Method for processing tobacco material and processed tobacco material

Abstract

A method for treating a tobacco material, the method comprising: providing a tobacco material; and fermenting the tobacco material to obtain a fermented tobacco material, the fermenting step comprising: #incubating the tobacco material under anaerobic conditions; #under the following conditions: #the content of lactic acid is more than 10 times, preferably more than 20 times, more preferably more than 50 times, more preferably more than 70 times, preferably more than 80 times, of an initial amount of lactic acid in the tobacco material; #the content of reducing sugar is less than 0.5, preferably less than 0.4, more preferably less than 0.2, more preferably less than 0.1, of an initial amount of reducing sugar in the tobacco material; #the content of indole-3 lactic acid is more than 5 times, preferably more than 10 times, preferably more than 20 times, of an initial amount of indole-3 lactic acid in the tobacco material; #the content of caffeic acid is more than 4 times, preferably more than 10 times, preferably more than 20 times, of an initial amount of caffeic acid in the tobacco material; #the content of quinic acid is more than 2 times, preferably more than 4 times, of an initial amount of quinic acid in the tobacco material; # the initial amount of asparagine in the material is less than 0.5, preferably less than 0.4, preferably less than 0.3; # the glutamine content is less than 0.5, preferably less than 0.4, of the initial amount of glutamine in the tobacco material; # the L-ornithine content is more than 10 times, preferably more than 20 times, preferably more than 50 times, preferably more than 100 times, of the initial amount of L-ornithine in the tobacco material; # the L-leucine content is more than 2 times, preferably more than 4 times, of the initial amount of L-leucine in the tobacco material; # the L-lysine content is more than 2 times, preferably more than 6 times, of the initial amount of L-lysine in the tobacco material; # a fermentation index is more than 50, preferably more than 100, more preferably more than 250, more preferably more than 400, wherein the fermentation index is obtained by dividing the ratio of the lactic acid content in the treated tobacco material to the lactic acid content of the non-fermented tobacco material by the ratio of the reducing sugar content in the treated tobacco material to the reducing sugar content of the non-fermented tobacco material.

Description

[Technical field]

[0001] The present invention relates to a method for processing tobacco, and to a tobacco material processed by fermentation. The present invention also relates to an aerosol-generating article containing a tobacco material processed by fermentation. In particular, the fermentation is anaerobic fermentation. [Background technology]

[0002] Various processing methods and additives have been proposed in the art to change the overall characteristics or properties of tobacco materials utilized in tobacco products. For example, tobacco materials have been treated with additives. In addition, the processing conditions used during the processing of these tobacco materials have been controlled to change the chemical or sensory properties of tobacco products produced from these tobacco materials, and to change the chemical or sensory properties of mainstream smoke or aerosols generated by smoking articles incorporating these tobacco materials.

[0003] Processing to enhance or add flavor and aroma to tobacco materials at later stages of tobacco processing often involves adding one or more additives to the tobacco, which can require additional processing steps and equipment, which can be costly and time consuming. Moreover, the addition of additives to tobacco may be viewed unfavorably by some consumers.

[0004] Thus, there is a need to provide tobacco materials that have good and improved organoleptic properties without any additives. Thus, there is also a need for methods of improving the organoleptic properties of tobacco materials that do not involve adding external flavorants to the tobacco itself. Furthermore, there is a need for tobacco materials that exhibit these different organoleptic materials without the need for complex processing.

[0005] There is also a need to provide tobacco materials that simultaneously have high organoleptic properties and reduced concentrations of harmful compounds.There is also a need to provide tobacco materials that simultaneously have high organoleptic properties produced upon use by a user and reduced concentrations of harmful compounds in the aerosol produced. Summary of the Invention

[0006] According to one aspect, the present invention relates to a method for processing tobacco material, the method comprising providing a tobacco material and fermenting the tobacco material to obtain a fermented tobacco material. Preferably, the fermentation step comprises incubating the tobacco material under anaerobic conditions.

[0007] Due to fermentation, the specific compounds present in the tobacco material may change, as well as the organoleptic properties of the tobacco material. Furthermore, during the fermentation process, the content of some compounds in the tobacco material decreases, and the concentration of some additional compounds increases.

[0008] At a sensory level, the fermented tobacco material consistently delivers a higher smoothness characteristic compared to the corresponding unfermented, dry-processed tobacco material. Additionally, the fermented tobacco material delivers a floral note or aroma characteristic upon use. The tobacco fermented material has a smoother characteristic that reduces the harsh sensation. Thus, the organoleptic properties of the tobacco material are increased, improving the experience for the user.

[0009] It is known that tobacco materials can be fermented. Tobacco plants can host microorganisms, which in turn can include bacteria, molds, and actinomyces. Studies have shown that bacteria make up the majority of the microorganisms present in tobacco, while molds and actinomyces are in minority. Yeasts are low in concentration or not detectable at all. Fermented tobacco can be made by a variety of suitable techniques known in the art, such as those described in "Research Progress in Tobacco Fermentation" by Yang Yang et al., Journal of Biosciences and Medicines 2018,6,105-114, available at http: / / www.scirp.org / journal / jbm, or in U.S. Pat. No. 5,372,149, or in U.S. Pat. No. 4,528,993, and elsewhere. Generally, tobacco fermentation involves adjusting the moisture content of dried and aged tobacco to a moisture content of about 20 percent to about 60 percent, and layering and fermenting the moistened tobacco. Fermentation can be terminated, for example, by drying or cold storage. As noted above, tobacco fermentation does not require the addition of microorganisms, as microorganisms are generally naturally occurring on tobacco plants.

[0010] In the present invention, fermentation is carried out under anaerobic conditions.

[0011] 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 conditions in which oxygen is not available for redox reactions, both as a result of chemical equilibrium and biochemical activity. Instead, other oxidized compounds may be present that can be used by the microorganism for certain types of energy metabolism.

[0012] Anaerobic conditions may coexist with aerobic conditions, and oxygen in gas form may be unavailable to microorganisms in a microenvironment (e.g., aggregates of debris suspended in water) while simultaneously being present in the macroenvironment (water).

[0013] In anaerobic fermentation of tobacco, without being bound by theory, the main energy extraction pathway may come from glycolysis, and some amino acids are also used as carbon / nitrogen sources. Preferred nitrogen compounds usually include glutamine, alanine, serine, threonine, aspartate, asparagine, urea, and arginine.

[0014] In the following, the definition of "fermentation conditions" means that the tobacco is subjected to anaerobic conditions. Although a certain amount of oxygen may be present in the fermentation environment, the oxygen is not available for oxidation-reduction reactions.

[0015] The anaerobic fermentation conditions do not affect the alkaloid, and in particular the nicotine, content of the tobacco material, and at the same time, the organoleptic properties of the tobacco material are increased. Preferably, a method provides for stopping the fermentation process when at least one desired condition is met.

[0016] In this way it is possible to obtain a processed tobacco material having certain desired properties, and it is also possible to avoid a too long fermentation process, thus preserving the quality of the tobacco material.

[0017] Preferably, the method comprises stopping the fermentation process when the content of lactic acid is greater than 10 times the initial amount of lactic acid in the tobacco material, preferably greater than 20 times, more preferably greater than 50 times. Preferably, the method comprises stopping the fermentation process when the content of lactic acid is greater than 70 times, preferably greater than 80 times, the initial amount of lactic acid in the tobacco material.

[0018] The increase in lactic acid is a reliable indicator of the fermentation of the tobacco material. The amount of lactic acid in the tobacco material after fermentation may also depend on the type of tobacco material or the initial amount of lactic acid in the tobacco material. Furthermore, the amount of lactic acid in the tobacco material after fermentation may also depend on the content of other compounds in the tobacco material before fermentation. Conversely, the increase in lactic acid in the tobacco material is a reliable indicator of the degree of fermentation of the tobacco material. This parameter does not depend on the type of tobacco material or the composition of the tobacco material and is not affected by further external parameters.

[0019] Lactic acid has two enantiomers, L-lactic acid and D-lactic acid. For the purposes of the present invention, the term lactic acid is intended to mean the sum of the two enantiomers, L-lactic acid and D-lactic acid. Nevertheless, in fermented tobacco, the main enantiomer L-lactic acid is present, but some D-lactic acid is also produced. Compared to L-lactic acid, D-lactic acid can be toxic to humans, i.e., the LD50 value level per orally poisoned rat is about 4.5 g / kilogram (Pohanka, 2020). However, D-lactic acid and L-lactic acid are non-volatile and therefore not transferred to the aerosols generated in tobacco materials.

[0020] The content of lactic acid in tobacco materials is determined spectrophotometrically according to MP 0309 rev 5 2012, Chelab Srl.

[0021] The method preferably comprises stopping the fermentation process when the content of reducing sugars is less than 0.5 of the initial amount of reducing sugars in the tobacco material, preferably less than 0.4, more preferably less than 0.2, more preferably less than 0.1.

[0022] Reducing sugars are converted to pyruvate by anaerobic fermentation, and pyruvate is a precursor of many other flavor compounds and provides the sugar and acid characteristics of caramel brown color. Therefore, the reduction in the content of reducing sugars in tobacco materials subjected to anaerobic fermentation is a good indicator of the extent of fermentation of the tobacco material. The amount of reducing sugars in tobacco materials may depend on different factors, such as tobacco type, harvest area, etc., but the reduction rate of reducing sugars indicates the extent of the fermentation process. The most abundant naturally occurring sugars in tobacco leaves are glucose, fructose, and sucrose. Differences in sugar content may exist between tobacco types. For example, Virginia is characterized by a high sugar content (generally in the range of 8 percent to 30 percent), while Burley is characterized by a low sugar content (generally in the range of 1 percent to 2 percent). However, regardless of the tobacco type used for the tobacco material, a reduction in the content of reducing sugars during fermentation under the fermentation conditions of the present invention was found. The change in the amount of reducing sugars may change the organoleptic properties of the tobacco material and the organoleptic properties of the smoke or aerosol produced with the tobacco material.

[0023] The content of reducing sugars in the tobacco material is preferably determined by a continuous flow analyzer according to CORESTA recommended method N°38.

[0024] The method preferably comprises stopping the fermentation process when the content of indole-3 lactic acid is more than 5 times the initial amount of indole-3 lactic acid in the tobacco material, preferably more than 10 times, preferably more than 20 times. Preferably, the absolute content of indole-3 lactic acid is determined by UPLC-MS, calculated based on a standard curve extracted.

[0025] Indole-3 lactic acid is present only in trace amounts in tobacco leaves, but is produced in the dried treated leaf material during anaerobic fermentation, indicating that the tobacco material had been subjected to anaerobic fermentation.

[0026] The method preferably includes stopping the fermentation process when the caffeic acid content is greater than 4 times the initial amount of caffeic acid in the tobacco material, preferably greater than 10 times, and more preferably greater than 20 times.

[0027] The presence of caffeic acid enhances the organoleptic properties of the tobacco material and improves the user experience. The content of caffeic acid in the tobacco material is preferably estimated by metabolomic analysis using UPLC-MS and comparing the caffeic acid content in non-fermented tobacco material with that in processed tobacco material, i.e. fermented or partially fermented tobacco material.

[0028] The method preferably includes stopping the fermentation process when the content of quinic acid is more than twice the initial amount of quinic acid in the tobacco material, preferably more than four times.

[0029] The content of quinic acid in a tobacco material is preferably estimated by metabolomic analysis using UPLC-MS and comparison of caffeic acid in non-fermented tobacco material and processed tobacco material, i.e. fermented or partially fermented tobacco material.

[0030] Quinic and caffeic acids were significantly increased after fermentation as a result of cinnamoyl esterase catabolizing chlorogenic acid. The increase in these compounds is independent of the tobacco substrate used for fermentation. Besides its strong antioxidant activity, increased collagen production, and prevention of premature aging, caffeic acid exhibits antibacterial activity and may be promising for the treatment of skin diseases (Magnani et al., 2014). Moreover, quinic acid is also a strong drug candidate to fight prostate cancer (Inbathamizh et al., 2013).

[0031] The method preferably includes stopping the fermentation process when the asparagine content is less than 0.5 of the initial amount of asparagine in the tobacco material, preferably less than 0.4, preferably less than 0.3. The asparagine content is preferably determined by ion exchange chromatography according to MP 2442 rev 0 2021, Chelab Srl.

[0032] A reduction in asparagine means a reduction in its conversion to acrylamide. Tobacco materials contain a certain amount of amino acids. The amino acids can contribute substantially to the level of certain components in the smoke or aerosol produced by the final product in which the fermented tobacco material is contained, and to the organoleptic properties of the smoke or aerosol. Different types of tobacco can contain different amounts of amino acids. In addition, there can be differences (mainly quantitative) in the amino acid composition between tobacco leaves or tips or stems. Also, depending on the tobacco growing region, the ratio of the levels of various amino acids can vary, but a fairly similar composition is generally maintained for the same tobacco amino acids. It was observed that the asparagine content in tobacco materials is reduced during fermentation under the fermentation conditions of the present invention, regardless of the tobacco type and origin. This suggests that the fermentation bacteria in the fermentation of the present invention produce a specific asparaginase to assimilate C and N from the amino acid resources. Asparagine can be thermally converted to acrylamide. Acrylamide is considered to be a potentially harmful substance. Reducing the content of asparagine in tobacco materials makes it possible to obtain a reduction in acrylamide formation.

[0033] The method preferably comprises stopping the fermentation process when the glutamine content is less than 0.5, preferably less than 0.4, of the initial amount of glutamine in the tobacco material. The glutamine content is preferably determined by ion exchange chromatography according to MP 2442 rev 0 2021, Chelab Srl.

[0034] A reduction in glutamine may increase the flavour of the tobacco material, as this means that the umami flavour characteristic is released from the tobacco material during use.

[0035] The method preferably comprises stopping the fermentation process when the content of L-ornithine is more than 10 times the initial amount of L-ornithine in the tobacco material, preferably when it is more than 20 times, preferably when it is more than 50 times, preferably when it is more than 100 times. The content of L-ornithine is preferably determined by ion exchange chromatography according to MP 2442 rev 0 2021, Chelab Srl.

[0036] An increase in L-ornithine in a fermented tobacco material is a good indicator of the extent of fermentation of the tobacco material, regardless of the type of tobacco material subjected to fermentation and its initial composition. It has been found that fermentation causes an increase in L-ornithine in the tobacco material, regardless of the type of tobacco material.

[0037] The method preferably comprises stopping the fermentation process when the L-leucine content is more than twice the initial amount of L-leucine in the tobacco material, preferably more than four times. The L-leucine content is preferably determined by ion exchange chromatography according to MP 2442 rev 0 2021, Chelab Srl.

[0038] The method preferably comprises stopping the fermentation process when the L-lysine content is more than twice the initial amount of L-lysine in the tobacco material, preferably more than 6. The L-lysine content is preferably determined by ion exchange chromatography according to MP 2442 rev 0 2021, Chelab Srl.

[0039] An increase in L-leucine and L-lysine is a good indicator of the extent of fermentation of a tobacco material, regardless of the type of tobacco material subjected to fermentation and its initial composition.

