A kit for determining starch and cellulose in tobacco and its application

Abstract

The present invention provides a kit for determining starch and cellulose in tobacco and its application. The kit for determining starch and cellulose in tobacco of the present invention comprises a determination reagent, and the determination reagent comprises an iodine-containing solution, a dilute sulfuric acid solution, an anthrone solution, a concentrated sulfuric acid solution, a starch standard stock solution and a cellulose standard stock solution. The kit of the present invention has low cost, simple and fast operation, high repeatability and accuracy, and can be used for the determination of starch and cellulose in a large number of tobacco samples.

Description

Technical Field

[0001] The invention relates to the technical field of tobacco chemical component detection, in particular to a kit for determining starch and cellulose in tobacco and application thereof. Background Art

[0002] Starch is an important energy storage substance in tobacco. It is a polysaccharide composed of multiple D-glucose bonds, including two parts: amylose and amylopectin. The degree of conversion of starch to sugar in tobacco leaves directly determines the quality of tobacco in a certain sense. Too high or too low starch content in tobacco leaves will have an adverse effect on the smoking quality of tobacco leaves. Cellulose is the basic substance that constitutes the cytoskeleton. It is a macromolecular polysaccharide composed of glucose, which provides structural support and protection for plants. The cellulose content is closely related to the grade of tobacco leaves. The compounds produced by the decomposition of polysaccharides such as cellulose can coordinate the aroma of tobacco and increase the aroma concentration. However, too high a cellulose content in tobacco leaves will have a bad effect on the sensory quality of tobacco, causing the smoke to produce strong irritation, choking, astringent, and burnt gas. Therefore, the determination of starch and cellulose in tobacco is an important content and data basis for conducting research on tobacco macromolecules.

[0003] At present, the starch content in tobacco is mainly determined by the tobacco industry standard YC / T 216-2013 "Continuous Flow Method for Determination of Starch in Tobacco and Tobacco Products". This method mainly uses 80% ethanol-saturated sodium chloride solution to ultrasonically remove interfering substances in tobacco samples, uses 40% perchloric acid to extract starch, and uses continuous flow analysis to determine starch. The pretreatment process of this method requires the use of a large amount of strong acid, which is complicated to operate and takes a long time to test. In addition, methods for determining starch also include enzymatic hydrolysis, high-performance liquid chromatography, etc. The enzymatic hydrolysis method uses the specific binding of starch and amylase to determine the starch content. However, this method has high requirements for the activity of amylase and high experimental costs. High-performance liquid chromatography hydrolyzes starch into monosaccharides and further calculates the starch content using the monosaccharide content. This method has high accuracy, but it requires the use of a high-performance liquid chromatograph, which has high instrument costs.

[0004] The cellulose content in tobacco is mainly determined by the tobacco industry standard YC / T 347-2010 "Determination of neutral detergent fiber, acid detergent fiber and acid-washed lignin in tobacco and tobacco products - detergent method". This method is cumbersome to operate, and the pre-treatment process requires a large amount of concentrated sulfuric acid and toxic reagents such as benzene and acetone, which is seriously polluting. In addition, a fiber analyzer is required during the detection process, the instrument cost is high, and each batch can only detect 6 samples, which takes a long time and is not suitable for large-scale promotion and use. In addition, the determination methods of cellulose also include Van Soest method (paradigm method), Wang Yuwan method and enzymatic method. The pre-treatment of cellulose in Van Soest method is complicated and difficult to filter. The determination of cellulose in Wang Yuwan method requires overnight hydrolysis, which takes a long time and the determination result is high. The enzymatic method requires the use of cellulase, which is costly and not conducive to the determination of large quantities of tobacco leaf samples.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a kit for determining starch and cellulose in tobacco and its application. The kit has low cost, simple and fast operation, high repeatability and accuracy, and can be used for the determination of starch and cellulose in large quantities of tobacco samples.

[0007] The invention provides a kit for determining starch and cellulose in tobacco, comprising determination reagents, wherein the determination reagents include iodine-containing solution, dilute sulfuric acid solution, anthrone solution, concentrated sulfuric acid solution, starch standard stock solution and cellulose standard stock solution.

[0008] Furthermore, the assay reagent may also include distilled water; the volume of the distilled water may be 5-15 mL, for example, 10 mL.

[0009] In the present invention, tobacco refers to tobacco leaves, tobacco shreds or other tobacco products, and is usually measured and weighed in the form of crushed and mixed tobacco powder.

