JPWO2020021599A1 - Tobacco Material Manufacturing Method, Tobacco Material, Tobacco Flavor Liquid Manufacturing Method, Tobacco Flavor Liquid, and Heated Flavor Aspirator - Google Patents

Abstract

A first tobacco pulverized product having a β-D-glucosidase activity of 30 [nkat / g] or more, and a second tobacco pulverized product containing a glycoside and having a β-D-glucosidase activity of 25 [nkat / g] or less. A method for producing a tobacco material, which comprises mixing with a pulverized leaf tobacco product to prepare a leaf tobacco mixture, and storing the leaf tobacco mixture under humidified conditions to obtain a tobacco material having an increased tobacco flavor.

Description

The present invention relates to a method for producing a tobacco material, a tobacco material, a method for producing a tobacco flavor liquid, a tobacco flavor liquid, and a heated flavor aspirator.

The leaves of cultivated and harvested tobacco plants are subjected to various processing processes such as drying process at the farm, long-term aging process for one to several years at the raw material factory, and subsequent blending and cutting at the manufacturing factory. After that, it is used in the manufacture of tobacco products. Tobacco leaf raw materials used in the manufacture of tobacco products are referred to in the art as "leaf tobacco".

Leaf tobacco is known to contain various glycoside components. Glycosides contained in leaf tobacco include glucosides such as scopoletin 7-glucoside (scopoletin) and quercetin 3-β-D-glucoside (isoquercetin); naringenin 7-ramnoglucoside (naringin) and quercetin 3-ramnoglucoside ( Lamnoglucoside (lucinoside) such as rutin); sophoroside such as ricitin β-sophoroside and quercetin 3-β-D-sophoroside have been identified, but there are many that have not yet been identified.

Glycosides contained in leaf tobacco are considered to have a function as precursors of tobacco flavor components. Specifically, when leaf tobacco is burned during smoking, the glycoside component contained in the leaf tobacco is decomposed into a sugar portion and a non-sugar portion (that is, aglycone), and the non-sugar portion is considered to function as a tobacco flavor component. Has been done.

On the other hand, attempts have been made to increase the tobacco flavor component contained in leaf tobacco. For example, Japanese Patent Application Laid-Open No. 56-51976 improves the flavor and taste of leaf tobacco by adding ethyl alcohol to tobacco leaves before a long-term aging process at a raw material factory and then performing an aging process. I am reporting that.

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

According to the first aspect
A first tobacco pulverized product having a β-D-glucosidase activity of 30 [nkat / g] or more, and a second leaf tobacco pulverized product containing a glycoside and having a β-D-glucosidase activity of 25 [nkat / g] or less. Provided is a method for producing a tobacco material, which comprises mixing with a crushed leaf tobacco to prepare a leaf tobacco mixture, and storing the leaf tobacco mixture under humidified conditions to obtain a tobacco material having an increased tobacco flavor. Will be done.

According to the second aspect, the tobacco material produced by the method is provided.
According to the third aspect, there is provided a method for producing a tobacco flavor liquid, which comprises extracting a tobacco flavor component from the tobacco material to obtain a tobacco flavor liquid.

According to the fourth aspect, the tobacco flavoring liquid produced by the above method is provided.
According to the fifth aspect, a heated flavor aspirator containing the tobacco material or the tobacco flavor liquid is provided.

According to the present invention, various glycosides contained in the second crushed tobacco product are decomposed by various glycoside-degrading enzymes contained in the first crushed leaf tobacco to produce various tobacco flavor components. This makes it possible to provide a tobacco material having an increased tobacco flavor and a tobacco flavor liquid having an increased tobacco flavor.

FIG. 1 is a flowchart showing the method of the present invention. FIG. 2 is a partial incision view showing an example of a heated flavor aspirator containing a tobacco material. FIG. 3 is a cross-sectional view showing an example of a heated flavor aspirator containing a tobacco flavor liquid. FIG. 4 is a graph showing the analysis results of the glycoside contents of the samples D, H, I and J for analysis. FIG. 5 is a graph showing the analysis result of the glycoside content. FIG. 6 is a graph showing the analysis result of the glycoside content. FIG. 7 is a GC-MS chromatogram of a tobacco flavor liquid. FIG. 8 is a GC-MS chromatogram of a tobacco flavor liquid. FIG. 9 is a graph showing the effect of the temperature during storage on the glycoside content. FIG. 10 is a graph showing the effect of the storage period on the glycoside content. FIG. 11 is a graph showing the effect of the water content in storage on the glycoside content. FIG. 12 is a graph showing the effect of the water content in storage on the glycoside content.

Hereinafter, the present invention will be described in detail, but the following description is intended to explain the present invention and is not intended to limit the present invention.

<1. Tobacco material manufacturing method>
The manufacturing method of tobacco material is
A first tobacco pulverized product having a β-D-glucosidase activity of 30 [nkat / g] or more, and a second leaf tobacco pulverized product containing a glycoside and having a β-D-glucosidase activity of 25 [nkat / g] or less. It involves mixing with a crushed leaf tobacco to prepare a leaf tobacco mixture, and storing the leaf tobacco mixture under humidified conditions to obtain a tobacco material with increased tobacco flavor. A flowchart of a method for producing a tobacco material is shown in FIG.

(Crushed leaf tobacco)
First, the first crushed leaf tobacco product and the second crushed leaf tobacco product will be described. In the following description, the term "crushed leaf tobacco" is used to refer to both the first crushed leaf tobacco and the second crushed leaf tobacco.

The crushed leaf tobacco product is obtained by crushing the leaf tobacco. The crushed leaf tobacco has a maximum diameter of, for example, 1 mm or less. A crushed leaf tobacco product having a maximum diameter of 1 mm or less can be obtained, for example, by crushing it with a commercially available crusher (mill) and sieving it on a 1.0 mm mesh. The crushed leaf tobacco has a maximum diameter of, for example, 0.5 to 1 mm. The use of crushed leaf tobacco results in the transfer of glycoside-degrading enzymes and glycosides between the first crushed leaf tobacco and the second crushed leaf tobacco, as compared to the case of using uncrushed leaf tobacco. It can be promoted and the efficiency of glycoside decomposition reaction can be increased. As a result, glycoside decomposition products are efficiently produced, and the tobacco flavor can be increased.

"Leaf tobacco", which is the raw material for crushed leaf tobacco, is made from the leaves of cultivated and harvested tobacco plants, which are dried by farmers, then aged for a year or several years at the raw material factory, and then manufactured at the manufacturing plant. It is obtained by performing various processing treatments such as blending and engraving in. That is, "leaf tobacco" refers to tobacco ticks that are ready for the manufacture of tobacco products. For example, as "leaf tobacco", a cigarette carving ready for the cigarette hoisting process can be used.

The first pulverized leaf tobacco has a β-D-glucosidase activity of 30 [nkat / g] or more. The first pulverized leaf tobacco generally has a β-D-glucosidase activity of 30 to 1000 [nkat / g]. The first ground leaf tobacco preferably has a β-D-glucosidase activity of 100-1000 [nkat / g]. On the other hand, the second pulverized leaf tobacco product contains a glycoside and has a β-D-glucosidase activity of 25 [nkat / g] or less. The second leaf tobacco pulverized product generally has a β-D-glucosidase activity of 0 to 25 [nkat / g]. The second leaf tobacco milling preferably has a β-D-glucosidase activity of 0 to 15 [nkat / g].

"Β-D-glucosidase" is one of the glycoside-degrading enzymes possessed by leaf tobacco. In the present specification, the "β-D-glucosidase activity" of the pulverized leaf tobacco refers to the degrading activity of 4-nitrophenyl β-D-glucopyranoside (Glc-β-pNP), which is a model substrate, and specifically, It refers to the enzyme activity determined by the measurement method of Example 1 described later. The "β-D-glucosidase activity" of crushed leaf tobacco is obtained by collecting partial samples from three locations of crushed leaf tobacco, measuring the enzyme activity of the partial samples, and calculating the average of the obtained measured values. Can be done.

In general, the enzymes contained in leaf tobacco are inactivated when exposed to high temperatures during the leaf tobacco manufacturing process (especially the drying process in farmers), and inactivated when not exposed to high temperatures during the leaf tobacco manufacturing process. The activity is maintained without. Therefore, if the "β-D-glucosidase activity" of the crushed leaf tobacco is maintained, other enzyme activities are also maintained. Therefore, the "β-D-glucosidase activity" of the crushed leaf tobacco can be used as an index of the activity of various glycoside-degrading enzymes contained in the crushed leaf tobacco.

