A method for determining lignin content in tobacco stems

Abstract

The present invention discloses a method for determining the lignin content in tobacco stems, belonging to the field of lignin content analysis in tobacco products. The method comprises pre-treating a tobacco stem sample to obtain a tobacco stem filter residue, wherein the pre-treatment includes sequentially removing fat-soluble substances and pigments; soluble sugars and phenolic substances; starch; cellulose, pectin, and protein from the tobacco stem sample. A NaOH / urea solution is then added to the tobacco stem filter residue to obtain a filter residue. A sulfuric acid solution is then added to the filter residue to obtain a supernatant and a lower filter residue. The acid-soluble lignin content of the supernatant is determined spectrophotometrically. The lower filter residue is treated until neutral, dried, and then dissolved in a NaOH / urea solution. The NaOH / urea solution is removed, and the residue is dried and weighed to determine the acid-insoluble lignin content. This method can more accurately determine the lignin content in tobacco stems and other products, has good reproducibility, and the lignin extracted from the tobacco stems using this method has a loose structure, which is conducive to further analysis and utilization of the lignin.

Description

Technical Field

[0001] The present invention relates to the technical field of lignin detection, in particular to a method for determining the lignin content in tobacco stems. Background Art

[0002] Tobacco belongs to the genus Nicotiana in the Solanaceae family and is an annual herb. Tobacco stems, the coarse veins within tobacco leaves, account for approximately 25% of the total leaf weight. As part of the tobacco leaf, they primarily support the leaves. Currently, one of the primary ways to utilize tobacco stems is to process them into cut stems and add them to cigarettes. Tobacco is a dicotyledonous plant, and the lignin in tobacco is a mixture of guaiacyl lignin and syringyl lignin. Lignin is one of the main cell wall substances in tobacco, contributing to the heavy, woody smell of cut stems and a burning throat when burned. Lignin is the primary aromatic component in tobacco stems and, as such, may be the primary source of harmful substances such as polycyclic aromatic hydrocarbons and aromatic amines in tar. The products of lignin pyrolysis contain catechols and alkylcatechols, which not only cause a harsh taste but also have carcinogenic activity, negatively impacting the quality of cut stems.

[0003] Analysis of lignin content requires extraction. Currently, the most commonly used methods include dissolution extraction using organic solvents such as dioxane. However, the tobacco stem matrix is ​​complex, with substances such as cellulose, protein, and pectin binding to and encapsulating lignin. This makes it difficult for these extraction methods to completely dissolve and extract the lignin. Furthermore, other interfering substances will dissolve into the extraction solution, resulting in large measurement errors. Therefore, it is necessary to establish a more accurate method for analyzing the lignin content in tobacco stems. This method can be used to evaluate the quality of tobacco stem raw materials from different sources, thereby providing a reference for improving the sensory quality of cut stems and cigarettes during tobacco stem processing. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method for more effectively removing interferences in the complex matrix of tobacco and more accurately determining the lignin content in products such as tobacco stems.

[0005] To achieve the above and other related objectives, the present invention provides a method for determining the lignin content in tobacco stems, comprising the following steps:

[0006] S1. Pre-treating the tobacco stem sample to obtain tobacco stem filter residue, wherein the pre-treatment includes sequentially removing fat-soluble substances and pigments; soluble sugars and phenolic substances; starch macromolecules; cellulose, pectin, and protein from the tobacco stem sample;

[0007] S2. Adding NaOH / urea solution to the tobacco stem residue in step S1 to dissolve the residue and then separate to obtain a residue;

[0008] S3, adding sulfuric acid solution to the filter residue obtained in step S2 to dissolve it and then separating the layers to obtain an upper clear liquid and a lower filter residue;

[0009] S4. Determine the content of acid-soluble lignin in the supernatant by spectrophotometry;

[0010] S5. Wash the lower filter residue to make it neutral and then dry it. Then add NaOH / urea solution to dissolve it and separate it to obtain the filter residue to be measured. Then, dry and weigh the filter residue to be measured to obtain the content of acid-insoluble lignin.

[0011] The beneficial technical effect of the present invention is: a method for determining the lignin content by low-temperature dissolution with NaOH / urea solution after pre-treatment is invented, which effectively removes other impurities in the tobacco stems and is more conducive to the separation, extraction and content determination of lignin in the tobacco stems. The average content of lignin in the tobacco stems measured by this method is 7.92%, and the relative standard deviation (RSD) is 11.06%. The lignin extracted from the tobacco stems using this method has a relatively loose structure, which is conducive to the further analysis and utilization of lignin. This method has good repeatability and high accuracy, is suitable for the determination of cellulose in tobacco stems, and can provide a reference for improving the quality of stems and cigarettes. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The concentration-absorbance standard curve of acid-soluble lignin was plotted. DETAILED DESCRIPTION

[0013] The present invention provides a method for determining lignin content by pre-treating tobacco stems and then dissolving them at low temperature using a NaOH / urea solution. This method more effectively removes interfering substances from the complex tobacco matrix and can more accurately determine the lignin content in tobacco stems and other products. The acid-soluble lignin content is measured spectrophotometrically, while the acid-insoluble lignin content is measured gravimetrically. Furthermore, the lignin prepared using this process has a loose structure, facilitating further analysis and utilization of the lignin. Based on this foundation, the present invention has been completed.

