Patent Description:
Leaves of cultivated and harvested tobacco plants are subjected to various processes, including a drying process in a farm house, subsequently one to several years of a long-term aging process in a leaf processing facility, and thereafter blending and cutting processes in a manufacturing facility, and then the processed tobacco leaves are used to produce tobacco products. The processed tobacco leaf materials used to produce tobacco products are referred to as "leaf tobacco" in the art.

Leaf tobacco is known to contain various glycoside components. As glycosides contained in the leaf tobacco, glucosides such as scopoletin <NUM>-glucoside (scopoletin) and quercetin <NUM>-β-D-glucoside (isoquercetin); rhamnoglucosides (rutinosides) such as naringenin <NUM>-rhamnoglucoside (naringin) and quercetin <NUM>-rhamnoglucoside (rutin); and sophorosides such as rishitin β-sophoroside and quercetin <NUM>-β-D-sophoroside have been identified, but there are many glycosides that have not yet been identified.

The glycosides contained in the leaf tobacco are considered to function as precursors of the tobacco flavor components. Specifically, it is considered that when the leaf tobacco is combusted during smoking, the glycoside components contained in the leaf tobacco are degraded into a sugar part and a non-sugar part (i.e., aglycone), and the non-sugar part functions as a tobacco flavor component.

Meanwhile, attempts have been made to increase the tobacco flavor components contained in the leaf tobacco. For example, Jpn. KOKAI Publication No. <CIT> reports that a smoking flavor of leaf tobacco is improved by adding, before a long-term aging process in a leaf processing facility, ethyl alcohol to tobacco leaves, and performing the aging process thereafter.

Furthermore, <CIT> is concerned with a tobacco material and a method for the production thereof. In the method, a first tobacco material is contacted with a second tobacco material to form a tobacco blend. The first tobacco material has a higher content of nitrogen source and a lower content of sugar source than the second tobacco material. The moisture content in the first and second materials of the tobacco blend is increased to at least about <NUM>%. The tobacco blend having an increased moisture content is then subjected to heat treatment at <NUM> (<NUM>°F) or more in a substantially atmospheric environment for generating flavorful and aromatic substances by a Maillard reaction. According to an embodiment, the first tobacco material may be burley tobacco and the second tobacco material may be flue cured tobacco.

In <CIT>, a method of preparing a tobacco material is described, which is supposed to suppress the formation of acryl amide in the mainstream smoke. The method includes (i) mixing a tobacco material, water, and an additive such as asparaginase; (ii) heating the mixture, preferably to more than <NUM>; and (iii) incorporating the heat-treated mixture into a smoking article as a smokable material.

An object of the present invention is to provide a technique relating to a tobacco material having an enhanced tobacco flavor, and a tobacco flavor liquid having an enhanced tobacco flavor.

According to the first aspect, there is provided a tobacco material production method, comprising:.

According to the second aspect, there is provided a tobacco material obtainable by the above-described method.

According to the third aspect, there is provided a tobacco flavor liquid production method, comprising extracting a tobacco flavor component from the above-described tobacco material to obtain a tobacco flavor liquid.

According to the forth aspect, there is provided a tobacco flavor liquid obtainable by the above-described method.

According to the fifth aspect, there is provided a heating-type flavor inhaler, comprising the above-described tobacco material, or the above-described tobacco flavor liquid.

According to the present invention, various glycosides contained in the second ground leaf tobacco are degraded by various glycoside-degrading enzymes contained in the first ground leaf tobacco to produce various tobacco flavor components, thereby providing a tobacco material having an enhanced tobacco flavor and a tobacco flavor liquid having an enhanced tobacco flavor.

Hereinafter, the present invention will be described, but the following description is for the purpose of detailed explanation of the present.

A tobacco material production method includes:.

The tobacco material production method is shown in <FIG> by way of a flowchart.

First, a description will be given of first ground leaf tobacco and second ground leaf tobacco. In the following description, the term "ground leaf tobacco" is used to refer to both the first ground leaf tobacco and the second ground leaf tobacco.

The ground leaf tobacco is obtainable by grinding the leaf tobacco. The ground leaf tobacco has a maximum diameter of, for example, <NUM> or less. The ground leaf tobacco having a maximum diameter of <NUM> or less is obtainable by, for example, grinding the leaf tobacco with a commercially available grinder (mill) and passing it through a <NUM> mesh sieve. The ground leaf tobacco has a maximum diameter of, for example, <NUM> to <NUM>. When the ground leaf tobacco is used, as compared to when non-ground leaf tobacco is used, it is possible to promote movements of glycoside-degrading enzymes and glycosides between the first ground leaf tobacco and the second ground leaf tobacco, and to enhance the efficiency of the glycoside degradation reaction. Thereby, glycoside degradation products are efficiently produced, and a tobacco flavor can be enhanced.

"Leaf tobacco" as a raw material of the ground leaf tobacco is obtainable by subjecting leaves of cultivated and harvested tobacco plants to various processes including a drying process in a farm house, subsequently one to several years of a long-term aging process in a leaf processing facility, and thereafter blending and cutting processes in a manufacturing facility. That is, "leaf tobacco" refers to cut tobacco ready for use in production of tobacco products. For example, as "leaf tobacco", cut tobacco ready for use in a cigarette making process can be used.

The first ground leaf tobacco has β-D-glucosidase activity of <NUM> [nkat/g] or more. The first ground leaf tobacco generally has β-D-glucosidase activity of <NUM> to <NUM> [nkat/g]. The first ground leaf tobacco preferably has β-D-glucosidase activity of <NUM> to <NUM> [nkat/g]. On the other hand, the second ground leaf tobacco contains glycosides, and has β-D-glucosidase activity of <NUM> [nkat/g] or less. The second ground leaf tobacco generally has β-D-glucosidase activity of <NUM> to <NUM> [nkat/g]. The second ground leaf tobacco preferably has β-D-glucosidase activity of <NUM> to <NUM> [nkat/g].

The "β-D-glucosidase" is one of the glycoside-degrading enzymes contained in the leaf tobacco. In the present specification, the "β-D-glucosidase activity" of the ground leaf tobacco refers to the degradation activity of <NUM>-nitrophenyl β-D-glucopyranoside (Glc-β-pNP), which is a model substrate, and specifically refers to enzyme activity determined by the measurement method of Example <NUM> described below. The "β-D-glucosidase activity" of the ground leaf tobacco is obtainable by collecting partial samples from three portions of the ground leaf tobacco, measuring the enzyme activity for each of the partial samples, and calculating an average of the obtained measurement values.

General enzymes contained in leaf tobacco are inactivated when exposed to a high temperature during a leaf tobacco production process (particularly, a drying process in a farm house), whereas the activities of the enzymes are maintained without inactivation when they are not exposed to a high temperature during the leaf tobacco production process. Therefore, if the "β-D-glucosidase activity" of the ground leaf tobacco is maintained, other enzyme activities are also maintained. Accordingly, the "β-D-glucosidase activity" of the ground leaf tobacco can be used as an index of activities of various glycoside-degrading enzymes contained in the ground leaf tobacco.

