JP2003294626A - Method for analyzing tissue construction of multi- components dispersion food - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for stereoscopically analyzing a tissue construction of a multi-components dispersion food. <P>SOLUTION: In the method for stereoscopically analyzing the tissue construction of the multi-components dispersion food, the food which contains at least a kind of component among a starch, a protein and a lipid, is stained by using a fluorescent pigment, and then images are obtained by using at least two different values of wavelength in a wavelength region lower than a value equal to the maximum absorption wavelength of the used fluorescent pigment minus 40 nm, and the obtained images are composed. <P>COPYRIGHT: (C)2004,JPO

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for three-dimensionally analyzing the tissue structure of a multi-component dispersion food containing at least one component of starch, protein and lipid. . [0002] Starches, proteins and lipids are said to be three major nutrients in foods. Secondary processed foods such as breads, noodles, cooked rice, confectionery, and tempura are mainly made of flour and rice, and taste is determined by the form of the three major nutrients contained in these foods. It is considered that the texture changes greatly. Conventionally, the evaluation of taste and texture has been performed exclusively by sensory tests, but in recent years, it has been attempted to elucidate the tissue structure of food using equipment and determine the texture and the like from the tissue structure. . However, even when the tissue structure is observed with these instruments, the boundary of each component is unclear or the obtained image is planar, so that satisfactory evaluation cannot be performed at present. [0003] For example, in the case of using an electron microscope, the shape of the sample is observed by detecting electrons reflected by skipping electrons on the sample (scanning electron microscope) or the difference between electrons transmitted through the sample and untransmitted electrons. Specimens are detected and the shape of the specimen is observed (transmission electron microscope). In each of these methods, the specimen can be observed with extremely high resolution, but it is difficult to distinguish specific components due to the inability to stain. And
The structural analysis of the boundary cannot be performed. When using an optical microscope, a specific component of the sample is stained and observed.But the border is still unclear, and multiple staining can be performed to some extent, but the specificity due to staining is small, and the overlapping of stains Did not substantially determine the tissue structure. The laser confocal microscope employs laser light and can concentrate strong light having excellent straightness on one point. By using the laser light, an image only at the focal point can be displayed, and a cross-sectional image with a large depth of focus can be obtained, so that an image with high contrast can be obtained. On the other hand, it is difficult to determine a specific component from the obtained image because it cannot be stained, and it is difficult to analyze the structure of the boundary portion. SUMMARY OF THE INVENTION [0004] The present inventors have developed starch,
As a result of various studies on an image analysis method capable of clearly distinguishing the complex tissue structure of a multi-component dispersion food containing protein and lipid, the present invention has been completed. [0005] That is, the present invention relates to a food containing at least one component of starch, protein and lipid, which is used for a fluorescent protein dye, a fluorescent lipid dye, and a fluorescent dye. After staining with at least one or more dyes from starch dyeing dyes or fluorescent non-characteristic dyes,
-40 nm of the maximum absorption wavelength of at least one fluorescent dye
This is a method of observing at least two different wavelengths in a lower wavelength region and analyzing the tissue structure of a multi-component dispersed food in which at least two or more acquired images are synthesized. Next, the method of the present invention will be described. A tissue specimen of a food containing at least one component of starch, protein and lipid (hereinafter referred to as a multi-component dispersion food) is prepared. As a method for preparing this tissue specimen, a freeze fixing method, an immersion fixing method, a microwave processing fixing method, or the like can be adopted. Next, the freeze fixing method will be described. The multi-component dispersion food is frozen and fixed on the cooling stage of the microtome. Next, the frozen and fixed sample is cut into a certain thickness. The cut section is placed on a slide, dried and fixed with a heater, and fixed. Separately, a fluorescent dye solution is prepared, and the fluorescent dye solution and the sample section are kept for a certain time (for example, 1 hour).
After the reaction, the reaction is dried for up to 30 minutes, and in the longest case, for 1 to 3 hours. A tissue specimen can be obtained by enclosing the dried sample section with soft Canadian balsam. In the case of staining a sample section with a plurality of fluorescent dyes in preparing the tissue specimen, using a plurality of fluorescent dyes by repeating an operation of reacting with a fluorescent dye and drying and then reacting with another fluorescent dye and drying. Can be dyed. [0008] In the immersion fixing method, a multi-component dispersion food is added to a formalin solution or a glutaraldehyde solution in 12 to 12 hours.
Let soak for 24 hours. Next, the multi-component dispersion food is washed in running water for several hours (for example, 1 to 12 hours). 80%, 95%, 9
The immersion treatment is performed successively for 2 to 4 hours with a 9.5% and 100% ethanol solution. Next, the multi-component dispersion food is immersed in a xylene-soft paraffin solution for 12 hours and embedded.
