A method for processing tobacco material and the processed tobacco material

Abstract

This invention relates to a method for processing tobacco material, the method comprising: ο fermenting the tobacco material to obtain processed tobacco material, comprising: # culturing the tobacco material under anaerobic conditions; # applying a pressure between 1000 kg / m² and 4000 kg / m² to the tobacco material; # maintaining the moisture content of the tobacco material between 25% by weight and 40% by weight of the total weight of the tobacco material; ο wherein the fermentation lasts for at least one month.

Description

[0001] This invention relates to a method for treating tobacco by fermentation and to a fermented tobacco product. In particular, the fermentation is anaerobic fermentation.

[0002] Various treatment methods and additives for altering the overall characteristics or properties of tobacco materials used in tobacco products have been proposed in this art. For example, tobacco materials have been treated with additives. Furthermore, the treatment conditions used in the processing of these tobacco materials have been controlled to alter the chemical or sensory properties of tobacco products derived from such tobacco materials, as well as the chemical or sensory properties of the mainstream smoke or aerosol generated from smoking articles incorporating such tobacco materials.

[0003] Later stages of tobacco processing, such as enhancing tobacco materials or adding flavor and aroma, often involve adding one or more additives and may require additional processing steps and equipment, which can be both expensive and time-consuming. Furthermore, some consumers may not be very perceptible to the additives being added to tobacco.

[0004] Therefore, it is necessary to modify the sensory properties of tobacco materials without involving the addition of external flavorings to the tobacco itself. Furthermore, there is a need for tobacco materials that exhibit such distinct sensory qualities without requiring complex processing.

[0005] According to one aspect, the present invention relates to a method for processing tobacco material, the method comprising fermenting the tobacco material. Preferably, the fermentation step comprises: culturing the tobacco material under anaerobic conditions. Preferably, the fermentation step comprises: applying a pressure between 1000 kg / m² and 4000 kg / m² to the tobacco material. Preferably, the fermentation step comprises: maintaining the moisture content of the tobacco material between 25% and 40% by weight of the total weight of the tobacco material. Preferably, the fermentation step lasts for at least one month.

[0006] According to the method of the present invention, tobacco fermentation is carried out under protected conditions. Due to fermentation, certain chemical compounds present in the tobacco material may change, and consequently, the sensory properties of the tobacco material may also differ. Furthermore, tobacco material fermented according to the method of the present invention may exhibit lower levels of asparagine.

[0007] It is known that tobacco material can be fermented. Tobacco plants may contain microorganisms, which may include bacteria, molds, and actinomycetes. Studies have shown that the majority of microorganisms present in tobacco are bacteria, while molds and actinomycetes are in the minority. Yeast is rarely or not detected at all. Fermented tobacco can be prepared using various suitable techniques known in the art, such as “Research Progress in Tobacco Fermentation”, Yang Yang et al., Journal of Biosciences and Medicines 2018, 6, 105-114, online address: http: / / www.scirp.org / journal / jbm Or as described in US 5372149 or US 4528993, etc. Typically, tobacco fermentation involves adjusting the moisture content of aged tobacco to approximately 20% to approximately 60% and allowing the moist tobacco to pile and ferment. Fermentation can be terminated, for example, by drying or refrigeration. Tobacco fermentation does not require the addition of microorganisms because, as mentioned above, microorganisms are normally present naturally in the tobacco plant.

[0008] Unlike those known in the art, in this invention, fermentation is carried out under anaerobic conditions.

[0009] Anaerobic fermentation is defined as the conversion of complex organic compounds into smaller molecules in the absence of oxygen. The term can also be defined as a condition where oxygen is unavailable for redox reactions due to both chemical equilibrium and biochemical reactivity. Instead, other compounds may be present that can be oxidized by microorganisms for specific types of energy metabolism.

[0010] Anaerobic conditions can coexist with aerobic conditions: microorganisms in the microenvironment (such as aggregates of debris suspended in water) may not be able to obtain oxygen in gaseous form, but at the same time it may be present in the macroenvironment (water).

[0011] In anaerobic tobacco fermentation, without being bound by theory, the main energy extraction pathway is likely to be glycolysis, and some amino acids are also used as carbon / nitrogen sources. Preferred nitrogen-containing compounds typically include glutamine, alanine, serine, threonine, aspartate, asparagine, urea, and arginine.

[0012] Preferably, anaerobic conditions are achieved by placing tobacco material in a container and sealing the container. Preferably, anaerobic conditions are achieved by placing tobacco material in a container, removing air from the container, and sealing the container in an airtight manner.

[0013] More preferably, pressure is applied to remove air from the tobacco material. The applied pressure "squeezes" air out of the tobacco material, so that after the container is sealed, there is no oxygen left in the container, or only a very small amount of oxygen remains.

[0014] The tobacco material is placed in a container and the container is sealed. After the air is removed from the sealed container, anaerobic conditions are rapidly achieved. This method of achieving anaerobic conditions is preferred because it is cost-effective and easy to implement.

[0015] Alternatively, place the tobacco material in a container, remove the air, and replace it with water.

[0016] The container for holding the tobacco material is, for example, a barrel. Preferably, the barrel is made of wood, or concrete, or metal, or any combination of these three materials.

[0017] To maintain the anaerobic conditions for the required fermentation duration.

[0018] To achieve the desired fermentation according to the invention, the tobacco material preferably has a moisture content of between 25% and 40% by weight of the total weight of the tobacco material. More preferably, the tobacco material has a moisture content of between 25% and 35% by weight of the total weight of the tobacco material. More preferably, the tobacco material has a moisture content of between 28% and 32% by weight of the total weight of the tobacco material. More preferably, the tobacco material has a moisture content of 30% by weight of the total weight of the tobacco material. Preferably, to achieve this moisture content, the tobacco material is moistened with water. Water is added to the tobacco material. Preferably, the tobacco material is moistened before being introduced into a container in which anaerobic conditions are obtained and maintained.

[0019] Furthermore, this moisture content is maintained during the fermentation process. Therefore, it is preferable to monitor the moisture content of the tobacco material during fermentation. For example, if the tobacco material is introduced into a fermentation container, the container can be opened, and the moisture content of the tobacco material can be measured when the container is opened. Preferably, the container is opened at regular time intervals to measure the tobacco moisture content.

[0020] Moisture content can be measured by a moisture sensor located inside the container. This allows for moisture measurement even when the tobacco material is in a closed container.

