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Abstract [Objectives] This study was conducted to improve the sensory quality and industrial availability of tobacco extracts. [Methods] The L9（34） design was adopted to carried out an extraction experiment， in which formula tobacco was extracted using supercritical CO2， and the extract was concentrated by vacuum distillation. Through sensory evaluation and chemical analysis， the function determination and chemical composition analysis of the tobacco extracts were carried out， and the optimal supercritical fluid extraction process was finally determined. [Results] The obtained optimal supercritical fluid extraction conditions were as follows： extraction temperature 55 ℃， extraction pressure 25 MPa， CO2 flow rate of 20 L/h， and entrainer of 95% ethanol. The tobacco extract obtained under the optimal conditions endowed the cigarettes with full and delicate aroma， less irritation and clean aftertaste and made the flavor of the cigarettes overall coordinated and softer， so the sensory quality was significantly improved. [Conclusions] The tobacco extract obtained by the supercritical CO2 extraction method from formula tobacco can effectively improve the quality of cigarettes.
Key words Supercritical extraction method; Aroma component; Orthogonal test; Tobacco; Flavor and fragrance
Most of the volatile aroma components in tobacco are weakly polar or non-polar compounds， and their solubility in supercritical CO2 is very large. The super-extract obtained by supercritical CO2 extraction technology from tobacco contains aroma components that are much higher in types and percentages than traditional solvent extraction methods[1-3]. Supercritical CO2 extraction can achieve separation below the boiling point of a material， and the heating time of the material is short， which reduces the possibility of thermal decomposition and transformation of the material[4-7]. The endogenous substances of tobacco can be separated by supercritical CO2 extraction and molecular distillation to obtain different grades of distillate products. There are many studies on the separation and application of tobacco extracts at home and abroad. Zhang et al.[8] used gel permeation chromatography to separate tobacco extracts， evaluated the taste characteristics of each separated fraction， and determined the degree of contribution of different sugars to sweetness. Zhang et al.[9] studied the saponification test of waste tobacco extract to improve the extraction rate of solanesol. Xiong et al.[10] used supercritical CO2 extraction combined with molecular distillation to extract the flavor components from tobacco leaves and added them to shredded tobacco to improve the sensory quality of cigarettes. Materials and Methods
Experimental materials
Raw materials
Based on the sensory quality evaluation， five tobacco leaf raw materials were initially selected from Yunnan （C3F-2014， C3F-2013， C2F-2013） and Zimbabwe raw materials （LLBL-2014， L1L-2015）. Because the quality of tobacco products is greatly affected by the regional climate， the use of a single variety of tobacco as raw materials may result in poor product quality stability. In order to ensure the stability of the raw material product quality， the preliminarily screened 5 tobacco leaf raw materials were combined based on the 1∶2∶1∶2∶1 formula， and shredded for later use.
Materials
CO2 used as the extraction fluid in the experiment was purchased from Qingdao Oxygen Plant; 95% ethanol used as the entrainer was purchased from Weifang Ensign Industry; and propanediol used as the entrainer was purchased from the United States.
Equipment
Supercritical extraction device （Applied Separations， USA）; KQ-500DB CNC ultrasonic cleaner （Kunshan Ultrasonic Instrument Co.， Ltd.）; 7890A-5975C gas chromatography-mass spectrometer （Agilent， USA）; V-850 rotary evaporation Instrument （BUCHI， Germany）.
Experimental methods
Flavor and fragrance evaluation method
The sensory evaluation was carried out in accordance with the national cigarette standard GB YC/T 498-2014， using the method of comparative evaluation， with no less than 7 professional evaluation personnel for each evaluation.
Research method of supercritical fluid extraction technique
The solubility and selectivity of supercritical CO2 on the components in tobacco materials are affected by extraction temperature， extraction pressure， extraction time， CO2 flow rate， entrainer and many other factors. Based on the influencing factors， the orthogonal experiment method was used to design the experimental conditions with the sensory evaluation quality of the extract as the investigation index. With extraction temperature （A）， extraction pressure （B）， CO2 flow rate （C） and entrainer （D） as the factors， a 4-factor 3-level experiment was designed （Table 1）.