Fix the embedded paraffin on the stock. The fixed multi-component dispersion food is cut into a certain thickness using a microtome. The cut section is floated on a hot water bath (35 to 40 ° C.) to remove wrinkles, and then scooped up on a slide glass and dried. Separately prepare a fluorescent dye solution,
The fluorescent dye solution and the section are allowed to react for a certain period of time (for example, 1 to 30 minutes, and in a long case, 1 to 3 hours), and then dried. Tissue specimens can be obtained by enclosing the dried sections in soft Canadian balsam. When staining with multiple fluorescent dyes, multiple dyeing is performed by staining with a fluorescent dye in the same manner as the freeze-fixing method, drying, staining again with another fluorescent dye, drying, and repeating this operation. It can be performed. Further, in the microwave treatment fixing method, the multi-component dispersion food is immersed in, for example, a glutaraldehyde solution for 12 to 24 hours, and microwaves are applied for 10 to 24 hours.
Irradiate for 60 seconds and fix. Thereafter, a tissue specimen can be obtained by performing the same treatment as in the immersion fixing method. Fluorescent dyes used as a dye in the method of the present invention include fluorescein-isothiocyanate, eoxin-isothiocyanate and Lucifer Yellow C
H, resorufin, tetramethylrhodamine-isothiocyanate, coumarinmaleimide, 7-amino-
4-methylcoumarin-3-acetic acid, fluorescamine, dansyl chloride, dansyl hydrazine, acid green,
Acridine orange, acridine yellow, congo red, acid fuchsin, nile blue, fast green, calcein, calcein blue, acriflavine,
Evans Blue, Rhodamine 123, Sudan Black B, Periodic Acid Schiff, Basic Pararoseaniline, Pararoseaniline Acetate, Basic Rubin, Magenta III, Aniline Blue, Alphazurin A, Alphazurin FG, Patent Blue VF, Bird -(P-aminophenyl) amine, tri- (p-isothiocyanatephenyl) amine and the like. Among these fluorescent dyes, the fluorescent dyes that specifically stain starch include Congo Red and Schiff periodate. The fluorescent dyes that specifically stain proteins include fluorescamine and acidic dyes. Fuchsin, basic pararose aniline, pararose aniline acetate,
Basic rubin, magenta III, aniline blue, alphazurin A, alphazurin FG, patent blue VF, tri- (p-aminophenyl) amine, tri-
(P-isothiocyanate phenyl) amine and the like. Nyl blue, Sudan black B and the like can be mentioned as fluorescent dyes that specifically stain lipids. Next, the fluorescently-stained food is observed at two or more wavelengths within a lower wavelength range of -40 nm, which is the maximum absorption wavelength of the fluorescent dye used. On the other hand, when this observation is performed in a wavelength region higher than the maximum absorption wavelength of the fluorescent dye of -40 nm, the fluorescence intensity becomes stronger. For example, the discrimination becomes difficult, and the object of the present invention is not achieved. The "observation" means in the present invention is carried out by an observation method using visible light or a method of observing in an infrared wavelength region or an ultraviolet wavelength region depending on the type of fluorescent dye used. When observing in the infrared or ultraviolet wavelength region, it is possible to use a specific observation instrument, convert observation data into electric signals, and perform computer processing to observe. The light source used for the fluorescence observation of the present invention is a mercury lamp (100 V) or a mercury lamp (200 V).
V), xenon lamp (75 V), xenon lamp (1
50V), halogen lamp (12V100W), tungsten lamp (6V30W). Other than this, a xenon lamp [250 to 1,000 nm], a tungsten lamp [250 to 1,000 nm], a Cr: LiSAF lamp [430 nm], a helium-cadmium laser [32
5,442 nm], argon ion laser [488,5
An excitation light source such as 14 nm], a Nd: YAG laser [532 nm], a helium neon laser [543, 594, 633 nm], and a krypton ion laser [568, 647 nm] is used. In the present invention, when analyzing the tissue structure of a multi-component dispersed food, it is usually performed by fluorescence observation, but by using it together with a phase difference, it can be observed as a more three-dimensional tissue structure. Examples of the phase difference means include bright field observation, phase difference observation, differential interference observation, and the like, and it is particularly preferable to use the phase difference observation or differential interference observation together. An excitation filter, a dichroic filter, an absorption filter or a mirror unit in which these are set are used as filters used for the above-mentioned fluorescence observation. These mirror units include U-MWU [excitation filter (330 to 385 nm), absorption filter (420 nm), absorption filter (400 nm).
m)], U-MWIB [excitation filter (460-49)
0 nm), absorption filter (515 nm), absorption filter (505 nm)], U-MWIG [excitation filter (52
0-550 nm), absorption filter (580 nm), absorption filter (565 nm)], U-MWIY [excitation filter (545-580 nm), absorption filter (610 n)
m), absorption filter (600 nm)] (Olympus Optical Industry Co., Ltd. product name) and the like. When both the fluorescence observation and the bright field observation are used, it is preferable to suppress the brightness transmittance of the illumination lamp to 6% or less of the whole. Examples of the ND filter used for this observation include an LBD filter (Olympus Optical Industry Co., Ltd. product name), an ND25 filter (Olympus Optical Industry Co., Ltd. product name), an ND6 filter (Olympus Optical Industry Co., Ltd. product name) and the like. In the present invention, when performing the above-mentioned fluorescence observation, it is necessary to observe at two or more wavelength points in a wavelength region lower than the maximum absorption wavelength of −40 nm of the fluorescent dye used. Next, the fluorescence observation of the present invention will be described.
