Tobacco cell wall macromolecular three-dimensional mode compound standard substance as well as preparation method and application thereof
By preparing high-layer-by-layer discrimination and resolution, the three-dimensional mode compound standard for large-molecule cell walls of tobacco leaf is solved, and the accurate analysis and processing optimization of large-molecule components of tobacco leafs is achieved.
Patent Information
- Application Number
- CN202510770644.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-10
- Publication Date
- 2025-09-05
AI Technical Summary
Traditional fluorescence staining method cannot provide three-dimensional data on cell walls of tobacco leaves when analyzing dried dead cells, resulting in a decrease in specificity and sensitivity of cell wall structure and composition analysis, and cannot accurately reveal the distribution of cell wall macromolecules and their interactions in primary tobacco leaves.
Using a specific preparation method, three-dimensional mode compound standards with high layer-by-layer discrimination and resolution were prepared by gradually modifying and mixing the macromolecule dispersion of cell walls of tobacco leaves to simulate the natural cross-linking structure of cell walls of large molecules in primary tobacco leaves.
It provides metrable and regulating three-dimensional mode compounds, which can simulate the three-dimensional internal microscopic components and natural cross-linked structure of tobacco leaf cell walls, support quantitative analysis and structural analysis of tobacco leaf macromolecular components, and optimize the tobacco leaf processing process.
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Figure CN120594576A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of tobacco leaf analysis, and in particular to a tobacco leaf cell wall macromolecular three-dimensional model compound standard product, a preparation method and application thereof. Background Art
[0002] The tobacco leaf cell wall is composed of macromolecules such as cellulose, hemicellulose, lignin, and pectin, and has a complex spatial structure. The structure of the tobacco leaf cell wall directly affects the burning properties and taste of the tobacco leaf. Accurate three-dimensional structural modeling can help optimize tobacco processing.
[0003] For example, model construction can be used to improve the hygroscopicity and permeability of tobacco leaves, and even to regulate the aroma components of tobacco. This not only improves tobacco processing efficiency and the quality of finished products, but also provides the tobacco industry with more refined production strategies. However, traditional fluorescent staining methods have limitations when analyzing dry dead cells and are unable to provide three-dimensional cell wall data. For freshly cured tobacco leaves, the structure and composition of the cell wall may have undergone irreversible changes, making it difficult for fluorescent dyes to effectively bind or label. This is because after cell death, the integrity of the cell membrane and the chemical properties of the cell wall may change, resulting in reduced permeability of the dye or reduced affinity of certain components in the cell wall, thereby affecting the staining effect. In addition, certain biomolecules in dead cells may be degraded or inactivated, reducing the specificity and sensitivity of the staining reaction.
[0004] Therefore, developing methods for analyzing and 3D-constructing macromolecular components in freshly flue-cured tobacco leaf cell walls will help more accurately reveal the distribution and interactions of these components within the cell wall. However, before developing these systematic analytical techniques, it is necessary to generate tobacco leaf cell wall macromolecular model compounds as a standard baseline data source. Currently, there are no reports on the preparation of tobacco leaf cell wall macromolecular model compound standards, and the development and application of such methods is necessary. Summary of the Invention
[0005] To address the above technical issues, the present invention provides a three-dimensional model compound standard for tobacco cell wall macromolecules, as well as its preparation method and application. Through a specific preparation method, the resulting standard exhibits high layer-by-layer differentiation and resolution. By simulating the naturally cross-linked structure of cell wall macromolecules in freshly flue-cured tobacco leaves, it provides a foundation for quantitative and structural analysis of tobacco leaf macromolecules.
