Cut tobacco perfuming uniformity evaluation method based on PCA nuclear density and additional marker

Abstract

The invention discloses a tobacco shred flavoring uniformity evaluation method based on PCA nuclear density analysis and an additional marker. The evaluation method comprises the following steps: S1, preparing a tobacco shred sample with the additional marker; s2, pretreating a tobacco shred sample; s3, performing gas chromatography-mass spectrometry analysis; and S4, performing perfuming uniformity evaluation based on PCA kernel density analysis. According to the method, the problems that the marker is difficult to select, the marker is easily interfered by a tobacco shred matrix and does not have universality when the essence is used as the marker are solved, meanwhile, the defect that the perfuming uniformity results represented by multiple indexes are inconsistent is overcome, and a rapid, accurate and effective method is provided for evaluating the uniformity of the cigarette perfuming process.

Description

Technical Field

[0001] The invention belongs to the technical field of cigarette flavoring, and in particular relates to a method for evaluating tobacco flavoring uniformity based on PCA kernel density and external markers. Background Art

[0002] Cigarette flavoring is a key process in tobacco production. The accuracy and uniformity of flavoring directly affect the stability of product sensory quality and is an important factor affecting the quality of cigarette products. Therefore, the uniformity evaluation of the cigarette flavoring process has always been a focus and problem for the tobacco industry.

[0003] According to literature research, the tobacco industry currently evaluates uniformity by calculating the coefficient of variation of the content of one or a few flavoring components added to tobacco. This method of evaluating uniformity has two main problems: (1) The chemical composition of tobacco and flavors is complex and diverse, and the tobacco and flavors formulas of different cigarette manufacturers are different. The selected flavoring components have large differences in content and are easily interfered by the tobacco matrix. Therefore, using flavoring components as markers for flavoring uniformity evaluation is not universally applicable; (2) Using a single marker for evaluation cannot provide an objective and comprehensive evaluation; and when using the coefficient of variation of multiple markers to evaluate flavoring uniformity, the evaluation results of different markers vary greatly.

[0004] Therefore, a series of compounds other than flavors and tobacco components are selected as external markers to eliminate the interference of tobacco matrix, which can more accurately and scientifically evaluate the flavoring uniformity of cigarettes. At the same time, the flavoring uniformity evaluation method combined with principal component analysis overcomes the problem of inconsistent evaluation of the coefficient of variation of multiple markers and the problem that the difference in flavoring uniformity between different production batches (batch to batch) cannot be quantified, which has practical application value for improving the accuracy and uniformity of flavoring in the cigarette shred process.

[0005] The invention patents with application numbers 201910153034.7 and 201910153041.7 disclose a method for evaluating the uniformity of tobacco flavoring based on PCA average distance and elliptical area after headspace solid phase microextraction quality analysis. However, headspace solid phase microextraction is susceptible to interference from complex tobacco matrix, resulting in low repeatability. In addition, the calculation of PCA average distance and elliptical area has complex formulas, large amount of calculation, and easy calculation errors. Summary of the invention

[0006] In order to solve the above technical problems existing in the prior art, the present invention provides a method for evaluating the uniformity of flavoring of shredded tobacco based on PCA kernel density and external markers. The present invention selects compounds such as ethyl benzoate, 2-phenylethyl formate, phenylethyl acetate, benzyl carbinol butyrate and 2-phenylethyl phenylacetate as external markers in shredded tobacco. These five markers are included in YQ 52-2015 "List of Additives Permitted for Use in Tobacco Products" and YQ 53-2015 "List of Additives Temporarily Permitted for Use in Tobacco Products", and are all non-volatile spices and common fixatives, so they can be tried for the investigation of flavoring uniformity. The present invention extracts the above markers in the shredded tobacco after flavoring by ultrasonic extraction and then analyzes them by conventional gas chromatography-mass spectrometry. The detection data is then analyzed by principal component analysis using origin software to draw a PCA score graph; then kernel density analysis is performed according to ArcGIS desktop10.8 software to quantify the flavoring uniformity, and a method for evaluating the uniformity of flavoring of shredded tobacco based on PCA kernel density analysis and ultrasonic extraction of external markers is established.

