Method for detecting key macromolecules in reconstructed cut stem and application thereof

Abstract

The application provides a detection method for key macromolecules in reconstituted stem and application thereof, and the detection method comprises the following steps: (1) mixing a to-be-detected sample with a neutral detergent, then mixing with amylase and defoaming agent, and filtering to obtain a pretreated sample; (2) mixing the pretreated sample with acid for acidolysis, and obtaining supernatant and filter residue; (3) performing a derivatization reaction on the supernatant, then performing liquid chromatography detection, obtaining monosaccharide content in the supernatant, and calculating cellulose and hemicellulose content; (4) performing absorbance detection on the supernatant, calculating acid-soluble lignin content according to the detection result; drying and ashing the filter residue, and calculating acid-insoluble lignin content according to the mass change before and after ashing. The detection method provided by the application can simultaneously detect the content of three key macromolecules in reconstituted stem, and realizes integrated analysis of cellulose, hemicellulose and lignin.

Description

Technical Field

[0001] This invention belongs to the field of natural compound content detection, specifically relating to a method for detecting key macromolecules in regenerated stem fibers and its application, and particularly to a simple and efficient method for detecting key macromolecules in regenerated stem fibers and its application. Background Technology

[0002] Cigarette stems are a crucial component of cigarette formulation. Well-processed stems not only increase the filling power of tobacco, reduce cigarette consumption per unit, and improve combustibility and smoke permeability, but also reduce tar content and enhance the color, aroma, and flavor of cigarettes. However, the quality of smoke produced after burning stems is significantly inferior to that of shredded tobacco, primarily manifested as insufficient aroma, excessive off-flavors, a bland taste, weak strength, and higher irritation. Reconstituted stems are a new type of stem product that has emerged in recent years. They are produced by removing components from tobacco stems that contribute little or even negatively to the flavor, and then replacing them with components that contribute to the flavor. Reconstituted stems are characterized by controllable chemical composition, high filling power, and a superior smoking experience. This product avoids the shortcomings of ordinary stems, such as insufficient aroma, bland taste, weak strength, and excessive off-flavors, thus expanding the application range of tobacco stems.

[0003] The compositional analysis and applications of reconstituted stem fibers are influenced by various factors, such as analytical methods, operating parameters, and interactions between different components. These factors involve multiple disciplines, including fluid mechanics, organic chemistry, inorganic chemistry, materials structure, and engineering control. Although we have some preliminary understanding of the processing methods and physical properties of reconstituted stem fibers, systematic and in-depth basic research is still lacking, especially in the analysis of the key macromolecular components of reconstituted stem fibers, which requires further refinement.

[0004] Therefore, the key to analyzing the composition of key macromolecules in reconstituted stem fibers lies in clearly dissecting the physicochemical properties of key macromolecular compounds such as cellulose / hemicellulose and lignin, and combining this with the selection of analytical methods and the separation and screening of key components, to conduct a systematic and comprehensive analysis. Exploring how to efficiently detect the content of key macromolecules in reconstituted stem fibers has become an urgent problem to be solved. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting key macromolecules in reconstituted cellulose fibers and its application, particularly a simple and efficient method for detecting key macromolecules in reconstituted cellulose fibers and its application. The detection method provided by this invention can simultaneously detect the content of three key macromolecules in reconstituted cellulose fibers, achieving integrated analysis of cellulose, hemicellulose, and lignin. The testing method is simple and efficient, yields accurate results, effectively improves detection efficiency, and has broad market prospects.

[0006] To achieve the object of the present application, the present application adopts the following technical solutions:

[0007] In one aspect, the present application provides a detection method of key macromolecules in reconstituted cut stem, including cellulose, hemicellulose and lignin.

[0008] The detection method comprises the following steps:

[0009] (1) mixing the sample to be tested with a neutral detergent, then mixing with amylase and defoaming agent, and filtering to obtain a pretreated sample;

[0010] (2) acid hydrolysis of the pretreated sample with acid to obtain supernatant and residue;

[0011] (3) derivatization reaction of the supernatant, then liquid chromatography detection to obtain monosaccharide content in the supernatant, and calculate cellulose and hemicellulose content;

[0012] (4) absorbance detection of the supernatant, and calculate acid-soluble lignin content according to the detection result; drying and ashing the residue, and calculate acid-insoluble lignin content according to the mass change before and after ashing.

[0013] Steps (3) and (4) are not distinguished in order.

