Method for determining soluble sugar, starch, cellulose and hemicellulose in sample

Abstract

The invention belongs to the field of detection and analysis, and particularly relates to a method for determining soluble sugar, starch, cellulose and hemicellulose.The method comprises the steps that a saturated salt water solution containing ethanol is used for extracting the sample, solid-liquid separation is conducted, and a first liquid-phase matter and a first solid-phase matter are obtained; extracting the first solid-phase substance by using a dimethyl sulfoxide solution, and carrying out solid-liquid separation to obtain a second liquid-phase substance and a second solid-phase substance; performing acidolysis on the second liquid-phase substance and a dilute sulfuric acid solution to obtain a first acidolysis product; carrying out two-step acidolysis on the second solid-phase substance, and carrying out solid-liquid separation to obtain a third liquid-phase substance; respectively detecting the first liquid-phase substance, the first acidolysis product and the third liquid-phase substance by adopting ion chromatography, and respectively calculating the content of soluble sugar, the content of starch, the content of cellulose and the content of hemicellulose in the sample according to spectrograms. The method disclosed by the invention is high in accuracy, high in sensitivity, good in repeatability and high in detection speed.

Description

Technical Field

[0001] This invention belongs to the field of detection and analysis, specifically relating to a method for determining soluble sugars, starch, cellulose and hemicellulose in a sample. Background Technology

[0002] Tobacco contains a rich variety of carbohydrate compounds, including monosaccharides (such as glucose and fructose), oligosaccharides (such as sucrose, maltose, and trehalose), polysaccharides (such as cellulose and starch), complex polysaccharides, glycosides, and glycolipids. These diverse carbohydrate compounds play a crucial role in balancing nitrogenous compounds, improving the strength and irritation of tobacco smoke, increasing fill power, and enhancing flexibility and processing resistance. Water-soluble sugars in tobacco are among the most important chemical components affecting the flavor of tobacco. Fructose, glucose, sucrose, and maltose are the main components of water-soluble sugars in flue-cured tobacco, accounting for over 99% of the total water-soluble sugar content.

[0003] Starch is an important substance affecting tobacco quality and a key indicator for tobacco quality evaluation. Appropriate starch content can improve the aroma and flavor of cigarettes; however, excessive starch can affect the combustion performance of tobacco, producing irritating, burnt, and off-flavors, negatively impacting the color, aroma, and taste of the tobacco.

[0004] The fibers in tobacco mainly consist of cellulose and hemicellulose, which have significantly different structural compositions. Cellulose is a linear polysaccharide composed of 1,000 to 10,000 D-pyranose glucose units linked by β-1,4 glycosidic bonds. Its basic unit is cellobiose, and the chains are bundled together along their long axes to form microfiber bundles, exhibiting high stability and resistance to chemical degradation. Hemicellulose has a more complex composition, including many high-molecular-weight sugars such as pentosans and hexoses, and it is insoluble in water.

[0005] Therefore, there is an urgent need for a method that can accurately and quickly determine the content of soluble sugars, starches, cellulose and hemicellulose in tobacco samples. Summary of the Invention

[0006] The purpose of this invention is to provide a method for determining the content of soluble sugars, starch, cellulose and hemicellulose in a sample. This method can quickly and accurately determine the content of soluble sugars, starch, cellulose and hemicellulose in a sample, and it has high sensitivity and good repeatability.

[0007] To achieve the above objectives, the present invention provides a method for determining soluble sugars, starch, cellulose, and hemicellulose in a sample, comprising:

[0008] The sample is first extracted using a saturated saline solution containing ethanol, followed by solid-liquid separation to obtain a first liquid phase and a first solid phase. Optionally, the first solid phase is washed with a saturated saline solution containing ethanol, and the collected washing liquid is added to the first liquid phase. The sample is tobacco raw material and / or tobacco product.

[0009] The first liquid phase or its dilution was detected by ion chromatography 1, and the soluble sugar content in the sample was calculated based on the spectrum.

[0010] The first solid phase or the washed first solid phase is extracted a second time using a dimethyl sulfoxide solution to separate the solid and liquid phases, resulting in a second liquid phase and a second solid phase. Optionally, the second solid phase is washed with a dimethyl sulfoxide solution, and the collected washing liquid is incorporated into the second liquid phase.

[0011] The second liquid phase is subjected to a first acid hydrolysis with a sulfuric acid solution of 2%-4% by mass (e.g., 2% by mass, 3% by mass) at 40°C-60°C (e.g., 45°C, 50°C, 55°C) for 1.5-2.5 hours (e.g., 2 hours) to obtain the first acid hydrolysis product.

[0012] The first acid hydrolysis product or its dilution was detected by ion chromatography 2, and the starch content of the sample was calculated based on the chromatogram.

[0013] The second solid phase or the washed second solid phase is subjected to a second acid hydrolysis with a sulfuric acid solution of 30%-50% by mass (e.g., 35% or 40% by mass) at 40°C-60°C (e.g., 50°C) for 1.5-2.5 hours (preferably 1-2 hours, e.g., 1.5 hours) to obtain the second acid hydrolysis product.