[0007] The technical solution adopted by the present invention is:

[0008] A method for evaluating tobacco flavoring uniformity based on PCA kernel density analysis and external markers, characterized in that the evaluation method comprises the following steps:

[0009] S1. preparing a tobacco sample with an external marker;

[0010] S2, pre-treating tobacco samples;

[0011] S3, gas chromatography-mass spectrometry analysis;

[0012] S4. Evaluation of flavoring uniformity based on PCA kernel density analysis.

[0013] Furthermore, in step S1, the specific process of preparing the tobacco sample with added markers is as follows:

[0014] Ethyl benzoate, 2-phenylethyl formate, phenylethyl acetate, benzyl carbinol butyrate and 2-phenylethyl phenylacetate are added to flavors to prepare labeled flavors with mass concentrations of each marker ranging from 0.2% to 2.0%, which are used for flavoring cigarettes in a tobacco workshop. After the flavoring equipment runs stably, tobacco samples are collected at a fixed position behind the outlet of the flavoring machine drum, 30 samples are collected for each batch, with an interval of 1 minute each time and a sampling mass of 100g each time. The taken tobacco samples are placed in sealed bags for standby use.

[0015] Furthermore, in step S2, the specific process of pre-treating the tobacco sample is as follows:

[0016] The tobacco sample flavored in step S1 is placed under the conditions of temperature (20±1)°C and relative humidity (60±3)% for equilibration for 24 hours; after grinding, 3.0 g of the sample is weighed and placed in a 250 mL conical flask, 100 mL of n-hexane extract containing 5 μg / mL of internal standard is added, ultrasonic extraction is performed at 30°C for 30 minutes, the upper layer of the extract is filtered through a 0.45 μm filter membrane and placed in a chromatographic injection bottle for testing.

[0017] Furthermore, in step S3, the specific process of gas chromatography-mass spectrometry analysis is as follows:

[0018] Gas chromatography / mass spectrometry was used for detection, mass spectrometry database retrieval for qualitative analysis, and internal standard method for quantitative analysis.

[0019] Furthermore, in step S4, the specific process of flavoring uniformity evaluation based on PCA kernel density analysis is as follows:

[0020] The detection data was analyzed by principal component analysis using origin software, and a PCA score graph was drawn;

[0021] Kernel density analysis was performed on the PCA score map to quantify the flavoring uniformity according to ArcGIS desktop 10.8 software.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. The kernel density analysis of the present invention is a spatial data analysis method that can be used to calculate and reflect the relative concentration of the spatial distribution of elements; this method can intuitively reflect the distribution of discrete measurement values ​​in a continuous area, and the data can be directly processed by the software ArcGIS desktop.

[0024] 2. The present invention avoids the problems of using the essence's own components as markers, such as difficulty in selecting markers, markers being easily interfered by the tobacco matrix and lacking universality. At the same time, it overcomes the inconsistency of the results of multiple indexes characterizing the uniformity of flavoring, and provides a fast, accurate and effective method for evaluating the uniformity of the cigarette flavoring process.

[0025] 3. The present invention avoids the problems of using the essence's own components as markers, such as difficulty in selecting markers, markers being easily interfered by the tobacco matrix and lacking universality, and can more objectively and accurately evaluate the uniformity of the cigarette flavoring process.

[0026] 4. Compared with the invention patent (application number 201910698824.3) that also selects an external marker, the ultrasonic extraction method used in the present invention is simpler than the accelerated solvent extraction method selected in the patent and does not require a special pretreatment device; in addition, the gas chromatography-mass spectrometry analyzer used in the present invention is more economical and popularizable than the expensive tandem mass spectrometer.