Specifically, when the ground leaf tobacco has β-D-glucosidase activity of <NUM> [nkat/g] or more, the ground leaf tobacco also has activities of predetermined values or more for glycoside-degrading enzymes other than the β-D-glucosidase, and has a sufficient ability to degrade various glycosides to produce tobacco flavor components. On the other hand, when the ground leaf tobacco has β-D-glucosidase activity of <NUM> [nkat/g] or less, the ground leaf tobacco also has activities of predetermined values or less for glycoside-degrading enzymes other than the β-D-glucosidase, and does not have a sufficient ability to degrade various glycosides to produce tobacco flavor components.

The "β-D-glucosidase activity" of the ground leaf tobacco refers to a value measured by the measurement method of Example <NUM> described later, immediately before the first ground leaf tobacco and the second ground leaf tobacco are mixed. Similarly, the "presence or absence of glycosides" in the ground leaf tobacco is determined by whether or not there is a peak identified as a glycoside according to the analysis method of Example <NUM> described later, immediately before the first ground leaf tobacco and the second ground leaf tobacco are mixed.

The first ground leaf tobacco may or may not contain glycosides, and the content thereof is not particularly limited; however, since the first ground leaf tobacco exhibits high glycoside-degrading enzyme activity, the glycoside content is generally low. On the other hand, the second ground leaf tobacco contains glycosides serving as a substrate of a glycoside degradation reaction. Since the second ground leaf tobacco exhibits low glycoside-degrading enzyme activity, the glycoside content is generally high. Because a large amount of glycosides contained in the second ground leaf tobacco leads to production of a larger amount of glycoside degradation products, it is preferable that the glycoside content of the second ground leaf tobacco be large.

Therefore, preferably, the first ground leaf tobacco contains glycosides at a smaller content per unit mass of the ground leaf tobacco than the second ground leaf tobacco. More preferably, the content of each component of glycoside components contained in the first ground leaf tobacco is smaller than that of the corresponding component of glycoside components contained in the second ground leaf tobacco. In the present specification, the glycoside content of the ground leaf tobacco refers to a content per unit mass of the ground leaf tobacco.

In the present specification, the "glycosides" contained in the ground leaf tobacco refer to all glycoside components contained in the leaf tobacco, and specifically refers to all components determined to be glycosides by the analysis method described in <CIT>. More specifically, "glycosides" contained in the ground leaf tobacco refer to all components determined to be glycosides according to the analysis method of Example <NUM> described later. The analysis method described in <CIT> prepares a leaf tobacco extraction liquid sample treated with β-D-glucosidase and a leaf tobacco extraction liquid sample not treated with β-D-glucosidase, analyzes each sample by LC-MS/MS to compare analysis results, and determines that peaks eliminated or reduced in intensity by β-D-glucosidase are derived from glycosides. The "glycosides" contained in the ground leaf tobacco can be, as described in <CIT>, quantified using an internal standard.

As described in the Background section above, leaf tobacco contains many kinds of glycoside components, many of which have not been identified. Therefore, in the present specification, the "glycosides" contained in the ground leaf tobacco include not only those identified as glycosides contained in the leaf tobacco but also those contained in the leaf tobacco but not identified.

The "first ground leaf tobacco" is typically of a variety in which drying of tobacco plant leaves in a farm house is performed by air-curing. Since the typical first ground leaf tobacco is exposed to natural temperature and humidity and natural aeration conditions in a drying process in a farm house, inactivation of enzymes does not easily occur, and most of the glycosides are degraded. As a result, the typical first ground leaf tobacco has high glycoside-degrading enzyme activity, and a low glycoside content.

The first ground leaf tobacco is, for example, of at least one variety selected from burley tobacco, domestic tobacco, dark fire-cured tobacco, dark air-cured tobacco, and dark sun-cured tobacco. The first ground leaf tobacco may be ground leaf tobacco of one variety, or a mixture of ground leaf tobaccos of multiple varieties.

The "second ground leaf tobacco" is typically of a variety in which drying of tobacco plant leaves in a farm house is performed by flue-curing accompanied by a heating process, or air-circulating curing performed by circulating heated air, or sun-curing. Since the typical second ground leaf tobacco is exposed to a high temperature in a drying process in a farm house, inactivation of enzymes easily occurs, and many of the glycosides remain without being degraded. As a result, the typical second ground leaf tobacco has low glycoside-degrading enzyme activity and a high glycoside content.

The second ground leaf tobacco is, for example, of at least one variety selected from flue-cured tobacco, oriental tobacco, sun-cured tobacco, and light sun-cured tobacco. The second ground leaf tobacco may be ground leaf tobacco of one variety, or a mixture of ground leaf tobaccos of multiple varieties.

As described above, the first ground leaf tobacco may be ground leaf tobacco of a variety in which drying of tobacco plant leaves in a farm house is performed by air-curing. Alternatively, the first ground leaf tobacco may be ground leaf tobacco obtainable by:
subjecting a leaf tobacco raw material to a treatment for activating a glycoside-degrading enzyme, and subsequently grinding the leaf tobacco raw material.

For the leaf tobacco raw material used herein, the content of glycosides is discretionary as long as the leaf tobacco raw material has glycoside-degrading enzyme activity. As the leaf tobacco raw material, for example, at least one variety selected from burley tobacco, domestic tobacco, dark fire-cured tobacco, dark air-cured tobacco, dark sun-cured tobacco, flue-cured tobacco, oriental tobacco, sun-cured tobacco, and light sun-cured tobacco may be used. The activation treatment can be performed by, for example, treating the leaf tobacco raw material with a buffering agent so as to adjust the pH of the leaf tobacco raw material to an optimum pH of the glycoside-degrading enzyme, or by placing the leaf tobacco raw material under an optimum temperature of the glycoside-degrading enzyme.

When the glycoside-degrading enzyme of the first ground leaf tobacco is activated in advance, the efficiency of the glycoside degrading reaction can be enhanced.

As described above, the second ground leaf tobacco may be ground leaf tobacco of a variety in which drying of tobacco plant leaves in a farm house is performed by flue-curing accompanied by a heating process, air-circulating curing performed by circulating heated air, or sun-curing. Alternatively, the second ground leaf tobacco may be ground leaf tobacco obtainable by:.

For the leaf tobacco raw material used herein, a value of the glycoside-degrading enzyme activity is discretionary as long as the leaf tobacco raw material contains glycosides. As the leaf tobacco raw material, for example, at least one variety selected from burley tobacco, domestic tobacco, dark fire-cured tobacco, dark air-cured tobacco, dark sun-cured tobacco, flue-cured tobacco, oriental tobacco, sun-cured tobacco, and light sun-cured tobacco may be used. Preferably, as the leaf tobacco raw material, at least one variety selected from flue-cured tobacco, oriental tobacco, sun-cured tobacco, and light sun-cured tobacco may be used. The inactivation treatment can be performed by, for example, putting the leaf tobacco in a fluidized bed having an air flow temperature of <NUM> to <NUM>, and treating the leaf tobacco for <NUM> to <NUM> seconds.