[0010] The iodine-containing solution is a mixed solution of iodine and potassium iodide, wherein the mass content of iodine in the iodine-containing solution is 0.5-0.6 mg / mL, for example, 0.52 mg / mL; the mass content of potassium iodide is 0.8-1.2 mg / mL, for example, 1 mg / mL. The preparation method of the iodine-containing solution comprises: grinding the iodine and mixing it with potassium iodide, adding distilled water to the mixture, stirring until it is completely dissolved and then constant volume to obtain the iodine-containing solution. The volume of the iodine-containing solution in the kit can be 0.4-0.6 mL, for example, 0.5 mL.

[0011] The mass content of the dilute sulfuric acid solution is 40-60%, for example 40-50%. The preparation method of the dilute sulfuric acid solution includes: mixing concentrated sulfuric acid and distilled water uniformly to prepare the dilute sulfuric acid solution. The volume of the dilute sulfuric acid solution in the kit can be 5-10mL, for example 10mL.

[0012] The mass content of anthrone in the anthrone solution is 1-3%, for example, 1.5%. The preparation method of the anthrone solution comprises: dissolving anthrone in ethyl acetate to obtain the anthrone solution. The volume of the anthrone solution in the kit can be 0.4-0.6 mL, for example, 0.5 mL.

[0013] In the present invention, the mass concentration of the concentrated sulfuric acid solution is 98%; the volume of the concentrated sulfuric acid solution in the kit can be 4-6 mL, for example, 5 mL.

[0014] The mass content of starch in the starch standard stock solution is 1-3 mg / mL, for example 2 mg / mL. The preparation method of the starch standard stock solution comprises: adding an appropriate amount of distilled water to the soluble starch, stirring and heating in boiling water until boiling, maintaining for 4-6 minutes until the soluble starch is completely dissolved, cooling and then constant volume, to obtain the starch standard stock solution.

[0015] The mass content of cellulose in the cellulose standard stock solution is 200-300 μg / mL. The preparation method of the cellulose standard stock solution comprises: mixing cellulose with a dilute sulfuric acid solution and reacting, and then fixing the volume with a dilute sulfuric acid solution to obtain a reaction solution; absorbing the reaction solution and fixing the volume with distilled water to obtain a cellulose solution; wherein the mass content of the dilute sulfuric acid solution is 40-60%, preferably 40-50%; the ratio of cellulose to the dilute sulfuric acid solution is 0.02g:5-10mL; and the reaction time is 40-60min, preferably 40-50min.

[0016] Furthermore, the kit also includes a starch standard working solution, in which the mass content of starch is 0-150 μg / mL, for example, 20-150 μg / mL; the starch standard working solution (also called a starch gradient solution) includes a series of solutions with starch concentrations from low to high, and the starch concentrations of a series of solutions in the starch standard working solution can be reasonably set according to actual needs, for example, set to 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, and 150 μg / mL in sequence.

[0017] Furthermore, the kit also includes a cellulose standard working solution, in which the mass content of cellulose is 0-200 μg / mL, for example, 40-200 μg / mL; similarly, the cellulose standard working solution (also called cellulose gradient solution) includes a series of solutions with cellulose concentrations from low to high, and the cellulose concentrations of a series of solutions in the cellulose standard working solution can be reasonably set according to actual needs, for example, set to 0 μg / mL, 40 μg / mL, 80 μg / mL, 120 μg / mL, 160 μg / mL, and 200 μg / mL, respectively.

[0018] Furthermore, the kit also includes a starch standard curve and a cellulose standard curve; the method for drawing the starch standard curve and the cellulose standard curve includes:

[0019] A) adopt starch standard stock solution to prepare starch standard working solution, add iodine solution to the starch standard working solution, adopt spectrophotometer to measure starch content at 560-570nm place, and draw starch standard curve;

[0020] B) Prepare a cellulose standard working solution using a cellulose standard stock solution, add anthrone solution and concentrated sulfuric acid solution to the cellulose standard working solution, heat in a boiling water bath, cool after heating, measure the cellulose content at 610-630 nm using a spectrophotometer, and draw a cellulose standard curve.

[0021] In step A), the preparation method of the starch standard working solution comprises: taking 0 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, and 0.75 mL of the starch standard stock solution, respectively, and diluting the volume to 10 mL with distilled water to prepare the starch standard working solution; the ratio of the starch standard working solution to the iodine-containing solution is 2 mL: 0.4-0.6 mL, for example 2 mL: 0.5 mL.

