Method of extracting sucrose esters from oriental tobacco

Abstract

The invention provides a method of extracting sucrose esters from Oriental tobacco, the method comprising: providing a Oriental tobacco material; contacting the solid Oriental tobacco material with a supercritical fluid, such as supercritical carbon dioxide, for a time and under conditions sufficient to extract sucrose esters from the Oriental tobacco into the supercritical fluid; and separating the supercritical fluid comprising extracted sucrose esters from the Oriental tobacco. The resulting extract can comprise more than 90% by weight of tetra-acyl sucrose esters, and can be used as a flavor additive for tobacco materials used in smoking articles.

Description

FIELD OF THE INVENTION

The invention relates to methods of processing Oriental tobacco, and in particular, to methods for extracting sucrose esters from Oriental tobacco.

BACKGROUND OF THE INVENTION

Popular smoking articles, such as cigarettes, have a substantially cylindrical rod shaped structure and include a charge, roll or column of smokable material such as shredded tobacco (e.g., in cut filler form) surrounded by a paper wrapper thereby forming a so-called “tobacco rod.” Normally, a cigarette has a cylindrical filter element aligned in an end-to-end relationship with the tobacco rod. Typically, a filter element comprises plasticized cellulose acetate tow circumscribed by a paper material known as “plug wrap.” Certain cigarettes incorporate a filter element having multiple segments, and one of those segments can comprise activated charcoal particles. Typically, the filter element is attached to one end of the tobacco rod using a circumscribing wrapping material known as “tipping paper.” It also has become desirable to perforate the tipping material and plug wrap, in order to provide dilution of drawn mainstream smoke with ambient air. A cigarette is employed by a smoker by lighting one end thereof and burning the tobacco rod. The smoker then receives mainstream smoke into his/her mouth by drawing on the opposite end (e.g., the filter end) of the cigarette.

The tobacco used for cigarette manufacture is typically used in a so-called “blended” form. For example, certain popular tobacco blends, commonly referred to as “American blends,” comprise mixtures of flue-cured tobacco, burley tobacco and Oriental tobacco, and in many cases, certain processed tobaccos, such as reconstituted tobacco and processed tobacco stems. The precise amount of each type of tobacco within a tobacco blend used for the manufacture of a particular cigarette brand varies from brand to brand. However, for many tobacco blends, flue-cured tobacco makes up a relatively large proportion of the blend, while Oriental tobacco makes up a relatively small proportion of the blend. See, for example, Tobacco Encyclopedia, Voges (Ed.) p. 44-45 (1984), Browne, The Design of Cigarettes, 3^rd Ed., p.43 (1990) and Tobacco Production, Chemistry and Technology, Davis et al. (Eds.) p. 346 (1999).

Oriental tobaccos are desirable components of the tobacco blends of smoking products because Oriental tobaccos yield smoke possessing certain unique and desirable flavor and aroma characteristics. Most Oriental tobaccos possess relatively low nicotine content, and possess relatively high levels of certain reducing sugars, acids and volatile flavor compounds. Some of the distinct flavors and aromas characteristic of Oriental tobacco smoke are attributed to the presence of sucrose esters in Oriental tobaccos, and the pyrolysis products of those sucrose esters. However, the sucrose ester concentrations in some types of Oriental tobaccos are relatively high, and can introduce so-called “off-notes” to the flavor and aroma of smoke that results from the burning of those tobaccos. Thus, there have been constraints upon the amount of certain Oriental tobaccos traditionally used in tobacco blends, because the desirable flavor and aroma characteristics of the smoke of those tobaccos become overpowering and undesirable when relatively high levels of those tobaccos are used in tobacco blends.

The types of sucrose esters that are present in Oriental tobaccos are sugar derivatives possessing covalently bound ester groups. See, Tobacco Production, Chemistry and Technology, Davis et al. (Eds.) p. 294 (1999). Sucrose esters thermally decompose (e.g., such as when the Oriental tobacco incorporating those sucrose esters is burned) to yield branched chain low molecular weight carboxylic acids, including 2-methylpropionic acid, 3-methylbutyric acid and 3-methylpentanoic acid. Many of the off-notes characteristic of the smoke of Oriental tobaccos are associated with relatively high levels of those carboxylic acids.

Different methods have been used to extract and isolate sucrose esters from green tobacco leaf. Severson et al. developed a gel filtration-partition chromatography method using SEPHADEX LH-20 to isolate individual groups of sucrose esters. In this method, tobacco leaf was washed with CH₂Cl₂ followed by a series of evaporation and extraction steps using different solvents. The final extract in CHCl₃ was fractionated first on a LH-20 column with i.d. of 2.54 cm and bed length of 58 cm. The sucrose ester gel fractions (GF #75-140) were evaporated to dryness and re-dissolved in 3 mL of CHCl₃. This fraction was separated once again on a 1.37 cm i.d. column (bed length 110 cm). Elution with CHCl₃ produced another fraction (GF #45-65, 95+ % sucrose esters). This fraction was then used with another GF column to isolate each SE. Severson et al. were able to isolate 6 groupings of ester isomers, differing by 14 amu. See Severson et al., Isolation and characterization of sucrose esters of the cuticular waxes of green tobacco leaf, J. Agric. Food Chem., 1985, 33, 870-875.