Aroma composition analysis method
Chromatographic column： HP-5ms quartz capillary column （30 m×0.25 mm×0.25 μm）; inlet temperature： 210 ℃; carrier gas： helium; injection volume： 1.0 μl; split ratio： 5∶1; column flow rate： 0.7 ml/min; programmed heating： column temperature 50 ℃， which was kept for 4 min and then increased at a rate of 2 ℃/min to 150 ℃， which was kept for 1 min and increased at a heating rate of 5 ℃/min to 180 ℃， which was kept for 5 min and increased at heating rate of 10 ℃/min to 280 ℃， which was kept for 30 min. Mass spectrometry conditions： MS interface temperature： 220 ℃; EI ionization energy： 70 eV; ion source temperature： 200 ℃; solvent delay： 2.8 min; scanning ion mass range： 35-550 amu.
Results and Analysis
Analysis of orthogonal experiment results
The experiment was conducted in accordance with the orthogonal experimental factor level table under "Research method of supercritical fluid extraction technique"， and 7 sensory evaluation judges from Shandong China Tobacco were organized to evaluate the extracts under each process condition according to the "Flavor and fragrance evaluation method". The data obtained is shown in Table 2.
According to the characteristics of the orthogonal design， range R can be used to judge the order of the primary and secondary factors. The larger the R value， the greater the influence of the change in the level of the factor on the test index and the more important the factor. The sensory evaluation results of the samples showed that the order of the factors was D>B>C>A. The entrainer had the greatest impact on the sensory evaluation of the product， followed by the extraction pressure and the CO2 flow rate， while the extraction temperature had a relatively small effect on the sensory evaluation of the product. The level with the largest k value of each factor was selected as the optimal level， and the optimal combination was determined as A1B3C1D2： extraction temperature 55 ℃， extraction pressure 25 MPa， CO2 flow rate 20 L/h， and entrainer 95% ethanol. The tobacco extract obtained under the optimal conditions endowed the cigarettes with full and delicate aroma， less irritation and clean aftertaste and made the flavor of the cigarettes overall coordinated and softer， so the sensory quality was significantly improved.
Analysis results of aroma composition
The composition analysis of aroma components provides theoretical support and application basis for the research of extraction technology. Through the analysis of aroma composition of the tobacco extracts， we provided a theoretical basis for future extraction formula adjustment， cigarette product flavoring， new tobacco research and development and storage of aroma chemicals.
The supercritical CO2 extract was analyzed according to "Aroma composition analysis method"， and 41 flavor components were found， all of which are commonly used monomer aroma chemicals for cigarette flavors and fragrances. Among them， there were 21 kinds of effective heterocyclic aromatic components， accounting for 51.2% of all aroma components; there were 10 kinds of effective ketone aroma components， accounting for 24.4% of all aroma components; there were 7 effective hydroxyl aroma components， accounting for 17% of all aroma components （Table 3）. Conclusions and Discussion
In this study， supercritical CO2 extraction was applied to extract the effective aroma components， and the aroma composition of formula tobacco was investigated. The L9（34） design was adopted to carried out an extraction experiment， in which formula tobacco was extracted using supercritical CO2， and the extract was concentrated by vacuum distillation. Through sensory evaluation and chemical analysis， the function determination and chemical composition analysis of the tobacco extracts were carried out， and the optimal supercritical fluid extraction process was finally determined as follows： extraction temperature 55℃， extraction pressure 25 MPa， CO2 flow rate of 20 L/h， and entrainer of 95% ethanol. The tobacco extract obtained under the optimal conditions endowed the cigarettes with full and delicate aroma， less irritation and clean aftertaste and made the flavor of the cigarettes overall coordinated and softer， so the sensory quality was significantly improved. Through this study， the sensory quality of tobacco extracts could be effectively improved， which lays a foundation for the next step in the application of cigarette flavoring and new tobacco research and development.