[0040] The method preferably includes stopping the fermentation process when the content of 3-sec-butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione is greater than 20 times the initial amount of 3-sec-butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione in the tobacco material, preferably when it is greater than 40 times, more preferably when it is greater than 60 times, and more preferably when it is greater than 80 times.

[0041] Although 3-sec-butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (BHHPPD) is only detected in trace amounts in cured tobacco materials, the content of BHHPPD in tobacco materials increases with the degree of fermentation, independent of the type of tobacco material. Thus, an increase in BHHPPD in tobacco materials is an imilligramss good indicator of the degree of fermentation of the tobacco material.

[0042] The method preferably includes stopping the fermentation process when the content of secoisolariciresinol (SECO) is greater than 10 times the initial amount of secoisolariciresinol (SECO) in the tobacco material, preferably when it is greater than 20 times, more preferably when it is greater than 40 times, more preferably when it is greater than 50 times.

[0043] Although only trace amounts of secoisolariciresinol are detected in dried tobacco materials, the content of SECO in tobacco materials increases with fermentation of the tobacco materials, regardless of the type of tobacco material. Therefore, the increase in SECO in tobacco materials is a reliable indicator of the degree of fermentation of the tobacco materials.

[0044] Preferably, the method comprises stopping the fermentation process when the fermentation index is greater than 50, preferably greater than 100, more preferably greater than 250, more preferably greater than 400. The fermentation index is obtained by dividing the ratio of the content of lactic acid in the treated tobacco material to the content of lactic acid in the non-fermented tobacco material by the ratio of the content of reducing sugars in the treated tobacco material to the content of reducing sugars in the non-fermented tobacco material. The fermentation index may be obtained by the following formula: Fr=(F LA / NF LA ):(F RS / NF RS ), where F LA = content of lactic acid in fermented tobacco material, NF LA = content of lactic acid in non-fermented tobacco material, F RS = content of reducing sugars in fermented tobacco material, NF RS = initial content of reducing sugars in the non-fermented tobacco material. Also, in this case, lactic acid refers to the sum of L-lactic acid and D-lactic acid, as described above.

[0045] The fermentation index (Fr) also makes it possible to detect slight changes in the composition of the tobacco material during fermentation. Moreover, this parameter is highly reliable, since it changes during fermentation and independently of the type of tobacco material being fermented.

[0046] Stopping the fermentation process when at least one of the above conditions is met makes it possible to obtain a treated tobacco material having the desired organoleptic properties. The tobacco material treated by the method of the present invention can be fully fermented or partially fermented. Regardless of the degree of fermentation of the tobacco material, by stopping the fermentation process when one of the above conditions is met, it is possible to obtain a tobacco material having specific characteristics.

[0047] For the purposes of the present invention, treated tobacco material refers to fermented or partially fermented tobacco material.

[0048] Preferably, the method further comprises an initial measuring step for measuring an initial content of at least one of lactic acid, or reducing sugars, or indole-3 lactic acid, or quinic acid, or caffeic acid, or asparagine, or glutamine, or L-ornithine, or L-leucine, or L-lysine, or butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or secoisolariciresinol (SECO), or a fermentation index, in the tobacco material before the fermentation step, to obtain an initial amount of lactic acid, or reducing sugars, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or secoisolariciresinol (SECO), or a fermentation index, respectively.

[0049] This feature makes it possible to obtain an accurate value of the initial content of at least one compound in the tobacco material before it is subjected to a fermentation process. The initial measurement step serves to measure a characteristic of the tobacco material before fermentation, in other words a characteristic of the non-fermented tobacco material is measured in the initial measurement step. The initial amount of the measured characteristic corresponds to the value of the characteristic of the non-fermented tobacco material.

[0050] Preferably, the method further comprises a measuring step for measuring the content of at least one of lactic acid, or reducing sugars, or indole-3 lactic acid, or quinic acid, or caffeic acid, or asparagine, or glutamine, or L-ornithine, or L-leucine, or L-lysine, or butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or secoisolariciresinol, or a fermentation index in the fermenting tobacco material.

[0051] This feature makes it possible to obtain an accurate value of at least one of the above mentioned characteristics within the tobacco material, making it possible to more accurately monitor the fermentation process and more accurately adjust the properties of the resulting tobacco material, in this way making it possible to obtain a very reliable indication of the degree of fermentation of the tobacco material in real time.

[0052] When the tobacco material contains dark tobacco, the method is preferably applied to stop the fermentation process when the content of 2,3 butanediol exceeds 5 times the initial amount of 2,3 butanediol in the tobacco material, and preferably applies to stop the fermentation process when it exceeds 10 times. The presence of 2,3 butanediol imparts the natural odor of cocoa butter. The presence of 2,3 butanediol contributes to the good flavorful volatiles present in the anaerobically fermented tobacco material.

[0053] When the tobacco material contains dark tobacco, the method is preferably applied to stop the fermentation process when the content of diacetyl exceeds 5 times the initial amount of diacetyl in the tobacco material, and preferably applies to stop the fermentation process when it exceeds 10 times. The presence of diacetyl imparts some organoleptic properties to the tobacco material. The presence of diacetyl contributes to the presence of good flavorful volatiles in the anaerobically fermented tobacco material.

[0054] The method preferably includes an initial measuring step for measuring an initial content of 2,3 butanediol or diacetyl in the tobacco material prior to fermentation so as to obtain an initial amount of 2,3 butanediol or diacetyl, respectively, in the tobacco material.

[0055] Preferably, the method further comprises a measuring step for measuring the content of 2,3 butanediol or diacetyl in the tobacco material during fermentation.

[0056] Preferably, the method includes providing a database comprising the amount of at least one of lactic acid, or reducing sugars, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or secoisolariciresinol (SECO), or a fermentation index, or butanediol, or diacetyl in the non-fermented tobacco material.

[0057] Preferably, the method includes providing a database comprising, for a plurality of different tobacco materials, an amount of at least one of lactic acid, or reducing sugars, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or secoisolariciresinol (SECO), or a fermentation index, or butanediol, or diacetyl, in the non-fermented tobacco material.

[0058] Thus, at least one database can be created for at least one type of tobacco material, each database may contain, for each type of tobacco material under non-fermented conditions, the typical amount of lactic acid, or reducing sugars, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or secoisolariciresinol (SECO), or a fermentation index, or butanediol, or diacetyl.

[0059] Preferably, the method provides for recovering from the at least one database an initial amount of at least one of lactic acid, or reducing sugars, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or secoisolariciresinol (SECO), or butanediol, or diacetyl, or a fermentation index in the tobacco material before fermentation.

[0060] Preferably, the method includes providing a database comprising an amount of at least one of lactic acid, or reducing sugars, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or secoisolariciresinol (SECO), or a fermentation index, or butanediol, or diacetyl in the fermented tobacco material and / or the partially fermented tobacco material.

[0061] Preferably, the method includes providing a database comprising an amount of at least one of lactic acid, or reducing sugars, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or secoisolariciresinol (SECO), or a fermentation index, or butanediol, or diacetyl, in the fermented tobacco material and / or the partially fermented tobacco material for a plurality of different tobacco materials.

[0062] Preferably, the method provides for recovering from at least one database an amount of at least one of lactic acid, or reducing sugars, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or secoisolariciresinol (SECO), or a fermentation index, or butanediol, or diacetyl, and comparing the recovered value with a corresponding measured value in the fermenting tobacco material.

[0063] Preferably, the method provides for stopping or continuing the fermentation process based on the result of the comparison step.Preferably, the method provides for continuing the fermentation process if the measured values ​​of lactic acid, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or L-leucine, or L-lysine, or butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or secoisolariciresinol (SECO), or fermentation index, or butanediol, or diacetyl are lower than the corresponding recovered values, and / or if the values ​​of reducing sugars, or asparagine, or glutamine, or fermentation index are higher than the corresponding recovered values.

[0064] According to another aspect, the present invention relates to a method for processing tobacco material, the method comprising providing a tobacco material and fermenting the tobacco material to obtain a fermented tobacco material. Preferably, the fermentation step comprises incubating the tobacco material under anaerobic conditions and stopping the fermentation step when at least one of the following conditions is met: - the tobacco material contains, on a total dry weight basis, at least 20 milligrams / gram, preferably at least 50 milligrams / gram, of lactic acid; - the tobacco material contains less than 3 percent total reducing sugars on a total dry weight basis; - the tobacco material contains, on a total dry weight basis, less than 300 milligrams per kilogram of asparagine; - the tobacco material contains less than 70 milligrams per kilogram of glutamine on a total dry weight basis; - the tobacco material contains more than 10,000 milligrams per kilogram of asparagine by total dry weight of total free amino acids; - the tobacco material contains more than 10 times, preferably more than 20 times, the initial amount of L-ornithine in the tobacco material; the tobacco material contains, on a total dry weight basis, more than 50 milligrams / kilogram, preferably more than 80 milligrams / kilogram, of L-ornithine.

[0065] The anaerobic conditions are preferably achieved by placing the tobacco material in a container and closing the container. The anaerobic conditions are preferably achieved by placing the tobacco material in a container, removing air from the container and closing the container. Preferably, the container is not closed in a completely airtight manner, so that CO2 or other gases produced during fermentation are allowed to escape from the container. In this way, accumulation of these substances in the container is avoided. The container is preferably tightly separated from the external environment and anaerobic conditions are maintained inside the container. The container is preferably closed with a closure that avoids overpressure being achieved in the container.

[0066] More preferably, pressure is applied to remove air from the tobacco material, the applied pressure forcing the air out of the tobacco material, such that oxygen is no longer present, or is present only in minimal amounts, in the container after it is closed.

[0067] Placing the tobacco material in a container and closing the container after air has been removed from the sealed container allows anaerobic conditions to be reached quickly. This method of achieving anaerobic conditions is preferred as it is cost-effective and easy to implement.

[0068] Preferably, the fermentation process is carried out at a yield of 1000 kilograms per square meter (kilograms / m 2 ) ~ 15,000 kg / m2 (kg / m2 ), preferably from 3000 kilograms / m² to 12000 kilograms / m², more preferably from 5000 kilograms / m² to 10000 kilograms / m², on the tobacco material.

[0069] The pressure applied to the tobacco material is maintained within the above ranges during the fermentation process.

[0070] Pressure may be applied to the tobacco material by any means. Pressure may be applied by pumping an inert gas into the container. Pressure may be applied by placing a weight on the tobacco material such that a desired pressure range is applied to the tobacco material. For example, the container may be filled with moist tobacco material and a weight is placed in contact with the tobacco material as a "lid" for the container until water seeps out of the container. Preferably, pressure is applied by a hydraulic actuator that pressurizes a closure of the container and / or the tobacco material contained therein to establish a desired pressure within the container.

[0071] A hydraulic actuator is preferably used to apply the desired pressure to the tobacco material. The hydraulic actuator may be selected depending on the level of pressure to be achieved in the container. Hydraulic actuators are known in the art for applying pressure to containers, for example wine presses. The tobacco material is inserted into the container and a weight is preferably positioned on or above the tobacco material to exert the desired pressure. The container is then preferably closed, leaving the weight inside the container so that the weight can continue to apply pressure to the tobacco material.

[0072] The fermentation step preferably includes maintaining the moisture content of the tobacco material during fermentation in an amount comprised between 10 and 50 percent by weight of the total weight of the tobacco material (weight by weight percentage). More preferably, the fermentation step includes maintaining the moisture content of the tobacco material during fermentation in an amount comprised between 35 and 45 percent by weight of the total weight of the tobacco material (weight by weight percentage). More preferably, the fermentation step includes maintaining the moisture content of the tobacco material at about 40 percent by weight during fermentation (weight by weight percentage). After drying, the moisture of the tobacco material is generally low. Therefore, it is preferred that water is added to the tobacco material to reach the desired moisture level. More preferably, water is also added during the fermentation process to maintain the moisture content of the tobacco material in an amount comprised between 10 and 50 percent by weight of the total weight of the tobacco material, preferably between 35 and 45 percent by weight, more preferably about 40 percent by weight, for at least one month, more preferably at least two months, preferably at least six months, even more preferably at least eight months, preferably at least twelve months, preferably at least twenty-four months.

[0073] To reach this moisture content, the tobacco material is preferably moistened with water. Water is added to the tobacco material. The tobacco material is preferably moistened with water before being introduced into a container where anaerobic conditions are created and maintained.

[0074] Furthermore, this moisture content is maintained during the fermentation process. Therefore, it is preferred that the moisture content of the tobacco material is monitored during the fermentation process. For example, when the tobacco material is introduced into a vessel in which fermentation occurs, the vessel may be opened and the moisture of the tobacco material may be measured when the vessel is opened. It is preferred that the vessel is opened at regular intervals to perform the tobacco moisture measurement.

[0075] The moisture may be measured by a moisture sensor located inside the container. In this way, the moisture may be measured even when the tobacco material is in the sealed container.

[0076] The moisture content of the tobacco material is preferably measured at regular intervals.

[0077] Preferably, the fermentation process lasts for a fermentation time of at least 1 month, preferably at least 2 months, more preferably at least 4 months, more preferably at least 6 months, even more preferably at least 8 months, preferably at least 10 months. Preferably, the fermentation process lasts for at least 12 months. Preferably, the fermentation process lasts for at least 24 months. Preferably, the tobacco material is subjected to fermentation conditions for a fermentation time of less than 36 months, more preferably for a fermentation time of less than 24 months. Preferably, the fermentation process comprises multiple fermentation stages, each fermentation stage lasting for the fermentation period. The overall fermentation process lasts for a fermentation time of at least 1 month, preferably at least 2 months, more preferably at least 4 months, more preferably at least 6 months, even more preferably at least 8 months, preferably at least 10 months, preferably at least 12 months, preferably at least 24 months.

[0078] The fermentation time may depend on at least one of the type of tobacco material, or characteristics of the tobacco material, or parameters of the fermentation process, such as, for example, temperature, pressure, relative humidity, etc.

[0079] The application of the fermentation conditions may continue for all the claimed time (e.g., more than one month, or more than two months, or more than six months, or more than twelve months, or more than twenty-four months). Alternatively, the fermentation process includes multiple fermentation stages lasting a certain fermentation period. In this case, the fermentation conditions may be applied during multiple time intervals to form a series of time intervals. The different fermentation stages are separated from each other by interruptions during which the fermentation conditions are not applied. An interruption is considered to be a period during which one or more of the following conditions are interrupted: the presence of anaerobic conditions, a moisture content comprised between 25 percent and 40 percent by weight of the total weight of the tobacco material, application of a pressure comprised between 1000 kilograms per square meter and 4000 kilograms per square meter. The interruption may occur to examine the tobacco material. For example, the moisture of the tobacco material may be measured during the interruption. The interruption may occur to rotate or mix the tobacco material so that a uniform processed tobacco material may be obtained. The interruption may last up to six hours.

[0080] The fermentation time is therefore the total period during which the tobacco is subjected to the fermentation conditions described above, which is calculated by adding up all the fermentation periods for each fermentation stage during which the fermentation conditions are actually applied. Alternatively, the fermentation time can be calculated starting from the moment the fermentation conditions are applied for the first time and ending when the fermentation conditions are applied for the last time, minus any periods of interruption thereafter.

