Method for distinguishing honey-sweet flue-cured tobacco and fresh-sweet flue-cured tobacco based on tobacco leaf alkaloid

Abstract

The invention discloses a method for distinguishing honey sweet flue-cured tobacco and fresh sweet flue-cured tobacco based on tobacco leaf alkaloid, which comprises the following steps: S1, obtaining the amount of alkaloid in tobacco leaves, the alkaloid comprising neonicotinine and nicotine; s2, calculating the ratio of the neonicotinine to the nicotine; s3, when the ratio is larger than or equal to a fixed value, the tobacco leaves are judged to be fresh and sweet, otherwise, the tobacco leaves are judged to be honey-sweet. And an orthogonal partial least square discriminant analysis model of eight alkaloids is further adopted, so that the judgment accuracy can be further improved. According to the method, relatively simple alkaloid is introduced, a simple, rapid and stable method is used for detecting fresh and sweet flavor type tobacco leaves and honey sweet flavor type tobacco leaves in Guizhou, the flavor type distinguishing model is constructed by combining the ratio of the neobisatin to nicotine with an orthogonal partial least square distinguishing method, the distinguishing accuracy of the model reaches 93.9%, the model is simple and easy to understand, and the method is suitable for large-scale popularization and application. The tobacco leaves with different aroma types in Guizhou can be accurately distinguished, and reference is provided for cigarette industry enterprise formula and raw material utilization.

Description

Technical Field

[0001] The invention relates to a method for distinguishing flue-cured tobacco with honey sweet aroma and fresh sweet aroma based on tobacco leaf alkaloids, and belongs to the field of qualitative and quantitative analysis and detection methods and discrimination of agricultural product style characteristics. Background Art

[0003] There are many factors that influence and determine the aroma of tobacco leaves. In addition to genetic characteristics, it is also closely related to factors such as ecology (soil, topography, climate) and cultivation measures. However, what ultimately determines the aroma of flue-cured tobacco is the chemical composition of flue-cured tobacco. The substances currently used to distinguish aroma types include aroma volatile substances, amino acids, polyphenols, etc., especially aroma volatile substances, which are closely related to aroma and taste and are the most widely used. For example, through the sensory evaluation of aroma - aroma style characteristic profile map - flue-cured tobacco aroma area index, the strong-aroma type, intermediate-aroma type and light-aroma type are discriminated and analyzed; simultaneous distillation and extraction of aroma components - gas chromatography-mass spectrometry detection - stepwise regression discriminant analysis of strong-aroma type, intermediate-aroma type and light-aroma type, support vector machine discriminant model analysis of strong-aroma type, intermediate-aroma type and light-aroma type, guided regularized random forest method - support vector machine discriminant model strong-aroma type, intermediate-aroma type and light-aroma type, random forest classification discriminant model strong-aroma type, intermediate-aroma type and light-aroma type; near-infrared spectroscopy - multi-algorithm fusion (projection method + partial least squares discriminant + support vector machine) discriminant model analysis of strong-aroma type, intermediate-aroma type and light-aroma type; ultrasound-assisted extraction of polyphenols - high performance liquid chromatography detection - multi-algorithm fusion (principal component analysis + clustering + discriminant analysis method) discriminant model analysis of strong-aroma type, intermediate-aroma type and light-aroma type. The only discriminant analysis of sweet and honey-sweet tobacco leaves in Guizhou is the petroleum ether extraction method-three-dimensional fluorescence spectrum-system clustering discriminant analysis model. The above technologies have the following shortcomings: the detection methods of aroma components are complex, the stability and repeatability are relatively low; the samples are mainly for the distinction of major aroma types in the country; the subjective factors of smoking evaluation have a great influence; and the model construction is complex. Summary of the invention

[0004] Based on the above, the present invention provides a simple, rapid and stable method for distinguishing between honey-sweet-flavored and light-sweet-flavored flue-cured tobacco based on tobacco leaf alkaloids, so as to overcome the deficiencies of the prior art.