References
[1] ZHAO XH， ZENG J， GAO HY. Optimization and composition of volatile oil from Polygonatum odoratum （Mill Druce） using supercritical fluid extraction[J]. Trop J Pharm Res， 2014， 13（5）： 779-786.
[2] SHAO QS， HUANG YQ， ZHOU AC. Application of response surface methodology to optimize supercritical carbon dioxide extraction of volatile compounds from Crocus sativus[J]. J Sci Food Agric， 2014， 94（7）： 1430-1436.
[3] XU QQ， XU JQ， CHEN Y， et al. Optimization of supercritical carbon dioxide fluid extraction process for aroma components of tobacco[J]， Fine Chemicals， 2017， 34（4）： 431-436.
[4] TANG ZL， WANG H. Application and development of molecular distillation[J]Application and development of molecular distillation， 2014， 34（10）： 44-48.
[5] XIA Y， CUI XM， YANG HW， et al. Studies on the new characteristics style of tobacco extract by extraction and separation technology[J]. The Food Industry， 2016（7）： 33-39.
[6] NIAN XK， LIU YY， LI XS， et al. Optimization of supercritical CO2 extraction conditions for Honghuadajinyuan tobacco essence and analysis of its volatile components[J].Tobacco Science & Technology， 2011， 48（4）： 46-52.
[7] WEI Q， SHI JZ， ZHENG B， et al. The study of the relationship between the chemical composition and sensory quality about paper-making reconstituted tobacco material[J]. Chinese Agricultural Science Bulletin， 2012， 28（12）： 264-268.
[8] ZHANG QD， LIU JH， ZHANG WJ， et al. Sensoryoriented analysis of key sweet taste components in cured tobacco extract[J]. Tobacco Science & Technology， 2016， 49（6）： 58-64.
[9] ZHANG XF， CAO D， DENG GD. Saponification experiments for solanesol extraction from waste and inferior tobacco concrete[J]. Tobacco Science & Technology， 2007（5）： 50-52.
[10] XIONG GX， ZHU W， YU ST， et al. Application of extracting the aroma constituents in tobacco with supercritical CO2 fluid extraction and molecular distillation technology[J]. HUBEI AGRICULTURAL SCIENCES， 2010， 49（7）： 1690-1693.
Key words Supercritical extraction method; Aroma component; Orthogonal test; Tobacco; Flavor and fragrance
Most of the volatile aroma components in tobacco are weakly polar or non-polar compounds， and their solubility in supercritical CO2 is very large. The super-extract obtained by supercritical CO2 extraction technology from tobacco contains aroma components that are much higher in types and percentages than traditional solvent extraction methods[1-3]. Supercritical CO2 extraction can achieve separation below the boiling point of a material， and the heating time of the material is short， which reduces the possibility of thermal decomposition and transformation of the material[4-7]. The endogenous substances of tobacco can be separated by supercritical CO2 extraction and molecular distillation to obtain different grades of distillate products. There are many studies on the separation and application of tobacco extracts at home and abroad. Zhang et al.[8] used gel permeation chromatography to separate tobacco extracts， evaluated the taste characteristics of each separated fraction， and determined the degree of contribution of different sugars to sweetness. Zhang et al.[9] studied the saponification test of waste tobacco extract to improve the extraction rate of solanesol. Xiong et al.[10] used supercritical CO2 extraction combined with molecular distillation to extract the flavor components from tobacco leaves and added them to shredded tobacco to improve the sensory quality of cigarettes. Materials and Methods
Experimental materials
Raw materials
Based on the sensory quality evaluation， five tobacco leaf raw materials were initially selected from Yunnan （C3F-2014， C3F-2013， C2F-2013） and Zimbabwe raw materials （LLBL-2014， L1L-2015）. Because the quality of tobacco products is greatly affected by the regional climate， the use of a single variety of tobacco as raw materials may result in poor product quality stability. In order to ensure the stability of the raw material product quality， the preliminarily screened 5 tobacco leaf raw materials were combined based on the 1∶2∶1∶2∶1 formula， and shredded for later use.