[0006] To achieve this object, the present invention adopts the following technical solutions:
[0007] In a first aspect, the present invention provides a method for preparing a three-dimensional model compound standard of a tobacco cell wall macromolecule, the preparation method comprising any one of the methods for preparing binary or higher standard products; the tobacco cell wall macromolecule comprises cellulose, hemicellulose, lignin or pectin;
[0008] The preparation method of binary standards includes the following steps:
[0009] Mixing any one of the tobacco cell wall macromolecules with a solvent to obtain a dispersion; freezing and drying the dispersion to obtain a monobasic standard; hydrophobically modifying the monobasic standard to obtain a modified sample; mixing any one of the tobacco cell wall macromolecules other than the tobacco cell wall macromolecules contained in the monobasic standard with a solvent to obtain a dispersion; mixing the modified sample and the dispersion, and then freezing and drying to obtain a binary standard;
[0010] The preparation method of the ternary standard comprises the following steps:
[0011] The binary standard is hydrophobically modified to obtain a modified sample; any one of the tobacco cell wall macromolecules other than the tobacco cell wall macromolecules contained in the monobasic standard and the binary standard is mixed with a solvent to obtain a dispersion; the modified sample and the dispersion are mixed, and then frozen and dried to obtain a ternary standard;
[0012] The preparation method of the quaternary standard comprises the following steps:
[0013] The ternary standard is hydrophobically modified to obtain a modified sample; any one of the tobacco cell wall macromolecules other than the tobacco cell wall macromolecules included in the monobasic standard, the dibasic standard, and the ternary standard is mixed with a solvent to obtain a dispersion; the modified sample and the dispersion are mixed, and then frozen and dried to obtain a quaternary standard;
[0014] The preparation method of five-element and above standard samples is similar, and the steps of hydrophobic modification, preparing dispersion, mixing the modified sample and dispersion, freezing, and drying are repeated.
[0015] The preparation method provided by the present invention can be used to prepare binary or higher-level standards, for example, binary, ternary, quaternary, quinary, and hexaary standards. The number of quinary species refers to the number of tobacco cell wall macromolecules present in the standard. For example, binary refers to the presence of two tobacco cell wall macromolecules in the standard, and so on.
[0016] The present invention does not limit the compounding order of tobacco cell wall macromolecules in the preparation process of the multi-component standard. For example, a mono-component standard of hemicellulose is first prepared, and then compounded with cellulose to obtain a hemicellulose / cellulose binary standard; then compounded with lignin to obtain a hemicellulose / cellulose / lignin ternary standard; and then compounded with pectin to obtain a hemicellulose / cellulose / lignin / pectin quaternary standard.
[0017] The three-dimensional model compound standard provided by the present invention can be compounded with multiple macromolecules through gradual modification and mixing with a dispersion of tobacco cell wall macromolecules. The resulting standard has an overall gas-condensed structure with numerous internal pores, and the internal structure gradually thickens during the compounding process. Through a specific preparation method, the internal structure of the resulting standard has high layer-by-layer differentiation and resolution. By simulating the naturally cross-linked structure of cell wall macromolecules in freshly cured tobacco leaves, this standard provides a foundation for quantitative and structural analysis of tobacco leaf macromolecular components.
[0018] The three-dimensional model compound standards prepared by this method can not only be used to analyze dried tobacco leaves, but also simulate their three-dimensional internal microscopic components and natural cross-linked structure. This provides a foundation for the development of technologies for analyzing the components of freshly cured tobacco leaves and helps evaluate the cell wall properties of different tobacco varieties. This method provides important support for in-depth research and practical applications in tobacco science, not only optimizing tobacco production and processing, but also promoting technological innovation in the field of plant cell walls.
[0019] The cellulose, hemicellulose, lignin or pectin used in the present invention can be extracted from primary flue-cured tobacco leaves according to conventional methods, or commercially available products can be used. The extraction method can be, for example, acid extraction or alkaline extraction.
[0020] Preferably, the solvents independently include any one of water, methanol or ethanol, or a combination of at least two of them.
[0021] Preferably, the concentration of tobacco cell wall macromolecules in the dispersion is independently 5 to 70 wt% (for example, 5 wt%, 10 wt%, 20 wt%, 30 wt%, 40 wt%, 50 wt%, 60 wt%, 70 wt%, etc.).