[0022] In step B), the preparation method of the cellulose standard working solution comprises: taking 0 mL, 0.4 mL, 0.8 mL, 1.2 mL, 1.6 mL, and 2.0 mL of the cellulose standard stock solution, respectively, and diluting the volume to 2 mL with distilled water to prepare a starch standard working solution; the ratio between the cellulose standard working solution, the anthrone solution and the concentrated sulfuric acid solution is 2 mL: 0.4-0.6 mL: 4-6 mL, for example 2 mL: 0.5 mL: 5 mL; the heating time is 5-15 min, preferably 10-15 min.

[0023] Furthermore, the test kit also includes a sample holding device, and the sample holding device includes a 15mL centrifuge tube.

[0024] The invention also provides application of the kit in determining starch and cellulose in tobacco.

[0025] The present invention also provides a method for determining starch and cellulose in tobacco, which is carried out using the above-mentioned kit, and the method comprises the following steps:

[0026] S1: Mix the tobacco sample with distilled water and heat it in a boiling water bath, then cool, fix the volume, and centrifuge to obtain a supernatant and a precipitate;

[0027] S2: adding iodine solution to the supernatant and measuring the starch content using a spectrophotometer at 560-570 nm;

[0028] S3: After drying the precipitate, add a dilute sulfuric acid solution and react in an ice-water bath. After the reaction, fix the volume, centrifuge, and fix the volume again to obtain a reaction solution;

[0029] S4: adding anthrone solution and concentrated sulfuric acid solution to the reaction solution, heating in a boiling water bath, cooling after heating, and measuring the cellulose content at 610-630 nm using a spectrophotometer.

[0030] In step S1, the ratio of tobacco sample to distilled water is 0.02 g: 5-15 mL, for example, 0.02 g: 6 mL; the heating time is 10-20 min, preferably 10-15 min, for example, 15 min.

[0031] In step S2, the ratio of the supernatant to the iodine-containing solution is 2 mL:0.4-0.6 mL, for example, 2 mL:0.5 mL.

[0032] In step S3, the ratio of tobacco sample to dilute sulfuric acid solution is 0.02 g: 5-10 mL, for example, 0.02 g: 6 mL; the reaction time is 40-60 min, preferably 40-50 min.

[0033] In step S4, the ratio of the reaction solution, the anthrone solution and the concentrated sulfuric acid solution is 2 mL: 0.4-0.6 mL: 4-6 mL, for example 2 mL: 0.5 mL: 5 mL; the heating time is 5-15 min, preferably 10-15 min.

[0034] The implementation of the present invention has at least the following advantages:

[0035] 1) The kit of the present invention can quickly, efficiently and accurately detect the starch and cellulose content in tobacco, thereby providing a basis for tobacco quality evaluation.

[0036] 2) The method of the present invention uses boiling water to extract starch from tobacco and uses a dilute sulfuric acid solution of a specific concentration to extract cellulose from tobacco. The method has a good extraction effect on starch and cellulose from tobacco, has less pollution to the environment, and is in line with the concept of green development.

[0037] 3) The pretreatment method of the present invention is easy to operate and has simple reagents. The starch and cellulose contents in tobacco can be detected simultaneously with only one pretreatment, which has the advantages of convenience, high efficiency, cost saving, etc.

[0038] 4) The present invention adopts the color development reaction of starch and iodine / potassium iodide and the reaction of anthrone and cellulose to determine the content of starch and cellulose, has high repeatability and accuracy, and can be used for the determination of starch and cellulose in large quantities of tobacco samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 is the starch standard curve;

[0041] Figure 2 is the cellulose standard curve;

[0042] Figure 3 This is a process flow chart for determining starch and cellulose in tobacco. DETAILED DESCRIPTION

[0043] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0044] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this description, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0045] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] Example 1

[0047] The kit for determining starch and cellulose in tobacco of the present embodiment comprises a sample holding device and a determination reagent, wherein the sample holding device comprises a 15 mL centrifuge tube, and the determination reagent comprises 10 mL of distilled water, 0.5 mL of iodine-containing solution, 10 mL of a dilute sulfuric acid solution with a mass content of 50%, 0.5 mL of anthrone solution with a mass content of 1.5%, 5 mL of a concentrated sulfuric acid solution with a mass content of 98%, 2 mg / mL of a starch standard stock solution, 200 μg / mL of a cellulose standard stock solution, 0-150 μg / mL of a starch standard working solution, 0-200 μg / mL of a cellulose standard working solution, a starch standard curve, and a cellulose standard curve; the preparation method of each solution and the drawing method of each standard curve are as follows:

[0048] 1) Prepare iodine solution

[0049] Weigh 0.052 g of iodine and 0.10 g of potassium iodide, grind the iodine in a mortar, then put it into a small beaker together with potassium iodide, add distilled water and stir continuously until completely dissolved, then transfer to a 100 mL volumetric flask, rinse the beaker glass rod twice with distilled water, and make up to 100 mL with distilled water to prepare an iodine-containing solution.