[0014] The second acid hydrolysis product is diluted to a sulfuric acid concentration of 1%-3% by mass (e.g., 2% by mass), and a third acid hydrolysis is performed at 100℃-120℃ (e.g., 110℃) for 1.5-4 hours (e.g., 3 hours). The resulting third acid hydrolysis product is then separated into solid and liquid phases to obtain a third liquid phase.

[0015] The third liquid phase or its dilution was detected by ion chromatography, and the content of cellulose and hemicellulose in the sample was calculated based on the chromatogram.

[0016] Regardless of theoretical limitations, saturated saline solutions containing ethanol can dissolve soluble sugars but hardly dissolve starch; therefore, using saturated saline solutions containing ethanol is beneficial for dissolving and separating soluble sugars for testing.

[0017] In any embodiment, the operating conditions of ion chromatography 1 and ion chromatography 2 each independently include one or more of the following:

[0018] (A) The chromatographic column was a Carbo PAC PA 10;

[0019] (B) The detection mode is integral pulse amperometric detection;

[0020] (C) The working electrode is an Au electrode, and the reference electrode is an AgCl / Ag electrode;

[0021] (D) The mobile phases include: mobile phase A is water, mobile phase B is a 150-250 mmol / L NaOH aqueous solution (e.g., 200 mmol / L NaOH aqueous solution), mobile phase C is an aqueous solution containing 0.5-1.5 mol / L NaAc (e.g., 1.0 mol / L NaAc) and 50-150 mmol / L NaOH (e.g., 100 mmol / L NaOH), and mobile phase D is a 5-15 mmol / L NaOH aqueous solution (e.g., 10 mmol / L NaOH aqueous solution).

[0022] (E) The flow rate of the mobile phase is 0.25 mL / min;

[0023] (F) The detection temperature is 20 ℃;

[0024] (G) The injection volume is 25 μL;

[0025] (H) The scan potential is the pulse point waveform shown in the table below;

[0026]

[0027] In any embodiment, the mobile phase elution program for ion chromatography 1 is shown in the table below:

[0028]

[0029] In any embodiment, the mobile phase elution program for the ion chromatography 2 is shown in the table below:

[0030]

[0031] In any embodiment, the soluble sugar concentration in the first liquid phase or its dilution is obtained by external standard analysis based on the spectrum of the first liquid phase or its dilution, and then the soluble sugar content in the sample is calculated according to the following formula;

[0032]

[0033] in, This indicates the soluble sugar content in the sample; Indicates the concentration of soluble sugars in the first liquid phase or its dilution, for example, in g / mL; Indicates the total volume of the first liquid phase or its dilutions, for example, in mL; Indicates the mass of the sample, for example, in grams.

[0034] In any embodiment, the starch concentration in the first acid hydrolysis product or its dilution is determined by external standard analysis based on the spectrum of the first acid hydrolysis product or its dilution, and then the starch content in the sample is calculated according to the following formula, wherein the starch content is expressed as glucose content.

[0035]

[0036] in, This indicates the starch content in the sample; Indicates the starch concentration in the first acid hydrolysis product or its dilution, for example, in g / mL; Indicates the volume of the first acid hydrolysis product or its dilution, for example, in mL; Indicates the mass of the sample, for example, in grams.

[0037] In any embodiment, the concentrations of arabinose, galactose, glucose, xylose, and mannose in the third liquid phase or its dilution are calculated using external standard analysis based on the spectrum of the third liquid phase or its dilution. Then, the cellulose and hemicellulose content in the sample is calculated according to the following formula:

[0038] Ci = Cms × Dilution factor / Ri

[0039] Cellulose content in the sample = C 葡萄糖 ×0.9×V / m,

[0040] Hemicellulose content in the sample = [(C 阿拉伯糖 +C 木糖 )×0.88+(C 半乳糖 +C 甘露糖 [0.90] × V / m

[0041] Where Ri represents the recovery rate of a single sugar; Cms represents the content of a single sugar in the diluted third liquid phase product, with the dilution factor being the dilution factor of the third liquid phase product, or Cms × dilution factor representing the content of a single sugar in the third liquid phase product, for example, in mg / L; Ci represents the converted content of a single sugar in the third liquid phase product, for example, in mg / L; C 葡萄糖 This refers to the glucose content in the converted third liquid phase product, for example, in mg / L; C 半乳糖This refers to the galactose content in the converted third liquid phase product, for example, in mg / L; C 甘露糖 This refers to the mannose content in the converted third liquid phase product, for example, in mg / L; C 木糖 This refers to the xylose content in the converted third liquid phase product, for example, in mg / L; C 阿拉伯糖 The value represents the arabinose content in the converted third liquid phase product, for example, in mg / L; V represents the volume of the third liquid phase product, for example, in L; and m represents the mass of the sample, for example, in mg.

