye; when the dye is used for dyeing starch and fat, the concentration of the starch dye is 5-10 times of that of the fat dye; when the dye is used for dyeing protein and fat, the concentration of the protein dye is 5-10 times of that of the fat, the dye is washed for 1-2 times by water after dyeing, and the light-proof treatment is needed in the processes of dye preparation, dye storage, dyeing and washing.

In one embodiment of the present invention, the dyeing is performed by the following specific steps: respectively weighing fluorescein isothiocyanate, methyl orange, rhodamine, methyl red and nile red dyes, dissolving the dyes in an acetone solution, wherein the concentration of each dye is 0.2-4 mg/mL, and the starch is dyed by the fluorescein isothiocyanate or the methyl orange to show green fluorescence; the protein is dyed by rhodamine or methyl red and shows red fluorescence; fat is dyed with nile red and shows orange fluorescence; when the compound dye is used for dyeing starch and protein, the concentration of the starch dye is 10-20 times higher than that of the protein dye; when the dye is used for dyeing starch and fat, the concentration of the starch dye is 5-10 times of that of the fat dye; when the dye is used for dyeing protein and fat, the concentration of the protein dye is 5-10 times of that of the fat, the dye is washed for 1-2 times by water after dyeing, and the light-proof treatment is needed in the processes of dye preparation, dye storage, dyeing and washing.

In one embodiment of the present invention, the observation is subjected to a component staining treatment according to the desired purpose of observation, followed by observation using a fluorescence microscope or a laser confocal microscope (CLSM).

A third object of the invention is the use of the method according to the invention in the food field.

[ advantageous effects ]

(1) The invention expands a new approach for grain research, and establishes an exploration method for the internal component structure and distribution of various mature hard grains from the viewpoint of dynamic monitoring. The invention can be used for comparing the change of the cooking process among different varieties of grains by monitoring and sampling each temperature section of the grains in the cooking process, explaining the difference of the cooking taste quality among the varieties from a microscopic angle and providing a theoretical basis for the formation of the grain quality. According to the invention, multiple exploration is carried out on the surface microstructure of the internal components of the grains, the component distribution and other multiple visual angles, and the visual sense is enhanced from plane to three-dimensional.

(2) The invention improves the traditional slicing method, improves the traditional stationary liquid, and improves the softening effect by mixing the stationary liquid; the dehydration time is shortened, and the conditions that the grains are excessively hard and fragile during slicing are prevented; the low-temperature paraffin is used for soaking wax, so that the influence of overhigh temperature on the internal structure of the sample is prevented; different staining solutions and slicing effects are adopted according to different requirements, and the discrimination between components is improved. The method has strong operability, is simple and convenient, is economical and applicable, can solve the problem that the grain slices are fragile in the later development stage, and provides new ideas and guidance for the grain research in China.

(3) According to the invention, paraffin with softer texture is used as an embedding agent, and particularly, two kinds of paraffin with different melting points are selected as wax dipping and embedding according to requirements, so that the influence of overhigh temperature on the structure of grain kernels is prevented; and the fixing liquid is improved, and the toughness of the mature grains can be improved to a certain extent by adopting the fixing liquid compounded with glutaraldehyde and paraformaldehyde.

Drawings

Fig. 1 is an observation of starch and protein in raw polished rice of south japonica 9108 and south japonica 46, wherein a: nanjing 9108; b: and 4. Nanjing 46.

FIG. 2 shows the microstructure of starch and protein in Nanjing 9108 rice during cooking; wherein A is raw rice; b, soaking for 30 min; c is cooking to 50 ℃; d is cooking to 70 ℃; e is cooking to 90 ℃; f is cooking to 100 ℃; g is cooked rice.

FIG. 3 shows the microstructure of starch and protein in Nanjing 46 rice during cooking; wherein A is raw rice; b, soaking for 30 min; c is cooking to 50 ℃; d is cooking to 70 ℃; e is cooking to 90 ℃; f is cooking to 100 ℃; g is cooked rice.

FIG. 4 is a cross-section of a scanning electron microscope of example 1 taken at a natural cut-off; wherein A is a cross-sectional view of Nanjing 9108 with magnification of 40 times; b is an internal tissue structure observed under the high power of Nanjing 9108.

FIG. 5 is a cross-sectional view taken through a scanning electron microscope of comparative example 1, in which freeze-drying was carried out without quenching; wherein A is the cross section form of freeze-dried Nanjing 9108; b is starch form of Nanjing 9108 amplified by 400 times.

FIG. 6 is a sectional view of a rice slice of Nanjing 9108 in example 2; wherein A is slices of Nanjing 9108 cooked to 50 deg.C; b is sliced from Nanjing 9108 by steaming to 70 deg.C.