[0050] 2) Prepare dilute sulfuric acid solution

[0051] Measure 50 mL of 98% concentrated sulfuric acid into 50 mL of distilled water, add while stirring to prepare a dilute sulfuric acid solution.

[0052] 3) Preparation of anthrone solution

[0053] Weigh 1.5 g of anthrone and dissolve it in 100 mL of ethyl acetate to prepare anthrone solution.

[0054] 4) Prepare starch standard stock solution

[0055] Accurately weigh 0.2 g of soluble starch and put it into a beaker. Add an appropriate amount of distilled water, stir and heat in boiling water until boiling, keep for 5 minutes, until the soluble starch is completely dissolved, cool and then dilute to 100 mL to prepare the starch standard stock solution.

[0056] 5) Preparation of cellulose standard stock solution

[0057] Accurately weigh 20 mg of pure cellulose in a beaker, slowly add 6 mL of 50% dilute sulfuric acid solution along the beaker wall in an ice-water bath, react in an ice-water bath for 50 minutes, then dilute to 10 mL with 50% dilute sulfuric acid solution, shake well. Take 1 mL of the above solution, dilute to 10 mL with distilled water in an ice-water bath to prepare a cellulose standard stock solution.

[0058] 6) Prepare starch standard working solution

[0059] Take 0mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, and 0.75mL of starch standard stock solution into 10mL volumetric flasks, respectively, and make up to 10mL with distilled water to prepare starch standard working solutions with starch concentrations of 0μg / mL, 20μg / mL, 40μg / mL, 60μg / mL, 80μg / mL, 100μg / mL, and 150μg / mL, respectively.

[0060] 7) Prepare cellulose standard working solution

[0061] Take 6 15mL centrifuge tubes, add 0mL, 0.4mL, 0.8mL, 1.2mL, 1.6mL, and 2.0mL of cellulose standard stock solution, respectively, make up to 2.0mL with distilled water, shake well, and prepare cellulose standard working solutions with cellulose concentrations of 0μg / mL, 40μg / mL, 80μg / mL, 120μg / mL, 160μg / mL, and 200μg / mL, respectively.

[0062] 8) Draw a starch standard curve

[0063] Take 2 mL of starch standard working solution, add 0.5 mL of iodine solution, use a spectrophotometer to determine the starch content at 565 nm, and draw a starch standard curve.

[0064] The standard curve is recorded as: y = ax + b

[0065] Where: y is equivalent to absorbance value ΔA = A 样品 -A 空白 , x is the concentration of starch.

[0066] Results Figure 1 .

[0067] 9) Draw a cellulose standard curve

[0068] Add 0.5 mL of anthrone solution to 2 mL of cellulose standard working solution, and slowly add 5 mL of concentrated sulfuric acid solution along the tube wall. After careful mixing, heat in a boiling water bath for 10 min, cool to room temperature, and use a spectrophotometer to determine the cellulose content at 620 nm to draw a cellulose standard curve.

[0069] The standard curve is recorded as: y = ax + b

[0070] Where: y is equivalent to absorbance value ΔA = A 样品 -A 空白 , x is the concentration of cellulose.

[0071] Results Figure 2 .

[0072] Example 2

[0073] Combination Figure 3 As shown, the method for determining starch and cellulose in tobacco in this embodiment is carried out using the kit of Example 1, and the steps are as follows:

[0074] 1) Extraction

[0075] A tobacco leaf sample (referred to as sample 1) was selected, ground and passed through a 100-mesh sieve for later use.

[0076] Weigh 0.02 g of sample 1 into a 15 mL centrifuge tube, add 6 mL of distilled water, heat in a boiling water bath for 15 min, cool to room temperature, make up to 10 mL, and centrifuge at 7000 rpm for 5 min to separate the precipitate and the supernatant.

[0077] 2) Starch determination

[0078] Take 2 mL of the supernatant from step 1), add 0.5 mL of iodine solution, and measure the starch content at 565 nm using a spectrophotometer.