A fluorescent dye A (λmaxa)
When staining is carried out using, for example, observation is performed at wavelengths of “a-45 nm” and “a-60 nm”. When a tissue specimen of a multi-component dispersion food is stained using a plurality of fluorescent dyes, for example, a B fluorescent dye (λ
When performing fluorescence observation using the maxb) and C fluorescent dyes (λmaxc), observation at “b-50 nm” and “b-80 nm”, observation at “c-60 nm” and “c-70 nm”, and “ observation at "b-50 nm" and "c-60 nm",
Observation at “b-80 nm” and “c-60 nm”, “b-
Observation at "50 nm", "b-80 nm" and "c-60 nm", "b-50 nm", "b-80 nm", "c-60 nm"
Various observation methods such as observation at "c-70 nm" can be adopted. The present invention will be described in more detail with reference to the following Examples, which by no means limit the present invention. Example 1, Comparative Examples 1-3 Bread dough is rapidly frozen on a cooling stage. The frozen section is sliced to a certain thickness with a microtome, and dried and fixed on a microscope preparation to prepare a section. Next, the sections are stained with aniline blue (λmax = 605 nm), which is a protein dye, and dried sufficiently. Next, the obtained tissue specimen was subjected to fluorescence observation using a fluorescence microscope (BX51 manufactured by Olympus Optical Industrial Co., Ltd.) using a filter for fluorescence observation shown in Table 1 below. In addition, fluorescence observation was performed in combination with phase difference observation or differential interference observation. The evaluation was performed according to the evaluation criteria shown in Table 2 by ten panelists on a photograph obtained by superimposing images of the tissue structure taken by observation. Table 1 shows the test results. [Table 1] [Table 2] Examples 2-4, Comparative Examples 4-6 Udon noodle strings are immersed and fixed. After the immersion-fixed noodle strings are frozen and fixed on a cooling stage, they are sliced to a certain thickness with a microtome, and dried and fixed on a microscope slide to prepare sections. Next, the sections are stained with pararose aniline acetate (λmax = 545 nm), which is a protein staining dye, and dried. Next, the sections are stained with a non-selective fluorescent dye, Fast Green FCF (λmax = 622 nm), and dried. Next, the obtained tissue specimen was subjected to fluorescence observation using a fluorescence observation filter shown in Table 3 below with a fluorescence microscope (BX51, manufactured by Olympus Optical Industries, Ltd.). In addition, fluorescence observation was performed in combination with phase difference observation or differential interference observation. The evaluation was performed in the same manner as in Example 1 for a photograph obtained by superimposing the tissue structures taken by observation in the same manner as in Example 1. Table 3 shows the test results. [Table 3] Example 5 and Comparative Examples 7 to 9 Tempura batter is rapidly frozen on a cooling stage together with a compound (Sankyo Copout CTC). The frozen section is sliced to a certain thickness with a microtome, and dried and fixed on a microscope preparation to prepare a section. The sections are then stained with Sudan Black B (λmax = 598 nm) with a lipid staining dye and dried. Next, the sections are stained with acidic fuchsin (λmax = 545 nm), which is a protein staining dye, and dried. Next, the obtained tissue specimen was subjected to fluorescence observation using a fluorescence observation filter shown in Table 4 below with a fluorescence microscope (BX51, manufactured by Olympus Optical Industries). Also, fluorescence observation,
The phase difference observation or the differential interference observation was performed in combination. The evaluation was performed in the same manner as in Example 1 for a photograph obtained by superimposing the tissue structures taken by observation in the same manner as in Example 1. Table 4 shows the test results. [Table 4] According to the method of the present invention, the tissue structure of a multi-component dispersion food containing starch, protein, lipid and the like can be analyzed as a three-dimensional image.

   ────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Shoji Yamada             5-3-1 Tsurugaoka, Oi-machi, Iruma-gun, Saitama               Nisshin Seifun Group Inc.Basic research             Inside (72) Inventor Koji Takeya             5-3-1 Tsurugaoka, Oi-machi, Iruma-gun, Saitama               Nisshin Seifun Group Inc.Basic research             Inside F term (reference) 2G043 AA03 BA14 CA05 DA01 EA01                       FA01 FA02 FA06 HA02 JA02                       KA02 KA03 KA05 KA09

Claims (1)

Claims 1. After dyeing a food containing at least one component of starch, protein and lipid with at least one fluorescent dye, the maximum absorption wavelength of the used fluorescent dye is -4.
A method for analyzing a tissue structure of a multi-component dispersion food, characterized by obtaining images at at least two different wavelengths in a wavelength region lower than 0 nm and synthesizing the obtained images.