JP2013088328A - Preparation method for transmission electric microscope sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preparing a transmission electric microscope sample without changing a tissue structure with respect to food and drink in a variety of shapes.SOLUTION: The method for preparing a transmission electric microscope sample of liquid and gelatinous food includes freeze-substitution of the food after quick-freezing. Also, the method is related to an evaluation method for tissue structure of liquid and gelatinous food which uses the transmission electric microscope to observe the sample prepared by the method.

Description

  The present invention relates to a method for preparing a sample for a transmission electron microscope.

  Several methods for observing the tissue structure of food using a transmission electron microscope have been proposed (Non-patent Document 1, Non-patent Document 2, Non-cited Document 3). A small amount of sample is prepared by cutting a certain ice cream with a knife or by attaching a thick semi-solid whipped cream to the tip of a triangular filter. It was not applicable to the preparation of a gel-like sample that easily collapsed. In addition, for liquid foods, for example, a method for observing casein micelles in milk using a transmission electron microscope has been proposed (Non-Patent Document 4). In this method, when preparing a milk sample, Because milk is diluted, the actual tissue structure of milk cannot be observed.

  A method for observing the tissue structure of gelled yogurt has also been proposed (Non-Patent Document 5). However, in the chemical fixation in this method, the tissue structure changes because a water-soluble substance flows out when the sample is fixed. It was difficult to accurately observe the tissue structure of the actual sample.

In addition, in a method for observing the intracellular structure of yeast using a transmission electron microscope, a sample preparation method using rapid freezing and freeze replacement has been proposed (Non-patent Documents 6 and 7). However, these methods are intended for observation of the individual intracellular structure of yeast, and are accompanied by centrifugation for the concentration of yeast solutions such as yeast cultures. When applying, the concentration of the liquid food and the tissue structure change, so it is difficult to accurately observe the actual tissue structure of the sample.
As described above, a sufficiently satisfactory means (sample preparation method) for accurately observing the structure of a liquid food or a gel food using a transmission electron microscope has not yet been developed.

International Dairy Journal, Vol. 9, P. 817-829 (1999) "A study of fat and air structures in ice cream", H.D.Goff, A.K.Smith Journal of Microscopy, Vol.213, Pt 1, P. 63-69 (January 2004) “Freeze-sucstitution and low-temperature embedding of dairy products for transmission electron microscopy”, A.K.Smith, H.D.Goff Food Research International, Vol. 33, P. 697-706 (2000) "Changes in protein and fat structure in whipped cream caused by heat treatment and addition of stabilizer to the cream", A.K.Smith, H.D.Goff Protein Nucleic Acid Enzyme 1989-09, 34, 11 Issue, P. 1359-1369 (1989) "Electron Microscopic Image of Casein", Toshiaki Kimura and Shinichi Taneya Journal of the Society of Dairy Technology, Vol 49, No 1, P. 1-10 (February 1996) `` The effect of starch based fat substitutes on the microstructure of set-style yogurt made from reconstituted skimmed milk powder '', AY Tamime, E Barrantes and AM Sword Microscope Vol.45, No.4, P. 212-217 (2010) “Structure analysis of yeast by freeze replacement method and serial section method”, Yamaguchi, Masanori Methods in Enzymology, Vol.451, P. 133-149 (2008) “Electron Microscopy In Yeast”, Mizue Baba

  Therefore, the objective of this invention is providing the preparation method of the sample for transmission electron microscopes which does not change the whole structure | tissue structure about various food-drinks including liquid food and gel-like food.

  In order to solve the above-mentioned problems, the present inventors have found that the quick freezing method and the freeze replacement method can be applied to foods and drinks of all shapes while repeating researches. It came to complete.

  That is, the present invention relates to the following method for preparing a sample for a transmission electron microscope, a sample observation method using a transmission electron microscope using the sample, and a sample evaluation method using observation using a transmission electron microscope.

