A method for measuring guar gum content

Abstract

The present invention belongs to the field of detection and relates to a method for determining guar gum content. The method comprises: extracting guar gum from reconstituted tobacco leaf base by soaking and shaking with an aqueous solution; evaporating the extract to dryness; decomposing the guar gum into mannose and galactose by acid hydrolysis; and then detecting the mannose and galactose contents by ion chromatography, thereby converting the guar gum content. The method can accurately determine the guar gum content in reconstituted tobacco leaf base and is simple to operate.

Description

Technical Field

[0001] The invention belongs to the field of detection and relates to a method for measuring guar gum content, and more particularly to a method for measuring guar gum content in reconstituted tobacco leaf base. Background Art

[0002] Reconstituted tobacco, a sheet-shaped reconstituted product made from raw materials such as tobacco stems, shredded tobacco, and tobacco dust using a papermaking process, is a crucial component of the tobacco leaf group. The refining process produces a large number of fine components, along with tobacco water-soluble substances and fillers, which are present in the slurry system. These components have a significant negative impact on retention and water filtration. The "2022 Report on Green and Low-Carbon Work in the Tobacco Industry" states that chemical oxygen demand (COD) emissions from reconstituted tobacco companies are relatively high, reaching 264.2 tons in 2022, an increase of 49.4 tons, or 23.0%, year-on-year, accounting for 25% of the industry's total COD output. This upward trend necessitates accelerated improvements in wastewater treatment capacity. Therefore, appropriate retention and drainage aids can, on the one hand, increase the retention of fillers and fine fibers in the sheet base, improve whitewater circulation, and reduce pollution; on the other hand, they can enhance the slurry's water filtration properties, improve paper machine dewatering efficiency, and increase paper machine speed, thereby improving the efficiency of reconstituted tobacco sheet papermaking.

[0003] Guar gum, a natural polysaccharide, has been included as a retention and drainage aid in the "List of Permitted Substances for Reconstituted Tobacco" (YQ 44-2014). Guar gum is a linear macromolecule whose hydroxyl groups can form hydrogen bonds with hydrophilic groups. The linear chain lacks non-polar groups, and most primary and secondary hydroxyl groups are located on the outside. The galactose side chains do not block the active alcoholic hydroxyl groups, resulting in a strong hydrogen bonding ability. Guar gum's most significant characteristic is its molecular configuration, which closely resembles that of fiber. This similarity gives it a strong affinity for fibers, allowing it to easily adsorb onto them, providing retention, drainage, and reinforcement. It is an important wet-end retention and drainage aid in the papermaking process of reconstituted tobacco. Appropriate use of guar gum improves sheet base yield while reducing the content of fillers and fine fibers in white water, contributing to improved profitability and green, low-carbon recycling for reconstituted tobacco companies. However, excessive use can cause the sheet base to become too tight, affecting the subsequent coating liquid absorption and the softness of the finished product.

[0004] Guar gum has a molecular weight of 200,000 to 3 million. Its molecular structure is a non-ionic polysaccharide. The main chain is composed of mannose units connected together, and the side chain is composed of a single galactose and connected to the main chain with a (1-6) bond, making it difficult to detect using traditional methods.

[0005] Prior art CN115856107A discloses a method for determining the guar gum content in cigarette paper. The method comprises the following steps: chopping a sample and mixing it uniformly; weighing 0.1-0.5 g of the sample and placing it in a conical flask; adding 2-5 mL of a 50-78% aqueous sulfuric acid solution and hydrolyzing the sample at 20-40° C. for 20-60 min; then diluting the solution with water to a sulfuric acid concentration of 1-5%; and continuing the hydrolysis in an autoclave at 110-130° C. for 20-60 min. After the reaction, 0.1-2% of a purifying agent (polyaluminum chloride, polyacrylamide) is added, and the hydrolyzate is fixed to an appropriate volume. After filtering through a water membrane, the hydrolyzate is analyzed by ion chromatography or liquid chromatography-mass spectrometry. The galactose and mannose contents in the solution are quantitatively determined by an internal standard method or an external standard method, and the guar gum content in the cigarette paper is calculated. This method involves directly adding sulfuric acid to the sample for hydrolysis, which also hydrolyzes the hemicellulose in the cigarette paper. The hydrolysis products include galactose and mannose. Furthermore, the hemicellulose content in cigarette paper is typically 3 to 6 times that of guar gum. Therefore, using the method disclosed in CN115856107A to detect guar gum, the test results far exceed the actual amount used, making it impossible to accurately determine the guar gum content. Using this method to detect a guar gum standard sample that does not contain hemicellulose, the recovery rate was extremely low, far below the disclosed 100% recovery rate, which is further speculated to be due to interference from hemicellulose.