Materials
CO2 used as the extraction fluid in the experiment was purchased from Qingdao Oxygen Plant; 95% ethanol used as the entrainer was purchased from Weifang Ensign Industry; and propanediol used as the entrainer was purchased from the United States.
Equipment
Supercritical extraction device （Applied Separations， USA）; KQ-500DB CNC ultrasonic cleaner （Kunshan Ultrasonic Instrument Co.， Ltd.）; 7890A-5975C gas chromatography-mass spectrometer （Agilent， USA）; V-850 rotary evaporation Instrument （BUCHI， Germany）.
Experimental methods
Flavor and fragrance evaluation method
The sensory evaluation was carried out in accordance with the national cigarette standard GB YC/T 498-2014， using the method of comparative evaluation， with no less than 7 professional evaluation personnel for each evaluation.
Research method of supercritical fluid extraction technique
The solubility and selectivity of supercritical CO2 on the components in tobacco materials are affected by extraction temperature， extraction pressure， extraction time， CO2 flow rate， entrainer and many other factors. Based on the influencing factors， the orthogonal experiment method was used to design the experimental conditions with the sensory evaluation quality of the extract as the investigation index. With extraction temperature （A）， extraction pressure （B）， CO2 flow rate （C） and entrainer （D） as the factors， a 4-factor 3-level experiment was designed （Table 1）.
Aroma composition analysis method
Chromatographic column： HP-5ms quartz capillary column （30 m×0.25 mm×0.25 μm）; inlet temperature： 210 ℃; carrier gas： helium; injection volume： 1.0 μl; split ratio： 5∶1; column flow rate： 0.7 ml/min; programmed heating： column temperature 50 ℃， which was kept for 4 min and then increased at a rate of 2 ℃/min to 150 ℃， which was kept for 1 min and increased at a heating rate of 5 ℃/min to 180 ℃， which was kept for 5 min and increased at heating rate of 10 ℃/min to 280 ℃， which was kept for 30 min. Mass spectrometry conditions： MS interface temperature： 220 ℃; EI ionization energy： 70 eV; ion source temperature： 200 ℃; solvent delay： 2.8 min; scanning ion mass range： 35-550 amu.
Results and Analysis
Analysis of orthogonal experiment results
The experiment was conducted in accordance with the orthogonal experimental factor level table under "Research method of supercritical fluid extraction technique"， and 7 sensory evaluation judges from Shandong China Tobacco were organized to evaluate the extracts under each process condition according to the "Flavor and fragrance evaluation method". The data obtained is shown in Table 2.
According to the characteristics of the orthogonal design， range R can be used to judge the order of the primary and secondary factors. The larger the R value， the greater the influence of the change in the level of the factor on the test index and the more important the factor. The sensory evaluation results of the samples showed that the order of the factors was D>B>C>A. The entrainer had the greatest impact on the sensory evaluation of the product， followed by the extraction pressure and the CO2 flow rate， while the extraction temperature had a relatively small effect on the sensory evaluation of the product. The level with the largest k value of each factor was selected as the optimal level， and the optimal combination was determined as A1B3C1D2： extraction temperature 55 ℃， extraction pressure 25 MPa， CO2 flow rate 20 L/h， and entrainer 95% ethanol. The tobacco extract obtained under the optimal conditions endowed the cigarettes with full and delicate aroma， less irritation and clean aftertaste and made the flavor of the cigarettes overall coordinated and softer， so the sensory quality was significantly improved.