Fig. 7 is CLSM graph of starch and protein distribution of polished rice of nanjing 9108 and nanjing 46, wherein a: nanjing 9108; b: and 4. Nanjing 46.

FIG. 8 is CLSM graph of starch and protein in Nanjing 9108 and Nanjing 46 rice at different temperatures, wherein A: nanjing 9108; b: and 4. Nanjing 46.

FIG. 9 is a section prepared in comparative example 2; wherein A is slices of Nanjing 9108 cooked to 50 deg.C; b is sliced from Nanjing 46 by steaming to 50 deg.C.

FIG. 10 is a slice prepared in comparative example 3, wherein A is a slice prepared by steaming Nanjing 9108 to 70 deg.C; b is CLSM picture of Nanjing 9108 cooked to 70 deg.C.

FIG. 11 is a section prepared in comparative example 4.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto. The solutions in the examples do not show that the solvents are deionized water.

Example 1

A method for observing the microstructure change of cross section in the grain cooking process comprises sampling, quenching, naturally cutting off, and observing;

the method specifically comprises the following steps:

(1) pretreatment: selecting rice (Nanjing 9108, Nanjing 46) with complete and plump grain shape, placing into a sealed bag, and storing at 4 deg.C for a long time;

(2) boiling and sampling: washing rice for 3 times, soaking at 25 deg.C for 35min, steaming for 35min, and stewing for 10 min; meanwhile, a thermocouple is inserted into the center area of the grain which starts to be cooked after soaking is finished, the temperature shown by a display screen is observed, and a rice sample is taken out at intervals of 10 ℃;

(3) quenching: immediately putting the rice in liquid nitrogen for quenching for 3min after taking out the rice in each temperature section, and drying the rice to be less than 5%;

(4) naturally cutting off: clamping the quenched seeds by two tweezers, breaking off the seeds at the middle part to obtain the seed sections, and adhering the sections on the conductive adhesive;

(5) and (4) gold plating observation: and (3) carrying out gold spraying treatment on the cross section of the grain by using an ion sputtering instrument, and observing the component structure and distribution of the grain at different cooking temperatures in a scanning electron microscope.

FIG. 1 is the observation of amyloplast and proteosome inside the polished rice of Nanjing 9108 and Nanjing 46, and can be seen from FIG. 1: starch is distributed in a large area of the rice seeds, and the starch is formed by gathering a plurality of polygonal starch particles to form compound starch, so that the whole structure is compact. The protein is distributed in the junction gap of partial starch bodies and is in a spherical shape with different sizes. Under the same magnification, the Nanjing 9108 and the Nanjing 46 have denser protein bodies with high protein content, and the Nanjing 46 has fewer protein bodies, but samples with different protein contents show the same distribution rule.

Fig. 2 and 3 show the microstructure changes of the amyloplasts and proteosome in the south japonica 9108 and the south japonica 46 rice in the cooking process, and can be seen from fig. 2 and 3: the rice is subjected to the whole process of cracking, structural disintegration, water absorption expansion, gelatinization and complete gelatinization after undergoing a complete starch body structure in the cooking process, and the change rule of protein is from distribution among starch bodies to gelatinization to form a net structure, but the rice still has a round grain shape. Different samples show different rapid and slow processes, and the gelatinization process of the Nanjing 9108 with slightly high protein content is slower than that of the Nanjing 46 with low protein content. After the Nanjing 9108 sample is soaked for 30min, amyloid is disintegrated, and proteosome existing in gaps of the amyloid is exposed. At 50 ℃, single-grain starch disintegrated from the composite starch can be obviously observed in the Nanjing 9108 and the Nanjing 46, while the Nanjing 46 with less protein content gradually absorbs water and the edge becomes round; at 70 ℃, the starch body of the Nanjing 46 is obviously pasted and mutually extruded, the periphery of the starch body has obvious expansion segmentation lines, and the Nanjing 9108 shows the tendency of further expansion. Finally, it is clearly visible that the protein bodies are squeezed at the edges, forming a "network-like" structure.

FIG. 4 is a cross-section of a naturally truncated SEM image of a quenched sample, as can be seen in FIG. 4: under the condition of amplifying by 40 times, the cross section state is natural, the tissue arrangement is compact, the structure of the internal components of the Nanjing 9108 can be clearly reflected, and the continuous arrangement of substances such as starch and the like is observed; further improving the magnification, and under the magnification of 50k, the distribution state of starch and protein in the Nanjing 9108 can be observed, the structure is complete, and the component differentiation is clear.

