Transparentizing reagent and transparentizing method

Abstract

Provided are: a reagent for transparentizing a carbohydrate- or protein-containing material, the reagent including a compound selected from the group consisting of a benzene carboxylic acid compound, a hydroxybenzene compound, a benzene sulfonic acid compound, a homosulfamine compound, and a benzenesulfohydroxamic acid compound; and a method for transparentizing said material, including processing the carbohydrate- or protein-containing material in said reagent.

Description

Clearing reagent and clearing method

[0001] The present invention relates to a reagent for making a carbohydrate- or protein-containing material transparent, a method for making the material transparent, a method for visualizing carbohydrates or proteins in the material, and a kit or device for use in these methods.

[0002] Gluten, which accounts for 85% of the endosperm protein in wheat, is considered to be a key factor determining the quality of wheat-based foods. During wheat dough production, the packing of gluten molecules leads to the formation of large structures exceeding the millimeter scale. However, despite extensive research on gluten over the past three centuries, imaging techniques for this complex system of macromolecules remain lacking, and the overall gluten structure remains unclear.

[0003] Recently, mechanical sectioning and reconstruction have been developed to visualize three-dimensional structures in biological samples, such as mammalian brains. This method involves serially slicing a sample using a microtome to obtain a set of consecutive two-dimensional images, which are then reconstructed into three dimensions using computer software. While this method allows access to deeper structures, it requires sophisticated techniques and labor and can sometimes cause the destruction of microstructures. On the other hand, optical sectioning, which utilizes fluorescent labels, is thought to enable simple and rapid imaging of three-dimensional reconstructions. However, light scattering caused by mismatches in refractive index (RI) between sample components and the surrounding solvent hinders the imaging of deep structures. Therefore, several clearing methods and reagents have been developed to reduce the amount of light scattering in biological samples, such as mammalian brains and plants (e.g., U.S. Patent No. 5,629,299; U.S. Patent No. 5,629,299; Non-Patent Document 1). These studies aimed to remove or denature high-RI components, such as proteins, lipids, and chlorophyll, and to replace the solvent with a high-RI reagent. For example, Patent Document 1 describes a method for clarifying biological materials of animal and plant origin using a solution containing fructose, and Patent Document 2 and Non-Patent Document 1 describe a clarifying reagent for biological materials of animal and plant origin, which contains urea as an active ingredient. However, to date, no research has been reported aimed at clarifying food materials, including wheat-based products.

[0004] Patent No. 5967528 Patent No. 6044900

[0005] Kurihara D. et al., Development 142, 4168-4179 (2015)

[0006] The inventors set themselves the task of developing an optical clearing reagent for carbohydrate- or protein-containing materials, including wheat-based products.

[0007] Wheat flour contains 80-85% starch, 8-14% protein, and 1% lipid, suggesting that starch is the primary source of light scattering in wheat-based samples. Therefore, attempts to clarify wheat-based samples have been made by removing starch or adjusting the RI difference between starch and surrounding materials. However, because starch exists as large particles in wheat flour, removing starch would inevitably damage the sample. Therefore, the inventors attempted to adjust the RI of the sample. After extensive research, they surprisingly found that treatment with certain aromatic compounds, such as benzenecarboxylic acid compounds, can clarify carbohydrate- or protein-containing materials, including wheat-based materials. Furthermore, by combining this with fluorescent labeling, they were able to successfully visualize and image the three-dimensional structures of carbohydrates or proteins in the materials at millimeter-scale sizes and submicron resolution. Thus, the present invention was completed.

[0008] That is, the present application provides the following aspects: [1] A reagent for clarifying a carbohydrate- or protein-containing material, comprising a compound selected from the group consisting of a benzenecarboxylic acid compound, a hydroxybenzene compound, a benzenesulfonic acid compound, a homosulfamine compound, and a benzenesulfohydroxamic acid compound; [2] A reagent for clarifying a carbohydrate- or protein-containing material, comprising a benzenecarboxylic acid compound, wherein the benzenecarboxylic acid compound is represented by Formula I: [In the formula, R １ ~R ５ are independently a hydrogen atom, a hydroxy group, an amino group, a nitro group, a carboxyl group, a C1 to C4 alkyl group, a C2 to C4 alkenyl group, a C2 to C4 alkynyl group, a C1 to C4 alkoxy group, or a halogen atom, wherein the C1 to C4 alkyl group, the C2 to C4 alkenyl group, the C2 to C4 alkynyl group, and the C1 to C4 alkoxy group are optionally substituted with a halogen atom, M is a monovalent or divalent metal atom, or a hydrogen atom, wherein R １ ~R ５ When either of R is a carboxyl group, the carboxyl group may form a salt with a monovalent metal atom, or R １ or R ５[3] The reagent for clarifying a carbohydrate- or protein-containing material according to [1], wherein R is a carboxyl group and M is a divalent metal atom, the carboxyl group may form a salt with M. １ ~R ５ [4] The reagent according to [2], wherein one or two of the following are hydroxy groups. [4] The reagent according to [2], wherein the benzenecarboxylic acid compound is a salt of benzoic acid, 2-, 3-, or 4-hydroxybenzoic acid, or phthalic acid with a monovalent or divalent metal atom, or a hydrate thereof. [5] The reagent according to any one of [2] to [4], wherein M is lithium, sodium, potassium, or calcium. [6] The reagent according to any one of [2] to [4], wherein the hydroxybenzene compound is represented by Formula II: [In the formula, R １ ~R ５ are independently a hydrogen atom, a hydroxy group, an amino group, a nitro group, a sulfonic acid group, a sulfonamide group, an acetamide group, a 1-hydroxy-2-(methylamino)ethyl group, a methoxycarbonyl group, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 alkoxy group, or a halogen atom, and the C1-C4 alkyl group, the C2-C4 alkenyl group, the C2-C4 alkynyl group, and the C1-C4 alkoxy group may be substituted with a halogen atom.] or a hydrate or hydrochloride thereof. [7] The reagent for clarifying a carbohydrate- or protein-containing material according to [1], １ ~R ５ [8] The reagent according to [6], wherein one or two of the hydroxybenzene compound are a hydroxy group. [9] The reagent according to [6], wherein the hydroxybenzene compound is catechol, resorcinol, or pyrogallol, or a hydrate thereof.

[10] The reagent according to [6], wherein the hydroxybenzene compound is a benzenesulfonic acid compound represented by formula III: [In the formula, R １ ~R ５are independently a hydrogen atom, a hydroxy group, a carboxyl group, an amino group, a nitro group, a sulfonic acid group, an aldehyde group, a C1 to C4 alkyl group, a C2 to C4 alkenyl group, a C2 to C4 alkynyl group, a C1 to C4 alkoxy group, or a halogen atom, wherein the C1 to C4 alkyl group, the C2 to C4 alkenyl group, the C2 to C4 alkynyl group, and the C1 to C4 alkoxy group are optionally substituted with a halogen atom, M is a monovalent or divalent metal atom, or a hydrogen atom, wherein R １ ~R ５ When either of R is a sulfonic acid group, the sulfonic acid group may form a salt with a monovalent metal atom, or R １ or R ５

[10] The reagent for clarifying a carbohydrate- or protein-containing material according to [1], wherein R is a sulfonic acid group and M is a divalent metal atom, the sulfonic acid group may form a salt with M. １ ~R ５wherein one or two of M are hydroxy groups.

[11] The reagent according to [9] or

[10] , wherein M is lithium, sodium, potassium, calcium, or nickel.

[12] The reagent according to any one of [1] to

[11] , further comprising a fluorescent dye.

[13] A method for making a carbohydrate- or protein-containing material transparent, comprising treating the carbohydrate- or protein-containing material with the reagent according to any one of [1] to

[12] .

[14] The method according to

[13] , comprising defatting and / or decolorizing the material by pretreatment, followed by treatment with the reagent.

[15] The method according to

[14] , wherein the material is pretreated with a decolorizing agent containing 1,2-hexanediol and / or a degreasing agent containing limonene.

[16] A method for visualizing carbohydrates or proteins in a sugar material, comprising making the material transparent by the method according to any one of

[13] to

[15] .

[17] The method according to

[16] , further comprising treating the material with a fluorescent dye.

[18] The method according to

[16] or

[17] , further comprising imaging.

[19] The method according to any one of

[16] to

[18] , wherein the material is a flour-based food material or a flour-processed food, and gluten proteins are visualized.

[20] A kit for making carbohydrate- or protein-containing materials transparent, comprising the reagent according to any one of [1] to

[12] .

[21] The kit according to

[20] , further comprising one or more selected from the group consisting of a fixative, a degreasing agent, and a decolorizing agent.

[22] The kit according to

[21] , further comprising a decolorizing agent containing 1,2-hexanediol and / or a degreasing agent containing limonene.

[23] A kit for visualizing carbohydrates or proteins in a material, comprising: (a) the reagent according to any one of [1] to

[11] , and a fluorescent dye, or (b) the reagent according to

[12] .

[24] The kit according to

[23] , further comprising one or more selected from the group consisting of a fixative, a degreasing agent, and a decolorizing agent.

[25] The kit according to

[24] , which comprises a decolorizing agent containing 1,2-hexanediol and / or a degreasing agent containing limonene.

[26] An apparatus for visualizing carbohydrates or proteins in a material, comprising: (a) the reagent according to any one of [1] to

[11] , a fluorescent dye, and an optical microscope, or (b) the reagent according to

[12] , and an optical microscope.

[27] A clarified carbohydrate- or protein-containing material comprising the reagent according to any one of [1] to

[12] .

[28] A decolorizing agent comprising 1,2-hexanediol.

[29] A degreasing agent comprising limonene.

[0009] By using the clarifying reagent or method of the present application, carbohydrate- or protein-containing materials can be easily and quickly made transparent. Furthermore, by using the visualization method of the present application, the structure of carbohydrates or proteins in carbohydrate- or protein-containing materials can be visualized, and the structure can be observed using microscopy and / or imaging techniques. For example, it can be revealed how the three-dimensional structure of gluten in wheat noodles changes from a honeycomb-like network structure to sparse large clumps upon the addition of salt during dough production, thereby changing the texture of the noodles, for example.

[0010] The present clearing reagent can clear carbohydrate- or protein-containing materials within a few days to a few weeks, enabling visualization and imaging of the three-dimensional structure of carbohydrates or proteins in the material at millimeter-scale sizes and submicron resolution. One advantage of the present clearing reagent is the ease with which clearing can be achieved by immersion alone. Furthermore, imaging devices for materials treated with the present clearing reagent can be used, including two-photon excitation microscopy (2PEM) and confocal laser scanning microscopy (CLSM), making the present technology easily accessible to many researchers. Furthermore, optical sectioning applied to the present clearing reagent or clearing method allows images of desired regions and planes to be viewed in any direction, thereby helping to better understand the structure of the material (e.g., gluten structure).