[1] A method for preparing a sample for a transmission electron microscope of a food, comprising freezing and replacing a liquid or gel food after rapid freezing.
[2] The method according to [1], comprising placing a liquid or gel food on a sample support and rapidly freezing it.
[3] The method according to [2], comprising cutting a sample at a position of 5 to 10 μm from the sample support.
[4] The method according to any one of [1] to [3], comprising freeze-replacement using a fixative containing water.

[5] A method for evaluating the tissue structure of a liquid or gelled food, comprising observing the sample prepared by the method according to any one of [1] to [4] using a transmission electron microscope.
[6] The method according to [5], wherein the storage stability of the liquid or gel food is evaluated.
[7] A liquid or gel food evaluated as having high storage stability by the method according to [6].
[8] A high-concentration liquid food having a protein particle size of 500 nm or less and at least 1 kcal / ml.

  According to the present invention, preparation of a sample for a transmission electron microscope can be performed while maintaining the tissue structure of foods and drinks of various shapes, including liquid foods and gel foods, which have been difficult to prepare. It becomes possible. Furthermore, since the original structure of the food and drink can be observed, the storage stability of the food and drink and the texture and the like can be evaluated by understanding the fine structure of the food and drink.

It is the transmission electron micrograph of set type yogurt which prepared the sample by the chemical fixation method. It is a transmission electron micrograph of a set-type yogurt prepared by a rapid freezing / freezing replacement method. It is the transmission electron micrograph of milk which prepared the sample by the agar fixation and the chemical fixation method. It is the transmission electron micrograph of the milk which prepared the sample by the quick freezing and the freeze substitution method. It is the transmission electron micrograph of the liquid food (nutrient composition) which prepared the sample by the protein adsorption method. It is the transmission electron micrograph of the liquid food which prepared the sample by the quick freezing and freeze substitution method. It is the transmission electron micrograph of the liquid food which prepared the sample by the quick freezing and freeze substitution method. It is the transmission electron micrograph of the liquid food which prepared the sample by the quick freezing and freeze substitution method.

  In the present invention, “liquid food” means liquid beverages / food such as solutions, emulsions, colloids, etc., for example, fruit juice, milk, processed milk, cream, vegetable cream, milk drink, drink yogurt, lactic acid bacteria drink, These include health drinks, liquid foods, soups, sauces, mayonnaise, and dressings that contain nutritional and functional ingredients.

  In the present invention, “gel food” means food that has been gelated and solidified by the action of a protein and / or a gelling agent due to heat and acid. For example, yogurt; pudding, mousse, bavaroa , Jelly, apricot tofu, water candy, etc .; agar; tofu; strawberry; gel-like / jelly-like dietary supplements.

  As a method for preparing a sample for a transmission electron microscope according to the present invention, a general method may be used other than rapid freezing and freeze replacement. Typically, after replacement with a fixing solution, a dehydration step, a resin embedding step, an ultrathin section manufacturing step, and the like are performed. Moreover, in order to observe the original tissue structure of food and drink, it is preferable not to include steps such as dilution / concentration, separation / purification of samples used in conventional methods.

“Quick freeze” refers to a method of putting a sample into liquid propane, slush nitrogen, liquid ethane or the like, a method of attaching a sample to a metal cooled with liquid propane, liquid nitrogen, or the like. From the viewpoint of freezing the sample in a shorter time, a method of introducing the sample into liquid propane cooled to −150 ° C. or less is preferable.
In quick freezing, generally, the time taken to pass through the maximum ice crystal formation zone of -1 to -5 ° C is less than 30 minutes, or the distance that the freezing front of food and drink travels from the surface to the inside in one hour Represents the freezing interface advance speed V, and the case of V = 5 to 20 cm / h is set as the processing condition. On the other hand, in “rapid freezing” in electron microscope observation, it is considered preferable to suppress the size of ice crystals to 10 nm or less so as not to hinder the observation of the fine structure. To obtain this frozen state, −3 It is necessary to pass through a temperature range of ˜−80 ° C. in a short time, specifically, a cooling rate of 10 ⁴ ° C./sec or more is required (reference book: “Understanding electron microscope technology”, P.137, Hiroshi Hirano, Shichiro Miyazawa, Asakura Shoten).