[0081] For example, if a fermentation time T (where the total period during which the tobacco material is subjected to fermentation conditions is referred to as the "fermentation time") is selected, the following cases are possible: the fermentation conditions are applied continuously for a total duration equal to T, in which case there is only one fermentation stage; or multiple fermentation stages are provided, in which the fermentation conditions are applied over N fermentation periods: t1, t2, ..., t N where t1+t2+...+t N = T. Fermentation period t j and the following time interval t j+1and the time gap between them is an interruption. This total fermentation time T is at least one month, or at least two months, or at least six months, or at least twelve months, or at least twenty-four months). Preferably, there is an interruption between two successive time intervals during which the fermentation conditions are applied. The interruption does not last more than six hours. Preferably, each fermentation stage lasts for about 15 days, more preferably for about 30 days. Preferably, each fermentation stage lasts for between about 30 days and about 60 days.

[0082] Preferably, the method involves drying the tobacco material to obtain a dried tobacco material having a moisture content comprised between 5 percent and 10 percent by weight of the total weight of the tobacco material. The drying step is preferably carried out after the fermentation step under fermentation conditions is completed. After the fermentation step is completed, the treated tobacco material is preferably removed from the container in which it was placed and the pressure applied to the tobacco is preferably reduced. The treated tobacco material is then dried to a moisture content between 1 percent and 15 percent by weight of the total weight of the tobacco material, more preferably between 5 percent and 10 percent by weight. The drying is carried out such that the treated tobacco material can be easily processed in subsequent steps.

[0083] The method preferably includes a step of drying the tobacco material prior to the fermentation step. The tobacco material processed by the method of the present invention may include cured tobacco. As used herein, the term "cured tobacco" refers to tobacco that has been cured. The drying of the tobacco is preferably achieved by standard procedures and may depend on the type of tobacco contained in the tobacco material. The tobacco material may include different types of tobacco, and tobacco that has been subjected to different curing. Different types of tobacco may be blended and then processed by the present invention.

[0084] Alternatively, the tobacco material is placed in a container and the air is removed and replaced with water.

[0085] The container in which the tobacco material is placed is, for example, a barrel, which is preferably made of wood, or concrete, or metal, or any combination of these three materials.

[0086] The anaerobic conditions are maintained for the duration of the desired fermentation process.

[0087] Preferably, the method provides for assessing the color of the tobacco material. The assessment of the color of the tobacco material may be performed in addition to the measurement of desired chemicals. The color of the tobacco material changes during the fermentation process. The assessment of the color of the tobacco material makes it possible to easily obtain an indication regarding the degree of fermentation.

[0088] Preferably, the method provides for stopping the fermentation process when the color of the tobacco material reaches a desired color. For example, fermentation conditions can be applied until the desired color of the tobacco material is obtained. In this way, it is possible to improve the effectiveness of the method of the present invention.

[0089] Preferably, the method provides for maintaining the temperature of the tobacco material during the fermentation step at a temperature comprised between 21°C and 35°C, preferably between 25°C and 31°C. Preferably, the temperature of the tobacco material during the fermentation step (while fermentation conditions are applied) remains within the range of 21°C and 35°C, more preferably between 25°C and 31°C. The temperature of the tobacco material is substantially maintained within this range during the entire fermentation step. The temperature is maintained by the fermentation itself, without the need to provide or subtract heat to the tobacco material. This temperature of the fermenting tobacco material is obtained when the ambient temperature around where the tobacco material is located is preferably comprised between 15°C and 25°C.

[0090] Preferably, the method includes a step of rotating the tobacco material during the fermentation step. Rotating the tobacco material may provide improved homogenization. Rotating the tobacco material may mean turning the tobacco material upside down. Rotating the tobacco material may mean turning the tobacco material upside down. The interruption of the fermentation conditions caused by the rotation may also be used to measure certain parameters of the tobacco material, such as the moisture content. During the rotation of the tobacco material, the fermentation conditions may no longer be applied. During the rotation of the tobacco, the fermentation process may be interrupted, in the sense that the tobacco material may not be subjected to anaerobic conditions during the rotation. After the rotation, the fermentation conditions are preferably applied again to the tobacco material. Preferably, the method includes a step of rotating the tobacco material for a time interval of about 30 days. Preferably, the method includes a step of rotating the tobacco material for a time interval of about 15 days.

[0091] The different fermentation stages of the fermentation process are preferably interrupted by a rotation process.

[0092] The method preferably includes securing the tobacco material in a moisture-retaining material. This step of securing the tobacco material preferably occurs before the tobacco material is subjected to fermentation conditions. The moisture-retaining material desirably resists degradation during the tobacco treatment process (fermentation). The moisture-retaining material may comprise a flexible material. This flexible material may be wrapped around the tobacco material. The moisture-retaining material preferably comprises a plastic material. Alternatively, or additionally, the moisture-retaining material may comprise a rigid material. The container into which the tobacco material is introduced may function as the moisture-retaining material. In this case, the material of the container may comprise, for example, metal, wood, plastic, or concrete.

[0093] Preferably, the method further comprises mixing a quantity of the non-fermented tobacco material with a desired quantity of the fermented or partially fermented tobacco material to obtain a tobacco material in which the amount of fermented tobacco material in the tobacco material comprises 5 weight percent to 10 weight percent of the total tobacco material. Preferably, the mixing step is provided before the fermentation step. It is preferably provided after the mixing step to subject the tobacco material to the fermentation step.

[0094] This improves the efficiency of the fermentation process, reduces the time required for the fermentation process, and improves the properties of the fermented tobacco material resulting from the fermentation process.

[0095] The method preferably includes a cutting step, in which the tobacco material is cut into pieces having dimensions comprised between about 0.3 mm and about 1.4 mm. The cutting step is preferably carried out before the fermentation step. This improves the efficiency of the fermentation step. This also reduces the duration of the fermentation step, i.e. the time required to obtain the desired change in the composition of the tobacco material.

[0096] Preferably, a method is further provided for maintaining the pH of the tobacco material during fermentation at a pH comprised between 4.5 and 5.5, preferably between about 4.8 and 5.4. The treated tobacco material is preferably at least 100 times more acidic than the untreated tobacco material. The pH of the treated tobacco material and the pH of the untreated tobacco material may differ by at least 2 pH units. The pH may remain substantially unchanged for the different tobacco materials.

[0097] Alternatively, or in addition, the tobacco material treated by the method of the present invention may include regraded tobacco, raw leaf blended tobacco, conditioned tobacco, stemmed or destemmed tobacco (or not in the case of whole leaf), cured tobacco, or stuffed tobacco.

[0098] According to another aspect, the present invention relates to a tobacco material obtained by a process comprising fermenting a tobacco material to obtain a treated tobacco material, the method comprising incubating the tobacco material under anaerobic conditions.

[0099] The treated tobacco material preferably contains lactic acid. The treated tobacco material preferably has a lactic acid content of more than 10 times, preferably more than 20 times, more preferably more than 50 times, more preferably more than 70 times, more preferably more than 80 times the initial amount of lactic acid in the tobacco material. In anaerobic fermentation, lactic acid is known to be a relevant catabolic product. Lactic acid may have a "smoothing effect" on the astringency of nicotine. Lactic acid may be responsible for the decrease in pH of the treated tobacco material.

[0100] The treated tobacco material preferably has a content of reducing sugars that is less than 0.5, preferably less than 0.4, more preferably less than 0.2, more preferably less than 0.1 of the initial amount of reducing sugars in the tobacco material. The treated tobacco material preferably contains an amount of total reducing sugars that is at least 50 percent, more preferably 60 percent, and even more preferably 85 percent lower than the amount of total reducing sugars contained in the same tobacco material before treatment by the method of the previous embodiment. At the end of the fermentation process, the amount of reducing sugars is preferably at least 50 percent, more preferably 60 percent, and even more preferably 85 percent lower than the amount of reducing sugars contained in the same tobacco material before treatment. Reducing sugars are the sum of glucose, fructose, sucrose, and maltose. The majority of the reducing sugars in the treated tobacco material can be converted. Reducing sugar resources such as glucose and fructose present in the starting tobacco material can be used as an energy source by anaerobic bacteria. In the absence of oxygen, the glycolytic pathway converts glucose (or fructose) to pyruvate. Changes in the levels of these compounds can contribute to the desirable taste and aroma of the treated tobacco material.

[0101] The treated tobacco material preferably has a content of indole-3 lactic acid that is greater than 5 times, preferably greater than 10 times, preferably greater than 20 times the initial amount of indole-3 lactic acid in the tobacco material.

[0102] It is preferred that the treated tobacco material has a caffeic acid content of more than four times, preferably more than ten times, the initial amount of caffeic acid in the tobacco material.

[0103] It is preferred that the treated tobacco material has a quinic acid content that is greater than two times, and preferably greater than four times, the initial amount of quinic acid in the tobacco material.

[0104] The treated tobacco material preferably has an asparagine content of less than 0.5, preferably less than 0.4, preferably less than 0.3 of the initial amount of asparagine in the tobacco material.

[0105] It is preferred that the treated tobacco material has a glutamine content of less than 0.5, preferably less than 0.4, of the initial amount of glutamine in the tobacco material.

[0106] The treated tobacco material preferably has a content of L-ornithine that is more than 10 times, preferably more than 50 times, preferably more than 100 times the initial amount of L-ornithine in the tobacco material.

[0107] It is preferred that the treated tobacco material has an L-leucine content that is greater than two times, preferably greater than six times, the initial amount of L-leucine in the tobacco material.

[0108] It is preferred that the treated tobacco material has an L-lysine content that is greater than two times, preferably greater than six times, the initial amount of L-lysine in the tobacco material.

[0109] The treated tobacco material preferably has a fermentation index greater than 50, preferably greater than 100, more preferably greater than 250, more preferably greater than 400. The fermentation index is obtained by dividing the ratio of the content of lactic acid in the tobacco material to the initial content of lactic acid by the ratio of the content of reducing sugars in the tobacco material to the initial content of reducing sugars.

[0110] According to a further aspect, the present invention relates to a tobacco processing material comprising at least one of the following characteristics: - lactic acid in an amount greater than 10 times, preferably greater than 20 times, more preferably greater than 50 times, more preferably greater than 70 times, more preferably greater than 80 times the initial amount of lactic acid in the tobacco material; - reducing sugars in an amount that is less than 0.5, preferably less than 0.4, more preferably less than 0.2, more preferably less than 0.1 of the initial amount of reducing sugars in the tobacco material; - indole-3 lactic acid in an amount greater than 5 times, preferably greater than 10 times, preferably greater than 20 times the initial amount of indole-3 lactic acid in the tobacco material; - caffeic acid in an amount greater than 4 times, preferably greater than 10 times, the initial amount of caffeic acid in the tobacco material; - quinic acid in an amount greater than two times, preferably greater than four times, the initial amount of quinic acid in the tobacco material; - asparagine in an amount less than 0.5, preferably less than 0.4, preferably less than 0.3 of the initial amount of asparagine in the tobacco material; - glutamine in an amount less than 0.5, preferably less than 0.4, of the initial amount of glutamine in the tobacco material; - L-ornithine is more than 10 times, preferably more than 50 times, preferably more than 100 times the initial amount of L-ornithine in the tobacco material; - L-leucine in an amount greater than twice, preferably greater than four times, the initial amount of L-leucine; - L-lysine in an amount greater than twice, preferably greater than six times, the initial amount of L-lysine; a fermentation index greater than 50, preferably greater than 100, more preferably greater than 250, more preferably greater than 400, where the fermentation index is obtained by dividing the ratio of the content of lactic acid in the tobacco material to the content of lactic acid in the non-fermented tobacco material by the ratio of the content of reducing sugars in the tobacco material to the content of reducing sugars in the non-fermented tobacco material.

[0111] A processed tobacco material having one or more of the aforementioned parameters has improved organoleptic properties. The aforementioned parameters make it possible to discern the quality and possible evaluation of the tobacco material from users. Furthermore, the aforementioned parameters influence the organoleptic properties and thus the final taste of the tobacco material. Based on the values ​​of the aforementioned parameters, it is therefore possible to predict the user group to which the tobacco material is preferably directed. Based on the values ​​of one or more of the indicated parameters, it is in fact possible to predict which users will appreciate the tobacco material more highly.

[0112] Preferably, the treated tobacco material has a content of 3-sec-butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione that is more than 20 times, preferably more than 40 times, more preferably more than 60 times, more preferably more than 80 times the initial amount of 3-sec-butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione in the tobacco material.

[0113] Preferably, the treated tobacco material has a content of secoisolariciresinol (SECO) that is greater than 10 times, preferably greater than 20 times, more preferably greater than 40 times, more preferably greater than 50 times the initial amount of secoisolariciresinol (SECO) in the tobacco material.

[0114] According to another aspect, the present invention relates to a tobacco material containing at least 20 milligrams / gram, preferably at least 50 milligrams / gram, of lactic acid on a total dry weight basis.

[0115] According to another aspect, the present invention relates to a tobacco material containing less than 3 percent total reducing sugars on a total dry weight basis.

[0116] According to another aspect, the present invention relates to a tobacco material comprising less than 300 milligrams per kilogram of asparagine on a total dry weight basis.

[0117] According to another aspect, the present invention relates to a tobacco material comprising less than 70 milligrams per kilogram of glutamine on a total dry weight basis.

[0118] According to another aspect, the present invention relates to a tobacco material comprising greater than 10,000 milligrams / kilogram of asparagine by total dry weight of total free amino acids.

[0119] According to another aspect, the present invention relates to a tobacco material comprising more than 10 times, preferably more than 20 times, the initial amount of L-ornithine in the tobacco material.

[0120] According to another aspect, the present invention relates to a tobacco material containing more than 50 milligrams / kilogram, preferably more than 80 milligrams / kilogram, of L-ornithine on a total dry weight basis.

[0121] The tobacco material of any one of the preceding aspects may be obtained by a process comprising fermenting the tobacco material, the method comprising incubating the tobacco material under anaerobic conditions.

[0122] The tobacco material is preferably dried. The tobacco material is preferably dried before being subjected to the fermentation process.

[0123] According to another aspect of the present invention, there is provided a dark tobacco material having a content of 2,3 butanediol that is greater than 5 times, preferably greater than 10 times, the initial amount of 2,3 butanediol in the tobacco material.

[0124] According to another aspect of the present invention, a dark tobacco material is provided having a diacetyl content of more than 5 times, preferably more than 10 times, the initial amount of diacetyl in the tobacco material.

[0125] According to another aspect of the present invention, there is provided a Virginia tobacco material as the tobacco material.

[0126] According to a further aspect of the present invention, there is provided an aerosol-generating article comprising tobacco material containing from about 2.5 weight percent on a total dry weight basis to 100 weight percent on a total dry weight basis, preferably at least about 4 weight percent on a total dry weight basis, preferably at least about 10 weight percent on a total dry weight basis, the percentages being given by weight on a total dry weight basis of tobacco fermentation material according to any one of the preceding aspects.