[0014] The above detection method can simultaneously detect the content of three key macromolecules in reconstituted cut stem, realize integrated analysis of cellulose, hemicellulose and lignin, and has simple and efficient test method, accurate determination result, effectively improves detection efficiency, and has broad market prospect.

[0015] Preferably, the neutral detergent in step (1) comprises any one or a combination of at least two of disodium ethylenediaminetetraacetate, sodium tetraborate, sodium dodecyl sulfate, ethylene glycol ether, disodium hydrogen phosphate, ethoxylated surfactant, TO-12, nonylphenol polyoxyethylene ether, alcohol ether or superwet-300, preferably a combination of sodium tetraborate, sodium dodecyl sulfate, nonylphenol polyoxyethylene ether and alcohol ether.

[0016] The above specific neutral detergent can reduce the interference of lipid substances in the sample, emulsify the oil and fat in the sample, fully dissolve the oil and fat in the detergent, and reduce experimental interference.

[0017] Preferably, the alcohol ether comprises any one or a combination of at least two of polyoxyethylene lauryl ether, hydrogenated lauryl ether or stearyl amide polyoxyethylene ether.

[0018] Preferably, the ratio of the sample to be tested to the neutral detergent in step (1) is 1: (10-500) g / mL, such as 1:10 g / mL, 1:50 g / mL, 1:100 g / mL, 1:150 g / mL, 1:200 g / mL, 1:250 g / mL, 1:300 g / mL, 1:350 g / mL, 1:400 g / mL, 1:450 g / mL, or 1:500 g / mL, etc., but not limited to the above-mentioned values, and other values not mentioned in the above-mentioned value range are also applicable.

[0019] Preferably, the amylase in step (1) includes any one or a combination of at least two of α-amylase, β-amylase, γ-amylase, or isoamylase, preferably a combination of α-amylase and isoamylase.

[0020] The above-mentioned amylase effectively eliminates starch in the sample, avoiding the influence of glucose produced by starch in the acidolysis process on the determination of cellulose content.

[0021] Preferably, the antifoaming agent in step (1) includes any one or a combination of at least two of polyethylene glycol, propylene glycol, glycerol, n-octanol, propylene oxide, or ethylene oxide.

[0022] Preferably, the ratio of the sample to be tested to the amylase in step (1) is (20-100):1, such as 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1, etc., but not limited to the above-mentioned values, and other values not mentioned in the above-mentioned value range are also applicable.

[0023] Preferably, the ratio of the sample to be tested to the antifoaming agent in step (1) is 5:1-1:10 g / mL, such as 5:1 g / mL, 4:1 g / mL, 3:1 g / mL, 2:1 g / mL, 1:1 g / mL, 1:2 g / mL, 1:3 g / mL, 1:4 g / mL, 1:5 g / mL, 1:6 g / mL, 1:7 g / mL, 1:8 g / mL, 1:9 g / mL, or 1:10 g / mL, etc., but not limited to the above-mentioned values, and other values not mentioned in the above-mentioned value range are also applicable.

[0024] Preferably, the specific process of acidolysis in step (2) is to mix the pretreated sample with acid and then heat.

[0025] Preferably, the acid includes any one or a combination of at least two of hydrochloric acid, sulfuric acid, or nitric acid, preferably a combination of hydrochloric acid and sulfuric acid.

[0026] The above-mentioned specific acid combination can further improve the effect of acidolysis and improve the accuracy of detection.

[0027] Preferably, the concentration of the acid is 0.5-1.5M, such as 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M or 1.5M, etc., but not limited to the above listed values, other values not listed in the above range are also applicable.

[0028] Preferably, the temperature of the heating is 90-110℃, and the time is 4-8h, wherein the temperature can be 90℃, 95℃, 100℃, 105℃ or 110℃, etc., and the time can be 4h, 5h, 6h, 7h or 8h, etc., but not limited to the above listed values, other values not listed in the above range are also applicable.

[0029] Preferably, the derivatization reagent of the derivatization reaction in step (3) is PMP (1-phenyl-3-methyl-5-pyrazolone).

[0030] Preferably, the mobile phase of the liquid chromatography detection in step (3) comprises acetonitrile and PBS buffer, and the volume ratio of the acetonitrile and PBS buffer is (1-4):(6-9), such as 1:9, 2:8, 3:7 or 4:6, etc., but not limited to the above listed values, other values not listed in the above range are also applicable.