[0006] Currently, there is no standard method for determining the guar gum content in reconstituted tobacco leaf base in the industry standards, and there is an urgent need to establish a method that can accurately determine the guar gum content. Summary of the Invention

[0007] The present invention provides a method for determining guar gum content. The method comprises the following steps: extracting guar gum from reconstituted tobacco leaf base by immersing and shaking with an aqueous solution; evaporating the extract to dryness; decomposing the guar gum into mannose and galactose by acid hydrolysis; and detecting the contents of mannose and galactose by ion chromatography, thereby converting the guar gum content.

[0008] The present invention relates to a method for determining the content of guar gum in reconstituted tobacco leaf base, which comprises the following steps:

[0009] (1) Weigh a reconstituted tobacco leaf sample, soak the sample in water for more than 1.5 hours, and separate the solid and liquid to obtain a supernatant;

[0010] (2) removing water from the supernatant obtained in step (1), adding acid, and reacting at 25° C. to 35° C. for 3.5 h to 5.5 h (preferably 4 to 5 h) to perform an acid hydrolysis, wherein the acid is sulfuric acid with a concentration of 60% to 80% or trifluoroacetic acid with a concentration of 75% to 85%;

[0011] (3) diluting the product of step (2) with water to an acid concentration of 3 to 5% (preferably 3.5 to 4.5%, for example 4%), and reacting at 110° C. to 130° C. for 3 to 5 hours (preferably 3.5 to 4.5 hours, for example 4 hours) for secondary acid hydrolysis;

[0012] (4) detecting the concentrations of galactose and mannose produced by acid hydrolysis in step (3);

[0013] (5) Calculate the guar gum content in the sample based on the galactose and mannose concentrations measured in step (4).

[0014] In certain embodiments, in step (1), the weight of the sample is 0.15 g to 0.4 g.

[0015] In certain embodiments, in step (1), the ratio of soaking water to sample is greater than 75 mL:1 g.

[0016] In certain embodiments, in step (1), the ratio of soaking water to sample is greater than 90 mL:1 g.

[0017] In certain embodiments, in step (1), the soaking time is 1.5 h to 3 h.

[0018] In certain embodiments, in step (1), a shaking operation is further included after soaking.

[0019] In certain embodiments, in step (1), the solid-liquid separation is selected from centrifugation and filtration operations.

[0020] In certain embodiments, in step (1), the weight of the sample is 0.2 g to 0.3 g.

[0021] In certain embodiments, in step (1), the ratio of soaking water to sample is 100 mL to 150 mL: 1 g.

[0022] In certain embodiments, in step (1), the soaking time is 2 to 3 hours.

[0023] In certain embodiments, in step (1), the soaking time is 2 to 2.5 hours.

[0024] In certain embodiments, in step (1), the shaking time is 2 to 10 minutes.

[0025] In certain embodiments, in step (1), the shaking time is 4 to 6 minutes.

[0026] In certain embodiments, in step (1), the shaking time is 5 minutes.

[0027] In certain embodiments, the acid is sulfuric acid at a concentration of 60% to 80%.

[0028] In certain embodiments, the acid is 72% sulfuric acid.

[0029] In certain embodiments, the ratio of the acid to the sample is greater than 5 mL:1 g.

[0030] In certain embodiments, the ratio of the acid to the sample is 5 mL to 10 mL: 1 g.

[0031] In certain embodiments, the ratio of the acid to the sample is 6-9 mL:1 g.

[0032] In certain embodiments, the ratio of the acid to the sample is 7-8 mL:1 g.

[0033] In certain embodiments, the ratio of the acid to the sample is 7.5 mL:1 g.

[0034] In certain embodiments, the primary acid hydrolysis is carried out at 30° C. for 4 to 5 hours.

[0035] In certain embodiments, the secondary acid hydrolysis is carried out at 120° C. for 3.5 to 4.5 hours.

[0036] In certain embodiments, step (3) further comprises: fixing the volume of the sample after the secondary acid hydrolysis is completed.