[0005] The technical solution of the present invention is: a method for distinguishing honey-sweet-scented and light-sweet-scented flue-cured tobacco based on tobacco leaf alkaloids, comprising the following steps:

[0006] S1, obtaining the amount of alkaloids in tobacco leaves, wherein the alkaloids include anatabine and nicotine;

[0007] S2, calculation of the ratio of anatabine to nicotine;

[0008] S3, when the ratio is ≥ the fixed value, the tobacco leaf is judged to be of light sweet aroma type, otherwise it is judged to be of honey sweet aroma type.

[0009] Preferably, it also includes:

[0010] S4, the alkaloids are substituted into the orthogonal partial least squares discriminant model for analysis, and the judgment result in step S3 is reviewed. The alkaloids also include nornicotine, myosamine, anabasine, diene nicotine, 2,3-bipyridine and cotinine. Specifically, nicotine, nornicotine, myosamine, anabasine, diene nicotine, anatabine, 2,3-bipyridine and cotinine are substituted into the online analysis software (https: / / www.metaboanalyst.ca / ) for orthogonal partial least squares discriminant model analysis to further confirm the judgment.

[0011] Preferably, in step S1, the specific method for obtaining the amount of alkaloids in tobacco leaves is:

[0012] S11 extraction: add the internal standard to the ground and sieved tobacco leaf sample, add alkaline solution to convert all nicotine into molecular state, vortex mix, add non-chlorinated solvent, vortex mix and then shake to extract, vortex mix again after extraction, centrifuge and take the supernatant to pass through an organic filter membrane, and perform chromatographic analysis on the filtrate;

[0013] S12 analysis: chromatographic analysis was performed using a gas chromatography-nitrogen phosphorus detector, with the retention time of the standard sample used for qualitative analysis and the internal standard method standard curve used for quantitative analysis to obtain the amount of the alkaloid.

[0014] Preferably, in step S11, the internal standard is quinoline and the like, the alkaline solution is an aqueous sodium hydroxide solution, and the non-chlorinated solvent is methyl tert-butyl ether.

[0015] Preferably, in step S12, the chromatographic conditions are: chromatographic column: DB-17 capillary chromatographic column (30mm×0.25mm id×0.25μm df); carrier gas: high-purity helium (99.999%); column flow rate: 1.50mL / min; inlet temperature: 230°C; injection volume: 1.00μL; injection mode: split; split ratio: 10:1; nitrogen-phosphorus detector temperature: 260°C; air flow rate: 60mL / min; hydrogen flow rate: 2.3mL / min; hydrogen delay time: 6min; programmed temperature rise: 110°C for 1min, then increase to 180°C at 4°C / min for 1min, increase to 250°C at 20°C / min for 7min, and run for a total of 30min.

[0016] Preferably, in step S3, the fixed value of anatabine and nicotine of the flavor is determined to be 5.1%.

[0017] Tobacco alkaloids (nicotine and seven other trace alkaloids) are important chemical components of tobacco leaves. Their composition and content determine the quality characteristics of tobacco. For example, the main alkaloids in tobacco leaves have a significant effect on the smoke and taste characteristics of tobacco leaves and their tobacco products. At the same time, the conversion rate of anatabine and anabasine in tobacco leaf trace alkaloids is not easy to change with the variety, and the change in their conversion rate mainly reflects the differences in ecology and cultivation measures. Based on the content ratio of anatabine and nicotine in tobacco leaves, these two alkaloids are important chemical components of tobacco leaves. Their composition and content determine the quality characteristics of tobacco, especially have a significant impact on the smoke and taste characteristics of tobacco leaves and their tobacco products. The applicant discovered that there are specific differences in the ratio of anatabine to nicotine in tobacco leaves of different aroma types, especially anatabine, which increases the most and is most indicative of light-aroma tobacco leaves, while anatabine is relatively less in sweet-aroma tobacco leaves. Therefore, the ratio of anatabine to nicotine can be used as a chemical marker to distinguish between sweet-aroma tobacco leaves and sweet-aroma tobacco leaves, thereby simplifying the aroma discrimination model and making the aroma judgment faster and more accurate. This method is not only scientific and reasonable, but also easy to operate, and can effectively distinguish and identify tobacco leaves of different aroma types.