In a later development, Danehower used a silica gel solid phase cartridge to clean the CH₂Cl₂ extract of leaf surface. First, the extract was loaded into the solid phase extraction (SPE) cartridge and washed with CH₂Cl₂. It was demonstrated that most of the polar compounds, including sucrose esters, were retained on the column and were not washed out with CH₂Cl₂. Next, methanol was used to quantitatively remove the sucrose esters from the silica column. It was shown by gas chromatography (GC) that 50% of the total material from the leaf surface was cleaned up using this technique, while 99% of the sucrose esters was retained on the SPE column. See Danehower, A rapid method for the isolation and quantification of the sucrose esters of tobacco, Tobacco Int., 1987, 189, 30-33.

In a later study by Kandra et al., CH₃CN was used as an extraction solvent in order to wash the leaf surface. They did not use the more common CH₂Cl₂ solvent, in order to avoid extraction of cuticular hydrocarbons that would have interfered with the sucrose esters separation. After CH₃CN extraction, the sucrose ester-enriched sample was extracted with CHCl₃/H₂O (2/1, v/v) to remove H₂O-soluble materials. The CHCl₃ phase was dried and later was separated by high performance liquid chromatography (HPLC) using a Cyano column. The workers were able to isolate four fractions from the HPLC separation with the last fraction being sucrose esters. See Kandra et al., Studies of the site and mode of biosynthesis of tobacco trichome exudates components, Arch. Biochem. Biophys., 1988, 265, 425-432.

It would be desirable to provide a method for efficient extraction of sucrose esters from Oriental tobacco materials in a manner that minimizes extraction of other components of the tobacco, thereby providing a relatively pure sucrose ester extract that can be used as a flavorful additive for tobacco materials used in smoking articles.

SUMMARY OF THE INVENTION

The present invention provides a method of extracting sucrose esters from Oriental tobacco using a fluid in a supercritical state as a solvent. The supercritical fluid extraction of the invention is more selective for desirable sucrose ester compounds in Oriental tobacco as compared to prior art methods. It has been discovered that raising the extraction temperature enhances the selectivity of the extraction for sucrose esters when coupled with relatively high fluid pressure, which enhances the solvating power of the fluid.

Thus, in one aspect, the present invention provides a method of extracting sucrose esters from Oriental tobacco, wherein the method comprises providing an Oriental tobacco material and contacting the Oriental tobacco material with a supercritical fluid for a time and under conditions sufficient to extract sucrose esters from the Oriental tobacco into the supercritical fluid. The supercritical fluid comprising the extracted sucrose esters is then separated from the Oriental tobacco. Following separation, the supercritical fluid can be evaporated in order to isolate the tobacco extract. In preferred embodiments of the invention, the isolated tobacco extract is at least about 90% by weight of sucrose esters, based on the total weight of the isolated extract. In certain embodiments, the isolated Oriental tobacco extract comprises at least about 95% by weight of sucrose esters. The resulting tobacco extract rich in sucrose esters can be used as a flavorful and aromatic additive for other tobacco materials, particularly where it is desirable to impart the character of Oriental tobacco to non-Oriental tobacco materials. For instance, the isolated extract can be applied to a tobacco material to be used in a smokable article, for example, as a casing or top dressing ingredient. The isolated extract can be applied to the tobacco material in the neat form or can be dissolved in a solvent and applied as a liquid.

The Oriental tobacco material that is subjected to the extraction process can take a variety of forms. Exemplary forms include whole leaf, laminae, cut filler, volume expanded, stems, cut-rolled stems, cut-puffed stems, reconstituted tobacco, and particulate forms. Preferably, in order to increase the available surface area and thus enhance the extraction process, the Oriental tobacco is in a cut filler or particulate form. It is preferable to use a pure Oriental tobacco unblended with other tobacco materials.

A variety of supercritical fluids could be used to practice the present invention. It is preferred that the supercritical fluid have a relatively moderate critical temperature, such as a temperature between about 30 and about 100° C. Exemplary supercritical fluids include carbon dioxide, sulfur hexafluoride, nitrous oxide, and halogenated hydrocarbons including one to four carbon atoms.

The temperature and pressure of extraction can greatly affect the selectivity of the extraction and the yield of sucrose esters. Preferably, the temperature of extraction is between about 30° and 150° C. and the extraction pressure is between about 200 and about 1,000 atm. In preferred embodiments, higher temperatures and pressures are used. For instance, in one embodiment, the extraction is conducted at a temperature of at least 60° C., and more preferably at least about 100° C. In preferred embodiments, the extraction pressure is at least about 300 atm, more preferably at least about 400 atm. In one preferred embodiment, the extraction temperature is about 100° C. and the extraction pressure is about 450 atm. The length of time during which the Oriental tobacco is contacted with the supercritical fluid can vary, but is typically from about 30 minutes to about 240 minutes.