[0037] In certain embodiments, ion chromatography is used to detect the concentrations of galactose and mannose produced by the acid hydrolysis in step (3).

[0038] In certain embodiments, the ion chromatography is anion chromatography.

[0039] In certain embodiments, the detection conditions include: the mobile phase is an aqueous NaOH solution.

[0040] In certain embodiments, the detection conditions include a flow rate of 0.2 to 0.3 mL / min.

[0041] In certain embodiments, the detection conditions include a flow rate of 0.25 mL / min.

[0042] In certain embodiments, the detection conditions include a temperature of 18-22°C.

[0043] In certain embodiments, the detection conditions include a temperature of 20°C.

[0044] In certain embodiments, the detection conditions include: an injection volume of 20 to 30 μL.

[0045] In certain embodiments, the detection conditions include: an injection volume of 25 μL.

[0046] In certain embodiments, the detection conditions are:

[0047] The mobile phase was NaOH aqueous solution.

[0048] The flow rate is 0.2-0.3 mL / min.

[0049] The temperature is 18-22℃, and

[0050] The injection volume is 20-30 μL.

[0051] In certain embodiments, the mobile phase is a 1.5-2.5 mmol / L NaOH aqueous solution.

[0052] In certain embodiments, the mobile phase is a 1.5-2 mmol / L NaOH aqueous solution.

[0053] In certain embodiments, the calculation formula of step (4) is:

[0054]

[0055] in,

[0056] C gal Indicates the measured mass concentration of galactose, in μg / mL;

[0057] C man It represents the measured mass concentration of mannose, in μg / mL;

[0058] V represents the volume after acid hydrolysis, mL;

[0059] F represents the dilution factor;

[0060] R gal represents galactose recovery, %;

[0061] R man represents mannose recovery, %;

[0062] m0 represents the sample weight, g;

[0063] w represents the moisture content of the sample, %.

[0064] In certain embodiments, the monosaccharide recovery is determined by the following steps:

[0065] (i) preparing a mixed solution of galactose and mannose standards;

[0066] (ii) treating the standard mixed solution according to steps (1) to (4) of the method of the present invention and detecting the mass concentrations of galactose and mannose in the treated sample;

[0067] (iii) Calculate the monosaccharide recovery rate using the formula:

[0068]

[0069] in,

[0070] C i represents the mass concentration of galactose or mannose measured after the standard mixed solution is treated with steps (1) to (4) of the method described in the present invention, in μg / mL;

[0071] C i0 Indicates the prepared mass concentration of galactose or mannose in the standard mixed solution, in μg / mL.

[0072] The beneficial effects achieved by the present invention are:

[0073] (1) The present invention can accurately determine the guar gum content in reconstituted tobacco leaf base: According to the method verification results, the two hydrolyzed monosaccharides of guar gum have a very good linear relationship in the concentration range of 0.1 to 10 μg / mL, and the correlation coefficient R 2 ≥0.9996, the detection limit of galactose is 0.0022μg / mL, and that of mannose is 0.0036μg / mL, which meet the detection requirements; the RSD of the guar gum detection result is 1.58%, which is less than 2%, and has good repeatability; the recovery rate is ≥95.8%, and has high accuracy.

[0074] (2) The present invention has the advantages of rapid and batch determination: the present invention uses a small number of reagents, only water and acid (such as sulfuric acid or trifluoroacetic acid); the present invention is simple to operate, and only acid hydrolysis is required after water extraction; the present invention can be operated in batches. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 , ion chromatogram of guar gum acidolysis to sugar when the concentration of NaOH eluent is 2 mmol / L.

[0076] Figure 2 , the influence of added water volume on the determination results of guar gum.

[0077] Figure 3 , the influence of standing time on the determination results of guar gum.

[0078] Figure 4 , effects of acid hydrolysis time and temperature on guar gum yield. DETAILED DESCRIPTION

[0079] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0080] Example 1

[0081] 1 Materials and Methods

[0082] 1.1 Materials, reagents, and instruments

[0083] Concentrated sulfuric acid (98%, AR) (Sinopharm Group); D-galactose (99%) and D-mannose (99%) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; NaOH solution (50%, Fluka, USA); sodium acetate (99%, J&K); the water used in the experiments met the requirements of GB / T6682 first-class water.