When a sample is rapidly frozen by loading the sample into liquid propane, slush nitrogen, liquid ethane, etc., from the viewpoint of freezing the sample in a shorter time, the sample is supported by a material (material) with high thermal conductivity. It is preferable to carry out with the sample sandwiched between the tables. In the present invention, the “sample support base” is a disk having a flat surface.
When the sample support is used, it is not particularly limited as long as it is made of a material having high thermal conductivity. For example, materials such as copper, silver, and carbon nanotube are preferable. Regarding the dimensions of the sample support, the thickness is preferably 25 μm or less and the area is preferably 10 mm ² or less from the viewpoint of easy handling and freezing of the sample in a shorter time.

  If the sample is a liquid food, and especially if it is a low-viscosity and high-fluid liquid food, the cooling efficiency is increased while preventing the original structure of the food from being destroyed, and the sample (liquid From the viewpoint of freezing the food) and ensuring the thickness and amount of the sample at a predetermined value or more, an appropriate amount of the sample is placed on one sample support table (first sample table), and then a plurality of fine samples are placed. It is preferable that a grid with a hole (mesh-like) is placed, and another sample support table (second sample table) is lightly placed on the grid, and then rapidly frozen. That is, when the sample is a liquid food, a plurality of sample support tables (the first sample table and the second sample table) are provided between the two sample support tables (first sample table and second sample table) from the viewpoint of securing the thickness and amount of the sample at a predetermined value or more. It is preferable to sandwich a grid with fine pores, fix (hold) an appropriate amount of sample there, and freeze quickly. When a sample is held in the pores using a grid, the sample can be fixed with the same size as the pores of the grid. At this time, in order to rapidly freeze the sample, it is preferable that the sample is smaller, for example, about 0.1 μl, but considering that it is actually easy to handle, it is preferably about 1 μl. .

  When the sample is a liquid food, if a sample is processed only with two sample support bases without using a grid, an appropriate amount of sample is not stably held between the two sample support bases. Variation in sample thickness occurs. When preparing a sample multiple times, it is difficult not only to observe the time for rapid freezing (freezing speed), but also to allow the sample to flow out during the cleaning process if the sample is too thin. There is also sex.

In the case of using a grid, the material is not particularly limited as long as it is made of a material having high thermal conductivity, like the sample support, but for example, a material such as copper, silver, or carbon nanotube is preferable. Further, regarding the dimensions of the grid, from the viewpoint of easy handling in practice and the viewpoint of freezing the sample in a shorter time, the thickness is preferably in the range of 25 μm or less, and the shape and dimensions equivalent to the sample support are actually easy to handle. Therefore, the area is preferably in the range of 10 mm ² or less. In addition, regarding the fine holes of the grid, the same shape and dimensions are always preferable from the viewpoint of unifying the time for quick freezing (freezing speed) when preparing a sample multiple times.

  When the sample is a gel food, the cooling efficiency is increased while suppressing the collapse of the original structure of the gel food, and one sample is supported from the viewpoint of freezing the sample (gel food) in a shorter time. It is preferable that an appropriate amount of sample is placed on the table (first sample table), and then another sample support table (second sample table) is lightly mounted on the table (first sample table) for quick freezing. At this time, in order to rapidly freeze the sample, it is preferable that the sample is smaller, for example, about 0.1 μl, but considering that it is actually easy to handle, it is preferably about 1 μl. . The size of the sample is preferably smaller and can be reduced to about 0.5 mm square. However, considering that it is actually easy to handle, it is preferably about 1 mm square.