[0018] Beneficial effects of the present invention: The present invention introduces relatively simple alkaloids, uses a simple, fast and stable method to detect Guizhou sweet and honey-sweet flavor tobacco leaves, and uses the ratio of anatabine to nicotine combined with the orthogonal partial least squares discrimination method to construct a flavor discrimination model, and the model discrimination accuracy rate reaches 93.9%. The model is simple and easy to understand, and can accurately distinguish Guizhou tobacco leaves with different flavors. The present invention is relatively simple in terms of detection method and data analysis method, and is particularly suitable for the discrimination of Guizhou tobacco flavors. It can provide data-based chemical judgment for flavor gradient classification, tobacco procurement, maintenance and research and development of cigarette brand style characteristic leaf group formulas, and the matching of flavors and spices. In addition, the quantitative use of alkaloids in the present invention simplifies the detection method, and the method is stable, accurate and reliable. The use of methyl tert-butyl ether to extract alkaloids increases the service life of the detector, the extract is easy to obtain and not easily contaminated, and the nitrogen-phosphorus detector is easy to obtain and simple to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Non-chlorinated solvent extraction-gas chromatography-nitrogen-phosphorus detector analysis and the steps of distinguishing between sweet and sweet tobacco leaves (weighing tobacco leaf samples-adding internal standard-adding 5% sodium hydroxide solution-vortex mixing-adding methyl tert-butyl ether and mixing-oscillating extraction for 50 minutes-vortex mixing again-centrifugation at 3000 rpm-gas chromatography-nitrogen-phosphorus detector analysis-obtaining chromatograms and quantification by internal standard method-anatabine to nicotine ratio analysis-orthogonal partial least squares discriminant analysis);

[0020] Figure 2Different volumes (0.4-0.8-1.2-1.6mL) of 5% sodium hydroxide solution were optimized (A-nicotine; B-trace organisms, nornicotine, anatabine and anabasine; C-trace alkaloids-myosamine, dienicotine, 2,3-bipyridine, cotinine); Different volumes (3-4-6-8mL) of methyl tert-butyl ether were optimized (D-nicotine; E-trace organisms, nornicotine, anatabine and anabasine; F-trace alkaloids-myosamine, dienicotine, 2,3-bipyridine, cotinine);

[0021] Figure 3 GC-NPD chromatograms of eight alkaloids in typical flue-cured tobacco samples; A overall chromatogram B enlarged chromatogram (quinoline -7.82min nicotine -9.15min nornicotine -12.29min myosamine -12.77min anabasine -13.52min nicotine diene -14.67min anatabine -15.09min 2,3-bipyridine -16.21min cotinine -21.49min);

[0022] Figure 4 A: Ratio of anatabine to nicotine in Guizhou sweet and fragrant flue-cured tobacco (<5.1%); B: Ratio of anatabine to nicotine in Guizhou sweet and fragrant flue-cured tobacco (≥5.1%);

[0023] Figure 5 A The data distribution of the content of 8 alkaloids after normalization (Log10 transformation and Pareto standardization); B The orthogonal partial least squares discriminant analysis of 8 alkaloids to distinguish Guizhou honey sweet and sweet tobacco leaves. DETAILED DESCRIPTION

[0024] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0025] Example 1: Extraction and detection of 8 alkaloids in tobacco leaves

[0026] The method comprises the following steps:

[0027] S1 Extraction of alkaloids in tobacco leaves: Weigh 200 mg of tobacco powder into a 50 mL plastic centrifuge tube, add 50 μL of 5.326 mg / mL quinoline internal standard using a 100 μL syringe, add 0.8 mL of 5% NaOH solution, vortex mix, and finally add 6 mL of methyl tert-butyl ether (MTBE) extraction solution, vortex mix, vortex at 2500 rpm for 5 min, oscillate on an oscillator at 260 rpm for 50 min, vortex at 2500 rpm for 5 min, centrifuge at 3000 rpm for 5 min, remove the upper organic phase, filter through an organic filter membrane into a chromatographic bottle, and analyze by GC-NPD.