[0079] 3) Cellulose determination

[0080] The precipitate of step 1) is naturally dried, 6 mL of a 50% by mass dilute sulfuric acid solution is slowly added along the wall of the centrifuge tube in a cold water bath, and the reaction is carried out in an ice water bath for 50 minutes; after the reaction is completed, the solution is diluted to 10 mL with a 50% by mass dilute sulfuric acid solution, centrifuged at 7800 rpm for 5 minutes, 1 mL of the above solution is transferred and diluted to 10 mL with distilled water, 2 mL of the solution is drawn into a 15 mL centrifuge tube, 0.5 mL of anthrone solution is added to the centrifuge tube, and 5 mL of concentrated sulfuric acid solution is slowly added along the wall of the tube, carefully mixed, heated in a boiling water bath for 10 minutes, cooled to room temperature, and the cellulose content is measured at 620 nm using a spectrophotometer.

[0081] 4) Calculation

[0082] Calculate the starch and cellulose content according to the formula:

[0083]

[0084] Where: x is the concentration of starch or cellulose, V is the sample measurement volume (mL), n is the dilution factor, and m is the sample weight (g).

[0085] The results of three parallel measurements are shown in Tables 1 and 2; the measurement results show that the starch and cellulose contents in the tobacco leaf sample of this example are 48.34 mg / g and 90.15 mg / g, respectively.

[0086] Example 3

[0087] The method for determining starch and cellulose in tobacco in this embodiment is carried out using the kit of Example 1, and the steps are as follows:

[0088] 1) Extraction

[0089] A tobacco leaf sample (referred to as sample 2) was selected, ground and passed through a 100-mesh sieve for later use.

[0090] Weigh 0.02 g of sample 2 into a 15 mL centrifuge tube, add 6 mL of distilled water, heat in a boiling water bath for 15 min, cool to room temperature, make up to 10 mL, centrifuge at 7000 rpm for 5 min, and separate the precipitate and supernatant.

[0091] 2) Starch determination

[0092] Take 2 mL of the supernatant from step 1), add 0.5 mL of iodine solution, and measure the starch content at 565 nm using a spectrophotometer.

[0093] 3) Cellulose determination

[0094] The precipitate of step 1) is naturally dried, 6 mL of a 50% by mass dilute sulfuric acid solution is slowly added along the wall of the centrifuge tube in a cold water bath, and the reaction is carried out in an ice water bath for 50 minutes; after the reaction is completed, the solution is diluted to 10 mL with a 50% by mass dilute sulfuric acid solution, centrifuged at 7800 rpm for 5 minutes, 1 mL of the above solution is transferred and diluted to 10 mL with distilled water, 2 mL of the solution is drawn into a 15 mL centrifuge tube, 0.5 mL of anthrone solution is added to the centrifuge tube, and 5 mL of concentrated sulfuric acid solution is slowly added along the wall of the tube, carefully mixed, heated in a boiling water bath for 10 minutes, cooled to room temperature, and the cellulose content is measured at 620 nm using a spectrophotometer.

[0095] The calculation was performed according to the standard curve and calculation method of Example 2. The results of three parallel measurements are shown in Tables 1 and 2. The measurement results show that the starch and cellulose contents in the tobacco leaf sample of this example are 32.25 mg / g and 101.96 mg / g, respectively.

[0096] Table 1 Determination results of starch content in tobacco leaf samples

[0097]

[0098] Table 2 Tobacco leaf sample cellulose determination results

[0099]

[0100] The above results show that:

[0101] The RSDs of starch and cellulose in tobacco leaf samples were both less than 5%, and the test results were stable, meeting the inspection requirements.

[0102] Example 4

[0103] The kit of this embodiment is basically the same as that of embodiment 1 except that a dilute sulfuric acid solution with a mass content of 40% is used, and the preparation of the cellulose solution and the drawing of the cellulose standard curve are different.

[0104] 1) Preparation of cellulose solution

[0105] Accurately weigh 20 mg of pure cellulose in a beaker, slowly add 6 mL of 40% dilute sulfuric acid solution along the beaker wall in an ice-water bath, react in an ice-water bath for 40 minutes, then dilute to 10 mL with 40% dilute sulfuric acid solution, shake well. Take 1 mL of the above solution, dilute to 10 mL with distilled water in an ice-water bath to prepare a cellulose solution.

[0106] 2) Draw a cellulose standard curve

[0107] Add 0.5 mL of anthrone solution to 2 mL of cellulose standard working solution, and slowly add 5 mL of concentrated sulfuric acid solution along the tube wall. After careful mixing, heat in a boiling water bath for 15 min, cool to room temperature, and use a spectrophotometer to determine the cellulose content at 610 nm to draw a cellulose standard curve.