  When the sample is a gel food, only one sample support base (for example, the first sample base) is used in the quick freezing operation from the viewpoint of suppressing the collapse of the original tissue structure of the gel food. Is preferably grabbed with tweezers and charged into liquid propane or the like.

  “Freeze replacement” refers to a method of immersing a frozen sample in a cooled medium (replacement medium) to replace the moisture and fat content of the sample with the replacement medium. Specifically, a rapidly frozen sample is placed in a sample bottle containing a replacement medium containing a fixative, and the sample bottle is maintained at a state below the recrystallization point of ice crystals to reduce the moisture and fat content of the sample. A method of replacing with a replacement medium. At this time, in order to increase the efficiency of replacement in the sample, when the sample is a liquid food, one sample support table sandwiching the sample is removed, the grid is also removed, and then the sample support table to which the sample is attached When the sample is a gel food, one of the sample support tables sandwiching the sample is peeled off, and then the sample support table to which the sample is attached is subjected to freeze replacement.

Although the substitution medium is not particularly limited, for example, a water-soluble solvent that is liquid at −80 ° C., such as methanol, ethanol, acetone, tetrahydrofuran, etc., can be used, and the viscosity is low even at low temperatures, and the substitution efficiency is excellent. Acetone is preferred from the standpoint of, for example. The fixing agent to be added to the substitution medium is not particularly limited, but is preferably one that dissolves in the substitution medium, and osmium tetroxide, tannic acid, glutaraldehyde, KMnO _{4 and the} like can be used from the viewpoint of preserving the sample form. Osmium tetroxide is preferable, and tannic acid and glutaraldehyde are preferable from the viewpoint of immunolabeling on a sample section. Preparation of transmission electron microscope samples of foods and drinks containing lactic acid bacteria and bifidobacteria such as fermented milk (set type yogurt, soft type yogurt, drink type yogurt, lactic acid bacteria beverages, etc.) In order to observe the structure of the cell membrane, especially the structure of the cell membrane, it is preferable to add a small amount of water, particularly distilled water, to the replacement medium. 1-5% by volume, more preferably 2-3% by volume.

  In the present invention, the temperature and time for freezing and replacement depend on the composition of the food or drink. For example, fermented milk (set-type yogurt, soft-type yogurt, drink-type yogurt, lactic acid bacteria beverage, etc.) contains lactic acid bacteria with cell membranes. It is preferable to do. Typically, rapidly frozen samples and sample bottles containing displacement media are held at about −80 ° C. (deep freezer, dry ice acetone, liquid nitrogen, etc.) for 12-72 hours, preferably 14-48 hours. After holding at −25 to −20 ° C. (freezer) for 2 to 12 hours, preferably 2 to 4 hours, at about 4 ° C. (refrigerator) for 1 to 2 hours, preferably 1 to 1 hour. The temperature is gradually increased by holding in half and finally returning to room temperature. And the sample returned to room temperature is wash | cleaned by hold | maintaining for 10-15 minutes x 3 times with anhydrous acetone.

  It is preferable to replace the sample washed with anhydrous acetone with anhydrous ethanol having a low water absorption from the viewpoint of increasing the efficiency of dehydration. Actually, if moisture exists in the sample, the polymerization of the resin becomes unstable in the resin embedding in the next step. Therefore, the sample washed with anhydrous acetone is replaced by holding in anhydrous ethanol for 10-15 minutes × 3 times. For samples that are difficult to dehydrate, the third hold time is set to about 12 hours.

  “Resin embedding” means embedding in a resin such as an epoxy resin in order to stably hold a sample. In the present invention, the resin embedding can be performed by a commonly used method. Specifically, the sample substituted with absolute ethanol is further replaced (washed) by holding it for about 15 minutes × twice with a solvent (such as propylene oxide) that can dissolve both the organic solvent and the resin. And this solvent (propylene oxide etc.) and resin are mixed and poured into a sample bottle. Thereafter, the solvent (such as propylene oxide) is volatilized, and then the sample is embedded in the resin.