[0014] The technical solution adopted by the present invention provides a method for determining the lignin content in tobacco stems, comprising the following steps:

[0015] S1. Pre-treating the tobacco stem sample to obtain tobacco stem filter residue, wherein the pre-treatment includes sequentially removing fat-soluble substances and pigments; soluble sugars and phenolic substances; starch macromolecules; cellulose, pectin, and protein from the tobacco stem sample;

[0016] Step S1 specifically includes the following steps in sequence:

[0017] In a specific embodiment, the specific method of pre-processing in the first step includes the following:

[0018] a) Removal of fat-soluble substances and pigments from the tobacco stem sample: The tobacco stem sample is pulverized to obtain tobacco stem powder. To ensure more complete pulverization and a small, uniform particle size, the tobacco stem sample is freeze-dried before pulverization. The particle size of the tobacco stem powder is 30-120 mesh, preferably 30-60 mesh. After obtaining the tobacco stem powder, the fat-soluble substances and pigments in the tobacco stem sample are removed using an organic solvent. A lipid solvent is typically used, preferably ethyl acetate. The specific removal method involves repeatedly washing the tobacco stem powder with an organic solvent to remove the fat-soluble substances and pigments, yielding a first filter residue. The ratio of organic solvent (mL) to tobacco stem sample (g) used is 1:10-30. This ratio has been verified through multiple experiments to achieve good results without excessive use of organic solvent. The repeated washing process is combined with ultrasonic treatment, centrifugation, and other treatments to remove fat-soluble substances and pigments. Ethyl acetate is preferably used as the organic solvent. After multiple ultrasonic and centrifugal washing operations, the resulting precipitate is the tobacco stem sample from which the fat-soluble substances have been removed. The ratio of tobacco stem sample g: ethyl acetate mL is 1:10-30, preferably the ratio of tobacco stem sample g: ethyl acetate mL is 1:15-25.

[0019] b) Removal of soluble sugars and phenolic compounds from the tobacco stem sample: The first filter residue obtained in step a) above is further treated with an alcohol solution to remove soluble sugars and phenolic compounds from the tobacco stem sample. The specific method is to repeatedly wash the filter residue with an alcohol solution to remove the soluble sugars and phenolic compounds from the tobacco stem sample. The washing method is ultrasonication for 30 minutes (2 times) and centrifugation (4000 rpm for 5 minutes) to obtain a filter residue. The ratio of tobacco stem sample g: alcohol solution mL is 1:10-50. The alcohol solution is generally an ethanol solution (the mass fraction of ethanol is 30%-100%, preferably 75%-95%). After the tobacco stem sample is treated with the alcohol solution, a filter residue is obtained. The filter residue is repeatedly washed with water, and the washing water is monitored using a DNS reagent (dinitrosalicylic acid) to determine the degree of removal of soluble sugars from the filter residue. The operation method is to repeatedly wash the filter residue with cold water, add a DNS reagent to the washing water, and heat it in a boiling water bath for 5 minutes. The removal of soluble sugars from the filter residue is determined by whether a color is developed. When no soluble sugar is detected, the treatment is completed. Preferably, 85% ethanol is used, ultrasonic treatment is performed for 30 minutes (twice), and centrifugation is performed (4000 rpm, 5 minutes) to obtain a filter residue, which is repeatedly washed with water to obtain a second filter residue from which soluble sugars and phenolic substances are removed.

[0020] c) Removal of starch macromolecules from the tobacco stem sample: α-amylase and / or saccharifying enzyme are used to remove starch macromolecules from the second filter residue obtained in b) above. Specifically, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer and α-amylase are added to the tobacco stem filter residue from which fat-soluble substances and pigments, soluble sugars and phenolic substances have been removed, and hydrolysis is carried out; after the hydrolysis in step S12 is completed, acetic acid-sodium acetate buffer solution and saccharifying enzyme are added, and hydrolysis is carried out to obtain a filter residue, which is then post-treated to obtain a tobacco stem sample from which starch has been removed. The pH of the sodium dihydrogen phosphate-disodium hydrogen phosphate buffer is 5 to 6.5, preferably pH 5.5 to 6. The amount of α-amylase used is 1000u / g to 3000u / g, preferably 1500u / g to 2000u / g. The hydrolysis conditions of α-amylase are hydrolysis in a water bath at 50°C to 70°C for 0.5h to 5h, preferably hydrolysis in a water bath at 55°C to 60°C for 2h to 3h. The pH of the acetic acid-sodium acetate buffer solution is 3.5 to 5.5, preferably pH 4 to 4.5. The dosage of saccharifying enzyme is 5000u / g to 20000u / g, preferably 8000u / g to 12000u / g. The hydrolysis conditions of saccharifying enzyme are hydrolysis in a water bath at 50 to 70°C for 0.5h to 5h, preferably hydrolysis in a water bath at 55°C to 60°C for 1h to 2h. After hydrolysis, centrifugation (4000r / min, 5min) is performed, and then the residue is repeatedly washed and hydrolyzed with water to obtain a third residue from which the macromolecular starch is removed.

