Patent Publication Number: US-2013239981-A1

Title: Method for supercritical extraction of characteristic fragrant substances in different fragrant tobacco leaves

Description:
BACKGROUND OF THE INVENTION 
     1. Technical Field of the Invention 
     The present invention relates to a method for extraction of characteristic fragrant substances in tobacco leaves, and in particular, to a method for supercritical CO 2  fluid extraction of characteristic fragrant substances in different fragrant tobacco leaves, and belongs to the tobacco field. 
     2. Description of the Related Art 
     The fragrances of flue-cured tobacco leaves are mainly classified into strong fragrance, mild fragrance and intermediate fragrance, and further include intermediary fragrances such as strong-to-intermediate fragrance, intermediate-to-strong fragrance, mild-to-intermediate fragrance, intermediate-to-mild fragrance, mild-over-strong fragrance, and strong-over-mild fragrance. After long-term development, characteristic types of tobacco leaves with certain geographical features have been formed in production of tobacco leaves in China; for example, mild fragrance flue-cured tobacco in Jiangchuan and Yuxi, Yu&#39;nan, mild fragrance flue-cured tobacco in Yongding, Fujian, intermediate fragrance flue-cured tobacco in Guiding, Guizhou, and strong fragrance flue-cured tobacco in Xiangcheng and Jiaxian, He&#39;nan. In summary, the fragrance style of flue-cured tobacco leaves in China has gradually changed from intermediate fragrance and strong fragrance to mild fragrance as a whole from northeast to southwest. For example, over 90% of tobacco leaves in Kaiyuan, Liaoning belong to intermediate fragrance, 51% of tobacco leaves in He&#39;nan belong to intermediate-to-mild fragrance, 50% of tobacco leaves in Meitan, Guizhou belong to intermediate fragrance, and the other 50% belongs to intermediate-to-mild fragrance, 68% of tobacco leaves in Ningnan, Sichuan belong to intermediate-to-mild fragrance, over 70% of tobacco leaves in Yongding, Fujian belong to intermediate-to-mild and mild fragrances, 51% of tobacco leaves in Wenshan, Yunan belong to intermediate-to-mild fragrance, and so on. Although the classification of the three types of fragrances of tobacco leaves has been used, the evaluation mainly depends on sensory evaluation through smoking, and qualitative and quantitative indicators are absent. 
     The basis of the style of cigarette lies in the raw tobacco leaves, while the key of the raw tobacco leaves lies in the fragrance style of the tobacco leaves. Studies show that chemical ingredients are the material basis for the formation of fragrance features of tobacco leaves. In-depth analysis of the chemical composition of different fragrant tobacco leaves and research on the correlation between important chemical ingredients and the fragrance of tobacco leaves have important practical significance for understanding and improving the intrinsic quality and availability of tobacco leaves, enhancing the regulation and control capability on cigarette formulation, and guiding procurement of tobacco leaves. 
     Supercritical fluid extraction (SFE) is an extraction and separation technology that has developed rapidly at home and abroad in recent years. The supercritical fluid is a high-density fluid that is subject to a temperature and pressure higher than the critical temperature and the critical pressure, and has a high dissolving capability, high extraction rate, and good transmission performance. Small changes in temperature and pressure may result in a large change in solubility of the solute. By changing physical parameters (temperature and pressure), the dissolving capability of the fluid can be easily changed, thereby achieving the purpose of selective extraction and separation. 
     Commonly used supercritical fluid includes methanol, ethanol, ethane, propane, ethylene, and CO 2 . CO 2  has the following advantages: being capable of working at a low temperature, causing no changes of thermally unstable substances, having a low critical pressure that can be easily achieved in actual operation, being non-toxic, colorless, and odorless, having no pollution to products, being chemically stable, inexpensive and easily available. Consequently, in processing of natural products, CO 2  is usually used as a working medium. 
     Extraction of nicotine in tobacco leaves with a supercritical fluid has been reported, but a method for extraction of characteristic fragrant substances in different fragrant tobacco leaves through SFE has not been reported. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to a method for extraction of characteristic fragrant substances in tobacco leaves, which is applicable to different fragrant tobacco leaves, so as to establish the foundation for providing a unified method for evaluating fragrance styles of tobacco leaves. 