[0042] In any implementation, the recovery rate Ri of a single type of sugar is calculated by the following steps:

[0043] A mixture of various sugars is mixed with a sulfuric acid solution of 30%-50% by mass (e.g., 35% or 40% by mass) to obtain a solution of the mixed sugars before acid hydrolysis.

[0044] The solution before acid hydrolysis of the mixed sugar was operated sequentially according to the steps of the second acid hydrolysis and the third acid hydrolysis described above. After solid-liquid separation, the obtained acid hydrolysis product was collected as the mixed sugar acid hydrolysis solution.

[0045] The mixed sugar solution before acid hydrolysis and the mixed sugar solution after acid hydrolysis were detected by ion chromatography 2 (the operating conditions of ion chromatography 2 are as described above). The contents of various sugars in the mixed sugar solution before acid hydrolysis and the mixed sugar solution after acid hydrolysis were calculated by external standard analysis based on the spectra.

[0046] The recovery rate Ri of a single sugar is obtained by dividing the content of a single sugar in the acid hydrolysis solution of the mixed sugar by the content of the corresponding sugar in the solution before acid hydrolysis of the mixed sugar.

[0047] In any embodiment, during the second extraction step, the ratio of the dimethyl sulfoxide solution used for extraction to the sample is 100-333 mL / g, preferably 125-267 mL / g, for example 180 mL / g.

[0048] In any embodiment, during the first extraction, the ratio of saturated saline containing ethanol to the sample is 100-333 mL / g, preferably 125-267 mL / g, for example 160 mL / g.

[0049] In any implementation, the first extraction takes 1.5-3 hours, for example, 2 hours.

[0050] In any embodiment, the first extraction is performed under ultrasonic conditions; preferably, the ultrasonic power is 1500W; preferably, the ultrasonic frequency is 50Hz.

[0051] In any embodiment, the ethanol concentration in the saturated saline solution containing ethanol used for the first extraction and washing of the first solid phase is independently 45%-60% by mass, for example 50% or 55% by mass.

[0052] In any embodiment, the ratio of the saturated saline solution containing ethanol to the sample used for washing the first solid phase is 5-20 mL / g, for example, 10 mL / g or 15 mL / g.

[0053] In any embodiment, after the first extraction, the first solid phase is allowed to stand for 20-40 minutes (e.g., 30 minutes) before washing.

[0054] In any implementation, the second extraction takes 30-90 minutes, for example, 60 minutes, 70 minutes, or 80 minutes.

[0055] In any embodiment, the second extraction is performed under ultrasonic conditions; preferably, the ultrasonic power is 1500W; preferably, the ultrasonic frequency is 50Hz.

[0056] In any embodiment, the concentration of the dimethyl sulfoxide solution used for the second extraction and washing of the second solid phase is independently 50%-75% by mass, for example 55% by mass, 60% by mass, 65% by mass, and 70% by mass.

[0057] In any embodiment, the ratio of dimethyl sulfoxide solution to sample used for washing the second solid phase is 5-15 mL / g, for example, 10 mL / g.

[0058] In any embodiment, after the second extraction, the second solid phase is allowed to stand for 5-25 minutes (e.g., 10 minutes, 15 minutes, 20 minutes) before washing.

[0059] In any embodiment, during the first acidolysis step, the volume ratio of sulfuric acid solution to the second liquid phase is 3.6:1 to 22.5:1, for example, 5:1, 9:1, 10:1, 12:1, 15:1, 18:1, 20:1, 21:1, or 22:1.

[0060] In any embodiment, the first acid hydrolysis step further includes cooling the first acid hydrolysis product.

[0061] In any embodiment, the ratio of sulfuric acid solution to sample used in the second acid hydrolysis is 6:1-10:1 mL / g, for example 7:1 mL / g, 8:1 mL / g, or 9:1 mL / g.

[0062] In any embodiment, the second and third acidolysis are carried out in a high-pressure resistant device.

[0063] In any embodiment, cooling is performed before solid-liquid separation during the third acid hydrolysis step.

[0064] In any embodiment, solid-liquid separation in the first extraction, second extraction, and third acid hydrolysis steps is carried out using a sand core funnel.

[0065] In any embodiment, the soluble sugar is selected from one or more of arabinose, rhamnose, galactose, glucose, xylose, sucrose, mannose, fructose, and maltose.

[0066] The present invention has achieved at least one of the following beneficial effects:

[0067] 1. The method of the present invention can quickly and accurately determine the content of soluble sugars, starches, cellulose and hemicellulose in a sample.

[0068] 2. The method of this invention has high sensitivity and good repeatability in determining soluble sugars, starches, cellulose and hemicellulose in samples, providing a reliable analytical means for quality evaluation, process improvement and product development of tobacco raw materials and their products. Attached Figure Description

[0069] Figure 1 This is an ion chromatogram of the solution obtained by adjusting the volume of the filtrate from tobacco sample 1# in step (1.2) of Example 1 of the present invention.

[0070] Figure 2 This is an ion chromatogram of the acid hydrolysis product obtained from tobacco sample 1# after volume adjustment in step (2.2) of Example 1 of the present invention.