[0027] 5. The present invention combines principal component analysis and kernel density analysis to evaluate flavoring uniformity, thus overcoming the problem of inconsistent evaluation of coefficients of variation of multiple markers and the problem that the differences in flavoring uniformity between different production batches and different flavoring methods cannot be quantified. The evaluation method has practical significance for improving the accuracy and uniformity of flavoring in the cigarette silk making process. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The chromatograms of the mixed standard solution and the tobacco extract sample of the present invention;

[0029] Figure 2 This is the effect of extraction temperature on recovery rate;

[0030] Figure 3 This is the effect of extraction time on recovery rate;

[0031] Figure 4 This is the effect of sample amount on recovery rate;

[0032] Markings in the accompanying drawings:

[0033] 1. 2-Phenylethyl formate, 2. Phenylethyl acetate, 3. Ethyl benzoate, 4. Phenylethyl propionate (internal standard), 5-Benzyl carbinol butyrate, 6. 2-Phenylethyl phenylacetate. DETAILED DESCRIPTION

[0034] The specific implementation of the embodiment of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the embodiment of the present invention, and is not used to limit the embodiment of the present invention.

[0035] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0036] The present invention will be described in detail below in conjunction with exemplary embodiments with reference to the accompanying drawings.

[0037] The materials, reagents and instruments used in this example are as follows:

[0038] 7890A-5975C gas chromatograph-mass spectrometer, Agilent, USA;

[0039] AG104 electronic balance (sensitivity 0.1 mg), Mettler-Toledo, Switzerland;

[0040] DS-7510DTH ultrasonic instrument, Shanghai Shengxi Ultrasonic Instrument Co., Ltd.

[0041] n-Hexane, isopropanol, methanol, acetonitrile, dichloromethane: chromatographic grade, Anpu Company;

[0042] Phenethyl acetate, purity (mass fraction) is 98%;

[0043] 2-Phenylethyl formate, purity (mass fraction) is 95%;

[0044] Ethyl benzoate, purity (mass fraction) is 98%;

[0045] Phenylethyl propionate, purity (mass fraction) is 98%;

[0046] Benzyl methanol butyrate, purity (mass fraction) is 98%;

[0047] 2-Phenylethyl phenylacetate, purity (mass fraction) is 97%:

[0048] All of the above were purchased from Aladdin Reagent Company.

[0049] In this embodiment, the extraction solution, the mixed standard stock solution, and the mixed standard working solution are prepared as follows:

[0050] (1) Extraction solution: 5 μg / mL phenylethyl propionate (internal standard) in dichloromethane.

[0051] (2) Mixed standard stock solution: Accurately weigh 0.1 g each of ethyl benzoate, 2-phenylethyl formate, phenylethyl acetate, benzyl methanol butyrate and 2-phenylethyl phenylacetate, and dilute to volume in a 1000-mL volumetric flask with the extraction solution to prepare mixed standard stock solutions with a concentration of 100 μg / mL for ethyl benzoate, 2-phenylethyl formate, phenylethyl acetate, benzyl methanol butyrate and 2-phenylethyl phenylacetate, respectively.

[0052] (3) Mixed standard working solution: Accurately pipette 0.25, 0.50, 2.50, 5.0, and 10.0 mL of the mixed standard stock solution and dilute to volume in a 50-mL volumetric flask with the extraction solution to prepare mixed standard solutions with concentrations of 0.5, 1.0, 5.0, 10.0, and 20.0 μg / mL for ethyl benzoate, 2-phenylethyl formate, phenylethyl acetate, benzyl carbinol butyrate, and 2-phenylethyl phenylacetate, respectively.

[0053] Sample pretreatment: add 100g each of ethyl benzoate, 2-phenylethyl formate, phenylethyl acetate, benzyl carbinol butyrate and 2-phenylethyl phenylacetate to 10kg of modern flavor, stir evenly, prepare labeled flavors with a mass concentration of each marker of 1.0%, and use them for two flavoring lines of cigarettes in the silk workshop respectively; the production batch of the double-nozzle flavoring line is batch I, and the production batch of the single-nozzle flavoring line is batch II. After the flavoring equipment runs stably, collect tobacco samples at a fixed position behind the outlet of the flavoring machine drum. Take samples 30 times for each batch, with an interval of 1min each time. The mass of each sample is 100g, and they are stored in sealed bags.