In another aspect, the present invention provides a tobacco material that has been treated by application of the sucrose ester rich extract formed by the above-described process. In yet another aspect, the invention provides a smoking article comprising tobacco material having the sucrose ester enriched extract applied thereto, the sucrose ester extract having been formed using their above-described process.

In yet another aspect, the invention provides Oriental tobacco material having been treated by supercritical fluid extraction as described above. The treated Oriental tobacco with have a greatly reduced concentration of sucrose esters, and will thus exhibit a milder flavor and aroma. The treated Oriental tobacco can be mixed with other Oriental tobaccos or non-Oriental tobaccos as desired during production of smoking articles.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to assist understanding of embodiments of the invention, reference will now be made to the appended drawings, wherein:

FIG. 1 is a schematic representation of two prior art methods of isolating sucrose esters from Oriental tobacco and one embodiment of the method of the invention;

FIGS. 2A-2C illustrate GC/MS separation of sucrose ester extract from Oriental tobacco using each of the three extraction methods of FIG. 1, wherein 2A is the supercritical fluid extraction (SFE) method, 2B is the Severson method, and 2C is the Kandra method;

FIG. 3 illustrates normal phase HPLC/UV separation of tobacco extract using the Severson extraction technique (HPLC conditions: 85/15% isooctane/ethanol, 0.8 mL/min, Cyano column (250×4.6 mm, 5 μm dp), detection 214 nm, injection volume 40 μL);

FIG. 4 is a GC/MS profile of sucrose esters of the Severson extract isolated via semi-preparative HPLC/UV prior to derivatization;

FIGS. 5A-5D are GC/MS profiles of isolated sucrose esters (derivatized) originating from fractionation of Oriental tobacco via SFE at different pressures (150, 200, 350, and 450 atm) and 60° C. using 2 mL/min of liquid CO₂ (extraction time of 75 minutes); and

FIGS. 6A-6D are GC/MS profiles of isolated sucrose esters (derivatized) originating from fractionation of Oriental tobacco via SFE at different pressures (150, 200, 350, and 450 atm) and 100° C. using 2 mL/min of liquid CO₂ (extraction time of 75 minutes).

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

The Oriental tobacco used in the invention can vary. Descriptions of Oriental-type tobaccos, growing practices, harvesting practices and curing practices are set forth in Wolf, Aromatic or Oriental Tobaccos (1962), Akehurst, Tobacco (1968), Tobacco Encyclopedia, Voges (Ed.) (1984), Tobacco Production, Chemistry and Technology, Davis et al. (Eds.) (1999). Oriental-type tobaccos also are referred to as Greek, aromatic and Turkish tobaccos. Representative Oriental-type tobaccos include the Izmir, Basma, Mavra and Samsun varieties. Other representative Oriental-type tobaccos include Trabzon, Thesalian, Tasova, Sinop, Izmit, Hendek, Edirne, Semdinli, Adiyanman, Yayladag, Iskenderun, Duzce, Macedonian, Katerini, Prilep, Krumovgrad, Bafra, Bursa, Bucak, Bitlis and Balikesir tobaccos, as well as the so-called semi-Oriental tobaccos such as Sebinkarahisar, Borgka and East Balkan tobaccos. Although Oriental-type tobaccos that are employed in accordance with the present invention can be grown in a variety of locations throughout the world, typical Oriental tobaccos are grown in eastern Mediterranean regions such as Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy, Yugoslavia, and Romania. Preferred Oriental tobaccos are sun-cured. Preferred sun-cured Oriental tobaccos are aged for at least one year after curing is complete.

Oriental-type tobaccos that are used in carrying out the present invention possess relatively high levels of sugar esters. Although the level of sucrose esters in Oriental tobaccos can vary considerably from growing region to growing region, and even within growing regions, Oriental tobacco material used in carrying out the method of the invention typically exhibits a sucrose ester concentration (expressed as methyl ester equivalents) of at least about 1,600 ppm, usually at least about 2,000 ppm, often at least about 3,000 ppm, frequently at least about 4,000, or even at least about 5,000 ppm, based on the dry weight of that Oriental tobacco.

As used herein, the term “sucrose esters” refers to sucrose molecules comprising three acyl groups on the glucose ring, each hydrocarbon chain of the acyl group comprising 3 to 8 carbon atoms and optionally including one or more double bonds. The sucrose esters also typically include an acetyl group on either the glucose ring or the fructose ring, which gives rise to the common reference to these esters as tetra-acyl sucrose esters. Although the exact structure of the sucrose esters isolated in the method of the invention can vary somewhat as to placement, chain length, and saturation of the acyl groups, most sucrose esters of Oriental tobacco are encompassed by the following structure:
wherein each R is an independently selected C3-C8 hydrocarbon, which can be straight chain or branched and saturated or unsaturated, and both R₁ are H or one R₁ is H and the other is acetyl (—C(O)—CH₃). Most common R groups include butyl, pentyl, and hexyl.