  In the present invention, an “ultra-thin section” is preferably prepared from a portion having a short time required for freezing in a sample prepared by the method of the present invention in order to observe a more accurate tissue structure. Specifically, the ultrathin section is preferably prepared from a sample at a position of 5 to 10 μm from the contact surface with the sample support. In order to prepare a slice with the contact surface with the sample support horizontal, in the resin embedding, the sample is embedded in the resin together with the sample support, and the sample support is removed from the resin immediately before preparing the slice. Is preferred. Since the surface of the sample is not exposed to the embedding process by embedding the resin in a state where the sample is attached to the sample support base, an ultrathin section having an appropriate shape can be produced from the desired position of the sample. .

  The ultrathin section may be of a size usually used for observation with a transmission electron microscope, and typically has a thickness of 60 to 100 nm. The ultrathin section actually produced is electronically stained using a commonly used staining agent such as launyl acetate or lead staining solution.

  About the sample obtained in this way, the structure of food-drinks can be observed by observing the image image | photographed using the transmission electron microscope. At this time, since the tissue structure of the food and drink can be accurately observed, for example, the physical properties of the food and drink can be evaluated from a new aspect, and the storage stability of the food and drink, the texture and flavor, etc. Can be evaluated.

  Hereinafter, the present invention will be further described based on examples, but these examples are illustrative of the present invention and do not limit the present invention.

<Comparative Example 1>
1. Preparation of set-type yogurt sample for transmission electron microscope by chemical fixation method Pre-fixation (1-1) Using a cutter, cut out the 1 mm x 1 mm x 1 mm square, taking care not to disrupt the tissue of the set-type yogurt.
(1-2) A prefix solution (0.1 M phosphate buffer containing 2% GA (glutaraldehyde) and 2% PFA (paraformaldehyde)) is placed in a sample bottle, and the cut sample is Pickle.
(1-3) Put the sample bottle into the refrigerator (about 4 ° C.), hold it for 2 hours, and fix it in advance.

2. Post-fixation (2-1) Discard the pre-fix solution and wash with 0.1 M phosphate buffer for 30 minutes x 3 times while changing the solution.
(2-2) Discard the 0.1M phosphate buffer and put the post-fix solution (0.1M phosphate buffer containing 2% OsO ₄ (osmium tetroxide)) into the sample bottle.
(2-3) Put the sample bottle into the refrigerator (about 4 ° C.), hold it for 2 hours, and fix it later.

3. Dehydration (3-1) After discarding the fixative, lightly wash with 0.1 M phosphate buffer.
(3-2) 50% ethanol (10 minutes, about 4 ° C.), 70% ethanol (10 minutes, about 4 ° C. → about 20-25 ° C. (room temperature)), 90% ethanol (10 minutes, room temperature) , 99.5% ethanol (10 minutes, room temperature), absolute ethanol (30 minutes x 2 times, room temperature) in this order, and the sample water is replaced with ethanol.

4). Embedding (4-1) After maintaining in propylene oxide (PO) for 30 minutes × twice and replacing ethanol, a mixed solution of propylene oxide: resin (Quetol-812) = 7: 3 was placed in a sample bottle. Put in and let the resin penetrate.
(4-2) In a desiccator, propylene oxide is volatilized and embedded in a resin (Quetol-812).

5. Section Preparation and Staining (5-1) An ultrathin section of about 70 nm is prepared from a sample embedded in a resin.
(5-2) Proteins and phospholipids are electronically stained with uranyl acetate stain and lead stain.

  FIG. 1 shows the result of observing and photographing the sample with a TEM (JEM 1200EX) at a magnification of 1740 times.

<Example 1>
1. Preparation of set-type yogurt sample for transmission electron microscope by rapid freezing / freezing replacement method Quick Freezing (1-1) Take care not to disturb the tissue of the set-type yogurt, cut it into 1 mm x 1 mm x 1 mm square using a cutter.
(1-2) Place the cut sample on the disk of MAXTAFORM Grid II HF51 (Pyser / SG Energy, UK), and place another disk on it.
(1-3) The lower disk is picked with precision tweezers and dropped into liquefied propane (about -175 ° C).