[0054] After flavoring, the tobacco cut sample was placed in a temperature of (20±1)℃ and a relative humidity of (60±3)% for 24 hours. After grinding, 3.0g of the sample was weighed and placed in a 250mL conical flask, and 100mL of n-hexane extract containing 5μg / mL of internal standard was added. Ultrasonic extraction was performed at 30℃ for 30min, and the upper layer of the extract was filtered through a 0.45μm filter membrane and placed in a chromatographic injection bottle for testing.

[0055] Instrument working conditions:

[0056] The chromatographic column was an HP-5MS column (dimensions were 30m×0.25mm×0.25μm); the carrier gas was helium, with a column flow rate of 1mL / min; the split injection was 1μL, with a split ratio of 10:1; ionization was performed using electron impact ionization (EI), with an electron energy of 70ev; the injection port temperature was 280°C, the ion source temperature was 250°C, the quadrupole temperature was 150°C, the transfer line temperature was 280°C, and the temperature program was “50°C for 3min→heated to 200°C at a rate of 5°C / min, held for 2min→heated to 290°C at a rate of 10°C / min, held for 5min”; the scanning mode was SIM (selective ion detection) mode; and the solvent delay was 10min.

[0057] This example studies and tests the chromatographic separation of external markers and internal standards on non-polar HP-5MS non-polar columns and HP-INNOWax polar columns. It was found that polar chromatographic columns could not effectively separate these ester compounds from the complex matrix of tobacco shreds, while using HP-5MS chromatographic columns, under the above instrument working conditions, the components eluted successively and all peaks were baseline separated. The full scan ion flow chromatograms of the mixed standard solution and tobacco shreds flavored with external markers obtained under these conditions are shown in Figure 1. Figure 1 The retention time and selected ions of the target compounds are shown in Table 1.

[0058] Table 1 Retention time and selected ions of target compounds

[0059]

[0060] In this embodiment, isopropanol, methanol, acetonitrile, n-hexane and dichloromethane were selected to extract the flavored tobacco samples. The results showed that the extraction recovery rates of some markers in n-hexane, isopropanol and acetonitrile were too low, resulting in the chromatographic peaks not being detected. When methanol was extracted, the impurities in the tobacco samples were extracted the most, and the chromatographic peak baseline was too high. Dichloromethane extracted less impurities in the tobacco, and the recovery rate of all added markers was high, so dichloromethane was selected as the extraction solvent.

[0061] This example experiment compares the effects of oscillation extraction and ultrasonic extraction on the recovery rate and relative standard deviation of the measured values ​​of 5 external markers, and the results are shown in Table 2. The effect of ultrasonic extraction is better than that of oscillation extraction, and ultrasonic extraction is selected as the extraction method.

[0062] Table 2 Comparison of recovery rates between mechanical oscillation extraction and ultrasonic extraction

[0063]

[0064] This example experiment compares the recovery rate of markers in tobacco shreds extracted by ultrasonic extraction at 20, 30, 40, and 50°C. Figure 2 As shown in the figure, from 20 to 30 °C, more markers were extracted. As the temperature increased, the recovery rate of the markers decreased due to the matrix effect. Therefore, 30 °C was selected as the ultrasonic extraction temperature.

[0065] This embodiment experiment investigated the extraction recovery rate of the marker in the tobacco shreds when the ultrasonic extraction temperature was 30°C and the extraction time was 10, 20, 30, 40, and 50 minutes. Figure 3 As shown in the figure, from 10 to 30 minutes, the recovery rate of the marker increased; from 30 to 50 minutes, the recovery rate of the marker decreased, which should also be due to the more serious matrix effect caused by the increase in extraction time. Therefore, 30 minutes was selected as the ultrasonic extraction time.