The physical form of the Oriental tobacco material used in the invention can vary. Most preferably, the Oriental tobaccos are those that have been appropriately cured and aged. The tobacco can be used in whole leaf form or in the form of laminae or strip. The tobacco also can have a shredded or cut filler form. The Oriental tobacco can have a processed form, such as processed tobacco stems (e.g., cut-rolled or cut-puffed stems), volume expanded tobacco (e.g., puffed tobacco, such as dry ice expanded tobacco (DIET), preferably in cut filler form), or reconstituted tobacco (e.g., reconstituted tobaccos manufactured using paper-making type or cast sheet type processes, preferably in strip or cut filler form). Tobacco stems or particulate tobacco material can also be used (e.g., fines, dust, or scrap). The Oriental tobacco is preferably used in solid particulate form, wherein the solid pieces are either pieces of whole Oriental tobacco or a portion thereof, or pieces of a reconstituted Oriental tobacco.

The supercritical fluid used in the invention may vary. As used herein, “supercritical fluid” refers to a fluid at a temperature and pressure above its critical point. Supercritical extraction is a technique in which gases are compressed under supercritical conditions to form a fluid, which is then used to remove components from a matrix. Supercritical fluids have good extracting power because of their density, which can be controlled by changes in pressure or temperature, and low viscosity, high diffusivity and low surface tension, which enhance mass transfer inside a solid matrix. A supercritical fluid has properties that are intermediate between those of gases and liquids. Increasing pressure of a supercritical fluid (i.e., increasing fluid density) increases its solvent power.

Exemplary supercritical fluids include carbon dioxide, sulfur hexafluoride, nitrous oxide, and halogenated hydrocarbons including 1 to 4 carbon atoms, such as CF₄, CHF₃—CClF₃, CBrF₃, CF₂═CH₂, CF₃—CF₂—CF₃, CHClF₂, CCl₂F₂, CHCl₂F, CCl₃F, CBrF₃, and CH₃—CF₃. Preferred supercritical fluids have a critical temperature in the range of about 30 to about 100° C.

Carbon dioxide is a preferred supercritical fluid because it has a manageable critical point (i.e., critical pressure of about 72 atm and critical temperature of 31° C.) and is non-toxic, non-flammable, inexpensive and relatively easy to separate from extracts. The solvent power of supercritical CO₂ can be readily tailored by adjusting the temperature and pressure of the supercritical phase such that the desired chemical compounds (i.e., sucrose esters) are selectively dissolved and removed from the tobacco.

The method of the invention involves extraction of sucrose esters from Oriental tobacco by contacting, and preferably intimately mixing, the Oriental tobacco material with a fluid in a supercritical state for a time and under conditions (i.e., suitable pressure and temperature) sufficient to extract sucrose esters from Oriental tobacco.

Any extraction temperature that is the same as, or higher, than the critical temperature of the supercritical fluid used can be used. For carbon dioxide, the extraction temperature is preferably at least about 60° C., more preferably at least about 80° C., and most preferably at least about 100° C. The extraction temperature is typically about 30 to about 150° C., more preferably about 50 to about 120° C., and most preferably about 70 to about 110° C. In one embodiment, the extraction temperature is about 100° C. It has been determined, as discussed in greater detail in the experimental section below, that higher extraction temperatures can enhance the selectivity of the extraction for sucrose esters as opposed to other species present in the tobacco material.

The extraction pressure can be any pressure above the critical pressure of the supercritical fluid employed in the method. For carbon dioxide, the pressure is preferably at least about 200 atm, more preferably at least about 300 atm, and most preferably at least about 400 atm. The extraction pressure is typically about 200 to about 1,000 atm, more preferably about 300 to about 800 atm, and most preferably about 400 to about 600 atm. In one embodiment, the extraction pressure is about 450 atm. Increasing extraction pressure increases the solvating power of the supercritical fluid, which in turn, improves the effectiveness of the extraction process.

The amount of time that the supercritical fluid must remain in contact with the Oriental tobacco can vary. Typically, the fluid will contact the tobacco material for about 30 minutes to about 240 minutes, although longer or short time periods could be used without departing from the invention.

The amount of supercritical fluid used in the extraction can vary, but is typically between about 30 grams and about 240 grams per gram of tobacco to be extracted. As would be understood, smaller amounts of fluid could be used, but may result in a reduced extraction efficiency and reduced sucrose ester yield. Likewise, greater amounts of fluid could be used, but would not be expected to enhance extraction.