[0011] Figure 1 shows the clarification performance of SoROCS. Photographs a–h were obtained using ImageQuant for transmission. The scale indicates 5 mm. Untreated noodle samples (a) were fixed in 4% PFA / PBS for 1.5 hours (b) and then immersed in water (with 0.5% (wt / wt) sodium azide added to prevent spoilage) (c, f), ClearSee (d, g), or SoROCS (e, h). Opacity curves for the fixation process (light blue ◇) and the clarification process using water (yellow □), ClearSee (orange △), or SoROCS (green ◯) were calculated from transmission images of noodle samples without a grid-pattern-printed film. Data are shown as mean ± SD (N = 3 independently prepared noodle samples). Photographs j–l were obtained using ImageQuant for transmission. Images j-l show the same samples as images f-h in Figures 1-2, respectively, with a grid-patterned film used as the background. The scale indicates 5 mm. Figures m-o provide plot profiles of noodle samples treated with water (m), ClearSee (n), or SoROCS (o). Red lines (j-l) indicate the corresponding positions of the plot profiles (m-o). The contrast values ​​in the plot profiles for the regions indicated in curly brackets (m-o) are shown. Figure 2 shows the expansion characteristics during clarification. The linear expansion curves for the immobilization process (light blue ◇) and the clarification process using water (yellow □) or SoROCS (green ◯) are shown. Data are presented as mean ± SD (N = 3 independently prepared noodle samples). Figure b shows a CLSM image of gluten in a non-clarified noodle sample section mechanically sliced ​​using a cryostat. Figure c shows a 2PEM image of gluten in a noodle sample clarified by SoROCS. d and e show images after protein network analysis using AngioTool (blue points: junctions, red lines: gluten backbone, yellow lines: gluten outline). Red arrowheads indicate enlarged images of sections outlined by red boxes. Scale bars indicate 100 μm. One of these eight different images, obtained using CLSM and 2PEM, is shown in b and c, respectively.Comparison of variables calculated by AngioTool for non-clarified noodles (blue) and clarified noodles (green): gluten percentage area (f), junction density (g), average gluten length (h), endpoint ratio (i), and average E-lacunarity (j). Noodle samples were prepared four times independently (N = 4), and two different section samples were prepared from each noodle preparation (N = 2). Data for f-j are shown as mean ± SEM. SEM was calculated from the mean of N = 2. P values ​​were calculated using a two-tailed unpaired Student's t-test (f: P = 0.92, g: P = 0.76, h: P = 0.38, i: P = 0.31, j: P = 0.76). All variables showed no significant differences at the P = 0.05 level. Figure 3 shows two-photon excitation microscopy images of gluten. a-e: After fixation, Thilite. ＴＭ Figure 3 shows the 2D gluten structure of a noodle sample treated with SoROCS and mixed with Green. To improve visibility, imaging processing was performed in the same way for each set (Figures 3-1 a-e, 3-3 g-k). The scale bar indicates 200 μm. 2D images were excited at 920 nm (a), 930 nm (b), 940 nm (c), 950 nm (d), and 960 nm (e). This experiment was repeated three times using independently prepared samples, with similar results as shown in Figure 3-2 f. The fluorescence intensity histogram in f was generated using 12-bit images obtained from a two-photon excitation microscope (2PEM). Histograms for 920 nm excitation (orange), 930 nm excitation (green), 940 nm excitation (red), 950 nm excitation (blue), and 960 nm excitation (purple) are shown in f. Fluorescence intensities up to ∼1500 are shown. gk are immobilized and then AlexaFluor ＴＭ 633C ５Figure 1 shows the 2D gluten structure of a noodle sample treated with SoROCS mixed with Maleimide. The scale bar indicates 200 μm. 2D images were excited at 820 nm (g), 830 nm (h), 840 nm (i), 850 nm (j), or 860 nm (k). This experiment was repeated three times using independently prepared samples, with similar results as shown in l (Figures 3-4). The fluorescence intensity histograms in l were generated using 12-bit images obtained from a two-photon excitation microscope (2PEM). Histograms for 820 nm excitation (orange), 830 nm excitation (green), 840 nm excitation (red), 850 nm excitation (blue), and 860 nm excitation (purple) are shown in l. Fluorescence intensities up to 3000 are shown. m is a merged image of gluten (magenta) and fluorescent microspheres (green) obtained using 2PEM. n is a magnified image of the area marked with a red square in m. The scale bars indicate 50 μm (m) and 5 μm (n), respectively. This experiment was repeated three times using independently prepared samples (the mean and SD of FWHM were 0.54 and 0.03 μm, respectively). o shows the plot profile (point spread function) of fluorescence intensity for fluorescent microspheres along the red line shown in n (Figures 3-5). The plot and line shown in o represent experimental data for fluorescence intensity and calculated data using the Gaussian function shown in equation (2) (Io = 156.2, X ｃ= 5.16 μm, A = 1346.9, w = 0.509). Two opposing black arrows indicate the FWHM. This experiment was repeated three times using independently prepared samples (the mean and SD of FWHM were 0.54 and 0.03 μm, respectively). Figure 4 shows 3D reconstructions of gluten in whole noodles. a) shows the experimental setup for 2PEM imaging. b) shows a comparison of deep imaging of noodles treated with water (left) and SoROCS (right). When rendering with FluoRender, gamma change (3.2) and alpha blending (500) were applied, and the threshold was set to 146 and 90 for water and SoROCS, respectively. c) shows 3D reconstructions of gluten for noodles treated with water (left) and SoROCS (right). When rendering with FluoRender, gamma change (3.2) and alpha blending (500) were applied, and the thresholds were set to 146 and 90 for water and SoROCS, respectively. (d) shows the quality of the gluten fluorescence images depending on the depth from the noodle surface. Data are shown as mean ± SD (N = 3 independently prepared noodle samples). 2D imaging conditions using 2PEM were optimized for each depth. (e–h) show slice images obtained from three independently prepared noodle samples. 2D imaging conditions using 2PEM were optimized for each depth. Optical sections were obtained at several depths: z = 550 μm (e), z = 1050 μm (f), z = 1550 μm (g), and z = 2050 μm (h). Scale bars indicate 100 μm. Figure 5 shows the 3D structure of gluten and stress-strain curves for various noodle samples. ＴＭThe gluten skeletal structure labeled with Green was imaged using 2PEM. The 3D structures of gluten in control noodles (a), 10% gluten noodles (b), and 20% gluten noodles (c) are shown. d, f, and h are optical section images of a-c, and e, g, and f are binarized images obtained using Otsu's method. The percentages of gluten calculated by Otsu's method for control noodles, 10% gluten noodles, and 20% gluten noodles were 13.27 ± 1.01, 21.69 ± 1.02, and 29.60 ± 0.68, respectively (values ​​represent the mean ± SD for N = 3 independently prepared noodle samples). The scale bar indicates 100 μm. Alpha blending (500) was applied when rendering with FluoRender (a-c). Thiolite ＴＭThe gluten skeletal structure, labeled with green, was imaged with 2PEM. The 3D structures of gluten in 3% NaCl (j), 6% NaCl (k), 9% NaCl (l), and 12% NaCl (m) are shown. The scale bar indicates 100 μm. Alpha blending (500) was applied when rendering with FluoRender (j-m). n shows stress-strain curves for control (black), 10% gluten (green), 20% gluten (orange), 3% NaCl (blue), 6% NaCl (yellow), 9% NaCl (purple), and 12% NaCl (red). Strain was normalized by the thickness of each sample, and stress values ​​in 0.1 increments from 0.1 to 0.9 were calculated by the interpolation complement method. Data are presented as mean ± SD (N = 20 different samples obtained from the same noodle preparation). Figure 6 shows volumetric analysis of the 3D images. a) shows 3D rendering of 12% NaCl noodles. The 3D data shown in this figure is the same as that in m of Fig. 5-2. b) shows surface rendering by binarizing the fluorescence intensity in a). Each gluten mass three-dimensionally isolated from the others is represented by a different color in b. c) is an image rendered from a different viewpoint of the 3D image shown in b. The scale bar indicates 50 μm (a, b) and 20 μm (c). d) shows the volume distribution of gluten shown in b of Fig. 6-1. Fig. 7) shows 3D reconstruction of gluten in noodles observed by confocal laser scanning microscopy. a) shows the experimental setup for confocal laser scanning microscopy (CLSM) imaging. b) shows the Thilite ＴＭFigure 7 shows a 3D reconstruction of gluten labeled with Green. When rendering with FluoRender, gamma change (3.2), alpha blending (500), and threshold (90) were applied. Optical sections at several depths are shown: Z = 250 μm (c) and Z = 550 μm (d). Scale bars indicate 100 μm. The 2D imaging conditions in the CLSM were adjusted to optimize each depth. Figure 7 shows the quality of the gluten fluorescence image as a function of depth from the noodle surface. Data are shown as mean ± SD (N = 3 independently prepared noodle samples). The 2D imaging conditions in the CLSM were adjusted to optimize each depth. The images used to calculate the VAR value in Figure 7-3 are shown in Figures 7-3c and 7-3d. Figure 8 shows the effect of clearing reagents on sample brittleness. Cleared images of noodles cleared with ClearSee (a) and SoROCS (b) are shown. Images a and b are the same as those in Figure 1-1 g and h, respectively, but both images were processed in the same way to improve visibility. The scale bar indicates 5 mm. The red arrows indicate visible cracks. The opacity measurements of noodle samples treated with various clarifying agents of the present invention are shown. Values ​​in the figure are the mean ± SD of three independent experiments.

[0012] [Clarifying Reagent] The clarifying reagent of the present application contains, as an active ingredient, a compound selected from the group consisting of a benzenecarboxylic acid compound, a hydroxybenzene compound, a benzenesulfonic acid compound, a homosulfamine compound, and a benzenesulfohydroxamic acid compound. The clarifying reagent of the present application may contain two or more compounds selected from the above group.

[0013] As used herein, the term "benzenecarboxylic acid compound" includes benzenecarboxylic acid and salts of benzenecarboxylic acid with monovalent or divalent metal atoms (hereinafter also referred to as benzenecarboxylic acid salts), as well as hydrates or hydrochlorides thereof. The benzenecarboxylic acid is an aromatic compound containing one benzene ring, in which a hydrogen atom at at least one of the six carbon atoms forming the benzene ring is substituted with a carboxyl group, and one or more of the remaining carbon atoms may have a substituent. From the viewpoint of solubility, benzenecarboxylic acid salts or their hydrates or hydrochlorides are preferably used. The benzenecarboxylic acid salt may be a salt of one molecule of benzenecarboxylic acid with a monovalent metal atom, or a salt of one or two molecules of benzenecarboxylic acid with a divalent metal atom.

[0014] Examples of the substituent that the benzene ring of the benzenecarboxylic acid compound may have include, but are not limited to, a hydroxy group, an amino group, a nitro group, a carboxyl group, a sulfonic acid group, a sulfonamide group (—S(O ２ ) NH ２ ), acetamide group (-CH ２ C(O)NH ２ Examples of the hydrocarbon group include alkyl, alkenyl, or alkynyl groups, alkoxy, alkoxycarbonyl, aldehyde, and halogen atoms. The hydrocarbon group may be linear, branched, or cyclic. The hydrocarbon and alkoxy groups may be substituted, for example, but not limited to, with one or more groups selected from the group consisting of an amino group, an alkylamino group, a carbonyl group, an aminocarbonyl group, a hydroxy group, and a halogen atom.

