Methods for observing food

Abstract

To provide an improved food observation method.SOLUTION: A method for observing food includes the steps of: preparing a food sample by collecting a part of the food; electronically staining the food sample; discharging an unobservable component from the electronically stained food sample; and observing the food sample from which the unobservable component has been discharged with an electron microscope.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for observing food using an electron microscope.

Background Art

[0002] Conventionally, food has been observed using an electron microscope. Since an electron microscope can observe the fine structure of food tissue, it is used as one of the methods for objectively evaluating the properties and quality of food by associating the microscope image with differences in raw materials, preparation methods, texture, and appearance of the obtained food.

[0003] Regarding the method for observing food using an electron microscope, for example, Patent Document 1 discloses an invention of a method for preparing a sample for a transmission electron microscope of the food, including rapid freezing and freeze substitution of a liquid or gel-like food. According to this method, it is said that the original tissue structure of yogurt or milk can be observed using a transmission electron microscope.

[0004] Also, for example, Non-Patent Document 1 describes observing the tissue of baked bread using an electron microscope. That is, using a scanning electron microscope (SEM) (acceleration voltage 10 KV), after dehydration, the sample subjected to critical point drying was cut and attached to the sample stage, and observed after ion coating with gold. Also, using a transmission electron microscope (TEM) (acceleration voltage 75 KV), after dehydration treatment, it was embedded in a low-viscosity epoxy resin, made into a 60-nm ultra-thin section, and observed after double staining with uranyl acetate and lead.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] According to the study by the present inventors, in the observation of foods such as bakery products under an electron microscope, there was a problem that the observation of the skeletal structure of the food tissue was hindered by starch grains and the like contained in the starchy raw materials. Therefore, pretreatment of washing the sample to be observed under an electron microscope with water made it possible to conduct a certain degree of observation. However, there was an aspect that due to the influence of such pretreatment of washing the sample with water, the skeletal structure of the food tissue itself was damaged, making it difficult to observe the original structure.

[0008] An object of the present invention is to provide an improved method for observing foods in which such problems are solved.

Means for Solving the Problems

[0009] The present inventors have conducted intensive studies to achieve the above object and have completed the present invention.

[0010] That is, the present invention provides a step of preparing a food sample by collecting a part from a food, a step of electron-staining the food sample, a step of causing components other than the observation target to flow out from the electron-stained food sample, a step of observing the food sample after causing the components other than the observation target to flow out with an electron microscope, and provides a method for observing foods, characterized by including the above steps.

[0011] According to the food observation method of the present invention, since observation is performed using an electron microscope, the fine structure of the food tissue can be observed. Furthermore, by taking a step to remove non-observable components that interfere with observation after electron staining the sample to be observed, the original structure of the food tissue can be effectively observed.

[0012] In the food observation method according to the present invention, the food may contain starchy raw materials, and the components not to be observed may also contain the starchy raw materials. This makes it possible to prevent the observation of the skeletal structure of the food tissue from being obstructed by starch granules, etc., contained in the starchy raw materials when observing food such as bakery products that contain starchy raw materials.

[0013] In the food observation method according to the present invention, the electron staining may be platinum blue staining. With this method, since platinum blue staining binds to the nitrogen atom portion of the protein, the staining protects the skeletal structure such as gluten, and also makes it easier for components not being observed to detach from the skeletal structure.

[0014] In the method for observing food according to the present invention, the observation using an electron microscope may be performed using a scanning electron microscope. This allows for more effective observation of the skeletal structure of the food tissue.

[0015] In the food observation method according to the present invention, the food sample may be prepared by taking a portion of the food, freezing it, and then cutting it. This allows for more effective observation of the skeletal structure of the tissue not only on the surface but also inside the food.

[0016] In the food observation method according to the present invention, the step of flushing out components not to be observed may be performed by immersing the electron-stained food sample in an aqueous solution. In this case, ultrasonic treatment may be performed while immersed. This allows for more efficient flushing out of components not to be observed.