[0022] Preferably, the modified sample and the dispersion are mixed for an independent period of 2 to 36 hours (eg, 2 hours, 6 hours, 12 hours, 16 hours, 24 hours, 30 hours, 36 hours, etc.).
[0023] Preferably, the modified sample and the dispersion are mixed by immersing the modified sample in the dispersion.
[0024] The present invention strictly controls and precisely modulates the concentration and mixing time of the tobacco leaf cell wall macromolecular components during the preparation process, thereby enabling the preparation of quantifiable and controllable three-dimensional model compounds. By simulating the natural cross-linked structure of the cell wall macromolecular components in freshly cured tobacco leaves, the present invention provides a basis for the quantitative and structural analysis of the tobacco leaf macromolecular components.
[0025] Preferably, the freezing temperature is independently -30 to -5°C (for example, -30°C, -25°C, -20°C, -15°C, -10°C, -5°C, etc.).
[0026] Preferably, the drying temperature is independently -30 to -20°C (for example, -30°C, -28°C, -25°C, -22°C, -20, etc.).
[0027] Preferably, at least one of the freezing or drying is subjected to an orientation operation so that the standard product has any one of the shapes of a cube, a cuboid or a cylinder.
[0028] In the present invention, when freezing and drying occur simultaneously, it is necessary to select one of them for directional operation.
[0029] Preferably, the modifiers used for the hydrophobic modification independently include any one of silane, phenolic compounds or terpenoid compounds, or a combination of at least two of them.
[0030] Preferably, the mass ratio of the standard to the modifier used for hydrophobic modification is independently 1:(0.5-1) (for example, it can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, etc.).
[0031] Preferably, the silane comprises gamma-aminopropyltriethoxysilane.
[0032] Preferably, the phenolic compound includes any one or a combination of at least two of octylphenol polyoxyethylene ether, methylparaben, propylparaben, tannic acid, sulfonated lignin grafted with long-chain alkyl, or bisphenol A diglycidyl ether.
[0033] Preferably, the terpenoid compound comprises trimethylsilyllimonene.
[0034] Preferably, the modifier used for the hydrophobic modification includes a combination of octylphenol polyoxyethylene ether and bisphenol A diglycidyl ether.
[0035] Preferably, the mass ratio of the combination of octylphenol polyoxyethylene ether and bisphenol A diglycidyl ether is 1:(0.5-1) (for example, it can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, etc.).
[0036] In the present invention, a better layering effect can be achieved by compounding the modifier, and the layer-by-layer distinction and resolution are higher.
[0037] Preferably, the hydrophobic modification is carried out in a solvent.
[0038] Preferably, the solvents independently include any one of methanol, acetone or water, or a combination of at least two of them.
[0039] Preferably, the temperature of the hydrophobic modification is independently 20-40°C (for example, 20°C, 25°C, 30°C, 35°C, 40°C, etc.), and the time of the hydrophobic modification is independently 0.5-4h (for example, 0.5h, 1h, 2h, 3h, 4h, etc.).
[0040] In a second aspect, the present invention provides a standard product of a three-dimensional model compound of a tobacco cell wall macromolecule prepared by the preparation method described in the first aspect.
[0041] In a third aspect, the present invention provides a use of the tobacco leaf cell wall macromolecular three-dimensional model compound standard as described in the second aspect in tobacco leaf analysis or simulation.
[0042] Compared with the prior art, the present invention has at least the following beneficial effects:
[0043] The present invention successfully provides a quantifiable and controllable three-dimensional model compound of tobacco cell wall macromolecules through a strictly controlled and precisely modulated preparation method. The internal structure of the three-dimensional model compound has a high layer-by-layer differentiation and resolution. The three-dimensional model compound prepared by the present invention can not only be used for quantitative analysis of tobacco leaves after initial curing, but also simulate its three-dimensional internal microscopic components and natural cross-linked structure, providing a basis for the correction and development of quantitative and structural analysis of macromolecular components of different varieties of tobacco leaves, and promoting technological innovation in the field of plant cell walls. In addition, the preparation process of the present invention is simple, low-cost, and easy to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS
[0044] Figure 1 This is a three-dimensional structure self-set component design diagram of the tobacco cell wall macromolecular three-dimensional model compound standard (1-1) prepared in Example 1.