Comparative example 1

The liquid nitrogen drying process in the step (3) in the embodiment 1 is adjusted to be freeze drying, the liquid nitrogen drying process is carried out at the temperature of minus 60 ℃, the liquid nitrogen drying process is placed in a closed container with high vacuum of 30Pa, the liquid nitrogen drying process is carried out for 90min, the rice is selected from Nanjing 9108, and the rest is the same as the embodiment 1.

FIG. 5 is a cross-sectional view of a scanning electron microscope which is observed after direct freeze-drying without quenching with liquid nitrogen. As can be seen from fig. 5: because under the environment of high vacuum, the moisture of the frozen food material is directly sublimated into steam from ice solid without melting ice, the obtained Nanjing 9108 sample is hollow, the internal structure is not compact, the number of holes is large, and the self structure of the starch is damaged to a certain extent. Further improving the magnification, observing that the gelatinized starch connected into slices is cut off to form a plurality of connecting wires caused by sudden cutting, and being incapable of reflecting the real influence of the cooking process on the change of the internal components of the Nanjing 9108.

Example 2

A method for dynamically monitoring the internal component structure and distribution change of grains in a cooking process comprises the steps of cooking sampling, fixing, washing, dehydrating, transparentizing, waxing, embedding, slicing, spreading, dyeing and observing;

the method specifically comprises the following steps:

(1) boiling and sampling:

washing rice (Nanjing 9108, Nanjing 46) for 3 times, soaking at 25 deg.C for 35min, steaming for 35min, and stewing for 10 min; meanwhile, a thermocouple is inserted into the center area of the grain which starts to be cooked after soaking is finished, the temperature shown by a display screen is observed, and a rice sample is taken out at intervals of 10 ℃;

(2) fixing

Selecting grain seeds at various temperatures, after water is balanced, soaking the grains in a fixing solution obtained by compounding a paraformaldehyde solution with the volume concentration of 4% and a glutaraldehyde solution with the volume concentration of 2.5% according to the volume ratio of 1:1 for 24 hours, and adding and updating the fixing solution appropriately according to the conditions of the grains every 2 hours;

(3) rinsing

Placing the fixed sample into an embedding box, washing the embedding box for 3min by using water, and draining the surface water after washing away the redundant fixing liquid;

(4) dewatering

Immersing the washed sample into ethanol solution with gradient concentration of 30%, 50%, 70%, 90% and 100% in volume for 5min for dehydration, so that the water content is reduced to 5%;

(5) is transparent

Soaking dehydrated rice in mixed solution of 50% ethanol and 50% xylene for 20min, and transferring to 100% xylene for 20 min; wherein% is volume percent;

(6) wax dipping

Soaking the rice after transparent treatment in 55 deg.C low temperature paraffin for 30 min;

(7) embedding

Putting the rice after being waxed into a high-temperature groove of an embedding instrument, and pouring liquid paraffin into the high-temperature groove at the temperature of 58 ℃ for embedding;

(8) slicing

Placing the embedded rice on a freezing table for 30min for demolding, and placing in a refrigerator at 4 ℃ for 12h after demolding; then, a paraffin slicer is used for paraffin slicing, the thickness of 15 mu m is selected, the paraffin slicing is firstly carried out, and the paraffin slicing is selected when complete sections continuously appear;

(9) exhibition piece

Taking a sliced surface by using a pair of tweezers, spreading the sliced surface in an ethanol solution with the volume fraction of 8%, spreading the sliced surface in water at 42 ℃ after the sliced surface is completely spread, fishing out the slices, and selecting a flat grain section sample to be adhered to a glass slide with high adhesiveness; then placing the glass slide in an oven at 37 ℃ to bake for 12h until the glass slide is completely dried;

(10) dyeing:

observing the distribution of starch and protein components in the cooking process by adopting a counterstaining method, preparing Fluorescein Isothiocyanate (FITC) with the mass concentration of 0.4% and rhodamine B (RhB) with the mass concentration of 0.04% by using acetone in a dark environment, and storing in the dark after preparation; mixing FITC solution and RhB solution according to the volume ratio of 1:1 under the conditions of keeping out of the sun and 25 ℃ to obtain mixed dye solution; then adding 50 mu L of mixed dye solution on the slice, dyeing for 5min, washing the dye on the slice with deionized water, and inverting the slice on a laser confocal microscope for observation after the surface moisture is dried.

FIG. 6 is a cross-sectional view of a rice slice prepared by steaming Nanjing 9108 to different temperature stages, as can be seen from FIG. 6: the complete rice grain structure of different cooking temperature sections can be obtained, the texture is compact, no fragmentation or fine seam exists, and the shape keeping state is good.