2. Freeze replacement (2-1) A fixative solution (acetone containing 2% OsO ₄ and 2.5% distilled water) is put into a sample bottle, and the quick frozen sample is put therein.
(2-2) Hold the sample bottle with dry ice acetone (about −80 ° C., 48 hours) and freeze-replace.
(2-3) After raising the temperature in the order of a freezer (about −25 ° C., 4 hours) and a refrigerator (about 4 ° C., 1 hour), the temperature is taken out from the refrigerator and returned to room temperature.
(2-4) After washing with anhydrous acetone (15 minutes × 3 times), replacement with absolute ethanol (15 minutes × 3 times) is performed.

3. Embedding (3-1) After holding for 30 minutes x 2 times with propylene oxide (PO) to replace ethanol, a mixed solution of propylene oxide: resin (Quetol-812) = 7: 3 was placed in a sample bottle. Put in and let the resin penetrate.
(3-2) The propylene oxide is volatilized in a desiccator and embedded in a resin (Quetol-812).

4). Preparation and Staining of Section (4-1) A position of 5 to 10 μm is cut from the sample support of the sample embedded in the resin to prepare an ultrathin section of about 70 nm.
(4-2) Stain proteins and phospholipids with uranyl acetate stain and lead stain.

FIG. 2 shows the result of observing and photographing the sample with TEM (JEM 1200EX) at a magnification of 1740 times.
The observation result by the chemical fixation method (Fig. 1) shows that the connection of the black colored protein is very loose and the relationship with the fat part that has become blank cannot be understood, but the observation by the quick freeze / freeze substitution method In the result (FIG. 2), it was confirmed that the protein was closely bound, and the original tissue structure of yogurt could be observed.

<Comparative example 2>
Preparation of Transmission Electron Microscope Sample of Milk by Agar Coagulation / Chemical Fixation Method (1) Heat homogenized milk to 25 ° C and add a predetermined amount of agar previously dissolved in hot water . The liquid thus obtained is cooled and solidified, and then cut into 1 mm × 1 mm × 1 mm square using a cutter.
(2) The following steps are the same as the steps in Comparative Example 1.
FIG. 3 shows the result of observing and photographing the sample with a TEM (JEM 1200EX) at a magnification of 10200 times.

<Example 2>
1. Preparation of Transmission Electron Microscope Sample of Milk by Rapid Freezing and Freezing Replacement Method (1-1) About 1 μl of milk was dropped on the disk of MAXTAFORM Grid II HF51 (Pyser / SG Energy, UK), and EM-Fine Grid F-400mesh (Nisshin EM Co., Ltd.) is placed, and another disc is placed on it and sandwiched.
(1-2) The grid sandwiched between the three layers is picked with precision tweezers and dropped into liquefied propane (about -175 ° C).
(1-3) Remove one of the MAXTAFORM Grid II HF51 discs and EM-Fine Grid F-400mesh with tweezers and leave the remaining one disc.

2. Freeze replacement (2-1) Fix solution (acetone containing 2% OsO4 and 2.5% distilled water) is placed in a sample bottle and the rapidly frozen sample is placed.
(2-2) Hold the sample bottle with dry ice acetone (about −80 ° C., 24 hours) and freeze-replace.
(2-3) After raising the temperature in the order of a freezer (about −20 ° C., 2 hours) and a refrigerator (about 4 ° C., 1 hour), the temperature is taken out from the refrigerator and returned to room temperature.
(2-4) Wash with anhydrous acetone (10 minutes × 3 times).