[0017] In the method for observing a food according to the present invention, the food may be a bakery product containing a starchy raw material or its dough. According to this, the skeletal structure of the tissue of the bakery product containing the starchy raw material or its dough can be effectively observed.

Advantages of the Invention

[0018] According to the method for observing a food according to the present invention, since it is observed by an electron microscope, the fine structure of the food tissue can be observed. And, after electron-staining the sample to be observed, components other than the observation target that interfere with the observation are allowed to flow out, so that the original structure of the food tissue can be effectively observed.

Brief Description of the Drawings

[0019] [Figure 1] In Test Example 1, it is an example of a microscopic image showing the result of observing baked bread with a scanning electron microscope. In Fig. 1(A), an example of the microscopic image of a sample without performing water washing is shown. In Fig. 1(B), an example of the microscopic image of a sample prepared by staining the sample with a platinum blue staining solution, immersing it in water, and subjecting it to ultrasonic treatment is shown. In Fig. 1(C), an example of the microscopic image of a sample prepared by washing with water is shown. [Figure 2] In Test Example 2, it is an example of a microscopic image showing the result of observing bread dough with a scanning electron microscope. In Fig. 2(A), an example of the microscopic image of a sample without performing water washing is shown. In Fig. 2(B), an example of the microscopic image of a sample prepared by staining the sample with a platinum blue staining solution, immersing it in water, and subjecting it to ultrasonic treatment is shown. [Figure 3] In Test Example 3, it is an example of a microscopic image showing the result of observing crackers with a scanning electron microscope. In Fig. 3(A), an example of the microscopic image of a sample without performing water washing is shown. In Fig. 3(B), an example of the microscopic image of a sample prepared by staining the sample with a platinum blue staining solution, immersing it in water, and subjecting it to ultrasonic treatment is shown. [Figure 4]In Test Example 4, this is an example of a microscopic image showing the result of observing a roll cake with a scanning electron microscope. In FIG. 4(A), an example of a microscopic image of a sample without performing water washing is shown. In FIG. 4(B), an example of a microscopic image of a sample prepared by staining the sample with a platinum blue staining solution, immersing it in water, and subjecting it to ultrasonic treatment is shown. [Figure 5] In Test Example 5, this is an example of a microscopic image showing the result of observing Danish pastry with a scanning electron microscope. In FIG. 5(A), an example of a microscopic image of a sample without performing water washing is shown. In FIG. 5(B), an example of a microscopic image of a sample prepared by staining the sample with a platinum blue staining solution, immersing it in water, and subjecting it to ultrasonic treatment is shown.

Embodiments for Carrying Out the Invention

[0020] The present invention provides a method for observing food with an electron microscope. Here, the "food" can be any food such as processed food (food composition composed of arbitrary food raw materials) that an evaluator wants to evaluate. Further, "observing" is synonymous with the meaning of electron microscope observation generally recognized by those skilled in the art, and can be an evaluation from any viewpoint that can be selected and set by an evaluator.

[0021] In the present invention, it includes a step of collecting a part from the food to be observed to prepare a food sample. In this step, the food is prepared into a size that is easy to handle for electron staining and subsequent observation with an electron microscope. The shape of the food sample may be any shape such as a slice shape or a cube shape, but preferably, when placed on a sample stage for observation with an electron microscope, it has a certain area as an observation surface, typically for example 1 to 40000 mm 2 , preferably 5 to 150 mm 2 and is preferably prepared to have an area.

[0022] The present invention includes a step of electron staining the food sample prepared as described above. In this step, care is taken to prevent the components to be observed from being washed away in a subsequent step (a step to remove components not to be observed). Here, "electron staining" refers to a treatment to enhance contrast by adding heavy elements in the preparation of a sample for electron microscopy observation. That is, in a sample mainly composed of light elements such as H, C, and O, the scattering energy in response to incident electrons is weak, but by adding heavy elements to the sample, the scattering energy in response to incident electrons increases, thereby obtaining high contrast. Specific examples of electron staining materials used for electron staining include platinum blue, methenamine silver periodate, osmium tetroxide, ruthenium tetroxide, phosphotungstic acid, uranium acetate, and lead citrate. Platinum blue is particularly preferred. Among electron staining materials, platinum blue has relatively high safety in handling and has the advantage of easily discerning the skeletal structure because it binds to the skeletal structure of food tissues containing proteins and reflects electrons during electron microscopy observation.