If the enzyme of the second ground leaf tobacco is inactivated in advance, the leaf tobacco subjected to the inactivation treatment can completely suppress subsequent degradation of glycosides, and the leaf tobacco raw material can be stored while a high glycoside content is maintained. Therefore, by performing the inactivation treatment, it is possible to stably supply a leaf tobacco raw material having a high glycoside content as a raw material of the "second ground leaf tobacco".

A leaf tobacco mixture is prepared by mixing the above-described first ground leaf tobacco and the above-described second ground leaf tobacco. The mixing ratio of the first ground leaf tobacco and the second ground leaf tobacco is discretionary, but they may be mixed preferably at a mass ratio of <NUM>:<NUM> to <NUM>:<NUM>, for example, at a mass ratio of <NUM>:<NUM>.

The leaf tobacco mixture may be composed of only the first ground leaf tobacco and the second ground leaf tobacco. The present invention merely requires that the glycoside-degrading enzymes contained in the first ground leaf tobacco act on the glycosides contained in the second ground leaf tobacco to produce glycoside degradation products; thus, there is no problem if the leaf tobacco mixture contains no components other than the ground leaf tobacco. However, the present invention does not exclude the case where the leaf tobacco mixture contains components other than the ground leaf tobacco, and the leaf tobacco mixture may contain components that promote the glycoside degradation reaction such as water or a pH buffering agent as additives.

The above-described leaf tobacco mixture is stored under a humidification condition to produce a tobacco material having an enhanced tobacco flavor.

The humidification condition can be, in general, a condition in which moisture is added to the leaf tobacco mixture so that a moisture content of the leaf tobacco mixture is <NUM> to <NUM>% by mass. The "moisture content (% by mass) of the leaf tobacco mixture" used herein refers to the ratio of the total moisture content of the moisture content originally possessed by the leaf tobacco mixture and the added moisture content to the total mass of the mass of the leaf tobacco mixture and the added moisture content.

The "moisture content originally possessed by the leaf tobacco mixture" can be obtained by the following method.

Based on a method of analyzing food moisture (heating and drying method), a leaf tobacco sample is heated at <NUM> for <NUM> hour under normal pressure and allowed to cool in a desiccator for <NUM> minutes, and moisture is determined from a difference in weight before and after heating. A specific procedure is as follows.

The "moisture content originally possessed by the leaf tobacco mixture" is generally <NUM> to <NUM>% by mass. For example, in order to change a moisture content of <NUM>% by mass possessed by a leaf tobacco mixture (<NUM>) to a moisture content of <NUM>% by mass, <NUM> of water needs to be added. After the required amount of moisture is added to the leaf tobacco mixture, the leaf tobacco mixture may be stirred so as to distribute the moisture throughout the leaf tobacco mixture.

Preferably, the humidification condition is a condition in which moisture is added to the leaf tobacco mixture so that the moisture content of the leaf tobacco mixture is <NUM> to <NUM>% by mass. More preferably, the humidification condition is a condition in which moisture is added to the leaf tobacco mixture so that the moisture content of the leaf tobacco mixture is <NUM> to <NUM>% by mass.

Moisture plays a role of a reactant because the glycoside degrading reaction is a hydrolysis reaction. Moisture further functions as a medium, and plays a role of promoting the movements of the glycoside-degrading enzymes and the glycosides between the first ground leaf tobacco and the second ground leaf tobacco, and enhancing the efficiency of the glycoside degradation reaction.

Therefore, it is preferable that the "moisture content of the leaf tobacco mixture" be a content sufficient to fulfill the above-described roles. Further, considering that the leaf tobacco mixture is used as a tobacco filler of a tobacco product or as a raw material of a tobacco flavor liquid after storage, it is preferable that the "moisture content of the leaf tobacco mixture" be not excessively high as long as it is sufficient to fulfill the above-described roles. Moreover, in terms of the risk of the occurrence of mold, it is preferable that the "moisture content of the leaf tobacco mixture" be not excessively high.

The leaf tobacco mixture is stored at a temperature suitable for the glycoside-degrading enzymes to function, and for a period of time necessary to produce a sufficient amount of glycoside degradation products.

The leaf tobacco mixture is stored at a temperature of <NUM> to <NUM>. The leaf tobacco mixture is stored preferably at a temperature of <NUM> to <NUM>, and more preferably at a temperature of <NUM> to <NUM>.

The leaf tobacco mixture is stored over a period of <NUM> to <NUM> hours. The leaf tobacco mixture is stored preferably over a period of <NUM> to <NUM> hours. The storage period may be determined based on a period up to the point at which the production amount of glycoside degradation products peaks in order to reduce the risk of disappearance of the produced glycoside degradation products due to volatilization or further degradation.

The leaf tobacco mixture is stored preferably under a sealed condition. The sealed condition can be prepared by placing the leaf tobacco mixture in a container having airtightness. The airtight container may have any capacity, but is preferably one in which the temperature therein can be controlled. The leaf tobacco mixture can be placed in the container to occupy, for example, approximately <NUM> to <NUM>% of the volume of the container. Storage under a sealed condition is preferred because the glycoside degradation products produced easily remain on the ground leaf tobacco.

The leaf tobacco mixture may be incorporated into a tobacco product after the above-described storage, or may be incorporated into a tobacco product after being dried to have a moisture content equivalent to that of ordinary leaf tobacco (i.e., <NUM> to <NUM>% by mass). Drying may be performed using a dryer or by natural drying. Natural drying may be performed by allowing the leaf tobacco mixture to stand for <NUM> to <NUM> days under conditions of a temperature of <NUM> to <NUM> and a humidity of <NUM> to <NUM>%. When a dryer is used, drying may be performed by reduced-pressure drying for <NUM> to <NUM> hours while avoiding heating and keeping the temperature at <NUM> or below.

A tobacco material produced according to the method of the present invention contains produced glycoside degradation products, and therefore has an enhanced tobacco flavor. It is preferable that a tobacco material produced according to the method of the present invention be promptly incorporated into a tobacco product (e.g., a flavor inhalation article) in order to reduce the risk of disappearance of the produced glycoside degradation products due to volatilization, further degradation, and the like. In other words, it is preferable that the method of the present invention be performed on leaf tobacco immediately before being incorporated into a tobacco product (e.g., a flavor inhalation article).

Further, in the method of the present invention, none of the "first ground leaf tobacco", "leaf tobacco mixture", and "tobacco material having an enhanced tobacco flavor" is exposed to a high temperature of <NUM> or more. The "first ground leaf tobacco" is required to have glycoside-degrading enzyme activity, and therefore, it is desirable that the first ground leaf tobacco be not exposed to a high temperature leading to inactivation of enzymes, for example, a temperature of <NUM> or higher, before or after being mixed with the second ground leaf tobacco. It is desirable that the "leaf tobacco mixture" be not exposed to a high temperature leading to inactivation of enzymes, for example, a high temperature of <NUM> or higher, so that the glycoside-degrading enzymes contained in the leaf tobacco mixture can function. It is desirable that the "tobacco material having an enhanced tobacco flavor" be not exposed to a high temperature of, for example, <NUM> or higher before or after being incorporated into a tobacco product, in order to reduce the risk of disappearance of the generated glycoside degradation products due to volatilization, further degradation, or the like.