[0021] d) Removal of cellulose, pectin and protein from the tobacco stem sample: Cellulase, pectinase and protease are used to remove cellulose, pectin and protein from the third filter residue obtained in c) above. Citric acid-trisodium citrate buffer is added to the tobacco stem filter residue from which fat-soluble substances and pigments, soluble sugars and phenolic substances, and starch macromolecules have been removed. Cellulase, pectinase and protease are added separately for enzymatic hydrolysis, and post-treatment is performed to obtain the tobacco stem filter residue from which cellulose, pectin and protein have been removed. The pH of the citric acid-trisodium citrate buffer is 3.5-5, preferably pH 4-4.7. The amount of cellulase used is 100u / g-300u / g, preferably 150u / g-300u / g. The amount of pectinase used is 1000u / g-8000u / g, preferably 4000u / g-6000u / g. The amount of protease used is 1000 to 8000 u / g, preferably 4000 to 6000 u / g. The enzymatic hydrolysis is performed in a shaking incubator at 35 to 55°C (90 to 120 rpm) for 5 to 15 hours. Finally, the enzymatic hydrolysis solution is centrifuged (4000 rpm for 5 minutes), and the filter residue is repeatedly washed with cold water to obtain the tobacco stem filter residue.

[0022] S2. Add NaOH / urea solution to the tobacco stem filter residue in step S1 for dissolution treatment, and then remove NaOH / urea to obtain filter residue; the method of solvent treatment is generally stirring, oscillating or ultrasonic, etc.; the method of removing NaOH / urea is generally to centrifuge the above solution, wash with pure water until the supernatant is neutral, and the lower layer is obtained as the filter residue. Add a certain volume of NaOH / urea solution to the filter residue, stir at a certain temperature for a certain period of time, and then centrifuge, wash with pure water until the supernatant is neutral, and obtain the filter residue in the lower layer. The mass of tobacco stem sample g: the volume of NaOH / urea solution added mL is 1:15~50, preferably the volume of NaOH / urea solution added mL is 1:20~30, and the volume of NaOH / urea solution added can be 20mL, 22mL, 24mL, 25mL, 26mL, 28mL, 30mL. The mass fraction of NaOH added to the NaOH / urea solution is 5% to 10%, preferably 7% to 9%; the mass fraction of urea is 6% to 20%, preferably 10% to 15%, more preferably 8% of NaOH and 12% of urea. The dissolution treatment is performed by stirring at -12°C to -8°C for 25-35 minutes. The stirring temperature can be -12°C, -11°C, -10°C, -9°C, or -8°C, and the stirring time can be 25 minutes, 27 minutes, 29 minutes, 30 minutes, 31 minutes, 33 minutes, or 35 minutes. Using NaOH / urea to dissolve substances such as cellulose and hemicellulose makes the tissue looser.

[0023] S3. Add sulfuric acid solution to the filter residue obtained in step S2 for dissolution treatment, and then obtain an upper clear liquid and a lower filter residue after treatment. The mass fraction of sulfuric acid in the sulfuric acid solution is 15% to 20%, preferably 16% to 18%. The mass of the tobacco stem sample g: the volume of the added sulfuric acid solution mL is 1:10 to 30, preferably 1:18 to 22. After adding the sulfuric acid solution, the reaction conditions are: stirring at 90 to 110 ° C for 15 min to 45 min. Preferably, stirring at 99 to 101 ° C for 25 min to 35 min.

[0024] S4. The content of acid-soluble lignin in the supernatant was determined by spectrophotometry. The absorbance corresponding to the maximum absorption peak of acid-soluble lignin in the supernatant was measured (generally at 325 nm). The absorbance was substituted into the acid-soluble lignin concentration-absorbance standard curve to obtain the acid-soluble lignin content. Specifically, 1 mL of the supernatant was diluted 3 times, and its absorbance at 325 nm was measured. The concentration of acid-soluble lignin was obtained by the acid-soluble lignin standard curve. The preparation method of the acid-soluble lignin concentration-absorbance standard curve is as follows:

[0025] Weigh 0.5g corncob lignin standard, add 20mL of 17.5% sulfuric acid solution, stir and dissolve at 100℃ for 30min, then dilute with 17.5% dilute sulfuric acid solution to form a series of solutions with different concentrations in the range of 0.06-0.34mg / mL. Measure the absorbance at 325nm, and fit the standard curve of acid-soluble lignin based on the relationship between lignin concentration and absorbance.

[0026] S5, process the lower filter residue in step S3 until it is neutral, wash it with acetone for many times, add NaOH / urea solution again to dissolve the filter residue after drying, then remove NaOH / urea to obtain the filter residue to be measured, dry and weigh to obtain the acid-insoluble lignin content. Use acetone to wash out some small molecular organics after acid treatment, and the filter residue can be dried faster in addition. Specifically, process the lower filter residue until it is neutral, wash it with acetone for many times, and then dry it in an oven at a certain temperature for a period of time. Add a certain volume of NaOH / urea solution, stir it for a period of time at a certain temperature, then centrifuge, wash it with pure water until the supernatant is neutral, freeze-dry the filter residue, and after weighing, the acid-insoluble lignin content can be obtained by calculation. The ratio of tobacco stem sample mass g: added NaOH / urea solution volume mL is 1:15-50, the mass fraction of NaOH added to the NaOH / urea solution is 5%-10%, preferably 7%-9%; the mass fraction of urea is 6%-20%, preferably 10%-15%, more preferably the mass fraction of NaOH is 8%, and the mass fraction of urea is 12%. The dissolution treatment is stirring at -12°C to -8°C for 25-35 minutes. Drying is done in an oven, and the temperature required for drying is 49-51°C. The oven drying time can be 15-45 minutes, preferably 15-25 minutes.