[0108] The method for measuring starch and cellulose in tobacco using the kit of this embodiment comprises the following steps:

[0109] 1) Extraction

[0110] Select a tobacco leaf sample (sample 1), grind it, and pass it through a 100-mesh sieve for later use.

[0111] Weigh 0.02 g of sample 1 into a 15 mL centrifuge tube, add 6 mL of distilled water, heat in a boiling water bath for 10 min, cool to room temperature, make up to 10 mL, and centrifuge at 7000 rpm for 5 min to separate the precipitate and the supernatant.

[0112] 2) Starch determination

[0113] Take 2 mL of the supernatant from step 1), add 0.5 mL of iodine solution, and measure the starch content at 560 nm using a spectrophotometer.

[0114] 3) Cellulose determination

[0115] The precipitate of step 1) is naturally dried, 6 mL of a 40% dilute sulfuric acid solution is slowly added along the wall of the centrifuge tube in a cold water bath, and the reaction is carried out in an ice water bath for 40 minutes; after the reaction is completed, the solution is diluted to 10 mL with a 40% dilute sulfuric acid solution, centrifuged at 7800 rpm for 5 minutes, 1 mL of the above solution is transferred and diluted to 10 mL with distilled water, 2 mL of the solution is drawn into a 15 mL centrifuge tube, 0.5 mL of anthrone solution is added to the centrifuge tube, and 5 mL of concentrated sulfuric acid solution is slowly added along the wall of the tube, carefully mixed, heated in a boiling water bath for 15 minutes, cooled to room temperature, and the cellulose content is measured at 610 nm using a spectrophotometer.

[0116] 4) Draw the starch standard curve

[0117] According to the above step 2), the starch content was determined using the starch standard working solution, and a starch standard curve was drawn.

[0118] 5) Draw a cellulose standard curve

[0119] According to the above step 3), the cellulose content was determined using the cellulose standard working solution, and a cellulose standard curve was drawn.

[0120] The calculation method of Example 2 was used for calculation, and the results of three parallel measurements are shown in Tables 3 and 4. The measurement results show that the starch and cellulose contents in the tobacco leaf sample of this example are 47.64 mg / g and 88.75 mg / g, respectively.

[0121] Example 5

[0122] The kit of this embodiment is basically the same as that of embodiment 1 except that a dilute sulfuric acid solution with a mass content of 40% is used, and the preparation of the cellulose solution and the drawing of the cellulose standard curve are different.

[0123] 1) Preparation of cellulose solution

[0124] Accurately weigh 20 mg of pure cellulose in a beaker, slowly add 6 mL of 40% dilute sulfuric acid solution along the beaker wall in an ice-water bath, react in an ice-water bath for 40 minutes, then dilute to 10 mL with 40% dilute sulfuric acid solution, shake well. Take 1 mL of the above solution, dilute to 10 mL with distilled water in an ice-water bath to prepare a cellulose solution.

[0125] 2) Draw a cellulose standard curve

[0126] Add 0.5 mL of anthrone solution to 2 mL of cellulose standard working solution, and slowly add 5 mL of concentrated sulfuric acid solution along the tube wall. After careful mixing, heat in a boiling water bath for 15 min, cool to room temperature, and use a spectrophotometer to determine the cellulose content at 610 nm to draw a cellulose standard curve.

[0127] The method for measuring starch and cellulose in tobacco using the kit of this embodiment comprises the following steps:

[0128] 1) Extraction

[0129] Select a tobacco leaf sample (sample 2), grind it, and pass it through a 100-mesh sieve for later use.

[0130] Weigh 0.02 g of sample 2 into a 15 mL centrifuge tube, add 6 mL of distilled water, heat in a boiling water bath for 10 min, cool to room temperature, make up to 10 mL, centrifuge at 7000 rpm for 5 min, and separate the precipitate and supernatant.

[0131] 2) Starch determination

[0132] Take 2 mL of the supernatant from step 1), add 0.5 mL of iodine solution, and measure the starch content at 560 nm using a spectrophotometer.