[0021] Preferably, the moisture content of the tobacco material is measured at regular time intervals.

[0022] During fermentation, the tobacco material is subjected to pressure. The preferred pressure is 1000 kg / m². 2 Up to 4000 kg / m² 2 )between.

[0023] During fermentation, the pressure applied to the tobacco material is maintained within the aforementioned range.

[0024] Pressure can be applied to tobacco material by any means. Pressure can be applied by pumping inert gas into a container. Pressure can also be applied by placing a weight on the tobacco material, thus applying the desired pressure range. For example, a container can be filled with wet tobacco material, and a weight can be placed in contact with the tobacco material as a "lid" until water seeps out of the container.

[0025] Preferably, the tobacco material is inserted into the container and the weight is positioned on top of or above the tobacco material to apply the desired pressure. Preferably, the container is then closed, leaving the weight inside so that the weight can continue to apply pressure to the tobacco material.

[0026] In the following text, the term "fermentation conditions" refers to all three conditions: anaerobic conditions, a moisture content in the tobacco material between 25% and 40% by weight of the total weight of the tobacco material, and an applied pressure between 1000 kg / m² and 4000 kg / m². Therefore, stating that tobacco material undergoes fermentation conditions means that the tobacco raw material undergoes anaerobic conditions, a moisture content in the tobacco material between 25% and 40% by weight of the total weight of the tobacco material, and an applied pressure between 1000 kg / m² and 4000 kg / m².

[0027] The tobacco material is subjected to the above fermentation conditions for at least one month. Preferably, the tobacco material is subjected to fermentation conditions for at least two months. Preferably, the tobacco material is subjected to fermentation conditions for at least six months. Preferably, the tobacco material is subjected to fermentation conditions for at least 12 months. Preferably, the tobacco material is subjected to fermentation conditions for at least 24 months. Preferably, the tobacco material is subjected to fermentation conditions for less than 36 months.

[0028] Fermentation conditions can be applied continuously for all claimed periods (e.g., more than one month, or more than two months, or more than six months, or more than 12 months, or more than 24 months). Alternatively, fermentation conditions can be applied at multiple time intervals, forming a series of time intervals. These time intervals are separated from each other by “interruptions.” For example, one or more of the following interruptions may occur: the presence of anaerobic conditions, a moisture content between 25% and 40% by weight of the total weight of the tobacco material, and an applied pressure between 1000 kg / m² and 4000 kg / m². Interruptions can be made to examine the tobacco material. For example, the moisture content of the tobacco material can be measured during an interruption. Interruptions can be made to rotate or mix the tobacco material to obtain a homogeneously treated tobacco material.

[0029] The disruption could last up to 6 hours.

[0030] Therefore, the total time period during which the tobacco is subjected to the aforementioned fermentation conditions should be calculated by adding up the durations of all time intervals during which the fermentation conditions were actually applied. Alternatively, the total time period can be calculated from the moment the fermentation conditions were first applied to the moment they were last applied, and then the duration of any interruptions can be "removed".

[0031] For example, if fermentation time T is selected, where "fermentation time" refers to the total time period during which the tobacco material is subjected to fermentation conditions, the following is possible.

[0032] Fermentation conditions are applied continuously for a total duration equal to T.

[0033] Fermentation conditions are applied for N fermentation time intervals t1, t2, ..., t N , where t1+t2+…+t N =T. Fermentation time interval t j With the subsequent time interval t j+1 The time interval between them is called an interruption.

[0034] The total time period T is at least one month, or at least two months, or at least six months, or at least 12 months, or at least 24 months.

[0035] Preferably, there is an interruption between two consecutive time intervals of applying fermentation conditions. The interruption lasts no more than 6 hours.

[0036] Fermentation conditions are applied to tobacco material for at least one month to observe the desired chemical modifications. For example, fermentation conditions may be applied until a desired amount of one or more chemical substances is reached in the tobacco material. For example, the chemical substances contained in the tobacco material may decrease or increase due to the fermentation conditions. Therefore, the fermentation conditions are stopped when the desired amount of chemical substances is reached. For example, fermentation conditions may be applied until the desired color of the tobacco material is obtained.

[0037] When tobacco materials are under fermentation conditions, anaerobic fermentation occurs.

[0038] During fermentation (when fermentation conditions are applied), the temperature of the tobacco material can be maintained between 25°C and 35°C, more preferably between 27°C and 31°C. Throughout the fermentation process (when fermentation conditions are applied), the temperature of the tobacco material remains essentially within this range. The temperature is maintained by the fermentation itself; no heat needs to be supplied to or deducted from the tobacco material. This temperature of the tobacco material will be achieved during fermentation when the ambient temperature of the environment in which the tobacco material is located is preferably between 15°C and 25°C.

[0039] During fermentation under the specified conditions, the levels of reducing sugars and free amino acids present in the tobacco material were monitored. The most abundant naturally occurring sugars in tobacco leaves are glucose, fructose, and sucrose. Differences in sugar content may exist between tobacco varieties. For example, Virginia tobacco has a high sugar content (typically ranging from 8% to 30%), while Burley tobacco is characterized by a low sugar content (typically ranging from 1% to 2%). However, regardless of the type of tobacco used in the tobacco material, a decrease in reducing sugar content has been observed during fermentation under the conditions of this invention.

[0040] Changes in the amount of reducing sugars can alter the sensory properties of tobacco materials and the smoke or aerosols produced from them.

[0041] Furthermore, tobacco materials contain certain levels of amino acids. Amino acids may significantly contribute to the levels of certain components in the smoke or aerosol produced from the final product containing fermented tobacco materials, as well as to the sensory properties of said smoke or aerosol. Different types of tobacco may contain different amounts of amino acids. Moreover, the distribution of amino acids may differ between tobacco leaves, tips, or stems, primarily in quantitative terms. Additionally, the growth location of tobacco can alter the ratio of different amino acid levels, but the same tobacco typically maintains a fairly similar distribution of amino acids. Regardless of tobacco type and origin, a decrease in asparagine content in tobacco materials has been observed during fermentation under the fermentation conditions of this invention.

[0042] This indicates that the fermenting bacteria in the fermentation process of the present invention produce specific asparaginases to absorb C and N from amino acid resources.