[0027] The beneficial effects of the present invention are further illustrated below with reference to the examples.

[0028] In order to make the invention objectives, technical solutions and beneficial technical effects of the present invention clearer, the present invention is further described in detail below with reference to the examples. However, it should be understood that the examples of the present invention are only for the purpose of explaining the present invention and are not intended to limit the present invention, and the examples of the present invention are not limited to the examples given in the specification. In the examples, where no specific experimental conditions or operating conditions are specified, the products were prepared under conventional conditions or under the conditions recommended by the material supplier.

[0029] Furthermore, it should be understood that the one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise specified. It should also be understood that the combination connection relationship between one or more devices / apparatuses mentioned in the present invention does not exclude the presence of other devices / apparatuses before or after the combination device / apparatus, or the insertion of other devices / apparatuses between two explicitly mentioned devices / apparatuses, unless otherwise specified. Furthermore, unless otherwise specified, the numbering of each method step is merely a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of the present invention. Changes or adjustments to their relative relationships, without substantially changing the technical content, should also be considered within the scope of the present invention.

[0030] In the following examples, all reagents, materials and instruments used are commercially available unless otherwise specified.

[0031] Example 1

[0032] (1) Remove fat-soluble substances and pigments

[0033] The tobacco stems were freeze-dried to remove moisture, crushed into powder by a high-speed grinder, and passed through a 60-mesh sieve; 1.005 g of the powdered sample was weighed, 25 mL of ethyl acetate was added, and ultrasonicated for 30 min, centrifuged (4000 r / min, 5 min), and the resulting precipitate was ultrasonicated a second time using the above method. The precipitate is the tobacco stem filter residue from which the fat-soluble substances have been removed.

[0034] (2) Ethanol ultrasonic extraction to remove soluble sugars and phenolic substances

[0035] Add 25 mL of 85% ethanol to the above precipitate, sonicate for 30 minutes (2 times), centrifuge (4000 r / min, 5 minutes), wash the filter residue repeatedly with cold water, add DNS reagent to the supernatant, heat in a boiling water bath for 5 minutes, and judge whether the soluble sugar in the filter residue has been completely removed by whether color is developed.

[0036] (3) Enzymatic hydrolysis with α-amylase and saccharifying enzyme to remove starch macromolecules

[0037] To the above filter residue, 40 mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 6) and 2000 u / g of α-amylase were added, and hydrolysis was carried out in a 60°C constant temperature water bath for 2.5 h. Subsequently, 40 mL of acetic acid-sodium acetate buffer (pH 4.5) and 10,000 u / g of saccharifying enzyme were added, and hydrolysis was carried out in a 60°C constant temperature water bath for 1.5 h. The mixture was centrifuged (4000 rpm, 5 min), and the filter residue was repeatedly washed with cold water.

[0038] (4) Cellulase, pectinase, and protease enzymes remove cellulose, pectin, protein and other macromolecules

[0039] To the filter residue, 40 mL of citric acid-trisodium citrate buffer (pH 4.7) was added, followed by cellulase, pectinase, and protease (at 150 u / g, 5000 u / g, and 5000 u / g, respectively). The mixture was shaken and placed in a constant temperature shaking incubator at 45°C (90 rpm) for enzymatic hydrolysis for 10 h. After enzymatic hydrolysis, the hydrolyzate was centrifuged (4000 r / min, 5 min) and the filter residue was repeatedly washed with cold water.

[0040] (5) Add 26 mL of NaOH / urea solution to the filter residue in step (4), wherein the mass fraction of sodium hydroxide is 8% and the mass fraction of urea is 12%; stir at -9°C for 27 minutes, then centrifuge and wash with pure water until the supernatant is neutral to obtain the lower layer filter residue.

[0041] (6) adding 19 mL of a 17.5% sulfuric acid solution to the filter residue of step (5), stirring the mixture at 101° C. for 35 min, and centrifuging the mixture to obtain a supernatant and a lower filter residue. The supernatant was retained to determine the acid-soluble lignin content in the filtrate.

[0042] (7) The lower filter residue from (6) was washed with pure water until neutral, then washed three times with acetone, and then dried in an oven at 55°C for 21 minutes. 23 mL of NaOH / urea solution was added, wherein the mass fraction of sodium hydroxide in the solution was 8% and the mass fraction of urea was 12%. The mixture was stirred at -12°C for 31 minutes, then centrifuged and washed with pure water until the supernatant was neutral. The filter residue was freeze-dried and weighed. The acid-insoluble lignin content was calculated.