[0084] ICS-3000 ion chromatograph [equipped with an integrating pulsed amperometric detector], CarboPac PA10 chromatographic column (2 mm × 250 mm, with a PA10 guard column (2 mm × 50 mm)) (Dionex, USA); 0.45 μm polyethersulfone microporous filter membrane (Agilent, USA); Milli-Q ultrapure water machine (Millipore, USA); BSA2245-CW electronic balance (sensitivity 0.0001 g, Sartorius, Germany); DWG-9423A drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.); centrifuge tubes (100 mL, Sigma); pressure-resistant glass tubes (140 mL, Shanghai Leigu Instrument Co., Ltd.); and electric heating plate (BIBBY / STUART, UK).

[0085] 1.2 Methods

[0086] 1.2.1 Preparation of standard solution

[0087] Dilute 15 mL of 72% sulfuric acid to 500 mL with pure water as the standard solution dilution solvent. Accurately weigh 50 mg (accurate to 0.1 mg) of each sugar standard sample (D-galactose, D-mannose) and dilute to 50 mL with dilution solvent to prepare a 1000 mg / L sugar standard stock solution. Use a pipette to transfer 10 mL of each sugar standard stock solution to a 100 mL volumetric flask and dilute to volume with the standard solution dilution solvent to prepare a 100 mg / L standard mixed solution. Then dilute the solution stepwise to prepare a series of standard mixed solutions with concentrations of 0.1, 0.5, 1.0, 2.5, 5.0, and 10.0 μg / mL.

[0088] 1.2.2 Sample processing

[0089] Accurately weigh 0.2g of reconstituted tobacco leaf base sample into a centrifuge tube, add 20mL of aqueous solution, let stand at room temperature for 2h, shake for 5min, and then centrifuge for solid-liquid separation. Heat the supernatant on an electric hot plate until nearly dry. Then add 1.5mL of 72% sulfuric acid, shake well, and heat in a 30°C water bath for 4h for primary acid hydrolysis. The sample is then diluted with water to a sulfuric acid concentration of approximately 4%, transferred to a pressure-resistant glass tube, tightly capped, and placed in a 120°C oven for a secondary acid hydrolysis of 4h. After cooling, the sample is diluted to 50mL and analyzed by ion chromatography. Two replicates are performed for each sample.

[0090] 1.2.3 Ion chromatography detection

[0091] Chromatographic column: CarboPac PA10 (2.0 mm × 250 mm, with PA10 guard column, 2 mm × 50 mm); detection mode: integrating pulsed amperometric detection; working electrode: Au electrode; reference electrode: AgCl / Ag; scanning potential: pulse point waveform recommended by Dionex (see Table 1); mobile phase: A: water, B: NaOH (200 mmol / L) solution, C: NaAc (1.0 mol / L)-NaOH (100 mmol / L) solution, D: NaOH (10 mmol / L) solution; flow rate: 0.25 mL / min; temperature: 20°C; injection volume: 25 μL. The mobile phase gradient elution program is shown in Table 2:

[0092] Table 1 Detection waveform of separated sugar

[0093]

[0094] Table 2 High performance anion chromatography gradient elution program

[0095]

[0096] Note: A: water; B: 200mmol / L NaOH; C: 1mol / L NaOAc-100mmol / LNaOH; D: 10mmol / LNaOH.

[0097] 1.2.4 Monosaccharide recovery rate

[0098] Since monosaccharides (D-galactose, D-mannose) will be further hydrolyzed into furfural, 5-hydroxymethylfurfural and furanaldehyde under acidic conditions, resulting in monosaccharide loss, a sugar recovery standard solution with a similar monosaccharide composition ratio needs to be prepared for each sample, and the acid hydrolysis reaction is carried out under the same reaction conditions. The recovery rate of monosaccharides is calculated to calibrate the concentration of monosaccharides obtained after acid hydrolysis of guar gum.

[0099] Prepare a 5.0 μg / mL galactose recovery standard solution according to the method in 1.2.1. Add D-galactose to the blank sample, then add 1.5 mL of 72% sulfuric acid, shake well, and heat in a 30°C water bath for 4 hours for primary acid hydrolysis. Dilute the sample with water to a sulfuric acid concentration of approximately 4%, transfer it to a pressure-resistant glass tube, tighten the lid, and heat in a 120°C oven for 4 hours for secondary acid hydrolysis. After cooling, dilute the sample to 50 mL and analyze by ion chromatography using the method in 1.2.3. Perform two replicates for each sample. The calculated galactose recovery rate is 94.5%.