[0045] Figure 2 This is a multi-hole design diagram of the three-dimensional structural section of the tobacco cell wall macromolecular three-dimensional model compound standard (1-1) prepared in Example 1.
[0046] Figure 3 This is a physical picture of the tobacco leaf cell wall macromolecular three-dimensional model compound standard (3-5) prepared in Example 3.
[0047] Figure 4 This is a scanning electron microscope (SEM) image of the internal microstructure of the tobacco leaf cell wall macromolecular three-dimensional model compound standard (5-5) prepared in Example 5. DETAILED DESCRIPTION
[0048] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.
[0049] Example 1
[0050] This embodiment provides a method for preparing a standard substance of a three-dimensional model compound of a tobacco cell wall macromolecule, comprising the following steps:
[0051] (1) Cellulose was ultrasonically and stirred in water to a concentration of 10 wt %, directionally frozen at -20°C, and then freeze-dried at -30°C in a vacuum to obtain a three-dimensional model compound monobasic standard of pure cellulose (designated as Standard 1-1). The standard was cylindrical, 6 cm in height, and 3 cm in diameter. The microfibrils inside the standard had a diameter of 3-5 μm and a length of 4-8 μm.
[0052] (2) The cellulose three-dimensional columnar model compound standard 1-1 was hydrophobically modified using γ-aminopropyltriethoxysilane, specifically, the standard 1-1, γ-aminopropyltriethoxysilane and methanol were mixed at a dosage ratio of 1 g:1 g:20 mL, treated at 30°C for 3 h, and then immersed in a hemicellulose aqueous dispersion, a lignin aqueous dispersion and a pectin aqueous dispersion (concentration of 35 wt%) for 7 h, and then frozen and dried (parameters were all referred to step (1)), to obtain cellulose / hemicellulose, cellulose / lignin and cellulose / pectin three-dimensional model compound binary standards (named standard 1-2, standard 1-3, standard 1-4, respectively); all were cylindrical, 6 cm in height, 3 cm in diameter, and 40 to 80 μm in thickness layer by layer;
[0053] (3) The cellulose three-dimensional columnar model compound standard 1-1 was hydrophobically modified (for specific steps, refer to step (2)), immersed in a hemicellulose aqueous dispersion (concentration of 35 wt%) for 10 h, and then frozen and dried (for parameters, refer to step (1)) to obtain a cellulose / hemicellulose three-dimensional model compound standard; the obtained standard was hydrophobically modified (for specific steps, refer to step (2)), immersed in a lignin aqueous dispersion (concentration of 35 wt%) for 10 h, and then frozen and dried (for parameters, refer to step (1)) to obtain a cellulose / hemicellulose / lignin three-dimensional model compound standard; the obtained standard was hydrophobically modified (for specific steps, refer to step (2)), immersed in a pectin aqueous dispersion (concentration of 35 wt%) for 10 h, and then frozen and dried (for parameters, refer to step (1)) to obtain a cellulose / hemicellulose / lignin / fructose three-dimensional model compound quaternary standard (named standard 1-5), with a layer thickness of 50 to 100 μm.
[0054] The three-dimensional structure of the tobacco cell wall macromolecular three-dimensional model compound standard 1-1 prepared in Example 1 is designed by setting the components as shown in the figure. Figure 1 shown.
[0055] The porous design diagram of the three-dimensional structure cross section of the tobacco cell wall macromolecular three-dimensional model compound standard 1-1 prepared in Example 1 is as follows: Figure 2 shown.