[0043] (8) Take 1 mL of the supernatant and dilute it 3 times. Measure its absorbance at 325 nm and calculate the concentration of acid-soluble lignin using the acid-soluble lignin standard curve (the standard curve is as follows: Figure 1 As shown), the content of acid-soluble lignin was calculated to be 0.95%, the content of acid-insoluble lignin was 7.38%, and the total lignin content was 8.33%.

[0044] Example 2

[0045] (1) Remove fat-soluble substances and pigments

[0046] The tobacco stems were freeze-dried to remove moisture, crushed into powder using a high-speed grinder, and passed through a 60-mesh sieve. 1.004 g of the powder sample was weighed, added with 25 mL of ethyl acetate, and sonicated for 30 min. The mixture was then centrifuged (4000 rpm for 5 min). The resulting precipitate was sonicated a second time using the above method.

[0047] (2) Ethanol ultrasonic extraction to remove soluble sugars and phenolic substances

[0048] Add 25 mL of 85% ethanol to the above precipitate, sonicate for 30 min (2 times), centrifuge (4000 rpm, 5 min), wash the filter residue repeatedly with cold water, add DNS reagent to the supernatant, heat in a boiling water bath for 5 min, and judge whether the soluble sugar in the filter residue has been completely removed by whether color develops;

[0049] (3) Enzymatic hydrolysis with α-amylase and saccharifying enzyme to remove starch macromolecules

[0050] To the above filter residue, 40 mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH = 6) and 2000 u / g of α-amylase were added, and the mixture was hydrolyzed in a constant temperature water bath at 60°C for 2.5 h. Subsequently, 40 mL of acetic acid-sodium acetate buffer (pH = 4.5) and 10000 u / g of saccharifying enzyme were added, and the mixture was hydrolyzed in a constant temperature water bath at 60°C for 1.5 h. The mixture was centrifuged (4000 r / min, 5 min), and the filter residue was repeatedly washed with cold water.

[0051] (4) Cellulase, pectinase, and protease enzymes remove cellulose, pectin, protein and other macromolecules

[0052] To the above filter residue, 40 mL of citric acid-trisodium citrate buffer with a pH of 4.7 was added, and cellulase, pectinase, and protease (doses of 150 u / g, 5000 u / g, and 5000 u / g, respectively) were added, shaken well, and placed in a constant temperature shaking incubator at 45°C (90 rpm) for enzymatic hydrolysis for 10 h; after enzymatic hydrolysis, the enzymatic hydrolyzate was centrifuged (4000 r / min, 5 min), and the filter residue was repeatedly washed with cold water;

[0053] (5) adding 25 mL of NaOH / urea solution to the filter residue of step (4), wherein the mass fraction of sodium hydroxide is 8% and the mass fraction of urea is 12%; stirring at -10°C for 30 minutes, then centrifuging and washing with pure water until the supernatant is neutral to obtain the lower layer filter residue;

[0054] (6) adding 20 mL of a 17.5% sulfuric acid solution to the filter residue of step (5), stirring the mixture at 100° C. for 30 min, and centrifuging to obtain a supernatant and a lower filter residue. The supernatant was retained to determine the content of acid-soluble lignin in the filtrate.

[0055] (7) Wash the lower filter residue with pure water until neutral, then wash it three times with acetone, and then dry it in a 50°C oven for 20 minutes. Then add 25 mL of NaOH / urea solution, in which the mass fraction of sodium hydroxide is 8% and the mass fraction of urea is 12%, stir it at -10°C for 30 minutes, then centrifuge it, wash it with pure water until the supernatant is neutral, freeze-dry the filter residue, weigh it, and calculate the acid-insoluble lignin content;

[0056] (8) Take 1 mL of the supernatant and dilute it 3 times. Measure its absorbance at 325 nm. The concentration of acid-soluble lignin is obtained by the acid-soluble lignin standard curve. The calculated content of acid-soluble lignin is 1.16%, the content of acid-insoluble lignin is 8.15%, and the total lignin content is 9.31%.

[0057] Example 3

[0058] (1) Remove fat-soluble substances and pigments

[0059] The tobacco stems were freeze-dried to remove moisture, crushed into powder using a high-speed grinder, and passed through a 60-mesh sieve. 1.004 g of the powder sample was weighed, added with 25 mL of ethyl acetate, and sonicated for 30 min. The mixture was then centrifuged (4000 rpm for 5 min). The resulting precipitate was sonicated a second time using the above method.

[0060] (2) Ethanol ultrasonic extraction to remove soluble sugars and phenolic substances

[0061] Add 25 mL of 85% ethanol to the above precipitate, sonicate for 30 min (2 times), centrifuge (4000 rpm, 5 min), wash the filter residue repeatedly with cold water, add DNS reagent to the supernatant, heat in a boiling water bath for 5 min, and judge whether the soluble sugar in the filter residue has been completely removed by whether color develops;

[0062] (3) Enzymatic hydrolysis with α-amylase and saccharifying enzyme to remove starch macromolecules

[0063] To the above filter residue, 40 mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH = 6) and 2000 u / g of α-amylase were added, and the mixture was hydrolyzed in a constant temperature water bath at 60°C for 2.5 h. Subsequently, 40 mL of acetic acid-sodium acetate buffer (pH = 4.5) and 10000 u / g of saccharifying enzyme were added, and the mixture was hydrolyzed in a constant temperature water bath at 60°C for 1.5 h. The mixture was centrifuged (4000 r / min, 5 min), and the filter residue was repeatedly washed with cold water.