[0015] The benzenecarboxylic acid compound may have two or more carboxyl groups on the benzene ring, preferably one or two carboxyl groups. When the benzenecarboxylic acid compound has two or more carboxyl groups on the benzene ring, preferably at least one carboxyl group forms a salt with a monovalent or divalent metal atom. For example, each of the two carboxyl groups may form a salt with a monovalent metal atom, or the two carboxyl groups may form a salt with a divalent metal atom.

[0016] As used herein, the term "hydroxybenzene compound" refers to an aromatic compound containing one benzene ring, in which a hydrogen atom at at least one carbon atom among six carbon atoms forming the benzene ring is substituted with a hydroxy group, and one or more other carbon atoms may have a substituent, and also includes a hydrate or hydrochloride thereof.

[0017] Examples of the substituent that the benzene ring of the hydroxybenzene compound may have include, but are not limited to, a hydroxy group, an amino group, a nitro group, a sulfonic acid group, a sulfonamide group, an acetamide group, a hydrocarbon group such as an alkyl group, an alkenyl group, or an alkynyl group, an alkoxy group, an alkoxycarbonyl group, an aldehyde group, and a halogen atom. The hydrocarbon group may be linear, branched, or cyclic. The hydrocarbon group and the alkoxy group may be substituted, for example, but not limited to, with one or more groups selected from the group consisting of an amino group, an alkylamino group, a carbonyl group, an aminocarbonyl group, a hydroxy group, and a halogen atom.

[0018] The hydroxybenzene compound may have two or more hydroxy groups on the benzene ring, and preferably has one to three hydroxy groups.

[0019] As used herein, the term "benzenesulfonic acid compound" includes benzenesulfonic acid and salts of benzenesulfonic acid with monovalent or divalent metal atoms (hereinafter also referred to as benzenesulfonate salts), as well as their hydrates or hydrochlorides. The benzenesulfonic acid is an aromatic compound containing one benzene ring, in which a hydrogen atom at at least one of the six carbon atoms forming the benzene ring is substituted with a sulfonic acid group, and one or more of the remaining carbon atoms may have a substituent. From the viewpoint of solubility, benzenesulfonate salts or their hydrates or hydrochlorides are preferably used. The benzenesulfonate salt may be a salt of one molecule of benzenesulfonic acid with a monovalent metal atom, or a salt of one or two molecules of benzenesulfonic acid with a divalent metal atom, preferably a salt of one molecule of benzenesulfonic acid with a monovalent metal atom, or a salt of two molecules of benzenesulfonic acid with a divalent metal atom.

[0020] Examples of the substituent that the benzene ring of the benzenesulfonic acid compound may have include, but are not limited to, a hydroxy group, an amino group, a nitro group, a sulfonic acid group, a sulfonamide group, an acetamide group, a hydrocarbon group such as an alkyl group, an alkenyl group, or an alkynyl group, an alkoxy group, an alkoxycarbonyl group, an aldehyde group, a carboxyl group, and a halogen atom. The hydrocarbon group may be linear, branched, or cyclic. The hydrocarbon group and the alkoxy group may be substituted, for example, but not limited to, with one or more groups selected from the group consisting of an amino group, an alkylamino group, a carbonyl group, an aminocarbonyl group, a hydroxy group, and a halogen atom.

[0021] The benzenesulfonic acid compound may have two or more sulfonic acid groups on the benzene ring, preferably one or two sulfonic acid groups. When the benzenesulfonic acid compound has two or more sulfonic acid groups on the benzene ring, preferably at least one sulfonic acid group forms a salt with a monovalent or divalent metal atom. For example, two sulfonic acid groups may each form a salt with a monovalent metal atom, or two sulfonic acid groups may form a salt with a divalent metal atom.

[0022] As used herein, the term "homosulfamine compound" refers to an aromatic compound containing one benzene ring, which has an aminomethyl group and an aminosulfonyl group at the para-position on the benzene ring and optionally has substituents at one or more other carbon positions, and also includes hydrates or hydrochlorides thereof. Examples of the substituents include, but are not limited to, hydroxyl groups, amino groups, nitro groups, sulfonic acid groups, sulfonamide groups, acetamide groups, hydrocarbon groups such as alkyl groups, alkenyl groups, and alkynyl groups, alkoxy groups, alkoxycarbonyl groups, aldehyde groups, carboxyl groups, and halogen atoms. The hydrocarbon groups may be linear, branched, or cyclic. The hydrocarbon groups and alkoxy groups may be substituted, for example, with one or more groups selected from the group consisting of amino groups, alkylamino groups, carbonyl groups, aminocarbonyl groups, hydroxyl groups, and halogen atoms. The homosulfamine compound may be, for example, homosulfamine, or a hydrate or hydrochloride thereof.

[0023] As used herein, the term "benzenesulfohydroxamic acid compound" refers to an aromatic compound containing one benzene ring, in which a hydrogen atom at at least one carbon atom among six carbon atoms forming the benzene ring is substituted with an N-hydroxysulfonamide group (-S(O ２)NHOH), and may have a substituent at one or more other carbon positions, including their hydrates or hydrochlorides. Examples of the substituent include, but are not limited to, a hydroxy group, an amino group, a nitro group, a sulfonic acid group, a sulfonamide group, an acetamide group, a hydrocarbon group such as an alkyl group, an alkenyl group, or an alkynyl group, an alkoxy group, an alkoxycarbonyl group, an aldehyde group, a carboxyl group, and a halogen atom. The hydrocarbon group may be linear, branched, or cyclic. The hydrocarbon group and the alkoxy group may be substituted, for example, but not limited to, with one or more groups selected from the group consisting of an amino group, an alkylamino group, a carbonyl group, an aminocarbonyl group, a hydroxy group, and a halogen atom. The benzenesulfohydroxamic acid compound may be, for example, benzenesulfohydroxamic acid.

[0024] Examples of the halogen atom include fluorine, chlorine, bromine, and iodine.

[0025] The monovalent metal atom may be, for example, an alkali metal atom, such as lithium, sodium, potassium, rubidium, or cesium, preferably lithium, sodium, potassium, or cesium, and more preferably lithium, sodium, or potassium.

[0026] The divalent metal atom may be, for example, an alkaline earth metal atom, nickel, iron, or copper. Examples of alkaline earth metal atoms include calcium, strontium, barium, beryllium, magnesium, etc. Examples of the divalent metal atom preferably include calcium and nickel.

[0027] An example of the benzenecarboxylic acid compound is a compound of Formula I: [In the formula, R １ ~R ５are independently a hydrogen atom, a hydroxy group, an amino group, a nitro group, a carboxyl group, a C1 to C4 alkyl group, a C2 to C4 alkenyl group, a C2 to C4 alkynyl group, a C1 to C4 alkoxy group, or a halogen atom, wherein the C1 to C4 alkyl group, the C2 to C4 alkenyl group, the C2 to C4 alkynyl group, and the C1 to C4 alkoxy group are optionally substituted with a halogen atom, M is a monovalent or divalent metal atom, or a hydrogen atom, wherein R １ ~R ５ When either of R is a carboxyl group, the carboxyl group may form a salt with a monovalent metal atom, or R １ or R ５ is a carboxyl group and M is a divalent metal atom, the carboxyl group may form a salt with M. or a hydrate or hydrochloride thereof.

[0028] Further examples of the benzenecarboxylic acid compounds include compounds of formula I, wherein R １ ~R ５ wherein one or two of the groups are hydroxy groups, or a hydrate or hydrochloride thereof.

[0029] Further examples of the benzenecarboxylic acid compounds include compounds of formula I, wherein R １ ~R ５ are independently a hydrogen atom, a hydroxy group, an amino group, a nitro group, or a carboxyl group, or a hydrate or hydrochloride thereof.

[0030] Further examples of the benzenecarboxylic acid compound include compounds represented by formula I, in which M is a monovalent or divalent metal atom, or hydrates or hydrochlorides thereof. Further examples of the benzenecarboxylic acid compound include compounds represented by formula I, in which M is lithium, sodium, potassium, or calcium, or hydrates or hydrochlorides thereof.

[0031] Further examples of the benzenecarboxylic acid compounds include compounds of formula I, wherein R１ ~R ５ and M is lithium, sodium, potassium, or calcium, or a hydrate or hydrochloride thereof. Further examples of the benzenecarboxylate compound include compounds represented by formula I, wherein R １ ~R ５ are independently a hydrogen atom, a hydroxy group, an amino group, a nitro group, or a carboxyl group, and M is lithium, sodium, potassium, or calcium, or a compound or a hydrate or hydrochloride thereof.

[0032] Further examples of the benzenecarboxylic acid compound include, but are not limited to, salts of benzoic acid, hydroxybenzoic acid, aminobenzoic acid, nitrobenzoic acid, aminosalicylic acid, nitrosalicylic acid, or phthalic acid with a monovalent or divalent metal atom (hereinafter also referred to as benzoate, hydroxybenzoate, aminobenzoate, nitrobenzoate, aminosalicylate, nitrosalicylate, or phthalate, respectively), as well as hydrates and hydrochlorides thereof. Furthermore, preferred examples of the benzenecarboxylic acid compound include, but are not limited to, benzoate, 2-, 3-, or 4-hydroxybenzoate, phthalate, and hydrates thereof.

[0033] Furthermore, specific examples of the benzenecarboxylic acid compound include, but are not limited to, sodium benzoate, potassium benzoate, sodium 2-hydroxybenzoate (sodium salicylate), lithium salicylate, sodium 3-hydroxybenzoate, sodium 4-hydroxybenzoate, sodium 2-aminobenzoate, sodium 3-aminobenzoate, sodium 4-aminobenzoate, potassium 4-nitrobenzoate, sodium 5-aminosalicylate, sodium 3-nitrosalicylate, disodium phthalate, and calcium phthalate.

[0034] An example of the hydroxybenzene compound is a compound of Formula II: [In the formula, R １ ~R ５are independently a hydrogen atom, a hydroxy group, an amino group, a nitro group, a sulfonic acid group, a sulfonamide group, an acetamide group, a 1-hydroxy-2-(methylamino)ethyl group, a methoxycarbonyl group, a C1 to C4 alkyl group, a C2 to C4 alkenyl group, a C2 to C4 alkynyl group, a C1 to C4 alkoxy group, or a halogen atom, and wherein the C1 to C4 alkyl group, the C2 to C4 alkenyl group, the C2 to C4 alkynyl group, and the C1 to C4 alkoxy group are optionally substituted with a halogen atom.] or a hydrate or hydrochloride thereof.

[0035] Further examples of the hydroxybenzene compounds include compounds of formula II, wherein R １ ~R ５ wherein one or two of the groups are hydroxy groups, or a hydrate or hydrochloride thereof.

[0036] Further examples of the hydroxybenzene compounds include compounds of formula II, wherein R １ ~R ５ are independently a hydrogen atom, a hydroxy group, an amino group, a sulfonamide group, an acetamide group, a 1-hydroxy-2-(methylamino)ethyl group, a methoxycarbonyl group, or a halogen atom, or a hydrate or hydrochloride thereof.