Specifically, when the crushed leaf tobacco has a β-D-glucosidase activity of 30 [nkat / g] or more, the crushed leaf tobacco also has a predetermined value for glycoside-degrading enzymes other than β-D-glucosidase. It has the above activity and has sufficient ability to decompose various glycosides to produce tobacco flavor components. On the other hand, when the crushed leaf tobacco has a β-D-glucosidase activity of 25 [nkat / g] or less, the crushed leaf tobacco also has an activity of 25 [nkat / g] or less for glycoside-degrading enzymes other than β-D-glucosidase. And does not have sufficient ability to decompose various glycosides to produce tobacco flavor components.

The "β-D-glucosidase activity" of the crushed leaf tobacco product refers to a value measured by the measuring method of Example 1 described later immediately before mixing the crushed leaf tobacco product and the crushed leaf tobacco product. Similarly, the "presence or absence of glycoside" of the crushed leaf tobacco is identified as a glycoside according to the analysis method of Example 2 described later immediately before mixing the crushed leaf tobacco and the crushed second leaf tobacco. It is determined by whether or not there is a peak to be formed.

The first crushed leaf tobacco may or may not contain glycosides, and the content thereof is not particularly limited, but the crushed leaf tobacco has high glycoside-degrading enzyme activity. As shown, the glycoside content is generally low. On the other hand, the second crushed leaf tobacco contains a glycoside that serves as a substrate for the glycoside decomposition reaction. The second crushed leaf tobacco exhibits a low glycoside-degrading enzyme activity, and therefore has a generally high glycoside content. If the amount of glycoside contained in the second crushed leaf tobacco is large, it leads to the production of more glycoside decomposition products. Therefore, it is preferable that the crushed leaf tobacco has a large amount of glycoside. ..

Therefore, preferably, the first crushed leaf tobacco contains glycosides in a smaller content per unit mass of the crushed leaf tobacco as compared with the second crushed leaf tobacco product. More preferably, the content of any of the glycoside components contained in the first crushed leaf tobacco is lower than that of the glycoside component contained in the second crushed leaf tobacco. In the present specification, the glycoside content of the crushed leaf tobacco refers to the content per unit mass of the crushed leaf tobacco.

In the present specification, the "glycoside" contained in the crushed leaf tobacco refers to all of the glycosides contained in the leaf tobacco, and specifically, the glycoside is defined by the analysis method described in WO2018 / 038245. Refers to all of the determined ingredients. More specifically, the "glycoside" contained in the pulverized leaf tobacco product refers to all the components determined to be glycosides according to the analysis method of Example 2 described later. In the analytical method described in WO2018 / 038245, a leaf tobacco extract sample treated with β-D-glucosidase and a leaf tobacco extract sample not treated with β-D-glucosidase were prepared, and each sample was prepared by LC-MS. The analysis results are compared by analysis by / MS, and the peak whose disappearance or intensity is reduced by β-D-glucosidase is determined to be a glycoside. The "glycoside" contained in the pulverized leaf tobacco product can be quantified using an internal standard substance as described in WO2018 / 308245.

As described in the background technology column, leaf tobacco contains many types of glycoside components, but many of these glycoside components have not been identified. Therefore, in the present specification, the "glycoside" contained in the crushed leaf tobacco is not only the glycoside identified as the glycoside contained in the leaf tobacco, but also the glycoside contained in the leaf tobacco but not identified. Also contains glycosides.

The "first crushed leaf tobacco" is typically a crushed leaf tobacco of a variety in which the leaves of a tobacco plant are dried in a farm by air-curing. A typical first crushed leaf tobacco is exposed to natural temperature and humidity and aeration conditions during the farm drying process, so that enzyme inactivation is unlikely to occur and many glycosides are decomposed. As a result, a typical first leaf tobacco pulverized product has a high glycoside-degrading enzyme activity and a low glycoside content.

The first crushed leaf tobacco is, for example, at least one cultivar crushed leaf tobacco selected from Burley, Native, Dark Fire Dried, Dark Air Dried, and Dark Sun Dried. The first crushed leaf tobacco may be a crushed leaf tobacco of one variety or a mixture of crushed leaf tobacco of a plurality of varieties.

A "second leaf tobacco crusher" is typically a farmer's drying of tobacco plant leaves, either flue-curing with a heating process or air-circulating curing with heated air. ) Or sun-curing varieties of crushed leaf tobacco. A typical second leaf tobacco crush is exposed to high temperatures during the farmer's drying process, which is prone to enzyme inactivation and many glycosides remain undegraded. As a result, a typical second leaf tobacco pulverized product has a low glycoside-degrading enzyme activity and a high glycoside content.

The second crushed leaf tobacco is, for example, at least one cultivar crushed leaf tobacco selected from yellow, orient, sun-dried, and light-colored sun-dried varieties. The second crushed leaf tobacco may be a crushed leaf tobacco of one variety or a mixture of crushed leaf tobacco of a plurality of varieties.

As described above, the first crushed leaf tobacco product may be a crushed leaf tobacco product of a variety in which the leaves of the tobacco plant are dried by air-curing in the farm. Alternatively, the first crushed leaf tobacco is
The leaf tobacco raw material is treated to activate glycoside-degrading enzymes, and then
It may be a pulverized product obtained by pulverizing the leaf tobacco raw material. As long as the leaf tobacco raw material used here is a leaf tobacco raw material having glycoside-degrading enzyme activity, the content of glycosides is arbitrary. At least one selected from leaf tobacco raw materials, for example, Burley, Native, Dark Fired, Dark Air, Dark Sun, Yellow, Orient, Sun, and Light Sun. Two varieties can be used. The activation treatment is, for example, treating the leaf tobacco raw material with a buffer so as to adjust the pH of the leaf tobacco raw material to the optimum pH of the enzyme, or placing the leaf tobacco raw material at the optimum temperature of the enzyme. Can be done by.

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

As mentioned above, the second leaf tobacco crushed product is a farmer's drying of the leaves of the tobacco plant, either flue-curing with a heating step or air-circulating curing or air-circulating curing with heated air. It may be a crushed leaf tobacco of a variety performed by sun-curing. Alternatively, the second crushed leaf tobacco is
The leaf tobacco raw material is heated to inactivate the enzyme contained in the leaf tobacco raw material, and then
It may be a pulverized product obtained by pulverizing the leaf tobacco raw material. If the leaf tobacco raw material used here is a leaf tobacco raw material containing a glycoside, the value of the glycoside-degrading enzyme activity is arbitrary. At least one selected from leaf tobacco raw materials, for example, Burley, Native, Dark Fired, Dark Air, Dark Sun, Yellow, Orient, Sun, and Light Sun. Two varieties can be used. Preferably, as the leaf tobacco raw material, at least one variety selected from yellow varieties, orient varieties, sun-dried varieties, and light-colored sun-dried varieties can be used. The deactivation treatment can be carried out, for example, by putting leaf tobacco into a fluidized bed having an air flow temperature of 220 to 250 ° C. and treating it for 1 to 5 seconds.

When the enzyme of the second leaf tobacco pulverized product was inactivated in advance, the leaf tobacco after the inactivation treatment could completely suppress the subsequent decomposition of glycosides and maintained a high glycoside content. The raw material for leaf tobacco can be preserved as it is. Therefore, by performing such deactivation treatment, a leaf tobacco raw material having a high glycoside content can be stably supplied as a raw material for the "second leaf tobacco pulverized product".

(Preparation of leaf tobacco mixture)
The above-mentioned first crushed leaf tobacco product and the above-mentioned second crushed leaf tobacco product are mixed to prepare a leaf tobacco mixture. The mixing ratio of the first ground leaf tobacco product and the second ground leaf tobacco product is arbitrary, but is preferably 1:20 to 20: 1 in mass ratio, for example, 1: 1 in mass ratio. it can.

The leaf tobacco mixture may be composed only of the first crushed leaf tobacco and the second crushed leaf tobacco. In the present invention, it is sufficient that the glycoside-degrading enzyme contained in the first crushed leaf tobacco acts on the glycoside contained in the crushed leaf tobacco to produce the glycoside-degrading product, and thus the leaf tobacco is produced. The mixture does not have to contain any components other than the ground leaf tobacco product. However, the present invention does not exclude that the leaf tobacco mixture contains components other than the pulverized leaf tobacco product, and contains components that promote the glycoside decomposition reaction, such as water and a pH buffer, as additives. You may be.

(Storage of leaf tobacco mixture)
The above-mentioned leaf tobacco mixture is stored under humidified conditions to produce a tobacco material having an increased tobacco flavor.

The humidification condition can generally be a condition in which water is added to the leaf tobacco mixture so that the water content of the leaf tobacco mixture is 12 to 80% by mass. Here, the "moisture content (mass%) of the leaf tobacco mixture" is the total water content of the water content originally possessed by the leaf tobacco mixture and the added water content, which is the sum of the mass of the leaf tobacco mixture and the added water content. The ratio to the mass.