[0071] Figure 3 This is an ion chromatogram of the diluted solution of the filter residue acid hydrolysis filtrate obtained from tobacco sample 1# in step (3.2) of Example 1 of the present invention. Detailed Implementation

[0072] The embodiments of the present invention will now be clearly and completely described in conjunction with examples. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0073] Example 1

[0074] (1) Determination of soluble sugars

[0075] (1.1) Plotting the standard curve:

[0076] A mixed standard stock solution was prepared using water, consisting of 0.005 g arabinose, 0.005 g rhamnose, 0.005 g galactose, 0.01 g glucose, 0.01 g xylose, 0.01 g sucrose, 0.005 g mannose, 0.01 g fructose, and 0.01 g maltose. The concentrations of arabinose, rhamnose, galactose, glucose, xylose, sucrose, mannose, fructose, and maltose in the mixed standard stock solution were 50 μg / mL, 50 μg / mL, 50 μg / mL, 100 μg / mL, and 100 μg / mL, respectively. 100 μg / mL, 50 μg / mL, 100 μg / mL, and 100 μg / mL were taken from the mixed standard stock solution and diluted 200 times, 100 times, 50 times, 25 times, 10 times, and 5 times respectively with saturated saline solution containing 80% ethanol. The mixed standard solutions of the series concentrations were determined by ion chromatography [the operating conditions of ion chromatography are the same as in step (1.2)]. The standard curves of various sugars were obtained by plotting the concentrations of various sugars in the mixed standard solutions as the abscissa and the peak areas of various sugar responses as the ordinate.

[0077] (1.2) Determination of soluble sugar content:

[0078] Accurately weigh 0.5000g (accurate to 0.0001g) of tobacco sample, add 80mL of saturated saline solution containing 50% ethanol (w / w), and extract under ultrasonic conditions for 2h at an ultrasonic power of 1500W and an ultrasonic frequency of 50Hz. Filter using a sand core funnel and collect the filtrate. Let the filter residue stand in the sand core funnel for 30min, then wash with 5mL of saturated saline solution containing 50% ethanol (w / w) and collect the filtrate. Combine all filtrates, take 2mL of the filtrate, and dilute to 10mL with saturated saline solution containing 50% ethanol (w / w). Analyze the solution after dilution using ion chromatography. The ion chromatogram of the solution after dilution from tobacco sample #1 is shown below. Figure 1 As shown, the peaks, in elution order, are: arabinose, rhamnose, galactose, glucose, xylose, mannose, fructose, sucrose, and maltose. The operating conditions for ion chromatography include:

[0079] Chromatographic column: Carbo PAC PA 10 (2.0 mm × 250 mm, with a PA10 guard column of 2 mm × 50 mm); Detection mode: Integrating pulse amperometric detection; Working electrode: Au electrode; Reference electrode: AgCl / Ag electrode; Scan potential is the pulse point waveform shown in Table 1; Mobile phase: Mobile phase A is water, mobile phase B is 200 mmol / L NaOH aqueous solution, mobile phase C is an aqueous solution containing 1.0 mol / L NaAc and 100 mmol / L NaOH, mobile phase D is 10 mmol / L NaOH aqueous solution; Mobile phase flow rate: 0.25 mL / min; Detection temperature: 20 ℃; Injection volume: 25 μL; Elution program of mobile phase is shown in Table 2.

[0080] Table 1. Scan potentials in ion chromatography

[0081]

[0082] Table 2 Elution program of mobile phase (volume percentage)

[0083]

[0084] *: A negative retention time indicates the time required to rinse the ion chromatography column before injection.

[0085] Calculation process: Substitute the peak areas of various sugar responses into the standard curve to calculate the concentration of various soluble sugars in the solution after the filtrate is brought to a fixed volume. Then, calculate the soluble sugar content in the tobacco sample according to the following formula.

[0086]

[0087] in, This indicates the soluble sugar content (%) in the tobacco sample. This indicates the concentration of soluble sugars (g / mL) in the solution after the filtrate has been diluted to a fixed volume. This indicates the volume (mL) of the solution after the filtrate has been diluted to the desired volume. This indicates the volume ratio of the total filtrate to the volume of the filtrate taken. This indicates the mass (g) of the tobacco sample.

[0088] (2) Determination of starch

[0089] (2.1) Plotting the standard curve:

[0090] A stock solution of glucose standard with a concentration of 0.28 mg / mL was prepared using water. This stock solution was then serially diluted with 0.004% sulfuric acid aqueous solution to obtain standard solutions with concentration gradients of 0.14 μg / mL, 0.28 μg / mL, 0.56 μg / mL, 1.12 μg / mL, and 2.8 μg / mL. Ion chromatography was used to determine the concentration of each standard solution [the operating conditions for ion chromatography were the same as in step (2.2)]. A standard curve was plotted with the concentration of the standard solution on the x-axis and the glucose peak area on the y-axis.