[0043] Asparagine can be thermally converted into acrylamide. Acrylamide is considered a potentially harmful substance. Reducing the asparagine content in tobacco materials is desirable because it may simultaneously reduce acrylamide formation.

[0044] The aforementioned changes in the amount of asparagine and reducing sugars in the tobacco material can be observed one month after the start of the fermentation process. The fermentation conditions are applied to the tobacco material and maintained for at least one month.

[0045] In the method of this invention, anaerobic fermentation of the tobacco material occurs when the tobacco material is subjected to fermentation conditions. This anaerobic fermentation alters the amount of reducing sugars and asparagine in the tobacco material. Therefore, the reduction of certain potentially harmful substances can be achieved through natural processes such as fermentation without the addition of additives or external microorganisms to the tobacco material. These substances may include acrylamide. Additionally, fermentation can alter the sensory properties of the tobacco material. These sensory changes may occur because reducing sugars are converted into pyruvate and pyruvate, which are precursors to many other flavor compounds. This means that the sensory properties of the tobacco material may be significantly altered after fermentation according to this invention. The flavor characteristics of the tobacco material may change compared to the same tobacco material after conventional ripening without the fermentation conditions according to this invention.

[0046] As used herein, the terms “altered” or “altered” are used in the context of flavor or sensory properties to indicate a change from one overall taste or sensory characteristic to another, as identified by a professional smoker. This can include improvements.

[0047] Preferably, the method includes drying the tobacco material to obtain a dried tobacco material with a moisture content between 1% and 15% by weight of the total weight of the tobacco material. The drying step is preferably carried out after fermentation under fermentation conditions has ceased. After the total fermentation time T has elapsed, the treated tobacco material is preferably removed from the container where it is held, and the pressure applied to the tobacco is reduced. The treated tobacco material is then dried to a moisture content between 1% and 15% by weight of the total weight of the tobacco material, more preferably between 5% and 10%. Drying facilitates the processing of the treated tobacco material in subsequent steps.

[0048] Preferably, the method includes the step of maturing the tobacco material prior to fermentation. The tobacco material processed according to the method of the invention may contain maturated tobacco. As used herein, the term "matured tobacco" refers to tobacco that has already been maturated. The maturation of the tobacco is preferably carried out according to a standard procedure and may depend on the type of tobacco contained in the tobacco material. The tobacco material may include different types of tobacco that have already undergone different maturations. Different types of tobacco may be blended and then processed according to the invention.

[0049] Alternatively, the tobacco material processed according to the method of the invention may comprise tobacco that has been re-graded, blended with green leaves, conditioned, destemmed or threshed (or not in the case of whole leaves), dried or packaged.

[0050] Preferably, the method involves maintaining the temperature of the tobacco material between 25°C and 35°C. Maintaining the temperature of the tobacco material between 25°C and 35°C while subjecting it to fermentation conditions. The temperature of the tobacco material is automatically maintained within this range by the fermentation process. No additional equipment is required to cool or heat the tobacco material.

[0051] Preferably, the method includes the step of rotating the tobacco material. Rotating the tobacco material can provide improved homogenization. Rotating the tobacco material may mean inverting it. Rotating the tobacco material may mean flipping it. The interruption of fermentation conditions caused by rotation can also be used to measure certain parameters of the tobacco material, such as moisture content. During the rotation of the tobacco material, the fermentation conditions may no longer be applied. During rotation, all three fermentation conditions may not be applied, or only some of them may be applied. The fermentation process is thus "interrupted". After rotation, it is preferable to reapply the fermentation conditions to the tobacco material.

[0052] Preferably, the method includes: securing the tobacco material within a moisture-retaining material. This step of securing the tobacco material is preferably performed before the tobacco material is subjected to fermentation conditions. It is desired that the moisture-retaining material resists degradation during the tobacco processing (fermentation). The moisture-retaining material may include a flexible material. This flexible material may wrap around the tobacco material. The moisture-retaining material preferably includes a plastic material. Alternatively, the moisture-retaining material may include a rigid material. The container in which the tobacco material is introduced can act as the moisture-retaining material. In this case, the material of the container may include, for example, metal, wood, plastic, or concrete.

[0053] Preferably, the method includes wetting the tobacco material in water before fermentation to obtain a moisture content of 25% to 40% of the total weight of the tobacco material. After curing, the moisture content of the tobacco material is typically low. Therefore, it is preferable to add water to the tobacco material to achieve a moisture level of 25% to 35% by weight. More preferably, water is also added during the fermentation process to maintain the moisture content of the tobacco material at 25% to 40% of the total weight of the tobacco material for at least one month, more preferably at least two months, more preferably at least six months, more preferably at least twelve months, and more preferably at least 24 months.

[0054] According to another aspect, the present invention relates to tobacco material treated according to the method of the foregoing aspect, wherein the treated tobacco material contains an amount of asparagine that is at least 50%, more preferably 60%, and even more preferably 80% lower than that contained in the same tobacco material before treatment according to the foregoing aspect. Preferably, at the end of fermentation, the amount of asparagine is at least 50%, more preferably 60%, and even more preferably 80% lower than that contained in the same tobacco material before treatment. The treated tobacco according to the method of the present invention can alter its chemical composition relative to untreated tobacco. In this context, "treated tobacco material" means tobacco material that has undergone the treatment described in the preceding process, i.e., tobacco material that has been subjected to fermentation conditions for at least one month. In this context, "untreated tobacco material" means tobacco material that has not undergone the treatment described in the preceding process, i.e., tobacco material that has not been subjected to fermentation conditions. Untreated tobacco material is, for example, tobacco material inserted into a container before the start of the treatment of the present invention. The treated tobacco material is compared with the same tobacco material that has not undergone the treatment according to the present invention (untreated tobacco material). The reduction of asparagine may be associated with an increase in aspartate. This indicates that fermenting bacteria produce specific asparaginases to absorb C and N from amino acid resources. This reaction can produce ammonia.

[0055] Preferably, the treated tobacco material contains at least 50%, more preferably 60%, and even more preferably 80% less glutamine than the same tobacco material before treatment according to the methods described above. Preferably, at the end of fermentation, the amount of glutamine is at least 50%, more preferably 60%, and even more preferably 80% less than the amount of glutamine in the same tobacco material before treatment. The treated tobacco material according to the method of the invention can alter its chemical composition relative to untreated tobacco material. Comparing the treated tobacco material with the same tobacco material that has not undergone treatment according to the invention, the reduction in glutamine may be associated with an increase in glutamate. This indicates that the fermenting bacteria produce specific glutaminases to absorb C and N from amino acid resources. This reaction can produce ammonia.