The "water content originally possessed by the leaf tobacco mixture" can be determined by the following method.
The leaf tobacco sample is heated at 100 ° C. for 1 hour under normal pressure according to a method for analyzing the moisture content of food (heat drying method), allowed to cool in a desiccator for 40 minutes, and the moisture content is determined from the weight difference before and after heating. The specific procedure is as follows.

(1) Measure the mass (W0) of the sample container stored in the desiccator.
(2) Weigh the required amount of leaf tobacco sample, put it in the sample container, and cover it. Here, the total mass of the weighed sample mass and the sample container mass (WO) is defined as W1.
(3) Open the lid of the sample container, arrange it in a rotary dryer, and heat it at 100 ° C. for 1 hour.
(4) After 1 hour, close the lid of the sample container, take it out, and allow it to cool in a desiccator.
(5) After 40 minutes, the mass (W2) of the sample container containing the sample is measured. The water content (Mw) is calculated by the following formula.

Mw = {(W1-W2) / (W1-W0)} × 100
Mw: Moisture content (mass%)
W1: Total mass (g) of sample before drying and sample container
W2: Total mass (g) of sample and sample container after drying
W0: Mass of sample container (g)

The "water content inherent in the leaf tobacco mixture" is generally 12 to 14% by mass. For example, in order to make the water content of the leaf tobacco mixture (30 g) having a water content of 12% by mass 55%, it is necessary to add 29 g of water. After adding the required amount of water to the leaf tobacco mixture, the leaf tobacco mixture may be agitated so that the water is distributed throughout the leaf tobacco mixture.

Preferably, the humidification condition is a condition in which water is added to the leaf tobacco mixture so that the water content of the leaf tobacco mixture is 12 to 55% by mass. More preferably, the humidification condition is a condition in which water is added to the leaf tobacco mixture so that the water content of the leaf tobacco mixture is 12 to 45% by mass.

Moisture plays a role as a reactant because the glycoside decomposition reaction is a hydrolysis reaction. In addition, water acts as a medium, facilitating the transfer of glycoside-degrading enzymes and glycosides between the first crushed leaf tobacco and the second crushed leaf tobacco, and the efficiency of the glycoside degradation reaction. Play a role in enhancing.

Therefore, the "water content of the leaf tobacco mixture" is preferably an amount sufficient to fulfill the above role. Further, considering that the leaf tobacco mixture is used as a tobacco filler for tobacco products or as a raw material for a tobacco flavor liquid after storage, the "moisture content of the leaf tobacco mixture" plays the above role. If the amount is sufficient, it is preferable that the amount is not more than necessary. It is preferable that the "moisture content of the leaf tobacco mixture" is not too much in terms of the risk of mold growth.

Storage of the leaf tobacco mixture can be carried out at a temperature suitable for the glycoside-degrading enzyme to work and for the period required for a sufficient amount of glycoside-degrading product to be produced.

Storage of the leaf tobacco mixture is preferably carried out at a temperature of 0 to 60 ° C. Storage of the leaf tobacco mixture is more preferably carried out at a temperature of 0 to 50 ° C, even more preferably 20 to 50 ° C.

Storage of the leaf tobacco mixture is preferably carried out over a period of 24-72 hours. Storage of the leaf tobacco mixture is more preferably carried out over a period of 24-48 hours. The storage period may be determined based on the period until the amount of glycoside decomposition products produced reaches a plateau in order to reduce the risk that the produced glycoside decomposition products disappear due to volatilization or further decomposition. ..

Storage of the leaf tobacco mixture is preferably carried out under closed conditions. Sealing conditions can be created by placing the leaf tobacco mixture in an airtight container. The airtight container may have an arbitrary capacity, but it is preferable that the temperature inside the container can be controlled. The leaf tobacco mixture can be placed in the container, for example, to the extent that it occupies about 50-80% of the capacity of the container. Storage under closed conditions is preferable because the produced glycoside decomposition products tend to stay in the crushed leaf tobacco products.

The leaf tobacco mixture may be incorporated into the tobacco product after storage, or may be incorporated into the tobacco product after being dried to a water content comparable to that of normal leaf tobacco (that is, 12 to 14% by mass). The drying may be carried out by using a dryer or by natural drying. Air drying can be carried out by allowing the leaf tobacco mixture to stand for 1 to 7 days under conditions of a temperature of 5 to 40 ° C. and a humidity of 10 to 90%. When a dryer is used, drying can be performed by drying under reduced pressure for 1 to 5 hours while keeping the temperature below 40 ° C. while avoiding heating.

Tobacco materials produced according to the method of the present invention have an increased tobacco flavor because they contain the produced glycoside degradation products. In order to reduce the risk that the produced glycoside decomposition products disappear due to volatilization, further decomposition, etc., the tobacco material produced according to the method of the present invention is rapidly incorporated into a tobacco product (for example, a flavor suction article). Is preferable. In other words, the method of the present invention is preferably carried out on leaf tobacco immediately before it is incorporated into a tobacco product (eg, a flavor-sucking article).

Further, in the method of the present invention, it is desirable that none of the "first crushed leaf tobacco", "mixture of leaf tobacco", and "tobacco material with increased tobacco flavor" is exposed to a high temperature of, for example, 80 ° C. or higher. Since the "first leaf tobacco crushed product" is required to have glycoside-degrading enzyme activity, a high temperature that inactivates the enzyme, for example, 80, before and after mixing with the second leaf tobacco crushed product. It is desirable not to be exposed to temperatures above ° C. It is desirable that the "leaf tobacco mixture" is not exposed to a high temperature that inactivates the enzyme, for example, a high temperature of 80 ° C. or higher, so that the glycoside-degrading enzyme contained in the leaf tobacco mixture functions. "Tobacco material with increased tobacco flavor" reduces the risk of the produced glycoside decomposition products disappearing due to volatilization or further decomposition, so that the temperature is high, for example, 80 ° C or higher before and after being incorporated into tobacco products. It is desirable not to be exposed to.

(effect)
As mentioned above, leaf tobacco has "varieties with high glycoside-degrading enzyme activity and low glycoside content" and "low glycoside-degrading enzyme activity" depending on how farmers dry them. , Varieties with high glycoside content ”. This is because if both the activity of the glycoside-degrading enzyme and the glycoside are present in the same leaf tobacco, the glycoside is decomposed, and the activity of the glycoside-degrading enzyme and the glycoside content are incompatible. it is conceivable that. Focusing on this point, the present inventors newly pay attention to this point, and a large amount of glycoside-degrading enzyme contained in leaf tobacco having high activity of glycoside-degrading enzyme and leaf tobacco having low activity of glycoside-degrading enzyme are contained. We succeeded in increasing the tobacco flavor of leaf tobacco by reacting with glycosides.

The present invention effectively utilizes various types of glycoside-degrading enzymes contained in one leaf tobacco with high activity and various types of glycosides contained in a large amount in another leaf tobacco to decompose various glycosides. It is excellent in that it can generate things.

When an enzyme preparation is used instead of the enzyme contained in leaf tobacco, the enzyme identifies a specific structure of the substrate and acts only on the specific substrate. Therefore, for example, β-glucosidase is a glycoside having a β-glycosidic bond. Can only be disassembled. Therefore, in order to decompose various glycoside components contained in leaf tobacco, it is necessary to prepare various enzyme preparations. In addition, many glycoside components contained in leaf tobacco have not been identified, and many do not know what kind of enzyme should be prepared in the first place. Even if all the glycoside components contained in leaf tobacco can be identified, it is burdensome to prepare various kinds of enzymes capable of decomposing all these glycoside components.

On the other hand, according to the present invention, by utilizing various kinds of glycoside-degrading enzymes inherent in leaf tobacco, many kinds of glycoside components existing in leaf tobacco can be decomposed at once, and in this respect as well, the present invention The method is excellent.

<2. Tobacco material>
According to another aspect, a tobacco material produced by the above method is provided. As mentioned above, the tobacco material of the present invention has an increased 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 the production of a tobacco flavor liquid.

The tobacco material of the present invention can be incorporated into any flavor-sucking article that the user tastes the tobacco flavor. Specific examples of the flavor suction article include a combustion-type smoking article that provides the user with a tobacco flavor by burning the tobacco material, and a heating type that provides the user with the tobacco flavor by heating the tobacco material without burning it. Examples include a flavor aspirator and a non-heated flavor aspirator that provides the user with a tobacco flavor without heating or burning the tobacco material. As a heating type flavor aspirator, a direct heating type that provides the user with a tobacco flavor by heating the tobacco material with a heating device such as a heater, or a liquid aerosol source that heats a liquid aerosol source to generate an aerosol and turns the aerosol into a tobacco material. An indirect heating type that provides a user with a tobacco flavor by passing through is known.