[0127] According to a further aspect of the present invention there is provided an aerosol-generating article containing at least 5 milligrams / gram, preferably 10 milligrams / gram, of lactic acid on a total dry weight basis.

[0128] According to another aspect of the invention, there is provided an aerosol-generating article containing less than 300 milligrams / kilogram of asparagine on a total dry weight basis.

[0129] According to another aspect of the present invention, there is provided an aerosol-generating article containing less than 70 milligrams / kilogram of glutamine on a total dry weight basis.

[0130] According to another aspect of the present invention, there is provided an aerosol-generating article containing less than 10,000 milligrams / kilogram of asparagine by total dry weight of total free amino acids.

[0131] The advantages of the tobacco material of the present invention have already been outlined with reference to the previous embodiment and will not be repeated here.

[0132] The tobacco material preferably comprises hand stripped leaves with the veins removed.

[0133] Preferably, the tobacco material comprises Kasturi tobacco.

[0134] Preferably, the tobacco material comprises Virginia tobacco.

[0135] Preferably, the tobacco material comprises dark tobacco.

[0136] Tobacco treated by the method of the present invention may change its chemical composition with respect to untreated tobacco. "Treated tobacco material" in this context means tobacco material that has been treated as described in a previous process, i.e. tobacco material that has been subjected to fermentation conditions for at least one month. "Untreated tobacco material" in this context means tobacco material that has not been treated as described in a previous process, i.e. tobacco material that has not been subjected to a fermentation step. Untreated tobacco material is for example tobacco material that is inserted into a container before the treatment of the present invention begins. Treated tobacco material is compared to the same tobacco material that has not been treated according to the present invention (untreated tobacco material). The reduction in asparagine may be associated with an increase in aspartate. This suggests that fermentation bacteria produce specific asparaginases to assimilate C and N from amino acid resources. This reaction may produce ammonia.

[0137] Non-fermented tobacco material in this context means tobacco material that has not been treated as described in the previous method, i.e. tobacco material that has not been subjected to a fermentation process. Fermented or partially fermented tobacco material in this context means tobacco material that has been treated as described in the previous method, i.e. tobacco material that has been subjected to a fermentation process for a particular period of time.

[0138] As used herein, the term "change" or "changed" is used in the context of flavor or organoleptic properties and means that there is a modification from one overall taste or sensory property to another as identified by a skilled smoker, which may include an improvement.

[0139] For purposes of this disclosure, the term "initial" refers to the amounts of chemicals, or generally the values ​​of parameters of a tobacco material, before the tobacco material is subjected to a fermentation process. In this sense, the term initial may be considered synonymous with non-fermented.

[0140] As used therein, the definitions of "fermentation process," or "fermentation," or "fermentation conditions" all indicate that materials are subjected to suitable conditions for a desired period of time to cause fermentation of the materials.

[0141] The term "tobacco material" refers to any part of the tobacco plant or a mixture of different tobacco plants, including, but not limited to, tobacco waste, unprocessed tobacco waste, tobacco stems, tobacco dust generated during tobacco processing, tobacco prime lamina strips, and combinations thereof. The tobacco material may have the form of processed tobacco parts or pieces, dried and aged tobacco in essentially natural lamina or stem form, tobacco extracts, or mixtures of the above, such as a mixture of extracted tobacco pulp combined with granulated dried and aged natural tobacco lamina. The tobacco material may be in solid, liquid, semi-solid, or the like. The term "tobacco material" preferably includes any part and any associated by-products, such as the leaves or stems of any member of the genus Nicotiana. Tobacco material for use in the present invention is preferably derived from the species Nicotiana tabacum. Any type, style, or variety of tobacco may be processed. Examples of tobacco that may be used may include, but are not limited to, Virginia, Burley, Orient tobacco, and any blends of these types. Preferably the tobacco material comprises Kasturi tobacco. The tobacco material to be treated may comprise or consist of tobacco after curing.

[0142] As used herein, the term "cured tobacco" refers to tobacco that has been cured but has not been subjected to any further processing processes to alter the taste or aroma of the tobacco material. The cured tobacco may be blended with other styles, varieties, or types of tobacco. Alternatively, or additionally, the processed tobacco material may include or consist of re-graded tobacco, unprocessed leaf blended tobacco, conditioned tobacco, stemmed or destemmed tobacco (or not in the case of whole leaf), cured tobacco, or stuffed tobacco.

[0143] Preferably, the tobacco material comprises laminar tobacco material. The tobacco may comprise between about 70 percent and 100 percent laminar material.

[0144] When the tobacco material comprises lamina tobacco material, the lamina may be in the form of whole leaves. In some embodiments, the tobacco material comprises cured whole leaf tobacco. In some embodiments, the tobacco material substantially comprises cured whole leaf tobacco. In some embodiments, the tobacco material consists essentially of cured whole leaf tobacco.

[0145] In some embodiments, the tobacco material comprises stem tobacco material. The tobacco may comprise up to 30 percent stem material.

[0146] The process of "curing" green tobacco depends on the type of tobacco harvested. For example, Virginia Flue (Bright) tobacco is typically flue-cured, while Burley and certain dark strains are usually air-cured. Flue-curing of tobacco is typically carried out over a period of five to seven days, compared to air-curing which may take place over a period of one to two months. A number of major chemical and biochemical changes are initiated during the curing process and continue throughout the early stages of leaf drying. The transformation of tobacco from yellow to brown generally results in the formation and significant accumulation of nitrosamines, and an increase in microbial content.

[0147] Different types of curing are used for different types of tobacco.

[0148] Virginia tobacco is generally "air-cured": tobacco leaves are hung in drying barns where heated air is turned on to dry the leaves. As the leaves lose moisture, they develop their unique aroma, texture, and color. Farmers must carefully manage 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 can have a negative effect on the quality of the tobacco.

[0149] Burley and Orient tobacco are dried in different ways. Burley is "air-dried" in barns where heat and humidity come from natural ventilation. The drying process can take up to two months. Orient tobacco is "sun-dried" by hanging the leaves outside in the sun for around two weeks.

[0150] In this text, the verbs "comprise" and "include" are synonymous and both indicate a non-exhaustive list of features. The verb "consist" indicates an exhaustive list.

[0151] The present invention is defined in the claims. However, below is provided a non-exhaustive list of non-limiting examples. Any one or more of the features of these examples may be combined with any one or more features of any other example, embodiment, or aspect described herein.

[0152] The present invention can be defined inter alia by the following examples.

[0153] Example 1. 1. A method for processing tobacco material, comprising: - providing tobacco materials; - fermenting a tobacco material to obtain a fermented tobacco material, - incubating the tobacco material under anaerobic conditions; - and the following conditions: the content of lactic acid is more than 10 times, preferably more than 20 times, more preferably more than 50 times, more preferably more than 70 times, preferably more than 80 times the initial amount of lactic acid in the tobacco material; - the content of reducing sugars is less than 0.5, preferably less than 0.4, more preferably less than 0.2, more preferably less than 0.1 of the initial amount of reducing sugars in the tobacco material; - the content of indole-3 lactic acid is more than 5 times, preferably more than 10 times, preferably more than 20 times the initial amount of indole-3 lactic acid in the tobacco material; - the caffeic acid content is more than 4 times, preferably more than 10 times, the initial amount of caffeic acid in the tobacco material; - the content of quinic acid is more than twice, preferably more than four times, the initial amount of quinic acid in the tobacco material; - the content of asparagine is less than 0.5, preferably less than 0.4, preferably less than 0.3 of the initial amount of asparagine in the tobacco material; - the content of glutamine is less than 0.5, preferably less than 0.4, of the initial amount of glutamine in the tobacco material; - the content of L-ornithine is more than 10 times, preferably more than 50 times, preferably more than 100 times the initial amount of L-ornithine in the tobacco material; - the content of L-leucine is more than twice, preferably more than four times, the initial amount of L-leucine in the tobacco material; - the content of L-lysine is more than twice, preferably more than six times, the initial amount of L-lysine in the tobacco material; - fermenting a tobacco material, comprising: stopping the fermentation step when at least one of the following conditions is met: a fermentation index is greater than 50, preferably greater than 100, more preferably greater than 250, more preferably greater than 400, where the fermentation index is obtained by dividing the ratio of the content of lactic acid in the tobacco material to the initial content of lactic acid by the ratio of the content of reducing sugars in the tobacco material to the initial content of reducing sugars. Example 2. The method of example 1, further comprising an initial measurement step for measuring an initial content of at least one of lactic acid, or reducing sugars, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or a fermentation index in the tobacco material. Example 3. The method according to example 1 or 2, further comprising a measuring step for measuring the content of at least one of lactic acid, or reducing sugar, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or a fermentation index in the tobacco material during the fermentation process. Example 4. The method according to any one of Examples 1 to 3, wherein when the tobacco material contains dark tobacco, the method is provided for stopping fermentation when at least one of the following conditions is met: the content of 2,3 butanediol is more than 5 times, preferably more than 10 times, the initial amount of 2,3 butanediol in the tobacco material; or the content of diacetyl is more than 5 times, preferably more than 10 times, the initial amount of diacetyl in the tobacco material. Example 5. 5. The method of example 4, further comprising an initial measuring step for measuring an initial content of 2,3 butanediol or diacetyl in the tobacco material before fermentation to obtain an initial amount of 2,3 butanediol or diacetyl, respectively, in the tobacco material. Example 6. The method according to any one of Examples 4 to 5, further comprising a measuring step for measuring the content of 2,3 butanediol or diacetyl in the tobacco material during the fermentation step. Example 7. 7. The method according to any one of Examples 1 to 6, comprising applying a pressure of from 1000 kilograms / m2 to 15000 kilograms / m2, preferably from 3000 kilograms / m2 to 12000 kilograms / m2, more preferably from 5000 kilograms / m2 to 10000 kilograms / m2 to the tobacco material during the fermentation step. Example 8. The method according to any one of Examples 1 to 7, comprising, during the fermentation step, maintaining the moisture content of the tobacco material during fermentation in an amount comprised between 10 weight percent and 50 weight percent, preferably between 35 weight percent and 45 weight percent, and more preferably about 40 weight percent of the total weight of the tobacco material. Example 9. The method according to Examples 1 to 8, provided for continuing the fermentation process for a fermentation time of at least 1 month, preferably at least 2 months, more preferably at least 4 months, more preferably at least 6 months, even more preferably at least 8 months, preferably at least 10 months, more preferably at least 12 months. Example 10. The method according to any one of Examples 1 to 9, provided that the fermentation process continues for a fermentation time of at least 24 months. Example 11. 11. The method of any one of Examples 1-10, further comprising drying the tobacco material to obtain a dried tobacco material having a moisture content comprised between 5 weight percent and 10 weight percent of the total weight of the tobacco material. Example 12. 12. The method according to any one of Examples 1 to 11, further comprising a drying step for drying the tobacco material prior to the fermentation step. Example 13. 13. The method according to any one of Examples 1 to 12, comprising maintaining the temperature of the tobacco material at a temperature comprised between 21°C and 35°C, preferably between 25°C and 31°C. Example 14. The method according to one or more of Examples 1 to 13, comprising rotating the tobacco material at a time interval of preferably about 15 days, more preferably at a time interval of about 30 days, preferably at a time interval of from about 30 days to about 60 days. Example 15. The method of any one or more of Examples 1-14, comprising securing the tobacco material within a moisture-retaining material. Example 16. The method of one or more of Examples 1-15, comprising wetting the tobacco material in water prior to fermentation to achieve a moisture content of the tobacco material of about 10 weight percent to about 50 weight percent of the total weight of the tobacco material, preferably about 35 weight percent to about 45 weight percent of the total weight, preferably about 40 weight percent of the total weight. Example 17. The method according to any one of Examples 1 to 16, wherein a certain amount of non-fermented tobacco material is mixed with a desired amount of fermented tobacco material to obtain tobacco material, and then the tobacco material is subjected to a fermentation process, wherein the amount of fermented tobacco material in the tobacco material comprises 5 weight percent to 10 weight percent of the tobacco material. Example 18. The method according to any one of Examples 1 to 17, further comprising a final measurement step for measuring the content of at least one of lactic acid, or reducing sugar, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or a fermentation index in the tobacco material after the fermentation step. Example 19. The method according to any one of Examples 1 to 18, further comprising maintaining the pH of the tobacco material during fermentation at a pH comprised between 4.5 and 5.5, preferably between about 4.8 and 5.4. Example 20. 1. A method for processing tobacco material, comprising: - providing tobacco materials; - fermenting a tobacco material to obtain a fermented tobacco material, the fermentation step comprising: - fermenting, comprising incubating the tobacco material under anaerobic conditions, wherein the incubating provides for applying a pressure to the tobacco material during the fermentation step of between 1000 kilograms / square meter and 15000 kilograms / square meter, preferably between 3000 kilograms / square meter and 12000 kilograms / square meter, more preferably between 5000 kilograms / square meter and 10000 kilograms / square meter. Example 21. 1. A method for processing tobacco material, comprising: - providing tobacco materials; - fermenting a tobacco material to obtain a fermented tobacco material, the fermentation step comprising: - fermenting, including incubating the tobacco material under anaerobic conditions, wherein incubating is provided to continue the fermentation process for a fermentation time of at least 1 month, preferably at least 2 months, more preferably at least 4 months, more preferably at least 6 months, even more preferably at least 8 months, preferably at least 10 months, more preferably at least 12 months. Example 22. 1. A method for processing tobacco material, comprising: - providing tobacco materials; - fermenting a tobacco material to obtain a fermented tobacco material, the fermentation step comprising: - fermenting, including incubating the tobacco material under anaerobic conditions, wherein the incubating provides for maintaining the temperature of the tobacco material at a temperature comprised between 21°C and 35°C, preferably between 25°C and 31°C, during the fermentation process. Example 23. The method of any one of Examples 1 to 22, further comprising providing a database comprising the amount of at least one of lactic acid, or reducing sugars, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or secoisolariciresinol (SECO), or a fermentation index, or butanediol, or diacetyl in the fermented tobacco material and / or partially fermented tobacco material. Example 24. The method of any one of Examples 1 to 23, comprising providing a database comprising amounts of at least one of lactic acid, or reducing sugars, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or secoisolariciresinol (SECO), or a fermentation index, or butanediol, or diacetyl, in the fermented tobacco material and / or partially fermented tobacco material for a plurality of different tobacco materials. Example 25. The method of any one of Examples 26 and 27, comprising recovering from at least one database an amount of at least one of lactic acid, or reducing sugars, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or secoisolariciresinol (SECO), or a fermentation index, or butanediol, or diacetyl, and comparing the recovered value to a corresponding measured value in the fermenting tobacco material. Example 26. The method of example 28, further comprising continuing the fermentation process if the measured values ​​of lactic acid, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or L-leucine, or L-lysine, or butylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, or secoisolariciresinol (SECO), or the fermentation index, or butanediol, or diacetyl are lower than the corresponding recovered values, and / or if the values ​​of reducing sugars, or asparagine, or glutamine, or the fermentation index are higher than the corresponding recovered values. Example 27. A tobacco material obtained according to the method described in any one of Examples 1 to 26. Example 28. 1. A tobacco material obtained by a process comprising fermenting a tobacco material, the process comprising incubating the tobacco material under anaerobic conditions, wherein at least one of the following conditions is met in the tobacco material: the content of lactic acid is more than 10 times, preferably more than 20 times, more preferably more than 50 times, more preferably more than 70 times, more preferably more than 80 times the initial amount of lactic acid in the tobacco; - the content of reducing sugars is less than 0.5, preferably less than 0.4, more preferably less than 0.2, more preferably less than 0.1 of the initial amount of reducing sugars in the tobacco material; - the content of indole-3 lactic acid is more than 5 times, preferably more than 10 times, preferably more than 20 times the initial amount of indole-3 lactic acid in the tobacco material; - the caffeic acid content is more than 4 times, preferably more than 10 times, the initial amount of caffeic acid in the tobacco material; - the content of quinic acid is more than twice, preferably more than four times, the initial amount of quinic acid in the tobacco material; - the content of asparagine is less than 0.5, preferably less than 0.4, preferably less than 0.3 of the initial amount of asparagine in the tobacco material; - the content of glutamine is less than 0.5, preferably less than 0.4, of the initial amount of glutamine in the tobacco material; - the content of L-ornithine is more than 10 times, preferably more than 50 times, preferably more than 100 times the initial amount of L-ornithine in the tobacco material; - the content of L-leucine is more than twice, preferably more than four times, the initial amount of L-leucine in the tobacco material; - the content of L-lysine is more than twice, preferably more than six times, the initial amount of L-lysine in the tobacco material; a fermentation index greater than 50, preferably greater than 100, more preferably greater than 250, more preferably greater than 400, where the fermentation index is obtained by dividing the ratio of the content of lactic acid in the tobacco material to the content of lactic acid in the non-fermented tobacco material by the ratio of the content of reducing sugars in the tobacco material to the content of reducing sugars in the non-fermented tobacco material. Example 29. A tobacco material containing at least one of the following characteristics: - lactic acid in an amount greater than 10 times, preferably greater than 20 times, more preferably greater than 50 times, more preferably greater than 70 times, more preferably greater than 80 times the initial amount of lactic acid in the tobacco material; - reducing sugars in an amount that is less than 0.5, preferably less than 0.4, more preferably less than 0.2, more preferably less than 0.1 of the initial amount of reducing sugars in the tobacco material; - indole-3 lactic acid in an amount greater than 5 times, preferably greater than 10 times, preferably greater than 20 times the initial amount of indole-3 lactic acid in the tobacco material; - caffeic acid in an amount greater than 4 times, preferably greater than 10 times, the initial amount of caffeic acid in the tobacco material; - quinic acid in an amount greater than two times, preferably greater than four times, the initial amount of quinic acid in the tobacco material; - asparagine in an amount less than 0.5, preferably less than 0.4, preferably less than 0.3 of the initial amount of asparagine in the tobacco material; - glutamine in an amount less than 0.5, preferably less than 0.4, of the initial amount of glutamine in the tobacco material; - L-ornithine is more than 10 times, preferably more than 50 times, preferably more than 100 times the initial amount of L-ornithine in the tobacco material; - L-leucine in an amount greater than twice, preferably greater than four times, the initial amount of L-leucine; - L-lysine in an amount greater than twice, preferably greater than six times, the initial amount of L-lysine; a fermentation index greater than 50, preferably greater than 100, more preferably greater than 250, more preferably greater than 400, where the fermentation index is obtained by dividing the ratio of the content of lactic acid in the tobacco material to the content of lactic acid in the non-fermented tobacco material by the ratio of the content of reducing sugars in the tobacco material to the content of reducing sugars in the non-fermented tobacco material. Example 30. The tobacco material according to Examples 1 to 29, wherein the tobacco material is obtained by a process comprising fermenting the tobacco material to obtain a treated tobacco material, the process comprising incubating the tobacco material under anaerobic conditions. Example 31. A tobacco material containing at least 20 milligrams / gram, preferably at least 50 mg / gram, and more preferably at least 60 milligrams / gram, of lactic acid on a total dry weight basis. Example 32. Tobacco material containing, on a total dry weight basis, less than 3 percent total reducing sugars. Example 33. Tobacco material containing, on a total dry weight basis, less than 300 milligrams per kilogram of asparagine. Example 34. 1. A tobacco material containing, on a total dry weight basis, less than 70 milligrams per kilogram of glutamine. Example 35. 1. A tobacco material containing, on a total dry weight basis, greater than 10,000 milligrams per kilogram of total free amino acids. Example 36. A tobacco material comprising at least 1 microgram / gram, preferably at least 2 micrograms / gram, more preferably at least 2.5 micrograms / gram of indole-3 lactic acid. Example 37. The tobacco material according to one or more of Examples 27 to 36, wherein the tobacco material is dried. Example 38. The tobacco material of one or more of Examples 27 to 37, wherein the tobacco material is ground. Example 39. An aerosol-generating article comprising tobacco material, comprising from about 2.5 weight percent on a total dry weight basis to 100 weight percent on a total dry weight basis, preferably at least about 4 weight percent on a total dry weight basis, preferably at least about 10 weight percent on a total dry weight basis, preferably at least about 20 weight percent on a total dry weight basis of the tobacco material described in any one of Examples 27-38. Example 40. 1. An aerosol-generating article comprising a tobacco material containing at least 5 milligrams / gram, preferably 10 milligrams / gram, of lactic acid. Example 41. An aerosol-generating article, including tobacco material, containing less than 300 milligrams per kilogram of asparagine on a total dry weight basis. Example 42. An aerosol-generating article, including tobacco material, containing less than 70 milligrams per kilogram of glutamine on a total dry weight basis. Example 43. An aerosol-generating article, including tobacco material, containing more than 10,000 milligrams of asparagine per kilogram of total dry weight of total free amino acids.