As described above, for the leaf tobacco, there are "a variety having high glycoside-degrading enzyme activity and a low glycoside content" and "a variety having low glycoside-degrading enzyme activity and a high glycoside content", depending on the method of drying in the farm house. It is considered that this is because when both the glycoside-degrading enzyme activity and the glycosides are present in the same leaf tobacco, the glycosides are degraded, and the glycoside-degrading enzyme activity and the glycoside content are not compatible with each other. The present inventors have newly focused on this point, and succeeded in enhancing the tobacco flavor of the leaf tobacco by reacting the glycoside-degrading enzymes contained in the leaf tobacco having high glycoside-degrading enzyme activity with the glycosides contained in a large amount in the leaf tobacco having low glycoside-degrading enzyme activity.

The present invention is excellent in that many kinds of glycoside degradation products can be produced by effectively utilizing many kinds of glycoside-degrading enzymes contained with high activity in a certain leaf tobacco material and many kinds of glycosides contained in large amounts in another leaf tobacco material.

When an enzyme preparation is used instead of enzymes contained in leaf tobacco, since the enzyme preparation identifies a specific structure of a substrate and acts only on a specific substrate, then for example β-glucosidase can only degrade glycosides having a β-glycoside bond. Therefore, in order to degrade various kinds of glycoside components contained in leaf tobacco, it is necessary to prepare various kinds of enzyme preparations. Further, many of the glycoside components contained in leaf tobacco have not been identified, and there are many for which it is unknown what kind of enzyme should be prepared in the first place. Even if all glycoside components contained in leaf tobacco can be identified, it is a great burden to prepare various kinds of enzyme preparations capable of degrading all these glycoside components.

On the other hand, by utilizing various kinds of glycoside-degrading enzymes inherently contained in leaf tobacco according to the present invention, various kinds of glycoside components present in leaf tobacco can be degraded at once, and the method of the present invention is excellent also in this respect.

According to another aspect, there is provided a tobacco material obtainable by the above-described method. As described above, the tobacco material of the present invention has an enhanced tobacco flavor.

The tobacco material of the present invention may be incorporated into any tobacco product as a tobacco filler, or may be used as a raw material for producing a tobacco flavor liquid.

The tobacco material of the present invention can be incorporated into any flavor inhalation article through which a user tastes tobacco flavor. Specific examples of the flavor inhalation article include a combustion type smoking article that burns a tobacco material to provide a user with a tobacco flavor, a heating-type flavor inhaler that heats, without burning, a tobacco material to provide a user with a tobacco flavor, and a non-heating-type flavor inhaler that provides a user with tobacco flavor without heating or burning a tobacco material. As a heating-type flavor inhaler, there are known a direct heating-type in which a tobacco material is heated by a heating device such as a heater to provide a user with a tobacco flavor, and an indirect heating-type in which a liquid aerosol source is heated to generate aerosol and the aerosol passes through the tobacco material to provide a user with tobacco flavor.

The heating-type flavor inhaler or the non-heating-type flavor inhaler does not burn a tobacco material, and thus does not easily release a tobacco flavor component as compared to the combustion type smoking article. Therefore, the tobacco material of the present invention exhibits a particularly excellent effect in that an enhanced tobacco flavor can be provided to a user when used in a heating-type flavor inhaler or a non-heating-type flavor inhaler.

Examples of the combustion type smoking article include a cigarette, pipe, Kiseru (i.e., traditional Japanese pipe for fine cut tobacco), cigar, or cigarillo.

Examples of the heating-type flavor inhaler include:.

Examples of the non-heating-type flavor inhalation article include a non-heating-type tobacco flavor inhalation article including, in an inhalation holder, a refill type cartridge containing a tobacco material, in which a user inhales a tobacco flavor derived from the tobacco material at normal temperature (see, for example, <CIT>).

The proportion of the tobacco material of the present invention in the total tobacco materials (hereinafter also referred to as a tobacco filler) contained in the flavor inhalation article is not particularly limited. That is, the tobacco material of the present invention may be blended so as to occupy the entire tobacco filler (<NUM>% by mass), or may be blended so as to occupy a part of the tobacco filler, for example, <NUM> to <NUM>% by mass of the tobacco filler.

According to another aspect, there is a tobacco flavor liquid production method, including extracting a tobacco flavor component from the tobacco material of the present invention to obtain a tobacco flavor liquid. That is, the tobacco flavor liquid production method includes:.

<FIG> shows a flowchart of the tobacco flavor liquid production method.

For the processes until obtaining the tobacco material, reference can be made to the description of <<NUM>. Tobacco Material Production Method>. The process of extracting a tobacco flavor component from the tobacco material can be performed using a method generally known as a method of extracting an aroma component. The tobacco flavor component can be extracted by, for example, distillation, specifically, steam distillation, hot water distillation, atmospheric distillation, or vacuum distillation. It is preferable that the tobacco flavor component be extracted by steam distillation from the viewpoint of extraction efficiency.

According to another aspect, there is provided a tobacco flavor liquid obtainable by the above-described method. The tobacco flavor liquid of the present invention has an enhanced tobacco flavor because it is produced using a tobacco material having an enhanced tobacco flavor as a raw material.

The tobacco flavor liquid of the present invention can be incorporated into any tobacco product.

For example, in a "heating-type flavor inhaler", the tobacco flavor liquid of the present invention may be incorporated into a liquid containing portion alone, into a pod after being mixed with a solid tobacco flavor source such as cut tobacco or tobacco granules, or into a liquid-containing portion after being mixed with a liquid of an aerosol source (for example, propylene glycol or glycerin). In a "non-heating-type flavor inhaler", the tobacco flavor liquid of the present invention may be incorporated into a liquid-containing portion alone, or into a pod after being mixed with a solid tobacco flavor source such as cut tobacco or tobacco granules.

According to another aspect, there is provided a heating-type flavor inhaler including the tobacco material of the present invention.

According to one embodiment, there is provided a heating-type flavor inhaler including a tobacco flavor source, in which the tobacco flavor source includes the tobacco material of the present invention and an aerosol source mixed with the tobacco material. An example of the heating-type flavor inhaler is shown in <FIG>.

The heating-type flavor inhaler <NUM> shown in <FIG> is an electrical heating-type inhaler that heats a tobacco flavor source with electrical heat to generate aerosol. In the following description, the heating-type flavor inhaler <NUM> will be simply referred to as a flavor inhaler <NUM>.

The flavor inhaler <NUM> includes a main body <NUM> and a mouthpiece <NUM>. The flavor inhaler <NUM> has a shape extending along a direction in which the main body <NUM> and the mouthpiece <NUM> are connected, and includes a non-mouthpiece end (end of the main body <NUM> side) and a mouthpiece end (end of the mouthpiece <NUM> side).