[0100] Mannose recovery was performed according to the galactose recovery method, and the mannose recovery was calculated to be 92.4%.

[0101] 1.2.5 Moisture content

[0102] Accurately weigh 0.2g of reconstituted tobacco leaf base sample, place it in an oven, and dry it at 40°C. Record the weight after drying. Perform three replicates for each sample. Calculate the moisture content of the reconstituted tobacco leaf base sample to be 10.1%.

[0103] 1.2.6 Data Processing

[0104] The following data processing was performed with reference to the standard “Determination of chemical composition of lignocellulosic biomass raw materials Part 5: Determination of cellulose, hemicellulose, pectin and lignin” (NB / T 34057.5-2017) and other literature.

[0105] Calculate the recovery rate of each monosaccharide

[0106]

[0107] C i ——The mass concentration of each monosaccharide in the sugar recovery standard solution measured after acid hydrolysis, in μg / mL;

[0108] C i0 ——The prepared mass concentration of each monosaccharide in the sugar recovery standard solution before acid hydrolysis, unit: μg / mL.

[0109] Wherein: i represents each sugar, gal and man represent galactose and mannose respectively.

[0110] The amount of guar gum was calculated using the measured concentrations of galactose and mannose, with a dehydration correction factor of 0.90. The calculation formula is:

[0111]

[0112] C gal ——The mass concentration of galactose in the sample measured by the instrument, in μg / mL;

[0113] C man ——The mass concentration of mannose in the sample measured by the instrument, in μg / mL;

[0114] V——fixed volume after acid hydrolysis, mL;

[0115] F——dilution factor;

[0116] R gal ——Galactose recovery rate, %;

[0117] R man ——Mannose recovery rate, %;

[0118] m0——sample weight, g;

[0119] w——water content of sample, %.

[0120] 2 Condition Optimization

[0121] 2.1 Optimization of ion chromatography eluent concentration

[0122] CarboPac PA10 is an anion exchange column that has the characteristics of rapid separation of monosaccharides and good chromatographic resolution. PA10 column can be used stably at pH 0-14, and this column was selected for method development. This experiment investigated the effect of different NaOH eluent concentrations (2mmol / L, 3mmol / L, 4mmol / L, 5mmol / L) on the resolution and peak shape of monosaccharides. The results showed that ( Figure 1 ). When the NaOH eluent concentration was 2 mmol / L, the galactose and mannose peaks from the guar gum acid hydrolysis showed better peak shapes and were completely separated. Therefore, in subsequent experiments, a 2 mmol / L NaOH eluent was ultimately used to separate monosaccharides.

[0123] 2.2 Influence of sample weight

[0124] This experiment investigated the effects of varying sample weights on the determination of guar gum content in reconstituted tobacco leaves. Three replicate samples were weighed for each gradient, and the experiment was conducted according to the method in 1.2.2. The results are shown in Table 3. When the sample weight was 0.1 g, the relative standard deviation of the results was greater than 7%. When the sample weight was 0.2 g or greater, the relative standard deviation was less than 2%. To conserve sample and reagents, the sample weight was set at 0.2 g in this experiment.

[0125] Table 3 Effect of sample weight on guar gum determination results

[0126]

[0127]

[0128] 2.3 Effect of extraction volume

[0129] Since guar gum is easily soluble in water, it can be extracted from reconstituted tobacco leaves using water. This experiment investigated the effect of the volume of water added during extraction on the determination of guar gum content in reconstituted tobacco leaves. Referring to the method in 1.2.2, different volumes of water were used for extraction. The results showed that (see Figure 2 ), when adding too little water, guar gum extraction is incomplete in the reconstituted tobacco leaf. Along with the increase of the volume added with water, the guar gum assay result in the reconstituted tobacco leaf increases gradually. When the volume added with water is increased to more than 20mL, the assay result tends to be stable. Therefore, selecting the volume added with water is 20mL.

[0130] 2.4 Effect of standing time

[0131] Since guar gum is easily soluble in water, it can be extracted from the sheet base by standing, shaking, centrifuging and filtering. If ultrasound or other methods are used, the fibers in the sheet base will be broken and interfere with the experimental results. This experiment investigated the effect of standing time on the determination of guar gum in the sheet base of reconstituted tobacco. Referring to the method in 1.2.2, the standing time was adjusted. The results showed that ( Figure 3 ), with the extension of the standing time, the guar gum determination results gradually increased, when the standing time exceeded 2h, the determination results were almost unchanged. Therefore, the standing time of 2h was selected in this experiment.