[0091] (2.2) Determination of starch content:

[0092] Add 90 mL of 60% (w / w) dimethyl sulfoxide aqueous solution to the filter residue after rinsing in step (1.2), and extract under ultrasonic conditions for 1 h (ultrasonic power 1500 W, ultrasonic frequency 50 Hz). Filter using a sintered glass funnel and collect the filtrate. Let the filter residue stand in the sintered glass funnel for 10 min, then rinse the filter residue with 5 mL of 60% (w / w) dimethyl sulfoxide aqueous solution and collect the filtrate. Combine all the filtrates for later use. Rinse the filter residue with 5 mL of anhydrous ethanol, air dry, and set aside for later use.

[0093] Transfer 2 mL of the filtrate to a 100 mL Erlenmeyer flask, add 18 mL of 2% sulfuric acid aqueous solution, and acidify in a 50°C water bath for 2 h. Cool the acidification product to room temperature and dilute to 100 mL. Take a small amount of the diluted acidification product for ion chromatography detection. The ion chromatogram of the diluted acidification product obtained from tobacco sample #1 is shown below. Figure 2 As shown in Table 3, the elution procedure of the mobile phase for ion chromatography is as shown in Table 3, and the other ion chromatography operating conditions are the same as in step (1.2).

[0094] Table 3 Elution program of mobile phase (volume percentage)

[0095]

[0096] *: A negative retention time indicates the time required to rinse the ion chromatography column before injection.

[0097] Calculation process: Substitute the peak area of ​​glucose into the standard curve to calculate the starch concentration (calculated as glucose) in the acid hydrolysis product after volume adjustment. Then, calculate the starch content (calculated as glucose) in the tobacco sample according to the following formula.

[0098]

[0099] in, Indicates the starch content (as glucose, %) in the tobacco sample. This indicates the starch concentration (g / mL) in the acid hydrolysis product after volume adjustment. The volume (mL) of the acid hydrolysis product after adjusting the volume. This indicates the volume ratio of the total filtrate to the volume of the filtrate taken. This indicates the mass (g) of the tobacco sample.

[0100] (3) Determination of cellulose and hemicellulose

[0101] (3.1) Plotting the standard curve:

[0102] A mixed standard stock solution was prepared using water, consisting of 0.008 g arabinose, 0.012 g galactose, 0.07 g glucose, 0.007 g xylose, and 0.003 g mannose. The concentrations of arabinose, galactose, glucose, xylose, and mannose in the mixed standard stock solution were 0.032 mg / mL, 0.048 mg / mL, 0.28 mg / mL, 0.028 mg / mL, and 0.012 mg / mL, respectively. A portion of the mixed standard stock solution was transferred and diluted 2000, 1000, 500, 250, and 100 times with 0.004% sulfuric acid aqueous solution to obtain a series of mixed standard solutions with different concentrations. The concentrations of the mixed standard solutions were determined by ion chromatography [the operating conditions for ion chromatography are the same as in step (2.2)]. A standard curve for each sugar was plotted with the concentration of each sugar in the mixed standard solution as the abscissa and the peak area of ​​each sugar response as the ordinate.

[0103] (3.2) Determination of cellulose and hemicellulose content:

[0104] The filter residue obtained in step (2.2) was transferred into a high-pressure flask, and 4 mL of 40% sulfuric acid aqueous solution was added. The mixture was shaken well and subjected to a first acid hydrolysis at 50 °C for 1.5 h. After the first hydrolysis, ultrapure water was added to the high-pressure flask to dilute the solution to a sulfuric acid concentration of 2% by mass.

[0105] Prepare a mixed standard (composed of 0.008 g arabinose, 0.012 g galactose, 0.09 g glucose, 0.007 g xylose and 0.003 g mannose). Transfer the mixed standard to another high-pressure resistant bottle, add 4 mL of 40% sulfuric acid aqueous solution, and shake well to obtain the mixed standard solution before acid hydrolysis. Perform the first acid hydrolysis and dilution process described above on the mixed standard solution before acid hydrolysis.

[0106] The two high-pressure bottles, after dilution, were placed in an oven and subjected to a second acid hydrolysis at 110 °C for 3 hours. After cooling, they were filtered using a G3 sintered glass funnel. The collected filtrates were the acid hydrolysis filtrate of the filter residue and the acid hydrolysis solution of the mixed standard, respectively. The acid hydrolysis filtrate of the filter residue was diluted to 250 mL with ultrapure water to obtain a diluted solution of the acid hydrolysis filtrate of the filter residue. Then, ion chromatography was used to detect the diluted solution of the acid hydrolysis filtrate of the filter residue and the acid hydrolysis solution of the mixed standard, respectively. The operating conditions of ion chromatography were the same as in step (2.2). The ion chromatogram of the diluted solution of the acid hydrolysis filtrate of the filter residue obtained from tobacco sample 1# is shown below. Figure 3 As shown, the peaks, in order of elution, are: arabinose, galactose, glucose, xylose, and mannose.