[0056] Example 2
[0057] This embodiment provides a method for preparing a standard substance of a three-dimensional model compound of a tobacco cell wall macromolecule, comprising the following steps:
[0058] (1) Cellulose was ultrasonically and stirred in water to a concentration of 10 wt %, rapidly frozen in liquid nitrogen at -25°C, and then directionally dried at -20°C to obtain a three-dimensional model compound standard of pure cellulose (designated as standard 2-1). The standard was a cube with a side length of 5 cm. The diameter of the microfibrils inside the standard was 1-3 μm and the length was 5-10 μm.
[0059] (2) The cellulose three-dimensional columnar model compound standard 2-1 was hydrophobically modified using octylphenol polyoxyethylene ether, specifically, the standard 2-1, octylphenol polyoxyethylene ether and methanol were mixed at a dosage ratio of 1 g:0.5 g:15 mL, treated at 25°C for 1 hour, and then immersed in a hemicellulose aqueous dispersion, a lignin aqueous dispersion and a pectin aqueous dispersion (concentration of 40 wt%) for 10 hours, and then frozen and dried (parameters were all referred to step (1)), respectively, to obtain cellulose / hemicellulose, cellulose / lignin and cellulose / pectin three-dimensional model compound standard products (named standard 2-2, standard 2-3, standard 2-4 in sequence); all were cubes with a side length of 5 cm and a layer thickness of 50 μm;
[0060] (3) The cellulose three-dimensional columnar model compound standard 2-1 was hydrophobically modified (for specific steps, refer to step (2)), immersed in a hemicellulose aqueous dispersion (concentration of 40 wt%) for 12 h, and then frozen and dried (for parameters, refer to step (1)) to obtain a cellulose / hemicellulose three-dimensional model compound standard; the obtained standard was hydrophobically modified (for specific steps, refer to step (2)), immersed in a lignin aqueous dispersion (concentration of 40 wt%) for 12 h, and then frozen and dried (for parameters, refer to step (1)) to obtain a cellulose / hemicellulose / lignin three-dimensional model compound standard; the obtained standard was hydrophobically modified (for specific steps, refer to step (2)), immersed in a pectin aqueous dispersion (concentration of 40 wt%) for 12 h, and then frozen and dried (for parameters, refer to step (1)) to obtain a cellulose / hemicellulose / lignin / fructose three-dimensional model compound quaternary standard (named standard 2-5), with a layer thickness of 65 μm.
[0061] Example 3
[0062] This embodiment provides a method for preparing a standard substance of a three-dimensional model compound of a tobacco cell wall macromolecule, comprising the following steps:
[0063] (1) Cellulose was ultrasonically and stirred in water to a concentration of 22 wt %, directionally frozen at -20°C, and then dried at -25°C to obtain a three-dimensional model compound standard 3-1 of pure cellulose, which was a cube with a side length of 6 cm. The diameter of the microfibrils inside the standard was 1-2 μm and the length was 2-8 μm.
[0064] (2) The cellulose three-dimensional columnar model compound standard 3-1 was hydrophobically modified using γ-aminopropyltriethoxysilane (refer to Example 1), and then immersed in a hemicellulose aqueous dispersion, a lignin aqueous dispersion, and a pectin aqueous dispersion (concentration of 25 wt%) for 12 h, and then frozen and dried (parameters were all referred to step (1)) to obtain cellulose / hemicellulose, cellulose / lignin, and cellulose / pectin three-dimensional model compound standard products (named standard product 3-2, standard product 3-3, and standard product 3-4, respectively), which were cubes with a side length of 6 cm and a layer thickness of 55 μm;
[0065] (3) The cellulose three-dimensional columnar model compound standard 3-1 was hydrophobically modified (for specific steps, refer to step (2)), immersed in a hemicellulose aqueous dispersion (concentration of 25 wt%) for 15 h, and then frozen and dried (for parameters, refer to step (1)) to obtain a cellulose / hemicellulose three-dimensional model compound standard; the obtained standard was hydrophobically modified (for specific steps, refer to step (2)), immersed in a lignin aqueous dispersion (concentration of 25 wt%) for 15 h, and then frozen and dried (for parameters, refer to step (1)) to obtain a cellulose / hemicellulose / lignin three-dimensional model compound standard; the obtained standard was hydrophobically modified (for specific steps, refer to step (2)), immersed in a pectin aqueous dispersion (concentration of 25 wt%) for 15 h, and then frozen and dried (for parameters, refer to step (1)) to obtain a cellulose / hemicellulose / lignin / fructose three-dimensional model compound quaternary standard (named standard 3-5), with a layer thickness of 110 μm.