[0023] Platinum blue staining is achieved by first synthesizing a deep blue, liquid complex called Platinum Blue [Pt4(NH3)8(C6H] from cisplatin and thymidine. 13 O5)4] +5 This method involves immersing the sample in a solution to stain it. Traditionally, users had to prepare platinum blue themselves, but in recent years, synthetic and stabilized platinum blue staining solutions have become commercially available, and these can also be used. One example of a commercially available platinum blue staining solution is "TI Blue" (Nisshin EM Co., Ltd.).

[0024] The present invention includes a step of flushing out components not to be observed from a food sample that has been electron-stained as described above. In this step, the skeletal structure of the food tissue to be observed is separated from other components to make it easier to observe. For example, the food sample can be electron-stained by immersing it in an aqueous liquid containing an electron-staining agent and allowing a certain period of time to pass, and then the components not to be observed can be flushed out by immersing the stained food sample in another aqueous solution. In this case, physical operations such as shaking the food sample in the aqueous solution may be performed for the purpose of flushing out the components not to be observed. In the present invention, the electron-staining step and the step of flushing out components not to be observed from the electron-stained food sample may be the same or an inseparable series of operations. For example, the food sample can be immersed in an aqueous liquid containing an electron-staining agent, and after a certain period of time, the components not to be observed may be flushed out by shaking or other operations in the same aqueous liquid. Alternatively, the food sample can be immersed in an aqueous liquid containing an electron-staining agent and immediately after, shaking or other operations in the same aqueous liquid may be performed to flush out the components not to be observed. Through this procedure, electron staining and the leaching of non-target components can be performed as a single, inseparable operation.

[0025] In either case, it is preferable that the process includes immersing the sample in water to rinse off the staining solution and removing some of the remaining moisture before the next electron microscope observation step. Examples of methods for removing moisture include placing the sample on paper and allowing the paper to absorb some of the water, or vacuum drying the sample to remove moisture. However, if the chamber in which the sample stage is placed is depressurized during electron microscope observation, the above moisture removal step does not need to be performed.

[0026] The present invention includes a step of observing a food sample, which has been subjected to the above-described procedure for draining components not to be observed, using an electron microscope. In this step, observation with an electron microscope is performed according to a conventional method. Although not limited to this, observation is preferably performed using a scanning electron microscope. Furthermore, observation is preferably performed under low vacuum conditions, where the vacuum level in the chamber where the sample stage is installed is approximately 10 Pa to 500 Pa. This prevents excessive evaporation of moisture and oil from the food sample, thus preventing the breakdown of the original structure of the food tissue.

[0027] In any non-limiting embodiment of the present invention, it is preferable to prepare a food sample by taking a portion from the food, freezing it, and then cutting it. This embodiment allows for more effective observation of the skeletal structure of the tissue not only on the surface but also inside the food. In this embodiment, cutting can be performed by thoroughly freezing the sample, including the inside, and then applying an impact to the frozen sample. For example, the sample may be cut by touching the sample, which is immersed in liquid nitrogen, with the tip of a knife and then tapping the handle of the knife with pliers to transmit vibrations.

[0028] In other, less-than-limited embodiments of the present invention, the step of flushing out components not to be observed is preferably performed by ultrasonic treatment while the electron-stained food sample is immersed in an aqueous solution. This embodiment allows the objective of the step to be achieved in a shorter time compared to cases where ultrasonic treatment is not performed. Furthermore, compared to strong physical treatments such as shaking the solution in which the sample is immersed or rubbing the sample in water, the degree of damage to the skeletal structure of the food tissue being observed is less.