[0064] (4) Cellulase, pectinase, and protease enzymes remove cellulose, pectin, protein and other macromolecules

[0065] To the above filter residue, 40 mL of citric acid-trisodium citrate buffer with a pH of 4.7 was added, and cellulase, pectinase, and protease (doses of 150 u / g, 5000 u / g, and 5000 u / g, respectively) were added, shaken well, and placed in a constant temperature shaking incubator at 45°C (90 rpm) for enzymatic hydrolysis for 10 h; after enzymatic hydrolysis, the enzymatic hydrolyzate was centrifuged (4000 r / min, 5 min), and the filter residue was repeatedly washed with cold water;

[0066] (5) adding 20 mL of NaOH / urea solution to the filter residue of step (4), wherein the mass fraction of sodium hydroxide is 8% and the mass fraction of urea is 12%; stirring at -8°C for 25 minutes, then centrifuging and washing with pure water until the supernatant is neutral to obtain the lower layer filter residue;

[0067] (6) adding 18 mL of 17.5% sulfuric acid solution to the filter residue of step (5), stirring the mixture at 97° C. for 25 min, and centrifuging to obtain a supernatant and a lower filter residue. The supernatant was retained to determine the content of acid-soluble lignin in the filtrate.

[0068] (7) Wash the lower filter residue with pure water until neutral, then wash it three times with acetone, and then dry it in a 47°C oven for 15 minutes. Then add 20 mL of NaOH / urea solution, in which the mass fraction of sodium hydroxide is 8% and the mass fraction of urea is 12%, stir it at -8°C for 25 minutes, then centrifuge it, wash it with pure water until the supernatant is neutral, freeze-dry the filter residue, weigh it, and calculate the acid-insoluble lignin content;

[0069] (8) Take 1 mL of the supernatant and dilute it 3 times. Measure its absorbance at 325 nm. The concentration of acid-soluble lignin is obtained by the acid-soluble lignin standard curve. The calculated content of acid-soluble lignin is 1.16%, the content of acid-insoluble lignin is 5.28%, and the total lignin content is 6.44%.

[0070] Example 4

[0071] (1) Remove fat-soluble substances and pigments

[0072] The tobacco stems were freeze-dried to remove moisture, crushed into powder using a high-speed grinder, and passed through a 60-mesh sieve. 1.001 g of the powdered sample was weighed, added to 25 mL of ethyl acetate, and sonicated for 30 minutes. The mixture was then centrifuged (4000 rpm for 5 minutes). The resulting precipitate was sonicated a second time using the above method.

[0073] (2) Ethanol ultrasonic extraction to remove soluble sugars and phenolic substances

[0074] Add 25 mL of 85% ethanol to the above precipitate, sonicate for 30 min (2 times), centrifuge (4000 rpm, 5 min), wash the filter residue repeatedly with cold water, add DNS reagent to the supernatant, heat in a boiling water bath for 5 min, and judge whether the soluble sugar in the filter residue has been completely removed by whether color develops;

[0075] (3) Enzymatic hydrolysis with α-amylase and saccharifying enzyme to remove starch macromolecules

[0076] To the above filter residue, 40 mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH = 6) and 2000 u / g of α-amylase were added, and the mixture was hydrolyzed in a constant temperature water bath at 60°C for 2.5 h. Subsequently, 40 mL of acetic acid-sodium acetate buffer (pH = 4.5) and 10000 u / g of saccharifying enzyme were added, and the mixture was hydrolyzed in a constant temperature water bath at 60°C for 1.5 h. The mixture was centrifuged (4000 r / min, 5 min), and the filter residue was repeatedly washed with cold water.

[0077] (4) Cellulase, pectinase, and protease enzymes remove cellulose, pectin, protein and other macromolecules

[0078] To the above filter residue, 40 mL of citric acid-trisodium citrate buffer with a pH of 4.7 was added, and cellulase, pectinase, and protease (doses of 150 u / g, 5000 u / g, and 5000 u / g, respectively) were added, shaken well, and placed in a constant temperature shaking incubator at 45°C (90 rpm) for enzymatic hydrolysis for 10 h; after enzymatic hydrolysis, the enzymatic hydrolyzate was centrifuged (4000 r / min, 5 min), and the filter residue was repeatedly washed with cold water;

[0079] (5) adding 22 mL of NaOH / urea solution to the filter residue of step (4), wherein the mass fraction of sodium hydroxide is 8% and the mass fraction of urea is 12%; stirring at -11°C for 31 minutes, then centrifuging and washing with pure water until the supernatant is neutral to obtain the lower layer filter residue;

[0080] (6) adding 20 mL of 17.5% sulfuric acid solution to the filter residue of step (5), stirring the mixture at 95° C. for 29 min, and centrifuging to obtain a supernatant and a lower filter residue. The supernatant was retained to determine the content of acid-soluble lignin in the filtrate.