[0031] Preferably, the column temperature of the liquid chromatography detection in step (3) is 25-35℃, and the flow rate of the mobile phase is 0.6-1mL / min, wherein the column temperature can be 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃ or 35℃, etc., and the flow rate of the mobile phase can be 0.6mL / min, 0.7mL / min, 0.8mL / min, 0.9mL / min or 1mL / min, etc., but not limited to the above listed values, other values not listed in the above range are also applicable.

[0032] Preferably, the monosaccharide in step (3) comprises glucose, arabinose and xylose.

[0033] In another aspect, the present application also provides the use of the detection method as described above in the analysis of reconstituted cut component.

[0034] Compared with the prior art, the present application has the following beneficial effects:

[0035] The application provides a detection method for key macromolecules in reconstituted stem, effectively eliminates starch in the sample by adopting amylase, and avoids the influence of glucose generated by starch on cellulose content determination in the acidolysis process; the absorbance method and the weight method are organically combined, the contents of three key macromolecules in the reconstituted stem can be detected at the same time, integrated analysis of cellulose, hemicellulose and lignin is realized, the test method is simple and efficient, the determination result is accurate, the detection efficiency is effectively improved, and the method has a wide market prospect. DETAILED DESCRIPTION

[0036] In order to further illustrate the technical means adopted by the application and its effects, the technical solutions of the application will be further described below in combination with preferred embodiments of the application, but the application is not limited in the scope of the embodiments.

[0037] Example 1

[0038] The embodiment provides a detection method for key macromolecules in reconstituted stem, and the specific steps are as follows:

[0039] (1) Preparation of neutral detergent

[0040] 5g of polyoxyethylene lauryl ether (Shanghai Gaoming Chemical Co., Ltd., MOA, 99%) and 6.8g of sodium tetraborate are weighed into a 100ml beaker, dissolved in an appropriate amount of distilled water, and then 3g of sodium dodecyl sulfate is added; 4g of nonylphenol polyoxyethylene ether (Shanghai Mayreer Biochemical Technology Co., Ltd.) is weighed into another beaker, dissolved after adding distilled water, and then the two solutions are transferred into a 1000ml volumetric flask after cooling, and water is added to the mark.

[0041] (2) Neutral detergent washing

[0042] 1g of reconstituted stem is weighed and mixed with 10ml of neutral detergent, then 5ml of a mixed solution of alpha-amylase (5% of the mass of reconstituted stem) and isoamylase (5% of the mass of reconstituted stem) is added, and n-octanol is added as a defoaming agent, the pH value is adjusted to 7.1, and the reaction is carried out at 20℃ for 1 hour. After complete enzyme hydrolysis, the solution is heated to boiling, and the power is adjusted to maintain a state of slight boiling for 1 hour to ensure complete dissolution of the oil. After heating, immediately perform suction filtration, and rinse the tube wall and remaining material with 90℃ water. Rinse the remaining material with acetone 3 times to ensure that the remaining material is fully mixed with acetone until the filtrate is colorless. After suction drying, the remaining material is placed in an oven and dried to a constant weight.

[0043] (3) Acidolysis

[0044] The dried 20 mL of 0.5 M sulfuric acid and 0.5 M hydrochloric acid mixed solution was added to the prepared configuration and stirred to wet it, and then placed in a constant temperature oil bath at 100°C for 6 h to fully react. After the reaction was completed, filtration was performed to obtain filtrate and residue, 5 mL of filtrate was taken and reacted with excess dilute sulfuric acid until no bubbles were generated, and then centrifuged to take the upper liquid for PMP derivatization.

[0045] (4) PMP pre-column derivatization

[0046] 200 μL of the upper liquid in step (3) was taken in a 10 mL centrifuge tube, 200 μL of 0.5 mol / L NaOH solution was added, mixed, 500 μL of 0.5 mol / L PMP methanol solution was added, mixed, and derivatized at 70°C in a constant temperature water bath for 1 h, taken out, cooled to 20°C, 200 μL of 0.5 mol / L HCl solution was added to neutralize the NaOH solution, mixed, 1.0 mL of chloroform was added for extraction, the supernatant was taken, repeated multiple times, the excess PMP methanol solution was removed to avoid affecting the determination of liquid chromatography, 0.4 mL of deionized water was added for dilution, filtered through a 0.22 μm filter membrane, and then detected by a liquid chromatograph to determine the concentrations of arabinose, glucose and xylose, and calculate the contents of cellulose and hemicellulose, the calculation formula is as follows:

[0047]

[0048] wherein C 葡萄糖 is the glucose concentration, C 阿拉伯糖、木糖 is the sum of arabinose and xylose concentrations, and m0 is the mass of the reconstituted cut rag.