[0029] In this embodiment, sonication can be performed at room temperature, but is not limited to this method. It can also be performed in an aqueous liquid that allows components not under observation to be leached out, and such aqueous liquid may be, for example, distilled water or a buffer solution.

[0030] While there are no particular limitations on the foods to which the present invention can be applied, it is preferable to apply it to foods that are not fluid at room temperature and maintain a certain shape. By applying it to such foods, the skeletal structure of the tissue that exhibits shape retention can be observed without being washed away by the washing process described above. Furthermore, as shown in the examples described later, it is preferable to apply it to foods that contain starchy raw materials, as this allows for the effective removal of starch particles and the like introduced from the starchy raw materials as components not to be observed, and the original structure of the food tissue can be observed. Examples of foods containing starchy raw materials include bakery products. Examples of bakery products include bread (white bread, sweet bread, variety bread, French bread, brioche, Danish pastry, yeast donuts, muffins, pizza, scones, steamed bread, waffles, English muffins, buns, etc.) and baked goods (biscuits, crackers, hardtack, pretzels, wafers, pies, puffed cream puffs, butter cakes, sponge cakes, castella, cake donuts, pancakes, dorayaki, etc.). Also included are bakery doughs before baking. Examples of bakery doughs include bread doughs (white bread dough, sweet bread dough, variety bread dough, French bread dough, brioche dough, Danish pastry dough, yeast donut dough, pizza dough, scone dough, waffle dough, English muffin dough, bun dough, etc.) and baked goods doughs (biscuit dough, cracker dough, hardtack dough, pretzel dough, pie dough, etc.). Other examples include snack foods (potato snacks, corn puff snacks, etc.), cooked foods containing starchy ingredients (crepes, oyaki, okonomiyaki, takoyaki, chijimi, etc.), and noodles.

[0031] The present invention is not limited to the embodiments described above, and various combinations and modifications are possible within the scope of disclosure herein, and such embodiments are also included in the technical scope of the present invention. [Examples]

[0032] The present invention will be described in more detail below with reference to examples. However, these examples are not intended to limit the scope of the present invention.

[0033] <Test Example 1> A small piece measuring 10mm x 10mm x 10mm was cut from the center of a loaf of bread ("Chojuku" brand, Shikishima Baking Co., Ltd.), frozen by immersing it in liquid nitrogen, and while the sample was still immersed in the liquid nitrogen, the tip of a knife was touched to the sample, and the upper part of the knife handle was struck to transmit vibration, thereby cutting the sample.

[0034] Each test group was prepared by removing the sample from liquid nitrogen, allowing it to return to room temperature, and then subjecting it to either treatment A) or B) below. (Sample processing conditions) A) No processing B) Immerse the sample in platinum blue staining solution ("TI Blue," Nissin EM Co., Ltd.) for 12 hours, remove the sample from the staining solution, immerse it in 10 ml of water, and place the container in the water bath of an ultrasonic cleaner for 10 seconds of ultrasonic treatment.

[0035] In addition, a test plot was established in which the following treatment C) was applied. (Sample processing conditions) C) A small piece measuring 10mm x 10mm x 10mm was cut from the center of a slice of bread ("Chojuku" brand, Shikishima Baking Co., Ltd.), placed in a container of water, and shaken vigorously for 10 seconds to rinse with water. The sample was removed from the water and frozen by immersing it in liquid nitrogen. While the sample was still immersed in the liquid nitrogen, the tip of a knife was touched to the sample, and the upper part of the knife handle was struck to transmit vibrations, causing the sample to break.

[0036] The sample was placed on the apparatus's sample stage so that the above-mentioned cross-section would serve as the observation surface, and observed using a scanning electron microscope ("TM4000Plus," Hitachi High-Technologies Corporation). The observation temperature was -20 to -30°C, the vacuum mode was "charge reduction mode," and other measurement conditions are shown in the figure. Note that "Mix" indicates a composite image of backscattered electron images and secondary electron images, and "BSE" indicates a backscattered electron image.