[0107] Substitute the ion chromatographic peak areas of various sugars in the diluted solution of the acid hydrolysis filtrate of the filter residue and the acid hydrolysis solution of the mixed standard into the standard curve to calculate the content of various sugars in the diluted solution of the acid hydrolysis filtrate of the filter residue and the acid hydrolysis solution of the mixed standard. Then, calculate the cellulose content and hemicellulose content in the tobacco leaf sample according to the following formula.

[0108] Recovery rate Ri = Content of a single sugar in the acid hydrolysate of the mixed standard / Content of a single sugar in the solution before acid hydrolysis of the mixed standard

[0109] Ci = Cms × Dilution factor / Ri

[0110] Cellulose content = 100% × (C glucose × V × 0.9) / M0

[0111] Hemicellulose content = 100% × [(C galactose + C mannose) × 0.9 + (C xylose + C arabinose) × 0.88] × V / M0

[0112] in:

[0113] Ri represents the recovery rate of a single type of sugar;

[0114] Cms represents the content (mg / L) of a single type of sugar in the diluted solution of the acid hydrolysis filtrate of the filter residue.

[0115] Ci represents the content (mg / L) of a single type of sugar in the converted acid hydrolysis filtrate of the filter residue.

[0116] C glucose is the glucose content (mg / L) in the acid hydrolysis filtrate of the filter residue after conversion.

[0117] C-galactose refers to the galactose content (mg / L) in the acid hydrolysis filtrate of the filter residue after conversion.

[0118] C_mannose is the mannose content (mg / L) in the acid hydrolysis filtrate of the filter residue after conversion.

[0119] C xylose is the xylose content (mg / L) in the acid hydrolysis filtrate of the filter residue after conversion.

[0120] C arabinose is the arabinose content (mg / L) in the acid hydrolysis filtrate of the filter residue after conversion.

[0121] V is the volume (L) of the filtrate from the acid hydrolysis of the filter residue;

[0122] M0 represents the mass (g) of the tobacco sample.

[0123] In the above formula, "0.9" is the dehydration correction factor for dextran, galactan, or mannan; "0.88" is the dehydration correction factor for xylan or arabinogalactan.

[0124] The results of determining the mass content of soluble sugar, starch, cellulose and hemicellulose in tobacco samples 1# to 4# using the above method are shown in Table 4-5.

[0125] Table 4. Results of soluble sugar content (mg / g, n=5) in tobacco samples 1# to 4#

[0126]

[0127] Table 5. Results of the mass content of starch, cellulose, and hemicellulose in tobacco samples 1# to 4# (n=5)

[0128]

[0129] Example 2: Accuracy, sensitivity, and repeatability of the method

[0130] (1) Validation of the accuracy, sensitivity and repeatability of the method for determining soluble sugars in tobacco samples according to the present invention:

[0131] Using the standard curves of various sugars in step (1.1) of Example 1, the detection limits of the analytical method were calculated according to the signal-to-noise ratio S / N≥3.

[0132] Spiked recovery experiments were conducted using tobacco sample #4. Multiple 0.5g portions of tobacco sample #4 were weighed and various sugar standards were added to each sample. The amounts added are shown in Table 6. The content of each sugar was determined according to the method in step (1.2) of Example 1. Each determination was performed in parallel for 5 times and the average value of the results was taken. The average spiked recovery rate and RSD were then calculated. The results are shown in Table 6.

[0133] Table 6. Average spiked recoveries, repeatability, and limits of detection for the soluble sugar determination method.

[0134]

[0135] As shown in Table 6, the average spiked recovery rate of the method for determining soluble sugars in this invention is in the range of 90%-101%, indicating that the method for determining soluble sugars in this invention has high accuracy; the RSD of the determination results of the method in this invention is less than 5%, indicating that the method for determining soluble sugars in this invention has good repeatability; the detection limit of the method in this invention is low for various sugars, indicating that the method for determining soluble sugars in this invention has high sensitivity.

[0136] The accuracy, sensitivity, and repeatability of the method of this invention for determining starch in tobacco samples were verified as follows:

[0137] Using the standard curve of glucose in step (2.1) of Example 1, the detection limit of the analytical method was calculated according to the signal-to-noise ratio S / N≥3.

[0138] Spiked recovery experiments were conducted using tobacco sample #4. 0.5 g of tobacco sample #4 was weighed, and starch standard was added (see Table 7 for the amount added). The starch content was determined according to step (2.2) of Example 1. Five measurements were performed, and the average value was taken. The results are shown in Table 7.

[0139] Table 7. Average spiked recoveries, repeatability, and limits of detection for starch determination methods.

[0140]

[0141] As shown in Table 7, the average recovery rate of starch by the method of the present invention is higher than 99%, indicating that the method has high accuracy; the relative standard deviation (RSD) of the determination results is less than 3%, indicating good repeatability; and the limit of detection is 0.003 μg / g, proving that the method has excellent sensitivity.

[0142] (3) Verification of the accuracy, sensitivity and repeatability of the method of the present invention for determining cellulose and hemicellulose in tobacco samples:

[0143] Using the standard curves of various sugars in step (3.1) of Example 1, the detection limits of various sugars were calculated according to the signal-to-noise ratio S / N≥3. Then, the detection limits of various sugars were substituted into the aforementioned calculation formulas for cellulose content and hemicellulose content to obtain the detection limits of cellulose and hemicellulose respectively.