Since the heated flavor aspirator and the non-heated flavor aspirator do not burn the tobacco material, the tobacco flavor component is less likely to be released as compared with the burning type smoking article. Therefore, the tobacco material of the present invention exerts a particularly excellent effect in that it can provide an increased tobacco flavor to the user when used in a heated flavor aspirator or a non-heated flavor aspirator. ..

Combustion-type smoking articles include, for example, cigarettes, pipes, pipes, cigars, cigarettes, and the like.

As a heating type flavor aspirator, for example,
A carbon heat source type aspirator (see, eg, WO 2006/073065) that heats a tobacco material (eg, a tobacco chopped or tobacco compact) with the combustion heat of a carbon heat source to generate an aerosol containing flavor components; or An electroheated aspirator (eg, an electroheated aspirator) with a refillable tobacco pod containing the tobacco material together with an aerosol source (eg, propylene glycol or glycerin) and an aspirator body that heats the tobacco pod by electric heating to generate an aerosol. (See WO 2013/025921); or an aerosol generated in the first cartridge comprising a first cartridge having an atomizer that atomizes the aerosol source with the power supplied by the battery and a second cartridge containing the tobacco material. An electroheated aspirator that imparts a tobacco flavor to the aerosol by passing it through a second cartridge (see, eg, WO2016 / 075747).
Can be mentioned.

As a non-heated flavor suction article, a non-heated tobacco flavor suction device (for example, WO2010 /) in which a refillable cartridge containing a tobacco material is provided in a suction holder and the user sucks the tobacco flavor derived from the tobacco material at room temperature. 110226).

The ratio of the tobacco material of the present invention to the total tobacco material (hereinafter, also referred to as a tobacco filler) contained in the flavor suction 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 (100% by mass), or occupy a part of the tobacco filler, for example, 1 to 99% by mass of the tobacco filler. It may be blended.

<3. Tobacco flavor liquid manufacturing method>
According to another aspect, there is provided a method for producing a tobacco flavor liquid, which comprises extracting a tobacco flavor component from the tobacco material of the present invention to obtain a tobacco flavor liquid. That is, the method for producing the tobacco flavor liquid is
A first leaf tobacco pulverized product having a β-D-glucosidase activity of 30 [nkat / g] or more, and a second leaf tobacco pulverized product containing a glycoside and having a β-D-glucosidase activity of 25 [nkat / g] or less. To prepare a leaf tobacco mixture by mixing with crushed leaf tobacco,
The leaf tobacco mixture is stored under humidified conditions to obtain a tobacco material having an increased tobacco flavor, and the tobacco flavor component is extracted from the tobacco material to obtain a tobacco flavor liquid. The method for producing the tobacco flavor liquid is shown in FIG. 1 in a flowchart.

The process to obtain the tobacco material is <1. The description of the method for producing a tobacco material> can be referred to. The step of extracting the tobacco flavor component from the tobacco material can be performed by using a method generally known as a method for extracting the aroma component. The extraction of the tobacco flavor component can be carried out by, for example, distillation, specifically, steam distillation, hot water distillation, atmospheric distillation or vacuum distillation. The extraction of the tobacco flavor component is preferably performed by steam distillation in terms of extraction efficiency.

<4. Tobacco flavor liquid>
According to another aspect, a tobacco flavor liquor produced by the above method is provided. The tobacco flavor liquid of the present invention has an increased tobacco flavor because it is produced using a tobacco material having an increased tobacco flavor as a raw material.

The tobacco flavor liquid of the present invention can be incorporated into any tobacco product.
For example, the tobacco flavor liquid of the present invention may be incorporated into the liquid container alone in the "heated flavor aspirator", or may be mixed with a solid tobacco flavor source such as tobacco chopped or tobacco granules to form a pod (). It may be incorporated into the pod) or mixed with the liquid of the aerosol source (eg, propylene glycol or glycerin) and incorporated into the liquid compartment. The tobacco flavor liquid of the present invention may be incorporated into the liquid container alone in the "non-heated flavor aspirator", or may be mixed with a solid tobacco flavor source such as tobacco chopped or tobacco granules to form a pod. ) May be incorporated.

<5. Heated flavor aspirator containing tobacco material>
According to another aspect, a heated flavor aspirator containing the tobacco material of the present invention is provided.
According to one embodiment, there is provided a heated flavor aspirator comprising a tobacco flavor source comprising the tobacco material of the present invention and an aerosol source mixed with the tobacco material. An example of such a heated flavor aspirator is shown in FIG.

The heated flavor aspirator 10 shown in FIG. 2 is an electrically heated aspirator that heats a tobacco flavor source by electric heating to generate an aerosol. In the following description, the heated flavor aspirator 10 is simply referred to as a flavor aspirator 10.

The flavor aspirator 10 includes a main body 110 and a mouthpiece 120. The flavor aspirator 10 has a shape extending along the direction in which the main body 110 and the mouthpiece 120 are connected to each other, and has a non-mouthpiece end (end on the main body 110 side) and a mouthpiece end (end on the mouthpiece 120 side). have.

In the following description, when a certain part is referred to as "non-mouthpiece end side", the position specified by this "non-mouthpiece end side" is closer to the non-mouthpiece end side of the flavor aspirator 10 among the parts. The position of the edge. Further, in the following description, when a certain part is referred to as "mouthpiece end side", the position specified by this "mouthpiece end side" is the position of the end of the part closer to the mouthpiece end of the flavor aspirator 10. Is.

The main body 110 includes a tubular body 111, a battery 112, a control circuit 113, and a heater 114.

The tubular body 111 is a bottomed tubular body and is open on the mouthpiece end side so that the tobacco pod 130, which will be described later, can be replaced. The tubular body 111 may be a cylindrical body or a polygonal tubular body. A charging unit (not shown) for charging the battery 112 is provided at the non-suction end of the cylinder 111. Further, a power button (not shown) for turning on / off the power of the flavor aspirator 10 is provided on the side wall of the tubular body 111.

The battery 112 is installed in the cylinder 111. The battery 112 is, for example, a lithium ion secondary battery. The battery 112 supplies the electric power required for the operation of the flavor aspirator 10 to the electric and electronic components included in the flavor aspirator 10. For example, the battery 112 supplies power to the control circuit 113 and the heater 114.

The control circuit 113 is installed in the tubular body 111 between the opening thereof and the battery 112. The control circuit 113 may be installed at another position in the tubular body 111. The control circuit 113 controls the operation of the flavor aspirator 10. Specifically, the control circuit 113 controls the electric power supplied to the heater 114 based on the value output by the temperature sensor installed in the vicinity of the heater 114.

The heater 114 is installed on the suction port end side in the tubular body 111. That is, the heater 114 is installed in the tubular body 111 between the opening thereof and the control circuit 113. The heater 114 has a cup shape capable of accommodating the tobacco pod 130. The heater 114 is electrically connected to the battery 112 and the control circuit 113. The temperature of the heater 114 is controlled by the control circuit 113. The heater 114 is preferably surrounded by an insulator so that its heat is not transferred to the cylinder 111, the battery 112, the control circuit 113, and the like.

The main body 110 may further include, for example, a light emitting element on the side wall of the tubular body 111 for notifying the user of the heating status of the tobacco pod 130 and the remaining amount of the battery 112.

Here, the tobacco pod 130 will be described.
The tobacco pod 130 is installed in the main body 110 so as to be surrounded by the heater 114. The cigarette pod 130 is replaced by the user after a predetermined number of suctions.

The tobacco pod 130 contains a container 131 and a tobacco flavor source 132.
The container 131 is, for example, a container made of metal (for example, aluminum).
The tobacco flavor source 132 is housed in the container 131. The tobacco flavor source 132 contains the tobacco material and aerosol generator of the present invention. The aerosol generating liquid is a liquid for generating an aerosol by heating, and is, for example, propylene glycol, glycerin, or a mixture thereof. The tobacco flavor source 132 may or may not contain a tobacco material other than the tobacco material of the present invention as a tobacco filler.

The tobacco pod 130 is sealed with an aluminum foil lid before being attached to the main body 110. When attached to the main body 110, the tobacco pod 130 is opened so that the tobacco flavor from the tobacco flavor source can be sucked.

The mouthpiece 120 is detachably provided on the mouthpiece end side of the main body 110. The mouthpiece 120 is removed from the main body 110 by the user when the tobacco pod 130 is replaced.

The mouthpiece 120 has a protrusion on the non-mouthpiece end side thereof. When the mouthpiece 120 is attached to the main body 110, the protrusion penetrates the lid of the tobacco pod 130 to open the tobacco pod 130. The mouthpiece 120 does not have to have a protrusion. In this case, the tobacco pod 130 is opened by the user, for example, immediately before being attached to the main body 110.