[0132] 2.5 Effect of acid hydrolysis time and temperature

[0133] This experiment investigated the effect of acid hydrolysis temperature and time on the yield of guar gum (the ratio of the detected value to the true value * 100%). 0.2g of guar gum was treated with the method 1.2.2, and the acid hydrolysis time (0.5-6h) and acid hydrolysis temperature (30℃ and 40℃) were adjusted to investigate the effect of acid hydrolysis time and temperature on the yield of guar gum. The results are as follows Figure 4 As shown in the figure, when the acid hydrolysis temperature was 30°C, the guar gum yield gradually increased with increasing acid hydrolysis time. After the acid hydrolysis time was extended to 4 hours, the yield stabilized and decreased after the acid hydrolysis time exceeded 5 hours. When the acid hydrolysis temperature was 40°C, although the yield was higher at the same acid hydrolysis time, it stopped increasing after the acid hydrolysis time exceeded 2 hours. This may be due to reactions such as partial carbonization of guar gum at 40°C, resulting in the low result. Therefore, in this experiment, the acid hydrolysis temperature was 30°C and the acid hydrolysis time was 4 hours.

[0134] 3 Method validation

[0135] 3.1 Investigation of linear relationships

[0136] According to the method in 1.2.1, mixed solutions of galactose and mannose standards with concentrations of 0.1, 0.5, 1.0, 2.5, 5.0, and 10.0 μg / mL were prepared. After the mixed solutions of the standards at each concentration were injected separately under the same conditions, the concentration of sugar was used as the horizontal coordinate and the corresponding peak area was used as the vertical coordinate to obtain the corresponding linear equation and correlation coefficient, and the instrument detection limit was calculated based on the three-fold signal-to-noise ratio (S / N=3). The linear relationship and detection limit are shown in Table 4. The results show that the two sugars have a very good linear relationship in the concentration range of 0.1 to 10 μg / mL, and the correlation coefficient R 2 ≥0.9996, detection limit 0.0022μg / mL-0.0036μg / mL.

[0137] Table 4 Linear relationship, correlation coefficient and detection limit of monosaccharide standards

[0138]

[0139] 3.2 Repeatability Experiment

[0140] Select any substrate sample, weigh 8 copies of one sample in parallel, and perform repeatability analysis and test according to the method in 1.2.2. The data are shown in Table 5:

[0141] Table 5 Repeatability experimental data

[0142]

[0143]

[0144] As shown in Table 5, the RSD of guar gum content was 1.58%, which was less than 2%. The established detection method had good repeatability.

[0145] 3.3 Recovery rate experiment

[0146] A 0.2g sample of the film was spiked with guar gum standards at different concentrations, high, medium, and low, respectively. The spiked samples were then spiked using the method described in 1.2.2. The data are shown in Table 6. The results indicate a guar gum recovery rate of ≥95.8%, demonstrating high accuracy.

[0147] Table 6 Sample recovery test results (unit: %)

[0148]

[0149] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solutions of the present invention. They should all be included in the scope of the technical solutions claimed for protection by the present invention.

Claims

1. A method for determining the guar gum content in reconstituted tobacco leaf base, comprising the following steps: (1) Weigh the reconstituted tobacco leaf base sample, soak the sample in water for more than 1.5 hours, and separate the solid and liquid to obtain the supernatant; (2) removing water from the supernatant obtained in step (1), adding acid, and reacting at 25°C to 35°C for 3.5 hours to 5.5 hours to perform an acid hydrolysis, wherein the acid is sulfuric acid with a concentration of 60% to 80% or trifluoroacetic acid with a concentration of 75% to 85%; (3) diluting the product of step (2) with water to an acid concentration of 3-5%, and reacting at 110°C-130°C for 3 h-5 h for secondary acid hydrolysis; (4) detecting the concentrations of galactose and mannose produced by acid hydrolysis in step (3); (5) Calculate the guar gum content in the sample based on the galactose and mannose concentrations measured in step (4).