Analysis results of aroma composition
The composition analysis of aroma components provides theoretical support and application basis for the research of extraction technology. Through the analysis of aroma composition of the tobacco extracts， we provided a theoretical basis for future extraction formula adjustment， cigarette product flavoring， new tobacco research and development and storage of aroma chemicals.
The supercritical CO2 extract was analyzed according to "Aroma composition analysis method"， and 41 flavor components were found， all of which are commonly used monomer aroma chemicals for cigarette flavors and fragrances. Among them， there were 21 kinds of effective heterocyclic aromatic components， accounting for 51.2% of all aroma components; there were 10 kinds of effective ketone aroma components， accounting for 24.4% of all aroma components; there were 7 effective hydroxyl aroma components， accounting for 17% of all aroma components （Table 3）. Conclusions and Discussion
In this study， supercritical CO2 extraction was applied to extract the effective aroma components， and the aroma composition of formula tobacco was investigated. The L9（34） design was adopted to carried out an extraction experiment， in which formula tobacco was extracted using supercritical CO2， and the extract was concentrated by vacuum distillation. Through sensory evaluation and chemical analysis， the function determination and chemical composition analysis of the tobacco extracts were carried out， and the optimal supercritical fluid extraction process was finally determined as follows： extraction temperature 55℃， extraction pressure 25 MPa， CO2 flow rate of 20 L/h， and entrainer of 95% ethanol. The tobacco extract obtained under the optimal conditions endowed the cigarettes with full and delicate aroma， less irritation and clean aftertaste and made the flavor of the cigarettes overall coordinated and softer， so the sensory quality was significantly improved. Through this study， the sensory quality of tobacco extracts could be effectively improved， which lays a foundation for the next step in the application of cigarette flavoring and new tobacco research and development.
References
[1] ZHAO XH， ZENG J， GAO HY. Optimization and composition of volatile oil from Polygonatum odoratum （Mill Druce） using supercritical fluid extraction[J]. Trop J Pharm Res， 2014， 13（5）： 779-786.
[2] SHAO QS， HUANG YQ， ZHOU AC. Application of response surface methodology to optimize supercritical carbon dioxide extraction of volatile compounds from Crocus sativus[J]. J Sci Food Agric， 2014， 94（7）： 1430-1436.
[3] XU QQ， XU JQ， CHEN Y， et al. Optimization of supercritical carbon dioxide fluid extraction process for aroma components of tobacco[J]， Fine Chemicals， 2017， 34（4）： 431-436.
[4] TANG ZL， WANG H. Application and development of molecular distillation[J]Application and development of molecular distillation， 2014， 34（10）： 44-48.
[5] XIA Y， CUI XM， YANG HW， et al. Studies on the new characteristics style of tobacco extract by extraction and separation technology[J]. The Food Industry， 2016（7）： 33-39.
[6] NIAN XK， LIU YY， LI XS， et al. Optimization of supercritical CO2 extraction conditions for Honghuadajinyuan tobacco essence and analysis of its volatile components[J].Tobacco Science & Technology， 2011， 48（4）： 46-52.
[7] WEI Q， SHI JZ， ZHENG B， et al. The study of the relationship between the chemical composition and sensory quality about paper-making reconstituted tobacco material[J]. Chinese Agricultural Science Bulletin， 2012， 28（12）： 264-268.
[8] ZHANG QD， LIU JH， ZHANG WJ， et al. Sensoryoriented analysis of key sweet taste components in cured tobacco extract[J]. Tobacco Science & Technology， 2016， 49（6）： 58-64.
[9] ZHANG XF， CAO D， DENG GD. Saponification experiments for solanesol extraction from waste and inferior tobacco concrete[J]. Tobacco Science & Technology， 2007（5）： 50-52.
[10] XIONG GX， ZHU W， YU ST， et al. Application of extracting the aroma constituents in tobacco with supercritical CO2 fluid extraction and molecular distillation technology[J]. HUBEI AGRICULTURAL SCIENCES， 2010， 49（7）： 1690-1693.