In the following description, if a "non-mouthpiece end side" is referred to for a certain part, the position specified by this "non-mouthpiece end side" indicates the end position of the part closer to the non-mouthpiece end of the flavor inhaler <NUM>. Furthermore, in the following description, if a "mouthpiece end side" is referred to for a certain part, the position specified by this "mouthpiece end side" indicates the end position of the part closer to the mouthpiece end of the flavor inhaler <NUM>.

The main body <NUM> includes a tubular body <NUM>, a battery <NUM>, a control circuit <NUM>, and a heater <NUM>.

The tubular body <NUM> is a tubular body with a bottom, and is open on the mouthpiece end side so that a later-described tobacco pod <NUM> can be replaced. The tubular body <NUM> may be a circular section tubular body or a polygonal section tubular body. The non-mouthpiece end of the tubular body <NUM> is provided with a charging part (not shown) for charging the battery <NUM>. In addition, a side wall of the tubular body <NUM> is provided with a power supply button (not shown) to turn on or off the flavor inhaler <NUM>.

The battery <NUM> is placed inside the tubular body <NUM>. The battery <NUM> is, for example, a lithium ion secondary battery. The battery <NUM> supplies the power necessary to operate the flavor inhaler <NUM> to the electrical and electronic parts included in the flavor inhaler <NUM>. For example, the battery <NUM> supplies the power to the control circuit <NUM> and the heater <NUM>.

The control circuit <NUM> is placed in the tubular body <NUM> between its opening and the battery <NUM>. The control circuit <NUM> may be placed at another position in the tubular body <NUM>. The control circuit <NUM> controls the operation of the flavor inhaler <NUM>. Specifically, the control circuit <NUM> controls the power supplied to the heater <NUM> based on a value output by a temperature sensor placed in the vicinity of the heater <NUM>.

The heater <NUM> is placed on the mouthpiece end side in the tubular body <NUM>. Specifically, the heater <NUM> is placed in the tubular body <NUM> between its opening and the control circuit <NUM>. The heater <NUM> has a cup shape allowing the tobacco pod <NUM> to be accommodated. The heater <NUM> is electrically connected to the battery <NUM> and the control circuit <NUM>. The temperature of the heater <NUM> is controlled by the control circuit <NUM>. It is preferable that the heater <NUM> be surrounded by an insulator to avoid transmission of its heat to the tubular body <NUM>, the battery <NUM>, the control circuit <NUM>.

The main body <NUM> may further include a light-emitting device, for example, on a side wall of the tubular body <NUM>, to notify a user of a heating state of the tobacco pod <NUM> or a remaining amount of the battery <NUM>.

Here, the tobacco pod <NUM> will be described.

The tobacco pod <NUM> is placed in the main body <NUM> to be surrounded by the heater <NUM>. The tobacco pod <NUM> is replaced by the user after inhalation is performed a predetermined number of times.

The tobacco pod <NUM> includes a container <NUM> and a tobacco flavor source <NUM>.

The container <NUM> is made of, for example, a metal (e.g., aluminum).

The tobacco flavor source <NUM> is contained in the container <NUM>. The tobacco flavor source <NUM> includes the tobacco material of the present invention and an aerosol-generating liquid. The aerosol-generating liquid is a liquid for generating aerosol through heating, examples of which include propylene glycol, glycerin, and a mixture thereof. The tobacco flavor source <NUM> may or may not contain a tobacco material, as a tobacco filler, other than the tobacco material of the present invention.

Before being attached to the main body <NUM>, the tobacco pod <NUM> is sealed with an aluminum foil lid. When attached to the main body <NUM>, the tobacco pod <NUM> is unsealed in such a manner that the tobacco flavor can be inhaled from the tobacco flavor source.

The mouthpiece <NUM> is provided on the mouthpiece end side of the main body <NUM> in a detachable manner. The mouthpiece <NUM> is removed from the main body <NUM> by the user when the tobacco pod <NUM> is replaced.

The mouthpiece <NUM> includes a protrusion on its non-mouthpiece end side. When the mouthpiece <NUM> is attached to the main body <NUM>, this protrusion penetrates the lid of the tobacco pod <NUM> to open the tobacco pod <NUM>. The mouthpiece <NUM> may not include a protrusion. In this case, the tobacco pod <NUM> is opened by the user's hand, for example, right before being attached to the main body <NUM>.

The mouthpiece <NUM> includes a first gas flow path that leads external air of the flavor inhaler <NUM> into a space in the tobacco pod <NUM>. The first gas flow path has a gas flow inlet, for example, in the vicinity of the connection between the mouthpiece <NUM> and the main body <NUM>. The mouthpiece <NUM> also includes a second gas flow path that connects the space in the tobacco pod <NUM> and the external space of the flavor inhaler <NUM> so that the user can inhale the tobacco flavor from the tobacco flavor source <NUM>. The second gas flow path has a gas flow outlet, for example, on the mouthpiece end of the mouthpiece <NUM>.

According to another aspect, there is provided a heating-type flavor inhaler including the tobacco flavor liquid of the present invention.

According to one embodiment, there is provided a heating-type flavor inhaler including a tobacco flavor source, in which the tobacco flavor source includes the tobacco flavor liquid of the present invention and an aerosol source mixed with the tobacco flavor liquid. An example of the heating-type flavor inhaler is shown in <FIG>.

The heating-type flavor inhaler <NUM> shown in <FIG> is an electrical heating-type inhaler that heats a tobacco flavor source with electrical heat to generate aerosol. In the following description, the heating-type flavor inhaler <NUM> will simply be referred to as a flavor inhaler <NUM>.

The flavor inhaler <NUM> has a rod-like or columnar shape, extending from its mouthpiece end 13A to its distal end <NUM>. The flavor inhaler <NUM> includes a cylindrical housing <NUM> constituting an outer shell, a cylindrical mouthpiece <NUM>, a distal end <NUM> provided on the opposite side of the mouthpiece end 13A of the mouthpiece <NUM>, a battery <NUM> housed in the housing <NUM>, an aerosol source <NUM> housed in the housing <NUM>, a wick <NUM> connected to the aerosol source <NUM>, a heater <NUM> made of an electrically resistive metallic material wound around the wick <NUM>, wiring <NUM> connecting the heater <NUM> to the battery <NUM>, air-intake holes <NUM> provided in the housing <NUM>, a ventilation path <NUM> provided in a cylindrical shape at the center of the housing <NUM>, and a drive circuit <NUM> for controlling the supply of electric power to the heater <NUM>.

The mouthpiece <NUM> is made of a metallic material such as stainless steel or brass. The housing <NUM> is formed in a cylindrical shape with, for example, a resin material. The housing <NUM> has a first part 12A positioned on the mouthpiece end 13A side, and a second part 12B positioned on the distal end <NUM> side. The first part 12A is made of a metallic material similar to the material of the mouthpiece <NUM>. The second part 12B is made of a resin material having a low specific gravity. As this resin material, for example, polycarbonate, polyacetal, polypropylene, or fluororesin (Teflon (registered trademark)) can be used. The battery <NUM> constitutes a power supply of the flavor inhaler <NUM>.