The mouthpiece 120 has a first gas flow passage that guides the air outside the flavor aspirator 10 to the space inside the tobacco pod 130. The gas inlet of the first gas flow passage is provided, for example, in the vicinity of the connection portion of the mouthpiece 120 with the main body 110. Further, the mouthpiece 120 further has a second gas flow passage that connects the space inside the tobacco pod 130 and the external space of the flavor aspirator 10 so that the user can suck the tobacco flavor from the tobacco flavor source 132. ing. The gas outlet of the second gas flow passage is provided, for example, at the mouthpiece end of the mouthpiece 120.

<6. Heated flavor aspirator containing tobacco flavor liquid>
According to another aspect, a heated flavor aspirator containing the tobacco flavor liquid of the present invention is provided.
According to one embodiment, there is provided a heated flavor aspirator comprising a tobacco flavor source comprising the tobacco flavor liquid of the present invention and an aerosol source mixed with the tobacco flavor liquid. An example of such a heated flavor aspirator is shown in FIG.

The heated flavor aspirator 11 shown in FIG. 3 is an electrically heated aspirator that heats a tobacco flavor source by electric heating to generate an aerosol. In the following description, the heated flavor aspirator 11 is simply referred to as a flavor aspirator 11.

The flavor aspirator 11 has a rod shape or a columnar shape extending from the mouthpiece end 13A to the tip end 14. The flavor aspirator 11 is housed in a housing 12 having a cylindrical shape to form an outer shell, a cylindrical mouthpiece 13, a tip 14 provided on the side opposite to the mouthpiece end 13A of the mouthpiece 13, and a housing 12. A battery 15, an aerosol source 16 housed in a housing 12, a wick 17 connected to the aerosol source 16, a heater 18 made of an electrically resistant metal material wound around the wick 17, and a heater 18. A wiring 21 for connecting the heater 18 and the battery 15, an intake hole 22 provided in the housing 12, a cylindrical ventilation path 23 provided in the center of the housing 12, and a drive for controlling the supply of electric power to the heater 18. The circuit 24 and the like are provided.

The mouthpiece 13 is made of, for example, a metal material such as stainless steel or brass. The housing 12 is formed in a cylindrical shape by, for example, a resin material. The housing 12 has a first portion 12A located on the mouthpiece end 13A side and a second portion 12B located on the tip 14 side. The first portion 12A is made of the same metal material as the mouthpiece 13. The second portion 12B is formed of a resin material having a low specific gravity. As the resin material, for example, polycarbonate, polyacetal, polypropylene, a fluororesin (Teflon (registered trademark)) and the like can be used. The battery 15 constitutes a power source for the flavor aspirator 11.

The battery 15 is composed of, for example, a cylindrical lithium battery, but other batteries may be used. The battery 15 may be a rechargeable battery that can be used repeatedly.

The aerosol source 16 is composed of an absorber such as absorbent cotton or a porous body other than that impregnated with a mixed solution of the tobacco flavor liquid of the present invention and an aerosol generating liquid such as propylene glycol or glycerin. Alternatively, the aerosol source 16 may be composed of a sealable small solution tank filled with a mixed solution of the tobacco flavor liquid of the present invention and an aerosol generating liquid such as propylene glycol or glycerin.

The wick 17 is formed by bundling a plurality of glass fibers (fibers), and can supply (suck) the liquid in the aerosol source 16 to the position of the heater 18 by the capillary force acting between the glass fibers. ..

The heater 18 constitutes a heat source that generates an aerosol by heating the liquid supplied from the aerosol source 16.

At least one or more intake holes 22 are formed at regular intervals along the circumferential direction of the housing 12. Although the intake holes 22 are composed of a plurality of the intake holes 22 in the present embodiment, the intake holes 22 may be composed of one. Each intake hole 22 is composed of small circular holes provided so as to penetrate the housing 12.

The operation of the flavor aspirator 11 will be described. The flavor aspirator 11 is activated by pressing a switch such as a push button provided on the housing 12 or by detecting by a flow rate sensor that a person sucks air from the suction port 13. Alternatively, when the battery 15 is composed of a rechargeable battery, the flavor aspirator 11 may be activated by detecting that the battery 15 has been removed from the charger by a sensing unit provided in the flavor aspirator 11.

When the flavor aspirator 11 is activated, the drive circuit 24 supplies electric power to the heater 18. The method of supplying electric power is arbitrary, and electric power may be intermittently supplied to the heater 18 at regular time intervals, or a certain voltage is applied to the heater 18 after the flavor aspirator 11 is started. You may. Alternatively, a flow meter may be provided in the middle of the ventilation passage 23 to control the electric power to be increased or decreased in proportion to the flow rate of the gas passing through the ventilation passage 23. The liquid supplied from the aerosol source 16 is heated by the heater 18 and mixed with the air supplied from the intake hole 22 to generate an aerosol.

When the user sucks from the suction port 13, air is taken into the housing 12 from the intake hole 22. This air becomes an aerosol containing a tobacco flavor as it passes through the wick 17. This aerosol can be taken into the user's oral cavity to provide the user with a tobacco flavor.

<7. Preferred Embodiment>
The preferred embodiments of the present invention are summarized below.
[1] A first tobacco pulverized product having a β-D-glucosidase activity of 30 [nkat / g] or more, and a β-D-glucosidase activity of 25 [nkat / g] or less containing a glycoside. Production of a tobacco material, comprising mixing with a second crushed leaf tobacco to prepare a leaf tobacco mixture, and storing the leaf tobacco mixture under humidified conditions to obtain a tobacco material with increased tobacco flavor. Method.

[2] The method according to [1], wherein the first crushed leaf tobacco product and the crushed second leaf tobacco product have a maximum diameter of 1 mm or less, preferably 0.5 to 1 mm.
[3] The first crushed leaf tobacco has a β-D-glucosidase activity of 30 to 1000 [nkat / g], preferably a β-D-glucosidase activity of 100 to 1000 [nkat / g] [1]. Or the method according to [2].

[4] The second leaf tobacco pulverized product has a β-D-glucosidase activity of 0 to 25 [nkat / g], preferably a β-D-glucosidase activity of 0 to 15 [nkat / g] [1]. The method according to any one of [3].
[5] The first crushed leaf tobacco is any one of [1] to [4], which is a crushed leaf tobacco of a variety in which the leaves of a tobacco plant are dried by air-curing in a farmhouse. The method described in.

[6] The first crushed leaf tobacco is a crushed leaf tobacco of at least one variety selected from Burley species, native species, dark-colored fire-dried species, dark-colored air-dried species, and dark-colored sun-dried species [1]. ] To [5].
[7] The second leaf tobacco crushed product is dried in a farm of tobacco plant leaves by iron tube drying (flue-curing) involving a heating step or air-circulating curing or air-circulating curing in which heated air is circulated. The method according to any one of [1] to [6], which is a crushed leaf tobacco product of a variety performed by sun-curing.

[8] The second crushed leaf tobacco product is a crushed leaf tobacco product of at least one variety selected from yellow species, orient species, sun-dried species, and light-colored sun-dried species [1] to [7]. The method according to any one.
[9] Any of [1] to [8], wherein the mixing is performed by mixing the first crushed leaf tobacco product and the crushed second leaf tobacco product at a mass ratio of 1:20 to 20: 1. The method according to 1.

[10] The method according to any one of [1] to [9], wherein the leaf tobacco mixture is composed of only the first leaf tobacco crushed product and the second leaf tobacco crushed product.
[11] The method according to any one of [1] to [10], wherein the humidification condition is a condition in which water is added to the leaf tobacco mixture so that the water content of the leaf tobacco mixture is 12 to 80% by mass. ..

[12] The humidification condition is a condition in which water is added to the leaf tobacco mixture so that the water content of the leaf tobacco mixture is 12 to 55% by mass, preferably 12 to 45% by mass [1] to [11]. The method according to any one of.
[13] The method according to any one of [1] to [12], wherein the storage is carried out at a temperature of 0 to 60 ° C., preferably 0 to 50 ° C., more preferably 20 to 50 ° C.

[14] The method according to any one of [1] to [13], wherein the storage is carried out for a period of 24 to 72 hours, preferably 24 to 48 hours.
[15] The method according to any one of [1] to [14], wherein the storage is performed under closed conditions.

[16] The method according to any one of [1] to [15], wherein the storage is performed in an airtight container.
[17] The method according to any one of [1] to [16], wherein the first crushed leaf tobacco product contains a glycoside at a smaller content per unit mass than the crushed leaf tobacco product. Method.

[18] The content of any of the glycoside components contained in the first crushed leaf tobacco is smaller than that of the glycoside component contained in the second crushed leaf tobacco [1]. The method according to any one of [17].
[19] The second crushed leaf tobacco is
The leaf tobacco raw material is heated to inactivate the enzyme contained in the leaf tobacco raw material, and then
The method according to any one of [1] to [18], which is a pulverized product obtained by pulverizing the leaf tobacco raw material.