[0028] Analysis and detection of alkaloids in S2 tobacco leaves: Gas chromatography-nitrogen phosphorus detector was used for chromatographic analysis. The main chromatographic and detector conditions are shown in Table 1. The retention time of the standard was used for qualitative analysis. The retention times of the alkaloids were respectively. The internal standard method standard curve was used for quantitative analysis.

[0029] Table 1 Chromatographic and nitrogen-phosphorus detector conditions

[0030]

[0031] Example 2: Optimization of extraction of 8 alkaloids from tobacco leaves

[0032] The non-chlorinated solvent MTBE was used to extract 8 alkaloids in tobacco, and the volumes of NaOH solution and MTBE (methyl tert-butyl ether) solution were optimized. ① Extraction was performed using the non-chlorinated solvent MTBE, and different volumes (0.4-0.8-1.2-1.6 mL) of 5% NaOH solution were used for alkalization. The relationship between the nicotine / internal standard ratio and the volume of NaOH solution was obtained as follows: Figure 2 As shown in A. As can be seen from the figure, with the increase of the volume of 5% NaOH solution, the nicotine / internal standard ratio first increases and then decreases. When the volume is 0.8mL 5% NaOH, the peak area ratio of nicotine to internal standard is the largest, that is, the extracted nicotine content is high. Figure 2 B and 2C used the same method to optimize trace and trace alkaloids. The results showed that the ratio of trace alkaloids to internal standards remained basically unchanged. When the sodium hydroxide solution was 0.8 mL, the ratio of trace alkaloids myosamine, 2,3-bipyridine, and cotinine to the internal standard was the largest, while diene nicotine remained basically unchanged. In summary, when extracting with non-chlorinated solvents, the optimal volume of 5% sodium hydroxide solution is 0.8 mL. ② Use non-chlorinated solvents for extraction, and use different volumes (3-4-6-8 mL) of MTBE solvent as the extractant. The relationship between the nicotine / internal standard ratio and the MTBE volume is as follows: Figure 2 D. As shown in the figure, as the volume of MTBE increases, the ratio of nicotine to internal standard increases first and then decreases. When the volume is 6mL MTBE, the peak area ratio of nicotine to internal standard is the largest, that is, the extracted nicotine content is high. Figure 2 E and 2F used the same method to optimize trace alkaloids. The results showed that when the volume of MTBE increased from 3mL to 6mL, the ratio of trace alkaloids to internal standard reached the maximum value. When the volume of MTBE was increased to 8mL, the ratio of trace alkaloids to internal standard remained basically unchanged. At the same time, the extraction efficiency of the ratio of trace alkaloids such as mysamine, diene nicotine, 2,3-bipyridine, and cotinine to internal standard peak was the highest in 6mL MTBE. In summary, the optimal volume of non-chlorinated extractant MTBE solution is 6mL.

[0033] Example 3: Validation of qualitative and quantitative analysis methods for eight alkaloids in tobacco leaves

[0034] 1. Standard curve

[0035] According to the standard curve range, a series of gradient alkaloid standard solutions were prepared with MTBE and analyzed by GC-NPD. The standard working curve was drawn with the mass ratio of the eight alkaloids / internal standard as the horizontal axis and the peak area ratio as the vertical axis. The results are shown in Table 2 and Table 3. The results show that the linear range of the eight alkaloids is wide and the linear relationship of the standard curve is good (r≥0.9976)

[0036] Table 2 Nicotine linear equation, linear range, correlation coefficient

[0037]

[0038] Table 3 Linear equations, linear ranges, and correlation coefficients of 7 alkaloids

[0039]

[0040]

[0041] 2. Determination method precision and recovery rate

[0042] The intra-day precision determination results of the same tobacco alkaloid fixed value sample using non-chlorinated extraction are shown in Table 4. As can be seen from Table 4: the relative standard deviation (RSD) of nicotine is 0.664%; the RSD of trace alkaloids is between 2.085% and 4.324%; the RSD of trace alkaloids is between 3.101% and 4.013%; the RSD of total alkaloids is 0.675%. The RSD of each alkaloid is between 0.664% and 4.324%.