[0144] Spiked recovery experiments were conducted using tobacco sample #4. Multiple 0.5g portions of tobacco sample #4 were weighed and cellulose and hemicellulose standards were added to them respectively. The amounts added are shown in Table 8. The cellulose and hemicellulose contents were determined according to the method in step (3.2) of Example 1. Five parallel determinations were performed and the average value was taken. The results are shown in Table 8.

[0145] Table 8. Average spiked recoveries, repeatability, and limits of detection for the determination of cellulose and hemicellulose.

[0146]

[0147] As shown in Table 8, the average spiked recoveries of cellulose and hemicellulose determined by the method of the present invention are higher than 98%, indicating that the method has high accuracy; the relative standard deviation (RSD) of the determination results is less than 3%, indicating good repeatability; the detection limit is 0.002-0.004 mg / L, confirming that the method of the present invention has good sensitivity.

[0148] The effect of omitting the starch determination step in the comparative example

[0149] "(2) Determination of starch" is omitted. The filter residue after rinsing in step (1.2) is used directly for the determination of cellulose and hemicellulose. The rest of the operation is the same as in Example 1.

[0150] Spiked recovery experiments were conducted using tobacco sample #4. Multiple 0.5g portions of tobacco sample #4 were weighed, and cellulose and hemicellulose standards were added to each sample, as shown in Table 9. The cellulose and hemicellulose contents were determined using the method described above. Five parallel determinations were performed, and the average value was taken. The results are shown in Table 9.

[0151] Table 9. Average spiked recoveries, repeatability, and limits of detection for the determination of cellulose and hemicellulose content using comparative methods.

[0152]

[0153] As shown in Table 9, after omitting the starch determination step, the average recoveries of cellulose and hemicellulose were 80.3% and 79.4%, respectively, lower than the above 98% in Example 1; the relative standard deviations (RSDs) of the determination results increased to 4.2% and 5.0%, higher than 3%; and the limits of detection increased to 0.01 mg / L and 0.008 mg / L. This indicates that omitting the starch determination step significantly reduces the accuracy, repeatability, and sensitivity of the method, affecting the overall determination results.

[0154] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A method for determining soluble sugars, starch, cellulose, and hemicellulose in a sample, comprising: The sample was first extracted using a saturated saline solution containing ethanol, followed by solid-liquid separation to obtain a first liquid phase and a first solid phase. Optionally, the first solid phase is washed with a saturated saline solution containing ethanol, and the collected washing liquid is incorporated into the first liquid phase; the sample is tobacco raw material and / or tobacco product; The first liquid phase or its dilution was detected by ion chromatography 1, and the soluble sugar content in the sample was calculated based on the spectrum. The first solid phase or the washed first solid phase was extracted a second time using a dimethyl sulfoxide solution, and the solid and liquid phases were separated to obtain a second liquid phase and a second solid phase. Optionally, the second solid phase is washed with dimethyl sulfoxide solution, and the collected washing liquid is incorporated into the second liquid phase; The second liquid phase was subjected to a first acid hydrolysis with a sulfuric acid solution of 2%-4% by mass at 40℃-60℃ for 1.5-2.5 hours to obtain the first acid hydrolysis product. The first acid hydrolysis product or its dilution was detected by ion chromatography 2, and the starch content of the sample was calculated based on the chromatogram. The second solid phase or the washed second solid phase is subjected to a second acid hydrolysis with a sulfuric acid solution of 30%-50% by mass concentration at 40℃-60℃ for 1.5-2.5 hours to obtain the second acid hydrolysis product; The second acid hydrolysis product was diluted to a sulfuric acid concentration of 1%-3% by mass, and then subjected to a third acid hydrolysis at 100℃-120℃ for 1.5-4 hours. The resulting third acid hydrolysis product was then separated into solid and liquid phases to obtain a third liquid phase. The third liquid phase or its dilution was detected by ion chromatography, and the content of cellulose and hemicellulose in the sample was calculated based on the chromatogram.

2. The method according to claim 1, wherein, The operating conditions for ion chromatography 1 and ion chromatography 2 each independently include one or more of the following: (A) The chromatographic column was a Carbo PAC PA 10; (B) The detection mode is integral pulse amperometric detection; (C) The working electrode is an Au electrode, and the reference electrode is an AgCl / Ag electrode; (D) The mobile phases include: mobile phase A is water, mobile phase B is a 150-250 mmol / L NaOH aqueous solution, mobile phase C is an aqueous solution containing 0.5-1.5 mol / L NaAc and 50-150 mmol / L NaOH, and mobile phase D is a 5-15 mmol / L NaOH aqueous solution; (E) The flow rate of the mobile phase is 0.25 mL / min; (F) The detection temperature is 20 ℃; (G) The injection volume is 25 μL; (H) The scan potential is the pulse point waveform shown in the table below; 3. The method according to claim 1 or 2, wherein, The mobile phase elution program for ion chromatography 1 is shown in the table below:

4. The method according to any one of claims 1 to 3, wherein, The mobile phase elution program for ion chromatography 2 is shown in the table below:

5. The method according to any one of claims 1 to 4, wherein, The concentration of soluble sugars in the first liquid phase or its dilution is obtained by external standard analysis based on the spectrum of the first liquid phase or its dilution, and then the soluble sugar content in the sample is calculated according to the following formula; in, This indicates the soluble sugar content in the sample; Indicates the concentration of soluble sugars in the first liquid phase or its dilution; Indicates the total volume of the first liquid phase or its dilutions; Indicates the mass of the sample.