As the battery <NUM>, a cylindrical lithium battery is adopted, for example, but other batteries may be used. The battery <NUM> may be a rechargeable battery that can be repeatedly used.

The aerosol source <NUM> is composed of an absorbent, such as absorbent cotton, or other porous body impregnated with a mixture of the tobacco flavor liquid of the present invention and an aerosol-generating liquid such as propylene glycol or glycerin. Alternatively, the aerosol source <NUM> may be composed of a sealable small solution tank, in which a mixture of the tobacco flavor liquid of the present invention and an aerosol-generating liquid such as propylene glycol or glycerin is enclosed.

The wick <NUM> is formed by making a plurality of glass fibers (fibers) into one bundle, and the wick <NUM> is capable of supplying (sucking up) a liquid in the aerosol source <NUM> to the position of the heater <NUM> using the capillary force acting among the glass fibers.

The heater <NUM> constitutes a heat source for generating aerosol by heating a liquid supplied from the aerosol source <NUM>.

At least one air-intake hole <NUM> is formed at a constant interval along the circumferential direction of the housing <NUM>. In the present embodiment, a plurality of air-intake holes <NUM> are formed, but one air-intake hole <NUM> will suffice. Each air-intake hole <NUM> is constituted by a circular small hole penetrating the housing <NUM>.

The operation of the flavor inhaler <NUM><NUM> is described herein. The flavor inhaler <NUM> is activated by pushing a switch such as a push button provided in the housing <NUM>, or by sensing a user's inhalation of the air through the mouthpiece <NUM> using a flow sensor. Alternatively, if the battery <NUM> is a rechargeable-type battery, the flavor inhaler <NUM> may be activated when removal of the battery <NUM> from the charger is sensed by a sensing unit provided in the flavor inhaler <NUM>.

When the flavor inhaler <NUM><NUM> is activated, the drive circuit <NUM> supplies electric power to the heater <NUM>. Any method can be adopted to supply electric power; for example, electric power may be intermittently supplied to the heater <NUM> at a fixed time interval, or a certain voltage may be applied to the heater <NUM> after the flavor inhaler <NUM> is activated. Alternatively, a flow meter may be provided in the ventilation path <NUM>, so that the electric power is increased or decreased in proportion to the flow rate of the gas passing through the ventilation path <NUM>. The liquid supplied from the aerosol source <NUM> is heated by the heater <NUM> and mixed with the air supplied from the air-intake hole <NUM> to generate aerosol.

When the user inhales from the mouthpiece <NUM>, air is taken into the housing <NUM> from the air-intake holes <NUM>. This air becomes aerosol containing a tobacco flavor when passing through the wick <NUM>. This aerosol is taken into the oral cavity of the user, meaning the tobacco flavor can be provided to the user.

In Example <NUM>, the β-D-glucosidase activity of the leaf tobacco was measured. Burley tobacco, flue-cured tobacco, and oriental tobacco were used as the leaf tobacco.

Leaf tobacco was ground to <NUM> mesh or less to obtain ground leaf tobacco. The ground leaf tobacco (<NUM>±<NUM>) was weighed in a glass vial, and suspended in <NUM> of a <NUM> McIlvaine buffer (<NUM> citrate - <NUM> disodium hydrogen phosphate buffer, pH5. <NUM>) which had been cooled to <NUM>. The suspension was homogenized and further sonicated to extract enzyme proteins for <NUM> minutes. The extract was filtered using Whatmann #<NUM>, and the filtrate was centrifuged at <NUM>,<NUM>×g for <NUM> minutes. The supernatant was filtered using a cellulose acetate membrane (Whatmann) with a pore size of <NUM>. <NUM> of the filtrate was fractionated, and low-molecular components were removed using a <NUM> kDa ultrafiltration membrane (Amicon Ultra, centrifugal ultrafiltration tube × <NUM>), thereby obtaining a separation liquid of high-molecular components. A <NUM> acetate buffer (pH <NUM>) was added to the separation liquid of high-molecular components to dilute the separation liquid, and ultrafiltration was performed again to wash the high-molecular fraction (remove low-molecular components). The washing operation of the high-molecular fraction was further repeated two times. The washed separation liquid was adjusted to <NUM> with an acetate buffer adjusted to <NUM> and pH <NUM>. This adjusted solution is defined as a "crude enzyme solution". All processes for preparing the crude enzyme solution were performed at <NUM>.

It is calculated that in the crude enzyme solution prepared, a high-molecular crude product corresponding to <NUM> of the tobacco raw material dissolves in <NUM> (soluble macromolecules in <NUM> of tobacco raw material/<NUM>).

Five solutions were prepared by mixing <NUM>µL of the prepared crude enzyme solution and <NUM>µL of the <NUM> acetate buffer (pH5. <NUM>) in the Eppendorff tube, and each solution was heated in the heat block at <NUM> for <NUM> minutes. <NUM>µL of the <NUM> <NUM>-nitrophenyl β-D-glucopyranoside (Glc-β-pNP) substrate solution was added to four of the five heated mixed solutions, and <NUM>µL of the <NUM> sodium carbonate solution was added to one of them after <NUM> minutes, another one after <NUM> minutes, the other one after <NUM> minutes, and the last one after <NUM> minutes, and the reaction was stopped. One remaining mixed solution was, after incubation, supplied with <NUM>µL of the <NUM> sodium carbonate solution followed by <NUM>µL of the Glc-β-pNP substrate solution.

The concentration of <NUM>-nitrophenol (pNP) generated in each of the solutions was calculated from the absorbance at the <NUM> wavelength, and the reaction rate was determined from the change in the concentration with respect to the reaction time. For the enzyme activity, the level of enzyme that releases <NUM> [nmol] of pNP per second was defined as <NUM> [nkat], and ultimately, the enzyme concentration in the reaction solution was converted to an activity value per weight of the leaf tobacco raw material [nkat/g].

As a specific calculation formula, the β-D-glucosidase activity value per weight of the leaf tobacco raw material [nkat/g] was obtained by dividing a value calculated by the calculation formula of reaction rate [nmol/mL/s] × <NUM> [mL (final solution amount after activity measurement)] / <NUM> [mL (crude enzyme solution amount)], by the weight of the leaf tobacco raw material [g/mL].

The β-D-glucosidase activities measured in the burley tobacco, flue-cured tobacco, and oriental tobacco are shown in the table below.

In Table <NUM>, lot numbers A to D of the flue-cured tobacco are different in the country of origin, lot numbers E to J of the burley tobacco are different in the country of origin, and lot numbers K and L of the oriental tobacco are different in the country of origin.

The measurement results show that the activity of β-glucosidase, which is a glycoside-degrading enzyme, was higher in the burley tobacco as compared to the flue-cured tobacco and the oriental tobacco.

In Example <NUM>, the glycosides contained in the leaf tobacco were analyzed. The analysis was carried out according to the method described in <CIT>. For the leaf tobacco, lot numbers D, H, I and J in Table <NUM> were used.