The extraction step is performed in a pressure-controlled and temperature-controlled environment, which is typically provided by contacting the Oriental tobacco with the supercritical fluid in an air-sealed vessel or chamber. Typically, a pressure-controlled environment is provided using a pressure vessel or chamber which is capable of withstanding relatively high pressures, such as pressures up to about 1,000 atm. Preferred pressure vessels are equipped with an external heating source, and can also be equipped with means for agitation, such as an impeller. Examples of suitable vessels include a high pressure autoclave from Berghof/America Inc. of Concord, Calif. and Parr Reactor Model No. 4522 or Parr Reactor Model No. 4552 available from The Parr Instrument Co., as well as CEM Corporation Model XP-1500 and HP-500 pressure vessels. Operation of such exemplary vessels will be apparent to the skilled artisan.

Exemplary supercritical fluid extraction operating conditions and equipment are shown, for example, in U.S. Pat. No. 4,153,063 to Roselius et al.; U.S. Pat. No. 4,506,682 to Muller; U.S. Pat. No. 4,714,617 to Gahrs; U.S. Pat. No. 4,727,889 to Niven, Jr. et al.; U.S. Pat. No. 5,018,540 to Grubbs et al.; and U.S. Pat. No. 5,435,325 to Clapp et al.; and in U.S. Pub. Appl. Nos. 2004/0025891 to McAdam et al. and 2004/0016436 to Thomas.

Following the extraction step, the supercritical fluid containing the entrained dissolved extract from the tobacco is removed from the tobacco material and preferably evaporated to isolate the tobacco extract. In preferred embodiments, the resulting isolated extract is at least about 90% by weight sucrose esters, based on the total weight of the isolated extract, more preferably at least about 95% by weight. The isolated extract can be subjected to further purification steps known in the art, such as through various solvent extraction techniques, including solvent partitioning.

The extracted sucrose esters are useful as additives in the manufacture of smoking articles. Since the unique sensory aspects of Oriental tobacco are attributable, in part, to the presence of sucrose esters, the concentrated sucrose ester extract produced by the method of the invention can be used to endow non-Oriental tobacco or mixtures of tobacco with flavor and aroma associated with Oriental tobacco varieties. Thus, the extracts of the invention can be used for the manufacture of tobacco products, and most preferably, smoking articles such as cigarettes. For example, the extract can be added to a smokable material, such as tobacco lamina or cut filler, in the form of a casing ingredient or top dressing. The extract can be added to tobacco material in neat form or dissolved in a solvent and added in liquid form, such as by spraying. The amount of the extract employed per smoking article can vary. Typically, for cigarettes having about 0.6 g to about 1 g of smokable material per rod, about 0.01 to about 1.0 percent by weight, preferably about 0.1 to about 0.5 percent, of the extract is added, based on the total weight of the smokable material in the cigarette. Representative tobacco blends, representative cigarette components, and representative cigarettes manufactured therefrom, are set forth in U.S. Pat. No. 4,836,224 to Lawson et al.; U.S. Pat. No. 4,924,888 to Perfetti et al.; U.S. Pat. No. 5,056,537 to Brown et al.; U.S. Pat. No. 5,220,930 to Gentry; and U.S. Pat. No. 5,360,023 to Blakley et al.; US Pat. Application 2002/0000235 to Shafer et al.; and PCT WO 02/37990. Those tobacco materials also can be employed for the manufacture of those types of cigarettes that are described in U.S. Pat. No. 4,793,365 to Sensabaugh; U.S. Pat. No. 4,917,128 to Clearman et al.; U.S. Pat. No. 4,947,974 to Brooks et al.; U.S. Pat. No. 4,961,438 to Korte; U.S. Pat. No. 4,920,990 to Lawrence et al.; U.S. Pat. No. 5,033,483 to Clearman et al.; U.S. Pat. No. 5,074,321 to Gentry et al.; U.S. Pat. No. 5,105,835 to Drewett et al.; U.S. Pat. No. 5,178,167 to Riggs et al.; U.S. Pat. No. 5,183,062 to Clearman et al.; U.S. Pat. No. 5,211,684 to Shannon et al.; U.S. Pat. No. 5,247,949 to Deevi et al.; U.S. Pat. No. 5,551,451 to Riggs et al.; U.S. Pat. No. 5,285,798 to Baneijee et al.; U.S. Pat. No. 5,593,792 to Farrier et al.; U.S. Pat. No. 5,595,577 to Bensalem et al.; U.S. Pat. No. 5,816,263 to Counts et al.; U.S. Pat. No. 5,819,751 to Barnes et al.; U.S. Pat. No. 6,095,153 to Beven et al.; U.S. Pat. No. 6,311,694 to Nichols et al.; and U.S. Pat. No. 6,367,481 to Nichols, et al.; and PCT WO 97/48294 and PCT WO 98/16125. See, also, those types of commercially marketed cigarettes described Chemical and Biological Studies on New Cigarette Prototypes that Heat Instead of Burn Tobacco, R. J. Reynolds Tobacco Company Monograph (1988) and Inhalation Toxicology, 12:5, p. 1-58 (2000):

Following extraction, the treated Oriental tobacco could also be used in tobacco blends for smoking articles. The treated Oriental tobacco will exhibit milder flavor and aroma due to the significant reduction in sucrose ester content, and could find use as a diluent in a tobacco mixture comprising other Oriental tobaccos having high sucrose ester content. The treated tobacco could also be used in tobacco mixtures comprising non-Oriental tobaccos, such as flue-cured tobacco, burley tobacco, Maryland tobacco and the like, in order to introduce mild Oriental flavor and aroma to the mixture.