[0056] Preferably, the treated tobacco material contains at least 50%, more preferably 60%, and even more preferably 85% less total reducing sugar than the same tobacco material before treatment according to the methods described above. Preferably, at the end of fermentation, the amount of reducing sugar is at least 50%, more preferably 60%, and even more preferably 85% less than the same tobacco material before treatment. The reducing sugar is the sum of glucose, fructose, sucrose, and maltose. Most of the reducing sugar in the treated tobacco material can be converted. Reducing sugar resources present in the starting tobacco material, such as glucose and fructose, can be used as an energy source by anaerobic bacteria. Under anaerobic conditions, glycolysis converts glucose (or fructose) into pyruvate. The altered levels of these compounds may contribute to the desired flavor and aroma of the treated tobacco material.

[0057] Preferably, the treated tobacco material is at least 100 times more acidic than the untreated tobacco material. The pH of the treated tobacco material may differ from that of the untreated tobacco material by at least 2 pH units. The pH can remain substantially constant across different tobacco materials.

[0058] Preferably, the treated tobacco contains lactic acid. Lactic acid is known to be a relevant catabolite in anaerobic fermentation. Lactic acid may have a "mitigating" effect on the stimulant effect of nicotine. Lactic acid may cause a decrease in the pH of the treated tobacco material.

[0059] According to another aspect, the present invention relates to a tobacco material comprising: less than 3% total reducing sugar by total dry weight. More preferably, the tobacco material comprises less than 2% total reducing sugar by total dry weight. Even more preferably, the tobacco material comprises less than 1% total reducing sugar by total dry weight. Preferably, the tobacco material comprises less than 300 mg / kg of asparagine by total dry weight. The tobacco material is fermented tobacco material. The tobacco material is preferably tobacco material treated according to the method of the present invention.

[0060] The advantages of the tobacco material of the present invention have been summarized with reference to the foregoing aspects and will not be repeated here.

[0061] Fermentation can be achieved without adding any microorganisms other than those already present in the tobacco material before fermentation.

[0062] Preferably, the tobacco material contains less than 70 mg / kg of glutamine based on total dry weight.

[0063] Preferably, the tobacco material contains more than 10,000 mg / kg of total free amino acids based on total dry weight.

[0064] Preferably, the tobacco material comprises hand-peeled leaves from which the leaf ribs have been removed.

[0065] Preferably, the tobacco material is cured. Preferably, curing is carried out before fermentation.

[0066] Preferably, the tobacco material comprises Kasturi tobacco.

[0067] According to one different aspect, the present invention relates to an aerosol-generating article comprising tobacco material according to the foregoing aspect.

[0068] The term "tobacco material" refers to any part of a tobacco plant or a mixture of different tobacco plants, including but not limited to loose tobacco leaves, green tobacco leaves, tobacco stems, tobacco dust produced during tobacco processing, and nascent tobacco leaves and combinations thereof. Tobacco material may be in the form of processed tobacco parts or blocks, cured and aged tobacco in the form of substantially natural tobacco leaves or stems, tobacco extracts, or mixtures thereof, for example, a mixture of extracted tobacco pulp with granulated cured and aged natural tobacco leaves. Tobacco material may be in solid, liquid, semi-solid, etc. Preferably, the term "tobacco material" includes any part of any member of the genus *Nicotiana* and any associated byproducts, such as leaves or stems. The tobacco material used in this invention is preferably from the species *Nicotiana tabacum*. Any type, style, or variety of tobacco can be processed. Examples of tobacco that can be used include, but are not limited to, Virginia tobacco, Burley tobacco, and Oriental tobacco, and any blends of these types. Preferably, the tobacco material comprises *Kasturi* tobacco. The tobacco material to be processed may comprise cured tobacco or consist of cured tobacco.

[0069] As used herein, the term "cured tobacco" refers to tobacco that has been cured but has not undergone any further processing to alter the flavor or aroma of the tobacco material. Cured tobacco may have been blended with other styles, varieties, or types of tobacco. Alternatively, the tobacco material to be processed may comprise or consist of tobacco that has been re-graded, blended with green leaves, conditioned, destemmed or threshed (or not in the case of whole leaves), dried, or packaged.

[0070] Preferably, the tobacco material comprises sheet tobacco material. The tobacco may contain between about 70% and 100% sheet tobacco material.

[0071] When the tobacco material comprises sheet tobacco, the sheet tobacco can be in whole leaf form. In some embodiments, the tobacco material comprises cured whole leaves of tobacco. In some embodiments, the tobacco material substantially comprises cured whole leaves of tobacco. In some embodiments, the tobacco material consists essentially of cured whole leaves of tobacco.

[0072] In some implementations, the tobacco material comprises stem tobacco material. The tobacco may contain up to 30% stem material.

[0073] The method of "curing" green tobacco depends on the type of tobacco harvested. For example, Virginia Bread (bright) tobacco is typically cured in the flue, while Burley and certain darker varieties are often cured in the air. Flue curing of tobacco usually takes five to seven days, while air curing takes one to two months. Many major chemical and biochemical changes begin during the curing process and continue throughout the early stages of leaf drying. The color change of tobacco from yellow to brown often leads to the formation and accumulation of nitrosamines, as well as an increase in microbial content.

[0074] Different types of curing are used for different types of tobacco.

[0075] Virginia tobacco is typically cured in a curing chamber. The tobacco leaves are suspended in a curing chamber where heated air is generated to dry them. As the leaves lose moisture, they develop their distinctive aroma, texture, and color. Farmers must carefully guide this process, which can take up to a week, during which the temperature of the heated air must be constantly monitored and gradually increased. Too much or too little heat at any stage of the process will negatively impact the quality of the tobacco.

[0076] Burley tobacco and Oriental tobacco are aged differently. Burley tobacco undergoes "air aging" in an aging chamber where heat and humidity are supplied by natural ventilation. The aging process takes up to two months. Oriental tobacco undergoes "sun aging" by hanging the leaves outdoors in sunlight for about two weeks.

[0077] In this text, the verbs "comprise" and "include" are synonyms, and both refer to a non-exhaustive list of features. The verb "compose of" indicates an exhaustive list.