[0043] Table 4 Intra-day precision determination results of tobacco alkaloid content in fixed value samples

[0044]

[0045] Note: The units of alkaloid content and relative standard deviation in Table 2-3 are mg / g and %. The same applies to Table 2-4.

[0046] The results of the intra-day precision determination of the same tobacco alkaloid sample using non-chlorinated extraction are shown in Table 5. As can be seen from Table 5: the RSD of nicotine is 0.676%; the RSD of trace alkaloids is between 2.676% and 4.891%; the RSD of trace alkaloids is between 4.002% and 4.508%; the RSD of total alkaloids is 0.730%; the RSD of each alkaloid is between 0.676% and 4.891%, all less than 5%. In summary, the intra-day and inter-day precision of this method are good, and it can be used to determine the eight alkaloids in tobacco leaves.

[0047] Table 5 Results of inter-day precision determination of tobacco alkaloid content in fixed value samples

[0048]

[0049]

[0050] The same tobacco alkaloid quantification sample was extracted by non-chlorination, and the results were compared with the standard values ​​to obtain relative recoveries as shown in Table 6. The results showed that the relative recoveries of the eight alkaloids were between 91.4% and 97.6%, which was good and met the strict quantitative requirements.

[0051] Table 6 Determination values ​​and relative recoveries of tobacco alkaloids samples

[0052]

[0053] Example 4: Differentiation of Guizhou honey sweet and fragrant flue-cured tobacco from fresh sweet and fragrant tobacco based on alkaloids

[0054] In order to accurately distinguish between Guizhou's sweet and light-sweet tobacco leaves, the ratio of anatabine to nicotine was first used for discrimination. Among the 123 flue-cured tobacco samples collected in 2023, there were 82 sweet tobacco leaves, including 41 C3F tobacco leaves and 41 B2F tobacco leaves; there were 41 light-sweet tobacco leaves, including 23 C3F tobacco leaves and 18 B2F tobacco leaves. The content of 8 alkaloids in the 123 flue-cured tobacco samples was analyzed by non-chlorinated solvent extraction-gas chromatography-nitrogen-phosphorus detector. The ratio of anatabine to nicotine was less than 5.1% for sweet-sweet-scented tobacco leaves, and the ratio was ≥5.1% for light-sweet-scented tobacco leaves. Figure 4 A can get 82 sweet tobacco leaves, only 3 samples are judged as light fragrance tobacco leaves, mainly 1 Ziyun B2F, 1 Dafang B2F and 1 Meitan B2F tobacco leaves, and the correct rate of sweet tobacco leaves is 96.3%; Figure 4B shows that among the 41 sweet tobacco leaves, only 7 samples were judged as honey sweet tobacco leaves, mainly 1 Panzhou C3F tobacco leaves, 2 Pu'an C3F tobacco leaves, 2 Pu'an B2F tobacco leaves, and 2 Xingyi C3F tobacco leaves. The wrongly judged tobacco leaves are mainly in Southwest Guizhou, while the 24 sweet tobacco leaves in Weining are all correct, with a correct rate of 82.9% for sweet tobacco leaves. Through ratio judgment, the average judgment accuracy is 89.6%.

[0055] In order to further improve the accuracy of judging the sweet and refreshing tobacco leaves of Guizhou Honey, orthogonal partial least squares discriminant analysis was used to build a model, and the online data analysis website https: / / www.metaboanalyst.ca / was used for statistical analysis (one factor). Before building the model, the content data of the eight alkaloids needed to be normalized, and the data were analyzed using Logarithm. 10 Transformation, then use Pareto normalization After standardization, the data can be dimensionless, making variables of different properties comparable. At the same time, the variable data of different orders of magnitude are transformed to the appropriate range to avoid large-value variables covering up the fluctuations of small-value variables. Figure 5 A. After data normalization, orthogonal partial least squares discriminant analysis was performed on the eight alkaloids. The model results are shown in Figure 5 As shown in B, among the honey-sweet-flavor tobacco leaves, 1 out of 3 classified as light-sweet-flavor tobacco leaves was classified as honey-sweet-flavor tobacco leaves, and the accuracy rate was increased to 97.6%. Among the light-sweet-flavor tobacco leaves, 3 out of 7 classified as honey-sweet-flavor tobacco leaves were classified as light-sweet-flavor tobacco leaves, and the accuracy rate was increased to 90.2%. The model was constructed by orthogonal partial least squares discriminant analysis, which further improved the overall accuracy, and the average judgment accuracy was 93.9%. In summary, the detection method is simple, fast, stable, and the model is simple to understand and accurate in judgment, which can distinguish Guizhou honey-sweet-flavor and light-sweet-flavor flue-cured tobacco. The content of the invention can be widely used in the rapid analysis of tobacco alkaloids, improving the efficiency of flue-cured tobacco flavor discrimination, and providing a reference for the formulation and raw material utilization of cigarette industry enterprises.