[0154] The embodiments will now be further described with reference to the following figures: [Brief description of the drawings]

[0155] [Figure 1] 1 and 2 are histograms representing the amount of lactic acid in the tobacco materials of Examples 1 and 2, measured respectively before (0T) and after 6 months (3T) of fermentation according to the invention. [Diagram 2] Same as above. [Diagram 3]3 and 4 are histograms showing the amount of total alkaloid (TA) levels (percent of total dry weight basis DW) in the tobacco materials of Examples 1 and 2 measured before (0T) and during fermentation, respectively, according to the present invention. [Figure 4] Same as above. [Diagram 5] 5 and 6 are histograms showing the amounts of glutamine and glutamic acid, respectively, in the tobacco materials of Examples 1 and 2 (on a total dry weight basis, DW) measured before (0T) and during fermentation according to the present invention. [Figure 6] Same as above. [Figure 7] 7 and 8 are histograms depicting the amounts of asparagine and aspartic acid, respectively, in the tobacco materials of Examples 1 and 2 (on a total dry weight basis, DW) measured before (0T) and during fermentation in accordance with the present invention. [Figure 8] Same as above. [Figure 9] Figures 9 and 10 are histograms showing the amounts of total alkaloids (Figure 9) and reducing sugars (Figure 10), respectively, in the tobacco material of Example 3 (on a total dry weight basis, DW) measured before (VG-BF), during, and after fermentation (VG-AF) according to the present invention. [Figure 10] Same as above. [Figure 11] Figure 11 shows the TA (total alkaloids), RS (reducing sugars), and ammonia (NH3) in the unfermented Virginia (VG) material (SM) during the fermentation process (T1–T7) and after fermentation (AF) on a total dry weight basis, as a percentage of DW. [Figure 12] FIG. 12 shows the amounts of glucose, fructose, citrate, malate, pyruvate, and lactate during anaerobic fermentation of Virginia tobacco in non-fermented Virginia material (SM) and after fermentation (AF). [Figure 13]Figures 13A and 13B show the free amino acids consumed (Figure 13A) and produced (Figure 13B) before and after fermentation of Virginia tobacco. Ratio data presented from metabolomic analysis (n=3), statistics are paired t-test (*p<0.05; **p<0.01; ***p<0.001); [Figure 14] Figure 14 shows the production of quinic acid and caffeic acid in Virginia tobacco fermentation. Ratio data presented from metabolomic analysis (n=3), statistics are paired t-test (*p<0.05; **p<0.01; ***p<0.001); [Figure 15] Figure 15 shows the accumulation of indole-3-lactic acid in fermented tobacco (AF) compared to non-fermented Virginia tobacco (SM) from enzymatic L-tryptophan degradation. Ratio data (n=3) presented from metabolomic analysis, statistics are paired t-test (*p<0.05; **p<0.01; ***p<0.001). [Figure 16] 16 shows the percentages of TA, RS, NO3, and NH3 at the start (0 months) and after 3 and 6 months of fermentation for the Virginia tobacco material of Example 4. The analysis was performed by the scalar method. [Figure 17] FIG. 17 shows the change in indole-3-lactic acid between the non-fermented material (control) and the fermented material (HF) in the tobacco materials of Example 1 (RAJ) and Example 2 (HS). [Figure 17A] FIG. 17A shows the change in indole-3-lactic acid in the tobacco material of Example 4 (VG-CH) in the non-fermented tobacco material (NF) and the fermented tobacco material (F). [Figure 18] FIG. 18 shows the change in L-ornithine between the non-fermented material (control) and the fermented material (HF) in the tobacco materials of Example 1 (RAJ) and Example 2 (HS). [Figure 19] FIG. 19 shows the change in BHHPPD between the non-fermented material (control) and the fermented material (HF) in the tobacco materials of Example 1 (RAJ) and Example 2 (HS). [Figure 20]FIG. 20 shows the change in secoisolariciresinol between the non-fermented material (control) and the fermented material (HF) in the tobacco materials of Example 1 (RAJ) and Example 2 (HS).

[0156] First and second tobacco materials were prepared that were the same tobacco type but underwent different processing prior to fermentation. The tobacco materials were Kasturi tobacco. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0157] Example 1 Dark tobacco leaf material was thoroughly sun-dried for approximately 10 days. The sun-dried leaves were stripped to retain only the lamina (hand-stripped leaves). This tobacco material is called "HS".

[0158] The tobacco material was conditioned to obtain a moisture content of approximately 30 percent. This conditioned, but not yet fermented, sample of tobacco material is referred to as 0T ("starting material").

[0159] The conditioned tobacco material is then introduced into three barrels, with approximately 100 kilograms of tobacco material being present in each barrel. Prior to introduction, the tobacco material is wrapped in the resulting moisture retaining material.

[0160] Pressure is applied to each barrel. The pressure ranges from 1000 kg / m2 to 4000 kg / m2.

[0161] After 1 month (samples designated 1T), 2.5 months (samples designated 2T), 6 months (samples designated 3T), and 8.5 months (samples designated 4T), the barrels were opened and at least three samples were taken from each barrel before turning the tobacco and before readjusting the moisture content to approximately 30 percent ± 5 percent.

[0162] During the heavy fermentation process under fully anaerobic conditions, the temperature inside the barrel did not increase significantly (it remained within the range of 27°C to 31°C). Fermentation was stopped after 8.5 months.

[0163] Example 2 Dark tobacco leaf material was allowed to yellow for two days and then rapidly shredded with a cut filler. This tobacco material contained both lamina and veins. The shredded leaves, containing both lamina and midrib, were sun-dried for two days. This tobacco material sample is hereinafter designated "CC".

[0164] The tobacco material was conditioned to obtain a moisture content of approximately 30 percent. This conditioned, but not yet fermented, sample of tobacco material is referred to as 0T ("starting material").

[0165] The conditioned tobacco material is then introduced into three barrels, with approximately 100 kilograms of tobacco material being present in each barrel. Prior to introduction, the tobacco material is wrapped in the resulting moisture retaining material.

[0166] Pressure is applied to each barrel. The pressure ranges from 1000 kg / m2 to 4000 kg / m2.

[0167] After 1 month (samples designated 1T), 2.5 months (samples designated 2T), 6 months (samples designated 3T), and 8.5 months (samples designated 4T), the barrels were opened and at least three samples were taken from each barrel before turning the tobacco and before readjusting the moisture content to approximately 30 percent ± 5 percent.

[0168] During the heavy fermentation process under fully anaerobic conditions, the temperature inside the barrel did not increase significantly (it remained within the range of 27°C to 31°C). Fermentation was stopped after 8.5 months.

[0169] Visual Observation The initial tobacco material had already changed after 2.5 months of fermentation (sample 2T), the color of both HS and CC leaves had become darker and the tobacco odor had developed a nice caramelized buttery and fermented complex character. The dark color was more pronounced in the fermented HS leaves compared to the CC leaves at the end of the process (8.5 months, 4T), which is probably due to the presence of the mid-vein in the CC leaves.

[0170] chemical analysis In what follows, when values ​​relating to a sample are mentioned, the value given represents the average of several values ​​obtained for each sample of the same type.

[0171] After fermentation conditions were applied for 2.5 months (as found for sample 2T), the pH of the tobacco material samples (both CC and HS) became acidic, reaching 3.2, reflecting the process of anaerobic fermentation accompanied by sugar decomposition, which usually produces organic acids such as (acetic and / or) lactic acid. The starting pH of tobacco material is generally comprised between pH 5 and pH 6.

[0172] Figures 1 and 2 show the presence of lactic acid in tobacco materials. As shown by the figures (Figure 1 represents the lactic acid content of HS leaves and Figure 2 represents the lactic acid content of CC leaves), before fermentation, all samples are free of lactic acid (three samples 0T per tobacco material (CC or HS) are shown). After fermentation (in this case after 6 months, three samples for a tobacco material called 3T are shown for both tobacco materials (CC or HS)), all samples (both CC and HS leaves) show the presence of lactic acid, albeit in variable amounts.

[0173] Alkaloids did not degrade or degraded only slightly during fermentation. Total alkaloid (TA) content in percent of total dry weight basis (indicated as percent DW in the figures) is shown in Figure 3 (HS leaves) and Figure 4 (chopped leaves, CC leaves). Total alkaloid content remained fairly stable during fermentation. After 8.5 months (4T), only 4 percent was degraded in HS leaves and 9 percent in CC leaves. Although statistically relevant, such small variations may only result from sampling. Some limited alkaloid hydrolase activity may not be excluded. Total alkaloids were analyzed in samples collected at the beginning (0T, n=6 samples analyzed), after 1 month (1T, n=9), after 2.5 months (2T, n=9), after 6 months (3T, n=9), and after 8.5 months (4T, n=12) during the heavy fermentation process. T-tests (test statistics) were performed for comparison with the control unfermented cured tobacco (0T). The results are shown in Figures 3 and 4 showing the p-values, which are given as follows: *, p < 0.05; **, p<0.01 and ***, p<0.001.

[0174] Sample 4T of HS leaves and sample 3T of CC leaves have p-values ​​<0.01, and samples 1T and 4T of CC leaves have p-values ​​<0.001, indicating a statistically significant difference between the fermented and non-fermented tobacco materials.