[0037] As a result, as shown in Figure 1(A), if the sample is not washed with water, a smooth surface is formed due to components such as starch granules contained in the baked bread.

[0038] In contrast, as shown in Figure 1(B), when the sample was immersed in platinum blue staining solution and subjected to sonication, a film-like structure was revealed. Since the components containing nitrogen atoms reflect electrons and appear white due to platinum blue staining, it was presumed that the film-like structure was a gluten membrane.

[0039] On the other hand, as shown in Figure 1(C), when the sample was simply washed with water, the components that make up the structure of the baked bread were washed away, and the skeletal structure appeared to have collapsed.

[0040] <Test Example 2> The bread dough was prepared as follows using the proportions shown in Table 1.

[0041] [Table 1]

[0042] Specifically, the starter ingredients were placed in a mixer bowl and mixed using a hook in a vertical mixer to prepare the starter. After fermenting the starter at 27°C for 4 hours, it was placed in the mixer bowl and mixed with the remaining ingredients using a hook.

[0043] After allowing a floor time of 30 minutes for the obtained dough, small pieces measuring 5 mm x 5 mm x 2 mm were cut out and each test group was subjected to either treatment A) or B) below. (Sample processing conditions) A) No processing B) Immerse the sample in platinum blue staining solution ("TI Blue," Nissin EM Co., Ltd.) for 12 hours, remove the sample from the staining solution, immerse it in 10 ml of water, and place the container in the water bath of an ultrasonic cleaner for 10 seconds of ultrasonic treatment.

[0044] Each sample was placed on the sample stage of the apparatus, and the sample surface was observed using a scanning electron microscope, as in Test Example 1. As a result, as shown in Figure 2(A), it appeared that if the sample was not washed with water, a rough surface was formed due to components such as starch granules contained in the bread dough.

[0045] In contrast, as shown in Figure 2(B), when the sample was immersed in platinum blue staining solution and subjected to sonication, the skeletal structure of the bread dough tissue became apparent.

[0046] <Test Example 3> A small piece measuring 10mm x 10mm x 5mm was cut from the center of a cracker ("Ritz," Mondelez Japan Co., Ltd.), frozen by immersing it in liquid nitrogen, and while the sample was still immersed in liquid nitrogen, the tip of a knife was touched to the sample, and the upper part of the knife handle was struck to transmit vibration, thereby cleaving the sample.

[0047] Each test group was prepared by removing the sample from liquid nitrogen, allowing it to return to room temperature, and then subjecting it to either treatment A) or B) below. The treated samples were then observed using a scanning electron microscope. (Sample processing conditions) A) No processing B) Immerse the sample in platinum blue staining solution ("TI Blue," Nissin EM Co., Ltd.) for 12 hours, remove the sample from the staining solution, immerse it in 10 ml of water, and place the container in the water bath of an ultrasonic cleaner for 10 seconds of ultrasonic treatment.

[0048] As a result, as shown in Figure 3(A), if the sample is not washed with water, a smooth surface is formed due to components such as starch granules contained in the cracker.

[0049] In contrast, as shown in Figure 3(B), when the sample was immersed in platinum blue stain and subjected to sonication, the skeletal structure of the cracker tissue became apparent.

[0050] <Test Example 4> Small pieces measuring 10mm x 10mm x 5mm were cut from the sponge of a "roll cake cut into five pieces" (Yamazaki Baking Co., Ltd.), frozen by immersing in liquid nitrogen, and while the sample was still immersed in liquid nitrogen, the tip of a knife was touched to the sample, and vibrations were transmitted by tapping the upper part of the knife handle with pliers, causing the sample to break.

[0051] Each test group was prepared by removing the sample from liquid nitrogen, allowing it to return to room temperature, and then subjecting it to either treatment A) or B) below. The treated samples were then observed using a scanning electron microscope. (Sample processing conditions) A) No processing B) Immerse the sample in platinum blue staining solution ("TI Blue," Nissin EM Co., Ltd.) for 12 hours, remove the sample from the staining solution, immerse it in 10 ml of water, and place the container in the water bath of an ultrasonic cleaner for 10 seconds of ultrasonic treatment.