     The technical solution adopted by the present invention is a method for supercritical extraction of characteristic fragrant substances in tobacco leaves of different fragrances, which includes the following steps: 
     1) pretreatment of tobacco leaves: shredding the tobacco leaves and placing the tobacco shreds into an extraction vessel with supercritical CO 2  fluid; 
     2) setting the extraction pressure and the extraction temperature of the supercritical CO 2  fluid to 100 to 350 bar and 35° C. to 70° C., performing static extraction on the tobacco shreds for 10 to 35 min under the condition, then setting the dynamic extraction flow rate of the supercritical CO 2  fluid to 1.0 to 3.5 L/min, and performing dynamic extraction on the tobacco shreds for 5 to 15 min; and 
     3) collecting the obtained supercritical CO 2  fluid extract, and obtaining key fragrant substances that act as determinants after complete evaporation of CO 2 . 
     The used CO 2  is food grade CO 2 . 
     Preferably, the width of the tobacco shreds is 0.8 mm to 1.2 mm. The tobacco leaves are cut into shreds of 0.8 mm to 1.2 mm to fully infiltrate the tobacco leaves. 
     Preferably, in Step 2), the extraction pressure and the extraction temperature of the supercritical CO 2  fluid are set to 250 to 320 bar and 45° C. to 55° C. 
     Preferably, in Step 2), static extraction is performed on the tobacco shreds for 20 to 30 min. 
     Preferably, in Step 2) the dynamic extraction flow rate of the supercritical CO 2  fluid is set to 2.0 to 3.5 L/min. 
     Preferably, in Step 2), dynamic extraction is performed on the tobacco shreds for 10 to 15 min. 
     Said tobacco leaves include three types of typical fragrance tobacco leaves and intermediary fragrance tobacco leaves. The three typical fragrances are strong fragrance, mild fragrance and intermediate fragrance, and the intermediary fragrances include strong-to-intermediate fragrance, intermediate-to-strong fragrance, mild-to-intermediate fragrance, intermediate-to-mild fragrance, mild-over-strong fragrance, and strong-over-mild fragrance. 
     The key fragrant substances that act as determinants obtained through static extraction and dynamic extraction in combination are dissolved in anhydrous ethanol, and analyzed by gas chromatography-mass spectrometry. The results show that the types of fragrant ingredients contained in the different fragrant tobacco leaves are substantially the same, but the contents of the ingredients are different; the fragrant ingredients in the tobacco can be substantially extracted through one-time extraction. After the extract is dissolved in anhydrous ethanol, the solution is sprayed on the tobacco shreds after the supercritical CO 2  fluid extraction, and after ethanol evaporation, the tobacco shreds are rolled into cigarettes, which are then subjected to evaluation smoking by experts. The tobacco shreds are compared with tobacco shreds that are not subjected to supercritical CO 2  fluid extraction, and the result shows that the fragrances of the two types of tobacco shreds are fully the same. This comparison indicates that the supercritical CO 2  extraction method provided by the present invention is applicable in extraction of characteristic fragrant substances in different tobacco leaves. 
     In view that the fragrance style of tobacco leaves is currently determined primarily through evaluation smoking by experts, and unified objective standards are absent, the present invention provides a method for extraction of characteristic fragrant substances, which is applicable to different fragrant tobacco leaves. By analyzing the correlation between the fragrant ingredients in the extract obtained through the method and the fragrance of tobacco leaves, the internal relationship between the type and the content of the fragrant ingredient and the fragrance of tobacco leaves could be found, so that the analysis of fragrant ingredients extracted from the tobacco leaves can be used as an objective standard for evaluation of the fragrance of tobacco leaves. Therefore, the extraction method of the present invention is an efficient means for objectively estimating the fragrance of tobacco leaves, and establishes the foundation for providing a unified method for evaluation of fragrance styles of tobacco leaves. 