[0175] The nitrate content was not affected by the heavy fermentation process. However, some effects were observed on tobacco-specific nitrosamines (TSNAs): NNN (N'-nitrosonornicotine), NNK (nicotine-derived nitrosamine ketone), and NAT (N'-nitrosoanatabine). After 8.5 months of fermentation, no changes were measured for NNK and NAT. However, an increase in NNN was observed in both HS (3-fold increase) and CC (5-6-fold increase). As nornicotine, the precursor of NNN before nitration, did not increase accordingly. Thus, NAT and NNK may be partially degraded by bacteria during fermentation, but NNN may not be partially degraded. The reason is that NNK and NAT first increased 2-fold until 2.5 months of fermentation, then decreased and reached the initial value of non-fermented tobacco. This observation may imply that nitration of alkaloids occurs during heavy fermentation.

[0176] The evolution of sugars and free amino acids during heavy fermentation according to the invention has been analyzed. The values ​​of the measurements carried out on samples of tobacco material are collected in Table 1. Table 1 shows the evolution of sugars and amino acids during the heavy fermentation process, from an untreated tobacco material sample (sample 0T) to 8.5 months of the fermentation process (sample 4T) under fermentation conditions in barrels containing either hand-stripped (HS) or shredded (CC) leaves, as in Examples 1 and 2. All values ​​in the table are on a total dry weight basis. The units of reducing sugars are percent of the total dry weight basis, while free amino acids are in milligrams per kilogram of total dried tobacco material. The decrease in reducing sugars appeared after 2.5 months (2T, see Table 1), in sync with the color change and slurry acidification. Glucose and fructose are the two tobacco leaf substrates that can be metabolized by anaerobic bacteria in the fermentation barrel. Conversely, most amino acids increased during the process. Both asparagine and glutamine were greatly decreased. Overall, these observations may indicate that the main fermentation activity occurred between the first and third month. Proline was not degraded under anaerobic fermentation (see Table 1). Ornithine increased significantly (>100-fold) during fermentation in both HS and CC, and citrulline (data obtained from metabolomic analysis between 0T and 3T) increased 16-fold in HS and 2-fold in CC. This may indicate that (plant-derived) lactic acid bacteria are active in the tobacco fermentation barrels, since these bacteria have been described to produce ornithine and citrulline at high levels (Rakhimuzzaman et al.,Biol Pharm Bull.2019;42(9):1581-1589). [Table 1]

[0177] In Figures 5-8, the amounts of glutamine and asparagine in the tobacco materials are shown. As shown by Figures 5-8 and based on the data presented in Table 1, the deamination of glutamine and asparagine occurring during the heavy fermentation process of both HS and CC leaves can be correlated with the simultaneous increase of glutamate and aspartate, respectively. This suggests that the fermentation bacteria produce specific glutaminase and asparaginase to assimilate C and N from amino acid resources. Both reactions produce ammonia, which increased two-fold during the anaerobic fermentation process of both HS and CC leaves. Figures 5 and 6 show the levels of glutamine (white histograms) and glutamic acid (black histograms) in HS and CC leaves, respectively. It is clear from the figures that glutamine decreases and glutamic acid increases during fermentation. Figures 7 and 8 show the levels of asparagine (striped histograms) and aspartic acid (black histograms) in HS and CC leaves, respectively. It is clear from the figure that asparagine decreases and aspartic acid increases during fermentation.

[0178] A metabolomic study was conducted to identify marker molecules or pathways related to the tobacco leaf anaerobic fermentation process. Sugar resources such as glucose and fructose present in the starting materials (controls) of both HS and CC leaves can be used as energy sources by anaerobic bacteria (see Table 1). In the absence of oxygen, the glycolytic pathway converts glucose (or fructose) into pyruvate, producing two ATP and two NADH+H+. Other organic compounds and abundant carbon 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 the most abundant organic acids in plants. Citrate and malate are also metabolized during tobacco heavy fermentation, similar to reducing sugars. Chemical analysis of the samples shows that more than 60 percent of the glucose, fructose, citrate and malate present in the starting tobacco material (sample 0T), hand-stripped and shredded leaves are catabolized after six months of heavy fermentation (sample 3T). Another observation that can be linked to the consumption of these organic molecules is the increase in pyruvate (13-14-fold) in both HS and CC fermented tobacco materials. Pyruvate is a substrate for several reactions that can occur under anaerobic conditions: (1) the production of D-lactate to regenerate NAD+, mainly for glycolytic reactions, and (2) the production of acetate, diacetyl and 2,3-butanediol, which can contribute to the delivery of aromatic compounds and flavors in heavy fermented tobacco. Pyruvate, as a product of lactic acid bacteria, can lead to the generation of aromatic compounds such as 2,3-butanediol or lactic acid.

[0179] Two other pathways emerged from metabolomic analysis of heavily fermented tobacco: (1) tryptophan degradation and (2) chlorogenic acid catabolism.

[0180] Concerning tryptophan degradation, a pathway has been described by Ummadi and Weimer (2001, J. Dairy Sci. 84:1773-1782) for cheese bacteria and has been adapted accordingly. In this case, more than 78 percent of the tryptophan present in the starting tobacco material (sample 0T) is catabolized after 6 months of fermentation (sample 3T) in both HS and CC leaves. The pathway showed that the product resulting from these catabolic reactions is mainly indole-3-lactic acid, as indicated by a 14- and 28-fold increase in HS and CC leaves, respectively. Other compounds belonging to this pathway did not show such an increase. No specific aromatic properties were reported for this compound.

[0181] Chlorogenic acid (CGA), an important biologically active dietary polyphenol, is produced by certain plant species such as tobacco and is the main component of coffee. In heavily fermented tobacco leaves, CGA is completely degraded after the anaerobic fermentation process. Meanwhile, the products resulting from the catabolism of CGA, namely quinic acid and caffeic acid, increased after 6 months of fermentation in both HS and CC leaves. This is probably due to bacterial cinnamoyl esterase activity, as documented by Guglielmetti et al. (2008, Applied and Environmental Microbiology, 74, 4:1284-1288). Thus, a part of the quinic acid pool and the caffeic acid pool probably result from the hydrolysis of CGA, none of which was reported to have flavor properties.

[0182] The elevated presence of pyruvic acid, indole-3-lactic acid, and the lack of chlorogenic acid in heavily fermented tobacco compared to cured tobacco may make them useful as chemical markers.

[0183] For purposes of this specification and the appended claims, unless otherwise indicated, all numbers expressing amounts, quantities, percentages, and the like, should be understood in all instances as modified by the term "about." Also, all ranges include the maximum and minimum points disclosed, and include any intermediate ranges therein, which may or may not be specifically recited herein. Thus, in this context, the number A is understood as A±10 percent. Within this context, the number A may be considered to include a numerical value that is within the general standard error for the measurement of the property represented by the number A. The number A may deviate by the percentages recited above, in some cases as used in the appended claims, provided that the amount by which A deviates does not materially affect the basic and novel properties of the claimed invention. Also, all ranges include the maximum and minimum points disclosed, and include any intermediate ranges therein, which may or may not be specifically recited herein.

[0184] The tobacco materials of Examples 1 and 2 were also tested for some additional chemical compounds that may be associated with fermentation.

[0185] 17 shows the change in indole-3-lactic acid between the non-fermented material (control) and the fermented material (HF) in the tobacco materials of Example 1 (RAJ) and Example 2 (HS). These data show an increase in the content of indole-3-lactic acid in the fermented material.

[0186] Figure 18 shows the change in L-ornithine between the non-fermented (control) and fermented (HF) tobacco materials of Example 1 (RAJ) and Example 2 (HS), shown as the amount of L-ornithine and also the absolute change. These data show an increase in the content of L-ornithine in the fermented materials.

[0187] 19 shows the change in BHHPPD between the non-fermented material (control) and the fermented material (HF) in the tobacco materials of Example 1 (RAJ) and Example 2 (HS). These data show an increase in the content of BHHPPD in the fermented material.

[0188] 20 shows the change in secoisolariciresinol between the non-fermented material (control) and the fermented material (HF) in the tobacco materials of Example 1 (RAJ) and Example 2 (HS). These data show an increase in the content of secoisolariciresinol in the fermented material.

[0189] Example 3 Further testing was carried out in Asia on samples of Virginia tobacco. The tobacco leaf material was hot air dried as a standard procedure.

[0190] The tobacco dried material in the form of strips was then conditioned to obtain a moisture content of approximately 30 percent. This conditioned, but not yet fermented, sample of tobacco material is referred to as SM (unfermented starting material).

[0191] The conditioned tobacco material was then introduced into two barrels, with approximately 100 kilograms of tobacco material in each barrel. Prior to introduction, the tobacco material was wrapped with a resulting moisture-retaining material. The two barrels containing the Virginia tobacco material were subjected to anaerobic fermentation as described in Example 1. The temperature inside the fermentation barrels and the pH of the fermenting tobacco material were monitored throughout the experiment.

[0192] The barrels were opened after 1 month (a sample designated 1T), 2 months (a sample designated 2T), 3 months (a sample designated 3T), 4 months (a sample designated 4T), 5 months (a sample designated 5T), 6 months (a sample designated 6T), 7 months (a sample designated 7T), and 8 months (a sample designated AF, post-fermentation).

[0193] The tobacco material in the two barrels was rotated monthly during the seven month period of the experiment.

[0194] Samples were taken before fermentation (VG-SM: starting material, six replicates), during the fermentation process (every month from VG-T1 to VG-T7, three replicates per barrel), and after fermentation (VG-AF: post-fermentation, six replicates) as shown in Table 2 reported below. Sample characteristics were analyzed. [Table 2]

[0195] No significant temperature change was observed during the fermentation process under fully anaerobic conditions, which varied linearly from 30°C at the beginning of fermentation (VG-T1) to 26°C at the end of fermentation (VG-AF). Temperature was measured inside the barrel using a captor.

[0196] The pH of the tobacco material did not change significantly during the fermentation runs (T1-AF) and remained at 5.1 ± 0.3.

[0197] Visual Observation As seen in the case of the Kastori tobacco material, the color of the tobacco material at the end of the fermentation process (VG-AF) was significantly darker compared to the starting material (VG-SM). However, after 4 months of anaerobic fermentation (VG-T4), and therefore after 4 rotations, the Virginia tobacco material did not show a very dark coloration, suggesting that 4 months may not be sufficient to obtain complete fermentation of the Virginia tobacco material under the experimental conditions. After 8 months of anaerobic fermentation (VG-AF), and therefore after 8 rotations, the Virginia tobacco material had a darker color than after 4 months, and therefore suggesting that complete fermentation of the Virginia tobacco material had occurred. Furthermore, after 8 months, a pleasant and floral aroma was perceived.

[0198] chemical analysis In what follows, when values ​​relating to a sample are mentioned, the value given represents the average of several values ​​obtained for each sample, replicate, of the same type.

[0199] Figure 11 shows the evolution of total alkaloids (TA), reducing sugars (RS) and ammonia (NH3) during the fermentation process. These data confirm that the alkaloids, especially nicotine (not shown), are not affected by the anaerobic fermentation. The bacteria did not consume the main alkaloids as fermentation substrate. No increase in ammonia was observed when Virginia was used as the fermentation material, and nitrates, which were not present in this Virginia material, did not show any increase during and after the fermentation process. Figure 11 shows TA (total alkaloids), RS (reducing sugars), and ammonia (NH3) in the non-fermented Virginia (VG) material (SM) during the fermentation process (T1-T7) and after fermentation (AF). The data are expressed as DW percent. The data of TA (total alkaloids), RS (reducing sugars), and ammonia (NH3) in the non-fermented Virginia (VG) material (SM) after fermentation (AF) are also reported in the histograms of Figure 11, where the difference in values ​​between the non-fermented and fermented materials is immediately evident.

[0200] On the other hand, reducing sugars were used as substrate by the fermentation bacteria, as already observed in Examples 1 and 2. Thus, about 60 percent of the reducing sugars (RS) were oxidized during the 8-month fermentation, changing from 18.3 percent (VG-SM) to 7.4 percent (VG-AF) in dry weight (DW). A longer fermentation period may have led to a higher degradation rate of reducing sugars.

[0201] Further chemical analysis was carried out to compare the compounds in the starting material (VG-SM) and the fermented material (VG-AF).

[0202] The analysis shows a strong significant increase (about 10-fold) of lactate in the fermented material (VG-AF) (HS ID VG-19-20-NF, HS ID VG-19-20-F in Table 3) compared to the non-fermented material (VG-SM). Moreover, the amount of glucose and fructose in the fermented material (VG-AF) is considerably lower than the glucose and fructose content in the starting material (VG-BF). Glucose in the fermented material (VG-AF) is 0.6 times less than that in the non-fermented material (VG-SM), and fructose in the fermented material (VG-AF) is 0.4 times less than that in the non-fermented material (VG-SM). Thus, a reduction of about 60 percent can be observed after fermentation of reducing sugars. Moreover, the organic acids, citrate and malate, are also affected by fermentation, as also shown in Figure 1 for dark tobacco. Figure 12 shows the catabolism of tobacco reducing sugars, citrate, and malate followed by accumulation of pyruvate and lactate (lactic acid fermentation) during anaerobic fermentation of Virginia tobacco. Ratio data presented from metabolomic analysis (n=3), statistics are paired t-test (*p<0.05; **p<0.01; ***p<0.001).

[0203] Chemical analysis also confirmed that asparagine and glutamine were on the side of reducing sugars that were also consumed by anaerobic bacteria, and other free amino acids such as glutamate, histidine, proline, and tryptophan were also significantly degraded after fermentation of Virginia tobacco, whereas other amino acids, especially L-leucine and L-lysine, increased after fermentation of Virginia tobacco (see Figures 13A and 13B). Figures 13A and 13B show the consumed and produced free amino acids before and after fermentation of Virginia tobacco, respectively. Ratio data represented from metabolomic analysis (n=3), statistics are paired t-test (*p<0.05; **p<0.01; ***p<0.001). FIG. 14 shows the change in the content of quinic acid and caffeic acid. As already observed in Example 1 and Example 2, when the content of quinic acid and caffeic acid in fermented tobacco material was compared with that in non-fermented tobacco material, these compounds were significantly increased after fermentation. This is probably the result of cinnamoyl esterase catabolizing chlorogenic acid. Besides its strong antioxidant activity, increasing collagen production, and preventing premature aging, caffeic acid also exhibits antibacterial activity and may be promising for the treatment of skin diseases. Meanwhile, quinic acid is also a strong drug candidate to fight prostate cancer.

[0204] Indole-3-lactic acid (Figure 15) is significantly increased (>10-fold) during the tobacco fermentation process and originates from the catabolism of tryptophan. Thus, as lactic acid, the organic acid indole-3-lactic acid is also a good marker of lactic acid fermentation.

[0205] Example 4 This experiment was carried out in Switzerland under the same experimental conditions as used in Example 3.

[0206] In this experiment, a metal wine barrel is used that can pressurize moist tobacco substrates for two to ten months. Pressing equipment used for grapes and customized to the needs of the project is used. The barrel has a flat bottom, a conventional disk diameter, and a conventional pressure gauge. It can press at 0 to 8 kilograms per square centimeter. Pressing can be triggered manually or by an electric motor. As an optional part, a wooden disk is attached to the stainless steel pressing disk. The fermentation tank is modified for the test purposes, with a height of 70 centimeters, a diameter of 57 centimeters, a capacity of 100 kilograms, and an opening at the rear of the metal tank (barrel) that serves to unpack the substrate when turning the material in the middle or at the end of the fermentation run.