[0056] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for distinguishing between honey-sweet-scented and light-sweet-scented flue-cured tobacco based on tobacco leaf alkaloids, characterized in that: The following steps are involved: S1, obtaining the amount of alkaloids in tobacco leaves, wherein the alkaloids include anatabine and nicotine; S2, calculation of the ratio of anatabine to nicotine; S3, when the ratio is ≥ the fixed value, the tobacco leaf is judged to be of light sweet aroma type, otherwise it is judged to be of honey sweet aroma type.

2. The method for distinguishing between honey-sweet-flavored and light-sweet-flavored flue-cured tobacco based on tobacco leaf alkaloids according to claim 1, characterized in that: Also includes: S4, substituting the alkaloids into the orthogonal partial least squares discriminant model for analysis, and reviewing the determination result in step S3, wherein the alkaloids further include nornicotine, myosamine, anabasine, nicotine diene, 2,3-bipyridine and cotinine.

3. The method for distinguishing between honey-sweet-flavored and light-sweet-flavored flue-cured tobacco based on tobacco leaf alkaloids according to claim 1, characterized in that: In step S1, the specific method for obtaining the amount of alkaloids in tobacco leaves is: S11 extraction: add the internal standard to the ground and sieved tobacco leaf sample, add alkaline solution to convert all nicotine into molecular state, vortex mix, add non-chlorinated solvent, vortex mix and then shake to extract, vortex mix again after extraction, centrifuge and take the supernatant to pass through an organic filter membrane, and perform chromatographic analysis on the filtrate; S12 analysis: chromatographic analysis was performed using a gas chromatography-nitrogen phosphorus detector, with the retention time of the standard sample used for qualitative analysis and the internal standard method standard curve used for quantitative analysis to obtain the amount of alkaloids.

4. The method for distinguishing between honey-sweet-flavored and light-sweet-flavored flue-cured tobacco based on tobacco leaf alkaloids according to claim 3, characterized in that: In step S11, the internal standard is quinoline and the like, the alkaline solution is an aqueous sodium hydroxide solution, and the non-chlorinated solvent is methyl tert-butyl ether.

5. The method for distinguishing between honey-sweet-flavored and light-sweet-flavored flue-cured tobacco based on tobacco leaf alkaloids according to claim 3, characterized in that: In step S12, the chromatographic conditions are: chromatographic column: DB-17 capillary chromatographic column 30mm×0.25mm id×0.25μm df; carrier gas: high-purity helium; column flow rate: 1.50mL / min; inlet temperature: 230℃; injection volume: 1.00μL; injection mode: split; split ratio: 10:1; nitrogen-phosphorus detector temperature: 260℃; air flow rate: 60mL / min; hydrogen flow rate: 2.3mL / min; hydrogen delay time: 6min; programmed temperature rise: 110℃ for 1min, then increase to 180℃ at 4℃ / min for 1min, increase to 250℃ at 20℃ / min for 7min, and run for a total of 30min.

6. The method for distinguishing between honey-sweet-flavored and light-sweet-flavored flue-cured tobacco based on tobacco leaf alkaloids according to claim 1, characterized in that: In step S3, the fixed value of anatabine and nicotine of the flavor is determined to be 5.1%.

Images