[0037] Further examples of the hydroxybenzene compound include, but are not limited to, catechol, resorcinol, pyrogallol, trihydroxybenzene, dichlororesorcinol, hydroxybenzenesulfonamide, hydroxyphenylacetamide, amino-hydroxybenzenesulfonic acid, methyl iodosalicylate, diaminoresorcinol, adrenaline, and hydrates and hydrochlorides thereof. Furthermore, specific examples of the hydroxybenzene compound include, but are not limited to, catechol, resorcinol, pyrogallol, 1,2,4-trihydroxybenzene, 4,6-dichlororesorcinol, 4-hydroxybenzenesulfonamide, 4-hydroxyphenylacetamide, 3-amino-4-hydroxybenzenesulfonic acid hydrate, methyl 4-iodosalicylate, 4,6-diaminoresorcinol dihydrochloride, and DL-adrenaline hydrochloride.

[0038] An example of the benzenesulfonic acid compound is a compound represented by Formula III: [In the formula, R １ ~R ５ are independently a hydrogen atom, a hydroxy group, a carboxyl group, an amino group, a nitro group, a sulfonic acid group, an aldehyde group, a C1 to C4 alkyl group, a C2 to C4 alkenyl group, a C2 to C4 alkynyl group, a C1 to C4 alkoxy group, or a halogen atom, wherein the C1 to C4 alkyl group, the C2 to C4 alkenyl group, the C2 to C4 alkynyl group, and the C1 to C4 alkoxy group are optionally substituted with a halogen atom, M is a monovalent or divalent metal atom, or a hydrogen atom, wherein R １ ~R ５ When either of R is a sulfonic acid group, the sulfonic acid group may form a salt with a monovalent metal atom, or R １ or R ５ is a sulfonic acid group and M is a divalent metal atom, the sulfonic acid group may form a salt with M.] or a hydrate or hydrochloride thereof.

[0039] Further examples of the benzenesulfonic acid compounds include compounds of formula III, wherein R１ ~R ５ wherein one or two of the groups are hydroxy groups, or a hydrate or hydrochloride thereof.

[0040] Further examples of the benzenesulfonic acid compounds include compounds of formula III, wherein R １ ~R ５ are independently a hydrogen atom, a hydroxy group, a carboxyl group, an amino group, a nitro group, a sulfonic acid group, an aldehyde group, a C1 to C4 alkyl group (preferably a C1 to C3 alkyl group, more preferably a C1 to C2 alkyl group), a C1 to C4 alkoxy group (preferably a C1 to C3 alkoxy group, more preferably a C1 to C2 alkoxy group), a halogen atom, or a C1 to C4 alkyl group substituted with a halogen atom (preferably a C1 to C3 alkyl group substituted with a halogen atom, more preferably a C1 to C2 alkyl group substituted with a halogen atom, for example, 2-bromoethyl or 1-bromoethyl), or a hydrate or hydrochloride thereof.

[0041] Further examples of the benzenesulfonic acid compound include compounds represented by formula III, wherein M is a monovalent or divalent metal atom, or hydrates or hydrochlorides thereof. Further examples of the benzenesulfonic acid compound include compounds represented by formula III, wherein M is lithium, sodium, potassium, calcium, or nickel, or hydrates or hydrochlorides thereof.

[0042] Further examples of the benzenesulfonic acid compounds include compounds of formula III, wherein R １ ~R ５ and M is lithium, sodium, potassium, calcium, or nickel, or a hydrate or hydrochloride thereof. Further examples of the benzenesulfonic acid compound include compounds represented by formula III, wherein R １ ~R ５are independently a hydrogen atom, a hydroxy group, a carboxyl group, an amino group, a nitro group, a sulfonic acid group, an aldehyde group, a C1 to C4 alkyl group (preferably a C1 to C3 alkyl group, more preferably a C1 to C2 alkyl group), a C1 to C4 alkoxy group (preferably a C1 to C3 alkoxy group, more preferably a C1 to C2 alkoxy group), a halogen atom, or a C1 to C4 alkyl group substituted with a halogen atom (preferably a C1 to C3 alkyl group substituted with a halogen atom, more preferably a C1 to C2 alkyl group substituted with a halogen atom, for example, 2-bromoethyl or 1-bromoethyl), and M is lithium, sodium, potassium, calcium, or nickel, or a hydrate or hydrochloride thereof.

[0043] Further examples of the benzenesulfonic acid compounds include, but are not limited to, benzenesulfonate, chlorobenzenesulfonate, iodobenzenesulfonate, hydroxybenzenesulfonate, nitrobenzenesulfonate, formylbenzenesulfonate, ethylbenzenesulfonate, bromoethylbenzenesulfonate, benzenedisulfonate, dinitrobenzenesulfonate, formylbenzenedisulfonate, nitroaniline-sulfonate, amino-nitrobenzenesulfonate, aniline-disulfonate, toluidine-sulfonate, picryl sulfonate, dichloro-hydroxybenzenesulfonate, amino-methylbenzenesulfonate, benzenedisulfonate, hydroquinonesulfonate, guaiacolsulfonate, sulfobenzoic acid monosalt, sulfoisophthalic acid monosalt, tiron, and hydrates and hydrochlorides thereof.

[0044] Furthermore, specific examples of the benzenesulfonic acid compound include, but are not limited to, sodium benzenesulfonate, nickel (II) benzenesulfonate hexahydrate, sodium 4-chlorobenzenesulfonate, sodium 2-iodobenzenesulfonate, sodium 4-hydroxybenzenesulfonate, sodium 4-hydroxybenzenesulfonate dihydrate, sodium 3-nitrobenzenesulfonate, sodium 2-sulfobenzenealdehyde, sodium 4-ethylbenzenesulfonate, sodium 4-(2-bromoethyl)benzenesulfonate, disodium 1,3-benzenedisulfonate, sodium 2,4-dinitrobenzenesulfonate, 4-formylbenzene-1,3-disulfonate, disodium aniline, 2-nitroaniline-4-sulfonate, sodium 2-amino-5-nitrobenzenesulfonate, monosodium aniline-2,5-disulfonate, sodium o-toluidine-4-sulfonate tetrahydrate, sodium picrylsulfonate dihydrate, sodium 3,5-dichloro-2-hydroxybenzenesulfonate, nickel(II) 2-amino-5-methylbenzenesulfonate, potassium benzene-1,2-disulfonate, potassium hydroquinonesulfonate, potassium guaiacolsulfonate, monosodium 3-sulfobenzoate, monosodium 5-sulfoisophthalate, sodium 4-hydroxybenzenesulfonate dihydrate, and tiron monohydrate.

[0045] The clarifying reagent of the present application may be a compound selected from the group consisting of benzenecarboxylic acid compounds, hydroxybenzene compounds, benzenesulfonic acid compounds, homosulfamine compounds, and benzenesulfohydroxamic acid compounds, or may be a solution containing a compound selected from the group consisting of benzenecarboxylic acid salt compounds, hydroxybenzene compounds, benzenesulfonate compounds, homosulfamine compounds, and benzenesulfohydroxamic acid compounds.

[0046] When the clarifying reagent of the present application is in the form of a solution, the clarifying reagent contains, for example, water, a buffer solution, methanol, ethanol, propanol, ethyl acetate, isopropyl alcohol, etc. as a solvent. The clarifying reagent of the present application contains a hydroxybenzoate compound at a concentration of 0.5 M or higher, preferably 1 M or higher, more preferably 2 M or higher, even more preferably 3 M or higher, and even more preferably 4 M or higher. Higher concentrations are expected to produce greater effects, but due to the nature of the compounds, for example, resorcinol and catechol are thought to be unable to dissolve at concentrations of 9 M or higher. For example, the solubility of sodium salicylate in water at 20°C is up to about 4.125 M.

[0047] A compound selected from the group consisting of benzenecarboxylic acid compounds, hydroxybenzene compounds, benzenesulfonic acid compounds, homosulfamine compounds, and benzenesulfohydroxamic acid compounds can be dissolved in a solvent by, for example, stirring, heating or cooling, ultrasonic treatment, etc. When the compound is particularly difficult to dissolve, the compound may be added to the solvent in small amounts and then ultrasonic treatment, heating or cooling, etc. may be performed to dissolve the compound to the desired concentration.

[0048] The clearing reagent of the present application may further contain a fluorescent dye. The fluorescent dye is not particularly limited, and for example, commercially available fluorescent dyes or synthetic fluorescent dyes may be used. Furthermore, as used herein, the term "fluorescent dye" also encompasses fluorescent labels, fluorescent microspheres, and the like. Fluorescent labels are useful when using the clearing reagent of the present application to visualize the structure of carbohydrates or proteins in a material. For example, the use of fluorescent labels makes it possible to visualize yeast and other substances in a material, and visualize their three-dimensional distribution within the material. The fluorescent label is not particularly limited, and a fluorescent label that targets a desired carbohydrate or protein (e.g., a fluorescent substance that binds to carbohydrates or proteins) may be selected. Such fluorescent labels may be commercially available, or may be a substance that specifically binds to a desired target, such as an antibody, to which the fluorescent dye is bound. The binding of a substance that specifically binds to a target to a fluorescent dye can be performed using known methods, and those skilled in the art can appropriately prepare desired fluorescent labels. Examples of fluorescent labels and fluorescent dyes include, but are not limited to, Thiolite. ＴＭ (Trademark) Green (TG), Alexa Fluor ＴＭ 633 C5 Maleimide (AF), various Alexa Fluor ＴＭ Examples of the fluorescent dye include fluorescein isothiocyanate, rhodamine, and the like. The amount of the fluorescent dye contained in the clearing reagent of the present application is not particularly limited and can be determined appropriately by those skilled in the art. Depending on the type of fluorescent dye used, the amount may be, for example, about 0.0001 to 0.01% (wt / wt) of the fluorescent dye.

[0049] The clearing reagent of the present application may further contain a surfactant. Surfactants are considered to be effective in degreasing materials and improving the permeability of the clearing reagent into the cytoplasm and cell walls. Preferably, a nonionic surfactant may be used as the surfactant. Examples of nonionic surfactants include, but are not limited to, Triton X, Tween, and NP-40. The amount of surfactant contained in the clearing reagent of the present application is not particularly limited and may be determined appropriately by one skilled in the art. For example, the clearing reagent may contain, but is not limited to, approximately 1 to 30% (wt / wt) of surfactant.

[0050] The clearing reagent of the present application may further contain a degreasing agent. A degreasing agent is useful when the material contains lipids. Examples of degreasing agents include, but are not limited to, 1,2-hexanediol, limonene, methanol, ethanol, propanol, hexane, ethyl acetate, isopropyl alcohol, etc. The amount of degreasing agent contained in the clearing reagent of the present application is not particularly limited and may be determined appropriately by those skilled in the art. Note that the degreasing effect of limonene was discovered for the first time in the present application.

[0051] The clarifying reagent of the present application may further contain a decolorizing agent. A decolorizing agent is useful when the material is colored. Examples of decolorizing agents include, but are not limited to, 1,2-hexanediol, methanol, ethanol, propanol, hexane, ethyl acetate, isopropyl alcohol, etc. The amount of decolorizing agent contained in the clarifying reagent of the present application is not particularly limited and may be determined appropriately by one skilled in the art. Note that the fact that 1,2-hexanediol exhibits a decolorizing effect was discovered for the first time in the present application.