[0049] The calculated contents of cellulose and hemicellulose are 23.11% and 2.48%, respectively.

[0050] The high performance liquid chromatography analysis conditions include:

[0051] Chromatographic column: sepax techenologies HP-C18 (4.6 x 250 mm, 5 μm);

[0052] Detection wavelength: 250 nm;

[0053] Column temperature: 30°C;

[0054] Injection volume: 20 μL;

[0055] Flow rate: 0.8 mL / min;

[0056] Mobile phase: The mobile phase is a mixture of acetonitrile and PBS, and the volume ratio of the acetonitrile / PBS mixture is 18:82.

[0057] (5) Take the filtrate of step (3) and test and calculate its acid soluble lignin content at 0.35% at UV wavelength of 320 nm, then wash the residue of step (3) clean to neutral (pH = 6.8) with deionized water, dry and weigh, then (A) place it in a muffle furnace, burn at 575°C for 3 hours, after the organic matter is ashed, accurately weigh (B) and calculate (A-B) to obtain the acid insoluble lignin content of 1.36%, and the total content of lignin is calculated to be 1.71%.

[0058] Example 2

[0059] The present embodiment provides a method for detecting key macromolecules in reconstituted stem, and the specific steps are consistent with those of Example 1 except that sodium tetraborate is not added, and part of the proportion is allocated to sodium dodecyl sulfate, polyoxyethylene lauryl ether and nonylphenol polyoxyethylene ether.

[0060] Example 3

[0061] The present embodiment provides a method for detecting key macromolecules in reconstituted stem, and the specific steps are consistent with those of Example 1 except that sodium dodecyl sulfate is not added, and part of the proportion is allocated to sodium tetraborate, polyoxyethylene lauryl ether and nonylphenol polyoxyethylene ether.

[0062] Example 4

[0063] The present embodiment provides a method for detecting key macromolecules in reconstituted stem, and the specific steps are consistent with those of Example 1 except that polyoxyethylene lauryl ether is not added, and part of the proportion is allocated to sodium dodecyl sulfate, sodium tetraborate and nonylphenol polyoxyethylene ether.

[0064] Example 5

[0065] The present embodiment provides a method for detecting key macromolecules in reconstituted stem, and the specific steps are consistent with those of Example 1 except that nonylphenol polyoxyethylene ether is not added, and part of the proportion is allocated to sodium dodecyl sulfate, polyoxyethylene lauryl ether and sodium tetraborate.

[0066] Example 6

[0067] The present embodiment provides a method for detecting key macromolecules in reconstituted stem, and the specific steps are consistent with those of Example 1 except that α-amylase is not added, and part of the proportion is allocated to isoamylase.

[0068] Example 7

[0069] The present embodiment provides a method for detecting key macromolecules in reconstituted stem, and the specific steps are consistent with those of Example 1 except that isoamylase is not added, and part of the proportion is allocated to α-amylase.

[0070] Example 8

[0071] The embodiment provides a detection method of key macromolecules in reconstituted cut stem, and the specific steps are consistent with those in Embodiment 1 except that the isoamylase is replaced by an equal amount of β-amylase.

[0072] Embodiment 9

[0073] The embodiment provides a detection method of key macromolecules in reconstituted cut stem, and the specific steps are consistent with those in Embodiment 1 except that no sulfuric acid is added, and part of the distribution is reduced to an equal amount of hydrochloric acid.

[0074] Embodiment 10

[0075] The embodiment provides a detection method of key macromolecules in reconstituted cut stem, and the specific steps are consistent with those in Embodiment 1 except that no hydrochloric acid is added, and part of the distribution is reduced to an equal amount of sulfuric acid.

[0076] Test example:

[0077] The recovery rate of the detection method provided in Embodiments 1-10 is tested. A series of standard solutions of arabinose, glucose and xylose with different concentrations are prepared, then PMP pre-derivation is performed according to step (4), and liquid chromatography analysis is performed; the peak area (Y) is recorded, and a linear curve is drawn with the monosaccharide concentration (X), and a linear equation is fitted. A known concentration of reconstituted cut stem sample is accurately weighed, then arabinose, glucose and xylose standard are added, then neutral detergent washing, acid hydrolysis and PMP pre-derivation are performed, and liquid chromatography analysis is performed, and the peak area of each monosaccharide is recorded, and the recovery rate is calculated.