[20] The leaf tobacco raw material is selected from Burley species, native species, dark-colored fire-dried species, dark-colored air-dried species, dark-colored sun-dried species, yellow species, Orient species, sun-dried species, and light-colored sun-dried species. The method according to [19], wherein the leaf tobacco is at least one variety; preferably a ground leaf tobacco product of at least one variety selected from yellow, Orient, sun-dried, and light-colored sun-dried varieties.
[21] Any one of [1] to [20], wherein the method further comprises drying the tobacco material after the storage so that the water content of the tobacco material is 12 to 14% by mass. The method described in.

[22] A tobacco material produced by the method according to any one of the above [1] to [21].
[23] A method for producing a tobacco flavor liquid, which comprises extracting a tobacco flavor component from the tobacco material according to the above [22] to obtain a tobacco flavor liquid.

[24] The method according to [23], wherein the extraction is carried out by distillation, preferably by steam distillation, hot water distillation, atmospheric distillation or vacuum distillation, more preferably by steam distillation.
[25] A tobacco flavor liquid produced by the method according to the above [23] or [24].

[26] A heated flavor aspirator containing the tobacco material according to the above [22].
[27] A heated flavor aspirator containing the tobacco flavor liquid according to the above [25].

[28] The tobacco material according to the above [22] and
A heated flavor aspirator comprising a tobacco flavor source comprising the tobacco material and an aerosol source mixed with the tobacco material.
[29] The heated flavor aspirator according to [28], further comprising a heating device that heats a mixture of the tobacco material and the aerosol source to generate an aerosol.

[30] The tobacco flavor liquid according to the above [25] and
A heated flavor aspirator comprising a tobacco flavor source containing the tobacco flavor liquid and an aerosol source mixed with the tobacco flavor liquid.
[31] The heated flavor aspirator according to [30], further comprising a heating device that heats a mixture of the tobacco material and the aerosol source to generate an aerosol.

[32] The heating type flavor aspirator heats the tobacco material according to the above [22], an aerosol source for generating an aerosol for passing through the tobacco material, and a heating device for heating the aerosol source to generate an aerosol. The heated flavor aspirator according to [26], which is a heated aspirator provided with.
[33] The heated flavor aspirator heats the refillable tobacco pod containing the mixture of the tobacco material and the aerosol source according to the above [22] and the tobacco pod by electric heating to generate an aerosol. The heated flavor aspirator according to [26], which is an electrically heated aspirator including an aspirator main body.

[34] The heated flavor aspirator includes a first cartridge having an atomizing portion that atomizes an aerosol source by electric power supplied from a battery, and a second cartridge containing the tobacco material according to the above [22]. The heated flavor aspirator according to [26], which is an electrically heated aspirator for imparting a tobacco flavor to the aerosol by passing the aerosol generated in the first cartridge through the second cartridge.
[35] The heated flavor aspirator according to any one of [28] to [34], wherein the aerosol source is propylene glycol, glycerin, or a mixture thereof.

[36] A combustion-type smoking article containing the tobacco material according to the above [22].
[37] A non-heated flavor aspirator containing the tobacco material according to the above [22].
[38] A non-heated flavor aspirator containing the tobacco flavor liquid according to the above [25].

[Example 1: Measurement of β-D-glucosidase activity]
In Example 1, the β-D-glucosidase activity of leaf tobacco was measured. For leaf tobacco, Burley, Yellow, and Orient species were used.

1-1. Preparation of crude enzyme solution Leaf tobacco was pulverized to 1.0 mm mesh or less to obtain a crushed leaf tobacco product. 100 mL of 15 mM McIlvaine buffer (4.8 mM citric acid-10.2 mM disodium hydrogen phosphate buffer) weighed in glass vials and cooled to 4 ° C. , PH 5.4). The suspension was homogenized and then sonicated to extract the enzyme protein for 30 minutes. The extract was filtered through Whatmann # 60 and the filtrate was centrifuged at 12,000 xg for 10 minutes. The supernatant was filtered using a cellulose acetate membrane (Whatmann) having a pore size of 0.2 μm. 60 mL of the filtrate was separated, and low molecular weight components were removed using a 30 kDa ultrafiltration membrane (Amicon Ultra, centrifugal ultrafiltration tube × 4) to obtain a separation solution of high molecular weight components. A 5 mM acetate buffer (pH 5.5) was added to the separation solution of the polymer component to dilute it, and the polymer fraction was washed (removed small molecules) by performing ultrafiltration again. Further, the washing operation of the polymer fraction was repeated twice. The washed separation solution was scalpel-up to 12 mL with an acetate buffer solution adjusted to 5 mM and pH 5.5. This scalpel-up solution is defined as a "crude enzyme solution". All the operations for preparing the crude enzyme solution were carried out at 4 ° C.

The prepared crude enzyme solution is calculated to have a crude polymer product equivalent to 0.1 g of a tobacco raw material dissolved in 1 mL (soluble polymer in 0.1 g of raw material / 1 mL).

1-2. Measurement of Enzyme Activity Five solutions were prepared by mixing 100 μL of the prepared crude enzyme solution and 1000 μL of 10 mM acetate buffer (pH 5.5) in an Eppendorf tube, and each was heated at 45 ° C. for 2 minutes in a heat block. Add 500 μL each of 20 mM 4-nitrophenyl β-D-glucopyranoside (Glc-β-pNP) substrate solution to 4 of the 5 heated mixed solutions, one after 5 minutes and the other. After 15 minutes, another 30 minutes, and the last 60 minutes, 400 μL of 50 mM sodium carbonate solution was added to stop the reaction. The remaining one mixture was incubated, followed by 400 μL of 50 mM sodium carbonate solution and then 500 μL of Glc-β-pNP substrate solution.

The concentration of 4-nitrophenol (pNP) produced in each solution was calculated from the absorbance at a wavelength of 405 nm, and the reaction rate was determined from the change in concentration with respect to the reaction time. For enzyme activity, the amount of enzyme that releases 1 [nmole] of pNP per second is defined as 1 [nkat], and the activity value per weight of the leaf tobacco raw material is finally determined from the enzyme concentration in the reaction solution [nkat / g]. Converted to.

As a specific calculation formula, the reaction rate [nmol / mL / s] x 2.0 [mL (final liquid volume after activity measurement)] ÷ 0.1 [mL (crude enzyme liquid volume)] is used. The value obtained by further dividing the value by the weight of the leaf tobacco raw material [g / mL] was defined as the β-D-glucosidase activity value [nkat / g] per weight of the leaf tobacco raw material.

1-3. Measurement Results The β-D-glucosidase activity measured in Burley, Yellow and Orient species is shown in the table below.

In Table 1, the yellow lot numbers A to D have different countries of origin, the Burley lot numbers E to J have different countries of origin, and the Orient lot numbers K and L have different countries of origin. different.

From the measurement results, it was found that the activity of β-glucosidase, which is a glycoside-degrading enzyme, is higher in Burley species than in Yellow species and Orient species.

[Example 2: Analysis of glycosides]
In Example 2, glycosides contained in leaf tobacco were analyzed. The analysis was performed according to the method described in WO2018 / 038245. Lot numbers D, H, I and J in Table 1 were used for leaf tobacco.

2-1. Preparation of sample for analysis The dried leaf tobacco sample crushed using a mill (Melita Japan Co., Ltd.) was freeze-dried for 3 days. Weigh the freeze-dried raw materials (0.5 g) into screw tubes (volume 20 mL, Maruem), add 20 mL of methanol (Wako Pure Chemical Industries, Ltd., Japan), and add ultrasonic waves (AS ONE, US CLEANER US-). 1R) Extraction was performed for 90 minutes while processing. Subsequently, 100 μL (1 mg / mL methanol) of a solution of n-dodecyl-β-D-glucopyranoside (Sigma-Aldrich Japan) as an internal standard substance was added to each screw tube and shaken. A filtrate containing a glycoside was prepared by filtering using a PTFE membrane (Whatman, 25 mm GD / X Disposable Filter Device) having a pore size of 0.45 μm.

2 mL of each filtrate was dispensed into vials to prepare a sample for analysis. The filtrates obtained from the leaf tobacco of lot numbers D, H, I and J are referred to as analytical samples D, H, I and J, respectively.

2-2. Analysis of glycosides by LC-MS / MS Samples D, H, I and J for analysis were analyzed by LC-MS / MS under the following conditions, respectively.