[0207] FC tobacco strips (CX B) from Brazil were used as the fermentation substrate, which was preconditioned with water to reach a final humidity of about 30 weight percent to about 50 weight percent water. About 100 kilograms of tobacco material are loaded into the fermentation tank and pressed. The pressure is kept at about 0.5 kilograms per square centimeter to about 1 kilogram per square centimeter. The tobacco material is maintained at a relative humidity of about 50 weight percent and a temperature of about 22°C during the entire fermentation process (6 months). After three months, the tobacco material is removed from the barrel, separated, mixed, and turned. About 30 weight percent to about 50 weight percent water is then added to the tobacco material, and then the barrel is reloaded.

[0208] The pressure and water levels were monitored periodically and corrected to prevent deviation from the target and any aerobic fermentation. The temperature inside the tank did not change during the entire run, as previously observed.

[0209] After 6 months, the tobacco material was pre-dried on a belt oven for a total drying time of about 8 minutes at different temperature ranges. The tobacco material was first subjected to a temperature of about 40°C, then to a temperature of about (70°C), and then to a temperature of about 60°C. The pre-drying process allows obtaining tobacco material with a relative humidity OV [percent] of about 25 percent by weight, allowing the strips to be cut into finer particles. The tobacco material is then cut into particles with a cutting width of about 1 millimeter. The tobacco material is then dried to a final moisture content of 10 percent by weight to about 15 percent by weight OV [percent]. The drying process was carried out in a rotary dryer at a temperature comprised between about 90°C and about 100°C for about 10 minutes and at a pressure of about 0.6 bar. The tobacco material is then finally ground to a specification of 70 micrometers for short storage.

[0210] The tobacco material in the two barrels was rotated once every two months for the six month period of the experiment.

[0211] Samples were taken before fermentation (VG-SM: starting material, six replicates), during the fermentation process, three months after the start of the fermentation process, and after fermentation (VG-AF: post-fermentation, six replicates).

[0212] Visual Observation The color of the tobacco material at the end of the fermentation process (VG-AF) was significantly darker compared to the starting material (VG-SM). After six months of anaerobic fermentation, the Virginia tobacco material exhibited a dark color indicating that complete fermentation of the Virginia tobacco material had occurred.

[0213] chemical analysis In what follows, when values ​​relating to a sample are mentioned, the value given represents the average of several values ​​obtained for each sample, replicate, of the same type.

[0214] FIG. 16 shows the difference in the contents of total alkaloids (TA) during the fermentation process, reducing sugars (RS), nitrates (NO3), and ammonia (NH3) before fermentation, after three months of fermentation, and at the end of the fermentation process. Total alkaloids (TA) are not affected by the anaerobic fermentation process. Reducing sugars are consumed by anaerobic bacteria and their content in the tobacco material decreases with fermentation. At the end of fermentation, the level of reducing sugars in the tobacco material is very low. In this case, reducing sugars were almost completely consumed after six months of fermentation and only one rotation. These data correspond to those obtained in Examples 1, 2, and 3.

[0215] Chemical analysis shows that lactic acid is produced during heavy fermentation. According to the metabolic pathways present in lactic acid bacteria, lactic acid is derived from the catabolism of reducing sugars (glucose and fructose), pyruvate, malate and citric acid, as already discussed in Example 3.

[0216] FIG. 17A shows the change in indole-3-lactic acid in the tobacco material of Example 4 (VG-CH). The content of indole-3-lactic is shown in micrograms per gram. The graph shows a significant increase in the content of indole-3-lactic acid in the fermented tobacco material. The data shown above for indole-3-lactic acid are obtained using samples of non-fermented tobacco material (VG-NFHS CH VG-19-20-NF) and fermented tobacco material (HS CH VG-19-20-F) subjected to measurement in ultra-high performance liquid chromatography polar and lipid positive and GC-MS polar negative ionization modes. The samples were measured on a Waters ACQUITY reversed-phase ultra-performance liquid chromatography (RP-UPLC) coupled to a Thermo-Fisher Exactive mass spectrometer consisting of an electrospray ionization source (ESI) and an Orbitrap mass spectrometer, and an Agilent Technologies mass spectrometer consisting of an electron impact ionization source (EI) and a time-of-flight (TOF) mass spectrometer. UPLC-MS determination of the aqueous phase allowed the detection of polar and semi-polar primary and secondary metabolites, while the organic phase allowed the detection of lipid and lipophilic content. GC-MS determination allows the analysis of primary metabolites.

[0217] Sample preparation Sample preparation was performed by metaSysX standard procedure, modified protocol of Salem et al. (Salem et al., Plant. Methods. 2016 45(12)). 20 (± 2) milligrams of ground material were used for metabolite extraction. Samples were extracted with MTBE:MetOH:H2O two-phase extraction method. Total (650 μl) organic phase was collected and dried for LC-MS lipid measurements. 450 μl of polar phase was collected and dried for LC-MS polar metabolite measurements, and 150 μl of polar phase was dried and derivatized for GC-MS measurements.

[0218] Standard curve preparation solution A stock of 2 milligrams / ml of indole-3-lactic was dissolved in water. Standard mixtures were prepared at concentrations of 10, 5, 2, 1, 0.5, 0.25, 0.125, 0.062 μg / ml. In addition, 200 μl of the standard mixture was either dried and subjected to the same extraction procedure (example) as the samples ("example"), or directly subjected to LC-MS analysis, or dried, derivatized, and analyzed by GC-MS (no example). The extraction procedure and samples of the standards were identical with all identical volumes.

[0219] Absolute content calculation Compound concentrations are calculated based on the extracted standard curve, taking all points (average of technical replicates) for the calculation and expressed as micrograms per milligram of sample weight.

[0220] These data are reported below in Table 3, which shows the indole-3-lactic acid content of diluted samples (standards) of the treated tobacco material sample of Example 4 (VG_IDC_AF), of the non-fermented tobacco material of Example 4 (VG_IDC_BF) and from different tobacco materials not subjected to the fermentation process (K326_75, TN90_110, K326_G, K326_110, TN90_G, K326_Dry Treated, TN90_75). [Table 3]

[0221] C-MS measurement (hydrophilic and lipophilic analytes) Samples were measured on a Waters ACQUITY reversed-phase ultra-performance liquid chromatography (RP-UPLC) coupled to a Thermo-Fisher Exactive mass spectrometer. C8 and C18 columns were used for lipophilicity and hydrophilicity measurements, respectively. Chromatograms were recorded in full-scan MS mode (mass range [100–1500]). All mass spectra were acquired in positive and negative ionization modes.

[0222] LC-MS data processing (hydrophilic and lipophilic analytes) Extraction of LC-MS data was achieved using the software PeakShaper (metaSysX GmbH). Alignment and filtering of LC-MS data was completed using in-house software. After extraction from the chromatograms, the data is processed, aligned and filtered for redundant peaks. Alignment of the data extracted from each chromatogram was performed according to the criterion that a feature must be present in all repeats of at least one of the groups. At this stage, the average RT and m / z values ​​are given to the features. Alignment was performed independently for each type of measurement.

[0223] LC-MS data annotation (hydrophilic, hydrophilic and analyte) An in-house metaSysX database of chemical compounds was used to match features detected by the LC-MS lipophilicity platform. Annotation of compounds of interest was performed by matching against the MSX database and confirmed by measured standards.

[0224] metaSysX (MSX) database The metaSysX in-house database contains mass-to-charge ratio and retention time information for 7500 reference compounds available as pure compounds, run under the same chromatographic and spectroscopic conditions as the measured samples. Additionally, 1500 lipids and sugar esters that are putatively annotated based on precursor m / z, fragmentation spectra, and elution patterns. Match criteria for DGDG annotation were 5 parts per million and 0.085 min deviation from the reference compound mass-to-charge ratio and retention time, respectively.

[0225] GC-MS measurement Samples were run on an Agilent Technologies GC coupled to a Leco Pegasus HT mass spectrometer consisting of an EI ionization source and a TOF mass analyzer.

[0226] Column: 30 meters DB35, starting temperature: 85°C for 2 minutes, gradient: 15°C per minute up to 360°C.

[0227] GC-MS data processing and annotation NetCDF files exported from Leco Pegasus software were imported into R. The Bioconductor package TargetSearch [3] was used to convert retention times to retention indices (RIs), align chromatograms, extract peaks, and annotate them.

[0228] The analyses carried out on the samples show that the content of indole-3-lactic in the fermented tobacco material (VG_IDC_AF) is 0.002520006 micrograms / milligrams and in the fermented tobacco material (VG_IDC_BF) is 0.000113988 micrograms / milligrams. These data are reported in Figure 17A. Comparing the results obtained in the above mentioned experiments, it is clear that the chemical analysis confirms a significant increase in lactic acid after anaerobic fermentation in all the experiments carried out. Fully fermented tobacco material contains about 50 mg / g to about 100 mg / g of lactic acid, while less than 4 mg / g can be found in non-fermented tobacco. Lactic acid data for different types of tobacco are reported in Table 2, where Rajangan (RAJ) tobacco RAJ ID KS-18-19, hand stripped (HS) dark tobacco HS ID KS-18-19, hand stripped Virginia tobacco HS ID VG-19-20, and hand stripped Virginia tobacco HS CH VG-19-20 are reported.

[0229] Rajangan (RAJ) Tobacco ID KS-18-19 refers to the tobacco material of Example 2, HS ID KS-18-19 refers to the tobacco material of Example 1, HS ID VG-19-20 refers to the tobacco material of Example 3, and HS CH VG-19-20 refers to the tobacco material of Example 4. [Table 4]

[0230] Table 4 shows the amount of lactic acid in the fermented tobacco material (F) and the non-fermented tobacco material (NF). The amount of lactic acid is shown in milligrams per kilogram, with reference to the dry weight of the tobacco material. The data show that all tobacco materials have an increase in the content of lactic acid after fermentation. All tobacco samples have similar amounts of lactic acid, thereby indicating that both the fast-cured cut filler tobacco with midrib and the corresponding hand-stripped cured processed material reach similar levels of lactic acid. Virginia tobacco reached similar levels of lactic acid compared to dark tobacco (compare HS CH VG-19-20-F [68.7 milligrams per gram] with HS ID KS-18-19-F [79.2 milligrams per gram]).

[0231] This confirms that tobacco fermentation can be carried out with different tobacco types, i.e., dark and Virginia, and that sugars and amino acids can be sources of carbon and nitrogen for the lactic acid bacteria. Even if tests have not been carried out with Burley and Orient tobacco, Orient tobacco has a composition that is often closer to dark tobacco, which is usually lower in alkaloids, despite not being affected by the anaerobic fermentation process.

[0232] The majority of lactic acid measured in fermented tobacco is the enantiomer L-lactic acid, which is consistent with the biochemical pathway of anaerobic bacteria. However, some D-lactic acid is also produced. Compared to L-lactic acid, D-lactic acid can be toxic to humans, i.e., the LD50 level per orally poisoned rat is about 4.5 g / kilogram (Pohanka, 2020). However, D-lactic acid and L-lactic acid are non-volatile and therefore do not migrate into aerosols.

[0233] Additionally, the fermentation ratios of the tobacco materials of Examples 1-2 and 4-5 were measured, and the data are collected in Tables 4 and 5 below.

[0234] The tobacco fermentation ratio is given by the following formula: Fr = (F LA / NF LA ):(F RS / NF RS ) in the formula, Fr = Fermentation Ration, F LA = content of lactic acid in fermented tobacco material, NF LA = initial content of lactic acid in the tobacco material, F RS = content of reducing sugars in fermented tobacco material, NF RS = initial content of reducing sugars in the tobacco material. [Table 5] [Table 6]

[0235] The tobacco fermentation index gives a further indication of the fermentation of the tobacco material. The tobacco fermentation ration makes it possible to monitor the level of fermentation in a tobacco sample. As shown in Table 5, the fermentation ration increases significantly during fermentation. Therefore, the fermentation ration is a very efficient indicator of the fermentation of the tobacco material.

[0236] In conclusion, the metabolomic data suggest that the compounds produced during tobacco fermentation, namely lactic acid, indole lactic acid, caffeic acid, and quinic acid, are not directly linked to tobacco type, since they are produced with both dark or flue-cured tobacco matrices. However, they can certainly vary quantitatively with respect to the previous abundance of the substrate compounds found in the starting tobacco leaf material. Thus, anaerobic fermentation is a process applicable to different tobacco types and materials, causing changes in the content of some substances in tobacco materials, independent of the type of tobacco material.

[0237] Certain substances may be used as indicators of the degree of fermentation of a tobacco material, and these substances may be used as reliable indications of the degree of fermentation.

[0238] The use of a number of indicators, as well as the correlation between, for example, color change, RS consumption, and lactic acid production by anaerobic bacteria, demonstrates the extent of fermentation of the tobacco material and gives a very reliable indication of the extent of fermentation.

[0239] Furthermore, the anaerobic process does not adversely affect the alkaloid content of the tobacco material.

[0240] The experiments carried out lead to the definition that the following conditions have a positive effect on fermentation and may increase the efficiency of the process: - pre-treating the manually peeled leaf material with water to reach a final humidity of about 40 to 60 percent by weight, expressed as relative humidity; - filling closed barrels, tanks or containers (barrels) with pre-conditioned strips to reach a capacity of approximately 100 kilograms; - maintaining a relative humidity of about 30 to 50 percent by weight of water in the sealed container during the fermentation process; - fermentation is carried out at a temperature of about 22 ° C; - Maintaining the temperature fairly stable during the fermentation process; - performing at least one rotation, removing the tobacco material, mixing the tobacco material, and reloading the tobacco material into the fermentation device during the fermentation process in order to homogenize the anaerobic fermentation process in the vessel; - During the fermentation process, a pressure of approximately 0.5 kilograms per square centimeter to 1 kilogram per square centimeter is applied, except when the tobacco is turned. - Drying the fermented tobacco material in a two-step drying process - subjecting the tobacco material to a first drying step in which the tobacco material is subjected to a temperature of about 60°C for about 8 minutes to reach about 25 percent OV; - subjecting the tobacco material to a second drying step at a temperature comprised between about 90°C and 100°C, at a pressure of about 0.6 bar, for about 10 minutes to reach 10 to 15 percent OV.

[0241] Meanwhile, reducing sugars were used as substrate by the fermentation bacteria, as depicted in Figure 10 and as already observed in Examples 1 and 2. Thus, about 60 percent of the reducing sugars (RS) were oxidized during the 8-month fermentation, changing from 18.3 percent (VG-BF) to 7.4 percent (VG-AF) in dry weight (DW). A longer fermentation period may have led to a higher RS ​​degradation rate.