The dried leaf tobacco sample that was ground using a mill (Melitta Japan Limited) was freeze-dried for <NUM> days. Each of the freeze-dried materials (<NUM>) was weighed in a screw tube (<NUM> volume, Maruemu Corporation), <NUM> of methanol (Wako Pure Chemical Industries, Ltd. , Japan) was added thereto, and while ultrasonic treatment was performed (AS ONE Corporation, US CLEANER US-1R), extraction was performed for <NUM> minutes. Subsequently, <NUM>µL (<NUM>/mL methanol) of n-dodecyl-β-D-glucopyranoside (Sigma-Aldrich Japan) as an internal standard was added to each screw tube, and shaken. Filtration was performed using a PTFE membrane having a pore size of <NUM> (Whatman, <NUM> GD/X Disposable Filter Device) to prepare filtrate containing glycosides.

The filtrate was dispensed into vial containers by <NUM> to prepare analysis samples. The filtrates obtained from the leaf tobacco of lot numbers D, H, I and J are referred to as analysis samples D, H, I and J, respectively.

Analysis samples D, H, I and J were each analyzed by LC-MS/MS under the conditions below.

Based on the chromatogram obtained from each of the analysis samples, the peaks derived from the glycosides identified by the method described in <CIT> were compared. The results thereof are shown in <FIG> shows the analysis results of the glycoside contents of analysis samples D, H, I and J.

The results of <FIG> show that the glycoside content contained in the ground leaf tobacco of the flue-cured tobacco (analysis sample D) was high, whereas the glycoside content contained in the ground leaf tobacco of the burley tobacco (analysis samples H, I and J) was low. The results of <FIG> indicate that the ground leaf tobacco of burley tobacco contains glycosides at a smaller content per unit mass than the ground leaf tobacco of flue-cured tobacco. Further, the results of <FIG> indicate that the content of each component of the glycoside components contained in the ground leaf tobacco of the burley tobacco is smaller than that of the corresponding component of the glycoside components contained in the ground leaf tobacco of the flue-cured tobacco.

<NUM> of the burley leaf tobacco was ground to have a maximum diameter of <NUM> or less to prepare burley ground leaf tobacco. <NUM> of the flue-cured leaf tobacco was ground to have a maximum diameter of <NUM> or less to prepare flue-cured ground leaf tobacco.

Using the prepared ground leaf tobaccos, the following four types of tobacco materials were produced.

A leaf tobacco mixture A was prepared by mixing <NUM> of the burley ground leaf tobacco and <NUM> of the flue-cured ground leaf tobacco. Water was added to the leaf tobacco mixture so that the moisture content of the leaf tobacco mixture was <NUM>% by mass, and the leaf tobacco mixture was stirred. In this manner, the ground leaf tobacco was humidified to such an extent that the surface thereof was slightly wet. The humidified leaf tobacco mixture was placed in a <NUM> container, capped, and stored at <NUM> for <NUM> days, thereby producing a tobacco material A.

Enzymes of the flue-cured ground leaf tobacco were inactivated in the following manner. The flue-cured ground leaf tobacco was put into a fluidized bed that was set to an air flow temperature of <NUM> or higher, an absolute humidity of <NUM> to <NUM> vol%, and a linear velocity of <NUM> to <NUM>/s, and then heat treatment was performed so that the residence time in the fluidized bed was <NUM> seconds or less, thereby inactivating the enzymes.

A leaf tobacco mixture B was prepared by mixing <NUM> of the burley ground leaf tobacco and <NUM> of the heat-treated flue-cured ground leaf tobacco. A tobacco material B was produced in the same manner as the tobacco material A except that the leaf tobacco mixture B was used instead of the leaf tobacco mixture A.

A tobacco material C was produced in the same manner as the tobacco material A except that <NUM> of the burley ground leaf tobacco was used instead of the leaf tobacco mixture A.

A tobacco material D was produced in the same manner as the tobacco material A except that <NUM> of the flue-cured ground leaf tobacco was used instead of the leaf tobacco mixture A.

Enzymes of the burley ground leaf tobacco were inactivated in the following manner. The burley ground leaf tobacco was put into a fluidized bed that was set to an air flow temperature of <NUM> or higher, an absolute humidity of <NUM> to <NUM> vol%, and a linear velocity of <NUM> to <NUM>/s, and then heat treatment was performed so that the residence time in the fluidized bed was <NUM> seconds or less, thereby inactivating the enzymes.

Similarly, enzymes of the flue-cured ground leaf tobacco were inactivated in the following manner. The flue-cured ground leaf tobacco was put into a fluidized bed that was set to an air flow temperature of <NUM> or higher, an absolute humidity of <NUM> to <NUM> vol%, and a linear velocity of <NUM> to <NUM>/s, and then heat treatment was performed so that the residence time in the fluidized bed was <NUM> seconds or less, thereby inactivating the enzymes.

A leaf tobacco mixture E was prepared by mixing <NUM> of the heat-treated burley ground leaf tobacco and <NUM> of the heat-treated flue-cured ground leaf tobacco. A tobacco material E was produced in the same manner as the tobacco material A except that the leaf tobacco mixture E was used instead of the leaf tobacco mixture A.

The glycosides contained in the tobacco materials A, B, C, D and E were analyzed by LC-MS/MS as described in Example <NUM>.

<FIG> shows the total of glycoside contents of the tobacco materials C and D, and the glycoside content of the tobacco material A. In <FIG>, the horizontal axis represents the peak number of the glycosides, and the vertical axis represents the peak area per <NUM> of the tobacco material.

The results of <FIG> show that "the glycoside content of the tobacco material A" is smaller than "the total of glycoside contents of the tobacco materials C (burley tobacco) and D (flue-cured tobacco)". The tobacco material A was produced by mixing and storing the burley and flue-cured ground leaf tobaccos. Further, the results of Examples <NUM> and <NUM> demonstrate that the flue-cured tobacco exhibits low glycoside-degrading enzyme activity and contains various large amounts of glycosides, and that the burley tobacco exhibits high glycoside-degrading enzyme activity and contains small amounts of glycosides. Therefore, for the tobacco material A, it can be said that various glycosides contained in the flue-cured ground leaf tobacco were degraded by various glycoside-degrading enzymes contained in the burley ground leaf tobacco.

<FIG> shows the glycoside content of the tobacco material B, and the glycoside content of the tobacco material E. In <FIG>, the horizontal axis represents the peak number of the glycosides, and the vertical axis represents the peak area per <NUM> of the tobacco material.

The results of <FIG> show that the "glycoside content of the tobacco material B" is smaller than the "glycoside content of the tobacco material E". The tobacco material B was produced by inactivating the enzymes of the flue-cured ground leaf tobacco, and then mixing and storing the flue-cured and burley ground leaf tobaccos. On the other hand, the tobacco material E was produced by inactivating both the enzymes of the flue-cured ground leaf tobacco and the enzymes of the burley ground leaf tobacco, and then mixing and storing the flue-cured and burley ground leaf tobaccos. Therefore, for the tobacco material B, it can be said that various glycosides contained in the flue-cured ground leaf tobacco were degraded by various glycoside-degrading enzymes contained in the burley ground leaf tobacco.