[0052] In addition to the above, the clearing reagent of the present application may contain glycerol, carboxyvinyl polymer, hydroxypropylmethylcellulose, propylene glycol, macrogol, Percoll, Ficoll, polyethylene glycol, polyvinylpyrrolidone, sucrose, fructose, glucose, etc.

[0053] The clarifying reagent of the present application can clarify carbohydrate- or protein-containing materials. The carbohydrate- or protein-containing material is not limited as long as it contains carbohydrate or protein, and may be any material, for example, food-derived, biologically derived, or derived from animals (fish, birds, amphibians, reptiles, mammals, etc.), plants, insects, or microorganisms. As used herein, "food" encompasses food, food ingredients, and processed foods. Examples of carbohydrate- or protein-containing materials derived from food include, but are not limited to, starch-containing foods, starch-containing food ingredients, flour-based food ingredients, processed flour foods, rice-based food ingredients, processed rice foods, meat, processed meat foods, meat-containing food ingredients, fish, processed fish foods, fish-containing food ingredients, vegetables, fruits, and confectioneries. More specifically, examples include, but are not limited to, noodles, fried noodles, bread, bread dough, cooked rice, meat, sausages and hams, and fish sausages.

[0054] Depending on the type of material, carbohydrate- or protein-containing materials may or may not be pretreated, including fixation, degreasing, decolorization, enzyme treatment, moisture content adjustment, fluorescent dye treatment, etc.

[0055] Fixation has the effect of, for example, preserving the material and stabilizing the structure of cells and the like in the material. Fixation can be performed by a conventional method, for example, by immersing the material in a fixative such as formaldehyde, paraformaldehyde, glutaraldehyde, alcohol, dimethyl suberiminate, Bouin's fixative, or Carnoy's fixative. Furthermore, the fixed material may be embedded in an embedding medium such as OCT Compound or SCEM, and then frozen, or may be embedded in an embedding medium such as epoxy resin or paraffin.

[0056] When the carbohydrate- or protein-containing material contains lipids, the material may be preferably subjected to a degreasing treatment. The degreasing treatment can be carried out, for example, using a degreasing agent. The treatment method may be a conventional method such as immersion. For example, the degreasing agents listed above can be used.

[0057] When the carbohydrate- or protein-containing material contains a pigment, such as meat or fish meat such as salmon, the material may preferably be subjected to a decolorization treatment. The decolorization treatment can be carried out, for example, using a decolorizing agent. The treatment method may be a conventional method such as immersion. For example, the decolorizing agents listed above can be used.

[0058] When the carbohydrate- or protein-containing material is a material that cracks when immersed in a solution, such as rice, it is preferable to adjust the moisture content of the material to about 22% wt / wt (dry basis) or more before immersing the material in a solution of the clearing reagent. The moisture content can be adjusted by a conventional method, such as gas-phase adsorption.

[0059] When the carbohydrate- or protein-containing material has a cell wall, such as rice, it is preferable to treat it with an enzyme to create micropores in the cell wall. Examples of such enzymes that can be used include, but are not limited to, cellulase, pectolyase, pectinase, or mixtures thereof. The temperature and time of the enzyme treatment can be appropriately set depending on the type and concentration of the enzyme used, and are not particularly limited, but are, for example, about 5 minutes to 2 hours at room temperature to about 30°C.

[0060] The carbohydrate- or protein-containing material may also contain a fluorescent dye by pre-treating it with a fluorescent dye or by being genetically engineered to express a fluorescent protein. The fluorescent dye treatment can be carried out, for example, by mixing or injecting the fluorescent dye into the material. For example, if the material is noodles or bread, the fluorescent dye may be pre-dissolved in water that is added to flour during the noodle or bread-making process. The fluorescent dye may be, for example, any of the fluorescent dyes listed above.

[0061] Although the mechanism by which the clarifying reagent of the present application clarifies carbohydrate- or protein-containing materials is not fully understood, it is believed that in the case of carbohydrate-containing materials such as starch, clarification occurs probably because hydroxybenzoates promote gelatinization of the carbohydrates in the material. Gelatinization is a phenomenon in which carbohydrate particles such as starch begin to swell with a gradual increase in transparency and viscosity when heated in the presence of water. Salt is one of the substances that affect gelatinization, and the order in which salts promote gelatinization follows the Hofmeister series. The Hofmeister series order of anions and cations is "OH" － >C ６ H ４ (OH)COO － >SCN － >I － >Br － >Cl － >SO ４ ２－ " and "Ba ２＋ > Ca ２＋ > Mg ２＋ >Li ＋ >Na ＋ >K ＋ >Rb ＋ >NH ４＋ " Therefore, hydroxybenzoates are relatively strong gelatinization promoters. In fact, sodium hydroxide was a candidate for further gelatinization promotion, but its high pH made it unsuitable for use as a clarifying agent, since alkali causes protein denaturation. Furthermore, three-dimensional imaging by optical fragmentation requires fluorescent labeling of structures, but high pH solutions can induce fading fluorescence of the labeling reagent.

[0062] Additionally, in the case of protein-containing materials, the hydroxybenzoate compounds likely cause clearing by increasing the refractive index of the material (to about 1.4 or higher). For example, as shown in the Examples below, the present clearing reagent containing sodium salicylate at a concentration of 4 M has a high refractive index of 1.455 at room temperature.

[0063] [Clearing Method] The clearing method of the present application comprises treating the carbohydrate- or protein-containing material with the clearing reagent. The clearing treatment may be performed by any method of infiltrating the carbohydrate- or protein-containing material with the clearing reagent. For example, the material may be immersed in a solution of the clearing reagent. When the clearing reagent is in the form of a solution, it may be used as is. When the clearing reagent is in the form of a solid, a solution may be prepared by dissolving the clearing reagent in the solvent described above to achieve the above-mentioned concentration. The temperature and time of immersion may be adjusted as appropriate and are not particularly limited. Depending on the material, examples of suitable immersion times include 1 hour to 4 days at approximately 25°C to 30°C, preferably 1 day to 6 weeks at approximately 10°C to 30°C, and more preferably 3 days to 12 weeks at approximately 5°C to 30°C.

[0064] Thus, a transparent carbohydrate- or protein-containing material is obtained. The transparent material contains a transparent reagent, and the transparent reagent is infiltrated into the material. The transparent treatment using the transparent material of the present application is reversible, and the material made transparent by the transparent method of the present application returns to its pre-treatment opaque state when the transparent reagent is removed. For example, the transparent material can be returned to its opaque state by immersing the transparent material in water and dissolving the transparent reagent that has penetrated into the material in water.

[0065] As used herein, "transparency" refers to matching the refractive index of a material with that of a transparentizing reagent, and refers to a state in which the material has a higher transmittance of light (especially visible light) after treatment with the transparentizing reagent than the material before treatment, allowing the viewer to see through the material. Transparency can be evaluated, for example, by quantifying the opacity of a transmitted light image.

[0066] [Visualization Method] The visualization method of the present application is a method comprising making a carbohydrate- or protein-containing material transparent by the clearing method of the present application. The visualization method of the present application may further comprise the step of treating the material with a fluorescent dye.

[0067] If the carbohydrate- or protein-containing material has autofluorescence or if the clearing reagent used in the present application contains a fluorescent dye, the above-mentioned fluorescent dye treatment step is not necessary. If the carbohydrate- or protein-containing material has autofluorescence or if the clearing reagent used in the present application contains a fluorescent dye, the carbohydrate or protein in the material can be visualized by clearing the material. For example, plant materials such as wheat and rice may have autofluorescence. However, if the autofluorescence is weak, it is preferable to perform the above-mentioned fluorescent dye treatment step.

[0068] The visualization method of the present application makes it possible to observe deep portions of a material, even at depths of, for example, 1 to 2 mm from the surface of the material. The visualization method of the present application makes it possible to visualize carbohydrates or proteins within a material, as well as their three-dimensional distribution. Furthermore, the presence and distribution of bubbles within a material can be visualized based on differences in refractive index.

[0069] The step of treating the material with the fluorescent dye may occur before, after, or simultaneously with the step of treating the material with the clearing reagent.

[0070] The visualized material can be observed using an optical microscope. The type of optical microscope is not particularly limited, and examples that can be used include a two-photon excitation microscope (2PEM), a confocal laser scanning microscope (CLSM), a light sheet microscope, and a structured illumination microscope.

[0071] Furthermore, by using fluorescent labels for carbohydrates or proteins in the material, the structure of the carbohydrates or proteins in the material can be visualized.

[0072] Additionally, by using appropriate computer software and imaging devices such as cameras and scanners, visualized materials can be obtained as microscopic images.

[0073] [Kit] The kit for making carbohydrate- or protein-containing materials transparent according to the present application includes the above-described clearing reagent of the present application. Furthermore, the kit for visualizing carbohydrate- or protein-containing materials according to the present application includes the above-described clearing reagent of the present application and a fluorescent dye, or includes the clearing reagent of the present application that further includes a fluorescent dye. The clearing or visualization kit according to the present application may further include one or more selected from the group consisting of a fixative for fixing the material, a degreasing agent for degreasing the material, and a decolorizing agent for decolorizing the material. When the clearing reagent of the present application is solid, it may further include a solvent for dissolving the clearing reagent.

[0074] [Apparatus] The apparatus for making carbohydrate- or protein-containing materials transparent according to the present application comprises the above-described clearing reagent of the present application. Furthermore, the apparatus for visualizing carbohydrate- or protein-containing materials according to the present application may be an apparatus that comprises the above-described clearing reagent of the present application and a fluorescent dye, or the clearing reagent of the present application that further comprises a fluorescent dye, and further comprises an optical microscope. The visualization apparatus according to the present application may further comprise an imaging device. The optical microscope and imaging device are as described above. The clearing or visualization apparatus according to the present application may further comprise a container for the clearing treatment.

[0075] The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples.

[0076] [Materials and Methods] Unless otherwise specified, the reagents, samples (materials) and methods used in the examples are as follows.

[0077] Preparation of the Clarifying Reagent of the Present Application 1. Preparation of SoROCS Sodium salicylate (Nacalai tesque, 31821-45) was completely dissolved in Milli-Q water with stirring and heating at 70°C to prepare a 4M sodium salicylate solution. Triton X-100 (Nacalai tesque, 35501-02) was then added to the 4M sodium salicylate solution at a concentration of 0.1% (wt / wt) to prepare SoROCS. The RI was measured at 30°C using a refractometer (Atago, PAL-BX / RI). SoROCS had a pH of 7.5, a high RI of 1.455, and was nearly colorless at 30°C.

[0078] For fluorescent labeling of gluten, Thiolite™ Green (TG) (AAT Bioquest, 21508) and Alexa Fluor were dissolved at a concentration of 10 mM in dimethyl sulfoxide (Wako Chemicals, 043-07216). ＴＭ (Trademark) 633C ５ Maleimide (AF) (Invitrogen, A20342) was added to SoROCS to final concentrations of 0.0005% and 0.0002% (wt / wt), respectively.