[0066] The actual substance of the tobacco leaf cell wall macromolecular three-dimensional model compound standard 3-5 prepared in Example 3 is as follows Figure 3 shown.
[0067] Example 4
[0068] This embodiment provides a method for preparing a standard substance of a three-dimensional model compound of a tobacco cell wall macromolecule, comprising the following steps:
[0069] (1) Cellulose was ultrasonically and stirred dispersed in ethanol and water at a volume ratio of 1:1, with a dispersion concentration of 30 wt %. The dispersion was rapidly frozen in liquid nitrogen at -20°C and then dried at -25°C to obtain a three-dimensional model compound standard of pure cellulose (designated as standard 4-1). The standard was a rectangular parallelepiped with a length of 6 cm and a width of 3 cm. The microfibers inside the standard had a diameter of 4 μm and a length of 8-10 μm.
[0070] (2) The cellulose three-dimensional columnar model compound standard 4-1 was hydrophobically modified using trimethylsilyllimonene, specifically, the standard 4-1, trimethylsilyllimonene and acetone were mixed at a dosage ratio of 1 g:0.8 g:18 mL, treated at 40°C for 1 h, and then immersed in a hemicellulose aqueous dispersion, a lignin aqueous dispersion and a pectin aqueous dispersion (concentration of 25 wt%) for 9 h, and then frozen and dried (parameters were all referred to step (1)), to obtain cellulose / hemicellulose, cellulose / lignin and cellulose / pectin three-dimensional model compound standards (named standard 4-2, standard 4-3, standard 4-4, respectively), rectangular parallelepiped, length 6 cm, width 3 cm, layer thickness 85 μm;
[0071] (3) The cellulose three-dimensional columnar model compound standard 4-1 was hydrophobically modified (for specific steps, refer to step (2)), immersed in a hemicellulose aqueous dispersion (concentration of 25 wt%) for 24 h, and then frozen and dried (for parameters, refer to step (1)) to obtain a cellulose / hemicellulose three-dimensional model compound standard; the obtained standard was hydrophobically modified (for specific steps, refer to step (2)), immersed in a lignin aqueous dispersion (concentration of 25 wt%) for 24 h, and then frozen and dried (for parameters, refer to step (1)) to obtain a cellulose / hemicellulose / lignin three-dimensional model compound standard; the obtained standard was hydrophobically modified (for specific steps, refer to step (2)), immersed in a pectin aqueous dispersion (concentration of 25 wt%) for 24 h, and then frozen and dried (for parameters, refer to step (1)) to obtain a cellulose / hemicellulose / lignin / fructose three-dimensional model compound quaternary standard (named standard 4-5), with a layer thickness of 200 μm.
[0072] Example 5
[0073] This embodiment provides a method for preparing a standard substance of a three-dimensional model compound of a tobacco cell wall macromolecule, comprising the following steps:
[0074] (1) Cellulose was ultrasonically and stirred in water to a concentration of 30 wt %, directionally frozen at -25°C, and then naturally dried at -20°C to obtain a three-dimensional model compound standard of pure cellulose (designated as standard 5-1). The standard was cylindrical, 6 cm in height and 4 cm in diameter. The microfibrils inside the standard had a diameter of 1 to 4 μm and a length of 6 to 12 μm.