[0052] As a result, as shown in Figure 4(A), if the sample is not washed with water, a smooth surface is formed due to components such as starch granules contained in the sponge.

[0053] In contrast, as shown in Figure 4(B), when the sample was immersed in platinum blue staining solution and subjected to sonication, the skeletal structure of the sponge tissue became apparent.

[0054] <Test Example 5> The Danish pastry dough was prepared as follows using the proportions shown in Table 2.

[0055] [Table 2]

[0056] Specifically, the ingredients other than the margarine for the roll-in dough were placed in a mixer bowl and mixed using a hook in a vertical mixer to prepare the dough. After a 30-minute floor time, the dough was retarded at 1°C for 20 hours. The margarine for the roll-in dough, which had been warmed to 15°C, was placed on top of the dough and rolled in using the conventional method (folded in thirds twice and in quarters once) to a thickness of 5 mm.

[0057] Small pieces measuring 10 mm x 10 mm were cut from the obtained Danish pastry dough, and each test group was subjected to either treatment A) or B) below. (Sample processing conditions) A) No processing B) Immerse the sample in platinum blue staining solution ("TI Blue," Nissin EM Co., Ltd.) for 12 hours, remove the sample from the staining solution, immerse it in 10 ml of water, and place the container in the water bath of an ultrasonic cleaner for 10 seconds of ultrasonic treatment.

[0058] Each sample was cut from the dough, and its cross-section was used as the observation surface. It was then placed on the sample stand of the apparatus and observed using a scanning electron microscope, similar to the method used in Test Example 1. As a result, as shown in Figure 5(A), without staining and washing the samples, components such as starch granules contained in the Danish pastry dough covered the cut surface, making it impossible to clearly observe the tissue structure.

[0059] In contrast, as shown in Figure 5(B), when the sample was immersed in platinum blue staining solution and subjected to ultrasonic treatment, the fat layer (dark colored layer) folded into the Danish pastry dough was distinguished from the other layers (light colored porous layer), revealing the overall structure of the tissue. At the same time, the skeletal structure, which appears to be gluten, in the parts other than the fat layer was also revealed.

[0060] Considering the results of the above test examples 1-5, it was concluded that platinum blue binds to the nitrogen atoms of proteins, and therefore, in food tissues, it binds to the skeletal structure containing proteins, particularly gluten and egg-derived proteins. Although the mechanism of action is not clear, it was thought that when platinum blue binds to these protein-containing skeletal structures, the core of the skeletal structure is protected, while other components attached to the surface of the skeletal structure, especially starch, are easily detached. As a result, it was thought that components other than those being observed can be released without strong physical treatment.

Claims

1. A step of preparing a food sample by taking a portion from a food containing starchy raw materials, The process of electronically staining the aforementioned food sample, A step of draining non-observable components, including the starchy raw material, from the electronically stained food sample, A step of observing the food sample with an electron microscope after the components not subject to observation have been released, Includes, The aforementioned electronic staining is performed using platinum blue staining; this is a method for observing food.

2. The method for observing food according to claim 1, wherein the observation using the electron microscope is performed using a scanning electron microscope.

3. The method for observing food according to claim 1 or 2, wherein the preparation of the food sample is carried out by taking a portion from the food, freezing it, and cutting it.

4. The method for observing food according to any one of claims 1 to 3, wherein the step of draining the components not to be observed is performed by immersing the electron-stained food sample in an aqueous liquid.

5. The method for observing food according to any one of claims 1 to 4, wherein the step of draining the components not to be observed is performed by ultrasonic treatment while immersing the electron-stained food sample in an aqueous liquid.

6. The method for observing a food according to any one of claims 1 to 5, wherein the food is a bakery product containing a starchy raw material or its dough.

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