[0242] The present invention is also directed to the following embodiments:

[0243] Embodiment 1. 1. A method for processing tobacco material, comprising: - providing tobacco materials; - fermenting a tobacco material to obtain a fermented tobacco material, the fermentation step comprising: Incubating the tobacco material under anaerobic conditions; The following conditions: the content of lactic acid is more than 10 times, preferably more than 20 times, more preferably more than 50 times, more preferably more than 70 times, preferably more than 80 times the initial amount of lactic acid in the tobacco material; the content of reducing sugars is less than 0.5, preferably less than 0.4, more preferably less than 0.2, more preferably less than 0.1 of the initial amount of reducing sugars in the tobacco material; the content of indole-3 lactic acid is more than 5 times, preferably more than 10 times, preferably more than 20 times the initial amount of indole-3 lactic acid in the tobacco material; the caffeic acid content is more than 4 times, preferably more than 10 times, preferably more than 20 times the initial amount of caffeic acid in the tobacco material; the content of quinic acid is more than two times, preferably more than four times, the initial amount of quinic acid in the tobacco material; the asparagine content is less than 0.5, preferably less than 0.4, preferably less than 0.3 of the initial amount of asparagine in the tobacco material; the glutamine content is less than 0.5, preferably less than 0.4, of the initial amount of glutamine in the tobacco material; the content of L-ornithine is more than 10 times, preferably more than 20 times, preferably more than 50 times, preferably more than 100 times the initial amount of L-ornithine in the tobacco material; the content of L-leucine is more than twice, preferably more than four times, the initial amount of L-leucine in the tobacco material; the content of L-lysine is more than twice, preferably more than six times, the initial amount of L-lysine in the tobacco material; and stopping fermentation when at least one of the following is satisfied: a fermentation index is greater than 50, preferably greater than 100, more preferably greater than 250, more preferably greater than 400, where the fermentation index is obtained by dividing the ratio of the lactic acid content in the treated tobacco material to the lactic acid content in the non-fermented tobacco material by the ratio of the reducing sugar content in the treated tobacco material to the reducing sugar content in the non-fermented tobacco material. Embodiment 2. 2. The method of embodiment 1, further comprising an initial measuring step for measuring an initial content of at least one of lactic acid, or reducing sugars, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or a fermentation index in the tobacco material. Embodiment 3. 3. The method of embodiment 1 or 2, further comprising a measuring step for measuring the content of at least one of lactic acid, or reducing sugar, or indole-3 lactic acid, or quinic acid, or caffeic acid, or L-ornithine, or asparagine, or glutamine, or L-leucine, or L-lysine, or a fermentation index in the tobacco material during the fermentation process. Embodiment 4. The method according to any one of embodiments 1 to 3, wherein when the tobacco material contains dark tobacco, the method is provided for stopping fermentation when at least one of the following conditions is met: the content of 2,3 butanediol is more than 5 times, preferably more than 10 times, the initial amount of 2,3 butanediol in the tobacco material; or the content of diacetyl is more than 5 times, preferably more than 10 times, the initial amount of diacetyl in the tobacco material. Embodiment 5. 5. The method of embodiment 4, further comprising an initial measuring step for measuring an initial content of 2,3 butanediol or diacetyl in the tobacco material before fermentation to obtain an initial amount of 2,3 butanediol or diacetyl, respectively, in the tobacco material. Embodiment 6. 1. A method for processing tobacco material, comprising: - providing tobacco materials; - fermenting a tobacco material to obtain a fermented tobacco material, the fermentation step comprising: - fermenting, comprising incubating the tobacco material under anaerobic conditions, wherein the incubating provides for applying a pressure to the tobacco material during the fermentation step of between 1000 kilograms / square meter and 15000 kilograms / square meter, preferably between 3000 kilograms / square meter and 12000 kilograms / square meter, more preferably between 5000 kilograms / square meter and 10000 kilograms / square meter. Embodiment 7. 1. A method for processing tobacco material, comprising: - providing tobacco materials; - fermenting a tobacco material to obtain a fermented tobacco material, the fermentation step comprising: - fermenting, including incubating the tobacco material under anaerobic conditions, wherein incubating is provided to continue the fermentation process for a fermentation time of at least 1 month, preferably at least 2 months, more preferably at least 4 months, more preferably at least 6 months, even more preferably at least 8 months, preferably at least 10 months, more preferably at least 12 months. Embodiment 8. 1. A method for processing tobacco material, comprising: - providing tobacco materials; - fermenting a tobacco material to obtain a fermented tobacco material, the fermentation step comprising: - fermenting, including incubating the tobacco material under anaerobic conditions, wherein the incubating provides for maintaining the temperature of the tobacco material at a temperature comprised between 21°C and 35°C, preferably between 25°C and 31°C, during the fermentation process. EMBODIMENT 9. A tobacco material obtained according to the method according to any one of embodiments 1 to 8. EMBODIMENT 10. A tobacco material comprising at least one of the following compounds: - lactic acid in an amount greater than 10 times, preferably greater than 20 times, more preferably greater than 50 times, more preferably greater than 70 times, more preferably greater than 80 times the initial amount of lactic acid in the tobacco material; - reducing sugars in an amount that is less than 0.5, preferably less than 0.4, more preferably less than 0.2, more preferably less than 0.1 of the initial amount of reducing sugars in the tobacco material; - indole-3 lactic acid in an amount greater than 5 times, preferably greater than 10 times, preferably greater than 20 times the initial amount of indole-3 lactic acid in the tobacco material; - caffeic acid in an amount greater than 4 times, preferably greater than 10 times, the initial amount of caffeic acid in the tobacco material; - quinic acid in an amount greater than two times, preferably greater than four times, the initial amount of quinic acid in the tobacco material; - asparagine in an amount less than 0.5, preferably less than 0.4, preferably less than 0.3 of the initial amount of asparagine in the tobacco material; - glutamine in an amount less than 0.5, preferably less than 0.4, of the initial amount of glutamine in the tobacco material; - L-ornithine is more than 10 times, preferably more than 50 times, preferably more than 100 times the initial amount of L-ornithine in the tobacco material; - L-leucine in an amount greater than twice, preferably greater than four times, the initial amount of L-leucine; - L-lysine in an amount greater than twice, preferably greater than six times, the initial amount of L-lysine; a fermentation index greater than 50, preferably greater than 100, more preferably greater than 250, more preferably greater than 400, where the fermentation index is obtained by dividing the ratio of the content of lactic acid in the tobacco material to the content of lactic acid in the non-fermented tobacco material by the ratio of the content of reducing sugars in the tobacco material to the content of reducing sugars in the non-fermented tobacco material. Embodiment 11. 11. The tobacco material according to any one of embodiments 1 to 10, wherein said tobacco material is obtained by a process comprising fermenting the tobacco material to obtain a treated tobacco material, comprising incubating the tobacco material under anaerobic conditions. Embodiment 12. A tobacco material comprising, on a total dry weight basis, at least 20 milligrams / gram, preferably at least 50 milligrams / gram, and more preferably at least 60 milligrams / gram of lactic acid. Embodiment 13. A tobacco material comprising: o Less than 3 percent total reducing sugars on a total dry weight basis; and / or o Tobacco materials containing less than 300 milligrams per kilogram of asparagine on a total dry weight basis. Embodiment 14. 1. A tobacco material containing, on a total dry weight basis, less than 70 milligrams per kilogram of glutamine. EMBODIMENT 15. 1. A tobacco material containing, on a total dry weight basis, greater than 10,000 milligrams per kilogram of total free amino acids. EMBODIMENT 16. A tobacco material comprising at least 1 microgram / gram, preferably at least 2 micrograms / gram, and more preferably at least 2.5 micrograms / gram of indole-3 lactic acid on a total dry weight basis. EMBODIMENT 17. 28. The tobacco material according to any one of embodiments 19 to 27, wherein the tobacco material is dried. EMBODIMENT 18. The tobacco material according to any one of embodiments 19 to 28, wherein the tobacco material is ground. EMBODIMENT 19. An aerosol-generating article comprising tobacco material, comprising from about 2.5 weight percent on a total dry weight basis to 100 weight percent on a total dry weight basis, preferably at least about 4 weight percent on a total dry weight basis, preferably at least about 10 weight percent on a total dry weight basis, preferably at least about 20 weight percent on a total dry weight basis of the tobacco material according to any one of embodiments 9-18. EMBODIMENT 20. 1. An aerosol-generating article comprising tobacco material, the article containing at least 5 milligrams / gram, and preferably 10 milligrams / gram, of lactic acid on a total dry weight basis.

Claims

1. 1. A method for processing tobacco material, said method comprising: providing a tobacco material; fermenting the tobacco material to obtain a fermented tobacco material; wherein the fermentation step comprises: incubating the tobacco material under anaerobic conditions; The following conditions: the content of lactic acid is more than 10 times, preferably more than 20 times, more preferably more than 50 times, more preferably more than 70 times, preferably more than 80 times the initial amount of lactic acid in the tobacco material; the content of reducing sugars is less than 0.5, preferably less than 0.4, more preferably less than 0.2, more preferably less than 0.1 of the initial amount of reducing sugars in the tobacco material; the content of indole-3 lactic acid is more than 5 times, preferably more than 10 times, preferably more than 20 times the initial amount of indole-3 lactic acid in the tobacco material; the caffeic acid content is more than 4 times, preferably more than 10 times, preferably more than 20 times the initial amount of caffeic acid in said tobacco material; the content of quinic acid is more than two times, preferably more than four times, the initial amount of quinic acid in the tobacco material; the asparagine content is less than 0.5, preferably less than 0.4, preferably less than 0.3 of the initial amount of asparagine in the tobacco material; the glutamine content is less than 0.5, preferably less than 0.4, of the initial amount of glutamine in the tobacco material; the content of L-ornithine is more than 10 times, preferably more than 20 times, preferably more than 50 times, preferably more than 100 times the initial amount of L-ornithine in the tobacco material; the content of L-leucine is more than two times, preferably more than four times, the initial amount of L-leucine in the tobacco material; the content of L-lysine is more than two times, preferably more than six times, the initial amount of L-lysine in the tobacco material; a fermentation index greater than 50, preferably greater than 100, more preferably greater than 250, more preferably greater than 400, said fermentation index being calculated by dividing the ratio of the lactic acid content in the treated tobacco material to the lactic acid content in the non-fermented tobacco material by the ratio of the reducing sugar content in the treated tobacco material to the reducing sugar content in the non-fermented tobacco material; stopping the fermentation when at least one of A method comprising:

2. 2. The method of claim 1, further comprising an initial measuring step for measuring an initial content of at least one of lactic acid, reducing sugars, indole-3 lactic acid, quinic acid, caffeic acid, L-ornithine, asparagine, glutamine, L-leucine, L-lysine, or a fermentation index in the tobacco material.

3. 2. The method of claim 1, further comprising a measuring step for measuring the content of at least one of lactic acid, reducing sugars, indole-3 lactic acid, quinic acid, caffeic acid, L-ornithine, asparagine, glutamine, L-leucine, L-lysine, or a fermentation index in the tobacco material during the fermentation step.

4. 2. The method of claim 1, wherein when the tobacco material contains dark tobacco, the fermentation is stopped when at least one of the following conditions is met: the content of 2,3 butanediol is more than 5 times, preferably more than 10 times, the initial amount of 2,3 butanediol in the tobacco material; or the content of diacetyl is more than 5 times, preferably more than 10 times, the initial amount of diacetyl in the tobacco material.

5. 5. The method of claim 4, further comprising an initial measuring step for measuring an initial content of 2,3 butanediol or diacetyl in the tobacco material before the fermentation to obtain an initial amount of 2,3 butanediol or diacetyl, respectively, in the tobacco material.

6. 2. The method of claim 1, wherein a pressure of from 1,000 kilograms per square meter to 15,000 kilograms per square meter, preferably from 3,000 kilograms per square meter to 12,000 kilograms per square meter, more preferably from 5,000 kilograms per square meter to 10,000 kilograms per square meter, is applied to the tobacco material during the fermentation process.

7. 2. The method according to claim 1, provided for continuing the fermentation process for a fermentation time of at least 1 month, preferably at least 2 months, more preferably at least 4 months, more preferably at least 6 months, even more preferably at least 8 months, preferably at least 10 months, more preferably at least 12 months.

8. 2. The method of claim 1, wherein provision is made to maintain the temperature of the tobacco material at a temperature comprised between 21°C and 35°C, preferably between 25°C and 31°C, during the fermentation step.

9. A tobacco material comprising: a) the following compounds: - lactic acid in an amount greater than 10 times, preferably greater than 20 times, more preferably greater than 50 times, more preferably greater than 70 times, more preferably greater than 80 times the initial amount of lactic acid in the tobacco material; reducing sugars in an amount less than 0.5, preferably less than 0.4, more preferably less than 0.2, more preferably less than 0.1 of the initial amount of reducing sugars in said tobacco material; indole-3 lactic acid in an amount greater than 5 times, preferably greater than 10 times, preferably greater than 20 times the initial amount of indole-3 lactic acid in the tobacco material; - caffeic acid in an amount greater than 4 times, preferably greater than 10 times, the initial amount of caffeic acid in said tobacco material; quinic acid in an amount greater than two times, preferably greater than four times, the initial amount of quinic acid in said tobacco material; asparagine in an amount less than 0.5, preferably less than 0.4, preferably less than 0.3 of the initial amount of asparagine in said tobacco material; glutamine in an amount less than 0.5, preferably less than 0.4, of the initial amount of glutamine in said tobacco material; - L-ornithine is more than 10 times, preferably more than 50 times, preferably more than 100 times the initial amount of L-ornithine in said tobacco material; - L-leucine in an amount more than twice, preferably more than four times, the initial amount of L-leucine, - L-lysine in an amount greater than twice, preferably greater than six times, the initial amount of L-lysine, a fermentation index greater than 50, preferably greater than 100, more preferably greater than 250, more preferably greater than 400, said fermentation index being obtained by dividing the ratio of the lactic acid content in said tobacco material to the lactic acid content in a non-fermented tobacco material by the ratio of the reducing sugar content in said tobacco material to the reducing sugar content in a non-fermented tobacco material; At least one of the following, or b) less than 300 milligrams per kilogram of asparagine on a total dry weight basis; or c) Less than 70 milligrams / kilogram of glutamine on a total dry weight basis; or d) greater than 10,000 milligrams / kilogram of total free amino acids on a total dry weight basis; or e) at least 1 microgram / gram, preferably at least 2 micrograms / gram, more preferably at least 2.5 micrograms / gram of indole-3 lactic acid on a total dry weight basis; , comprising tobacco materials.

10. 10. The tobacco material of claim 9, wherein the tobacco material is obtained by a process comprising fermenting the tobacco material to obtain a treated tobacco material, the process comprising incubating the tobacco material under anaerobic conditions.

11. The tobacco material of claim 9, wherein the tobacco material is dried.

12. The tobacco material of claim 9 , wherein the tobacco material is ground.

13. An aerosol-generating article comprising tobacco material, comprising from about 2.5 weight percent on a total dry weight basis to 100 weight percent on a total dry weight basis, preferably at least about 4 weight percent on a total dry weight basis, preferably at least about 10 weight percent on a total dry weight basis, preferably at least about 20 weight percent on a total dry weight basis of the tobacco material described in any one of claims 9 to 12.

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