[0075] (2) The cellulose three-dimensional columnar model compound standard was hydrophobically modified using γ-aminopropyltriethoxysilane (refer to Example 1), and then immersed in a hemicellulose aqueous dispersion, a lignin aqueous dispersion, and a pectin aqueous dispersion (concentration of 50 wt%) for 6 h, and then frozen and dried (parameters were all referred to step (1)), to obtain cellulose / hemicellulose, cellulose / lignin, and cellulose / pectin three-dimensional model compound standard products (named standard product 5-2, standard product 5-3, and standard product 5-4, respectively), which were cylindrical, 6 cm in height, 4 cm in diameter, and 120 μm in thickness layer by layer;
[0076] (3) The cellulose three-dimensional columnar model compound standard 5-1 was hydrophobically modified (for specific steps, refer to step (2)), immersed in a hemicellulose aqueous dispersion (concentration of 50 wt%) for 18 h, and then frozen and dried (for parameters, refer to step (1)) to obtain a cellulose / hemicellulose three-dimensional model compound standard; the obtained standard was hydrophobically modified (for specific steps, refer to step (2)), immersed in a lignin aqueous dispersion (concentration of 50 wt%) for 18 h, and then frozen and dried (for parameters, refer to step (1)) to obtain a cellulose / hemicellulose / lignin three-dimensional model compound standard; the obtained standard was hydrophobically modified (for specific steps, refer to step (2)), immersed in a pectin aqueous dispersion (concentration of 50 wt%) for 18 h, and then frozen and dried (for parameters, refer to step (1)) to obtain a cellulose / hemicellulose / lignin / fructose three-dimensional model compound quaternary standard (named standard 5-5), with a layer thickness of 150 μm.
[0077] The internal microstructure of the tobacco cell wall macromolecular three-dimensional model compound standard 5-5 prepared in Example 5 characterized by scanning electron microscopy (SEM) is as follows: Figure 4 shown.
[0078] Example 6
[0079] This embodiment provides a method for preparing a standard product of a three-dimensional model compound of a tobacco cell wall macromolecule. The only difference from Example 2 is that the modifier in steps (2) and (3) is an equal amount of bisphenol A diglycidyl ether. The rest is based on Example 2, and standard products 6-1, 6-2, 6-3, 6-4, and 6-5 are obtained, respectively.
[0080] Example 7
[0081] This embodiment provides a method for preparing a standard product of a three-dimensional model compound of a tobacco cell wall macromolecule. The method differs from Example 2 only in that the modifiers in steps (2) and (3) are octylphenol polyoxyethylene ether and bisphenol A diglycidyl ether in a mass ratio of 1:1, and the total amount of the modifier remains unchanged. Other steps are similar to those in Example 2, and standard products 7-1, 7-2, 7-3, 7-4, and 7-5 are obtained, respectively.
[0082] Comparative Example 1
[0083] This comparative example provides a method for preparing a standard product of a three-dimensional model compound of a tobacco cell wall macromolecule. The method differs from Example 2 only in that no modification is performed in steps (2) and (3). The other steps are similar to those in Example 2, and standard products 8-1, 8-2, 8-3, 8-4, and 8-5 are obtained, respectively.
[0084] Test Case
[0085] Each sample was tested, scanned, and three-dimensionally reconstructed using micron CT. The quality of the sample was determined by the degree of differentiation and resolution of macromolecules within the three-dimensional model. The higher the layer-by-layer differentiation and resolution, the better. The results are shown in Table 1.
[0086] Table 1
[0087]
[0088]
[0089] Test results:
[0090] (1) It can be seen from Examples 1 to 7 that the specific preparation method of the present invention can achieve high layer-by-layer differentiation and resolution within the three-dimensional model compound standard.
[0091] (2) By comparing Example 7 with Examples 2 and 6, it can be seen that the present invention further optimizes the modifier formula and can achieve higher layer-by-layer differentiation and resolution of the standard product by compounding the two modifiers.
[0092] (3) By comparing Example 2 with Comparative Example 1, it can be seen that when no modification treatment is performed, the layer-by-layer distinction and resolution of the standard product are significantly reduced.
[0093] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.