[0079] 2. Preparation of Other Clarifying Reagents Each compound was completely dissolved in Milli-Q water with stirring and heating at 70°C to prepare 4 M sodium 3-hydroxybenzoate solution, 3.5 M sodium 4-hydroxybenzoate solution, 3 M sodium benzoate solution, 4 M lithium salicylate solution, 3.5 M disodium phthalate solution, 4 M catechol solution, 4 M resorcinol solution, and 4 M pyrogallol solution. If the compound was difficult to dissolve, it was dissolved in small amounts over time with ultrasonic treatment or stirring to prepare a solution of the desired concentration.

[0080] Noodle Sample Preparation Noodles without excess gluten powder or sodium chloride (referred to as "control noodles") were prepared as follows: 500 g of wheat flour (Nisshin Seifun, 8.5% protein, 0.34% ash) was mixed with 160 g of Milli-Q water and then kneaded for 20 minutes at 139 rpm using a mixer (Hobart, N50). The wheat dough was then placed in a pasta-making machine (Bottene) equipped with a vacuum pump (Ulvac, DTC21) and extruded through the opening of a 1.8 mm wide, 0.7 mm thick Teflon die (Tagliolini No. 17) at 11.0-15.0 kPa (abs) to produce noodles.

[0081] Noodles containing excess gluten powder (Nacalai tesque, 17011-95) or sodium chloride (Nacalai tesque, 31320-05) were produced as follows. Gluten powder was mixed with wheat flour at 10% and 20% weight ratios, followed by addition of Milli-Q water. Wheat dough was prepared as described above, and then noodles were made. The noodles thus produced are referred to as "10% gluten" and "20% gluten," respectively. Sodium chloride was dissolved in Milli-Q water at 3, 6, 9, and 12% weight ratios, followed by mixing with wheat flour. Wheat dough was prepared as described above, and then noodles were made. The noodles thus produced are referred to as "3%-NaCl," "6%-NaCl," "9%-NaCl," and "12%-NaCl," respectively. The amount of Milli-Q water was changed depending on the amount of sodium chloride. The composition of each noodle sample is shown in Table 1.

[0082]

[0083] Fixation and clearing. Samples were fixed in 4% (wt / wt) paraformaldehyde (PFA) in PBS for 1.5 h at 30°C, washed twice in D-PBS, and immersed in ClearSee, SoROCS, or water supplemented with 0.5% (wt / wt) sodium azide to prevent spoilage for 3 days at 30°C with shaking. ClearSee was prepared by mixing xylitol powder (Nacalai tesque, 36718-65; final concentration 10% (wt / vol)), sodium deoxycholate (Nacalai tesque, 10712-96; final concentration 15% (wt / vol)), and urea (Nacalai tesque, 35905-35; final concentration 25% (wt / vol)) in Milli-Q water.

[0084] Opacity Measurement. Sample opacity was measured using a cooled CCD camera system (GE Healthcare, ImageQuant LAS 4000). Samples were placed on the bottom of a flat-centered glass dish filled with ClearSee, SoROCS, or water supplemented with 0.5% (wt / wt) sodium azide to prevent spoilage. Using white light transillumination, the samples were then digitized by a CCD camera cooled to −25°C with a lens (F0.85 43 mm). Gray values ​​of 3,072 × 2,048-pixel 16-bit tiff images were obtained with an exposure time of 6 seconds and an iris of F2.8. Sample opacity was calculated by normalizing the sample gray value with the background gray value using free software (Fiji). The contrast C, which indicates the difference in gray levels, was calculated from an image of the sample placed on a film printed with a grid pattern according to the following equation (1): The plot profile was measured using Fiji software.

[0085] Formula (1): [In the formula, g ｍａｘ and g ｍｉｎ indicate the maximum and minimum values ​​of the gray level in a 5 mm long plot profile, respectively.]

[0086] Measurement of Sample Morphology To measure the linear expansion of the samples, a 5,472 × 3,648 pixel photograph was taken of the top of the sample placed on a glass dish with a central plane filled with each clarifying agent using a high-resolution digital camera (Canon, EOS 70D; ISO = 100, f-stop = 29, shutter speed = 0.5) with a lens (Canon, EFS 60 mm). Aberrations in imaging performance (vignetting, color blur, distortion) and loss of resolution resulting from diffraction phenomena in the photograph were then corrected using software (Canon, Digital Photo Professional) based on the design lens values ​​provided by the supplier. 14-bit raw data were recorded in 16-bit tiff format. Based on the image of the sample width, linear expansion was measured using software (Fiji).

[0087] Two-photon excitation microscopy imaging. Samples were imaged using an upright or inverted two-photon excitation microscope (2PEM) system (Olympus, upright: FV1200MPE-BX61WI, inverted: FV1000MPE-IX83) with a 25x immersion objective (XLSLPLN25XGMP, numerical aperture (NA) = 1.00, working distance (WD) = 8 mm, RI = 1.41-1.52, correction collar) for SoROCS, a 25x immersion objective (XLPlan N 25X, NA = 1.05, WD = 2 mm, correction collar) for water, or a 10x dry objective (UPLSAPO10X2, NA = 0.40, WD = 3.1 mm). Detector sensitivity and laser power were varied using the z-axis light control. Fluorescence signals were quantified using a normal PMT for AF or a GaAsP PMT for TG. Imaging was performed using Fluoview FV10-ASW software Ver. 4.2c (Olympus). TG and AF were excited at 940 nm and 840 nm, respectively, using InSight DeepSee (Spectra-Physics). Microscope settings and imaging conditions, such as objective lenses, used in the examples and figures are summarized in Tables 2-1 and 2-2.

[0088]

[0089] Point spread function analysis. Fluorescent microspheres (yellow green fluorescent FluoSpheres, diameter = 0.49 μm, Invitrogen, F8813) were diluted to a 0.01% (vol / vol) concentration in Milli-Q water and dispersed under ultrasonic waves for 15 minutes. Next, 160 g of water containing the fluorescent microspheres was mixed with 500 g of wheat flour to prepare noodles in the same manner as above. The noodles were immobilized in 4% (wt / wt) PFA / PBS at 30°C for 1.5 hours and then immersed in an AF-mixed SoROCS with shaking at 30°C for 3 days. Two-dimensional PEM xy images of the AF-labeled gluten and fluorescent microspheres at a depth of approximately 1 mm from the noodle surface were then acquired at 840 nm and 940 nm excitation, respectively, as described above. The experimental data (point spread function) for the fluorescence intensity I of the fluorescent microspheres was represented by the following Gaussian function (Equation (2)): The full width at half maximum (FWHM) value of the peak on the x-axis was obtained by Gaussian fitting using OriginPro 8.1J SR3 software (OriginLab, Ver. 8.1.34.90).

[0090] Formula (2): [In the formula, I ０ is the baseline fluorescence intensity, and x ｃ is the central position of the peak, A is the coefficient, and w is the full width at half maximum (FWHM) of the peak.

[0091] Fluorescence Image Quality Analysis To examine the quality of the gluten fluorescence images at different depths from the noodle surface, the variance (VAR) was calculated from the histogram according to equation (3). The 2D imaging conditions using 2PEM and CLSM were adjusted to be optimal for each depth, and the fluorescence intensity values ​​in blocked-up shadows and blown-out highlights were excluded when calculating the VAR value.

[0092] Formula (3): where I is the fluorescence intensity and I ｐis the fluorescence intensity at the peak of the histogram, and F(I) denotes the normalized frequency for each fluorescence intensity I.]

[0093] Confocal laser scanning microscope imaging: Samples were imaged using an inverted confocal laser scanning microscope (CLSM) system (Olympus, FV1000D IX81) with a 20x dry objective (UPLSAPO 20X, NA = 0.75, WD = 0.6). The detector sensitivity and laser power were varied using the light intensity control function in the z direction. Imaging was performed using Fluoview FV10-ASW software Ver. 3.0a (Olympus). TG and Alexa Fluor ＴＭ 594 was excited at 473 nm and 559 nm, respectively, using a diode laser. The imaging conditions for each figure are shown in Table 2-1 and Table 2-2.

[0094] Protein network analysis: Protein network features were analyzed for CLSM and 2PEM images of the gluten backbone structure using software (National Cancer Institute, National Institutes of Health, AngioTool64 Ver. 0.6a). To ensure reproducible quantification of the gluten network, the same settings and parameters were applied: the vessel thickness (here, corresponding to the thickness of the gluten backbone structure) was set to 8, the low and high intensity thresholds were set to 0 and 255, small particles were removed below 30 pixels, and the fill holes function was not used.

[0095] Immunohistochemical staining The samples were fixed in 4% (wt / wt) PFA / PBS at 30°C for 1.5 hours and washed twice in D-PBS. The samples were then embedded in OCT Compound and sectioned at 10 μm thickness using a cryostat (Leica, CM1860) at -20°C and collected on slides. The OCT Compound on the slides was then rinsed with distilled water. Subsequent immunohistochemical staining was performed at room temperature. The slides were washed twice in D-PBS and then washed with SuperBlock. ＴＭ The slides were blocked with PBS (1:4 (D-PBS); Pierce Thermo Scientific, 37515) for 30 minutes and incubated with primary antibody (1:1000 (D-PBS); anti-wheat gluten chicken polyclonal IgY, Agrisera AB, AS09-571) for 1 hour. The slides were then washed twice with D-PBS and incubated with secondary antibody (1:200 (D-PBS); goat anti-chicken IgY with Alexa Fluor 594, Invitrogen, A11042) for 1 hour. The slides were then washed twice in D-PBS and incubated with TG (10 mM in DMSO) diluted in D-PBS to a final concentration of 0.05% (wt / wt) for 30 minutes. The fluorescence signals of TG and Alexa Fluor 594 were acquired using CLSM at 473-nm and 559-nm excitation, respectively. The imaging conditions for each figure are shown in Table 2-1 and Table 2-2.

[0096] Image reconstruction and reslicing: Using software (FluoRender), 3D reconstructed images were visualized and various 2D stacked images were obtained from the entire 3D data set for Fig. 4-2(b), Fig. 4-3(c), Fig. 5-1(a-c), Fig. 5-2(j-m), and Fig. 7-2(b). Imaris® x64 software (Bitplane, Ver. 8.3.1) was used to render the 3D data set and analyze the volume distribution shown in Fig. 6-2.

[0097] Compression Test The mechanical properties were characterized using stress-strain curves, which show the relationship between stress and strain during compression of the sample. Stress-strain curves were generated at 0.1 mm / s using a creep meter (Yamaden, RE2-33005B Rheoner II) equipped with a V-shaped plunger (No. 49) and a 20 N load cell. A sample cut to a length of approximately 30 mm was placed on a cylindrical stand. The force and travel distance applied by the V-shaped plunger were then recorded on a computer while the cylindrical stand was raised. The strain, normalized by the sample thickness, was set to 99%.

[0098] Statistical Analysis Numerical data calculations and statistical analyses were performed using Microsoft 365 Excel. Data are expressed as mean ± SD or mean ± SEM, as indicated. P values ​​were calculated using Student's two-tailed unpaired t test. P < 0.05 was considered statistically significant.