[0066] Due to the serious matrix effect of tobacco, the recovery rate of markers when the sample size was 2, 3, 4, and 5 g was investigated. The results are shown in Figure 4 When the sample size was 3 g, the recovery rate of each marker was the highest, so the sample size of tobacco for ultrasonic extraction was selected to be 3 g.

[0067] Linear equation, method limit of detection (LOD) and limit of quantification (LOQ) for target analytes

[0068] The mixed standard working solution configured in this embodiment was injected according to the instrument working conditions, and a linear regression equation of the response ratio (y) and the concentration ratio (x) was established. The LOD and LOQ were calculated according to the signal-to-noise ratio (S / N) = 3 and 10, respectively. The results are shown in Table 3.

[0069] Table 3 Detection linear equations, LOD and LOQ of target analytes

[0070]

[0071] Take the blank tobacco samples without markers, add different concentrations of target analytes at three levels of low, medium and high, and measure them in parallel 6 times. The recovery rate and relative standard deviation (RSD) are calculated (see Table 4). The recovery rate is 89.5%-95.7%, and the relative standard deviation (RSD) is less than 5.1%, which shows that the repeatability and stability of this method are good and can meet the needs of flavoring uniformity detection.

[0072] Table 4 Precision and recovery test results (n = 6)

[0073]

[0074]

[0075] Uniformity evaluation based on individual flavoring characteristic compounds

[0076] The mean and standard deviation of the test data of the double-nozzle flavoring line production batch I and the single-nozzle flavoring production batch II were calculated, and then the coefficient of variation was calculated as follows: CV = (σ / μ) × 100%. Among them, σ represents the standard deviation and μ represents the mean. According to the coefficient of variation of the detection content of each external marker, the flavoring uniformity was evaluated, and the results are shown in Tables 5 and 6. It can be seen that when a single flavoring characteristic compound is used to evaluate the flavoring uniformity of the same batch of samples, the evaluation results of different characteristic compounds vary greatly, which also leads to the inability to quantitatively evaluate the flavoring uniformity of tobacco cuts from different batches and different flavoring methods.

[0077] Table 5 Content of external markers in batch I of cigarette shreds

[0078]

[0079]

[0080]

[0081] Table 6 Contents of external markers in batch II cigarette shreds and principal component analysis scores

[0082]

[0083]

[0084] Homogeneity evaluation and verification based on principal component analysis and kernel density analysis

[0085] The origin software was used to perform principal component analysis on the data in Table 5 and Table 6, and the dimension was reduced to 2 principal components to obtain PCA data, as shown in Table 7. Kernel density analysis is a spatial data analysis method that can be used to calculate the relative concentration of the spatial distribution of elements, reflecting the uneven distribution of point sets on the two-dimensional coordinate axis. This method can intuitively reflect the distribution of discrete measurement values ​​in a continuous area. The present invention uses ArcGIS desktop 10.8-Spatial analyst tools-Density-Kernel density tool to implement spatial kernel density analysis of the PCA score data in batch I and batch II in Table 7, and obtains the kernel density value of each PCA score in batch I and batch II through ArcGIS desktop 10.8-Spatial analyst tools-Extraction-Extract Values ​​toPoints tool, and then adds and calculates respectively: the total kernel density value of batch I is 94.55, and the total kernel density value of batch II is 108.82. According to the concept of kernel density analysis, it is obvious that the data of batch I is more focused, indicating that the flavoring uniformity of batch I is better than that of batch II, which is consistent with the comparison result that the coefficient of variation calculated by using the content of a single flavoring characteristic compound is generally greater for batch II than for batch I.