EXPERIMENTAL

The following examples are given to illustrate the invention, but should not be considered in limitation of the invention.

All air-dried ground Oriental tobacco samples are obtained from R. J. Reynolds Tobacco Co. (Winston-Salem, N.C.). Solvents are EM Science (Gibbstown, N.J.) HPLC grade and used as received. Dimethylformamide (DMF), sodium acetate, and sodium sulfate are obtained from Sigma-Aldrich (Milwaukee, Wis.). N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (Alltech Associate, Deerfield, Ill.) is silylation grade.

Tobacco extracts and sucrose esters (SE) are analyzed as their trimethylsilyl ether derivatives using an Agilent 5890 gas chromatograph (Wilmington, Del.) equipped with a 5972 Mass Selective Detector (MSD). All separations are obtained on a DB-5 MS capillary column (15 m×0.25 mm i.d., and 0.25 μm d_f). All supercritical fluid extractions are performed on an Isco-Suprex AP-44 extractor (Lincoln, Nebr.) using 5 mL extraction vessels. All HPLC separations are conducted using an Agilent series 1050 HPLC equipped with a multi-wavelength UV detector and 3396 integrator. All HPLC separations are obtained using analytical or semi-preparative Supelco (Bellefonte, Pa.) CN column (250 mm×4.6 mm and 250 mm×10 mm, 5 μm d_p).

EXAMPLE 1

Extraction Procedures

Three different extraction procedures are used to extract and isolate SE from tobacco, each of which is set forth in FIG. 1. Two of the extraction methods substantially replicate prior art methods of extracting SE from Oriental tobacco. In the first extraction method, listed as the Severson method in FIG. 1, 2 grams of tobacco are transferred into a 100-mL bottle fitted with a Teflon coated cap. Then, 20 mL of CH₂Cl₂ is added to the bottle and the sample was manually shaken for 3-5 minutes. The solution is filtered using a type 1 filter paper (Whatman Co., Maidstone, UK). Next, the residual tobacco and filter paper are transferred into the bottle where tobacco was re-extracted using an additional 20 mL of fresh CH₂Cl₂. Next, the combined CH₂Cl₂ extracts are evaporated to dryness using a nitrogen stream. The residue is then partitioned between 20 mL each of hexane and 80/20% MeOH—H₂O. The MeOH—H₂O solution is re-extracted a second time using an additional 20 mL hexane. Next, 10 mL of saturated KCl solution is added to the MeOH—H₂O solution followed by 15 mL of CHCl₃. The CHCl₃ solubles are removed and the aqueous phase is extracted with another 15 mL of CHCl₃. The combined CHCl₃ fractions are then washed with H₂O and filtered through a bed of anhydrous Na₂SO₄. After the solvent is removed, the extract is quantitatively re-dissolved in 10 mL of CHCl₃.

In the second extraction method, listed as the Kandra method in FIG. 1, 2 grams of tobacco is transferred into a 100 mL bottle equipped with a Teflon coated cap. Then, 20 mL of CH₃CN is added to the bottle and the sample is manually shaken for 3-5 minutes. The solution is filtered using a type 1 filter paper. Next, tobacco and filter paper are transferred into the 100-mL bottle and the tobacco is re-extracted using an additional 20 mL of fresh CH₃CN. Next, the combined CH₃CN extracts are evaporated to dryness using a nitrogen stream. The residue is partitioned between 20 mL CHCl₃ and 10 mL of H₂O (FIG. 2) in order to remove H₂O-soluble materials. The CHCl₃ phase is dried with anhydrous Na₂SO₄ and evaporated to dryness using a stream of N₂. The extract residue is then quantitatively re-dissolved in 10 mL of CHCl₃.

In the third extraction method according to the invention, listed as supercritical fluid extraction (SFE) method in FIG. 1, a 5 mL extraction vessel is filled with 2 grams of tobacco. Next, samples are extracted using 180 grams of supercritical CO₂ at different pressures and temperatures. Extracts are collected in a trap packed with stainless steel balls. After completion of each extraction step, the trap is rinsed with 5 mL of 50/50% MeOH/CH₂Cl₂ into a 12 mL vial. The trap rinse solvent is evaporated using a nitrogen gas stream. The resulting residue is next partitioned between 20 mL each of hexane and 80/20% MeOH—H₂O (FIG. 2) and cleaned like the Severson et al. method.