[0078] The invention is defined in the claims. However, a non-exhaustive list of non-limiting embodiments is provided below. Any one or more features of these embodiments may be combined with any one or more features of another embodiment, implementation, or aspect described herein.

[0079] Example Ex1: A method for processing tobacco material, the method comprising:

[0080] ○ Fermenting the tobacco material to obtain treated tobacco material, comprising:

[0081] ■ The tobacco material was cultured under anaerobic conditions;

[0082] ■ Apply a pressure between 1000 kg / m² and 4000 kg / m² to the tobacco material;

[0083] ■ The moisture content of the tobacco material is maintained between 25% and 40% by weight of the total weight of the tobacco material;

[0084] ○The fermentation process described herein lasts for at least one month.

[0085] Example Ex 2: The method according to Ex 1 includes the following steps:

[0086] ○Dry the tobacco material to obtain dried tobacco material with a moisture content between 5% and 10% by weight of the total weight of the tobacco material.

[0087] Example Ex 3: The method according to Ex 1 or Ex 2 includes the following steps:

[0088] ○ The tobacco material is matured before fermentation.

[0089] Example Ex 4: The method described in one or more of the preceding Ex 1–Ex 3, wherein the fermentation lasts for less than three years.

[0090] Example Ex 5: The method according to one or more of the preceding Ex 1–Ex 4 includes the following steps:

[0091] ○ Rotate the tobacco material.

[0092] Example Ex 6: The method according to one or more of the preceding Ex 1-Ex 5 includes:

[0093] ○ Add water to the tobacco material to maintain the moisture content of the tobacco material between 25% and 40% by weight of the total weight of the tobacco material.

[0094] Example Ex 7: The method described in one or more of the preceding Ex 1–Ex 6 includes maintaining the temperature of the tobacco material between 25 degrees Celsius and 35 degrees Celsius.

[0095] Example Ex 8: The method according to one or more of the preceding Ex 1–Ex 7 includes:

[0096] ○ Fix the tobacco material inside the moisture-retaining material.

[0097] Example Ex 9: The method according to one or more of the preceding Ex 1–Ex 8 includes:

[0098] ○ Before fermentation, the tobacco material is moistened in water to obtain a moisture content of between 25% and 40% of the total weight of the tobacco material.

[0099] Example Ex10: The method according to one or more of the preceding Ex 1–Ex 9, wherein the amount of asparagine in the treated tobacco material becomes at least 50% lower than the amount of asparagine contained in the same tobacco material before the treatment according to Ex 1–Ex 9.

[0100] Example Ex11: According to one or more of the methods described in Ex 1–Ex 10 above, the amount of asparagine in the treated tobacco material becomes at least 50% lower than the amount of asparagine contained in the same tobacco material before the treatment according to Ex 1–Ex 10.

[0101] Example Ex12: According to one or more of the methods described in Ex 1–Ex 11 above, the amount of reducing sugar in the treated tobacco material becomes at least 50% lower than the amount of reducing sugar contained in the same tobacco material before the treatment according to Ex 1–Ex 11.

[0102] Example Ex 13: Tobacco material treated according to any one of Ex 1–Ex 12, wherein the treated tobacco material contains at least 80% less asparagine than the amount contained in the same tobacco material prior to the treatment according to Ex 1–Ex 12. Example Ex 14: Tobacco material treated according to Ex 13, wherein the treated tobacco material contains at least 80% less glutamine than the amount contained in the same tobacco material prior to the treatment according to Ex 1–Ex 12.

[0103] Example Ex 15: The treated tobacco material according to Ex 13 or Ex 14, wherein the treated tobacco material contains at least 85% less total reducing sugar than the amount of total reducing sugar contained in the same tobacco material prior to treatment according to Ex 1–Ex 12.

[0104] Example Ex 16: The treated tobacco according to any one of Ex 13–Ex 15 above, wherein the treated tobacco material is at least 100 times more acidic than the same tobacco material before treatment according to Ex 1–Ex 12.

[0105] Example Ex 17: A tobacco material comprising:

[0106] ○ Total reducing sugars less than 3% by total dry weight;

[0107] ○ Asparagine with a total dry weight of less than 300 mg / kg.

[0108] Example Ex 18: The tobacco material according to Ex 17 comprises:

[0109] ○ Total reducing sugars less than 1% by total dry weight.

[0110] Example Ex 19: The tobacco material according to Ex 17 or Ex 18, comprising:

[0111] ○ Glutamine with a total dry weight of less than 70 mg / kg.

[0112] Example Ex 20: Tobacco material according to one or more of Ex 17-Ex 19, wherein the fermented tobacco material comprises:

[0113] ○ Total free amino acids with a total dry weight of more than 10,000 mg / kg.

[0114] Example Ex 21: The tobacco according to any one of Ex 17–Ex 20 above, which contains lactic acid.

[0115] Example Ex 22: Tobacco material according to one or more of Ex 17–Ex 21, wherein the tobacco material comprises hand-peeled leaves from which leaf ribs have been removed.

[0116] Example Ex 23: Tobacco material according to one or more of Ex 17-Ex 22, wherein the tobacco material is cured.

[0117] Example Ex 24: The tobacco material according to one or more of Ex 17–Ex 24 contains lactic acid.

[0118] Example Ex 25: An aerosol-generating article comprising tobacco material according to any one of Ex 17–Ex 24.

[0119] The embodiments will now be described further with reference to the accompanying drawings, in which:

[0120] ● Figure 1 and Figure 2 The bar charts showing the amount of lactic acid in the tobacco materials of Example 1 and Example 2 were measured before fermentation (0T) and 6 months later (3T) according to the present invention.

[0121] ● Figure 3 and Figure 4 The bar charts represent the levels of total alkaloids (TA) in the tobacco materials of Examples 1 and 2 (in percentage of total dry weight, DW), measured before (OT) and during fermentation according to the invention.

[0122] ● Figure 5 and Figure 6The bar charts showing the amounts of glutamine and glutamic acid (in total dry weight, DW) in the tobacco materials of Examples 1 and 2, respectively, were measured before (OT) and during fermentation according to the present invention.

[0123] ● Figure 7 and Figure 8 The bar charts showing the amounts of asparagine and aspartic acid (in total dry weight, DW) in the tobacco materials of Examples 1 and 2, respectively, were measured before (OT) and during fermentation according to the present invention.