[0099] Example 1: Clarification of Wheat Noodles Using the Clarifying Reagent of the Present Invention 1 A 4 M aqueous solution of sodium salicylate was prepared as the clarifying reagent of the present invention, and Triton X-100 was added at a concentration of 0.1% (wt / wt) as a surfactant. Specifically, as described in the Materials and Methods section above, sodium salicylate (Nacalai Tesque, 31821-45) was completely dissolved in Milli-Q water by stirring and heating to 70°C. Next, Triton X-100 (Nacalai Tesque, 35501-02) was added to the 4 M sodium salicylate solution at a concentration of 0.1% (wt / wt). The resulting solution (hereinafter referred to as "SoROCS") had a pH of 7.5, a high RI of 1.455, and was nearly colorless at room temperature.

[0100] Wheat noodle samples (control noodles) were prepared and fixed with 4% paraformaldehyde for 1.5 hours. To prevent spoilage, they were immersed in water containing 0.5% (wt / wt) sodium azide, ClearSee, or SoROCS. ClearSee is a clearing reagent for plant samples, described in Non-Patent Document 1, and is designed to remove chlorophyll autofluorescence from green leaves. To evaluate the transparency of the noodle samples, the opacity of transmitted light images was quantified.

[0101] When the noodle samples were fixed with 4% paraformaldehyde for 1.5 hours, the opacity increased from 3.54±0.03 to 5.58±0.05 (Figure 1-2(i)). The noodles then immersed in water remained opaque, while the noodles treated with ClearSee or SoROCS began to become transparent within 6 hours (Figure 1-1(a-h)). The opacity of the noodles treated with ClearSee or SoROCS for 3 days was 1.61±0.04 and 1.27±0.01, respectively (Figure 1-2(i)).

[0102] To further compare the effects of ClearSee and SoROCS treatments on noodle transparency, we placed a film with a printed grid underneath the noodle to examine how clearly the grid pattern penetrated the noodle. Compared to the ClearSee-treated noodle, the plot profile of the SoROCS-treated noodle showed a large difference in gray level between sections with and without the grid lines, and the gray-level peaks reflecting the lines were sharper (Figures 1-3(j-o)). The contrast C, which indicates the difference in gray level, was calculated using Equation (1). The plot profiles were measured using Fiji software. The contrast C (shown in brackets in Figures 1-3(n-o)) obtained for ClearSee and SoROCS values ​​in a 5-mm-long plot profile was 0.10 and 0.31, respectively. This high contrast indicates that SoROCS can further suppress light scattering, a common obstacle associated with deep imaging by optical sectioning. Notably, noodles treated with ClearSee had visible cracks, indicating disruption of the noodle structure (Figure 8). Previous studies have reported that mouse brains treated with a urea-containing clearing reagent became very fragile and difficult to handle because urea causes partial denaturation and loss of cellular proteins. In contrast, noodles treated with our urea-free clearing reagent, SoROCS, did not exhibit significant fragility (Figure 8).

[0103] Example 2: Evaluation of Swelling Caused by the Present Clarifying Reagent Previously reported clearing reagents for biological samples resulted in significant changes in sample volume. Therefore, we investigated the morphological changes caused by the present clearing reagent. We observed an increase in linear expansion for noodles immersed in water and SoROCS (Figure 2-1(a)). Specifically, linear expansion increased initially and then gradually approached a maximum value. After immersion in water and SoROCS for 3 days, the linear expansion values ​​were 1.28 ± 0.01 and 2.23 ± 0.08, respectively. Collectively, these results suggest that the increase in opacity of noodles immersed in water was due to noodle swelling. In contrast, noodles immersed in SoROCS swelled considerably, but their transparency also increased. This substantial expansion of the SoROCS-treated samples may be due to the swelling of starch induced by sodium salicylate.

[0104] Next, to investigate the effect of swelling on gluten structure, we performed protein network analysis, an image analysis that allows accurate quantification of the structural and morphological network characteristics of the complex gluten structure, on both clarified and unclarified noodles. First, we prepared noodles fixed in 4% paraformaldehyde for 1.5 hours, and then acquired 2D images of mechanically sliced ​​sections of the samples using a cryostat (Figure 2-2(b)). Gluten was then treated with thiolite, which reacts with thiol groups (cysteine ​​residues) on proteins. ＴＭThe noodles were fluorescently labeled with TG (Titanium Dioxide Green). The noodles were then cleared using TG-mixed SoROCS (the noodles were fixed in 4% paraformaldehyde for 1.5 hours before clearing), and the gluten was visualized by optical sectioning (Fig. 2-2(c)). The linear expansions of the noodles fixed in 4% paraformaldehyde for 1.5 hours and the noodles cleared in SoROCS for 3 days after fixation were 1.30 ± 0.1 and 2.23 ± 0.08, respectively (Fig. 2-1(a)). Mechanical section images of the uncleared noodles (Fig. 2-2(b)) and optical section images of the cleared noodles (Fig. 2-2(c)) were observed using 20x and 10x objective lenses, respectively. Protein network analysis was performed on both images using AngioTool (Fig. 2-2(d, e)). AngioTool was originally developed for quantitative analysis of angiogenesis, but given that the gluten network shares many commonalities with blood vessels, it has been successfully applied to accurately quantify the structural and morphological network properties of gluten structures. By calibrating the parameters used in AngioTool with the gluten backbone structure (Figures 2(b-e)), we obtained variables characterizing the morphology of gluten structures: gluten percentage area, junction density, average gluten length, endpoint rate, and average E lacunarity. No significant differences were observed in variables between mechanical section images of uncleared noodles and optical section images of clarified noodles (P > 0.05 for each; Figures 2(f-j)). Although swelling caused by the expansion of starch granules during the clarification process using SoROCS may pose a challenge for deep imaging, we found that swelling did not significantly affect the gluten backbone structure from a morphological perspective.

[0105] Example 3: Fluorescence imaging of gluten using the present clearing reagent. For imaging with 2PEM, the optimal wavelength for two-photon excitation was sought. TG-mixed SoROCS or Alexa Fluor ＴＭ 633 C ５Noodles were prepared using maleimide (AF)-mixed SoROCS and their fluorescence intensities were measured by varying the excitation wavelength from 920 nm to 960 nm (Figs. 3-1 and 3-2(a-f)) or from 820 nm to 860 nm (Figs. 3-3 and 3-4(g-l)) at 10-nm intervals. Fluorescence intensity histograms of the resulting images showed slightly higher counts in the high-fluorescence intensity range (approximately 500-1,000 when excited at 940 nm for TG staining and approximately 1,500-2,000 when excited at 840 nm for AF staining (Figs. 3-2 and 3-4(f,l)).

[0106] Next, to investigate the image resolution for the SoROCS-processed sample obtained with 2PEM, noodles were kneaded with fluorescent microspheres (0.49 μm diameter), immobilized, and immersed in AF-mixed SoROCS. The fluorescent microspheres, located approximately 1 mm deep from the noodle surface, were excited by a 940 nm laser, and the AF-labeled gluten was excited at 840 nm and then imaged (Figures 3-5(m,n)). The experimental data (point spread function) for the fluorescence intensity I of the fluorescent microspheres was well described by a Gaussian function shown in Equation (2). Because the full-width-at-half-maximum (FWHM) value of the obtained peak was similar to the diameter of the fluorescent microspheres, we determined that the SoROCS-processed sample could be imaged with a resolution of 0.54 ± 0.03 μm (values ​​represent the mean ± SD for FWHM obtained from three independently prepared noodle samples).

[0107] After establishing the clearing and imaging conditions, we performed imaging of whole noodles using the TG-mixed SoROCS (Figures 4-1 to 4-5). Although the SoROCS induced a linear expansion of approximately 2.23 times (Figure 2-1(a)), imaging depths exceeding 2 mm could be achieved using 2PEM (Figures 4-2 and 4-3(b, c)). In contrast, existing deep imaging without sample clearing is limited to a depth of approximately 50 μm (Figures 4-2 and 4-3(b, c)). Furthermore, clearing with the SoROCS enabled observation of the entire structure of gluten formed around starch granules, each approximately 30 μm in size. To further examine the quality of the gluten fluorescence image as a function of depth from the noodle surface, we characterized the distribution of fluorescence intensity by level and calculated the variance (VAR) from a histogram according to equation (3), which indicates that the fluorescence intensity is smaller when concentrated at a specific value. The VAR value decreased as the depth from the noodle surface increased (Fig. 4-4(d)). At a depth of 0.550 mm, the VAR value was 5.69 ± 0.31 × 10 ５ and when the depth exceeds 2 mm, the VAR value is 8.98 ± 3.81 × 10 ４ However, the gluten skeletal structure was visible even at depths of more than 2 mm (Fig. 4-4 and Fig. 4-5(d-h)). To our knowledge, this is the first report of rendering the 3D structure of gluten, especially at millimeter-scale size and submicron resolution.

[0108] Example 4: Observation of Dramatic Changes in Gluten Structure Using the Present Clarification Reagent Now that imaging of entire noodles is possible using SoROCS, we demonstrated the imaging of other gluten skeletal structures. Noodles were prepared by adding gluten powder or sodium chloride to wheat flour. The purpose of adding gluten powder to wheat flour at a weight ratio of 10% or 20% was to create additional support structures between the gluten skeletal structures formed in the wheat dough. The noodles thus produced are referred to as 10%-gluten noodles and 20%-gluten noodles, respectively. As expected, compared to noodles without either addition (referred to as control noodles), the addition of gluten powder did not change the primary skeletal structure composed of gluten, but the overall structure became unclear (Figures 5-1(a) to 5-1(c)). To explain this, optical section images were binarized and the percentage of gluten in the image was calculated. The results showed that the densities of the control, 10%-gluten, and 20%-gluten noodles were 13.27±1.01%, 21.69±1.02%, and 29.60±0.68%, respectively (values ​​represent the mean±SD for N=3 independently prepared noodle samples). These results indicate that increasing the amount of gluten powder caused increased formation of the gluten network (Figure 5-1(d-i)). In contrast, the addition of sodium chloride at weight ratios of 3, 6, 9, and 12% was intended to change the gluten skeletal structure, and the noodles thus produced are referred to as 3%-NaCl noodles, 6%-NaCl noodles, 9%-NaCl noodles, and 12%-NaCl noodles, respectively. Although there are several possible explanations for the mechanism by which sodium chloride changes the gluten skeletal structure, sodium chloride likely shields the charge on the gluten, thus limiting the electrostatic repulsion between gluten polymers and causing them to aggregate. As the sodium chloride concentration increased to 3% and 6%, the gluten structure changed dramatically from a relatively uniform honeycomb network to sparse large clumps (Fig. 5-2(j, k)). Then, when the sodium chloride concentration was further increased to 9% and 12% (though such concentrations are not realistic), the aggregated large clumps dispersed (Fig. 5-2(l, m)).