     In the present invention, food grade supercritical CO 2  fluid is used as the working medium, and the characteristic fragrant substances in different tobacco leaves are extracted through static extraction and dynamic extraction in combination. In the extraction method of the present invention, no entrainer is used, and the method has the advantages of being safe and non-toxic, is a simple and reproducible process, and has high reliability. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The present disclosure will become more fully understood from the detailed description given herein below for illustration only, and thus does not limit the present disclosure, wherein: 
         FIG. 1  is a process flow chart of an extraction method according to the present invention; 
         FIG. 2  is a gas chromatography/mass spectrometry analysis chromatogram of SFE extract of Guizhou intermediate-fragrance tobacco leaves; 
         FIG. 3  is a gas chromatography/mass spectrometry analysis chromatogram of SFE extract of Yunnan mild-fragrance tobacco leaves; 
         FIG. 4  is a gas chromatography/mass spectrometry analysis chromatogram of SFE extract of He&#39;nan strong-fragrance tobacco leaves; and 
         FIG. 5  shows results of a repeatability test of an extraction method. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is further described below with reference to the embodiments. It should be understood that, these embodiment are merely used to illustrate the present invention, but are not intended to limit the scope of the present invention. 
     Embodiment 1 
     Key fragrant ingredients in Guizhou intermediate-fragrance tobacco leaves, Yunnan mild-fragrance tobacco leaves, and He&#39;nan strong-fragrance tobacco leaves were each extracted by adopting the following method, including the following steps: 
     1) cutting tobacco leaves into shreds of 0.8 mm to 1.2 mm; 
     2) placing 5 g tobacco shreds into an extraction vessel with supercritical CO 2  fluid; 
     3) setting the static extraction pressure and the extraction temperature of the supercritical CO 2  fluid to 290 bar and 50° C.; 
     4) performing static extraction on the tobacco shreds for 30 min under a static extraction condition; 
     5) setting the CO 2  dynamic extraction flow rate to 2.5 L/min; 
     6) performing dynamic extraction on the tobacco shreds for 10 min under a dynamic extraction condition; 
     7) collecting the supercritical CO 2  fluid extract by using a clean glass bottle, and after complete evaporation of CO 2 , adding 3 mL anhydrous ethanol to dissolve the extract for analysis of the ingredients of the extract. 
     The ethanol solutes, obtained through supercritical CO 2  fluid extraction, of the three types of tobacco leaves were analyzed by using gas chromatography-mass spectrometry. The results are shown in  FIGS. 2 to 4 . The analysis results show that the SFE extract in different fragrant tobacco leaves contains organic acids such as 2-methyl butyric acid, 3-methyl valeric acid, n-nonanoic acid, oleic acid, linoleic acid, linolenic acid, and stearic acid, and further contains neutral fragrant ingredients such as solanone, norsolanone, phenethyl alcohol, megastigmatrienone, 3-oxo-α-ionol and 3-OH-β-damascone, 2-acetylpyrrole, and fragrant precursors such as cembratrienediol, thunbergol, and phytol. The types of fragrant ingredients contained in He&#39;nan strong-fragrance tobacco leaves, Guizhou intermediate-fragrance tobacco leaves, and Yunnan mild-fragrance tobacco leaves are substantially the same, but the contents of the fragrant ingredients are different. 
     Embodiment 2 
     By adopting the extraction method in Embodiment 1, the three types of tobacco shred samples were subjected to continuous five-time supercritical CO 2  fluid extraction, and the extract of each type of tobacco leaves obtained by each extraction was subjected to gas chromatography/mass spectrometry analysis. The analysis results show that, with the increase of the number of times of extraction, the content of the fragrant ingredients in the extracts of the three types of tobacco leaves (tobacco shreds) are significantly decreased, and the content of the fragrant ingredients in the first-time extract is much greater than those of the following extracts, indicating that the fragrant ingredients in the tobacco leaves can be substantially extracted through one-time extraction. 
     The three types of tobacco leaves (tobacco shreds) after the supercritical CO 2  fluid extraction were rolled into cigarettes, subjected to smoke panel tests, and compared with tobacco leaves (tobacco shreds) that are not subjected to SFE extraction. The results of the smoke tests are listed in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Results of the smoke panel tests of the different fragrant tobacco leaves after 
               