[0081] (7) Wash the lower filter residue with pure water until neutral, then wash it three times with acetone, and then dry it in a 45°C oven for 23 minutes. Then add 22 mL of NaOH / urea solution, in which the mass fraction of sodium hydroxide is 8% and the mass fraction of urea is 12%, stir it at -9°C for 29 minutes, then centrifuge it, wash it with pure water until the supernatant is neutral, freeze-dry the filter residue, weigh it, and calculate the acid-insoluble lignin content;

[0082] (8) Take 1 mL of the supernatant and dilute it 3 times. Measure its absorbance at 325 nm. The concentration of acid-soluble lignin is obtained by the acid-soluble lignin standard curve. The calculated content of acid-soluble lignin is 1.13%, the content of acid-insoluble lignin is 6.32%, and the total lignin content is 7.45%.

[0083] Example 5

[0084] (1) Remove fat-soluble substances and pigments

[0085] The tobacco stems were freeze-dried to remove moisture, crushed into powder using a high-speed grinder, and passed through a 60-mesh sieve. 1.006 g of the powder sample was weighed, added to 25 mL of ethyl acetate, and sonicated for 30 min. The mixture was then centrifuged (4000 rpm for 5 min). The resulting precipitate was sonicated a second time using the above method.

[0086] (2) Ethanol ultrasonic extraction to remove soluble sugars and phenolic substances

[0087] Add 25 mL of 85% ethanol to the above precipitate, sonicate for 30 min (2 times), centrifuge (4000 rpm, 5 min), wash the filter residue repeatedly with cold water, add DNS reagent to the supernatant, heat in a boiling water bath for 5 min, and judge whether the soluble sugar in the filter residue has been completely removed by whether color develops;

[0088] (3) Enzymatic hydrolysis with α-amylase and saccharifying enzyme to remove starch macromolecules

[0089] To the above filter residue, 40 mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH = 6) and 2000 u / g of α-amylase were added, and the mixture was hydrolyzed in a constant temperature water bath at 60°C for 2.5 h. Subsequently, 40 mL of acetic acid-sodium acetate buffer (pH = 4.5) and 10000 u / g of saccharifying enzyme were added, and the mixture was hydrolyzed in a constant temperature water bath at 60°C for 1.5 h. The mixture was centrifuged (4000 r / min, 5 min), and the filter residue was repeatedly washed with cold water.

[0090] (4) Cellulase, pectinase, and protease enzymes remove cellulose, pectin, protein and other macromolecules

[0091] To the above filter residue, 40 mL of citric acid-trisodium citrate buffer with a pH of 4.7 was added, and cellulase, pectinase, and protease (doses of 150 u / g, 5000 u / g, and 5000 u / g, respectively) were added, shaken well, and placed in a constant temperature shaking incubator at 45°C (90 rpm) for enzymatic hydrolysis for 10 h; after enzymatic hydrolysis, the enzymatic hydrolyzate was centrifuged (4000 r / min, 5 min), and the filter residue was repeatedly washed with cold water;

[0092] (5) adding 22 mL of H2O / urea solution to the filter residue of step (4), wherein the mass fraction of sodium hydroxide is 8% and the mass fraction of urea is 12%; stirring at -12°C for 35 minutes, then centrifuging and washing with pure water until the supernatant is neutral to obtain the lower layer filter residue;

[0093] (6) adding 22 mL of a 17.5% sulfuric acid solution to the filter residue of step (5), stirring the mixture at 103° C. for 27 min, and centrifuging the mixture to obtain a supernatant and a lower filter residue. The supernatant was retained to determine the acid-soluble lignin content in the filtrate.

[0094] (7) Wash the lower filter residue with pure water until neutral, then wash it three times with acetone, and then dry it in a 49°C oven for 25 minutes. Then add 28 mL of NaOH / urea solution, in which the mass fraction of sodium hydroxide is 8% and the mass fraction of urea is 12%, stir it at -11°C for 33 minutes, then centrifuge it, wash it with pure water until the supernatant is neutral, freeze-dry the filter residue, weigh it, and calculate the acid-insoluble lignin content;

[0095] (8) Take 1 mL of the supernatant and dilute it 3 times. Measure its absorbance at 325 nm. The concentration of acid-soluble lignin is obtained by the acid-soluble lignin standard curve. The calculated content of acid-soluble lignin is 1.18%, the content of acid-insoluble lignin is 7.01%, and the total lignin content is 8.19%.

[0096] The above embodiments are merely illustrative of the principles and effects of this application and are not intended to limit this application. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical concepts disclosed in this application shall be covered by the claims of this application.