[0133] 3) Cellulose determination

[0134] The precipitate of step 1) is naturally dried, 6 mL of a 40% dilute sulfuric acid solution is slowly added along the wall of the centrifuge tube in a cold water bath, and the reaction is carried out in an ice water bath for 40 minutes; after the reaction is completed, the solution is diluted to 10 mL with a 40% dilute sulfuric acid solution, centrifuged at 7800 rpm for 5 minutes, 1 mL of the above solution is transferred and diluted to 10 mL with distilled water, 2 mL of the solution is drawn into a 15 mL centrifuge tube, 0.5 mL of anthrone solution is added to the centrifuge tube, and 5 mL of concentrated sulfuric acid solution is slowly added along the wall of the tube, carefully mixed, heated in a boiling water bath for 15 minutes, cooled to room temperature, and the cellulose content is measured at 610 nm using a spectrophotometer.

[0135] 4) Draw the starch standard curve

[0136] According to the above step 2), the starch content was determined using the starch standard working solution, and a starch standard curve was drawn.

[0137] 5) Draw a cellulose standard curve

[0138] According to the above step 3), the cellulose content was determined using the cellulose standard working solution, and a cellulose standard curve was drawn.

[0139] The calculation method of Example 2 was used for calculation, and the results of three parallel measurements are shown in Tables 3 and 4. The measurement results show that the starch and cellulose contents in the tobacco leaf sample of this example are 31.77 mg / g and 100.59 mg / g, respectively.

[0140] Table 3 Tobacco leaf sample starch content determination results

[0141]

[0142] Table 4 Tobacco leaf sample cellulose determination results

[0143]

[0144] The above results show that:

[0145] When measured under different conditions, there was no significant difference in the starch and cellulose contents of tobacco leaves between Sample 1 and Sample 2 (RSD less than 5).

[0146] Example 6

[0147] 1) Spike recovery rate

[0148] Two tobacco leaf samples of Examples 2 and 3 were selected, and the contents of starch and cellulose after adding 80 mg / g starch and 80 mg / g cellulose were determined by the methods of Examples 2 and 3, respectively. The spiked recovery was calculated, and each sample was repeated 3 times. The results are shown in Tables 5 and 6.

[0149] Table 5 Spiked recovery of starch content determination in tobacco leaf samples

[0150]

[0151] Table 6 Spiked recovery of cellulose content in tobacco samples

[0152]

[0153]

[0154] The above results show that:

[0155] The spiked recoveries of starch in tobacco leaf samples were 100.94% and 101.75%, and the spiked recoveries of cellulose were 100.63% and 101.92%, which met the test requirements for spiked recoveries.

[0156] 2) Reproducibility

[0157] The starch and cellulose contents in the two tobacco leaf samples of Examples 2 and 3 were determined by the methods of Examples 2 and 3, respectively. Each sample was repeated 6 times to test the reproducibility. The results are shown in Tables 7 and 8.

[0158] Table 7 Reproducibility of determination of starch content in tobacco leaf samples

[0159]

[0160] Table 8 Reproducibility of cellulose content determination of tobacco leaf samples

[0161]

[0162] The above results show that:

[0163] The RSDs of the two tobacco leaf samples were both less than 5%, meeting the reproducibility requirement, indicating that the kit and the detection method of the present invention are relatively stable, and the detection data have good stability and reproducibility.

[0164] Comparative Example 1

[0165] Except that the heating time in the boiling water bath during the extraction process in step 1) is 30 min, the rest is basically the same as Example 2.

[0166] The measurement results show that the starch content in the tobacco leaf sample of this control example is 27.64 mg / g, which is much lower than that of Example 2.

[0167] Comparative Example 2

[0168] The kit of this comparative example is substantially the same as that of Example 1, except that 70% dilute sulfuric acid solution is used to replace 50% dilute sulfuric acid solution in the step of preparing the cellulose solution.

[0169] The kit of this control example was used to perform the test according to the method of Example 2, except that the 70% dilute sulfuric acid solution was used to replace the 50% dilute sulfuric acid solution in the cellulose determination step, and the rest was basically the same as Example 2.

[0170] The measurement results show that the cellulose content in the tobacco leaf sample of this control example is 50.59 mg / g, which is much lower than that of Example 2.

[0171] Comparative Example 3

[0172] The starch content of the two tobacco leaf samples (sample 1 and sample 2) of Examples 2 and 3 was tested using a common commercially available starch content test kit (article number: G0507F). The results are shown in Tables 9 and 10.

[0173] Comparative Example 4

[0174] The cellulose content of the two tobacco leaf samples (sample 1 and sample 2) of Examples 2 and 3 was tested using a commercially available cellulose content test kit (article number: G0715F). The results are shown in Tables 9 and 10.