Equipment Agilent 6410 Triple Quadrupole LC / MS
Chromatography conditions Column: YMC-Pack Pro C18 (YMC Co., Ltd.), inner diameter 2.0 mm x length 150 mm, particle size 3 μm
Injection volume: 5 μL
Flow rate: 0.15 mL / min Analysis time (run time): 60 minutes Elution method: Gradient elution
Eluent A: 1% formic acid, eluent B: acetonitrile gradient conditions: 15% B (0-5 minutes), 15-45% B (5-15 minutes), 45-90% B (15-45 minutes), 90% B (45-60 minutes)
Re-equilibrium time: 20 minutes Column temperature: 40 ° C.
Ion source parameters Ionization method: Electrospray ionization (ESI)
Nebulizer gas: Nitrogen Nebulizer gas temperature: 350 ° C
Nebulizer gas flow rate: 11 L / min Nebulizer pressure: 35 psi
Capillary voltage: 4000V
Mass spectrometer parameters Ion polarity: Positive Fragmentor voltage: 100V
Collision gas: Nitrogen Collision energy: 20V
Measurement mode: Scan MS scan range: m / z 250-500
Scan time: 500ms
Scan method: Constant loss scan Neutral loss mass set value (neutral loss off-set): 162u

Based on the chromatograms obtained from each analytical sample, the peaks derived from glycosides identified by the method described in WO2018 / 308245 were compared. The result is shown in FIG. FIG. 4 shows the analysis results of the glycoside contents of the analytical samples D, H, I and J.

2-3. Analysis Results From the results of FIG. 4, the glycoside content in the crushed leaf tobacco of the yellow species (sample D for analysis) is high, and it is contained in the crushed leaf tobacco of the Burley species (samples H, I and J for analysis). It was found that the glycoside content was low. The results in FIG. 4 show that the crushed leaf tobacco of Burley varieties contains glycosides at a lower content per unit mass than the crushed leaf tobacco varieties of yellow varieties. In addition, the result of FIG. 4 shows that the content of any of the glycoside components contained in the crushed leaf tobacco of Burley species is smaller than that of the glycoside components contained in the crushed leaf tobacco of yellow species. Show that.

[Example 3: Production of tobacco material]
3-1. Method for Producing Tobacco Material 15 g of Burley leaf tobacco was crushed so as to have a maximum diameter of 1 mm or less to prepare a Burley leaf tobacco crushed product. Similarly, 15 g of yellow seed leaf tobacco was crushed so as to have a maximum diameter of 1 mm or less to prepare a crushed yellow seed leaf tobacco product.

The following four types of tobacco materials were produced using the prepared crushed leaf tobacco.

Tobacco material A
A leaf tobacco mixture A was prepared by mixing 1.5 g of a crushed leaf tobacco of Burley and 1.5 g of a crushed leaf of yellow. Water was added to the leaf tobacco mixture so that the water content of the leaf tobacco mixture was 40% by mass, and the leaf tobacco mixture was stirred. As a result, the crushed leaf tobacco was humidified to the extent that its surface was slightly moist. The humidified leaf tobacco mixture was placed in a 20 mL container, covered, and stored at 37 ° C. for 3 days to produce tobacco material A.

Tobacco material B
The enzyme of yellow leaf tobacco crushed product was inactivated as follows. A crushed yellow leaf tobacco is put into a fluidized bed set at an air flow temperature of 220 ° C. or higher, an absolute humidity of 69 to 78 vol%, and a linear velocity of 30 to 34 m / s so that the residence time in the fluidized bed is 2 seconds or less. Was heat-treated to inactivate the enzyme.

A leaf tobacco mixture B was prepared by mixing 1.5 g of a crushed leaf tobacco of Burley seeds and 1.5 g of a crushed leaf tobacco of a heat-treated yellow seed. Tobacco material B was produced according to the same method as that for tobacco material A, except that the leaf tobacco mixture B was used instead of the leaf tobacco mixture A.

Tobacco material C
Tobacco material C was produced according to the same method as that for tobacco material A, except that 3.0 g of crushed Burley leaf tobacco was used instead of the leaf tobacco mixture A.

Tobacco material D
Tobacco material D was produced according to the same method as that for tobacco material A, except that 3.0 g of crushed yellow leaf tobacco was used instead of the leaf tobacco mixture A.

Tobacco material E
The enzymes of the crushed leaf tobacco of Burley seeds were inactivated as follows. Burley leaf tobacco crushed material is put into a fluidized bed set at an air flow temperature of 220 ° C. or higher, an absolute humidity of 69 to 78 vol%, and a linear speed of 30 to 34 m / s so that the residence time in the fluidized bed is 2 seconds or less. Was heat-treated to inactivate the enzyme.

Similarly, the enzyme of yellow leaf tobacco crushed product was inactivated as follows. A crushed yellow leaf tobacco is put into a fluidized bed set at an air flow temperature of 220 ° C. or higher, an absolute humidity of 69 to 78 vol%, and a linear velocity of 30 to 34 m / s so that the residence time in the fluidized bed is 2 seconds or less. Was heat-treated to inactivate the enzyme.

1.5 g of the heat-treated crushed Burley leaf tobacco and 1.5 g of the heat-treated yellow crushed leaf tobacco were mixed to prepare a leaf tobacco mixture E. Tobacco material E was produced according to the same method as that for tobacco material A, except that the leaf tobacco mixture E was used instead of the leaf tobacco mixture A.

3-2. Analysis Results of Glycosides The glycosides contained in the tobacco materials A, B, C, D and E were analyzed by LC-MS / MS as described in Example 2.

FIG. 5 shows the total of the glycoside content of the tobacco material C and the glycoside content of the tobacco material D, and the glycoside content of the tobacco material A. In FIG. 5, the horizontal axis represents the peak number of the glycoside, and the vertical axis represents the peak area per 1 g of the tobacco material.

From the results of FIG. 5, the "glycoside content of tobacco material A" is the sum of the glycoside content of tobacco material C (Burley type) and the glycoside content of tobacco material D (yellow type). It can be seen that there are few compared to. Tobacco material A is a tobacco material produced by mixing and storing Burley and yellow crushed leaf tobacco. In addition, from the results of Examples 1 and 2, the yellow species showed low glycoside-degrading enzyme activity and contained various large amounts of glycosides, and the Burley species showed high glycoside-degrading enzyme activity. It is active and has been demonstrated to contain small amounts of glycosides. Therefore, in the tobacco material A, it can be said that various glycosides contained in the crushed leaf tobacco of the yellow species were decomposed by various glycoside-degrading enzymes contained in the crushed leaf tobacco of the Burley species.

FIG. 6 shows the glycoside content of the tobacco material B and the glycoside content of the tobacco material E. In FIG. 6, the horizontal axis represents the peak number of the glycoside, and the vertical axis represents the peak area per 1 g of the tobacco material.

From the results of FIG. 6, it can be seen that the "glycoside content of the tobacco material B" is smaller than the "glycoside content of the tobacco material E". Tobacco material B is a tobacco material produced by inactivating the enzyme of the crushed leaf tobacco of the yellow species and then mixing and storing the crushed leaf tobacco of the yellow species and the Burley species. On the other hand, tobacco material E is produced by inactivating both the enzyme of crushed yellow leaf tobacco and the enzyme of crushed Burley leaf tobacco, and then mixing and storing the crushed yellow leaf tobacco and Burley crushed leaf tobacco. It is a tobacco material that has been milled. Therefore, in the tobacco material B, it can be said that various glycosides contained in the crushed leaf tobacco of the yellow species were decomposed by various glycoside-degrading enzymes contained in the crushed leaf tobacco of the Burley species.

[Example 4: Production of tobacco flavor liquid]
4-1. Method for Producing Tobacco Material The crushed yellow leaf tobacco was heat-treated under the same conditions as the heat treatment conditions used in the production of "Tobacco Material B" of Example 3 to inactivate the enzyme. Tobacco material F was produced according to the same method as in the production of "Tobacco material A" in Example 3 except that 3.0 g of the heat-treated yellow varieties of crushed leaf tobacco was used instead of the leaf tobacco mixture A.

4-2. Manufacturing method of tobacco flavor liquid (1) Steam distillation Put 1 L of water in a steam distillation device (Tokyo Seisakusho herb oil maker (standard type)) that cleaned the inside for about 1 hour with water and heat it with a heater (250 ° C). )did. After boiling, any (60 g) of the tobacco materials B and C prepared in Example 3 and the tobacco material F prepared in Example 4 was added and distillation was started. Distillation was continued and 500 mL of fraction was collected by distillation for 2 hours. The obtained fraction was collected in an Erlenmeyer flask and left in an ice bath (5 ° C.) for 2 hours.

(2) Extraction of Distillate with Organic Solvent Diethyl ether was used as the organic solvent.
15 g of sodium chloride was added to the Erlenmeyer flask containing the distillate and shaken. Next, 500 mL of the distillate (including the oil floating in the distillate) was placed in a 1 L separatory funnel, 150 mL of an organic solvent was added, and the mixture was shaken. After removing the aqueous phase, the organic phase was placed in a new Erlenmeyer flask. The operation of newly adding 150 mL of the organic solvent to the aqueous phase and recovering the organic phase was repeated twice. 20 g of anhydrous sodium sulfate was added to the Erlenmeyer flask from which the organic phase was recovered, and the mixture was dehydrated by leaving it at room temperature for 30 minutes.