6. The method according to any one of claims 1 to 5, wherein, The starch concentration in the first acid hydrolysis product or its dilution is determined by external standard analysis based on the spectrum of the first acid hydrolysis product or its dilution. Then, the starch content in the sample is calculated according to the following formula, wherein the starch content is expressed as glucose content. in, This indicates the starch content in the sample; Indicates the starch concentration in the first acid hydrolysis product or its dilution; Indicates the volume of the first acid hydrolysis product or its dilution; Indicates the mass of the sample.

7. The method according to any one of claims 1 to 6, wherein, The concentrations of arabinose, galactose, glucose, xylose, and mannose in the third liquid phase or its dilution were calculated using external standard analysis based on the spectral data. Then, the cellulose and hemicellulose content in the sample was calculated using the following formula: Ci = Cms × Dilution factor / Ri Cellulose content in the sample = C 葡萄糖 ×0.9×V / m, Hemicellulose content in the sample = [(C 阿拉伯糖 +C 木糖 )×0.88+(C 半乳糖 +C 甘露糖 [0.90] × V / m Where Ri represents the recovery rate of a single sugar; Cms represents the content of a single sugar in the diluted third liquid phase product, with the dilution factor being the dilution factor of the third liquid phase product, or Cms × dilution factor representing the content of a single sugar in the third liquid phase product; Ci represents the converted content of a single sugar in the third liquid phase product; C 葡萄糖 This represents the glucose content in the converted third liquid phase product; C 半乳糖 This represents the galactose content in the converted third liquid phase product; C 甘露糖 The mannose content in the converted third liquid phase product; C 木糖 The xylose content in the converted third liquid phase product; C 阿拉伯糖 V represents the arabinose content in the converted third liquid phase product; V represents the volume of the third liquid phase product; m represents the mass of the sample.

8. The method according to claim 7, wherein, The recovery rate Ri of a single type of sugar is calculated through the following steps: A mixture of various sugars is mixed with a sulfuric acid solution of 30%-50% by mass to obtain a solution of the mixed sugars before acid hydrolysis. The solution before acid hydrolysis of the mixed sugar was operated sequentially according to the steps of the second acid hydrolysis and the third acid hydrolysis. After solid-liquid separation, the liquid phase of the obtained acid hydrolysis product was collected as the mixed sugar acid hydrolysis solution. The mixed sugar solution before acid hydrolysis and the mixed sugar solution after acid hydrolysis were detected by ion chromatography 2. The contents of various sugars in the mixed sugar solution before acid hydrolysis and the mixed sugar solution after acid hydrolysis were calculated by external standard analysis based on the spectra. The recovery rate Ri of a single sugar is obtained by dividing the content of a single sugar in the acid hydrolysis solution of the mixed sugar by the content of the corresponding sugar in the solution before acid hydrolysis of the mixed sugar.

9. The method according to any one of claims 1 to 8, wherein, In the second extraction step, the ratio of dimethyl sulfoxide solution used for extraction to sample is 100-333 mL / g.

10. The method according to any one of claims 1 to 9, characterized in that... One or more of the following: 1) During the first extraction, the ratio of saturated saline solution containing ethanol to the sample is 100-333 mL / g; 2) The first extraction takes 1.5-3 hours; 3) The first extraction is performed under ultrasonic conditions; preferably, the ultrasonic power is 1500W; preferably, the ultrasonic frequency is 50Hz; 4) The ethanol concentration in the saturated saline solution containing ethanol used for the first extraction and washing of the first solid phase was independently 45%-60% by mass; 5) The second extraction should take 30-90 minutes; 6) The second extraction is performed under ultrasonic conditions; preferably, the ultrasonic power is 1500W; preferably, the ultrasonic frequency is 50Hz; 7) The concentration of the dimethyl sulfoxide solution used for the second extraction and washing of the second solid phase was independently 50%-75% by mass; 8) In the first acid hydrolysis step, the volume ratio of sulfuric acid solution to the second liquid phase is 3.6:1-22.5:1; 9) The ratio of sulfuric acid solution to sample used in the second acid hydrolysis is 6:1-10:1 mL / g; 10) The second and third acidolysis processes are carried out in high-pressure resistant equipment; 11) The soluble sugar is selected from one or more of arabinose, rhamnose, galactose, glucose, xylose, sucrose, mannose, fructose and maltose.

Images