[0124] ● Figure 9 and Figure 10 The bar charts showing the amounts of total alkaloids and reducing sugars (in total dry weight, DW) in the tobacco material of Example 3 were measured before fermentation (VG-BF), during fermentation, and after fermentation (VG-AF) according to the present invention.

[0125] First and second tobacco materials of the same tobacco type but with different treatments prior to fermentation were prepared. The tobacco material was Kasturi tobacco.

[0126] Example 1

[0127] The dark tobacco leaves have been fully sun-cured for about 10 days. The sun-cured leaves are then peeled off, leaving only the leaf tissue (hand-peeled leaves). This type of tobacco material is called "HS".

[0128] The tobacco material is adjusted to obtain approximately 30% moisture. This sample of adjusted but unfermented tobacco material is called OT (“starting material”).

[0129] The conditioned tobacco material was then introduced into three barrels, each containing approximately 100 kg of tobacco material. Before introduction, the tobacco material was wrapped in a material that retained the obtained moisture.

[0130] Pressure is applied to each barrel. This pressure ranges from 1000 kg / m² to 4000 kg / m².

[0131] After 1 month (samples are referred to as 1T), 2.5 months (samples are referred to as 2T), 6 months (samples are referred to as 3T), and 8.5 months (samples are referred to as 4T), the barrels were opened, and samples were taken from each barrel before rotating the tobacco, with at least three replicates, and the moisture content was readjusted to approximately 30% ± 5%.

[0132] During the intensive fermentation process under completely anaerobic conditions, the temperature inside the tank did not rise significantly (maintained within the range of 27°C to 31°C). Fermentation was stopped after 8.5 months.

[0133] Example 2

[0134] Dark tobacco leaf material, which had turned yellow for two days, was rapidly shredded in a shredding filler. This tobacco material contained both leaf sheets and midribs. The shredded leaves, containing both leaf sheets and midribs, were sun-dried for two days. The sample of this tobacco material is designated "CC" below.

[0135] The tobacco material is adjusted to achieve a moisture content of approximately 30%. This sample of adjusted but unfermented tobacco material is referred to as OT (“starting material”).

[0136] The conditioned tobacco material was then introduced into three barrels, each containing approximately 100 kg of tobacco material. Before introduction, the tobacco material was wrapped in a material that retained the obtained moisture.

[0137] Pressure is applied to each barrel. This pressure ranges from 1000 kg / m² to 4000 kg / m².

[0138] After 1 month (samples are referred to as 1T), 2.5 months (samples are referred to as 2T), 6 months (samples are referred to as 3T), and 8.5 months (samples are referred to as 4T), the barrels were opened, and samples were taken from each barrel before rotating the tobacco, with at least three replicates, and the moisture content was readjusted to approximately 30% ± 5%.

[0139] During the intensive fermentation process under completely anaerobic conditions, the temperature inside the tank did not rise significantly (maintained within the range of 27°C to 31°C). Fermentation was stopped after 8.5 months.

[0140] Visual observation

[0141] The initial tobacco material changed after 2.5 months of fermentation (sample 2T), with both HS and CC leaves darkening in color and exhibiting a pleasant caramel-buttery aroma and a complex fermented character. At the end of the process (8.5 months, 4T), the darker color was more pronounced in the fermented HS leaves compared to the CC leaves, likely due to the presence of midribs in the CC leaves.

[0142] Chemical analysis

[0143] In the following text, when referring to values ​​relative to a sample, the given value represents the average of several values ​​obtained for each sample of the same type.

[0144] After 2.5 months of fermentation, the pH of both CC and HS samples became acidic, reaching 3.2 (as seen in sample 2T). This reflects an anaerobic fermentation process involving sugar degradation, which typically produces organic acids such as (acetic acid and / or) lactic acid. The initial pH of tobacco materials is usually between pH 5 and pH 6.

[0145] Figure 1 and 2 The presence of lactic acid in tobacco materials is shown in the figure. Figure 1 This represents the lactic acid content in HS leaves. Figure 2 (Representing the lactic acid content in CC leaves), before fermentation, lactic acid was absent in all samples (three samples 0T are shown for each tobacco material - CC or HS). After fermentation (in this case, 6 months later, three tobacco material samples, referred to as 3T, are shown for both tobacco materials - CC or HS), all samples (both CC and HS leaves) showed the presence of lactic acid, although in varying amounts.

[0146] During fermentation, the alkaloids are not degraded or only slightly degraded. Figure 3 (HS leaf) and Figure 4 The percentage of total alkaloids (TA) content based on total dry weight is shown in the (CC leaves) (expressed as %DW in the figure). The total alkaloid content remained fairly stable during fermentation. At 8.5 months (4T), only 4% degradation was observed in HS leaves and only 9% in CC leaves. Although statistically relevant, such small variations are likely due to sampling. Limited alkaloid hydrolase activity may not be ruled out. Total alkaloids were analyzed in samples collected at the start of the intensive fermentation process (0T, n=6 samples analyzed), 1 month later (1T, n=9), 2.5 months later (2T, n=9), 6 months later (3T, n=9), and 8.5 months later (4T, n=12). T-tests (test statistics) were performed for comparison with the control unfermented mature tobacco (0T). Results are presented in... Figure 3 and 4 The diagram shows that it indicates the p-value, which is shown below:

[0147] *, p<0.05;

[0148] **, p<0.01 and

[0149] ***, p<0.001.

[0150] The p-values ​​for HS leaf sample 4T and CC leaf sample 3T were <0.01, while the p-values ​​for CC leaf samples 1T and 4T were <0.001. This indicates a statistically significant difference between fermented and unfermented tobacco materials.

[0151] Nitrate content was not affected by the intensive fermentation process. However, some effects were observed on tobacco-specific nitrosamines (TSNAs): NNN (N'-nitrosonornicotinamide), NNK (nicotine-derived nitrosamine), and NAT (N'-nitrosonechoic alkaloid). No changes were measured in NNK and NAT after 8.5 months of fermentation. However, increases in NNN were observed in both HS (3x increase) and CC (5-6x increase). Since nornicotinamide—the precursor to NNN prior to nitrosation—did not increase accordingly, NAT and NNK, rather than NNN, may have been partially degraded by bacteria during the fermentation run, as NNK and NAT initially increased twofold before decreasing to their initial values ​​in unfermented tobacco at 2.5 months of fermentation. This observation may indicate that alkaloid nitrosation occurred during intensive fermentation.