[0078]

[0079]

[0080] It can be found from the above data that the detection method provided by the application can simultaneously detect the content of three key macromolecules in reconstituted cut stem, and realizes integrated analysis of cellulose, hemicellulose and lignin; it can be found by comparing Embodiments 1-5 that by using a specific neutral detergent combination, the application can further reduce the interference of lipid substances in the reconstituted cut stem during the treatment process, and improve the detection accuracy; it can be found by comparing Embodiments 1, 6-8 that by using a specific enzyme combination, the application can further eliminate starch in the sample, avoid the influence of glucose produced by starch in the acid hydrolysis process on the determination of cellulose content, and improve the detection accuracy; it can be found by comparing Embodiments 1, 9-10 that by using a specific acid combination, the application can effectively hydrolyze the sample, and further improve the detection accuracy.

[0081] The applicant declares that the application is illustrated by the above-mentioned embodiments for the detection method of key macromolecules in reconstituted cut tobacco of the application and the application thereof, but the application is not limited to the above-mentioned embodiments, that is, it does not mean that the application must rely on the above-mentioned embodiments to be implemented. It should be understood by those skilled in the art that any improvement of the application, equivalent replacement of each raw material of the product of the application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the application.

[0082] The preferred embodiments of the application are described in detail above, but the application is not limited to the specific details in the above-mentioned embodiments, and various simple modifications can be made to the technical solutions of the application within the technical concept of the application, and these simple modifications all belong to the protection scope of the application.

[0083] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined by any suitable method without contradiction, and in order to avoid unnecessary repetition, the application will not further describe various possible combination methods.

Claims

1. A method for detecting key macromolecules in reconstituted cut, characterized by, The key macromolecules include cellulose, hemicellulose and lignin; The detection method comprises the following steps: (1) mixing the sample to be tested with a neutral detergent, then mixing with amylase and defoaming agent, and filtering to obtain a pretreated sample; (2) mixing the pretreated sample with acid for acidolysis to obtain supernatant and residue; (3) performing a derivatization reaction on the supernatant, then performing liquid chromatography detection to obtain the monosaccharide content in the supernatant, and calculating the cellulose and hemicellulose content; (4) performing absorbance detection on the supernatant, calculating the acid-soluble lignin content according to the detection result, drying and ashing the residue, and calculating the acid-insoluble lignin content according to the mass change before and after ashing; Steps (3) and (4) are not distinguished in order; In step (1), the neutral detergent is a combination of sodium tetraborate, sodium dodecyl sulfate, nonylphenol polyoxyethylene ether and polyoxyethylene lauryl ether, and the ratio of the sample to be tested to the neutral detergent is 1:(10-500) g / mL; In step (1), the amylase is a combination of alpha-amylase and isoamylase; In step (1), the defoaming agent includes any one or a combination of at least two of polyethylene glycol, propylene glycol, glycerol, n-octanol, propylene oxide or ethylene oxide; In step (2), the acid is a combination of hydrochloric acid and sulfuric acid, and the concentration of the acid is 0.5-1.5 M; In step (2), the specific process of acidolysis is: mixing the pretreated sample with acid, then heating, and the heating temperature is 90-110℃ and the heating time is 4-8h; In step (3), the derivatization reagent of the derivatization reaction is PMP; In step (3), the chromatographic column for liquid chromatography detection is a C18 column, the detection wavelength is 250 nm, and the mobile phase is a mixture of acetonitrile and PBS, and the volume ratio of the acetonitrile / PBS mixture is 18:

82.

2. The detection method according to claim 1, characterized in that, The alcohol ether includes any one or a combination of at least two of polyoxyethylene lauryl ether, hydrogenated lauryl ether or stearyl amide polyoxyethylene ether.

3. The method of claim 1, wherein, In step (1), the mass ratio of the sample to be tested to the amylase is (20-100):

1.

4. The method of claim 1, wherein, In step (1), the ratio of the sample to be tested to the defoaming agent is 5:1-1:10 g / mL.

5. The method of claim 1, wherein, In step (3), the column temperature for liquid chromatography detection is 25-35℃, and the flow rate of the mobile phase is 0.6-1 mL / min.

6. The method of claim 1, wherein, In step (3), the monosaccharide includes glucose, arabinose and xylose.

7. Use of the detection method according to any one of claims 1-6 in analysis of reconstituted cut stem components.

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