[0086] Table 7 PCA score data of batch I and batch II

[0087]

[0088]

[0089] In summary, the method for analyzing the uniformity of tobacco flavoring based on ultrasonic extraction and external markers combined with PCA score evaluation established in the present invention has good precision, a wide linear range, a recovery rate of 89.5% to 95.7%, and a relative standard deviation (RSD) of less than 5.1%, indicating that the method has good repeatability and stability and meets the needs of daily testing; it avoids the problems of difficulty in selecting markers when using flavors' own components as markers, markers being easily interfered by tobacco matrix and not having universality, and can more objectively and accurately evaluate the uniformity of the cigarette flavoring process; it is the first time to combine principal component analysis and kernel density analysis for flavoring uniformity evaluation, which directly With the help of software calculations to obtain quantitative results, the problem of inconsistent evaluation of coefficients of variation of multiple markers and the problem that the difference in flavoring uniformity between different production batches and different flavoring methods cannot be quantified is overcome. The evaluation method has practical significance for improving the accuracy and uniformity of flavoring in the cigarette making process. It adopts ultrasonic extraction suitable for large-scale processing and gas chromatography-mass spectrometry commonly used in laboratories. This method has good economy, strong operability and high scalability. It has stable physical and chemical properties, which are similar to various characteristic aroma components in cigarette flavors. A small amount of flavored tobacco samples will not affect the aroma style of the cigarette after smoking, which is of great significance for stabilizing product quality.

[0090] In conclusion, the present invention has certain significance for the evaluation of flavoring uniformity in the tobacco processing and cutting process.

[0091] The above is only a specific implementation case of the patent of the present invention, but the technical features of the patent of the present invention are not limited thereto. Any changes or modifications made by any technician in the relevant field within the field of the present invention are covered by the patent scope of the present invention.

Claims

1. A method for evaluating tobacco flavoring uniformity based on PCA kernel density analysis and external markers, characterized in that: The evaluation method comprises the following steps: S1. preparing a tobacco sample with an external marker; S2, pre-treating tobacco samples; S3, gas chromatography-mass spectrometry analysis; S4. Evaluation of flavoring uniformity based on PCA kernel density analysis.

2. The method for evaluating tobacco flavoring uniformity based on PCA kernel density analysis and external markers according to claim 1, characterized in that: In step S1, the specific process of preparing the tobacco sample with added markers is as follows: Ethyl benzoate, 2-phenylethyl formate, phenylethyl acetate, benzyl carbinol butyrate and 2-phenylethyl phenylacetate are added to flavors to prepare labeled flavors with mass concentrations of each marker ranging from 0.2% to 2.0%, which are used for flavoring cigarettes in a tobacco workshop. After the flavoring equipment runs stably, tobacco samples are collected at a fixed position behind the outlet of the flavoring machine drum, 30 samples are collected for each batch, with an interval of 1 minute each time and a sampling mass of 100g each time. The taken tobacco samples are placed in sealed bags for standby use.

3. The method for evaluating tobacco flavoring uniformity based on PCA kernel density analysis and external markers according to claim 1, characterized in that: In step S2, the specific process of pre-treating the tobacco sample is as follows: The tobacco sample flavored in step S1 is placed under the conditions of temperature (20±1)°C and relative humidity (60±3)% for equilibration for 24 hours; after grinding, 3.0 g of the sample is weighed and placed in a 250 mL conical flask, 100 mL of n-hexane extract containing 5 μg / mL of internal standard is added, ultrasonic extraction is performed at 30°C for 30 minutes, the upper layer of the extract is filtered through a 0.45 μm filter membrane and placed in a chromatographic injection bottle for testing.

4. The method for evaluating tobacco flavoring uniformity based on PCA kernel density analysis and external markers according to claim 1, characterized in that: In step S3, the specific process of gas chromatography-mass spectrometry analysis is as follows: Gas chromatography / mass spectrometry was used for detection, mass spectrometry database retrieval for qualitative analysis, and internal standard method for quantitative analysis.

5. The method for evaluating tobacco flavoring uniformity based on PCA kernel density analysis and external markers according to claim 1, wherein in step S4, the specific process of evaluating flavoring uniformity based on PCA kernel density analysis is as follows: The detection data was analyzed by principal component analysis using origin software, and a PCA score graph was drawn; Kernel density analysis was performed on the PCA score map to quantify the flavoring uniformity according to ArcGIS desktop 10.8 software.

Images