3. Embedding, preparation of sections and staining The following steps are the same as those in Example 1.

FIG. 4 shows the result of observing and photographing the sample with a TEM (JEM 1200EX) at a magnification of 10200 times.
In the observation results of the agar coagulation / chemical fixation method (Fig. 3), each component of milk can be observed, but because protein and fat are localized, the original tissue structure of milk cannot be observed. From the observation results by the freezing / freezing replacement method (FIG. 4), it was possible to understand the distribution of proteins and fats and to observe the original tissue structure of milk.

<Comparative Example 3>
Preparation of liquid food transmission electron microscope sample by protein adsorption method (1) The liquid food is diluted 4 times, fixed with 1% GA solution, and the protein is adsorbed on the sample support.
(2) Absorb the liquid with filter paper and dry.
(3) Shadow the germanium on the sample support on which the protein is adsorbed.
FIG. 5 shows the result of observing and photographing the sample with TEM (JEM 1200EX) at a magnification of 5000 times.

<Example 3>
Preparation of Sample for Liquid Electron Transmission Electron Microscope by Rapid Freezing / Freezing Replacement Method According to the same process as in Example 2, a sample for liquid crystal transmission electron microscope was prepared.
FIG. 6 shows the result of observing and photographing the sample with a TEM (JEM 1200EX) at a magnification of 10200 times.
In the observation result by the protein adsorption method (FIG. 5), the state in which the protein is present in the liquid food is unknown, and the state of fat not bound to the protein cannot be confirmed. According to the observation result (Fig. 6), the protein can be observed in the state of being present in the liquid food, and only the portion where the fat is present becomes a clear blank, so the state where the lipid is present can also be confirmed. .

  From the above results, it can be understood that according to the present invention, it is possible to prepare a transmission electron microscope sample capable of observing the tissue structure of foods and drinks of all shapes such as liquid foods and gel foods.

<Application Example 1: Relationship between the structure of liquid food and storage stability>
In order to observe the tissue structures of sample A (1 kcal / ml, Meiji Co., Ltd.) and sample B (1.5 kcal / ml, Meiji Co., Ltd.) of liquid foods each having a different composition, samples prepared in the same manner as in Example 3 were used. Observed. FIGS. 7 and 8 show the results obtained by observing and photographing each sample with a TEM (JEM 1200EX) at magnifications of 10200 and 30200 times.

From the above results, the following was found.
The protein particles of sample A are small and distorted. Sample A fat globules are smaller than protein particles.
The protein particles of sample B are larger and completely spherical than sample A. The fat globules of sample B were almost the same size as the sample A on average.
Sample B, which is a high-concentration liquid food, has larger protein particles and a perfect spherical shape than Sample A, which is a general-concentration liquid food, and the protein particles are not sufficiently dissolved or dispersed. it was thought. On the other hand, in samples A and B, the size of fat globules was not proportional to the size of protein particles, and the size was almost the same regardless of the concentration of liquid food.

Table 1 shows the physical property values of samples A and B of the liquid food.

Comparing the results of Table 1 with the observation results of TEM shows the following.
In the measurement of the particle diameter, Sample B showed a value about three times that of Sample A. In general, the particle size measured using a particle size distribution analyzer is often used as a reference for expressing the size (dimension) of fat globules. However, by observing a sample of a liquid food prepared by the method of the present invention, the particle size measured using a particle size distribution meter strongly reflects the size (dimension) of protein particles and expresses the size of protein particles. It was shown that.

  That is, in the particle size measurement using a laser diffraction type particle size distribution measuring device or the like, the sizes of protein particles and fat globules cannot be distinguished from each other. The size can be distinguished from each other, and the shape of protein particles can be visually understood. Whether the numerical value of the particle size measured using a laser diffraction type particle size distribution analyzer represents the size of the protein particle or reflects the size of the fat globule. Can be accurately determined.

  In this liquid food sample, assuming that the numerical value of the particle size measured using the method of the present invention represents the size of the protein particles, in sample A, the average size of the protein particles is about 300 nm. In Sample B, it can be determined that the average is about 800 nm. The size of fat globules is difficult to recognize as a particle size, but can be visually understood, and the size of fat globules in sample A is the same as that of protein particles in sample A. It can be determined that the average value of Sample B is about 300 nm.