Claims
1. A method for preparing a standard product of a three-dimensional model compound of a tobacco cell wall macromolecule, characterized in that: The preparation method includes any one of the preparation methods of binary and above standard products; the tobacco leaf cell wall macromolecules include cellulose, hemicellulose, lignin or pectin; The preparation method of binary standards includes the following steps: Mixing any one of the tobacco cell wall macromolecules with a solvent to obtain a dispersion; freezing and drying the dispersion to obtain a monobasic standard; hydrophobically modifying the monobasic standard to obtain a modified sample; mixing any one of the tobacco cell wall macromolecules other than the tobacco cell wall macromolecules contained in the monobasic standard with a solvent to obtain a dispersion; mixing the modified sample and the dispersion, and then freezing and drying to obtain a binary standard; The preparation method of the ternary standard comprises the following steps: The binary standard is hydrophobically modified to obtain a modified sample; any one of the tobacco cell wall macromolecules other than the tobacco cell wall macromolecules contained in the monobasic standard and the binary standard is mixed with a solvent to obtain a dispersion; the modified sample and the dispersion are mixed, and then frozen and dried to obtain a ternary standard; The preparation method of the quaternary standard comprises the following steps: The ternary standard is hydrophobically modified to obtain a modified sample; any one of the tobacco cell wall macromolecules other than the tobacco cell wall macromolecules included in the monobasic standard, the dibasic standard, and the ternary standard is mixed with a solvent to obtain a dispersion; the modified sample and the dispersion are mixed, and then frozen and dried to obtain a quaternary standard; The preparation method of five-element and above standard samples is similar, and the steps of hydrophobic modification, preparing dispersion, mixing the modified sample and dispersion, freezing, and drying are repeated.
2. The preparation method according to claim 1, characterized in that The solvents each independently include any one of water, methanol or ethanol or a combination of at least two thereof; Preferably, the concentration of tobacco cell wall macromolecules in the dispersion is independently 5 to 70 wt%; Preferably, the modified sample and the dispersion are mixed for a time independently of each other of 2 to 36 hours.
3. The preparation method according to claim 1 or 2, characterized in that The freezing temperature is independently -30 to -5°C; Preferably, the drying temperature is independently -30 to -20°C.
4. The preparation method according to any one of claims 1 to 3, characterized in that At least one of the freezing or drying is oriented so that the standard product has any one of the shapes of a cube, a cuboid or a cylinder.
5. The preparation method according to any one of claims 1 to 4, characterized in that The modifiers used for the hydrophobic modification independently include any one of silane, phenolic compounds or terpenoid compounds, or a combination of at least two thereof; Preferably, the mass ratio of the standard product to the modifier used for hydrophobic modification is independently 1:(0.5-1).
6. The preparation method according to claim 5, characterized in that The silane includes γ-aminopropyltriethoxysilane; Preferably, the phenolic compound comprises any one or a combination of at least two of octylphenol polyoxyethylene ether, methylparaben, propylparaben, tannic acid, sulfonated lignin grafted long-chain alkyl, or bisphenol A diglycidyl ether; Preferably, the terpenoid compound comprises trimethylsilyllimonene.
7. The preparation method according to claim 6, characterized in that The modifier used for the hydrophobic modification includes a combination of octylphenol polyoxyethylene ether and bisphenol A diglycidyl ether; Preferably, the mass ratio of the combination of octylphenol polyoxyethylene ether and bisphenol A diglycidyl ether is 1:(0.5-1).
8. The preparation method according to any one of claims 1 to 7, characterized in that The hydrophobic modification is carried out in a solvent; Preferably, the solvents each independently include any one of methanol, acetone or water, or a combination of at least two thereof; Preferably, the temperature of the hydrophobic modification is independently 20 to 40° C., and the time of the hydrophobic modification is independently 0.5 to 4 hours.
9. A tobacco leaf cell wall macromolecular three-dimensional model compound standard prepared by the preparation method according to any one of claims 1 to 8.
10. Use of the tobacco leaf cell wall macromolecular three-dimensional model compound standard according to claim 9 in tobacco leaf analysis or simulation.