[0152] The evolution of sugars and free amino acids during the intensive fermentation according to the invention has been analyzed. Table 1 summarizes the measurements taken in samples of tobacco material. Table 1 shows the evolution of sugars and amino acids during intensive fermentation in barrels containing hand-peeled (HS) or chopped (CC) leaves, as in Examples 1 and 2, from untreated tobacco material samples (Sample 0T) to a fermentation process under fermentation conditions for 8.5 months (Sample 4T). All values ​​in the table are in total dry weight. Reducing sugars are expressed as a percentage of total dry weight, while free amino acids are expressed as mg / kg total dry tobacco material. After 2.5 months (2T, see Table 1), a decrease in reducing sugars occurred, consistent with color changes and slurry acidification. Glucose and fructose are two tobacco leaf substrates that anaerobic bacteria can metabolize in the fermentation barrel. Conversely, most amino acids increased during this process. Asparagine and glutamine both decreased significantly. In summary, these observations likely indicate that the main fermentation activity occurs between the first and third months. Proline was not degraded under anaerobic fermentation (see Table 1). Ornithine increased dramatically (>100-fold) in both HS and CC fermentation, while citrulline (data obtained from metabolomics analysis between 0T and 3T) increased 16-fold in HS and 2-fold in CC. This may indicate that (plant-derived) lactic acid bacteria were active in tobacco fermentation tanks, as such bacteria have been described as producing ornithine and citrulline at high levels (Rakhimuzzaman et al., Biol Pharm Bull. 2019; 42(9):1581-1589).

[0153]

[0154] Table 1

[0155] exist Figure 5-8 The table shows the amounts of glutamine and asparagine in tobacco materials. (Example) Figure 5-8 As shown and based on the data presented in Table 1, the deamination of glutamine and asparagine occurring during the intensive fermentation processes of both HS and CC leaves is likely associated with the accompanying increases in glutamate and asparagine, respectively. This indicates that the fermenting bacteria produce specific glutaminases and asparaginases to absorb C and N from amino acid resources. Both reactions produce ammonia, which increased threefold during the anaerobic fermentation processes of both HS and CC leaves. Figure 5 and 6 The levels of glutamine (white bars) and glutamate (black bars) in HS and CC leaves are shown separately. It is clear from the graphs that glutamine decreases while glutamate increases during fermentation. Figure 7 and 8 The levels of asparagine (striped bar chart) and aspartic acid (black bar chart) in HS and CC leaves are shown separately. It is clear from the graph that asparagine decreases while aspartic acid increases during fermentation.

[0156] Metabolomics studies were conducted to identify marker molecules or pathways associated with the anaerobic fermentation process of tobacco leaves. Sugar resources such as glucose and fructose, present in the starting materials (controls) of both HS and CC leaves, could be used as energy sources by anaerobic bacteria (see Table 1). Under anaerobic conditions, the glycolysis pathway converts glucose (or fructose) to pyruvate, producing 2 ATP and 2 NADH+H+. Other organic and carbon-rich compounds that can be rapidly used by anaerobic bacteria are citrate and malate (Bintsis, T, 2018, AIMS Microbiology, 4(4):665–684), both of which are among the most abundant organic acids in plants. Like reducing sugars, citrate and malate are also metabolized during intensive tobacco fermentation: chemical analysis of the samples showed that more than 60% of the glucose and fructose, citrate, and malate present in the starting tobacco material (sample 0T) (hand-peeled and chopped leaves) were catabolized after 6 months of intensive fermentation (sample 3T). Another observation that may be related to the consumption of such organic molecules is the increase (13-14 times) of pyruvate in both HS and CC fermented tobacco materials. Pyruvate is a substrate for several reactions that may occur under anaerobic conditions, including: (1) the production of D-lactate, primarily for the regeneration of NAD+ in glycolysis; and (2) the production of acetate, diacetyl, and 2,3-butanediol, which may contribute to the delivery of aromatic compounds and flavors in heavily fermented tobacco. Pyruvate may lead to the formation of aromatic compounds such as 2,3-butanediol or lactate as products of lactic acid bacteria.

[0157] Metabolomics analysis of heavily fermented tobacco also revealed two other pathways: (1) degradation of tryptophan and (2) catabolism of chlorogenic acid.

[0158] Regarding tryptophan degradation, Ummadi and Weimer (2001, J. Dairy Sci. 84:1773–1782) have described and adapted the pathway for cheese bacteria. In this case, in both HS and CC leaves, over 78% of the tryptophan present in the starting tobacco material (sample 0T) was catabolized after 6 months of fermentation (sample 3T). This pathway indicates that the product generated by such catabolism is primarily indole-3-lactic acid. This is illustrated by increases of 14-fold and 28-fold in HS and CC leaves, respectively. No other compounds belonging to this pathway showed such an increase. No specific aromatic properties of this compound have been reported.

[0159] Chlorogenic acid (CGA) is an important bioactive dietary polyphenol produced by certain plant species, such as tobacco, and is a major component of coffee. In heavily fermented tobacco leaves, CGA is completely degraded after the anaerobic fermentation process. On the other hand, the products of CGA catabolism, namely quinic acid and caffeic acid, are increased in both HS and CC leaves after 6 months of fermentation. This may be due to bacterial cinnamyl esterase activity, as recorded by Guglielmetti et al. (2008, Applied and Environmental Microbiology, 74, 4: 1284–1288). Therefore, a portion of the quinic acid and caffeic acid pools may be produced by the hydrolysis of CGA, and neither has been reported to have flavor properties.

[0160] Compared to mature tobacco, the elevated levels of pyruvate and indole-3-lactic acid, and the lack of chlorogenic acid in heavily fermented tobacco, make them suitable as chemical markers.

[0161] For the purposes of this specification and the appended claims, unless otherwise stated, all figures representing quantities, quantities, percentages, etc., shall be understood to be modified by the term "about" in all cases. Furthermore, all ranges include the disclosed maximum and minimum points, and include any intermediate ranges therein, which may or may not be specifically listed herein. Thus, in this context, the number A is understood to be A ± 10% of A. In this context, the number A can be considered to include a value within the general standard error for the measurement of the property represented by the number A. In some instances, as used in the appended claims, the number A may deviate from the percentages listed above, provided that the amount of deviation from A does not significantly affect the fundamental and novel features of the claimed invention. Furthermore, all ranges include the disclosed maximum and minimum points, and include any intermediate ranges therein, which may or may not be specifically listed herein.