[0175] Table 9 Tobacco leaf sample starch content determination results

[0176]

[0177] Table 10 Tobacco leaf sample cellulose determination results

[0178]

[0179] The above results show that:

[0180] The starch content test kit and cellulose content test kit commonly purchased on the market not only have poor detection stability, but also the detection results of starch and cellulose content in tobacco leaf samples are far lower than those of the present invention.

[0181] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A kit for determining starch and cellulose in tobacco, characterized in that: The invention comprises measuring reagents, which include iodine solution, dilute sulfuric acid solution, anthrone solution, concentrated sulfuric acid solution, starch standard stock solution and cellulose standard stock solution.

2. The kit according to claim 1, characterized in that The iodine-containing solution is a mixed solution of iodine and potassium iodide. The mass content of iodine in the iodine-containing solution is 0.5-0.6 mg / mL, and the mass content of potassium iodide is 0.8-1.2 mg / mL.

3. The kit according to claim 1, characterized in that The mass content of the dilute sulfuric acid solution is 40-60%; the mass content of the anthrone in the anthrone solution is 1-3%.

4. The kit according to claim 1, characterized in that The mass content of starch in the starch standard stock solution is 1-3 mg / mL; the mass content of cellulose in the cellulose standard stock solution is 200-300 μg / mL.

5. The kit according to claim 1, characterized in that It also includes a starch standard working solution and a cellulose standard working solution; the mass content of starch in the starch standard working solution is 0-150 μg / mL; the mass content of cellulose in the cellulose standard working solution is 0-200 μg / mL.

6. The kit according to claim 1, characterized in that The preparation method of the cellulose standard stock solution comprises: mixing cellulose with a dilute sulfuric acid solution and reacting the mixture, and then fixing the volume with the dilute sulfuric acid solution to obtain a reaction solution; taking the reaction solution and fixing the volume with distilled water to obtain a cellulose solution; Preferably, the mass content of the dilute sulfuric acid solution is 40-60%; the ratio of cellulose to the dilute sulfuric acid solution is 0.02 g: 5-10 mL; and the reaction time is 40-60 min.

7. The kit according to claim 1, characterized in that It also includes a starch standard curve and a cellulose standard curve; the drawing method of the starch standard curve and the cellulose standard curve includes: A) adopt starch standard stock solution to prepare starch standard working solution, add iodine solution to the starch standard working solution, adopt spectrophotometer to measure starch content at 560-570nm place, and draw starch standard curve; B) preparing a cellulose standard working solution using a cellulose standard stock solution, adding anthrone solution and concentrated sulfuric acid solution to the cellulose standard working solution, heating in a boiling water bath, cooling after heating, measuring the cellulose content at 610-630 nm using a spectrophotometer, and drawing a cellulose standard curve; Preferably, in step A), the ratio of the starch standard working solution to the iodine-containing solution is 2 mL: 0.4-0.6 mL; Preferably, in step B), the ratio of the cellulose standard working solution, the anthrone solution and the concentrated sulfuric acid solution is 2 mL: 0.4-0.6 mL: 4-6 mL, and the heating time is 5-15 min.

8. Use of the kit according to any one of claims 1 to 7 in determining starch and cellulose in tobacco.

9. A method for determining starch and cellulose in tobacco, characterized in that: The method is carried out using the kit according to any one of claims 1 to 7, and the method comprises the following steps: S1: Mix the tobacco sample with distilled water and heat it in a boiling water bath, then cool, fix the volume, and centrifuge to obtain a supernatant and a precipitate; S2: adding iodine solution to the supernatant and measuring the starch content using a spectrophotometer at 560-570 nm; S3: After drying the precipitate, add a dilute sulfuric acid solution and react in an ice-water bath. After the reaction, fix the volume, centrifuge, and fix the volume again to obtain a reaction solution; S4: adding anthrone solution and concentrated sulfuric acid solution to the reaction solution, heating in a boiling water bath, cooling after heating, and measuring the cellulose content at 610-630 nm using a spectrophotometer.

10. The method according to claim 9, characterized in that In step S1, the ratio of tobacco sample to distilled water is 0.02 g: 5-15 mL, and the heating time is 10-20 min, preferably 10-15 min; in step S2, the ratio of supernatant to iodine-containing solution is 2 mL: 0.4-0.6 mL; in step S3, the ratio of tobacco sample to dilute sulfuric acid solution is 0.02 g: 5-10 mL, and the reaction time is 40-60 min, preferably 40-50 min; in step S4, the ratio of reaction solution, anthrone solution and concentrated sulfuric acid solution is 2 mL: 0.4-0.6 mL: 4-6 mL, and the heating time is 5-15 min.

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