The flue-cured ground leaf tobacco was heat-treated under the same conditions as those used in the production of "tobacco material B" in Example <NUM> to inactivate the enzymes. A tobacco material F was produced in the same manner as in the production of the "tobacco material A" of Example <NUM>, except that <NUM> of the heat-treated flue-cured ground leaf tobacco was used instead of the leaf tobacco mixture A.

A steam distillation apparatus (Herb Oil Maker (Standard Type) manufactured by Tokyo Seisakusho), the inside of which was cleaned with water for about <NUM> hours, was supplied with <NUM> of water and heated with a heater (<NUM>). After boiling, one of the tobacco material B or C prepared in Example <NUM> or the tobacco material F prepared in Example <NUM> (<NUM>) was put into the apparatus, and distillation was started. The distillation was continued, and <NUM> of the fraction was obtained by <NUM>-hour distillation. The obtained fraction was collected in an Erlenmeyer flask and left in an ice bath (<NUM>) for <NUM> hours.

As an organic solvent, diethyl ether was used.

<NUM> of sodium chloride was added to an Erlenmeyer flask containing the fraction, followed by shaking. Next, <NUM> of the fraction (including the oil floating in the fraction) was placed in a <NUM> separation funnel, and <NUM> of an organic solution was added, followed by shaking. After removal of an aqueous phase, an organic phase was placed in a new Erlenmeyer flask. The operation of newly adding <NUM> of the organic solvent to the aqueous phase and recovering the organic phase was repeated two times. <NUM> of anhydrous sodium sulfate was added to the Erlenmeyer flask in which the organic phase was recovered, and the mixture was left to stand at room temperature for <NUM> minutes for dehydration.

The dehydrated organic phase was filtered through a filter paper (ADVANTEC, No. <NUM>, <NUM>). The filtrate was evaporated to dryness under the reduced pressure in a hot water bath at <NUM> by a rotary evaporator, thereby obtaining a tobacco extract as a dried product. Propylene glycol was added at a weight <NUM> times the weight of the extract, thereby obtaining a "tobacco flavor liquid". Tobacco flavor liquids produced from the tobacco materials B, C and F are referred to as tobacco flavor liquids B, C and F, respectively.

The tobacco flavor liquids B, C and F were analyzed by GC/MS.

The following conditions can be used for GC/MS.

<FIG> is a GC-MS chromatogram when the tobacco flavor liquids C and F were mixed (at <NUM>:<NUM>). <FIG> is a GC-MS chromatogram of the tobacco flavor liquid B. The results of <FIG> and <FIG> show that the tobacco flavor liquid B has more flavor component peaks and richer flavors, as compared to the mixture of the tobacco flavor liquids C and F. Therefore, the results of <FIG> and <FIG> show that by performing the method of the present invention, it is possible to provide a tobacco flavor liquid having an enhanced flavor.

A leaf tobacco mixture was prepared by mixing <NUM> of the burley ground leaf tobacco and <NUM> of the flue-cured ground leaf tobacco. Water was added to the leaf tobacco mixture so that the moisture content of the leaf tobacco mixture was <NUM>% by mass, and the leaf tobacco mixture was stirred. In this manner, the ground leaf tobacco was humidified to such an extent that the surface thereof was slightly wet. The humidified leaf tobacco mixture was placed in a <NUM> container, sealed, and stored at a temperature of <NUM>, <NUM>, <NUM>, or <NUM> for <NUM> days, thereby preparing a tobacco material.

The glycosides contained in each of the tobacco materials stored at different temperatures were analyzed by LC-MS/MS as described in Example <NUM>. <FIG> shows the glycoside content at each storage temperature. In <FIG>, the sum of the glycoside content of the tobacco material C of Example <NUM> and the glycoside content of the tobacco material D of Example <NUM> is shown as a reference value.

The results of <FIG> show that when the leaf tobacco mixture was stored at <NUM>, the glycoside content was higher and the glycoside degradation amount was smaller than when it was stored at the lower temperatures. It is considered that this is because the activity of the glycoside-degrading enzymes was diminished due to the leaf tobacco mixture being exposed to the higher temperature. This result shows that a temperature range desirable for glycoside degradation is a temperature of <NUM> to <NUM>.

A leaf tobacco mixture was prepared by mixing <NUM> of the burley ground leaf tobacco and <NUM> of the flue-cured ground leaf tobacco. Water was added to the leaf tobacco mixture so that the moisture content of the leaf tobacco mixture was <NUM>% by mass, and the leaf tobacco mixture was stirred. In this manner, the ground leaf tobacco was humidified to such an extent that the surface thereof was slightly wet. The humidified leaf tobacco mixture was placed in a <NUM> container, sealed, and stored at <NUM> for <NUM>, <NUM> or <NUM> days, thereby producing a tobacco material.

The glycosides contained in each of the tobacco materials stored for different periods of time were analyzed by LC-MS/MS as described in Example <NUM>. <FIG> shows the glycoside contents when the storage periods were changed. In <FIG>, the sum of the glycoside content of the tobacco material C of Example <NUM> and the glycoside content of the tobacco material D of Example <NUM> is shown as a reference value.

The results of <FIG> show that approximately <NUM>% of the glycoside content of the reference value was degraded on storage day <NUM>. Degradation of the glycosides further progressed on storage day <NUM>. The glycoside content on storage day <NUM> was not significantly different from that of storage day <NUM>. This result shows that a storage period desirable for glycoside degradation is <NUM> hours to <NUM> hours.

A leaf tobacco mixture was prepared by mixing <NUM> of the burley ground leaf tobacco and <NUM> of the flue-cured ground leaf tobacco. Water was added to the leaf tobacco mixture so that the moisture content of the leaf tobacco mixture was <NUM>% by mass, <NUM>% by mass, <NUM>% by mass, <NUM>% by mass, <NUM>% by mass, <NUM>% by mass, <NUM>% by mass, or <NUM>% by mass, and the leaf tobacco mixture was stirred. The humidified leaf tobacco mixture was placed in a <NUM> container, capped, and stored at <NUM> for <NUM> days, thereby producing a tobacco material.

The glycosides contained in each of the tobacco materials having different moisture contents during storage were analyzed by LC-MS/MS as described in Example <NUM>. <FIG> and <FIG> show the glycoside contents when the moisture contents during storage were changed. In <FIG> and <FIG>, the sum of the glycoside content of the tobacco material C of Example <NUM> and the glycoside content of the tobacco material D of Example <NUM> is shown as a reference value.

Claim 1:
A tobacco material production method, comprising:
mixing first ground leaf tobacco having β-D-glucosidase activity of <NUM> [nkat/g] or more and second ground leaf tobacco containing glycosides and having β-D-glucosidase activity of <NUM> [nkat/g] or less to prepare a leaf tobacco mixture; and
storing the leaf tobacco mixture under a humidification condition at a temperature of <NUM> to <NUM> over a period of <NUM> to <NUM> hours to obtain a tobacco material having an enhanced tobacco flavor,
wherein none of the first ground leaf tobacco, the leaf tobacco mixture, and the tobacco material having the enhanced tobacco flavor is exposed to a temperature of <NUM> or more.