[0162] A third tobacco material of a different tobacco type than that used in Examples 1 and 2 was prepared. This tobacco material was Virginia tobacco.

[0163] Example 3

[0164] The tobacco leaf material has been fully sun-cured for approximately 10 days. This sun-cured leaf is considered the standard for Virginia tobacco.

[0165] The tobacco material is adjusted to obtain approximately 30% moisture. This sample of adjusted but unfermented tobacco material is called BF (starting material before fermentation).

[0166] The conditioned tobacco material was then introduced into two containers, each containing approximately 100 kg of tobacco material. Before introduction, the tobacco material was wrapped in a material that retained the obtained moisture.

[0167] Pressure is applied to each barrel. This pressure ranges from 1000 kg / m² to 4000 kg / m².

[0168] The tank was opened after 1 month (sample 1T), 2 months (sample 2T), 3 months (sample 3T), 4 months (sample 4T), 5 months (sample 5T), 6 months (sample 6T), 7 months (sample 7T), and 8 months (sample AF, after fermentation).

[0169] Each month, rotate the tobacco material in both buckets at least 6 times.

[0170] Samples were collected before fermentation (VG-BF: starting material, 6 replicates), during fermentation (3 replicates per barrel in all months from VG-T1 to VG-T7), and after fermentation (VG-AF: after fermentation, 6 replicates).

[0171] During sample collection, the tobacco material was rotated and its moisture content was readjusted to approximately 30% ± 5%.

[0172] During the intensive fermentation process under completely anaerobic conditions, no large temperature changes were observed: a linear change from 30 degrees Celsius at the start of fermentation (VG-T1) to 26 degrees Celsius at the end of fermentation (VG-AF). The temperature inside the tank was measured using a trap.

[0173] During the fermentation run (T1 to AF), the pH of the tobacco material did not change significantly, remaining at 5.1 ± 0.3.

[0174] Fermentation stops after 8 months.

[0175] Visual observation

[0176] As observed with the Kasturi tobacco material, the tobacco material at the end of fermentation (VG-AF) became significantly darker in color compared to the starting material (VG-BF). However, after 4 months of anaerobic fermentation (VG-T4), the Virginia tobacco material did not exhibit the same "dark color" as the Kasturi tobacco after the same amount of fermentation, suggesting that 4 months may not be sufficient for the Virginia tobacco material to fully ferment.

[0177] Chemical analysis

[0178] In the following text, when referring to values ​​relative to a sample, the given value represents the average of several values ​​obtained for each sample of the same type.

[0179] The behavior of lactic acid in tobacco materials over time and Figure 1 and 2 The results are very similar. Qualitatively, lactic acid was absent in all samples before fermentation. After fermentation, lactic acid was present, although in varying amounts.

[0180] Figure 9 The evolution of total alkaloids (TA) during fermentation is shown. These data confirm that alkaloids, particularly nicotine (not shown), are unaffected by anaerobic fermentation. The bacteria do not consume the main alkaloids as fermentation substrates.

[0181] On the other hand, such as Figure 10 As illustrated and as observed in Examples 1 and 2, reducing sugars were used as substrates by the fermenting bacteria. Therefore, approximately 60% of the reducing sugars (RS) were oxidized during the 8-month fermentation period, decreasing from 18.3% (VG-BF) on a dry weight basis (DW) to 7.4% (VG-AF). It is possible that longer fermentation times would result in an even higher percentage of RS degradation.

[0182] The mean (n=6) and SD are presented in Figure 9 and 10 In addition, a t-test (paired) was performed between BF and AF.

[0183] Further chemical analysis revealed that the starting material (VG-BF) had an asparagine content of 262 μg / g on a dry weight basis. The same tobacco material after fermentation (8 months, VG-AF) had an asparagine content of 19 μg / g on a dry weight basis.

[0184] The starting material (VG-BF) had a glutamine content of 185 μg / g on a dry weight basis. The same tobacco material after fermentation (8 months, VG-AF) had a glutamine content of 12 μg / g on a dry weight basis.

Claims

1. A method for processing tobacco material, the method comprising: o Fermenting the tobacco material to obtain treated tobacco material, comprising: The tobacco material was cultured under anaerobic conditions; A pressure between 1000 kg / m² and 4000 kg / m² is applied to the tobacco material; The moisture content of the tobacco material is maintained between 25% and 40% by weight of the total weight of the tobacco material. The fermentation process described therein lasts for at least two months. o The amount of asparagine in the treated tobacco material becomes at least 50% lower than the amount of asparagine in the same tobacco material before treatment.

2. The method according to claim 1, wherein the method comprises the steps of: After fermentation, the tobacco material is dried to obtain dried tobacco material with a moisture content between 5% and 10% by weight of the total weight of the tobacco material.

3. The method according to claim 1 or 2, wherein the method comprises the steps of: o The tobacco material is matured before fermentation.

4. The method according to claim 1 or 2, wherein the method comprises maintaining the temperature of the tobacco material between 25 degrees Celsius and 35 degrees Celsius.

5. The method according to claim 1 or 2, wherein the method comprises the steps of o Rotate the tobacco material.

6. The method according to claim 1 or 2, wherein the method comprises: o Fix the tobacco material within the moisture-retaining material.

7. The method according to claim 1 or 2, wherein the method comprises: o Before fermentation, the tobacco material is moistened in water to obtain a moisture content of between 25% and 40% of the total weight of the tobacco material.

8. The method according to claim 1 or 2, wherein the amount of reducing sugar in the treated tobacco material becomes at least 50% lower than the amount of reducing sugar in the same tobacco material before treatment.

9. A treated tobacco material obtained by the method of claim 1, the treated tobacco material comprising: o Total reducing sugars less than 3% by total dry weight; Asparagine with a total dry weight of less than 300 mg / kg.

10. The treated tobacco material according to claim 9, wherein the treated tobacco material comprises: o glutamine less than 70 mg / kg on total dry weight.

11. The treated tobacco material according to claim 9 or 10, wherein the treated tobacco material comprises: o Total free amino acids greater than 10,000 mg / kg based on total dry weight.

12. The treated tobacco material according to claim 9 or 10, wherein the tobacco material is cured prior to fermentation.

13. An aerosol-generating article comprising the treated tobacco material according to any one of claims 9 to 12.

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