Surprisingly, all extraction methods provide a similar extraction recovery of SE from the tobacco. However, it is important to note that the Kandra and Severson methods extract much more polar analytes than supercritical fluid CO₂, which no doubt accounts for the fact that the color of the solvent extracts are darker than the supercritical fluid extracts even after application of the cleanup procedure. Thus, it is apparent that the method of the invention provides better extraction selectivity for sucrose esters than the other two methods.

GC/MS Analysis

After completion of each extraction method, 1.0 mL of CHCl₃ solution from each extract is quantitatively transferred into a GC vial for derivatization. The solvent is evaporated to dryness using N₂ at room temperature. A 500 μL portion of 1:1 BSTFA-DMF is added to each vial for the purpose of forming silyl ethers of the four hydroxyl groups on each SE molecule. Each vial is purged with N₂ and capped with a Teflon-lined cap and heated at 70° C. for 30 minutes. After cooling, the sample is placed inside the 7673 Agilent autosampler for GC analysis. All GC runs are obtained with a DB-5 MS capillary column (15 m×0.25 mm i.d.) as described earlier using the following temperature program: initial temperature 80° C., hold for 2 minutes, ramp to 140° C. at a rate of 10° C./min and then ramp to 290° C. at a rate of 4° C./min., hold at 290° C. for 10 minutes.

HPLC Analysis

In order to obtain fractions of SE with higher purity, HPLC is applied to each of the extracts employing a cyano bonded-phase as described earlier. Separation is achieved via isocratic elution using isooctane: ethanol (85/15%) at room temperature flowing at 0.8 mL/min. UV detection is set at 214 nm.

FIG. 2 shows the GC/MS of each extract after derivatization using 1:1 BSTFA:DMF. As can be observed, all three extraction methods provide similar chromatograrns. However, it is important to note that the SFE extract exhibits a lighter color than the other two extracts.

It is important to determine the recovery of SE using each technique. For this purpose, an internal standard (50 μL of pyrene at a concentration of 100 ng/μL in isooctane/ethanol (85/15%) is added to each extract solution (1 mL) before derivatization. In order to compare extraction recovery of SE using each extraction method, 5 ions from a SE are spectrometrically extracted (443, 457, 471, 485 and 499 amu). These ions are fragments of the various SE tetramethylsilyl ethers. Table 1 shows the total concentration of each ion. As can be observed, all extraction methods provide similar extraction recovery of SE from the tobacco. Again, it is important to note here that Kandra's and Severson's extraction methods remove much more of the polar analytes than supercritical fluid CO₂, which makes the color of these extracts much darker after extraction even after the clean up procedure.

TABLE 1
		Conc. (μg/g)	Conc. (μg/g)	Conc. (μg/g)
Molecular	Extracted Ion	Severson	Kandra	SFE
Mass	from GC/MS	Method	Method	Method
622	443	13.8	11.5	16.4
636	457	57.4	62.0	58.9
650	471	152.9	163.6	162.9
664	485	184.4	192.4	192.9
678	499	77.8	80.5	83.9
Total Conc.		472.5	498.5	498.5

An HPLC method developed by Danehower (Tobacco International, 1987, 189, 30-33) is used to obtain practically pure fractions of SE preliminarily extracted by each of the three procedures. A total of three fractions are collected in the semi-preparative HPLC mode. FIG. 3 shows the HPLC/UV trace of the Severson extract with detection at 214 nm. The time of collection for the first fraction was from 3.5-8.1 min, second fraction from 8.1-17.5 min, and the third fraction from 17.5-30.0 min. GC/MS analysis of each fraction reveals that only the third fraction contained SE. As can be observed in FIG. 4, the GC/MS of the third fraction of the Severson extract shows that some impurities still were being eluted from the HPLC column.

EXAMPLE 2

In this example, SE from a Turkish tobacco is isolated via selective fractionation with no semi-preparative HPLC step. In order to show the feasibility of this process, 2 grams of tobacco is extracted at different pressures and temperatures. In this part of the study, extraction time for each fraction is 75 minutes using 2 mL/min of liquid CO₂. First, the tobacco sample is extracted at four pressures (150, 200, 350 and 450 atm) and two temperatures. No modifier is used since all SE are extractable with pure C0₂. FIG. 5 shows the GC/MS traces of the various derivatized fractions at 60° C. As can be observed, at 150 atm (FIG. 5A), no SE is extracted. When the CO₂ pressure is systematically increased, however, from 150 to 200 and 350 atm (FIG. 5B and 5C), a mixture of SE's begins to appear in the extract at retention time ˜35-40 minutes. At 450 atm, after continuous extraction of the same sample at the three lower pressures, a relatively small amount of SE is observed in this extract (note the much smaller axis in FIG. 5D relative to the same axis in FIGS. 5A-5C) and an even lower amount of co-extractives is observed.