FIG. 7 is CLSM graph of starch and protein distribution of polished rice of Nanjing 9108 and Nanjing 46. As can be seen from fig. 7: the starch is extruded into a block in the form of a polygonal complex to form starch bodies, and the starch bodies are connected compactly and connected into slices. The protein is distributed around the starch body in a dotted manner, which shows that the circular structure of the protein and the distribution of the protein body is not uniform inside the grains. The proteosome presents the same distribution rule among different samples.

FIG. 8 is CLSM graph of starch and protein in Nanjing 9108 and Nanjing 46 rice cooked to different temperatures. As can be seen from fig. 8: the starch in the soaked rice grains is looser than that of the raw and refined rice, which indicates the immersion of water. At 50 deg.C, the starch disintegrates to form gaps in the cut surface, and the protein is distributed in the gaps of the starch. At 90 ℃, the starch is no longer the structure of the starch body due to further gelatinization, and from the observation of slicing, the starch is nearly completely gelatinized and takes on a pasty structure, while the protein takes on a 'network-like' structure and exists around the gelatinized starch.

Comparative example 2

The dehydration time in step (4) of example 2 was adjusted to 20min, the temperature of the wax impregnation in step (6) was adjusted to 70 ℃, and the rest was kept the same as example 2.

The resulting slice is shown in fig. 9, from which fig. 9 it can be seen that: the Nanjing 9108 and the Nanjing 46 show that the slices prepared by the method are hard and fragile, the obtained slices are easy to hollow, and the complete seed structure cannot be obtained.

Comparative example 3

The 8% ethanol solution in the section (9) of example 2 was adjusted to water (the amount of ethanol added was 0), and the rest was the same as in example 2.

The resulting slice is shown in fig. 10, from which fig. 10 it can be seen that: the southern japonica 9108 slices cooked to 70 ℃ have uneven sections and folds, and further laser confocal microscope observation shows that uneven dyeing is caused by uneven sections, and the components in the southern japonica 9108 cannot be displayed on a plane, so that the observation effect is poor.

Comparative example 4

The slice in step (8) of example 2 was adjusted to an unmodified slice, specifically: after embedding, paraffin sections were immediately performed without storage for 12h at 4 ℃ and the thickness was selected to be 25 μm, which otherwise remained the same as in example 2.

The resulting slice is shown in fig. 11, and it can be seen from fig. 11 that: because the solid state is not carried out, the shape of the tissue just embedded in the paraffin section is easy to be changed by external force to cause the section shape to be damaged, and the section is uneven due to the over-thick thickness.

Comparative example 5

The procedure of example 2 was followed except that the fixative in step (2) was replaced with fixative 1 (glutaraldehyde), fixative 2 (paraformaldehyde), fixative 3 (glutaraldehyde/paraformaldehyde volume ratio 1:1 complex), fixative 4(FAA fixative (5 ml formalin; 5ml glacial acetic acid; 90ml 70% alcohol)), and fixative 5 (formalin, paraformaldehyde, glutaraldehyde, glacial acetic acid fixative in volume ratio 1:1: 1), respectively.

The comparison of the results of 5 fixative solutions can find that: fixing with fixing solution 4(FAA fixing solution) and fixing solution 5 (formalin, paraformaldehyde, glutaraldehyde and glacial acetic acid fixing solution at a volume ratio of 1:1:1: 1) to obtain hard and fragile tissue blocks, wherein the tissues are loose and easily broken during slicing, and complete slices cannot be obtained; and the fixing liquid 1 (glutaraldehyde) and the fixing liquid 2 (paraformaldehyde) can improve the hardness of the grain to a certain extent, and the ratio of 1:1 after compounding, the effect is better, and the grains samples at different temperature sections in the cooking process can be sliced with good effect.

Comparative example 6

The wax impregnation temperature in step (6) of example 2 was adjusted to 40 ℃, and the rest was kept the same as example 2.

The melting point of the paraffin is 45-65 ℃, when the wax immersion temperature is 40 ℃, the paraffin cannot completely permeate into grain seed blocks, and after the paraffin is embedded and cooled, the incompletely permeated paraffin cannot play a supporting role, so that the grain seeds deform and collapse, complete slices cannot be cut off, and the observation effect is poor.

Comparative example 7

Replacing the slide in step (9) of example 2 with a slide with a polymeric adhesive coated on the surface to attach the sample flat to the slide; the rest of the process was identical to that of example 2.

After slicing, the slices are firstly placed into an aqueous solution containing 5% ethanol and then transferred into warm water at 42 ℃, so that the slices are flat, wrinkles and bubbles are few, the sliced slices are directly adhered to the glass slide flatly by the glass slide coated with the polymerization viscose on the surface, the slices cannot be completely unfolded, wrinkles are easily caused, and the follow-up observation is not favorable.