Claims

1. A method for determining the lignin content in tobacco stems, comprising the following steps: S1. Pre-treating the tobacco stem sample to obtain tobacco stem filter residue, wherein the pre-treatment includes sequentially removing fat-soluble substances and pigments; soluble sugars and phenolic substances; and starch macromolecules from the tobacco stem sample; cellulose, pectin, and protein; Using ethyl acetate to remove fat-soluble substances and pigments in the tobacco stem sample to obtain a first filter residue; removing soluble sugars and phenolic substances from the first filter residue using an ethanol solution to obtain a second filter residue; Using α-amylase and saccharifying enzyme to remove starch macromolecules in the second filter residue to obtain a third filter residue; using cellulase, pectinase and protease to remove cellulose, pectin and protein in the third filter residue to obtain tobacco stem filter residue; S2. Adding a mixed solution of NaOH and urea to the tobacco stem residue obtained in step S1 to dissolve the mixture and then separate the mixture to obtain a residue; S3, adding sulfuric acid solution to the filter residue obtained in step S2 to dissolve it and then separating the layers to obtain an upper clear liquid and a lower filter residue; S4. Determine the content of acid-soluble lignin in the supernatant by spectrophotometry; S5. Wash the lower filter residue to make it neutral and then dry it. Then add a mixed solution of NaOH and urea to dissolve it and separate it to obtain the filter residue to be measured. Then, dry and weigh the filter residue to be measured to obtain the content of acid-insoluble lignin.

2. The method for determining the lignin content in tobacco stems according to claim 1, wherein The pre-processing method in step S1 includes the following technical features: A1. The tobacco stem sample is tobacco stem powder obtained by crushing.

3. The method for determining the lignin content in tobacco stems according to claim 2, wherein: A1 features include one or more of the following technical features: A11, the tobacco stem powder is freeze-dried and then crushed to obtain; A12. The particle size of the tobacco stem powder is 30 mesh to 120 mesh.

4. The method for determining the lignin content in tobacco stems according to claim 2, wherein: The step of removing fat-soluble substances and pigments from the tobacco stem sample using ethyl acetate to obtain a first filter residue; include Washing the tobacco stem samples with ethyl acetate to remove fat-soluble substances and pigments; or The ratio of tobacco stem sample g: ethyl acetate mL is 1:10~30.

5. The method for determining the lignin content in tobacco stems according to claim 2, wherein: The method of removing soluble sugars and phenolic substances from the first filter residue by using an ethanol solution to obtain a second filter residue comprises: Washing with an ethanol solution to remove soluble sugars and phenolic substances in the first filter residue; or; Tobacco stem sample g: alcohol solution mL is 1:10~50; or; The mass fraction of ethanol in the ethanol solution is 30% to 100%; or; The second filter residue is washed with water.

6. The method for determining the lignin content in tobacco stems according to claim 2, wherein: The method of using α-amylase and saccharifying enzyme to remove starch macromolecules in the second filter residue to obtain a third filter residue comprises: adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer and α-amylase to the second filter residue, performing a first hydrolysis, then adding acetic acid-sodium acetate buffer solution and saccharifying enzyme, performing a second hydrolysis, filtering, and washing to obtain the third filter residue.

7. The method for determining the lignin content in tobacco stems according to claim 6, wherein: Include one or more of the following conditions: B1, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution pH = 5~6.5; B2, the dosage of α-amylase is 1000 u / g ~3000 u / g; B3. The conditions for the first and second hydrolysis are 50℃~70℃ water bath hydrolysis for 0.5h~5h; B4, pH of acetic acid-sodium acetate buffer solution = 3.5~5.5; B5. The dosage of saccharifying enzyme is 5000 u / g ~20000 u / g.

8. The method for determining the lignin content in tobacco stems according to claim 2, wherein: The method of removing cellulose, pectin and protein from the third filter residue by using cellulase, pectinase and protease to obtain tobacco stem filter residue comprises: First, add citric acid-trisodium citrate buffer to the third filter residue, then add cellulase, pectinase and protease respectively for enzymatic hydrolysis, and then filter and wash with water to obtain tobacco stem filter residue.

9. The method for determining the lignin content in tobacco stems according to claim 8, wherein: Include one or more of the following technical features: C1, citric acid-trisodium citrate buffer pH = 3.5~5.5; C2, cellulase dosage is 100 u / g ~300 u / g; C3, the dosage of pectinase is 1000u / g ~8000u / g; C4, the dosage of protease is 1000u / g ~8000u / g; C5. The enzymatic hydrolysis conditions are to keep the enzyme in a shaking incubator at 35℃~55℃ for 5 h~15 h.

10. The method for determining the lignin content in tobacco stems according to claim 1, wherein Step S2 or S5 includes one or more of the following technical features: D1: In the mixed solution of NaOH and urea, the mass fraction of NaOH is 5%~10%; In the mixed solution of D2, NaOH and urea, the mass fraction of urea is 6%~20%; D3, dissolution treatment: stirring at -12℃~-8℃ for 25 min-35 min; D4, tobacco stem sample mass g: added NaOH and urea mixed solution volume mL is 1:15~50; D5, the separation comprises: performing centrifugal separation after dissolution to remove NaOH and urea; D6. Wash the filter residue and the filter residue to be measured with water until the washing water is neutral.

11. The method for determining the lignin content in tobacco stems according to claim 1, wherein: Step S3 includes one or more of the following technical features: E1. The mass fraction of sulfuric acid in the sulfuric acid solution is 15%~20%; E2, tobacco stem sample mass g: sulfuric acid solution volume mL added is 1:10~30; E3. After adding sulfuric acid solution, the reaction conditions are: stirring at 90℃~110℃ for 15 min-45 min.

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