  Samples A and B of the liquid food were placed flat and stored at room temperature for 2 months, 4 months, and 6 months. Each storage stability (precipitation dry weight [g / 100 ml]) is shown in Table 2. A smaller precipitation dry weight means higher storage stability.

From the results shown in Table 2, it can be seen that Sample A has high storage stability (small precipitation dry weight), and Sample B has somewhat low storage stability (precipitation dry weight is somewhat large). When the compositions of the precipitates of Samples A and B were examined, most of them were proteins. Compared to sample A, sample B was thought to have an effect on the amount of dry precipitate weight due to the large protein particles.
On the other hand, in sample A, the viscosity was 11 mPa · s, and in sample B, it was 20 mPa · s. In sample B, the viscosity was higher than in sample A. However, this difference in viscosity has a low effect of suppressing the formation of precipitates, and the size of protein particles greatly affects the formation of precipitates. It was thought that there was. During the storage of Samples A and B, no fat levitation was observed.

From the above results, it was found that the size of protein particles affects storage stability (precipitation amount). From this, by using the method of the present invention, by observing the tissue structure of food and drink, it is more accurate than using only the particle size measured using a laser diffraction particle size distribution measuring device as an index. It was derived that the storage stability of the product can be predicted.
In particular, it was shown that a liquid food having a protein particle size of 500 nm or less, preferably 300 nm or less, has a high storage stability like Sample A regardless of the viscosity.
In addition, the liquid food having fat globules of 500 nm or less, preferably 300 nm or less, showed high storage stability like Samples A and B regardless of the viscosity.

  ADVANTAGE OF THE INVENTION According to this invention, the sample for transmission electron microscopes of the state which hold | maintained the original structure | tissue structure of food-drinks as much as possible can be provided. Thereby, the original structure of the food and drink can be observed, and the storage stability of the food and drink and the texture and flavor can be evaluated. Furthermore, the numerical value of the particle size measured using a laser diffraction type particle size distribution measuring device represents the particle size of protein, the particle size of fat globule, or the particle size of other particles. It is possible to judge visually.

  In particular, liquid food has a long shelf life and is allowed to stand for a long time, so that there is a high possibility that precipitation occurs during long-term storage or fat rises. At this time, if sediment or suspended matter is generated, problems such as clogging of sediment or suspended matter may occur in the nasal tube or tube tube used at medical or nursing care sites. It is desired to provide a liquid food with high storage stability. Furthermore, it is desirable to be able to evaluate the storage stability of the liquid food in a short time immediately after the production of the liquid food (product). To meet such problems, measurement has been performed using a laser diffraction particle size distribution analyzer. The quality was controlled by using an index such as the particle size, but it was difficult to determine whether the protein particles were large or the fat globules were large only by the index of particle size. Therefore, by evaluating the liquid food using the method of the present invention, the storage stability of the liquid food can be predicted in detail, and the quality can be managed finely.

Claims (8)

  A method for preparing a sample for a transmission electron microscope of a food, comprising freezing and replacing a liquid or gel food after rapid freezing.   The method according to claim 1, comprising placing a liquid or gel food on a sample support and rapidly freezing.   The method according to claim 2, comprising cutting a sample at a position of 5 to 10 μm from the sample support.   The method according to claim 1, comprising freeze-replacement with a fixative containing water.   The evaluation method of the structure | tissue structure of a liquid or gel-like food including observing the sample prepared by the method in any one of Claims 1-4 using a transmission electron microscope.   The method according to claim 5, wherein the storage stability of the liquid or gel food is evaluated.   A liquid or gel food evaluated as having high storage stability by the method according to claim 6.   A high-concentration liquid food with a protein particle size of 500 nm or less and at least 1 kcal / ml.