               
                 SFE extraction 
               
            
           
           
               
               
               
               
            
               
                 Tobacco leaves 
                 Treatment 
                 Flavor 
                 Remark 
               
               
                   
               
               
                 Guizhou 
                 Untreated 
                 Intermediate 
                 Typical 
               
               
                 intermediate-fragrance 
                   
                 fragrance 
                   
               
               
                 tobacco leaves 
                 SFE extraction 
                 Intermediate 
                 Plain fragrance, not clear 
               
               
                   
                 one time 
                 fragrance, 
                   
               
               
                   
                   
                 unobvious 
                   
               
               
                   
                 SFE extraction 
                 Intermediate 
                 Poor fragrance 
               
               
                   
                 two times 
                 fragrance, 
                   
               
               
                   
                   
                 unobvious 
                   
               
               
                 Yunnan mild-fragrance 
                 Untreated 
                 Mild fragrance 
                 Fragrant, occasionally clear 
               
               
                 tobacco leaves 
                   
                   
                 fragrance 
               
               
                   
                 SFE extraction 
                 Mild fragrance, 
                 Flat fragrance, substantially no 
               
               
                   
                 one time 
                 unobvious 
                 fragrance 
               
               
                   
                 SFE extraction 
                 Mild fragrance 
                 Poor fragrance 
               
               
                   
                 two times 
                 unobvious 
                   
               
               
                 He&#39;nan 
                 Untreated 
                 Strong fragrance 
                 Typical 
               
               
                 strong-fragrance 
                 SFE extraction 
                 Intermediate 
                 Tenuous fragrance, having no 
               
               
                 tobacco leaves 
                 one time 
                 fragrance 
                 feature 
               
               
                   
                 SFE extraction 
                 Intermediate 
                 Poor fragrance 
               
               
                   
                 two times 
                 fragrance, obvious 
               
               
                   
               
            
           
         
       
     
     The results of the smoke panel tests indicate that, with one-time extraction by adopting the method of the present invention, the fragrances of the intermediate-fragrance tobacco leaves and the mild-fragrance tobacco leaves are not obvious, and the strong fragrance turns into the intermediate fragrance, and all the tobacco leaves lose the original fragrance features. It is indicated that the fragrant ingredients in the tobacco leaves can be substantially extracted through one-time SFE extraction method of the present invention. 
     Embodiment 3 
     In order to study the repeatability of the method of the present invention, by adopting the extraction method in Embodiment 1, certain fragrant tobacco leaf samples were subjected to continuous five-time supercritical CO 2  fluid extraction, and the extract of the tobacco leaves obtained by each extraction was subjected to gas chromatography/mass spectrometry analysis. The analysis results are shown in  FIG. 5 . The detection results show that the method of the present invention has good repeatability and high reliability. 
     Embodiment 4 
     Key fragrant ingredients in Shandong intermediate-fragrance tobacco leaves, Anhui strong-fragrance tobacco leaves, Hu&#39;nan strong-fragrance tobacco leaves, and Fujian mild-fragrance tobacco leaves were each extracted using the following method, which includes the following steps: 
     1) cutting tobacco leaves into shreds of 0.8 mm to 1.2 mm; 
     2) placing 7.5 g tobacco shreds into an extraction vessel with supercritical CO 2  fluid; 
     3) setting the static extraction pressure and the extraction temperature of the supercritical CO 2  fluid to 250 bar and 55° C.; 
     4) performing static extraction on the tobacco shreds for 20 min under a static extraction condition; 
     5) setting the CO 2  dynamic extraction flow rate to 2.0 L/min; 
     6) performing dynamic extraction on the tobacco shreds for 15 min under a dynamic extraction condition; 
     7) collecting the supercritical CO 2  fluid extract by using a clean glass bottle, and after complete evaporation of CO 2 , adding 2 mL anhydrous ethanol to dissolve the extract for analysis of the ingredients of the extract. 
     The ethanol solutes, obtained through supercritical CO 2  fluid extraction, of the four types of fragrant tobacco leaves were analyzed through gas chromatography-mass spectrometry. The analysis results are substantially the same as those in Embodiment 1. 
     Embodiment 5 
     Key fragrant ingredients in Heilongjiang intermediate-to-mild fragrance tobacco leaves, Sichuan mild-to-intermediate fragrance tobacco leaves, Jiangxi intermediate-to-strong fragrance tobacco leaves, He&#39;nan strong-to-intermediate fragrance tobacco leaves, Yunnan strong-over-mild fragrance tobacco leaves, and He&#39;nan mild-over-strong fragrance tobacco leaves were each extracted using the following method, which includes the following steps: 
     1) cutting tobacco leaves into shreds of 0.8 mm to 1.2 mm; 
     2) placing 5 g tobacco shreds into an extraction vessel with supercritical CO 2  fluid; 
     3) setting the static extraction pressure and the extraction temperature of the supercritical CO 2  fluid to 320 bar and 45° C.; 
     4) performing static extraction on the tobacco shreds for 25 min under a static extraction condition; 
     5) setting the CO 2  dynamic extraction flow rate to 3.5 L/min; 
     6) performing dynamic extraction on the tobacco shreds for 10 min under a dynamic extraction condition; 
     7) collecting the supercritical CO 2  fluid extract by using a clean glass bottle, and after complete evaporation of CO 2 , adding 3 mL anhydrous ethanol to dissolve the extract for analysis of the ingredients of the extract. 
     The ethanol solutes, obtained through supercritical CO 2  fluid extraction, of the six types of fragrant tobacco leaves were analyzed through gas chromatography-mass spectrometry. The analysis results are substantially the same as those in Embodiment 1.