[0109] Next, we conducted compression tests to investigate the effect of changes in the gluten skeletal structure on the mechanical properties (Fig. 5-3(n)). For 10%-gluten and 20%-gluten noodles, the stress-strain curves obtained when the noodles were compressed with a V-shaped plunger shifted upward. Conversely, the stress-strain curves for noodles containing sodium chloride shifted downward. These results suggest that reinforcement of the gluten skeletal structure increases structural strength, whereas severing the gluten network decreases it. In particular, dramatic changes in the primary gluten skeletal structure significantly affected the mechanical properties of wheat dough. These results are consistent with previous studies on the physicochemical and structural changes of gluten. High-resolution 3D reconstructions using SoROCS may facilitate structural insights into the mechanical properties of wheat dough.

[0110] As a further demonstration of the imaging method using SoROCS, gluten volume was quantified using 3D measurements. First, the fluorescence intensity was binarized for 3D images of gluten in 12%-NaCl noodles (Fig. 6-1(a) and (b)). Each gluten mass, three-dimensionally isolated from the others, is represented by a different color in Fig. 6-1(b). In particular, the spatial relationship of each gluten mass can be easily observed from all angles and directions (Fig. 6-1(c)). Next, when the gluten volume distribution was analyzed, it was found that a small number of large gluten masses accounted for the majority of the entire product (Fig. 6-2(d)). Information on volume distribution, which can only be obtained by this imaging method using SoROCS, will be useful for improving the industrial performance of wheat foods.

[0111] Example 5: Imaging Using Confocal Laser Scanning Microscopy. Although 2PEM is a powerful tool for deep imaging, the high cost of the equipment makes it unaffordable for all researchers. Therefore, to evaluate imaging performance, we demonstrated confocal laser scanning microscope (CLSM) observation of SoROCS-treated noodles. Images of clarified noodles were obtained using CLSM (Figures 7-1 and 7-2(a, b)). The xy image at a depth of 250 μm from the noodle surface revealed a clear gluten structure (Figure 7-3(c)). To examine the quality of the gluten fluorescence images, VAR was calculated from histograms for xy images at several depths (Figures 7-3 and 7-4(c-e)). Although no significant differences were observed, the VAR values ​​tended to decrease with increasing depth from the noodle surface (Figure 7-4(e)). The working distance of the objective lens used in this example limits the imaging depth to 600 μm. However, considering that gluten surrounding starch granules of approximately 30 μm size forms a honeycomb-like network, SoROCS can be imaged using CLSM as well as 2PEM.

[0112] Example 6: Clarification of Wheat Noodles Using the Clarifying Reagents of the Present Invention 2 Wheat noodle samples were treated in the same manner as in Example 1 using the following clarifying reagents: 4 M sodium 2-hydroxybenzoate (sodium salicylate) solution (SoROCS), 4 M sodium 3-hydroxybenzoate solution, 3.5 M sodium 4-hydroxybenzoate solution, 3 M sodium benzoate solution, 4 M lithium salicylate solution, 3.5 M disodium phthalate solution, 4 M catechol solution, 4 M resorcinol solution, and 4 M pyrogallol solution. Clarification was evaluated by treating the noodle samples in the same manner as in Example 1. The immobilized noodle samples were immersed in each clarifying reagent at 30°C for 3 days, and their opacity was measured. As controls, the opacity of a noodle sample immediately after immobilization and a noodle sample immersed in D-PBS instead of the clarifying reagent were measured. As a result, the samples were clarified regardless of the clarifying reagent used. The opacity measurement results are shown in Figure 9. The values ​​in the figure are the mean ± SD of three independent experiments.

[0113] Example 7: Clarification of various samples using the present clearing reagent. A 4 M sodium salicylate solution (SoROCS) was used as the clearing reagent to conduct a clearing experiment on various samples. Rice, pork, fish (salmon and sea bream), and vegetables (radish sprouts) were used as samples. Each sample was pretreated appropriately and then subjected to a clearing treatment.

[0114] Rice was pretreated by adjusting its moisture content to approximately 22% wt / wt (dry basis) or higher through gas-phase adsorption, then immobilized in 4% PFA / PBS and subsequently treated with 1 wt% cellulase and 0.5 wt% pectolyase. Pork, fish (salmon and sea bream), and vegetables (radish sprouts) were cut to appropriate sizes and pretreated with immobilization in 4% PFA / PBS, followed by degreasing and decolorization. Each pretreated sample was then immersed in a clarifying reagent. As a result, all samples were clarified.

[0115] Example 8: Testing of Decolorizing and Defatting Agents. Pasta samples made from durum wheat were fixed in 4% (wt / wt) PFA / PBS at 30°C for 1.5 hours and then washed twice in D-PBS. They were then soaked in 1,2-hexanediol or water (control) for 5 days. Both samples were then soaked in 4 M sodium 2-hydroxybenzoate (sodium salicylate) solution (SoROCS) for 3 days. The opacity of the water-treated pasta sample was 1.079 ± 0.002. Treatment with 1,2-hexanediol successfully removed the carotenoid pigments in durum wheat, achieving significant clarification with an opacity of 1.041 ± 0.002. Values ​​are the mean ± SD of three independent experiments.

[0116] Wheat noodles fried in vegetable oil were fixed in 4% (wt / wt) PFA / PBS at 30°C for 1.5 hours and then washed twice in D-PBS. Subsequently, samples were immersed in limonene or water (control) for 5 days. Both samples were then immersed in 4 M sodium 2-hydroxybenzoate (sodium salicylate) solution (SoROCS) for 3 days. The opacity of the control pasta sample treated with water was 1.120 ± 0.006, whereas limonene treatment removed the oil from the fried noodles, achieving significant clarification with an opacity of 1.089 ± 0.004. Values ​​are the mean ± SD of three independent experiments.

Claims

1. A reagent for clarifying carbohydrate- or protein-containing materials comprising a compound selected from the group consisting of benzene carboxylic acid compounds, hydroxybenzene compounds, benzene sulfonic acid compounds, homosulfamine compounds, and benzene sulfohydroxamic acid compounds.

2. 1. A benzene carboxylic acid compound having the formula I: 【Chemistry 1】 [In the formula, R １ ~R ５ are independently a hydrogen atom, a hydroxy group, an amino group, a nitro group, a carboxyl group, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 alkoxy group, or a halogen atom, wherein the C1-C4 alkyl group, the C2-C4 alkenyl group, the C2-C4 alkynyl group, and the C1-C4 alkoxy group are optionally substituted with a halogen atom; M is a monovalent or divalent metal atom or a hydrogen atom; Here, R １ ~R ５ When any of R is a carboxyl group, the carboxyl group may form a salt with a monovalent metal atom, or R １ Or R ５ is a carboxyl group and M is a divalent metal atom, the carboxyl group may form a salt with M.

2. The reagent for clarifying a carbohydrate- or protein-containing material according to claim 1, which is a compound represented by the following formula:

3. R １ ~R ５ The reagent according to claim 2, wherein one or two of the following are hydroxy groups:

4. 3. The reagent according to claim 2, wherein the benzenecarboxylic acid compound is a salt of benzoic acid, 2-, 3-, or 4-hydroxybenzoic acid, or phthalic acid with a monovalent or divalent metal atom, or a hydrate thereof.

5. The reagent according to any one of claims 2 to 4, wherein M is lithium, sodium, potassium, or calcium.

6. 1. The method of claim 1, further comprising the step of: 【Chemistry 2】 [In the formula, R １ ~R ５ are independently a hydrogen atom, a hydroxy group, an amino group, a nitro group, a sulfonic acid group, a sulfonamide group, an acetamide group, a 1-hydroxy-2-(methylamino)ethyl group, a methoxycarbonyl group, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 alkoxy group, or a halogen atom, and the C1-C4 alkyl group, the C2-C4 alkenyl group, the C2-C4 alkynyl group, and the C1-C4 alkoxy group may be substituted with a halogen atom.] 2. The reagent for clarifying a carbohydrate- or protein-containing material according to claim 1, which is a compound represented by the following formula:

7. R １ ~R ５ The reagent according to claim 6, wherein one or two of the following are hydroxy groups:

8. 7. The reagent according to claim 6, wherein the hydroxybenzene compound is catechol, resorcinol, or pyrogallol, or a hydrate thereof.

9. 1. The method of claim 1, further comprising the step of: 【Chemistry 3】 [In the formula, R １ ~R ５ are independently a hydrogen atom, a hydroxyl group, a carboxyl group, an amino group, a nitro group, a sulfonic acid group, an aldehyde group, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C1-C4 alkoxy group, or a halogen atom, wherein the C1-C4 alkyl group, the C2-C4 alkenyl group, the C2-C4 alkynyl group, and the C1-C4 alkoxy group are optionally substituted with a halogen atom; M is a monovalent or divalent metal atom or a hydrogen atom; Here, R １ ~R ５ When any of R is a sulfonic acid group, the sulfonic acid group may form a salt with a monovalent metal atom, or １ Or R ５ is a sulfonic acid group and M is a divalent metal atom, the sulfonic acid group may form a salt with M.

2. The reagent for clarifying a carbohydrate- or protein-containing material according to claim 1, which is a compound represented by the following formula:

10. R １ ~R ５ The reagent according to claim 9 , wherein one or two of the following are hydroxy groups:

11. 11. The reagent of claim 9 or 10, wherein M is lithium, sodium, potassium, calcium, or nickel.

12. The reagent according to any one of claims 1 to 11, further comprising a fluorescent dye.

13. A method for clarifying a carbohydrate- or protein-containing material, comprising treating the carbohydrate- or protein-containing material with a reagent according to any one of claims 1 to 12.

14. 14. The method of claim 13, comprising pretreating the material to degrease and / or decolorize it before treating with the reagent.

15. 15. The method of claim 14, wherein the material is pretreated with a decolorizing agent comprising 1,2-hexanediol and / or a degreasing agent comprising limonene.

16. A method for visualising carbohydrates or proteins in a sugar material, comprising making the material transparent by the method according to any one of claims 13 to 15.

17. 17. The method of claim 16, further comprising treating the material with a fluorescent dye.

18. 18. The method of claim 16 or 17, further comprising imaging.

19. The method according to any one of claims 16 to 18, wherein the material is a flour-based food material or a flour-processed food, and gluten proteins are visualized.

20. A kit for clarifying carbohydrate- or protein-containing materials, comprising the reagent according to any one of claims 1 to 12.

21. 21. The kit of claim 20, further comprising one or more selected from the group consisting of a fixative, a degreasing agent, and a decolorizing agent.

22. 22. The kit of claim 21, further comprising a decolorizing agent comprising 1,2-hexanediol and / or a degreasing agent comprising limonene.

23. A kit for visualizing carbohydrates or proteins in a material, comprising: (a) a reagent according to any one of claims 1 to 11, and a fluorescent dye; or (b) The reagent according to claim 12 Including the kit.

24. 24. The kit of claim 23, further comprising one or more selected from the group consisting of a fixative, a degreasing agent, and a decolorizing agent.

25. 25. The kit of claim 24, further comprising a decolorizing agent comprising 1,2-hexanediol and / or a degreasing agent comprising limonene.

26. 1. An apparatus for visualizing carbohydrates or proteins in a material, comprising: (a) a reagent according to any one of claims 1 to 11, a fluorescent dye, and an optical microscope; or (b) the reagent according to claim 12, and an optical microscope.

13. An apparatus comprising:

27. A clarified carbohydrate- or protein-containing material comprising a reagent according to any one of claims 1 to 12.

Images