2. The method according to claim 1, wherein Acid hydrolysis was performed at 25°C~35°C for 4~5 h.

3. The method according to claim 1, wherein The acid concentration is 3.5-4.5%.

4. The method according to claim 1, wherein The acid concentration is 4%.

5. The method according to claim 1, wherein The secondary acid hydrolysis was carried out at 110°C~130°C for 3.5~4.5 h.

6. The method according to claim 1, wherein The secondary acid hydrolysis was carried out at 110°C~130°C for 4 h.

7. The method according to any one of claims 1 to 6, wherein: Step (1) has one or more of the following technical features: (a) Weigh the sample to a weight between 0.15 g and 0.4 g; (b) The ratio of soaking water to sample is greater than 75 mL: 1 g; (c) Soaking time is 1.5 h to 3 h; (d) including a shaking operation after soaking; (e) Solid-liquid separation is selected from centrifugation and filtration operations.

8. The method according to claim 7, wherein: The ratio of soaking water to sample should be greater than 90 mL: 1 g.

9. The method according to claim 7, wherein: Step (1) has one or more of the following technical features: (a) The weight of the sample is 0.2 g to 0.3 g; (b) The ratio of soaking water to sample is 100 mL–150 mL: 1 g; (c) Soaking time is 2 to 3 hours; (d) The oscillation time is 2 to 10 minutes.

10. The method according to claim 9, wherein: Step (1) has one or more of the following technical features: (a) Soaking time is 2 to 2.5 hours; (b) The oscillation time is 4 to 6 minutes.

11. The method according to claim 9, wherein The shaking time was 5 minutes.

12. The method according to any one of claims 1 to 6, wherein the method has one or more of the following technical features: (a) the acid is sulfuric acid having a concentration of 60% to 80%; (b) the ratio of the acid to the sample is greater than 5 mL: 1 g; (c) Acid hydrolysis at 30°C for 4 to 5 hours; (d) Secondary acid hydrolysis: Acid hydrolysis at 120°C for 3.5 h to 4.5 h.

13. The method according to claim 12, which has one or more of the following technical features: (a) the acid is sulfuric acid having a concentration of 72%; (b) The ratio of the acid to the sample is 5 mL to 10 mL: 1 g.

14. The method according to claim 12, wherein: The ratio of the acid to the sample is 6-9 mL: 1 g.

15. The method according to claim 12, wherein: The ratio of the acid to the sample is 7-8 mL: 1 g.

16. The method according to claim 12, wherein: The ratio of the acid to the sample was 7.5 mL: 1 g.

17. The method according to any one of claims 1 to 6, wherein: Step (3) also includes: After the secondary acid hydrolysis is completed, the sample is fixed to volume.

18. The method according to any one of claims 1 to 6, wherein the concentrations of galactose and mannose produced by acid hydrolysis in step (3) are detected by ion chromatography.

19. The method according to claim 18, wherein The ion chromatography is anion chromatography.

20. The method according to claim 18, wherein The detection conditions include: (a) The mobile phase is NaOH aqueous solution; (b) Flow rate: 0.2–0.3 mL / min; (c) Temperature is 18-22°C; (d) The injection volume is 20–30 μL.

21. The method according to claim 20, wherein The detection conditions have one or more of the following technical features: (a) Flow rate: 0.25 mL / min; (b) a temperature of 20°C; (c) The injection volume is 25 μL.

22. The method according to claim 20, wherein The mobile phase is a 1.5-2.5 mmol / L NaOH aqueous solution.

23. The method according to claim 20, wherein The mobile phase is a 1.5-2 mmol / L NaOH aqueous solution.

24. The method according to any one of claims 1 to 6, wherein: The calculation formula for step (5) is: in, C gal Indicates the measured mass concentration of galactose, in µg / mL; C man It represents the measured mass concentration of mannose, in µg / mL; V represents the volume after acid hydrolysis, mL; F represents the dilution factor; R gal represents the galactose recovery rate, %; R man represents the mannose recovery rate, %; m0 represents the sample weight, g; w represents the moisture content of the sample, %.

25. The method according to claim 24, wherein Monosaccharide recovery was determined by the following steps: (i) preparing a mixed solution of galactose and mannose standards; (ii) treating the standard mixed solution according to steps (1) to (4) and detecting the mass concentrations of galactose and mannose in the treated sample; (iii) Calculate the monosaccharide recovery rate using the formula: in, C i represents the mass concentration of galactose or mannose measured after the standard mixed solution is treated in steps (1) to (4), in µg / mL; C i0 Indicates the prepared mass concentration of galactose or mannose in the standard mixed solution, in µg / mL.

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