Similar extractions are performed on a fresh sample while increasing the extraction temperature from 60 to 100° C. In this series of extractions (as can be observed via GC/MS chromatograms of each derivatized fraction), no SE is extracted at 150 atm or 200 atm (FIG. 6A and 6B). However, at 350 atm and 100° C., a relatively large amount of SE is extracted (FIG. 6C) with minimal yield of co-extractives. Increasing the pressure from 350 to 450 atm at 100° C., increased the solvating power of the fluid which causes the extraction of additional SE (FIG. 6D) with even less interference from co-extractives. Integration of the SE peak areas suggests that the majority of the SE's are removed at 350 atm with pure CO₂ if the temperature is 100° C.

Many modifications and other embodiments of the invention will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing description. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

1. A method of extracting sucrose esters from Oriental tobacco, the method comprising:

providing a Oriental tobacco material;

contacting the Oriental tobacco material with a supercritical fluid for a time and under conditions sufficient to extract sucrose esters from the Oriental tobacco into the supercriticar fluid; and

separating the supercritical fluid comprising extracted sucrose esters from the Oriental tobacco.

2. The method according to claim 1, wherein the Oriental tobacco material is in a form selected from the group consisting of whole leaf, laminae, cut filler, volume expanded, stems, cut-rolled stems, cut-puffed stems, reconstituted tobacco, and particulate.

3. The method according to claim 1, wherein the Oriental tobacco material is in cut filler or particulate form.

4. The method according to claim 1, wherein the Oriental tobacco is unblended with other tobacco materials.

5. The method according to claim 1, wherein the supercritical fluid has a critical temperature of between about 30 to about 100° C.

6. The method according to claim 1, wherein the supercritical fluid is selected from the group consisting of carbon dioxide, sulfur hexafluoride, nitrous oxide, and halogenated hydrocarbons including 1 to 4 carbon atoms.

7. The method according to claim 1, wherein said contacting step occurs at a temperature of about 30 to about 150° C. and a pressure of about 200 to about 1,000 atm.

8. The method according to claim 7, wherein said contacting step occurs at a temperature of about 50 to about 120° C. and a pressure of about 300 to about 800 atm.

9. The method according to claim 1, wherein said contacting step occurs at a temperature of about 70 to about 110° C. and a pressure of about 400 to about 600 atm.

10. The method according to claim 1, wherein the contacting step is conducted for a period of time from about 30 minutes to about 240 minutes.

11. The method according to claim 1, further comprising evaporating the supercritical fluid in order to isolate an Oriental tobacco extract, wherein the isolated Oriental tobacco extract comprises at least about 90% by weight of sucrose esters, based on the total weight of the isolated extract.

12. The method according to claim 11, wherein the isolated Oriental tobacco extract comprises at least about 95% by weight of sucrose esters, based on the total weight of the isolated extract.

13. The method according to claim 11, further comprising purifying the isolated extract by solvent extraction.

14. The method according to claim 11, further comprising re-dissolving the isolated extract in a solvent and applying the isolated extract to a tobacco material in the form of a casing or top dressing.

15. A smoking article comprising sucrose esters extracted according to the method of claim 1.

16. A method of extracting sucrose esters from Oriental tobacco, the method comprising:

providing a Oriental tobacco material in cut filler or particulate form;

contacting the Oriental tobacco material with supercritical carbon dioxide for a time and under conditions sufficient to extract sucrose esters from the Oriental tobacco into the supercritical carbon dioxide, wherein the pressure during said contacting step is at least about 300 atm and the temperature is at least about 60° C.; and

separating the supercritical carbon dioxide comprising extracted sucrose esters from the Oriental tobacco.

17. The method according to claim 16, further comprising evaporating the supercritical carbon dioxide in order to isolate an Oriental tobacco extract, wherein the isolated Oriental tobacco extract is at least about 90% by weight of tetra-acyl sucrose esters, based on the total weight of the isolated extract.

18. The method according to claim 17, wherein the isolated Oriental tobacco extract is at least about 95% by weight of tetra-acyl sucrose esters, based on the total weight of the isolated extract.

19. The method according to claim 17, further comprising purifying the isolated extract by solvent extraction.

20. The method according to claim 17, further comprising re-dissolving the isolated extract in a solvent and applying the isolated extract to a tobacco material in the form of a casing or top dressing.

21. A smoking article comprising sucrose esters extracted according to the method of claim 16.

22. A method of extracting sucrose esters from Oriental tobacco, the method comprising:

providing a Oriental tobacco material in cut filler or particulate form;

contacting the Oriental tobacco material with supercritical carbon dioxide for a time and under conditions sufficient to extract sucrose esters from the Oriental tobacco into the supercritical carbon dioxide, wherein the pressure during said contacting step is at least about 400 atm and the temperature is at least about 80° C.;

separating the supercritical carbon dioxide comprising extracted sucrose esters from the Oriental tobacco;

evaporating the supercritical carbon dioxide in order to isolate an Oriental tobacco extract comprising sucrose esters, the Oriental tobacco extract comprising at least about 90% by weight of tetra-acyl sucrose esters, based on the total weight of the isolated extract; and

applying the extracted sucrose esters to a tobacco material as a flavor additive.

23. A smoking article comprising sucrose esters extracted according to the method of claim 22.