(3) Removal of Organic Solvent from Organic Phase The organic phase after dehydration is filtered through filter paper (ADVANTEC, No. 2,150 mm), and the filtrate is evaporated under reduced pressure in a hot water bath at 35 ° C. by a rotary evaporator. The mixture was dried to obtain a tobacco extract as a dry matter. Propylene glycol having a weight 50 times the weight of the extract was added to obtain a "tobacco flavor liquid". Tobacco flavor liquids produced from tobacco materials B, C and F are referred to as tobacco flavor liquids B, C and F, respectively.

4-3. Analysis of Tobacco Flavor Liquids The above tobacco flavor liquids B, C and F were analyzed by GC / MS.
As for the GC / MS conditions, the following conditions can be used.

Equipment: Agilent gas chromatogram mass detector Column: Agilent HP-1MS (30m x 0.25mm (film thickness 0.25μm))
Injection volume: 1 μL
Flow velocity (Septum Page Flow): 1.3 mL / min Analysis time (run time): 52 minutes Heating conditions: 40 ° C (4 minutes) → 10 ° C / min, 150 ° C → 15 ° C / min, 190 ° C
→ 15 ° C / min, 240 ° C → 15 ° C / min, 250 ° C (30 minutes)
[MS condition]
Equipment: Agilent 5975C Inert XL MSD
Detection mode: SCAN
Ion source temperature: 230 ° C
Quadrupole temperature: 150 ° C

4-4. Results of Flavor Component Analysis of Tobacco Flavor Liquid FIG. 7 is a GC-MS chromatogram when tobacco flavor liquids C and F are mixed (mixed at a ratio of 1: 1). FIG. 8 is a GC-MS chromatogram of tobacco flavor liquid B. From the results of FIGS. 7 and 8, it can be seen that the tobacco flavor liquid B has more peaks of flavor components and has a rich flavor as compared with the mixed liquid of the tobacco flavor liquids C and F. Therefore, from the results of FIGS. 7 and 8, it can be said that the tobacco flavor liquid having an increased flavor can be provided by carrying out the method of the present invention.

[Example 5: Effect of temperature during storage on the effect]
5-1. Method for Producing Tobacco Material A leaf tobacco mixture was prepared by mixing 1.5 g of crushed leaf tobacco of Burley seeds and 1.5 g of crushed leaf tobacco of yellow seeds. Water was added to the leaf tobacco mixture so that the water content of the leaf tobacco mixture was 40% by mass, and the leaf tobacco mixture was stirred. As a result, the crushed leaf tobacco was humidified to the extent that its surface was slightly moist. The humidified leaf tobacco mixture was placed in a 20 mL container and sealed, and stored at a temperature of 27 ° C., 37 ° C., 47 ° C., or 57 ° C. for 3 days to prepare a tobacco material.

5-2. Analysis Results of Glycosides Glycosides contained in each tobacco material having different temperatures during storage were analyzed by LC-MS / MS as described in Example 2. FIG. 9 shows the glycoside content at each storage temperature. In FIG. 9, as a reference value, the total value of the glycoside content of the tobacco material C of Example 3 and the glycoside content of the tobacco material D of Example 3 is shown.

From the results of FIG. 9, it can be seen that when stored at 57 ° C., the glycoside content is higher and the glycoside decomposition amount is lower than when stored at a lower temperature. It is considered that this is because the activity of the glycoside-degrading enzyme decreased due to the exposure of the leaf tobacco mixture to the higher temperature. From this result, it can be seen that the desirable temperature range for glycoside decomposition is a temperature of 20 ° C to 50 ° C.

[Example 6: Effect of storage period on effect]
6-1. Method for Producing Tobacco Material A leaf tobacco mixture was prepared by mixing 1.5 g of crushed leaf tobacco of Burley seeds and 1.5 g of crushed leaf tobacco of yellow seeds. Water was added to the leaf tobacco mixture so that the water content of the leaf tobacco mixture was 40% by mass, and the leaf tobacco mixture was stirred. As a result, the crushed leaf tobacco was humidified to the extent that its surface was slightly moist. The humidified leaf tobacco mixture was placed in a 20 mL container and sealed, and stored at 37 ° C. for 1, 2, or 3 days to produce a tobacco material.

6-2. Analysis Results of Glycosides Glycosides contained in each tobacco material having different storage periods were analyzed by LC-MS / MS as described in Example 2. FIG. 10 shows the glycoside content when the storage period is changed. In FIG. 10, as a reference value, the total value of the glycoside content of the tobacco material C of Example 3 and the glycoside content of the tobacco material D of Example 3 is shown.

From the result of FIG. 10, it can be seen that about 50% of the glycoside content of the reference value is decomposed in one day of storage. On the 2nd day of storage, the decomposition of glycosides further progressed. In addition, the glycoside content on the third day of storage was not significantly different from the glycoside content on the second day of storage. From this result, it can be seen that the desirable storage period for glycoside decomposition is 24 hours to 48 hours.

[Example 7: Effect of the amount of water in the storage on the effect]
7-1. Method for Producing Tobacco Material A leaf tobacco mixture was prepared by mixing 1.5 g of crushed leaf tobacco of Burley seeds and 1.5 g of crushed leaf tobacco of yellow seeds. Water was added to the leaf tobacco mixture so that the water content of the leaf tobacco mixture was 12% by mass, 22% by mass, 26% by mass, 30% by mass, 40% by mass, 45% by mass, 50% by mass, or 55% by mass. The leaf tobacco mixture was stirred. The humidified leaf tobacco mixture was placed in a 20 mL container, covered, and stored at 37 ° C. for 3 days to produce a tobacco material.

7-2. Analysis Results of Glycosides Glycosides contained in each tobacco material having different water content in storage were analyzed by LC-MS / MS as described in Example 2. 11 and 12 show the glycoside content when the amount of water in the storage is changed. 11 and 12 show the total value of the glycoside content of the tobacco material C of Example 3 and the glycoside content of the tobacco material D of Example 3 as reference values.

From the results of FIGS. 11 and 12, it can be seen that the glycosides are decomposed at any water content. The glycoside content when the water content was 50% by mass and 55% by mass was almost the same as the glycoside content when the water content was 45% by mass. From this result, it can be seen that the water content in the storage (that is, the water content of the leaf tobacco mixture) desirable for glycoside decomposition is 12% to 45% by mass.

Claims (14)

A first tobacco pulverized product having a β-D-glucosidase activity of 30 [nkat / g] or more, and a second leaf tobacco pulverized product containing a glycoside and having a β-D-glucosidase activity of 25 [nkat / g] or less. A method for producing a tobacco material, which comprises mixing with a pulverized leaf tobacco product to prepare a leaf tobacco mixture, and storing the leaf tobacco mixture under humidified conditions to obtain a tobacco material having an increased tobacco flavor. The method according to claim 1, wherein the first crushed leaf tobacco product and the second crushed leaf tobacco product have a maximum diameter of 1 mm or less. The method according to claim 1 or 2, wherein the humidification condition is a condition in which water is added to the leaf tobacco mixture so that the water content of the leaf tobacco mixture is 12 to 80% by mass. The method according to any one of claims 1 to 3, wherein the storage is performed at a temperature of 0 to 60 ° C. The method according to any one of claims 1 to 4, wherein the storage is carried out over a period of 24 to 72 hours. The method according to any one of claims 1 to 5, wherein the storage is performed under closed conditions. The method according to any one of claims 1 to 6, wherein the first crushed leaf tobacco contains glycosides in a smaller content per unit mass than the crushed second leaf tobacco. The seventh aspect of claim 7, wherein the content of any of the glycoside components contained in the first crushed leaf tobacco is smaller than that of the glycoside component contained in the second crushed leaf tobacco. Method. The second crushed leaf tobacco
The leaf tobacco raw material is heated to inactivate the enzyme contained in the leaf tobacco raw material, and then
The method according to any one of claims 1 to 8, which is a pulverized product obtained by pulverizing the leaf tobacco raw material. A tobacco material produced by the method according to any one of claims 1 to 9. A method for producing a tobacco flavor liquid, which comprises extracting a tobacco flavor component from the tobacco material according to claim 10 to obtain a tobacco flavor liquid. A tobacco flavoring liquid produced by the method according to claim 11. A heated flavor aspirator containing the tobacco material according to claim 10 or the tobacco flavor liquid according to claim 12. The tobacco material according to claim 10 and
A heated flavor aspirator comprising a tobacco flavor source comprising the tobacco material and an aerosol source mixed with the tobacco material.

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